Physiology of Microglia

Physiol Rev 91: 461–553, 2011;
doi:10.1152/physrev.00011.2010.
Physiology of Microglia
HELMUT KETTENMANN, UWE-KARSTEN HANISCH, MAMI NODA, AND ALEXEI VERKHRATSKY
Max-Delbrück-Center for Molecular Medicine, Berlin-Buch; University of Göttingen, Institute of

Neuropathology, Göttingen, Germany; Laboratory of Pathophysiology, Graduate School of Pharmaceutical
Sciences, Kyushu University, Fukuoka, Japan; and Faculty of Life Sciences, The University of Manchester,
Manchester, United Kingdom
I. The Discovery and Definition of Microglia 462

II. Microglia: Origin and Development 466
III. Evolutionary Origins of Microglia 466
IV. Microglia: From the Warden (Survelliance) to Cleaner (Macrophage): The Process of Activation 468
V. Morphology and Identification of Microglia 471
A. Morphology 471
B. Identification 472
VI. General Physiology of Microglia 474
A. Membrane properties of microglial cells in normal tissue 474
B. Membrane properties of microglial cells in pathological tissue 474
C. Membrane properties of microglial cells in culture 475
VII. Calcium Signaling in Microglia 477
A. Intracellular Ca2⫹ stores 477
B. Store-operated Ca2⫹ entry 478
C. Calcium extrusion 480
D. Ca2⫹ homeostasis and microglial cell death 480
VIII. Ion Channels in Microglia 480
A. Sodium channels 480
B. Calcium-permeable channels 483
C. Potassium channels 484
D. Anion channels 487
E. Proton channels 488
F. Aquaporins 488
G. Connexons 489
IX. Microglial Receptors: Concept of “On” and “Off” Receptor-Mediated Signaling 489
X. Neurotransmitter Receptors 493
A. Purinoceptors 493
B. Glutamate receptors 501
C. GABA receptors 502
D. Cholinergic receptors 503
E. Adrenergic receptors 504
F. Dopamine receptors 504
XI. Receptors for Neurohormones And Neuromodulators 505
A. PAF receptors 505
B. Bradykinin receptors 505
C. Histamine receptors 505
D. Endothelin receptors 505
E. Cannabinoid receptors 506
F. Angiotensin II receptors 507
G. Somatostatin receptors 507
H. Glucocorticoid and mineralocorticoid receptors 507
I. Opioid receptors 507
J. Neurokinin (substance P) receptors 508
K. Vasoactive intestinal polypeptide receptors 508
L. Neurotrophin receptors 508
XII. Cytokine and Chemokine Receptors 508
A. Chemokine receptors 508
B. TNF-␣ receptors 510
www.prv.org 461
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462 KETTENMANN ET AL.
C. Interleukin receptors 511

XIII. Pattern-Recognition Receptors 511
A. Toll-like receptors 511
XIV. Other Receptor Systems 514
A. Calcium receptors 514
B. Leukotriene receptors 514
C. Notch receptors 515
D. Complement receptors 516
E. Thrombin receptors 517
F. Macrophage Colony-Stimulating factor receptors 517
G. Epidermal growth factor receptors 517
H. CD200 receptors 517
I. Lysophosphatidic acid receptors 518
J. Formyl peptide receptors 518
K. Sigma receptors 518
XV. Microglial Plasmalemmal Transporters 518
A. Xc transporters 518
B. Glutamate transporters 518
C. Glucose transporters 519
D. Monocarboxylate transporters 519
E. ATP-Binding cassette transporters 519
F. Chloride transporters 520
G. Bicarbonate transporters 520
H. H⫹/K⫹ pump 520
I. Proton pump 520
XVI. Migration and Motility 520
A. Control of processes motility in vivo 520
B. Control of microglial migration 521
C. Inhibitors of microglial migration 523
D. Microglial residence in the brain 524
XVII. Phagocytosis 524
XVIII. Neuronal-Microglial Interaction: Possible Role in Neural Plasticity? 525
XIX. Outlook and Future Directions 528
Kettenmann H, Hanisch U-K, Noda M, Verkhratsky A. Physiology of Microglia. Physiol Rev 91: 461–553, 2011;
doi:10.1152/physrev.00011.2010.—Microglial cells are the resident macrophages in the central nervous system.
These cells of mesodermal/mesenchymal origin migrate into all regions of the central nervous system,
disseminate through the brain parenchyma, and acquire a specific ramified morphological phenotype termed
“resting microglia.” Recent studies indicate that even in the normal brain, microglia have highly motile
processes by which they scan their territorial domains. By a large number of signaling pathways they can
communicate with macroglial cells and neurons and with cells of the immune system. Likewise, microglial cells
express receptors classically described for brain-specific communication such as neurotransmitter receptors
and those first discovered as immune cell-specific such as for cytokines. Microglial cells are considered the
most susceptible sensors of brain pathology. Upon any detection of signs for brain lesions or nervous system
dysfunction, microglial cells undergo a complex, multistage activation process that converts them into the
“activated microglial cell.” This cell form has the capacity to release a large number of substances that can act
detrimental or beneficial for the surrounding cells. Activated microglial cells can migrate to the site of injury,
proliferate, and phagocytose cells and cellular compartments.
I. THE DISCOVERY AND DEFINITION 2) These invading cells have amoeboid morphology and
oF MICROGLIA are of mesodermal origin. 3) They use vessels and
white matter tracts as guiding structures for migration
Pio del Rio-Hortega introduced the concept of mi- and enter all brain regions. 4) They transform into a
croglia (Fig. 1) as a defined cellular element of the branched, ramified morphological phenotype in the
central nervous system in a book chapter called “Mi- more mature brain (known today as the resting micro-
croglia” (208) written for the landmark publication Cy- glia). 5) In the mature brain, they are found almost
tology and Cellular Pathology of the Nervous System, evenly dispersed throughout the central nervous sys-
edited by Wilder Penfield in 1932. In this visionary tem and display little variation. 6) Each cell seems to
article, which we strongly recommend to read in orig- occupy a defined territory. 7) After a pathological
inal, del Rio-Hortega postulated the following: 1) mi- event, these cells undergo a transformation. 8) Trans-
croglia enter the brain during early development. formed cells acquire amoeboid morphology similar to
Physiol Rev • VOL 91 • APRIL 2011 • www.prv.org

PHYSIOLOGY OF MICROGLIA 463
FIG. 1. Microglial cells discovered by Pio del Rio-Hortega. A: Pio del Rio-Hortega (1882–1945). B: images of ramified microglial cells drawn by
Hortega. [From del Rio-Hortega (202).] C: evolution of microglia during its phagocytic activity. A, cell with thick, rough prolongations; B, cells with
short prolongations and enlarged cell body; C, hypertrophic cell with pseudopodia; D and E, amoeboid and pseudopodic forms; F, cell with
phagocytosed leukocyte; G, cell with numerous phagocytosed erythrocytes; H, fat-granule cell; I, cell in mitotic division. [Photomicrographs from
del Rio-Hortega (208).]
the one observed early in development. 9) These cells even referred to as “Hortega cells” (583). In the first two
have the capacity to migrate, proliferate and phagocy- glial books published after the second world war, these
tose. cells were already termed microglia cells (324, 992), the
All these statements are perfectly valid today and name which (being slightly modified to microglial cells)
could appear in a modern textbook of neuroscience with- has remained in the present day.
out the smallest change. Del Rio-Hortega had based his The concept of neuroglia was introduced by Rudolf
postulates on studies published in a series of articles Virchow (963, 964; for historical overview, see also Refs.
between 1919 and 1927 (202–207, 209, 210) in which he 443, 956), and his first drawings of glia do not resemble
used a modified silver carbonate impregnation to label the the modern image of an astrocyte or an oligodendrocyte,
microglial cells. The technique was tedious, varied in but look rather similar to an activated microglial cell. It is
efficiency between species, but when working yielded therefore unclear what Virchow has depicted. At that time
excellent images of these cells. and for the next 50 years or so, different types of glial
Del Rio-Hortega called the newly discovered cell cells were not well defined despite the introduction of the
class microglia and the individual cell “microgliocyte.” In term astrocyte in 1891 (516, 517). Nonetheless, patholo-
some of the contemporary publications, these cells were gists had noted the appearance of morphologically dis-

tinct cells in pathological tissue, cells which differed very scribed cells in patients with neurodegenerative diseases
much from those in the normal brain. The origin of these such as syphilitic paralysis, which they termed rod cells
“pathological” cells was heavily debated. In 1878, Carl (Stäbchenzellen), grid cells (Gitterzellen), or clearance
Frommann (300) identified cellular changes in defined cells (Abräumzellen).
areas of the brain and spinal cord when examining tissue The origin of these cells was debated. Conceptually,
from a deceased 22-yr-old multiple sclerosis patient glial cells were believed to be of ectodermal origin, although
(Fig. 2). Frommann had the most modern vision at the Nissl concluded that pia cells contribute to the formation of
time in postulating that the glial cells change their mor- rod cells and considered them as mesodermal (647). Rob-
phology. He described that their soma became larger and ertson, a Scottish pathologist, described a new form of glia
the cellular processes shorter and less numerous. From- termed mesoglial cells (762), which he also considered to be
mann also noted a higher density of glial cells in the of mesodermal origin. He also noted that unlike the macro-
“epicentres of damage” (Heerdsubstanz), and moreover, glial cells (most likely he referred to astrocytes), the me-
he found that some of the glial cells in these “epicenters” soglial cells do not contact blood vessels. He also mentioned
had a granulated soma containing inclusions. It seemed in his Textbook of Pathology (763) that mesoglial cells with-
obvious to him that the glial cells transformed in the draw their processes and transform into granule cells in
multiple sclerosis pathology. response to injury. He was close to unraveling the mystery
With later studies, the field became more unclear. In of the microglia, and therefore, Glees (324) suggested to
the late 19th to early 20th century, several scientists found name microglia the “Robertson-Hortega” cells.
and described microglial cells in the pathological brain Also with his developmental concept, del Rio-
(Figs. 2 and 3) without clearly understanding their origin. Hortega had predecessors. Campobianco and Fragnito
Nissl, Alzheimer, and Merzbacher (14, 15, 582, 647) de- (117) and Campobianco (116) reported that at the early
FIG. 2. Neuroglial cells in pathological context. A and B: different types of glial cells found in multiple sclerosis plaques of human cortex.
C: glial cell close to a 14-day-old hemorrhage in human white matter. Axons pass through the network of the cell. [A and B from Frommann (300);
C from Alzheimer (14).]

FIG. 3. Activated glial cells associ-

ated with neurons. Top left: amoeboid
microglial cells in different transition
forms from human pathologic tissue
(psychosis, status epilepticus). Top right:
diverse amoeboid glial cells of different
human pathological tissue (infection, ep-
ilepsy, psychosis). Bottom: examples of
association with amoeboid glial cells
with neurons in brains of humans with
different degenerative diseases. Aglz, ac-
tivated glial cell; Gaz, ganglion cell; Glz,
glial cell; Abp, degenerative material; Ez,
endothelium cell; Cap, capillary. [From
Alzheimer (15).]
embryonic stages a number of mesoblastic cells migrate separated from the vessel wall, became amoeboid, and
into the nervous system and are transformed into neuro- migrated away from the capillary. He concluded that the
glia. Hatai (362) described two types of glial cells in the brain contains two types of glial cells, one of ectodermal
early postnatal brain of mouse and rat. One form he ter- and the other of mesodermal origin. All in all, the time
med the type “a” cell, which he considered to be the was ripe for the discovery of microglia. What should,
ectodermal derived glial cells. The second type, the type however, be emphasized is the fact that it was the intro-
“b” cell, was morphologically distinct. Based on his mor- duction of a novel staining technique which enabled del
phologic studies, Hatai concluded that these “b”-type cells Rio-Hortega to finally convince his peers.

In the years to follow, del Rio-Hortega’s observations question has been experimentally addressed by irradiat-
were confirmed and further elaborated. Kershman (441) ing animals to remove monocytes with subsequent bone
studied the infiltration of microglia in embryonic human marrow transplantation with, for example, GFP-labeled
development and described hot spots of immigrations monocytes. Early reports indicated that monocytes in-
such as at the plexus choroideus and coined the term vaded the brain also in adulthood (376, 837). Recent stud-
microglia fountains for these sites. For many years, how- ies, however, demonstrated that the exchange of micro-
ever, the field did not advance beyond the statements of glial cells in the normal undisturbed brain is almost neg-
del Rio-Hortega formulated in 1932. A detailed historical ligible (10, 586). An essential step in the latter study was
account on the concept of mesodermal cells in the CNS the protection of the brain from irradiation. After blood-
can be found in References 38 and 758. brain barrier damage, a subpopulation of monocytes en-
The modern era of microglial research started in the ters the brain and transforms into microglia (586). The
late 1960s when Georg Kreutzberg introduced the facial obvious conclusion is that in a healthy, intact brain, the
nerve lesion model (71). This preparation allowed study- microglial cells exist as a stable population. As reviewed
ing microglial responses to injury in tissue with an intact by Chan (138), there is also an embryonic invasion of cells
blood-brain barrier. It also provided the possibility to that gives rise to a microglial population. This occurs
distinguish between responses of intrinsic microglia and from the middle of the first trimester and throughout the
invading monocytes and helped to establish the concept early part of the second trimester in human and between
that microglial cells are important for both de- and regen- embryonic days 10 and 19 in rodents. There is evidence
eration of the brain. Despite the fact that in vitro cultures for at least two separate populations of microglial cells.
of microglial cells were described as early as 1930 by One population is derived from progenitors that are of
Costero (172), their introduction as a wide-spread tool to myeloid/mesenchymal origin (not necessarily derived
study microglial properties and functions happened al- from monocytes). The second population represents a
most half a century later after the development of a developmental and transitory form of fetal macrophage,
method of obtaining large numbers of microglia from the and this cell is related to amoeboid microglial population
postnatal brain (319). This has led to an explosion of as described in the postnatal brain of rodents (757).
studies on cultured microglia. Unfortunately, microglial After invading the brain parenchyma, microglial cells
cells recognize the cell culture environment as alien with transform into the ramified phenotype. We know much
the consequence that all these in vitro studies may not more what controls the activation of microglia compared
faithfully reflect properties of microglia in the normal, with what converts them to the “resting,” ramified pheno-
non-pathological brain. The combination of advanced im- type. Some cell culture studies provide certain hints: as-
aging techniques with the use of genetically based cell- trocyte conditioned medium increases ramification of
specific markers has recently allowed investigations of blood monocytes in culture (835). ATP or adenosine mim-
microglia in the undisturbed tissue (190, 645). icked the astrocyte conditioned medium, but could not be
confined to a distinct purinoceptor subtype. Combining
astrocyte conditioned medium with ATP or adenosine
II. MICROGLIA: ORIGIN AND DEVELOPMENT
yielded a phenotype with more extensive ramification,
indicating that purines are not the only ramification-in-
The origin of microglia has been debated for a long
ducing factors of microglia (997). Further candidates are
time. There were arguments that these cells stem from the
cytokines released from astrocytes, namely, transforming
neuroectoderm (271). Today there is general consensus
growth factor-␤ (TGF-␤), macrophage colony-stimulating
that microglial cells are derived from progenitors that
factor (M-CSF), and granulocyte/macrophage colony-
have migrated from the periphery and are from mesoder-
stimulating factor (GM-CSF). The ramification in re-
mal/mesenchymal origin (for review, see Ref. 138). In
sponse to astrocyte medium was prevented by neutraliz-
rodents, these cells immigrate from the blood system as
ing antibodies against TGF-␤, M-CSF, or GM-CSF (804).
monocytic cells. They originate from bone marrow. Dur-
The activity of Cl⫺ channels seems to be a requirement
ing postnatal development, they immigrate into the brain
for the cells to make the morphological transformation.
commonly until postnatal day 10 in rodents. These immi-
Pharmacological blockade of Cl⫺ channels prevented the
grating cells are amoeboid and can thus be easily recog-
ramification process (242). It should, however, be noted
nized due to their morphology, for instance, in acute
that the Cl⫺ channel pharmacology is not very specific.
slices of the corpus callosum (86, 342; see also supple-
mentary videos 1 and 2).1
In the adult animals there seems to be very little III. EVOLUTIONARY ORIGINS OF MICROGLIA
exchange between blood and brain parenchyma. This
The evolutionary origins of microglia remain largely
1
The online version of this article contains supplemental materials. unexplored; however, it is conceivable to assume that

they appeared simultaneously with an emergence of com- ATP and its analogs, which induce migratory movement;
pact neuronal masses and were represented by myeloid effects of purines were antagonized by reactive blue 2,
cells entering the neuronal ganglia. The evidence for mi- suggesting the role for P2 purinoceptors (229).
croglial cells is available for leech (Annelids), bivalves Opioids have been postulated to modulate various
and snails (Molluscs), and insects (Arthropods). immune responses also in leech. Indeed, morphine and
Microglial cells of invertebrates were best studied in naloxone inhibited the microglial accumulation at the
the leech. Following injury, the leech nervous system has lesion site. There is evidence that mu and kappa opioid
the capacity to regenerate as evidenced by axonal sprout- receptors mediate the response (1015). The accumulation
ing. In the injured nervous system, small amoeboid cells of microglia is a prerequisite for axonal sprouting. When
that have the capacity to phagocytose and migrate had microglial accumulation was inhibited by 3 mM ATP or a
been observed. These cells can be labeled by weak silver NOS inhibitor, there was a significant reduction in total
carbonate, a classical stain for vertebrate microglia and, sprout lengths compared with controls when microglial
thus, these leech cells were also termed microglia. Within accumulation was reduced (640). Thus, in the leech ner-
24 h after the injury, these cells accumulate at the lesion vous system, microglial cells are important elements to
site, indicating that they respond to nerve injury (612). control regeneration (631).
Live imaging in leech nerve cords revealed that only a Similar mechanisms are also present in molluscs.
fraction of microglia, typically ⬍50%, moved at any time Microglial cells can be observed to egress from excised
with a speed of up to 7 ␮m/min. Cells were moving within ganglia of the mussel Mytilus edulis, a marine bivalve,
15 min of crushing directly toward the lesion along axons and this migratory activity was naloxone-sensitive. The
or axon tracts (565). The leech microglial cells were also authors conclude that morphine can stimulate NO re-
implicated in production of antimicrobial peptides in release, which counteracts the egress of microglia from the
sponse to infectious attack (800). ganglia (531). The morphine-induced NO release medi-
Cell culture experiments revealed that leech micro- ated by activation of the opiate alkaloid-selective, opioid
glial cells maintained on the plant lectin concanavalin A peptide-insensitive micro3 receptor, which is similar in
(Con A) had a rounded shape, remained stationary, and human monocytes and invertebrate immunocytes, indi-
were avoided by growth cones (50). In contrast, microglia cating that functional coupling of morphine to NO pro-
cultured on extracts of leech extracellular matrix were duction has been conserved during 500 million years of
mobile, spindle-shaped with long processes, and did not evolution (545). Invertebrates and vertebrates share even
influence neurite growth (557). Both types of microglia, more signaling pathways: anandamide, an endogenous
the round and the spindle-shaped, had similar membrane cannabinoid receptor ligand, is found in invertebrate im-
properties in culture. They were characterized by a high munocytes and mammalian monocytes. Anandamide trig-
input resistance similar to microglial cells from acutely gered the release of NO both from Mytilus edulis micro-
isolated slices of rodents and distinct from cultured roglia and human macrophages. The release was blocked by
dent microglia. A small cationic conductance, which has a NOS inhibitor and a cannabinoid antagonist (855). More-
not been described for rodents, was also present (865). over, recombinant human interleukin-10 (IL-10) inhibits
In response to tissue damage, leech microglia, simi- the migration of microglia from excised ganglia of Myti-
larly to vertebrates, migrate to the site of injury, change lus edulis. A substance immunoreactivity similar to hu-
their morphology (248, 556), and acquire phagocytic prop- man IL-10 was detected in ganglia homogenates. This
erties (706). When the leech nerve cord is injured, micro- IL-10 is another example of a signaling pathway in micro-
glia migrate towards the lesion and accumulate at the glia, which has evolved early in evolution. A third com-
damage site (145, 482, 556, 970). In response to injury, mon signaling pathway found in invertebrates and verte-
leech connective glia and some microglia express endo- brates is represented by 17␤-estradiol. This estrogen in-
thelial nitric oxide synthase (eNOS) as revealed by immu- hibits the migration of Mytilus edulis microglia from the
nolabeling. Minutes after the injury, eNOS activity could ganglia and is also coupled to the release of NO. Indeed,
be detected as indicated by a histochemical labeling for the authors could identify a fragment of the estrogen
NADPH diaphorase (822). The NO could mediate the receptor-beta gene with 100% sequence identity to the
accumulation of microglia at the lesion site in the leech: a human counterpart (856). The egress from the excised
NOS inhibitor significantly reduced microglial accumula- ganglia as an assay of microglial activity was also de-
tion. The nitric oxide (NO) donor spermine NONOate also scribed for the snail Planorbarius corneus and the insect
inhibited accumulation, reduced average microglial mi- Leucophaea maderae (847). Traumatic injury triggers pro-
gratory speed, and even reversibly arrested cell move- liferative response in microglial cells from Planorbarius
ment, indicating that NO is a stop signal for microglia and corneus (712); activation of microglia was associated with
results in accumulation (145). NO’s action on the migra- expression of inducible NOS (iNOS) (711). Evidence also
tory behavior of leech microglia may be mediated by exists that opiate signaling regulates microglial activities
cGMP (228). The leech microglia also has sensitivity to in this mollusc (848).

Similar to the vertebrate system, apoptosis occurs in neurotrophic factors and the physical association with
the nervous system during Drosophila development. Ap- endangered neurons.
optotic neurons are engulfed by a variety of glia including The stages of microglial activation were defined
midline glia, interface (or longitudinal tract) glia, and based on morphological, molecular, and functional char-
nerve root glia. However, the majority of apop- acteristics, with fully activated microglia presenting them-
totic cells in the CNS are engulfed by subperineurial glia selves like other macrophages (169, 192, 351). Yet these
in a fashion similar to the microglia of the vertebrate CNS. cells are embedded in a specialized tissue (brain paren-
In a Drosophila mutant, which lacks macrophages, sub- chyma) which also has to protect itself from the poten-
perineurial glia contain an abundance of apoptotic cells, tially damaging consequences of an immune reaction.
indicating that there is a cross-talk between macrophages This special condition has been described as the “immune
and subperineurial glia resulting in the removal of apop- privilege” of the CNS (306). The microglial activation is
totic cells (849). Inhibition of a receptor required for thus a highly regulated process. The CNS is a complex
macrophage-mediated engulfment of dead cells causes organ with regional variations in glial and neuronal cell
CNS defects, indicating that macrophage-mediated clear- populations as well as the biochemical milieu conditioned
ance of cell corpses is required for proper morphogenesis by and for them. Microglia may thus not necessarily rep-
of the Drosophila CNS (817). resent a homogeneous population. This has been impres-
sively shown in a study on the role of the chemokine/
receptor system CXCL10/CXCR3 for neuronal cell death
IV. MICROGLIA: FROM THE WARDEN and glial activation in the mouse hippocampus (951).
(SURVELLIANCE) TO CLEANER Based on organotypic slice cultures and N-methyl-D-as-
(MACROPHAGE): THE PROCESS OF partic acid (NMDA)-induced excitotoxicity, the authors
ACTIVATION found that the different vulnerability of CA1 versus CA3
neurons depends on microglia. Constitutive heterogeneity
Microglia in the healthy mature CNS, including the as a kind of diversity “to start with” may thus predestine
brain, spinal cord, as well as the eye and optic nerve, have microglial performance and overlay with the inducible
a ramified morphology, a small soma with fine cellular diversity upon stimulations (351).
processes. This typical appearance, which is quite differ- Until recently, microglia of the healthy adult CNS
ent from a classical macrophage, has been associated were considered quiescent, i.e., functionally dormant, due
with microglial “resting” state. Infection, trauma, ische- to the low or absent expression of activation-associated
mia, neurodegenerative diseases, or altered neuronal ac- molecules and their “immobile” ramified morphology
tivity, that is any disturbance or loss of brain homeostasis (479). In reality, however, these “resting” cells actively
indicating real or potential danger to the CNS can evoke scan their environment. With motile processes, and by
rapid and profound changes in the microglial cell shape, constant integration and interpretation of environmental
gene expression and the functional behavior which sum- cues, microglia monitor their extracellular space and cel-
marily is defined as “microglial activation” (72, 169, 192, lular neighborhood, always ready to transform to execu-
334, 351, 479, 869, 949). Phenotypically, the complexity of tive states of activity. The in vivo imaging studies employ-
the cellular processes is reduced, and microglia revert to ing mice with EGFP-expressing microglia demonstrated
an amoeboid appearance (Fig. 4). Microglia can become that their fine cellular processes are in constant motion
motile and actively move to a lesion or herd of infectious (190, 645). Moving the fine processes the territorially
invaders following chemotactic gradients. Local densities faithful microglia can scan the environment without dis-
of microglia can also increase by proliferation, to provide turbing the fragile neuronal circuitry. The “resting” micro-
more cells for the defense against invading germs and to glia thus actively survey the tissue, being ready to rapidly
organize for the protection and restoration of tissue ho- transform to “activated” states upon appearance of signs
meostasis. Induction and rearrangement of surface mole- indicating a threat to the CNS. “Activated” microglia will
cules for cell-cell and cell-matrix interactions, changes in then primarily serve for a support and protection of the
intracellular enzymes as well as release of multiple fac- structural and functional integrity of the CNS.
tors and compounds with proinflammatory and immuno- Nevertheless, while assuming that the surveillance
regulatory effects are additional elements of the activa- function of microglia is important for the maintenance of
tion process. Microglia can unfold their phagocytotic ac- CNS homeostasis, the housekeeping activity of microglia
tivities to clear tissue debris, damaged cells, or microbes. may remain largely unnoticed (351). A proper “physiolog-
Release of chemoattractive factors recruits and guides ical” performance of microglia could be simply over-
immune cell populations to the CNS, and presentation of looked. Microglia would, in case of a small vascular defect
antigens to T cells can subsequently aid the adaptive or an isolated neuronal impairment, act immediately to
immunity in fight against viral or bacterial invasion. The offer protection and trophic support, even reduce activat-
range of microglial activities also covers production of ing synaptic input by the phenomenon of synaptic strip-

FIG. 4. Microglial activity states throughout an activation process. The “resting” microglia constantly and actively scan their environment for
exogenous or endogenous signals indicating a threat to the homeostasis. Sudden appearance of “activating” signals or a loss of constitutively
“calming” inputs can then trigger transitions to alerted and activated states. Cells can commit to distinct reactive phenotypes depending on the
challenging stimuli and the situational context. Initial response profiles may further shift as instructed by additional influences. Not only resident
CNS cells, but also invading immune cells would exert such modulating influences. Initial reactive phenotypes with defense orientation may convert
to repair-orientated activity profiles. Cells may eventually return to a resting state or stay “experienced.” Experienced microglia could reveal altered
responsiveness and exert distinct responses upon rechallenge. [Based on Hanisch and Kettenmann (351).]
ping (923, 972). Such locally restricted and transient ac- for failure and harmful contributions as they have no
tivities would, however, not be recognized. In other balance for the beneficial outcome of microglial engage-
words, little is known about the impact of the daily func- ment. It would be important to focus in the future on the
tion of microglia. On the contrary, failure to offer protec- role of microglia in health to better unravel the deviations
tion, generation of maladapted responses, or excessive in cellular function that, occasionally, cascade into dis-
and chronic activation is detected when surfacing with ease.
clinical symptoms or, in the worst case, by histopatholog- The transition from the surveillance mode of a “rest-
ical evidence. The neuropathological records hence bias ing” cell to the executive states thereby represents more

of a shift in activities rather than “activation” per se. This 111, 128, 192, 247, 287, 327, 351, 438, 475, 485, 487, 488,
view acknowledges that microglia have a constant func- 552, 755, 809, 810, 825, 854, 872, 993). Heterogeneity was
tional importance. There are no periods of inactivity. also observed in microglia from the aging brain where
Moreover, the term activation does not contain any in- surprising differences between individuals were found
formation about the functional orientation, which decides (579, 834).
on the CNS consequences (919). Instead, there is increas- An influence on the expression profiles and func-
ing evidence that microglial activation is not an “all-or- tional properties could result from the major tissue archi-
none” process, neither is it a linear path with a fixed tecture. White and gray matter microglia were recently
uniform outcome, to the contrary, activated microglia can reported to differ in the expression of the immunoregu-
acquire distinct functional states (128, 166, 351, 705, 816). latory receptor Tim-3 (16). Rapid ex vivo methods now
There is ample evidence that microglia undergo allow for “snapshot” analyses of multiple protein expres-
changes in shape and expression patterns during the sions by microglia of different CNS regions (193). Here,
course of an activation in vivo. Findings derive from too, first dissociations in populational profiles seem to
responses to injury (152, 488, 743), ischemia (778), or follow a sorting by white matter index, indicating that the
autoimmune challenges (722). Microglial activation may myelin environment requires and/or dictates a special
start with an early emergency response, such as defense- microglial setting that is different from that in gray mat-
oriented functions, to fight off an infection or to limit ter. These differences could impact on development, nor-
further damage after an injury. The activated population mal functionality, as well as vulnerability to inflammation
with an initially chosen program may eventually convert (390). Interestingly, the heterogeneity of microglia is mir-
to a repair-oriented support for tissue restoration. The rored by heterogeneity of oligodendroglial subpopula-
scheme in Figure 4 highlights the major components of tions in subregions of gray and white matter (459).
such a dynamic response. The exposure to neurotransmitters, the proximity to
While still a lot has to be learned about the events blood vessels, and properties of the blood-brain barrier
affecting microglia throughout the activation process, controlling the microenvironment (3) may associate with
even less is known about the period after. A terminated “provincial” adaptations of microglia (192). Whether these
microglial response, in the best case successful and not specificities concern mainly housekeeping activities, such
even much noticed, may still leave traces (351). Are there as the scanning of neuronal structures or whether they
subtle alterations within a previously activated microglia? become important only upon further activation is not
The postactivated microglia may remain undistinguish- known. The more information will become available on
able by morphology or isolated criteria of detection (via the constitutive heterogeneity of “the” microglia by ana-
marker staining) from the “resting” cells in nearby popu- tomical divisions, the more studies on inducible diversity
lations, while still bearing long-lasting adjustments. Re- have to respect that microglial behavior can vary by lo-
ceptors with regulatory influences on the transcriptional cation, i.e., superimposing constitutive and inducible di-
control in macrophages have been reported, and these versity.
regulations could be selective for sets of genes (197, 289, Being consequent, the demonstration of a white-ver-
962). Epigenetic mechanisms organizing longer-lasting ad- sus-gray-matter distinction in receptor expression (16)
justments are already known. The experienced cell could could lead down the road to the hypothesis that special-
then behave differently when being challenged again. As a ized cells reside even among a regional population. Not or
local population, such a microglia could influence the only barely visualized by the conventional detection tool-
likelihood of a tissue region to develop age-related dys- kit, individual microglial cells could be predestined to
function or to succumb to neurodegenerative processes certain functions. Indeed, population heterogeneity was
(72, 170). Conceivably, this principle could also have reported already in one of the first papers dealing with
some protective effect, when the former activation pre- microglial protein expression by anatomical subdivisions
pares the affected population to respond faster or more (247). Neurotrophins were seen in microglia with a re-
efficient upon a second encounter. gion-specific pattern, but also within a region, only sub-
When discussing the process of activation, the func- populations contributed to their production. Strong sup-
tional heterogeneity of microglia has to be also consid- port for such individuality came more recently from
ered. Regional heterogeneity and populational segrega- studies on TREM-2 (triggering receptor expressed on my-
tion of microglia in tissues and preparations were eloid cells-2) (809, 810). Found as being expressed by
demonstrated by morphology, selective detection of con- inactivated microglia, and downregulated upon lipopolysac-
stitutive or inducible mRNAs and proteins [e.g., for charide (LPS) or interferon (IFN)-␥ encounter, TREM-2 var-
TNF-␣, IL-6, IGF-I, integrins, CD4, CD11c, CD34, CD40, ied in its expression by microglia not only from region to
CD45, CD86, major histocompatibility complex (MHC) region, but also within each. The authors interpreted these
class II, Fc␥RII, iNOS, and members of the neurotrophin observations as a sign for the role of the microenvironment
family], or differences in the proliferative potential (6, in instructing a microglial phenotype. Cell-to-cell variability,

indeed, is gaining attraction (842), and the novel technical microglial transformation from resting to activated states
approaches will certainly contribute to evaluate the extent is accompanied by marked morphological changes. Mi-
as applying to microglia. croglia reduce the complexity of their shape by shorten-
ing (retracting) the branches of their processes so that
they are resorbed into the cell body. Several steps and
V. MORPHOLOGY AND IDENTIFICATION OF
intermediate stages can be identified, including character-
MICROGLIA
istics of process withdrawal, transition or hyper-ramifica-
tion, and subsequent formation of new protrusions along
A. Morphology with motility and locomotion, i.e., microglial movement in
the tissue (539, 861, 872). Interestingly, in ageing, micro-
It was through the study of morphological changes glia gain morphological markers characteristic for senes-
that a microglial role in the diseased CNS was recognized cence and functional deterioration (587, 868, 872).
(351, 479). At first glance, the microglial appearance in the Microglial cells in vitro usually do not have the ram-
healthy matured tissues of the brain, spinal cord, or the ified structure typically seen in the normal CNS. They
retina does not suggest an immediate association with a show heterogeneous shapes, ranging from spindle and
macrophage nature (Fig. 5). The cellular processes rod-shaped or amoeboid versions with short thick pro-
branching off from the small soma with further distal cesses expanding as lamellipodia to even round cells, but
arborization are typical for the “ramified” microglia. This morphological reorganization can still be imposed by
term has been almost synonymously used with “resting” treatment with typical activating agents, such as bacterial
microglia, implying an intimate link between morphology LPS (6). Morphological responses differ depending on the
and function (872). Even though time-lapse analyses on type of insult (872, 873). Cells can acquire bushy or bi- and
microglial morphology had been conducted already (861), tripolar, spindle- or rod-shaped morphologies (635, 759).
it was the elegant application of in vivo two-photon im- The heterogeneous forms may interchange, while pro-
aging to the monitoring of resting microglia in the mouse gression from the ramified microglia to the amoeboid
neocortex which revealed, surprisingly, the dynamics of macrophage-like appearance is thought to occur as a
the processes (190, 645). Upon challenges, the process of stereotypic sequence (861).
FIG. 5. Morphology of resting and activated mi-

croglia. Mouse microglia in brain tissue and cultures.
A: staining for Iba1 in mouse cortex reveals microglia
with their processes. B: a microglial cell visualized in
a brain tissue slice by filling with Lucifer Yellow
through a micropipette. C: upon stimulation with bac-
terial cell wall components, such as LPS, microglia in
a primary culture present with the typical morphology
of activation. Cells were stained with FITC-conju-
gated ILB4. D: microglia in culture stained with FITC-
ILB4 and showing phagocytosed rhodamine-labeled
myelin (made by coupling of rhodamine to a mouse
myelin preparation). [Images are from Denise van
Rossum, University of Göttingen (A, C, and D) and
Clemens Boucsein, MDC Berlin (B).]

The remarkably fast and drastic transformations in cell clearance of opsonised and nonopsonized material (542).
shape indicate that they might be required for functional Antibodies against Iba1, a protein with a suggested role in
adjustments. For example, filopodia protrusion and dynamic calcium homeostasis (406, 407), have proven most helpful
rebuilding are needed for motility and directed migration. in visualizing microglia with details of their processes.
Similarly, appearance of “foamy” cells with lipid- or myelin- In the neuropathological routine and experimental re-
loaded organelles in brain tissue sections points to phago- search, immunoglobulin receptors (CD16/32/64, Fc␥RIII/II/
cytotic activity, for example, in a myelin lesion. Yet morphol- I), CD45 (leukocyte common antigen, LCA), CD68 (macro-
ogy is not always a reliable reflection of functional orienta- sialin), CD163 (scavenger receptor M130, ED2), CD169 (sia-
tion. Certain gene inductions or activities can occur in the loadhesin, siglec-1), CD204 (MSR), F4/80 antigen, ␤-glucan
absence of obvious morphological transitions (254), receptor dectin-1, or mannose receptor (CD206) are useful
whereas a ramified shape can be experimentally induced targets, with more or less prominent and overlapping recog-
even under conditions that do not support ramification (400, nition of other tissue macrophages/macrophage-like cells.
886). As a consequence, loss of ramified and gain of more The expression level of many of these molecules, such as for
amoeboid cell formats do not tell much about the actual example CD11b or Iba1, mostly increases with microglial
reactive phenotype (551). activation (420, 421). MHC class II structures are only ex-
pressed by activated microglia including the expression of
the accessory molecules for antigen presentation, namely,
B. Identification
B7.1 or B7.2 (CD80/86).
While microglial cells can be easily distinguished
The identification of microglial cells beyond cyto- from other brain cells, this is not always possible with
morphological criteria was facilitated by the development respect to non-CNS monocytes. Costaining of other CNS
of staining procedures that take advantage of the exclu- macrophage populations, like perivascular cells or the
sive expression of certain molecules in specific cell types macrophages of the meninges (912), comes with a (mi-
(Table 1). The suitability of the various markers and
cro)anatomical separation. The situation is, however, dif-
methods is determined by the ability 1) to discriminate
ferent when injured and diseased tissues are examined.
microglia from other CNS-resident cells, like neurons,
Virtually all common macrophage markers, e.g., CD11b,
astrocytes, oligodendrocytes, or endothelial cells; 2) to
Iba1, or F4/80, will simultaneously report all macrophages
distinguish parenchymal microglia from other resident or
present in the CNS tissue (128). Despite considerable
infiltrating monocytes/macrophages; and 3) to either re-
efforts, hitherto a clear distinction of (activated) micro-
veal the entire population of microglia or to display a bias
glia from infiltrating monocytes is hampered by the over-
for certain activity states. The mere issue of technical
lap in the expression of macrophage-associated factors
aspects that affect the success and quality of staining, like
tissue collection, fixation, and embedding as well as typically serving microglial staining. The CNS-invading
choice of staining tools and procedures, is out of the cells from the periphery share lineage origin and features
scope of this review. Below we may rather focus on with the resident microglia, and both major populations
selected molecules that have been valuable to identify intermingle in a CNS lesion by both morphological and
microglia and to reveal their morphology. biochemical properties. To some extent, the expression
Microglia can be visualized in human and animal level of CD45 has been exploited to discriminate between
brain tissue sections and in cultures by a variety of cell them. CD45⫹ cells in the CNS reveal a low (CD45lo, pa-
surface-associated or intracellular/cytosolic molecules. In renchymal microglia) or intermediate (CD45int, other
some cases, the exact identity and function of the respec- CNS-associated macrophages), but rarely a high (CD45hi)
tive protein or carbohydrate structure is not known, such expression level (1029). Infiltrating cells, on the contrary,
as for surface-expressed glycan moieties identified by seem to be rather of the CD45hi phenotype. Separation of
Griffonia simplicifolia isolectin B4 (ILB4) or tomato lec- the CD11b⫹CD45lo and CD11b⫹CD45hi populations, for
tin (84, 870). Nonetheless, these are useful markers in example, in FACS analyses, can thus be used to differen-
lectin-histochemical or antibody-based procedures. Al- tiate parenchymal microglia from the infiltrating macro-
though blood vessels can be stained by ILB4 with su- phages. However, with lesion progression, the clear de-
preme intensity as well, a differentiation is possible by marcation may fade, rendering an unequivocal identifica-
morphological criteria and even by automated image anal- tion more difficult. Other distinctions have been made by
ysis. In other cases, the molecules targeted by staining superoxide dismutase expression (250), whereas expres-
have an established function as receptors, adhesion mol- sion of the calcium binding macrophage related proteins
ecules, or enzymes in macrophages, for example, the MRP8/S100A8 and MRP-14/S100A9 is thought to identify
␣M␤2 integrin (CD11b/CD18, complement receptor 3, CR3, circulating and more recently invaded monocytes/macro-
MAC1), which binds complement C3b, plasma, and extra- phages (375, 490), while delayed expression in microglia
cellular matrix proteins and serves in the phagocytotic can occur as well (249).

TABLE 1. Markers of microglia
Molecular Structure/Antigen Detection Agent/Technique Properties and Functional Relevance Reference Nos.
Identification*
Surface carbohydrates Griffonia (Bandeiraea) Not known, also expressed by blood 84, 870, 1013
(oligosaccharides containing simplicifolia isolectin B4 vessels
␣-galactose residues)
Surface carbohydrates Lycopersicon esculentum Not known, also expressed by blood 82, 994
(oligosaccharides containing (tomato) lectin vessels
N-acetylglucosamine oligomers)
Iba1(ionized calcium-binding adaptor Antibodies against Iba1 Protein with suggested role in calcium 406, 407
molecule 1) homeostasis
CD11b/18 (␣M␤2 integrin) Antibodies against CD11b/ Complement receptor 3 (CR3) with 12, 509, 542, 774, 780, 819
CD18 (MAC1, OX42) additional binding properties for
other molecules, e.g., fibrinogen
CD45 (leukocyte common antigen, Antibodies against CD45, low Transmembrane protein tyrosine 148, 192, 701, 707, 819
LCA) expression compared with phosphatase, functions in signaling,
infiltrating leukocytes also roles for TCR and BCR activity
CD68 (macrosialin) Antibodies against CD68 Role in phagocytosis and as oxidized 148, 200, 333, 509, 998
LDL-binding protein
F4/80 antigen F4/80 antibody Member of the epidermal growth 148, 394, 537, 547, 946, 950
factor (EGF)-transmembrane 7
(TM7) family, suggested role in
immune tolerance
Functional orientation⫹
MHC II Antibodies against MHC II Antigen presentation by APC 564, 636, 666, 704
components, combinable
with functional assays for
antigen presentation to T
cells in vitro
CD11c (integrin ␣X) Antibodies against CD11c Cell adhesion, marker for dendritic 12, 113, 753, 754, 909
cells as APC
CD34 (mucosialin, sialomucin) Antibodies against CD34 Roles in adhesion, marker for 487
precursor cells of the myeloid
lineage
iNOS (inducible nitric oxide Antibodies against iNOS, Enzymatic synthesis of NO from 93, 615
synthase) in situ hybridization, arginine during inflammation (upon
indirectly by measurement gene induction), resulting also in
of NO production via citrulline generation
nitrite in vitro
Transgenic models
CX3CR1 Transgenic mice expressing Receptor for the chemokine 190, 645
enhanced green fractalkine (CX3CL1) with
fluorescent protein (EGFP) implications for the control of
in the Cx3cr1 locus microglial activity
(fractalkine receptor)
Iba1 Transgenic mice expressing Protein with suggested role in calcium 379
enhanced green homeostasis
fluorescent protein (EGFP)
under control of the Iba1
promoter
*For visualization of cells in vitro or in tissue sections, lectins and antibodies are directly (or via secondary antibodies) coupled to fluorescent
dyes or biotin for combination with strept/avidin-conjugated dyes, peroxidase, or other enzymes for staining-suitable substrate conversion. ⫹For
further examples, see the text.
More recently, new antigens for immunolabeling ripheral macrophages). Yet the suitability of the marker in
have been introduced which promise the selectivity for reliably identifying only microglia could be limited to
microglia among cells of the mononuclear phagocyte sys- human material. Discrete expression patterns of the var-
tem (790). The glucose transporter 5 (GLUT5), at least in ious GLUT isoforms seem to vary in other species. Rodent
human tissue, is suggested as a useful marker for resting macrophages may not be excluded from GLUT5 staining.
and activated microglia (387), as this isoform seems to be On the other hand, antibodies against the keratin sulfate
restricted to microglia (546, 952). Antibodies against epitope 5D4 can serve such discrimination in the rat (54).
GLUT5 can apparently stain human microglia to even More recently, generation of genetically modified an-
allow for a distinction from other related cell types (pe- imals expressing fluorescent proteins under the control of

macrophage/microglia-expressed factors, such as the Similar results were obtained in forebrain slices from
fractalkine receptor (CX3CR1), have been employed as adult (6 – 8 wk old) mice: cells commonly lacked voltage-
microglia-indicator mice. In this mouse line, EGFP was gated currents, while the membrane potential recordings
inserted into the fractalkine receptor locus, and heterozy- distinguished two subpopulation of cells with resting po-
gous mice express both EGFP and fractalkine (430). tential around ⫺52 and ⫺29 mV, respectively (82). In
Whether the missing gene copy may influence microglial ramified microglial cells in acute slices from juvenile
function could be a concern. A transgenic mouse line mice, the resting potential was around ⫺38 mV (802).
expresses the enhanced green fluorescent protein under Microglial cells during early postnatal development
control of the Iba1 promoter (379). The fluorescence of were studied in the corpus callosum of 6- to 9-day-old
this line is, however, not as bright as the one of the mice. This area is one of the “microglial fountains” (i.e.,
fraktalkine mouse. the sites of microglia progenitors invasion) as described
by Kershman (441). These cells had amoeboid morphol-
ogy; they rapidly migrated within the slice and commonly
VI. GENERAL PHYSIOLOGY OF MICROGLIA
accumulated at the slice surface within one to several
hours. The amoeboid cells were characterized by only
A. Membrane Properties of Microglial Cells in minute if any membrane currents with depolarizing volt-
Normal Tissue age steps, while the hyperpolarizing voltage steps induced
large inward currents (Fig. 7). These currents exhibited
The majority of studies on microglial physiology properties of the inwardly rectifying K⫹ current in that
were performed on cultured microglial cells. These cells the reversal potential depended on the transmembrane
exhibit amoeboid morphology and therefore cannot be K⫹ gradient, inactivation time constants decreased with
considered as a model for the resting microglial cells. The hyperpolarization, and the current was blocked by tetra-
cells in culture can be stimulated and thus triggered to ethylammonium (86). The membrane potential varied
acquire a proinflammatory phenotype. A classical tool to considerably between ⫺10 and ⫺70 mV (with the mean
activate microglial cells is LPS; within 24 h, microglial value of ⫺42 mV).
cells are considered as activated and release a number of
proinflammatory substances such as TNF-␣, IL-6, or nitric
oxide. Microglial activation is, however, not an all-or-none B. Membrane Properties of Microglial Cells in
process, but can vary depending on the stimulation con- Pathological Tissue
text (for review, see Ref. 351). Therefore, much of the
physiological recordings in vitro are from moderately to The changes in microglial membrane currents in re-
fully activated microglial cells. sponse to a pathological event have been studied in
There are also an ample number of investigations acutely isolated slices containing the facial nucleus. Using
performed on various types of microglial cells lines. the classical paradigm developed by the Kreutzberg group
These cell lines, however, cannot be considered as a (71), the facial nerve was cut, thus triggering microglial
proper model of microglial cells due to substantial and activation in the facial nucleus without the disturbance of
inconsistent modifications in physiological properties; the blood-brain barrier; microglial cells were studied 12 h,
therefore, in the present review we deliberately omitted 1 day, 3 days, and 7 days after the lesion. At the 12-h time
results obtained from cell line preparations. point, the cells expressed predominantly inward rectify-
So far, we have no physiological recordings from ing currents. One day after the lesion, the membrane
microglial cells in vivo, neither of their membrane poten- conductance was dominated by delayed rectifying out-
tial nor of currents nor of intracellular Ca2⫹ concentra- ward currents and inward rectifying currents. After 3
tion. The data that come closest to the in vivo situation days, these voltage-gated currents became smaller in am-
are recordings from acutely isolated brain slices (Fig. 6). plitude, and after 7 days, currents were only slightly larger
Microglial cells were studied in slices from cortex, stria- than in the control cells (81).
tum, and facial nucleus from young-adult (8 –12 wk old) A similar observation was obtained in microglial cells
rats. Cells were identified by specific labeling with toma- studied after an ischemic insult. To distinguish peripheral
to-lectin coupled to Texas Red. The labeled cells retained leukocytes from microglia, the blood cells were prelabeled
their ramified morphology within the first hours after slice in vivo with a fluorescent dye rhodamine. Six hours after
preparation. These cells were characterized by high input ischemia, the microglial cells were still lacking voltage-gated
resistance and little, if any, voltage-gated membrane cur- channels or exhibited only an inward rectifier current. After
rents and a very low membrane potential (⫺20 mV; range 48 h, the intrinsic microglial population expressed inward
⫺2 to ⫺40 mV) (81). Clamping the membrane potential of and outward currents similar to those observed 1 day after
microglial cells in slices at ⫺70 mV led to a rapid (10 s to facial nerve axotomy (540). This pattern was further con-
a couple of minutes) cell damage and loss of a giga-seal. firmed by a study using a mouse model of status epilepticus

FIG. 6. Membrane currents of mouse microglia in situ and in vitro. A: membrane currents from cells in the acute slice were recorded during
de- and hyperpolarizing voltage steps from a holding potential of ⫺20 mV (top graph) or ⫺70 mV (bottom graph). The current traces are displayed
on the left, and the resulting current-voltage curve (I-V curve) is in the middle. On the right, the average currents (mean ⫾ SE) from 89 cells are
shown. The picture below, scanned with a confocal microscope, shows a microglial cell after recording and filling with Lucifer Yellow. It displays
the typical morphology of a resting microglial cell. B: distribution of resting membrane potential values of microglial cells from the slice. Note the
two peaks at ⫺52 and ⫺23 mV. C: membrane current recordings were also obtained from cultured microglial cells. The holding potential was ⫺70
mV. The average current-voltage curve on the right indicates that the membrane conductance of the cultured cells is on average ⬃4 times larger
compared with cells in the slice (note the difference in current scale). D: distribution of resting membrane potential values of cultured cells. The
peaks are at slightly more negative values compared with cells from the slice (⫺61 and ⫺29 mV). [From Boucsein et al. (82).]
induced by intraperitoneal kainate injections. In 24 – 48 h (which generally were in resting ramified state) was ⬃12.4
after the injection, microglial cells in the hippocampal slices pF, which was significantly smaller than the capacitance of
were characterized by the appearance of voltage-activated activated microglial cells from the same slices organotypi-
inward and outward potassium currents (27). A study on cally cultured for 3–5 days (⬃16 pF) (802). The difference in
human microglia obtained from epilepsy patients who un- the input resistance was even greater: resting microglia in
derwent surgery indicated a nonactivated, normal pattern. acutely isolated slices had input resistance of ⬃2.4 G⍀,
These human microglia had a high input resistance, a low whereas in activated microglia in cultured slices, the input
resting membrane potential of ⫺16 mV, and lacked Na⫹ resistance increased to 4.0 G⍀ (802).
currents as well as inwardly rectifying and delayed rectifying
K⫹ currents similarly to a nonactivated microglia from
mouse and rat (77). C. Membrane Properties of Microglial Cells
The cell membrane capacity (which is proportional to in Culture
cell membrane surface area) of ramified microglia in acutely
prepared slices was ⬃12 pF; the activation of microglia by Most studies on cultured microglia follow the pro-
LPS resulted in significant increase in cell capacitance (to cedure introduced by Giulian and Baker (319, 320), in
⬃43 pF in 48 h) after triggering the activation process (42). which postnatal CNS tissue is used to isolate microglial
In the acutely prepared hippocampal slices from juvenile cells from rats and mice. Initial physiological experi-
(P5–P9) mice, the membrane capacity of microglial cells ments on these cultures indicated that inward rectify-

⫹
FIG. 7. Method of isolation of single amoeboid microglial cells from the surface of corpus callosum slice and inward rectifier K currents
recorded from these cells. A–D: combinations of images taken by means of infrared video microscopy (left panels) and schematic drawings showing
the method of cell isolation. Video images were captured with an intensified CCD camera (Hamamatsu, Japan) and digitized by a frame grabber
connected to the PC. A: initial position of amoeboid microglial cells situated on the surface of a corpus callosum slice. B: a single microglial cell
was approached with a micropipette, and a whole cell patch-clamp configuration was established. C: after 2–3 min, the cell partially spread over the
pipette, intensifying the cell-to-pipette contact. D: finally, the cell was lifted for 200 ␮m over the slice surface. Scale bar in D ⫽ 10 ␮m. E and
F: voltage-activated whole cell currents recorded from a single cell at stages indicated in B and D, respectively. Currents were activated by
depolarization and hyperpolarization voltage steps (duration 200 ms, increment 10 mV) from a holding potential of ⫺70 mV. The lifting of the cell
did not affect the ionic current pattern. During the experiments, slices were held in a recording chamber mounted on the stage of an upright
microscope (Axioscope, Zeiss, Oberkochen) and continuously superfused with HEPES-buffered salt solution, containing (in mM) 150 NaCl, 5.4 KCl,
2 CaCl2, 1 MgCl2, 5 HEPES, and 10 glucose, pH 7.4. Membrane currents of amoeboid microglial cells were recorded using a standard patch-clamp
technique in whole cell configuration. Current signals were amplified with conventional electronics (EPC-7 amplifier; HEKA, Germany), filtered at
3 kHz, and sampled at 3–5 kHz by an interface connected to an AT-compatible computer system, which also served as a stimulus generator.
Experiments were controlled by Wintida software (HEKA, Germany). Recording pipettes were fabricated from borosilicate capillaries (Hilgenberg,
Germany), with resistances of 5–10 M⍀. The pipette solution contained (in mM) 130 KCl, 0.5 CaCl2, 5 EGTA, 4 MgCl2, 10 HEPES, 2 Na2ATP, pH 7.3.
[From Haas et al. (342), with permission from Elsevier.]
ing K⫹ currents dominate the membrane conductance subpopulations at around ⫺48 and ⫺70 mV; the resting
of cultured microglial cells (442). The resting mem- membrane potential of ⫺48 mV is mostly determined by
brane potential of microglia cultured from rodents or the delayed rectifier K⫹ channels which have a “win-
humans was about ⫺50 mV (442, 658). Thus the cul- dow” current region around ⫺45 mV (159), whereas in
tured cells have a membrane current pattern different cells with the more negative membrane potential the
from microglial cells in slices from normal brain tissue. dominant K⫹ permeability was represented by the in-
This current pattern matches that of the amoeboid, ward rectifier (160). This pattern is similar to microglial
invading microglial cells in situ or that of microglia 12 cells 24 or 48 h after a pathological insult. Incidentally,
h after a pathological insult. Stimulation of cultured the acidification of an extracellular solution led to de-
microglial cells with bacterial LPS or IFN-␥ induced the polarization of resting potential of cultured rat micro-
expression of an additional outward current (656). The glia (from ⫺45 at pH 7.4 to ⫺29 at pH 6.4) probably due
resting membrane potential of LPS-activated cultured to the shift of delayed rectifier potassium current
rat microglial cells showed two levels in distinct cells (IKDR) activation curve (162).

The majority of subsequent studies on microglia con- cellular Ca2⫹ signals as the substrate for their excitability,
firmed this current pattern (for review, see Ref. 264). whereas propagating Ca2⫹ waves enable long-range signal-
Differences were described for human brain macro- ing in astroglial syncytia (954, 955, 958, 960). For electrically
phages (microglia) isolated from explants of neurosurgi- nonexcitable microglial cells, the primary route for Ca2⫹
cal adult human tissue in that some of these cells ex- signal generation is associated with Ca2⫹ release from the
pressed Na⫹ currents (658). intracellular stores and with Ca2⫹ entry through the plasma-
lemma via ligand-gated (see section X for detail) and store-
operated Ca2⫹-permeable channels (263, 600; and Fig. 8).
VII. CALCIUM SIGNALING IN MICROGLIA
Calcium signaling/homeostatic system, which is opera- A. Intracellular Ca2ⴙ Stores

tive in most living cells, is controlled by several evolutionary
conserved molecular cascades responsible for Ca2⫹ trans- Microglia contain at least two types of dynamic Ca2⫹
port across cellular membranes and intracellular Ca2⫹ buff- stores: the endoplasmic reticulum (ER) and mitochon-
ering (130, 713). Conceptually, intracellular Ca2⫹ signals are dria. The ER is one of the major cellular organelles,
shaped by electrochemically driven Ca2⫹ diffusion through formed by the endomembrane, which extends from the
membrane channels and Ca2⫹ transport against the concen- nuclear envelope down to the finest cellular processes.
tration gradient, which is accomplished by several families The ER lumen is internally continuous, thus providing the
of Ca2⫹ pumps and exchangers (120, 714). These channels intracellular communication system through which vari-
and transporters are differentially distributed within the cell, ous molecules are transported to their destinations (715).
thus enabling different intracellular compartments to handle The functions of the ER are diverse, from protein synthe-
Ca2⫹ in a distinct way. Generally, neuroglial cells use intra- sis and protein posttranslational folding to a generation of
2⫹
FIG. 8. Calcium signaling in microglia. The main calcium signaling cascades in microglial cells are represented by 1) InsP3-induced Ca release
from the ER. The InsP3 is generated by PLC coupled with multiple G protein-coupled receptors (GPCR). Depletion of the ER Ca2⫹ store activates
the store-operated channels (CRAC), which are likely to be formed through interactions between STIM1/Orai proteins. 2) Ca2⫹ influx through P2X7
and/or P2X4 receptors constitutively expressed in microglia. The extrusion of Ca2⫹ from the cytosol is accomplished by 1) Ca2⫹ uptake into the ER
via sarco(endo)plasmic reticulum Ca2⫹-ATPases (SERCA); 2) Ca2⫹ accumulation onto mitochondria through Ca2⫹-selective uniporter, and 3) Ca2⫹
extrusion to the extracellular space by plasmalemmal Ca2⫹-ATPase (PMCA) and Na⫹/Ca2⫹ exchanger (NCX).

various intracellular signaling molecules. The intra-ER which are discussed in detail in sections X–XIV of this
free Ca2⫹ concentration ([Ca2⫹]i) varies between 0.2 nM review.
and 0.8 mM thus providing electro-driving force for Ca2⫹ Apart from metabotropic receptors, which are the
release (598, 844, 846); the high [Ca2⫹]i is maintained by primary triggers for the activation of InsP3-induced Ca2⫹
sarco(endo)plasmic reticulum Ca2⫹-ATPases (SERCA), release, the InsP3 signaling cascade can be affected by
the activities of which are precisely controlled by free several other factors. For example, the exposure of pri-
Ca2⫹ in both cytosol and ER lumen (99). The release of mary cultured rat microglia to pure albumin or albumin
Ca2⫹ is accomplished by several families of endomem- associated with immunoglobulins and fatty acids (albu-
brane-resident Ca2⫹ channels (see Refs. 61, 473, 957, 958), min fraction V) triggered [Ca2⫹]i elevation, which was
from which the most characterized are Ca2⫹-gated Ca2⫹ mediated through Src tyrosine kinase, PLC, and InsP3 and
release channels, generally referred to as ryanodine re- was coupled to microglial proliferation (386). Interest-
ceptors (RyRs) and inositol 1,4,5-trisphosphate (InsP3)- ingly, albumin did not trigger Ca2⫹ signals in peritoneal
gated Ca2⫹ release channels commonly known as InsP3 macrophages (386). Similarly, the application of ammo-
receptors (InsP3Rs). Importantly, both RyRs and InsP3Rs nium to primary cultured mouse microglia triggered ER
are capable to undergo regenerative activation (52), Ca2⫹ release through direct activation of PLC/InsP3 sig-
which together with rapid intra-ER Ca2⫹ equilibration naling cascade (589).
(“Ca2⫹ tunneling”; Refs. 715, 845) create a substrate for Some chronic pathological conditions may also af-
intracellular propagating Ca2⫹ waves. fect the status of the ER Ca2⫹ store. In particular, the
Microglial cells contain both types of intracellular InsP3-mediated Ca2⫹ release [following the stimulation of
Ca2⫹ release channels. Microglial cultures obtained from purinoceptors or platelet-activating factor (PAF) recep-
the fetal human brains expressed mRNA for all three tors] was reduced by more than 50% in microglial cells
RyRs subunits, RyR1–3, whereas adult human microglia obtained from the brains of Alzheimer’s disease patients,
had only RyR1/2 mRNAs (462). Functionally, Ca2⫹ signal- possibly indicating chronic depletion of the ER store
ing in these cultured microglial cells can be induced by (569).
very high concentrations of 4-chloro-m-cresol (4-CmC; The inhibition of SERCA pumps by thapsigargin in-
1–5 mM), this effect being antagonized by 1,1’-diheptyl- duced morphological transformation of amoeboid micro-
4,4=-bipyridinium dibromide (DHBP, 10 ␮M). Both DHBP glia in culture to a ramified phenotype (1014).
(10 ␮M) and 4-CmC (100 ␮M) when administered together
with microglia-activating agents (LPS and IFN-␥ or IFN-␥
and ␣-synuclein) reduced neurotoxicity of conditioning B. Store-operated Ca2ⴙ Entry
media obtained from microglial cultures and applied to
cultured neuroblastoma (462). The Ca2⫹ release following Store-operated Ca2⫹ entry (SOCE) was initially con-
activation of the RyRs by natural agonist cyclic ADP templated by Jim Putney as a “capacitative Ca2⫹ influx”
ribose (cADPR) was also demonstrated in human cul- mechanism (732, 733), which coupled depletion of ER
tured microglia (826). In primary microglial cultures, the stores with the activation of the plasmalemmal Ca2⫹ entry
cADPR signaling cascade was involved in LPS-induced pathway. This initial hypothesis gained a universal ac-
activation process. The cADPR was produced by ectoen- knowledgment since. The SOCE pathway was identified
zyme CD38, and the cADPR-dependent events were inhib- in the majority of nonexcitable and many excitable cells
ited by ryanodine and cADPR antagonist 8-bromo-cADPR, (520, 521, 692, 731). The SOCE pathway is mediated
thus indicating possible involvement of RyRs (293, 562). through plasmalemmal channels, most probably of sev-
Nonetheless, the functional characterization of microglial eral subtypes. The most characterized are the calcium
RyRs is yet to be done, and so far it seems that RyRs are release-activated Ca2⫹ (CRAC) channels (389, 691), al-
not particularly important for Ca2⫹ signaling in these though other channels (e.g., TRPC) can have store-oper-
cells. ated gating (734). Properties of CRAC channels in micro-
The main route for the generation of microglial Ca2⫹ glia are discussed in detail in section VIII. Conceptually,
signals is associated with InsP3Rs. The InsP3Rs are part of the SOCE channels are open upon the depletion of intra-
a widely present intracellular signaling cascade, which cellular Ca2⫹ stores and aid in replenishing the Ca2⫹
starts from G protein-coupled metabotropic receptors stores. In different cell types, depletion of ER can activate
linked to phospholipase C (PLC); activation of the latter SOCE either in a graded or in a threshold manner; in the
produces two intracellular second messengers the diacyl- latter case, only a substantial depletion of the store (or a
glycerol (DAG) and the InsP3 (see Refs. 51, 53, 714, 957 for depletion of specific portion of the store) triggers plas-
review). The InsP3, in turn, activates InsP3Rs of the ER, malemmal Ca2⫹ influx (231, 358, 690). Essentially, the
thus producing Ca2⫹ release and [Ca2⫹]i elevation, which activation of SOCE often substantially outlasts the period
then effects on numerous cellular functions. Microglial of initial stimulation and Ca2⫹ release, therefore provid-
cells express a multitude of metabotropic receptors, ing a long-lasting Ca2⫹ influx. The mechanisms of ER to

SOCE coupling are not yet completely understood, al- Incidentally, activation of microglial cells in vitro by
though recently two molecules were discovered to be LPS leads to an elevation of basal [Ca2⫹]i which is asso-
major players in both the signaling to and permeation ciated with suppression of evoked calcium signaling, as
mechanisms of the store-operated channels. These mole- indicated by reduced [Ca2⫹]i transients in response to
cules are Stim1 and the Orai proteins, with Stim1 acting as UTP or complement factor 5a (382). Preventing the LPS-
an ER Ca2⫹ sensor and Orai as the pore-forming subunit induced increase in basal [Ca2⫹]i by BAPTA strongly at-
of CRAC channel (280, 723, 734, 772). tenuated the (LPS-induced) release of NO and certain
The first evidence for functional SOCE in microglial cytokines and chemokines. Ionomycin, an ionophore ele-
cells was obtained by [Ca2⫹] imaging experiments inves- vating basal [Ca2⫹]i, failed to induce release activity. El-
tigating the response of microglia to complement frag- evated [Ca2⫹]i is obviously required, but it is not sufficient
ments or endothelin (603, 605). In both sets of experi- to trigger the release of NO and certain cytokines and
ments, the plateau phase of the agonist-induced cytoplas- chemokines. Elevation of basal [Ca2⫹]i was suggested to
mic Ca2⫹ signal was completely dependent on the be a central element in the regulation of executive func-
presence of extracellular Ca2⫹. Subsequently, it became tions in activated microglia (382). It is not yet resolved
evident that activation of microglial metabotropic recep- whether the persistent elevation of basal [Ca2⫹]i in acti-
tors linked to Ca2⫹ release from internal stores activates vated microglial cells is associated with long-term activa-
SOCE, which can be recorded as a plateau phase of the tion of SOCE. In human embryonic cultured microglia,
ligand-induced Ca2⫹ response. Examples are many; the activation of metabotropic purinoceptors with adenosine
SOCE is activated following stimulation of metabotropic 5’-O-(2-thiodiphosphate) (ADP␤S) triggered a biphasic
purinoceptors, PAF receptors, lysophosphatidic acid re- [Ca2⫹]i elevation with the second plateau phase lasting
ceptors, etc. (See sects. X, XI, and XVI for detailed ex- several minutes. The plateau phase was sensitive to the
amples). The SOCE appear to play an important role in SOC inhibitor SKF96365. This Ca2⫹ elevation was due to
human microglia, where several reports indicated the store-operated Ca2⫹ entry and was instrumental in in-
creasing production of COX-2 (153).
strong dependency of metabotropic agonist-induced
The IFN-␥, another substance to activate microglia,
[Ca2⫹]i transients on external Ca2⫹ (326, 573, 982, 985,
led to slowly developing steady-state [Ca2⫹]i increase in
1031). However, the SOCE in human microglia is not only
cultured human microglia (rate of increase 0.8 nM/s and
coupled to P2Y purinoceptor-dependent depletion of in-
maximal amplitude 102 nM; Ref. 292). Likewise, elevated
ternal stores, but is also modulated by membrane depo-
basal [Ca2⫹]i was found in activated microglia isolated
larization either following the activation of Cl⫺ channels
from the human post mortem brains from Alzheimer’s
(570) or ionotropic P2X purinoceptors (568). Extracellu-
disease patients (569) or in cultured microglia exposed to
lar acidification (from pH 7.4 to 6.2) resulted in almost
A␤25–35 (472).
instant and pronounced (by 87%) inhibition of SOCE ac- There is a link between mitochondria and the SOCE
tivated following PAF-induced ER depletion (447), which in microglial cells. The SOCE induced following stimula-
may be explained by rapid cell depolarization triggered by tion of PAF receptors or the depletion of ER after expo-
pH lowering. sure to cyclopiazonic acid (CPA) can be effectively (IC50
The maximal depletion of ER Ca2⫹ store in cultured ⬃9 ␮M and 20 ␮M, respectively) blocked by PK11195, a
microglia by overstimulation of P2Y2/4 metabotropic puri- ligand of mitochondrial peripheral benzodiazepine recep-
noceptors resulted in the chronic activation of SOCE tor. This inhibition was also accompanied with a reduced
(914). In these experiments, the Ca2⫹ release was induced expression of COX-2. This peculiar pharmacology led to
when bathing cells in Ca2⫹-free external solution. After the suggestion that there is a direct link between mito-
the period of stimulation, the external Ca2⫹ was reintro- chondria and SOC entry pathways (385), which has been
duced, which resulted in strong rebound [Ca2⫹]i increase; considered previously as a general mechanism of CRAC
the Ca2⫹ remained at an increased level for tens of min- channels regulation (316, 689).
utes and reflected the persistent activation of SOCE, as The LPS was reported to trigger direct, transient
changes in extracellular Ca2⫹ were mirrored in rapid Ca2⫹ increases in 10 –12% of microglial cells from primary
changes of [Ca2⫹]i (914). During this period, the ER stores cultures of the rat area postrema and from rat brain. The
remained depressed as neither the stimulation of purino- LPS-induced Ca2⫹ signaling in cultured microglia was
ceptors nor administration of SERCA blocker thapsi- abolished by ruthenium red and preincubation with caf-
gargin induced any [Ca2⫹]i responses. The long-lasting feine, thus suggesting the involvement of RyRs and caf-
basal [Ca2⫹]i elevation can be also induced by treatment feine-sensitive ER Ca2⫹ store (29, 1007). This response
with phorbol ester (600), and with BDNF (597) that may was not confirmed by others including our own observa-
result from chronic activation of SOCE. Whether such a tions.
chronic activation ever occurs in vivo remains, however, Significant abnormalities are present in the Ca2⫹-
unknown. mediated signal transduction in microglia isolated from

Alzheimer’s disease (AD) patients; these microglial cells MCAO) significantly increased the expression of NCX1 in
were characterized by significantly higher (20%) basal microglial cells penetrating into the infarct core 3–7 days
(resting) Ca2⫹ relative to the cells from individuals not after the surgery (79). A similar increase in NCX1 expres-
suffering from dementia. The peak amplitude of ATP and sion was observed in microglial cells cultured from the
initial phase of PAF responses, which reflect rapid deple- infarction core (79).
tion of Ca2⫹ from the ER, were reduced by ⬃60% in AD
cells relative to amplitudes recorded from normal micro-
glia. Additionally, AD microglia showed diminished am- D. Ca2ⴙ Homeostasis and Microglial Cell Death
plitudes (by ⬃60%) of SOCE following PAF stimulation
and prolonged time courses (increase by 60%) of ATP Calcium ions are intimately involved not only in
responses compared with healthy cells (569). physiological signaling but also in triggering various types
of cell death (644). Disruption of microglial Ca2⫹ homeo-
stasis triggers activation of death programs, which are
C. Calcium Extrusion regulated by the microglia activation status. Treatment of
primary cultured microglial cells by thapsigargin or iono-
Calcium extrusion following [Ca2⫹]i elevation is ac- mycin induced apoptosis, whereas the same agents ap-
complished by plasmalemmal Ca2⫹ pumps of the PMCA plied to LPS-activated microglia resulted in necrotic cell
(plasmalemmal Ca2⫹-ATPase) family and the sodium/cal- death (625). Both apoptotic and necrotic pathways were
cium exchanger (NCX). The Na⫹/Ca2⫹ exchangers, repre- regulated by [Ca2⫹]i because the treatment of cultures
sented in mammals by three subtypes NCX1, NCX2, and with BAPTA-AM reduced cell death (625).
NCX3, are members of Ca2⫹/cation antiporter superfamily
(541). VIII. ION CHANNELS IN MICROGLIA
The microglial PMCAs have not been yet character-
ized in detail. Operational Na⫹-dependent Ca2⫹ uptake
(as assayed by 45Ca2⫹ technique) was detected in cultured A. Sodium Channels
microglial cells in 2001 (559). Subsequent experiments
revealed an expression of all three NCX isoforms, NCX1, The presence of sodium channels in microglial cells
NCX2, and NCX3, at both translational and protein levels is controversial. In microglial cells in situ and in the
in cultured rat microglia (624). At the mRNA level, ex- majority of studies in cultured microglia, rapid inward
pression of NCX1 was dominating and was larger than in currents typical for voltage-gated sodium channels were
neurons from the same animals; expression of NCX2 and not observed. Sodium currents (INa) were described in
NCX3 was weak, being much less that in nerve cells (624). cultured microglial cells prepared from rat (471, 812) and
Treatment with IFN-␥ caused an increase in Na⫹-depen- human (658) brains (see Table 2). Sodium currents in
dent 45Ca uptake (under conditions favoring the reverse cultured microglial cells were similar to a typical neuronal
mode of operation) and stimulated NCX protein synthe- Na⫹ channels; they were highly sensitive to TTX (com-
sis. This stimulation was governed by two distinct intra- plete inhibition at 2–5 ␮M), had a very rapid kinetics, and
cellular signaling cascades: the early increase in NCX had characteristic voltage dependence (threshold approx-
activity was sensitive to inhibitors of PKC (staurosporine imately ⫺40 mV and peak ⬃0 mV). With peak amplitude
and GF109203X) and tyrosine kinase (herbimycin A), of ⬃50 pA, they were much smaller than in neurons.
whereas delayed effects of NCX synthesis were in addi- Voltage-gated Na⫹ currents were expressed only in 20% of
tion sensitive to the protein synthesis inhibitors cyclohex- cultured rat microglial cells; the cells with ramified mor-
imide and actinomycin D, indicating de novo synthesis phology had a higher incidence of INa detection (471). In
(624), which affected all three NCX isoforms. The activity contrast, in human microglial cells, cultured from ex-
of microglial NCX was also positively regulated by NO, plants obtained during surgery on patients suffering from
the latter even causing depletion of the ER and subse- brain tumors, the majority of cells (30 of 32 recorded)
quent ER stress, which are somehow connected to the demonstrated TTX-sensitive INa (658). Incidentally, cocul-
NCX function (560) because inhibition of NCX prevented turing microglial cells with astrocytes, which promotes
NO-induced ER stress and microglial death (626). The ramified morphology of the former, significantly in-
NCX is also involved in NADPH-mediated respiratory creased the proportion of cells expressing INa (812). Very
burst induced by phagocytotic activity: inhibition of the recently, three types of sodium channels, namely, TTX-
reversed mode of Na⫹/Ca2⫹ exchanger with KB-R7943 sensitive Nav1.1 and Nav1.6 and TTX-resistant (TTX-R)
decreased the respiratory burst in dose-dependent man- sodium channel Nav1.5 were identified, by using specific
ner (639); similarly, the reverse mode of NCX action was antibodies, in purified rat microglial cultures (70). The
instrumental for bradykinin-induced microglial migration inhibition of Na⫹ channels in LPS-activated microglia
(397). Ischemic insult (middle cerebral artery occlusion, with use-dependent blocker phenytoin (1 ␮M) decreased

TABLE 2. Ion channels in microglia
Experimental Biophysical Properties and

Channel Type/Subunit Preparation/Technique Pharmacology functional relevance Reference Nos.
Sodium channels
TTX-sensitive INa Rat, human/cultured primary TTX In rat cultures, INa was detected 471, 658, 812
microglia/whole cell voltage in ⬃20% of cells; in human; in
clamp ⬃95%; human cultures
however were prepared from
patients with brain tumors.
Coculturing rat cells with
astrocytes increased
proportion of cells with INa.
Nav1.1, Nav1.5 (TTX- Rat/cultured primary microglia/ TTX, phenytoin Treatment of LPS-activated 70
sensitive), Nav1.6 specific antibodies microglia with TTX or
(TTX-resistant) phenytoin reduced IL-␣1, IL-
1␤, and TNF-␣ secretion and
decreased motility responses
to ATP
Nav1.6 EAE mice/postmortem MS Phenytoin The protein and mRNA for 180
human spinal Nav1.6 were detected in EAE
cord/immunocytochemistry/ mice spinal cord and optic
in situ hybridization nerve, as well as in MS-
affected human spinal cord.
Phenytoin reduced microglial
activation in EAE model.
Calcium channels
3⫹
ORAI1/CRAC Rat/cultured primary microglia/ Gd , SKF-96365, High Ca2⫹-selective channel 263, 655, 671,
whole cell voltage diethylstilbestrol (DES), activated following depletion 914
clamp/RT-PCR 2-aminoethoxydiphenyl borate of ER Ca2⫹ stores. Activation
(2-APB; 50 ␮M). of ICRAC participates in
formation of sustained phase
of metabotropically induced
Ca2⫹ signals. Profound ER
store depletion may cause
persisting (tens of minutes)
activation of ICRAC-mediated
store-operated Ca2⫹ entry.
TRPM7; TRPC6; TRPM2, Rat, mouse, microglial cell Activation of TRPs participates 42, 286, 425, 453,
TRPV1; TRPM4 and to lines/whole cell voltage in microglial Ca2⫹ signaling 476, 789, 801
a lesser extent other clamp/RT-PCR and may be linked to IL-6
TRPs release and initiation of
microglial cells death.
Potassium channels
Inward rectifier Rat, mouse, human/culture, Ba , Cs⫹, TEA, quinine
2⫹
Generally expressed in activated 86, 235, 238, 442,
potassium channels amoeboid microglia from microglia; very low densities 471, 572, 574,
IKIR, acute juvenile corpus in resting microglia. 657, 808
Kir2.1 callosum slices/whole cell
voltage clamp
IKDR; Kv1.1; Kv1.2; Rat, mouse/cultured primary Cd2⫹, Zn2⫹, Ba2⫹, TEA, 4-AP, The IKDR is upregulated 81, 134, 234, 235,
Kv1.3; Kv1.5 microglia; slices/whole cell CTX, KTX, NTX, MTX following microglial activation 238, 239, 241,
voltage clamp/RT-PCR/ in both in vitro and in situ 288, 474, 540,
immunocytochemistry preparations. The Kv1.3 and 654, 656, 657,
Kv1.5 channels are required 688, 735
for IKDR in adult activated
microglia. The Kv1.1 and
Kv1.2.channels are expressed
in amoeboid postnatal
microglia; hypoxic insults
increase Kv1.2 expression in
adult microglial cells. Both
Kv1.3 and Kv1.5 channels are
linked to various functional
responses of activated
microglia; from regulation of
motility (Kv1.3) to release of
NO (Kv1.5)
Continued

TABLE 2.—Continued
Biophysical Properties and

Channel Type/Subunit Experimental Preparation/Technique Pharmacology functional relevance Reference Nos.
IKDR; Kv1.1; Kv1.2; Rat, mouse/cultured primary Cd2⫹, Zn2⫹, Ba2⫹, TEA, 4-AP, The IKDR is upregulated 81, 134, 234, 235,
Kv1.3; Kv1.5 microglia; slices/whole cell CTX, KTX, NTX, MTX following microglial activation 238, 239, 241,
voltage clamp/RT-PCR/ in both in vitro and in situ 288, 474, 540,
immunocytochemistry preparations. The Kv1.3 and 654, 656, 657,
Kv1.5 channels are required 688, 735
for IKDR in adult activated
microglia. The Kv1.1 and
Kv1.2.channels are expressed
in amoeboid postnatal
microglia; hypoxic insults
increase Kv1.2 expression in
adult microglial cells. Both
Kv1.3 and Kv1.5 channels are
linked to various functional
responses of activated
microglia; from regulation of
motility (Kv1.3) to release of
NO (Kv1.5)
Ca2⫹-dependent Calf, rat, mouse, human, adult, Apamin (KCNN2), clotrimazole The BK channels were identified 77, 436, 446, 572,
potassium (KCa) and newborn rat /primary (KCNN4), based on their single-channel 574, 802, 807
channels cultures; slices/sections of conductance (140–240 pS).
striatum/whole cell voltage The SK channels are linked to
IK(Ca); BK/KCa1.1; clamp/RT-PCR/ Apamin (100 nM KCNN3), NO release, MAPK signaling
immunohistochemistry and respiratory burst. The
KCNN3/SK3 channels were
KCNN1/SK1 Tapamin (5 nM KCNN3) predominantly expressed in
both cultures and healthy
KCNN3/SK2 striatum tissue; LPS treatment
or ischemic insult increased
KCNN3/SK3 the level of expression.
Inhibition of KCNN3/SK3
affected microglial activation
KCNN4/SK4; and reduced microglial
neurotoxicity.
G protein-activated K⫹ Mouse/primary cultures/ 4-AP, PTX Currents were activated 401, 402
channels whole cell voltage clamp following stimulation of G
protein-coupled metabotropic
receptors.
Anion channels
Volume-regulated Cl⫺ Mouse; microglial cell lines/ Flufenamic acid; DIDS, SITS, Single-channel conductances 230, 242, 304,
channels, Best family primary cultures/whole cell DIOA, NPPB, IAA-94 1-3.5 pS. The volume- 638, 808, 1040
of channels voltage clamp/qRT-PCR activated channels are
involved in setting the resting
Vm, regulation of microglia
proliferation, phagocytosis
and morphological phenotype.
CLIC-1 chloride Rat; microglial cell IAA-94 Single-channel conductance 588, 662
channels lines/primary cultures/whole 6.5–8 pS. CLIC-1 channels are
cell voltage clamp involved in microglia
proliferation and ROS
generation.
Other channels
Proton channels Mouse, rat, human, microglial TEA, bi- and trivalent cations Single-channel conductance ⬃ 237, 238, 460,
Hv1 voltage-activated cell lines/primary cultures/ fS range. Highly sensitive to 461, 574, 802,
H⫹ channels whole cell voltage clamp extracellular pH; probably are 965
associated with generation of
respiratory burst. Disruption
of cytoskeleton leads to an
⬃50% reduction in H⫹ current
amplitude, whereas cell
swelling potentiates H⫹
currents.
Continued

Biophysical Properties and

Channel Type/Subunit Experimental Preparation/Technique Pharmacology functional relevance Reference Nos.
Aquaporins Rat/RT-PCR/Western blotting ? Expression of AQP4 was 915

AQP4 observed in activated
microglial cells following LPS
injection into substantia
nigra.
Connexins Rat, Human, mouse/RT-PCR, ? No indications for connexins in 220, 255, 554,
Cx43, Cx36, Cx32, Cx45 immunocytochemistry, resting microglia in situ. 898
imaging, whole cell voltage Traumatic brain lesion
clamp resulted in an appearance of
Cx43 immunoreactivity. Cx32,
Cx6, Cx43, and Cx45 were
identified in cultured
microglia.
See text for definitions.
phagocytosis and secretion of IL-1␣, IL-1␤, and TNF-␣ isolated brain slices failed to detect any signs of Na⫹
(70). Similarly, the inhibition of sodium channels with currents (77, 81). The lack of sodium currents is also
TTX or phenytoin reduced ATP-induced microglial motil- documented for microglia after pathology (81) or in amoe-
ity; microglial motility was also reduced in med mice, boid microglia during development (86). Finally, treat-
which lack Nav1.6 channels (70). ment with TTX (20 –50 ␮M) does not affect the motility of
In activated microglia in autoimmune encephalomy- microglial process in vivo (645).
elitis, generally employed as model for multiple sclerosis,
the expression of a sodium channel Nav1.6 (both at mRNA
and protein levels) was found to be increased (180). Fur- B. Calcium-permeable Channels
thermore, treatment of animals with Na⫹ channel blocker
phenytoin decreased the inflammatory cell infiltrate in 1. Voltage-operated Ca2⫹ channels
this model by 75%, and TTX at micromolar concentrations The evidence for microglial voltage-gated Ca2⫹ chan-
inhibited the phagocytic capacity of microglia (180). nels is limited to a single report, in which voltage-clamp
Each of these studies, however, has its weakness. experiments revealed, in ⬃30% of the cells tested, a very
1) In the rat culture (471), only a subpopulation of micro- small (if any) inward Ca2⫹ current, which was somewhat
glial cells with a ramified morphology expressed INa cur- enhanced by BAY K 8644 (168). Additional indirect evi-
rents. It needs to be emphasized that the individually dence comes from the observation that treatment of cul-
recorded cells in Korotzer’s study were not identified by tured rat microglial cells with high K⫹ (25 to 55 mM)
microglial markers and could potentially also be NG2 solutions enhanced superoxide anion production induced
cells. 2) Schmidtmayer (812) placed blood monocytes or by phorbol 12-myristate 13-acetate. This effect was
spleen macrophages onto an astrocyte monolayer and blocked by the voltage-operated Ca2⫹ channel (VOCC)
termed these cells microglia. 3) In the study on human antagonist nifedipine, whereas treatment with the VOCC-
microglial cells, the cells were cultured from explants positive modulator BAY K 8644 enhanced superoxide an-
obtained during surgery on patients suffering from brain ion production similarly to high-K⫹ solutions (168). The
tumors (658). This cell population therefore could be incubation of cultured microglia with ␤-amyloid A␤25–35
potentially contaminated with blood monocytes. Further- (836), prion protein PrP 106 –126, HIV-1 regulatory protein
more, the individual recorded cells were not identified Tat or CCR3 cytokine receptor agonist RANTES (367)
with cell-type specific markers. 4) The presence of a Na⫹ triggered [Ca2⫹]i elevation that was sensitive to L-type
channel protein as shown by Craner in a pathological Ca2⫹ channels antagonists verapamil, nifedipine, and dil-
model (180) does not necessarily indicate the presence of tiazem, thus indicating a possible role for VOC channels
the functional Na⫹ channel. (367, 836). The presence of VOCCs channels in microglial
A large number of other labs found no evidence for cells was not, however, confirmed by other groups (see
the presence of INa in a variety of different microglial Ref. 600).
preparations. Sodium channels were not detected in cul-
tured rat or mouse microglia neither unstimulated nor 2. Ca2⫹ release-activated Ca2⫹ current
activated with LPS or in cultured microglia from human
fetuses obtained at 12–20 wk of gestation (574). Voltage- The Ca2⫹ release-activated Ca2⫹ current (ICRAC) was
clamp experiments on ramified microglia in situ in acutely initially found in mast cells in which the ER Ca2⫹ store

was fully depleted (389). The CRAC channels are charac- was further confirmed by in situ hybridization in mouse
terized by extremely low conductance (⬃fS range). Whole brain (476). Exposure of cultured rat microglial cells to
cell patch-clamp experiments on cultured microglia iden- 0.1–5 mM H2O2 (which is known as TRPM2 activator, Ref.
tified a current very similar to ICRAC (655). This current 703) triggered Ca2⫹ influx and cationic currents that were
was activated by intracellular perfusion with 10 ␮M InsP3, significantly potentiated by LPS (476). Intracellular ad-
was very selective for Ca2⫹, and was responsible for a ministration of ADP-ribose (which is also acting on
Ca2⫹ influx following store depletion with InsP3 (655). TRPM2, Ref. 703) triggered large cationic currents in both
Similarly to many other Ca2⫹-selective channels (374, naive and LPS-treated microglial cells (476). The hydro-
796), the removal of extracellular divalent cations con- gen peroxide- and ADP-ribose induced currents and Ca2⫹
verted ICRAC channels into Na⫹-permeable ones (344). influx were attributed to the activation of TRPM2 chan-
The Na⫹ currents through store-operated channels dem- nels (476).
onstrated very slow activation kinetics; the unitary chan- The [Ca2⫹]i-activated TRPM4-like currents were re-
nel conductance was 42.5 pS, and the channels were cently recorded from mouse microglial cells; they were
positively modulated by PKA and negatively by PKC half-activated at 1.2 ␮M of intracellular Ca2⫹ (42). The
(344). Likely, these currents reflected the activation of TRPM7 transcripts were found in rat cultured microglia.
TRPM7 channels (671). The TRPM7-mediated currents were activated spontane-
The currents through CRAC channels were measured ously after establishment of whole cell configuration and
from both resting and activated mouse microglial cells. In were inhibited by increase in intracellular Mg2⫹ concen-
the resting conditions, ICRAC amplitude was ⬃1.2–1.3 pA/ tration and by tyrosine kinase inhibitors. Microglial acti-
pF, whereas LPS-induced activation significantly reduced vation did not affect TRPM7-mediated currents (425). Pri-
the amplitude of the current (to ⬃0.5 pA/pF in 48 h after mary rat cultured microglial cells express TRPV1 chan-
exposure to LPS; Ref. 42). nels as demonstrated by RT-PCR, Western blot analysis,
Recently, the molecular identity of ICRAC was ana- and immunocytochemsitry (453).
lyzed in cultured neonatal rat microglia. The qRT-PCR There is growing evidence that TRP channels control
revealed expression of all three Orai mRNAs with maxi- microglial functions. In the retina, elevated intraocular
mal expression of Orai3 (671). Further electrophysiolog- pressure triggers [Ca2⫹]i elevation and IL-6 release from
ical and pharmacological investigations allowed the au- microglial cells; both Ca2⫹ signaling and IL-6 release were
thors to suggest that highly Ca2⫹-selective (PCa/PNa partially inhibited by the TRPV1 specific antagonist iodo-
⬎1,000) Orai1/CRAC channels are responsible for ICRAC resiniferatoxin (I-RTX; 0.1–10 ␮M) (789). The activation
generation in microglia (671). of TRPV1 channels with capsaicin or resiniferatoxin
(RTX) induced microglia death, which was prevented by
3. TRP channels the TRPV1 antagonists capsazepine and I-RTX. The cell
death was due to TRPV1-mediated Ca2⫹ influx with sub-
TRP channels belong to an extended family of cat- sequent mitochondrial damage, release of cytochrome c,
ionic ion channels that were originally identified as tran- and upregulation of caspase-3 (453). Direct injections of
sient receptor potential (TRP)-generating channels in capsaicin or TRPV1 endogenous agonist 12-hydroper-
Drosophila photoreceptors (356, 591). Further experi- oxyeicosatetraenoic acid to substantia nigra produced
ments identified an extended superfamily of TRP chan- microglial damage, presumably through TRPV1 activation
nels, comprising seven major families of TRPC, TRPM, (453). Inhibition of TRPV1 with La3⫹, capsazepine, ruthe-
TRPV, TRPA, TRPP, TRPML, and TRPN channels (698). nium red, or I-RTX reduced ROS production on activated
Many TRP channels are expressed in the nervous system, cultured microglia (801). Ischemic insults produced by
in both neurons and glial cells. The TRP channels are occlusion of cerebral artery induced an increase in the
intimately involved in thermo- and chemosensitivity as expression of TRPM2 mRNA in rat brain, which was
well as in neural development and growth and may also suggested to be mainly microglial, but its functional role
play an important role in various types of neuropatholo- yet needs to be determined (286).
gies (1, 643, 899).
There is evidence for the expression of several TRP
C. Potassium Channels
channels in microglia. The qRT-PCR performed on neo-
natal cultured neonatal rat microglia revealed the follow-
1. Inward rectifier K⫹ channels
ing rank of mRNAs expression: TRPM7 ⬎ TRPC6 ⬎
TRPM2 ⬎ TRPC1 ⬎ TRPC3 ⱖ TRPC4 ⬎ TRPC7 ⬎ TRPC5 ⬎ The inward rectifier K currents (IKIR) can almost be
TRPC2 (671). The TRPM7 expression was very high and considered as a marker for nonresting microglial cells.
comparable with expression of housekeeping gene They are expressed in all microglial cells with the excep-
HPRT-1 (671). A strong expression of TRPM2 mRNA was tion of resting microglia. The current was first described
found in cultured rat microglia (286, 476); this expression in cultured rat microglia, and a single-channel conduc-

tance of 30 pS was reported (442); subsequently, similar polysaccharide, IFN-␥, or their incubation in hydrophobic
unitary currents were found in other in vitro microglial teflon bags (654, 657), which activate microglial cells in
preparations (234, 572, 812, 965). A second inward recti- culture, induced the expression of a delayed rectifying
fier channel with unitary conductance of 43 pS was also outward K⫹ current. Since cycloheximide, a blocker of
found in rat microglia cocultured with fibroblasts (812). protein synthesis, prevented the development of this con-
The current showed a strong inward rectification and an ductance, Norenberg et al. (656) concluded that the chan-
increased inactivation with more negative potentials. The nel protein was newly expressed. Activation of microglia
inward rectifier current can be recorded in all cultured in situ resulted in prominent upregulation of delayed K⫹
microglial cells. Activation of cultured microglial cells by currents in the facial nerve nucleus 24 h after axotomy
LPS results in a reduction of the current by 30% (728). (81) and 48 h after an ischemic insult (540).
When recording from cells in slices from normal Delayed rectifying K⫹ channels are mostly belonging
adult mouse or rat brain (see Fig. 6), the inward current to the large family of Kv channels (for review, see Ref.
was either small or undetectable (81, 82). It may even be 341). Cultured rat microglia express transcripts for Kv1.2,
possible that the small currents recorded in the acutely Kv1.3, and Kv1.5 and LPS-induced activation triggers up-
isolated slices are an early response to the slicing proce- regulation of Kv1.3 expression (288). In vivo analysis of
dure, since there is usually a 2-h delay between slicing and KDR channels expression has demonstrated that intracor-
recording, suggesting that resting microglia lack the in- tical injection of 2 ␮g LPS triggered microglial activation
ward rectifier entirely. as judged by OX42 immunostaining. The OX42-positive
These channels can thus be considered as an early cells showed increased immunoreactivity for Kv1.5 but
marker for activated microglia. Microglial cells in slices of not for Kv1.3 channels (428). Incidentally, treatment of
the facial nucleus strongly increase the functional expres- microglial cultures with “calming” signaling agent trans-
sion of this channel within 12 h after axotomy. The ex- forming growth factor-␤ (TGF-␤) induced a fivefold in-
pression of inward rectifier returned to control levels 7 crease in the Kv1.3 mRNA level and a sixfold increase in
days after the lesion (81). This increase in the inward delayed rectifying K⫹ current density (805). The expres-
current correlates with a more negative membrane poten- sion of Kv1.1 channels was identified in early postnatal
tial from ⫺15 mV before axotomy to about ⫺30 mV after (P1–P10) amoeboid microglia; these channels disap-
axotomy. The presence of the KIR channels and its poten- peared by P14 –P21 and were not detectable in ramified
tial influence on the membrane potential could affect microglial cells (1003).
microglial Ca2⫹ signaling, since it increases electric driv- The expression of both Kv1.5 and Kv1.3 message is
ing force for capacitative Ca2⫹ entry. For example, the necessary for the presence of the outward currents as
inhibition of KIR channels by Ba2⫹ significantly reduced shown by two strategies to interfere with channel expres-
the plateau phase of ATP-induced [Ca2⫹]i transients in sion, a Kv1.5 knockout (Kv1.5⫺/⫺) mouse and an antisense
cultured rat microglia (291). oligonucleotide approach against Kv1.5 and Kv1.3. In the
In situ, the inward rectifier is present in microglial rat brain, Kv1.2 channels were detected in amoeboid mi-
cells in a pathological context such as after ischemia croglia at early postnatal (P1-P10) stages; these channels
(540) or facial nucleus axotomy (81). During postnatal were still present in P14 ramified microglial cells but
development, invading microglial cells with amoeboid almost completely disappeared at P21. The exposure of
morphology can be studied in acutely isolated slices from postnatal rats brains to hypoxia significantly upregulated
6- to 8-day-old mouse corpus callosum. The IKIR was the the expression of Kv1.2 (522).
main component of K⫹ permeability of these invading There are a number of other agents besides LPS that
microglia (86). The IKIR in these cells were inhibited by trigger the activation of microglia and result in the acti-
TEA (50 mM), but not by 4-AP, and were almost com- vation of outward K⫹ currents. Norenberg et al. (656)
pletely blocked by 2–5 mM Ba2⫹ (86). reported that IFN-␥ triggered the induction of the outward
In hippocampal slices acutely isolated from juvenile current. Treatment of microglial cultures from newborn
mice (P5–P9), 74% of ramified microglial cells exhibited mice and rats with pneumococcal cell walls (derived from
relatively small (mean current density ⬃3.6 pA/pF) IKIR; Gram-positive Streptococcus pneumoniae) resulted in mi-
in activated microglia from the same organotypic slices croglial activation and the upregulation of IKDR very sim-
cultured for 3–7 days, the current was expressed in 91% of ilar to that observed after exposure to LPS and involved
cells and its density increased severalfold (to ⬃9.6 pA/pF) de novo synthesis of Kv1.3 channels (226). Interestingly,
(802). the embryonic microglia treated with pneumococcal cell
walls despite showing all signs of activation (such as
2. Delayed (outward) rectifier K channels morphological shift, release of TNF-␣, and downregula-
tion of IKIR) did not demonstrate an upregulation of IKDR
In 1992, Norenberg et al. (656) found that the treat- (226), suggesting a developmental remodeling of an ion
ment of cultured rat microglial cells with bacterial lipo- channel-related activation program (226). ␤-Amyloid pro-

tein fragment 25– 45 (A␤25– 45) as well as with full-length at more negative potentials in more alkaline pH in that it
␤-amyloid peptide (A␤1– 42) triggered the upregulation of differed by 20 mV comparing extracellular pH 5.8 to 7.8.
Kv1.3 and Kv1.5 channels in cultured rat microglial cells Additionally, extracellular pH affected IKDR kinetics,
(161). The amplitudes of IKDR (similar in their parameters slowing the inactivation kinetics in a more acidic environ-
to Kv1.3-generated currents) were ⬃5.6 times larger in ment (240).
activated microglial cells from organotypically cultured In microglial cultures treated with ACM to promote
(5–7 days) hippocampal slices compared with resting mi- ramification, patch-clamp recordings revealed IKDR very
croglia in acutely prepared slices (802). In contrast, in rat similar to those recorded from amoeboid microglia. These
cultured microglia subjected to oxygen/glucose depriva- potassium currents were effectively blocked by charyb-
tion, Kv1.3 was downregulated. This downregulation was dotoxin (CTX, IC50 ⬃1.13 nM), noxiustoxin (NTX, IC50 ⬃
mediated via protein tyrosine kinases of the src family 1.24 nM), and kaliotoxin (KTX, IC50 ⬃0.81 nM) and were
and was considered as a response to stress (134). The insensitive to dendrotoxin (DTX) (241).
Kv1.3-mediated currents were increased in the microglia
activated following experimentally induced (systemic kai- 3. Ca2⫹-dependent potassium channels
nate injection) status epilepticus (581).
The expression of Kv channels can be linked to mi- In inside-out patches obtained from cultured bovine
croglial function. The LPS-induced NO release was re- microglia, a calcium-dependent K⫹ channel (KCa) with
duced by the antisense deletion of Kv1.5 and completely unitary conductance of 240 pS in symmetrical 140 mM K⫹
absent in the Kv1.5-deficient animals; the antisense dele- was detected (572). In cultured microglia from human
tion of Kv1.3 had no effect (688). In contrast, proliferation embryos, the single KCa channels had a conductance of
was augmented with loss of both Kv1.3 or Kv1.5 channel 106 pS at physiological transmembrane K⫹ concentra-
expression. This is supported by the observation that the tions (574). Mean open times of KCa were increased with
proliferation rate was higher in Kv1.5-deficient animals patch depolarization due to an increase in both opening
after a facial nerve lesion (688). Similarly, LPS treatment frequency and mean open time (572, 574). Microglia in
of cultured microglia induced de novo synthesis of Kv1.5 hippocampal slices from patients who underwent surgery
channels; the inhibition of these channels with 4-AP sup- for pharmaco-resistant epilepsy showed a small compo-
pressed the NO release (735). At the same time, the for- nent of a Ca2⫹-sensitive outward current (77). The esti-
mation of peroxynitrite, which is formed from superoxide mated single-channel conductances varied between 149
and nitric oxide, requires Kv1.3 channel activity; Kv1.3 pS (in outside-out mode) and 187 pS (in cell-attached
channel blockers reduced the respiratory burst, but not configuration), indicating the activation of high-conduc-
NO production (288). Oxidative stress by itself, however, tance (BK) channels (77). The chemokine MIP1-␣ in-
may downregulate microglial Kv1.3 currents through src creased whole cell outward current amplitudes measured
family protein tyrosine kinase-dependent phosphoryla- at ⫹60 mV by a factor of 3.3 (77).
tion; moreover, it was suggested that Kv1.3 channel and Rat cultured microglia also express high densities of
the protein tyrosine kinases are linked into one functional small-conductance Ca2⫹/calmodulin-activated K⫹ chan-
complex through postsynaptic density protein 95 (PCD-95), a nels of KCNN4/KCa3.1/SK4/IK1 type (436), although an
scaffolding protein (134). Incidentally, the correlation be- expression of KCNN2/SK2 and KCNN3/SK3 mRNA was
tween Kv channels expression and microglial proliferation also reported (446). The inhibition of these channels by a
was detected in microglia that were tissue-printed and selective antagonist triarylmethane-34 (TRAM-34) signifi-
cultured from rat hippocampal slices. It turned out that cantly decreased the neurotoxic potency of microglia in a
proliferating microglia expressed predominantly Kv1.3 mixed culture system (436). In addition, KCNN4/SK4
currents, whereas Kv1.5 currents were identified in non- channels were involved in the control of microglia acti-
proliferating cells (474). Treatment of cultured microglia vation and contributed to the upregulation of iNOS and
with HIV-1 regulatory protein Tat (100 ng/ml to 1 ␮g/ml, the production of NO and peroxynitrite, with these being
24 h) induced three- to sixfold increase in Kv1.3 current directly implicated in neurotoxicity. On a signaling level,
density (from ⬃5 to ⬃15 pA/pF at 100 mg/ml and 30 pA/pF the KCNN4/SK4 channels were linked to the activation of
at 1 ␮g/ml Tat, respectively). This increase was mediated mitogen-activated protein kinase (MAPK) cascade (436).
through NF-␬B signaling pathway and most probably re- As a consequence of these signaling functions, an injec-
quired Tat entry into the cell (968). The inhibition of Kv1.3 tion of TRAM-34 into the rat optic nerve had significant
channels with agitoxin-2 reduced the respiratory burst in neuroprotective impact (436). The KCa channels are also
cultured microglial cells treated with phorbol 12- involved in controlling the microglial respiratory burst;
myristate 13-acetate (446). the treatment of microglial cells with apamin (selective
The gating properties of IKDR are regulated by extra- blocker of KCNN2/SK2 channels) or with clotrimazole
cellular pH in a concentration range that can occur under (KCNN4/SK4 channels inhibitors) markedly inhibited the
pathological conditions. Activation and inactivation occur respiratory burst (446). The messages for KCNN1/SK1,

KCNN2/SK2, and KCNN3/SK3 channels were identified in estimated to be very small, ranging between 1 and 3.5 pS
rat cultured microglia; the KCNN3/SK3 showed predomi- (230), which is much lower than that reported for volume-
nant expression, which increased ⬃1.7 times following sensitive Cl⫺ channels of other cell types (15– 40 pS; Refs.
LPS stimulation (807). The immunoreactivity for KCNN3/ 584, 980). Quantitative real-time PCR (and normalized vs.
SK3 was found in microglial cells in healthy adult rat housekeeping gene HPRT-1) detected mRNAs for several
striatum; this immunoreactivity substantially increased Cl⫺ channel genes in purified rat microglial cultures; the
after ischemic lesion (807). Finally, inhibition of KCNN3/ order of expression being CLIC1 ⬎ ClC3 ⬎ ICln ⱖ ClC2 ⬎
SK3 channels by 100 nM apamin or by 5 nM tamapin Best2 ⬎ Best1 ⱖ Best3 ⬎ Best4 (230). Out of these genes,
reduced microglial neurotoxicity and somewhat amelio- the family of Bestophine (Best) channels (359) is, accord-
rated microglial activation (807). ing to its biophysical properties, the most suitable candi-
date for the microglial volume-regulated Cl⫺ channels
4. G protein-activated K⫹ channels (230).
In addition, Cl⫺ channels in murine microglial cells
Activation of G proteins by either intracellular perfu- are involved in preserving the ramified morphology, as
sion with guanosine 5’-O-(3-thiotriphosphate) (GTP␥S) or pharmacological inhibition of these channels prevented
by stimulation of metabotropic receptors (by ATP, C5a, ramification (242). At the same time, Cl⫺ channels are
TNF-␣, or EGF) induced outward K⫹ currents. These also involved in phagocytosis and their inhibition by flufe-
currents were sensitive to 4-AP (⬃40% inhibition at 4 mM namic acid (200 ␮M) or NPPB (200 ␮M) almost com-
concentration) and did not show time-dependent inacti- pletely blocked phagocytic activity. Interestingly, SITS
vation (401, 402). The receptor-activated K⫹ currents dis- was less potent in respect to the inhibition of phagocyto-
appeared after treatment of cultures with pertussis toxin sis, whereas DIOA had no effect (304). Furthermore, the
(PTX). inhibition of swelling-activated Cl⫺ channels in primary
cultured microglia by flufenamic acid (200 ␮M) or DIOA
D. Anion Channels (10 ␮M) or the incubation of the cells in Cl⫺-free high K⫹
solution suppressed the formation of a lamellipodia
1. Volume-regulated Cl⫺ channels (1040). The volume-sensitive Cl⫺ channel blockers NPPB,
tamoxifen, and DIDS prevented the rapid process out-
Microglial cells express volume (or swelling)-regu- growth of microglial processes initiated in response to
lated Cl⫺ currents. Conceptually, the volume-regulated brain tissue damage. In contrast, filopodia extension was
Cl⫺ channels are implicated into a general cellular phe- resistant to Cl⫺ channel inhibitors, indicating that these
nomenon of regulatory volume decrease, which follows a motile processes have different cellular mechanisms
hypotonic shock and involves electroneutral extrusion of (378).
KCl (through Cl⫺ and K⫹ channels) with concominant
water loss needed to restore the cell volume (424). 2. CLIC-1 chloride channels
Outwardly rectifying, voltage- and time-independent
volume-sensitive Cl⫺ currents were detected in cultured The chloride intracellular channel-1 (CLIC-1), active
murine microglial cells (808) where they were suggested in both nuclear and plasma membranes, was found in
to regulate proliferation (808) and to be involved in set- several cell lines and was associated with activation of
ting the resting membrane potential (638). The swelling- macrophages (916, 944).
sensitive Cl⫺ currents are rapidly and reversibly activated The functional expression of CLIC-1 was identified in
by exposure to hyposmotic (up to 55% normal osmolarity) primary rat neonatal microglial cultures (662). The CLIC-1
extracellular solutions (230, 808). The selectivity of these channels were localized in the plasma membrane, and the
channels to anions is similar to other anion channels single-channel currents were recorded in a cell-attached
being I⫺ ⬎ Br⫺ ⬎ Cl⫺ ⬎ F⫺ ⬎ aspartate ⱖ glutamate configuration. The single-channel conductances were
(230). Incidentally, the swelling-activated Cl⫺ channels ⬃6.5– 8 pS. Treatment of microglia with A␤1– 42 or A␤25–35
were also permeable to gluconate (Pgluconate/PCl ⫽ 0.34; peptides potentiated expression of CLIC-1 proteins (as
Ref. 808). Microglial volume-sensitive Cl⫺ channels are determined by Western blot) and increased CLIC-1 single-
inhibited by flufenamic acid, 4-nitro-2-(3-phenylpropyl- channel open probability and mean open time (662), with
amino)-benzoic acid (NPPB), 6,7-dichloro-2-cyclopentyl- the latter effect being the result of A␤-dependent promo-
2,3-dihydro-2-methyl-1-oxo-1H-inden-5-yl(oxy)acetic acid tion of CLIC-1 translocation from the cytosol to the plas-
(IAA-94), and [(dihydroindenyl)oxy]acetic acid (DIOA) malemma (588). The activation of microglia with LPS or
(230, 242, 808) as well as by DIDS and SITS in a voltage- basic fibroblast growth factor affected neither the expres-
and time-dependent manner (242). sion nor the functional properties of these Cl⫺ channels.
The single-channel conductance of swelling-sensitive Inhibition of CLIC-1 channels with the specific blocker
Cl⫺ channels determined indirectly by noise analysis was IAA-94 (R(⫹)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-

methyl-1-oxo-1H-inden-5yl)-oxy]acetic acid) suppressed exceedingly small, being in a range of several fA, as

microglial proliferation and reduced neurotoxicity of A␤- estimated in noise analysis performed on human neutro-
treated microglial cells. This reduced neurotoxicity was phils (201).
primarily due to the inhibition of the ROS generation Proton currents in microglial cells are inhibited by
following the inhibition of CLIC-1-mediated Cl⫺ conduc- TEA (1 mM) and by micromolar concentrations of poly-
tance (588); this effect was present following the block- valent metals with the order of potency Zn2⫹ ⬎ La3⫹ ⬎
ade of CLIC-1 by pharmacological agents or anti-CLIC-1 Ni2⫹ ⬎ Cd2⫹ ⬎ Co2⫹ ⬎ Ba2⫹ (238). The inhibitory action
antibody, or by replacement of extracellular Cl⫺ with of these cations is greatly potentiated by acidification
impermeable anions. Ultimately, the downregulation of (237).
CLIC-1 synthesis with siRNA reduced A␤-potentiated re- The treatment of cultured microglia with either LPS
lease of TNF-␣ from microglial cells (662). Thus the or with an astrocyte-conditioning medium caused ⬃50%
CLIC-1 channels participate in various aspects of micro- decrease in the H⫹ current amplitude and deceleration in
glial activation during AD (588). their activation kinetic (461). Similar effects were caused
by incubation of microglial cultures with cytoskeletal dis-
3. Other Cl⫺ channels ruptive agents cytochalasin D (2 ␮M) or colchicine (1 ␮M,
both for 24 h), whereas the stabilization of cytoskeleton
A yet unidentified Cl⫺ current was triggered by ap-
by 20 ␮M phalloidin or 0.5 ␮M taxol did not affect proton
plication of the chemokine CCL21. Local application of
currents (460, 461). Changes in cell volume are reported
CCL21 for 30 s triggered a Cl⫺ conductance, which per-
to potentiate H⫹ currents: for example, in cultured rat
severed for tens of minutes. It was identified as mediated
microglial cells exposed to lactoacidosis or to intracellu-
by Cl⫺ channel by its sensitivity to the transmembrane
lar dialysis with acidic (pH 5.5–5.8) solutions (618). Aci-
Cl⫺ gradient and to the blockers DIDS and SITS (747).
dosis-induced cell swelling potentiated H⫹ currents by
High conductance (325 pS at symmetrical Cl⫺ con-
shifting their activation curve to more negative potentials
centrations) channels were detected in inside-out patches
and by accelerating activation kinetics. This potentiation
from cultured bovine microglial cells. These channels
required intracellular ATP and was prevented by cytoskel-
were activated in response to hyper- or depolarization of
eton-active agents phalloidin and cytochalasin D (618).
the patch membrane (572). In fetal human cultured mi-
The functional role of microglial H⫹ currents is not
croglia, the Cl⫺ channel with unitary conductance of 280
known, although they may play an important role in the
pS was also detected (574).
“respiratory burst” associated with phagocytosis (236,
368, 369, 610, 803), which represents rapid oxygen con-
E. Proton Channels sumption because of the activation of NADPH oxydase
with subsequent production of superoxide anion. As
Changes in extracellular pH accompany both physi- NADPH oxydase produced one proton per every super-
ological and pathological processes in the brain. Neuronal oxide ion the acidification of cytosol occurs; H⫹ channels
activity causes rapid alkalosis by a tenth of a pH unit, may provide a pathway for protons efflux. Microglia, and
which is followed by longer lasting acidosis by about especially activated microglia is known to produce super-
two-tenths of a pH unit [for example in the cerebellum oxide (167), and therefore this role for H⫹ channels seems
(477), in the cortex (942), in the hippocampus (431) or in to be likely (237). The activation of H⫹ channels produced
the spinal cord (423)]. In the optic nerve, a white matter a substantial efflux of protons, which decreases extracel-
tract, the onset of neuronal activity only triggers an acid lular pH and therefore reduces the H⫹ current during
shift (191). In pathology, the pH shifts are even larger as long-lasting activation (617).
in an ischemic insult where pH can drop to 5.0.
Voltage-gated currents carried by protons were ini-
tially discovered in snail neurons and oocytes of axolotl F. Aquaporins
(37, 911) and were subsequently detected in various cell
types, including neurons and glia (see Ref. 237 for re- Aquaporins form transmembrane channels perme-
view). In microglia, H⫹ currents were discovered in pri- able to water and some other molecules such as, for
mary cultures from mice, rats, and humans (239, 460, 461, example, glycerol and urea (8). In mammals, 11 types of
574, 965). Similarly to other cell types, microglial H⫹ aquaporins (designated as AQP0 –10) are described.
current have an exceptionally high selectivity to protons These channels are mainly responsible for water homeo-
(238). The voltage dependence of microglial H⫹ currents stasis, although they are also involved in the regulation of
strongly depends on extracellular pH (pHo), with an acid- other cell functions, including cell migration (961). In the
ification shifting the activation threshold to more positive brain, aquaporins are important for maintaining the extra-
potentials thus attenuating the current (237). The single- cellular volume, and they play an important role in the
channel conductance of proton channels is believed to be pathogenesis of the brain oedema (1026). The AQP1,

AQP4, and AQP9 are expressed in the brain (463); the was upregulated following treatment with Ca2⫹ iono-
AQP4 and AQP9 are expressed predominantly in astro- phore 4-bromo-A23187 and downregulated by PKC acti-
cytes (429, 838, 876), being the most abundant. An injec- vated with phorbol 12-myristate 13-acetate (554).
tion of LPS into substantia nigra of rats triggered an Another study indicates that microglial cells express
expression of AQP4 at both mRNA and protein level in the Cx36. The Cx36 mRNA and protein were found in micro-
activated microglia, with the latter being identified by glial cultures prepared from human and mouse, and Cx45
positive OX-6 staining (915). In the microglia from control mRNA was found in mouse microglial cultures only.
specimens, the expression of AQP4 was almost undetect- Functional coupling using electrophysiology was re-
able. Hitherto, however, this finding did not receive inde- ported for one-third of human or mouse microglial pairs.
pendent confirmation, and the possible role of aquaporins Low-strength electrical coupling was also reported be-
in microglial cells remains unexplored. tween cultured microglia and neurons. The expression of
Cx36 was not affected by microglial activation (220).
Cultured mouse microglial cells treated with TNF-␣ were
G. Connexons also shown to significantly upregulate surface expression
of Cx32; the latter reportedly function as hemichannels
The intercellular channels, generally known as con- (898) and may mediate microglial release of glutamate
nexons, form gap junctions, which are responsible for following cytokine stimulation. Increased expression of
cell-to-cell coupling important for spread of electrical Cx32 was detected in microglial cells deficient in MECP2
(e.g., in the heart muscle) or metabolic (e.g., in astroglial gene. The latter encodes the epigenetic factor methyl-
syncytia or in the liver) signals (60, 257, 776, 843). Molec- CpG-binding protein-2, and MECP2 mutations are linked
ularly, every connexon is composed of six subunits, to Rett syndrome and various forms of autism. Increased
named connexins (i.e., a full intercellular channel is com- expression of Cx32 was associated with elevated micro-
posed from 2 opposing connexons, which in turn are glial release of glutamate that exhibited neurotoxic action
made up from 12 connexins). At least 20 different con- (544). In conclusion, the expression pattern and physio-
nexins have been identified molecularly; they are classi- logical relevance of microglial connexons remain unre-
fied according to their molecular weigh as CxM.W., e.g., solved.
Cx26, Cx32, Cx43, etc. With the use of the appropriate
knockout mice, the differential cell-type specific (Cx43 in
astrocytes, ependymal, and vascular cells; Cx32 in oligo- IX. MICROGLIAL RECEPTORS: CONCEPT OF
dendrocytes; and Cx36 in neurons and microglia) expres- “ON” AND “OFF” RECEPTOR-MEDIATED
sion of connexins in the brain was identified (396). SIGNALING
Importantly, connexons may function not only as
fully assembled intercellular channels, but also as a “he- Multiple signals converge on microglial cells to ac-
michannels,” which may provide, for example, pathways tively maintain or alter their functional state and to or-
for release of relatively big molecules (853). chestrate the specific repertoire of microglial functions.
Dye injection into resting or activated microglia in Transitions between surveillance and activated states are
situ has not revealed any dye transfer to another cell, triggered when microglia perceives the sudden appear-
indicating that there is no evidence for intercellular cou- ance, abnormal concentration, or unusual molecular for-
pling. Resting microglia in situ were not immunoreactive mat of certain factors (72, 351). We can distinguish be-
for Cx43, yet Cx43 immunoreactivity markedly increased tween two principles of signaling, termed the “on” and
after applying the stab wound to the rat brain. The Cx43 “off” receptor-mediated signaling (67, 351). The “on” sig-
were mostly immunolocalized in aggregates of microglia naling is quite obvious: a novel signaling molecule that
around the insult area (255), and homocellular microglial appears in the brain is recognized by microglia and trig-
gap junctions may be formed under various pathological gers activation. An “off signal” may, instead, maintain a
conditions (448). persisting signaling to contain microglia. Much like a wire
In vitro, in primary cultures, mouse microglial cells in a shopping window conducting a constant current, the
showed low levels of Cx43, as well as low levels of dye constant signaling of “off signals” keeps the microglia in
(Lucifer yellow) coupling. Treatment of cultured micro- the default setting, and only its interruption represents
glia with mixture of LPS and INF-␥ or INF-␥ and TNF-␣ the alarm.
triggered a significant increase of Cx43 expression, spe- The biochemical nature and the sources of “on” sig-
cific accumulation of Cx43 at the cell contact sites, and nals are diverse, as the variety of receptors expressed by
increase in the probability of dye coupling to ⬃60%. How- microglia (see Tables 3–7 and sections X–XIV). Struc-
ever, in the image which is representatively shown for tures associated with bacterial cell walls, viral envelopes,
microglial dye coupling, only three cells are visible (255). or their respective DNAs and/or RNAs are typical exam-
In cultured rat newborn microglia, expression of Cx43 ples for signals that are usually not present in the micro-

TABLE 3. Neurotransmitter receptors in microglia
Experimental
Receptor Type Preparation/Technique Pharmacology Properties and Functional Relevance Reference Nos.
Purinoceptors
A1, A2A, A2B, A3 Rat, mouse/primary cultures/ Agonists: Adenosine receptors generally 282, 309, 347, 366,
adenosine receptors RT-PCR/ 2-chloro-adenosine (A1); mediate trophic effects, 484, 497, 506,
immunohistochemistry/ CGS 21680 (A2A); regulating microglial 670, 795, 889,
pharmacological assays 2-chloro-N6-(3-iodobenzyl-N- proliferation, survival, 947
methyl-5=-carbamoyladenosine, secretion of cytokines, and
Cl-IB-MECA (A3) expression of various genes.
Antagonists:
Caffeine, theophylline,
8-(p-sulphophenyl)-theophylline;
N-[9-chloro-2-(2-furanyl)
[1,2,4]triazolo[1,5-c]quinazolin-5-
yl]benzeneacetamide, MRS1523
(A3)
P2X1, P2X4, P2X7 Rat/brain sections/RT-PCR/ Embryonic microglia expressed 132, 1008
immunohistochemistry P2X1 and P2X4 subunits;
⬃30% of cells expressed P2X7
receptors. Adult microglia
expressed only P2X4 and
P2X7 subunits.
P2X4 Rat/whole brain, organotypic Inhibition by ATP-TNP; insensitive Expression of P2X4 receptors 44, 415–417, 633,
slices, spinal cord/ to PPADS; positively modulated specifically increased after 929, 930, 932
immunocytochemstry, by ivermectin spinal cord lesions;
pharmacological assays, pharmacological inhibition of
antisense expression P2X4 receptors alleviated
inhibition symptoms of neuropathic
pain in animal models
P2X7 Rat, mouse/primary cultures, Agonist: BzATP ATP in 1-5 mM concentrations 150, 218, 277, 279,
corpus callosum Inhibitors: oxATP, BBG triggered [Ca2⫹]i transients 342, 377, 590,
slices/microglial cell lines/ and transmembrane currents, 787, 851
whole cell voltage-clamp/ which were potentiated by
Ca2⫹ imaging/RT-PCR/ lowering extracellular
immunohistochemistry divalent cations
concentration, were
mimicked by BzATP and
inhibited by oxATP and BBG.
P2X7 receptors are linked to
microglial activation and
release of cytokines.
P2Y2, P2Y6, P2Y12, Mouse/primary culture/RT- Agonists: ADP; UDP; UTP Activation of P2Y receptors 365, 467, 527, 568,
P2Y13 PCR/Ca2⫹ imaging Antagonists: suramin (ATP, UDP) triggered [Ca2⫹]i 966
transients and [Ca2⫹]i
oscillations originating from
PLC/InsP3-mediated ER Ca2⫹
release. P2Y12 receptors are
critical for initiation of
microglial activation and
migration. In the spinal cord,
P2Y12 receptors are involved
in neuropathic pain.
Glutamate receptors
AMPA receptors Rat/primary culture/microglial Inhibitors: CNQX, LY300164 AMPA receptors were identified 156, 343, 529, 652
GluR1,GluR2, GluR3, cell line/whole cell Positive modulators: PEPA, CTZ in cultured cells only;
GluR4 voltage-clamp/RT-PCR activation of AMPA receptors
is linked to TNF-␣ release,
remodeling of cytoskeleton
and glutamate-induced
chemotaxis.
Metabotropic glutamate Rat/primary culture/Ca2⫹ Agonist: 1S,3R-ACPD Stimulation of with 1S,3R-ACPD 66
receptors imaging/RT-PCR triggered [Ca2⫹]i transient.
Group I - mGluR5a
Continued

Experimental
Group II - mGluRs2, Rat/primary culture/RT-PCR Agonist: 1S,3R-ACPD Stimulation of group II 906–908

mGluR3 receptors stimulated
Group III - mGlu4, microglial activation, release
mGlu6, and mGlu8 of TNF-␣, and neurotoxicity;
stimulation of group III
receptors reduced
neurotoxicity.
GABA receptors
GABAB(1a), GABAB(1b) Rat, mice/primary cultures/ Agonists: baclofen, SKF 97541 GABAB receptors were 142, 480
and GABAB(1c) acute slices/whole cell identified at mRNA and
voltage-clamp/RT-PCR/ protein levels as well as
immunocytochemistry/Western functionally by recording
blot GABAB-mediated whole cell
currents. Axotomy of facial
nerve triggered an increase in
number of microglial cells in
the facial nucleus that
expressed GABAB receptor.
Cholinergic receptors
␣7 nAChRs Mouse, rat/RT-PCR, Western Agonist: methyllycaconitine Stimulation of cultured 198, 833, 884
blot, microglia with nicotine
immunohistochemistry/Ca2⫹ triggered [Ca2⫹]i transients,
imaging which were blocked by
␣3, ␣5, ␣7, ␤4 nAChRs Human embryo/RT-PCR U73122 and xestospongin C, 765
␣6 or ␤4 nAChR Rhesus monkey/retina/ thus suggesting the functional 530
immunohistochemistry coupling between ␣7nAChRs
and PLC.
Adrenergic receptors
␣1A, ␣2A, ␤1, ␤2 Rat/primary culture/RT-PCR/ Agonists: norepinephrine, Stimulation of b receptors 302, 613, 727, 902,
biochemical assays/Ca2⫹ terbutaline increased cAMP level and 990
imaging induced Ca2⫹ release from
the ER. Stimulation of
adrenergic receptors
modulated release of
cytokines.
Agonists: salmeterol
Dopamine receptors
D1, D2 Rat, mouse/primary culture, Agonists: dihydrexidine (D1 Stimulation of dopamine 267, 555
acute slices/whole cell receptors), (⫺)-quinpirole (D2 receptors inhibited IKIR and
patch clamp receptors) activated outward potassium
currents. Chronic stimulation
enhanced migration and
decreased NO release. In
microglia from Parkinson
disease brains, the expression
of D1-4 receptors was
increased.
glial environment and that are thus identified as signs of partments or when they become (bio)chemically altered
infection. The activation signals are represented not only (350, 512, 513, 536, 777). Both pathogen- and damage/danger-
by infectious agents but also by molecules delivered fol- associated molecular patterns (PAMPs/DAMPs) can alert
lowing tissue damage so that even the smallest lesions microglia in a quite similar, yet distinct way, and even em-
can cause rapid microglial responses (645). ploy overlapping receptors of the Toll-like receptor (TLR)
Intracellular proteins or serum factors, which normally family. By virtue of multiple receptors (see sects. X–XIV),
carry diverse physiological functions, can acquire microglia- microglia can also sense deviations from the physiological
activating features when they are further induced upon concentration of neurotransmitter molecules. Beyond criti-
stress, when they leave their respective physiological com- cal levels, some neuro- and cotransmitters could indicate

TABLE 4. Microglial receptors for neurohormones and neuromodulators
Receptor Type Experimental Preparation/Technique Properties and Functional Relevance Reference Nos.
PAF receptors Rat, mouse, human/brain In the brain, PAF receptors were 385, 614, 794, 982
sections, primary cultures/in predominantly expressed in microglia.
situ hybridization, Northern Stimulation of PAF receptors triggers
blot, Ca2⫹ imaging [Ca2⫹]i elevation, resulted from ER Ca2⫹
release and long-lasting activation of
Ca2⫹ entry. The Ca2⫹ entry increases
IL-6 expression in cultured microglia.
Bradykinin receptors, Rat/primary cultures/RT-PCR, Resting microglia express only B2 397, 648–650
B1, B2 immunocytochemistry, whole receptors, LPS activation upregulates B1
cell voltage-clamp expression. Stimulation of B2 receptors
triggers IKCa following [Ca2⫹]i increase.
B1 receptors induce chemotaxis and
activate NCX.
Histamine receptors Rat/primary cultures/Ca2⫹ Histamine triggers Ca2⫹ release from the 29
imaging ER.
Endothelin receptors, Mouse, human/primary cultures/ Stimulation of microglia with ET-1 and ET- 573, 603, 1017
ETB single cell RT-PCR, Ca2⫹ 3 triggered Ca2⫹ release and SOCE in
imaging 13% of mouse and in 80% of human
cells. Ca2⫹ responses were inhibited by
ETB antagonist BQ788 and mimicked by
ETB agonist BQ3020.
Cannabinoid receptors, Rat, mouse, human/primary Resting microglia express CB1 receptors; 114, 259, 549, 744, 766
CB1, CB2 cultures/RT-PCR activation causes upregulation of CB2
receptors. High expression of CB2
receptors found in mice with
experimental autoimmune
encephalomyelitis. Stimulation of CB
receptors stimulates proliferation and
reduces neurotoxicity.
Angiotensin II receptors, AT1, AT2 Rat embryo/primary cultures/ Unstimulated microglia express AT2 596
RT-PCR, pharmacological receptors; LPS triggers upregulation of
assays AT1 receptor. Inhibition of AT1 receptors
by losartan suppresses microglial
activation and reduces production of NO
and IL-1␤.
Somatostatin receptors, Rat/primary cultures/RT-PCR Activation of sst receptors induced protein 272
sst2, sst3, sst4 phosphorylation and inhibited microglial
proliferation.
Glucocorticoid and Rat/primary cultures/binding Microglia express both glucocorticoid and 900
mineralocorticoid receptors assays/Western blot mineralocorticoid receptors; stimulation
of glucocorticoid receptors inhibited
proliferation of cultured microglia and
enhanced lysosomal formation.
Opioid receptors, Human embryo, cat, rat/primary Stimulation of MOR inhibits chemotaxis 140, 221, 388
KOR, cultures/RT-PCR, towards C5a, increases migration and
MOR immunohistochemistry upregulates P2X4 expression in rat.
binding assay,
pharmacological assay
Neurokinin (substance P) receptors, Human embryo, mouse/RT-PCR, Stimulation of NK-1 receptors triggers 491, 749
NK-1 immunocytochemistry activation of NF-␬B.
VIP receptors, VPAC1 Rat/primary cultures/RT-PCR, Stimulation of VPAC1 receptors inhibits 212–214, 330, 455
pharmacological assays LPS-induced activation and secretion of
proinflammatory factors TNF-␣, IL-1␤,
and NO.
Neurotrophin receptors, Rat/primary culture/RT-PCR; BDNF triggered sustained [Ca2⫹]i elevation 597
Trk-B1 Ca2⫹ imaging resulting from PLC/InsP3-medaietd Ca2⫹
release followed by a long-lasting
activation of SOCE. The Trk-B1
receptors were identified by RT-PCR.
impaired or excessive neuronal activity (82, 365), which are trol in the normal CNS. Such a principle is indicated for
perceived by microglia as a sign of lesion. the ligand-receptor pairs CD200-CD200R, CX3CL1-
The “off” receptor-mediated signaling is due to a loss CX3CR1, and SIRP␣(CD172a)-CD47 (33, 89, 123, 381,
of constitutive signaling. They may deliver a calming con- 1001). Here, disrupted signaling may set off the response.

Accordingly, triggers of microglial alert could be sorted has ancient evolutionary roots and is operative virtually in
by the mode of signaling as to “on” and “off” signals, every type of cells and in all tissues (100, 103, 104, 107,
independently of their chemical constitution (67, 351, 108). In the nervous system ATP (the principal purinergic
949). This distinction should not be mistaken as dividing signaling molecule) and its derivatives act both as a pri-
“activating” and “inhibiting” influences. Certain factors, mary transmitter and as a cotransmitter in the brain, in
such as IL-10, have suppressive effects on microglia, as the spinal cord, and in the peripheral nerves (5, 101, 107,
they have for other macrophages. These factors, however, 661). Neural cells release ATP through several mecha-
themselves depend on induction and can then develop nisms, which include exocytosis, diffusion through
their influences. Overlap between principles of “off sig- “maxi” plasmalemmal channels, through transporters and
nals” and microglial suppressors certainly exist. Some may be even by lysosomes (5, 686, 687, 851, 1034). Fur-
receptor systems, such as adrenergic receptors, will prob- thermore, massive ATP release invariably accompanies
ably prove to maintain some basal as well as inducible cell death and destruction, and therefore, ATP acts as a
influences. However, the principle of “off signals” comes universal “danger” signal; the excess of ATP in the brain
with another consequence for microglial recruitment. It parenchyma is neurotoxic (108, 781). Importantly, ATP
allows microglia to react to unknown signs of danger. released from the cells is rapidly degraded by extracellu-
Microbial structures are sensed by cells of the innate lar ectonucleotidases (5, 1041); ATP derivatives (ADP,
immunity through the pattern recognition receptors AMP, and adenosine) thus produced also act as purinergic
(PRR), such as the TLRs, which initiate first measures and signaling molecules. Physiological effects of purines and
also influence adaptive immune functions (350). Yet all of pyrimidines are mediated through an extended family of
the typical “on signals” require the expression of cognate purinoceptors, which are classified as metabotropic P1
receptors to be identified as a sign of threat to the CNS adenosine receptors, metabotropic P2Y purinoceptors,
homeostasis. In contrast, the “off signal,” when disruption and ionotropic P2X purinoceptors (102, 297, 660, 879),
of an input acts as a sign of alert, enables microglia to which are widely expressed in the majority of living cells
respond in situations where the actual source of distur- (106) and are particularly abundant in glia (283, 457, 458,
bance is not known. Loss of input, rather than input per 493, 959).
se, is the signal. Microglia may therefore recognize neu- The ATP-induced currents were, for the first time,
ronal stress and loss of homeostasis independent of pre- identified in microglial cultures prepared from embryonic
formed receptors. The “on” and “off signaling” concept mouse or newborn rat brains (976). The cells were posi-
may thus relate to the detection of known and unknown tively identified as microglia by specific staining with
indicators of danger (351). Upcoming research will dem- DiI-ac-LDL, as well as with Mac-1 and CD-45 antibodies.
onstrate whether microglial containment by this mecha- The application of 100 ␮M ATP evoked a biphasic current
nism plays a critical role in the steady-state functions and response comprising a fast cationic current (with reversal
whether loosening of calming signals occurs with ageing potential ⬃0 mV) and slower outward K⫹ current. The
(868). fast inward current rapidly reached the peak of ⬃100 pA
Receptor equipment and signaling outcomes may dif- and desensitized in the presence of the agonist. Subse-
fer among microglial populations, since CNS regions dif- quent Ca2⫹-imaging experiments have demonstrated that
fer, e.g., by the neurotransmitter microenvironment, ATP also induced [Ca2⫹]i elevation, which was entirely
blood-brain barrier properties, or myelin content. The produced by Ca2⫹ entry, most likely reflecting Ca2⫹ per-
surveillance features and the interpretation of environ- meability of ionotropic purinoceptors (976). These initial
mental cues could differ between microglial subsets that findings were subsequently confirmed in LPS-treated rat
are probably predestined for distinct reactive options cultured microglial cells (659) and in cultured human
(816). It will be in line with the differential induction of microglia (575). Pharmacological assays applied to these
diverse reactive phenotypes to characterize such a pre- preparations demonstrated that the two components of
formed specialization also by anatomical divisions. Be- the ATP-induced membrane current reflected activation
sides the mere density, the functional setup of microglia of ionotropic P2X (nonselective currents) and metabo-
could decide on the role of these cells in CNS pathologies. tropic P2Y (modulation of K⫹ channels) receptors (399,
575, 659). The very same pattern of the ATP current
X. NEUROTRANSMITTER RECEPTORS response was found in microglial cells voltage-clamped in
acute slices from an adult mouse brain (82).
A. Purinoceptors
2. Purinergic control of microglial function
1. Purinergic signaling system
The purinergic signaling system is specifically impor-
Purines and pyrimidines act as widespread extracel- tant for neuronal-glial interactions because almost every
lular signaling molecules. The purinergic signaling system type of glia is sensitive to ATP due to the functional

expression of various purinoceptors (283, 959). The dam- strates activation-dependent plasticity. Microglial activa-
age to the brain is invariably associated with a massive tion in vitro with LPS triggered significant remodeling of
release of ATP present in high concentrations in the cy- cells sensitivity to P1/P2 receptors agonists (604). Cul-
tosol of living cells. Experiments in vitro and in vivo have tured rat microglia exposed to hypoxic conditions signif-
demonstrated that ATP and its analogs trigger rapid func- icantly upregulated both P2Y and P2X7 receptors (616).
tional responses of microglial cells, comprising fast con- Treatment with LPS led to a significant increase in
verging movement of microglial processes towards the metabotropic ATP-induced Ca2⫹ responses, whereas the
lesion, membrane raffling, the outgrowth of microglial P2X7 ionotropic Ca2⫹ signaling was reduced (64).
processes, and the release of various biologically active Similar remodeling of purinoceptors expression was
substances, such as cytokines and inflammatory proteins observed in situ in various pathological models. That is,
(see, e.g., Refs. 65, 190, 265, 277, 384, 645, 788, 997). The epileptic seizures induced by kainate injections triggered
treatment of cultured rat microglia with 3 mM ATP trig- an activation of microglia as seen in acute hippocampal
gered rapid (within 2 h) morphological activation and slices (27). This activation resulted in the upregulation of
acquisition of the amoeboid phenotype (1009). In another the expression of mRNA specific for P2X1,4,7 and P2Y6,12,13
study, however, ATP and adenosine (0.6 –1 mM) had the receptors. Functionally this upregulation was manifested
opposite effect; when added to the medium, they induced by an increase in ATP-induced membrane currents and
the transformation of amoeboid microglia to a more ram- ATP-induced microglial motility (27). The upregulation of
ified (and hence less activated) phenotype (997). microglial P2X4, P2X7, and P2Y6 receptors was also ob-
The effects of purines on microglia are mediated served in superoxide dismutase 1 mutant expressing ani-
through purinoceptors of both metabotropic and iono- mals (these being an accepted model for amyotrophic
tropic subtypes, which are abundantly expressed in lateral sclerosis) (185). Changes in the P2X/P2Y mRNA
microglial cells throughout the nervous system (265). Ex- expression were also observed in microglial cultures pre-
pression of purinoceptors in microglia can vary consider- pared from animals of different (1–12 mo) age (179).
ably depending on the activation status (604). Further- Oxygen and glucose deprivation triggered an upregu-
more, microglia are endowed with ectonucleotidases, lation of P2X4 and P2X7 receptors in the hippocampal and
which degrade ATP and its derivatives. Microglial cells cortical/striatal/subventricular zone organotypic slices
were shown to express nucleoside triphosphatase (132, 133), which are linked to neuronal damage. Similarly
(NTPase), nucleoside diphosphatase (NDPase), 5=- the traumatic brain injury resulted in significant upregu-
nucleotidase (5=-Nase), and purine nucleoside phosphor- lation of microglial P2X4 receptors, which was sup-
ylase (PNPase), with this expression being dependent on pressed by systemic treatment with 1 mg/kg dexametha-
the activation and developmental stage (186). Microglial sone (1033). Injections of kainate into rat hippocampus
cells also express CD39 ectonucleotidase, which is criti- increased the expression of P2X6 receptors (467). The
cal for ATP-mediated signaling. In microglia deficient for mechanical trauma of a rat nucleus accumbens rapidly
CD39, migration cannot be stimulated by ATP (266). The triggered the appearance of immunoreactivity for
application of ATP in concert with adenosine or the ad- P2X1,2,4,7 and P2Y1,2,4,6,12 receptors in microglial cells; in
dition of exogenous soluble ectonucleotidase restored the control preparations, the specific signal was completely
ATP stimulated migration, thus indicating that P1 and P2 absent (295). It was also found that an intramyocardial
receptor stimulation are required and that the ectonucle- injection of formalin, which triggers acute heart ische-
otidases can provide the substrate to P1 receptors (266). mia, induced microglial activation in the locus coeruleus
(which is intimately involved in pathogenesis of heart
3. Plasticity of the microglial purinoceptive system diseases); this microglial activation was paralleled with a
significant increase in expression of P2X4 receptors
Microglial cells, both in cultures and in brain slices, (1030).
usually express several types of metabotropic and iono- The P2Y12 receptor has been identified as an impor-
tropic purinoceptors. An analysis of ATP-induced Ca2⫹ tant control element for the movement of microglial pro-
transients in various microglial cultures demonstrated cesses as indicated by imaging studies in live animals
that, as a rule, more that one type of purinoceptor is (365, 384). This receptor is downregulated when micro-
involved in the regulation of Ca2⫹ influx and Ca2⫹ release glia are activated following injury to the brain. The oppo-
(see, e.g., Refs. 527, 967, 984). Electrophysiological exper- site was reported for another P2Y receptor, the P2Y6
iments in vitro and in situ also showed that several types receptors. After injury, these receptors are upregulated,
of purinoceptors mediate ATP action (82). Even within and their activation triggers phagocytosis (467).
the same culture dish microglial cells demonstrate a vari- These plastic changes in the purinoceptors expres-
ability in pharmacological profiles of [Ca2⫹]i transients sion are important for orchestrating specific responses of
induced by different purinoceptor agonists (604). The microglia to purines and pyrimidines at different stages of
expression pattern of microglial purinoceptors demon- neuropathology. Particularly robust changes in expres-

sion are observed for P2X4, P2X7, and several types of P2Y 2-chloro-N6-(3-iodobenzyl-N-methyl-5=-carbamoyladenos-
receptors, which are discussed in detail below. ine) (Cl-IB-MECA) affected the phopshorylation of the
extracellular signal-regulated protein kinase 1/2 (ERK1/2).
4. Adenosine P1 receptors These effects were absent in cells obtained from trans-
genic mice with deleted A3 receptor gene (347). The dy-
Adenosine receptors of P1 type are represented by namic balance between A2A and A3 receptors controlled
classical seven-transmembrane spanning G protein-cou- the TLR-dependent activation of microglia and secretion
pled receptors and are subclassified into adenosine A1, of cytokines (947).
A2A, A2B, and A3 receptors with distinct pharmacological Finally, the treatment of cultured rat microglial cells
and functional properties. The A1 and A3 receptors are with broad A1 agonist 2-chloro-adenosine induced pro-
coupled to Gi/o proteins, whereas A2A and A2B receptors grammed cell death, which was not prevented by specific
usually act through Gs proteins (297, 298). adenosine receptors antagonists, suggesting the existence
All four types of adenosine receptors were identified of atypical adenosine regulated signaling pathway (670).
at the mRNA level in cultured rat microglia (282). Func-
tional P1 receptors were also identified in cultured micro-
5. P2X receptors
glial cells, where they exert several metabotropic effects.
The application of adenosine in concentrations 0.001–100 Ionotropic ATP receptors, named P2X in 1985 (105),
␮M triggered an outward current in cultured rat microglia are classic ligand-gated cationic (Na⫹/K⫹/Ca2⫹) channels,
voltage-clamped at 0 mV. This current had a reversal which are assembled from different subunits into homo-
potential at ⫺70 mV and was produced by the activation or heterotrimers (444, 642, 761). These trimers form a
of K⫹ channels. The effects of adenosine were antago- chalice-shaped structure as revealed by their crystal
nized by an incubation with PTX and inhibited by the structure (439). The P2X subunits are encoded by distinct
broad P1 receptors antagonist 8-(p-sulfophenyl)theophyl- genes, which have been classified as P2X1 to P2X7 accord-
line (497). A proliferative response of cultured rat micro-
ing to the historical order of cloning (445, 660, 879). The
glia required the simultaneous expression of A1 and A2
P2X receptors have substantial Ca2⫹ permeability, which,
receptors (309). The A1 adenosine receptors were partic-
depending on the subunit composition, may vary (PCa/
ularly concentrated in microglial cells surrounding glio-
Pmonovalent) between 1 and ⬎10 (684, 685) and could be
blastomas, and their activation suppressed tumor growth
even greater for the pore-forming P2X7 receptors (243).
by a yet unknown mechanism (889). There is also evi-
The P2X-mediated currents were identified in micro-
dence for a protective role of microglial A1 receptors
glial cultures prepared from human and rodent brains
following acute brain trauma (360). The activation of A2A
(976, 659, 575) and in resting microglial cells from adult
receptors stimulated the expression of NGF mRNA as
mouse acute brain slices (82). The P2X-mediated [Ca2⫹]i
well as the release of NGF in rat cultured microglial cells
(366) and potentiated release of NO from LPS-activated elevation was also described in cultured microglia from
microglia (795). The same A2A receptors also regulated retina; the ATP/Bz-ATP-induced Ca2⫹ entry was potenti-
the expression of COX-2 and synthesis and the release of ated after keeping the cultured cells with 30 mM glucose
prostaglandin E2 from cultured rat microglial cells (282). for 7 days. This increased Ca2⫹ influx was not inhibited by
In addition, the A2A receptors control retraction of micro- Brilliant Blue G, thus ruling out the participation of P2X7
glial processes under conditions of chronic neuroinflam- receptors (702).
mation (678). Selective stimulation of the A2A receptor in Using single- and double-labeling immunfluorescence
primary cultured rat microglia by 6 h of incubation with and RT-PCR techniques, Xiang and Burnstock (1008)
the specific agonist CGS 21680 increased expression of characterized developmental changes in the P2X subunits
mRNA for Kv1.3 channels and renal epithelial K⫹ channel expression in the rat brain. The majority of microglial
ROMK1 (Kir1.1) and increased levels of Kv1.3 protein cells stained positively for the marker ED1 at embryonic
(484). A2A receptor-induced upregulation of Kv1.3 chan- day 16 expressed P2X1 and P2X4 subunits, whereas only
nels was mediated through a cAMP-dependent pathway, 30% of these cells expressed P2X7 receptors. From post-
whereas upregulation of ROMK1 channels involved PKC natal day 7, the P2X4-positive microglia concentrated
(484). Finally, A2A receptors may be involved in microglial around blood vessels. At postnatal day 30, the cells ex-
activation in neuropathic pain, and intrathecal injection of pressing P2X1 receptors virtually disappeared; the P2X7-
A2A antagonists prevented the development of mechanical positive cells were distributed evenly through the fore-
allodynia and thermal hyperalgesia in rats with chronic brain, whereas cells bearing P2X4 receptors outlined
constriction injury neuropathic pain model (534). blood vessels and subarachnoid space (1008). In organo-
Pharmacological assays have also identified func- typic slices from the cortical/striatal/subventricular zone,
tional A3 receptors in primary mouse microglial cultures. the immunostaining for P2X4 and P2X7 receptors showed
The activation of A3 receptors by selective agonist the colocalization with microglial marker OX42 (132).

6. P2X4 receptors and neuropathic pain intraperitoneal injection of LPS triggered hyperalgesia
associated with the activation of microglia and the up-
Neuropathic (i.e., chronic and malignant) pain devel- regulation of P2X4 receptor expression in the spinal cord
ops following peripheral nerve damage of multiple aetiol- (338). A considerable increase in P2X4 receptor expres-
ogy (e.g., acute trauma, diabetic neuropathy, cancer, or sion, which paralleled the development of mechanical
surgery) and represents a severe medical problem (999). allodynia, was also identified in rats with experimental
Experiments of the recent decade demonstrated the pri- autoimmune neuritis (44), a commonly used model of
mary role of microglial purinergic signaling in the patho- acute inflammatory demyelinating polyradiculoneuropa-
genesis of neuropathic pain (see, e.g., Refs. 408, 409, 411, thy, the latter being the most common subtype of Guillain-
416, 921 for detailed reviews). In particular, two types of Barre syndrome (1035). Similarly a significant increase in
purinoceptors, the P2X4 and P2Y12, are critical for trigger- microglial expression of P2X4 receptors was observed
ing neuropathic remodeling in the spinal cord. following 4/5 overhemisection of the dorsal spinal cord of
Initially, it was found that peripheral nerve injury rats (815). The upregulation of P2X4 receptor expression
triggers rapid activation of microglial cells in the spinal can be mediated through fibronectin-integrin and integrin-
dorsal horn. The first signs of activation (rounding of the Lyn (tyrosine kinase) signaling cascades, with the possi-
soma, retraction of processes, and increase in immunore- ble involvement of phosphatidylinositol 3-kinase (PI3K)-
activity for OX42) became obvious 24 h after lesioning the Akt and mitogen-activated protein kinase, MAPK (932).
peripheral nerve (252, 887). In parallel, peripheral nerve The level of P2X4 receptor expression was significantly
injury induced microglial proliferation, which peaked increased in microglial cells cultured on fibronectin-
within 2–7 days after the lesion (311, 632). Microglial coated dishes (633), with this effect being inhibited by
activation paralleled the development of tactile allodynia peptide echistatin (selective blocker of ␤1- and ␤3-con-
(178), the latter being a primary symptom of the neuro- taining integrins) and with ␤1 integrin antibody (931, 933).
pathic pain. The activation of microglia was identified A rapid increase in the plasmalemmal presence of P2X4
also in animal models of bone cancer (1032), experimen- receptors can be achieved by the redistribution of the
tal autoimmune neuritis (44), and diabetes (934). Inciden- receptor molecules from the cytoplasm to the cell mem-
tally, activated microglia were also found in dorsal root brane (867). Such redistribution caused almost fourfold
ganglia following sciatic nerve lesion (696). increase in plasmalemmal P2X4 receptors in cultured mi-
Initial evidence for the role of purinoceptors in me- croglia treated with LPS for 3 h (83). There is also evi-
diating microglial activation in neuropathic pain was ob- dence indicating the concentration of microglial P2X4
tained by Tsuda et al. in 2003 (930), who used the rat receptors in lysosomes; the exocytosis of the latter (fol-
spinal nerve injury model in which L5 spinal nerve was lowing, e.g., Ca2⫹ signaling) may very rapidly increase the
surgically severed. Tsuda et al. found that intrathecal pool of plasmalemmal functional receptors and contrib-
injection of 2=,3=-O-(2,4,6-trinitrophenyl)adenosine 5-triphos- ute to an increase in P2X4 currents (741).
phate (TNP-ATP) reversed the decrease of the paw with- An increase in P2X4 expression was further corrob-
drawal threshold (the latter being a readout of the tactile orated by immunostaining, which demonstrated a signif-
allodynia). In contrast, an injection of pyridoxal- icant increase in P2X4 reactivity in the spinal cord ipsilat-
phosphate-6-azophenyl-2=,4=-disulfonic acid (PPADS) had eral to the site of nerve injury. Most importantly, in-
no effect. This peculiar sensitivity of tactile allodynia to creased P2X4 immunoreactivity was detected exclusively
P2 receptor antagonists (TNP-ATP inhibits P2X1– 4 recep- in OX42 positive microglial cells (930). The inhibition of
tors whereas PPADS blocks all P2X receptors but P2X4; P2X4 receptors expression by antisense prevented the
Refs. 98, 850) led them to suggest the specific role of P2X4 development of tactile allodynia following nerve injury,
subunits in triggering the neuropathic pain. whereas an intrathecal injection of activated cultured
Subsequent experiments showed a significant in- microglia expressing P2X4 receptors triggered allodynia
crease in the level of P2X4 subunit protein in homoge- without cutting the nerve (930). These results have dem-
nates obtained from the ipsilateral spinal cord of lesioned onstrated that P2X4 receptors are critically important for
animals; the P2X4 protein levels began to increase 1 day the initiation of neuropathic pain in the animal model
after surgery and reached maximal levels after 14 days; (417, 929, 930).
this time course matched the development of tactile allo- The mechanisms of how activated microglia cause
dynia (930). The microglial P2X4 expression was consid- neuropathic pain are probably many. Electrical substrate
erably increased following formalin injection into the spi- of neuropathic pain is believed to be represented by long-
nal cord (a common inflammatory pain model); maximal term potentiation of C-fiber-evoked field potentials in the
levels of P2X4 immunoreactivity were observed 7 days spinal dorsal horn, which occurs in response to injury and
after lesion (339). Incidentally, the formalin-induced acti- inflammation (785). Stimulation of P2X4 receptors of spi-
vation of microglia and hyperalgesia was blocked by the nal cord microglia induces long-term potentiation of C-fi-
broad P2 antagonist suramin (1006). Similarly, a single bers, by yet poorly understood mechanisms, which may

possibly involve p38 signaling cascades and release of desensitization in the presence of the agonist, and upon
TNF-␣ or IL-1 from microglial cells (329). In addition intense stimulation, they produce large transmembrane
activated microglia can cause neuronal hyperexcitability pores (699, 852, 879); the phenomenon initially described
by an increase in Cl⫺ concentration in spinal neurons, as an “ATP-dependent plasma membrane permeabiliza-
thus turning GABA-mediated responses from hyperpolar- tion” (163). The mechanism of the pore formation remains
izing to depolarizing (176). Such a shift of Cl⫺ equilibrium unclear (227); the pore formation may result from the
potential (ECl) in lamina I spinal neurons was shown to be dilatation of the channel or from the activation of other
caused by ATP-stimulated release of brain-derived neu- pore-forming proteins (699, 700) associated with P2X7
rotrophic factor (BDNF) from activated microglia; BDNF receptors. Several relatively specific pharmacological
in turn affected transmembrane distribution of chloride tools for probing for P2X7 receptors are available. They
ions by the activity of the potassium/chloride exporter include an agonist 2=,3=-(benzoyl-4-benzoyl)-ATP (BzATP)
KCC-2, responsible for maintaining low intraneuronal Cl⫺ which is ⬃30 times more potent than ATP at P2X7 recep-
concentration (175, 1027). The inhibition of BDNF synthe- tors (although BzATP can activate other P2X receptors
sis and the release by interfering RNA or blockade of and can be degraded to Bz adenosine, which stimulates
signaling via TrkB receptors (by using function- P1 receptors; Ref. 481), and several antagonists such as
blocking antibody against the TrkB receptor or BDNF- Brilliant Blue G (BBG) which blocks P2X7 receptors at
sequestering fusion protein) as well as the inhibition of 10 –200 nM concentrations and oxidized ATP (oxATP).
P2X receptors with TNP-ATP prevented both the ECl The latter, however, also inhibits P2X1 and P2X2 receptors
shift and development of tactile allodynia (175). Fur- (660).
thermore, BDNF secreted following stimulation of mi- P2X7 receptors are ubiquitously expressed in the
croglial P2X4 receptors may induce phosphorylartion of cells of the immune system (106, 147) and are believed to
NR1 subunit of NMDA receptors of dorsal horn neu- be responsible for numerous immune reactions, including
rons, which could also contribute to neuronal hyperex- the processing and the release of various cytokines (278,
citability (940). The release of BDNF triggered by activa- 700). Furthermore, intensive stimulation of P2X7 recep-
tion of P2X4 receptors takes place in two waves; the early tors associated with pore formation is involved in initiat-
peak of release occurs 5 min after ATP stimulation, ing apoptosis and cells death (935).
whereas the second peak of release is attained ⬃60 min Functional P2X7 receptors (which, at that time, were
later (922). This biphasic kinetic reflects the rapid release referred to as P2XZ receptors) were identified in micro-
of an already existing pool of BDNF, followed by an glial cells in 1996, both in situ, in amoeboid microglia
increase in BDNF synthesis. The BDNF release occurs (342) and in vitro, in freshly isolated mouse microglia
through Ca2⫹-regulated exocytosis, as both elimination of (279). In cultured microglia, the existence of P2X7 recep-
Ca2⫹ influx and inhibition of soluble N-ethylmaleimide- tors was deduced based on 1) detecting massive Ca2⫹
sensitive factor attachment protein receptor, SNARE, in- influx following exposure to ATP and Bz-ATP, 2) ATP-
hibits BDNF secretion, and regulation of BDNF synthesis induced uptake of ethidium bromide or Lucifer yellow,
involves p38-MAPK signaling pathway (922). 3) ATP-induced release of cytoplasmic markers such as
lactate dehydrogenase, and 4) identification of P2X7 pro-
7. P2X7 receptors and microglial function tein (218, 276, 279). The treatment of microglial cell lines
with high concentrations of ATP also induced rapid exci-
First indications for ATP opening large plasmalem- totoxicity associated most likely with plasmalemma per-
mal pores were found in cell cultures, in which brief meabilization (276). All these effects were blocked by the
application of ATP made cellular membranes permeable P2X7 receptor antagonist oxidized ATP (218, 279).
to inorganic phosphates (163, 775). Subsequently, the un- In the amoeboid microglia collected from the surface
derlying ATP-gated ion channel producing transmem- of corpus callosum slices of 5- to 7-day-old mice, the
brane pore upon activation was biophysically character- P2X7-mediated currents were recorded directly, using a
ized and named P2Z receptor (332). whole cell patch-clamp technique (Fig. 9). The P2X7 cur-
This P2Z receptor became P2X7 receptor, when it was rents in amoeboid microglia were activated by 1 mM ATP
cloned and molecularly characterized in 1996 (880). These in normal extracellular solution, or by 100 ␮M ATP after
receptors share the least homology with other members removal of extracellular divalent cations (342). Subse-
of P2X family and have several unique functional charac- quently, P2X7 receptor-mediated currents were detected
teristics. The P2X7 receptors demonstrate an exception- in ramified microglia in acute slices from adult (6 – 8 wk
ally low sensitivity to ATP (their activation requires mill- old) mice, as indicated by their sensitivity to BzATP and
imolar concentrations of ATP); in all probability, it is the current potentiation in Ca2⫹/Mg2⫹-free extracellular solu-
tetra-anionic form of ATP (ATP4⫺) that acts as a true tion (Fig. 9; Ref. 82).
agonist for P2X7 receptors (although this was never In the undisturbed brain, microglial cells expressing
proved experimentally). The P2X7 receptors show little P2X7 receptors are diffusely scattered throughout virtu-

FIG. 9. P2X7 receptor-mediated cur-

rents in microglial cells in situ. A: ATP-
induced membrane currents measured
from amoeboid microglial cells collected
from the acute young mouse corpus cal-
losum slices (see also Fig. 7) in control
conditions and Ca2⫹/Mg2⫹-free bath so-
lution. B: membrane currents were re-
corded from microglial cells in the acute
mouse forebrain slices when clamping
the membrane potential to ⫺20 mV. The
membrane was repetitively clamped for
100 ms to a series of de- (0, 20, 40, and 60
mV) and hyperpolarizing (⫺40, ⫺60,
⫺80, ⫺100, and ⫺120 mV) potentials.
The series of voltage steps were per-
formed every 5 s. In the condensed time
scale on each series of these voltage
jumps appears as a line. BzATP (□) and
ATP (〫) were applied in Ca2⫹/Mg2⫹-free
bath solution. Subsequently, ATP was ap-
plied in normal bath solution (Œ). The
current-voltage curves were constructed
from series of voltage jumps as indicated
by the different symbols and relate to the
current component induced by the ago-
nist application (after subtraction of cur-
rents without agonist). [A modified from
Haas et al. (342); B modified from Bouc-
sein et al. (82).]
ally all areas (1025). In depth analysis of the expression of reactivity was also increased in microglia surrounding the
various P2X receptor subunits have found prominent in- areas of cryogenic injury delivered to rat brain (1024). A
duction of P2X7 receptor expression in microglial cells pronounced increase in P2X7 mRNA was found in micro-
following brain damage (165). This initial observation was glial cells obtained from Alzheimer’s diseased brains, and
subsequently confirmed in many in vitro and in vivo neu- intense P2X7 immunostaining was observed in reactive
ropathological models, and the link between microglial microglial cells surrounding plaques in situ, in AD trans-
activation and P2X7 receptors expression was established genic model (571, 694). An increase in P2X7 immunostain-
(851, 852). Upregulation of P2X7 receptors is generally ing was also observed after an injection of 1 nM A␤1– 42
induced by ischemic and traumatic insults (294, 580). into rat hippocampus (571). Similarly, the exposure of
The MCAO triggered a pronounced increase in P2X7 fetal human microglia to A␤1– 42 (5 ␮M for 18 h) substan-
expression in microglia in striatum and a frontoparietal tially increased P2X7 expression and enlarged P2X7-de-
cortex in both infarcted and surrounding areas (the P2X7 pendent [Ca2⫹]i responses by 145% (571). An increase in
immunoreactivity was absent in microglia from control microglia-associated P2X7 immunoreactivity was also ob-
samples); the P2X7 immunoreactivity was also detected in served in an epileptic (kainate-induced seizures) rat
microglia in ipsi- and contralateral cingulate and medial model (748). Similarly, P2X7 receptors are upregulated in
frontal cortex (580). Injections of broad P2X antagonist brains of scrapie-infected mice and in microglial cell line
Reactive Blue 2 reduced the size of ischemic damage and persistently infected with mouse-adapted scrapie (894).
improved neurological deficit (580). Expression of P2X7 An injection of LPS into the rat striatum triggered micro-
receptors was significantly upregulated in immunocyto- glia activation accompanied by significant upregulation of
chemically identified microglia from specimens of spinal P2X7 expression as well as upregulation of variety of
cord obtained from humans with multiple sclerosis and proinflammatory mediators (154). Inhibition of P2X7 re-
amyotrophic lateral sclerosis (1022) and in post mortem ceptors with oxATP reduced expression of all inflamma-
brain of AD patients (897). The P2X7 receptors immuno- tory mediators and LPS-induced activation of p38 mito-

gen-activated protein kinase and NF-␬B and had a neuro- pled to the release of IL-1␤ (590). Pretreatment of cul-
protective effect (154). tured microglia with LPS or with A␤1– 42 significantly (1.5-
The activation of microglial P2X7 receptors has var- to 4-fold) increased P2X7-induced secretion of IL-1␣ and
ious functional consequences that comprise both trophic IL-1␤ (745).
and cytotoxic effects, the balance between these being The brief conditioning of microglial cells with ATP or
probably dependent on the degree and length of the re- BzATP sensitized microglial P2X7 receptors to ADP and
ceptor stimulation. The stimulation of P2X7 receptors in AMP, so the latter acquire an ability to activate the recep-
primary cultured microglia triggers cell death (90, 276), tors (albeit without pore formation) and stimulate the
which was absent in P2X7 knockout animals (90). Activa- release of IL-1␤ (136). Treatment of primary microglial
tion of P2X7 receptors in rat cultured microglia also in- cultures with an inflammatory phospholipid lysophos-
duced the release of superoxide, which may mediate mi- phatidylcholine potentiated P2X7-dependent Ca2⫹ influx
croglial cytotoxicity (694). Prominent microglial cell and pore formation (896). The P2X7-mediated signaling is
death was also observed in organotypic rat hippocampal involved in LPS-stimulated release of interleukins, as LPS
slices treated with a combination of LPS and ATP (1 mM) (as well as other bacterial endotoxins; Refs. 276, 277)
or BzATP (100 ␮M); this cell death was prevented by triggers the release of ATP from microglia, which, in turn
pharmacological blockade of P2X7 receptors (49). The activates purinoceptors in autocrine/paracrine fashion
P2X7 receptors are also involved in regulation of micro- (277). Similarly, the stimulation of P2X7 receptors with 1
glial autophagy through the release of autolysosomes mM ATP or BzATP triggered the release of TNF-␣ from
(893, 895). primary cultured rat microglia (377), this release being
At the same time, many experiments demonstrated a controlled by P2X7-mediated sustained [Ca2⫹]i elevation
prominent trophic role of microglial P2X7 receptors. In and ERK/p38 signaling pathway. The TNF-␣ release fol-
experiments in rat hippocampal cultures or in microglia- lowing stimulation of microglial P2X7 receptors can pro-
enriched cultures, the sole overexpression of P2X7 recep- tect neurons against glutamate-induced neurotoxicity
tors (without any additional stimulation) was found to
(883). The stimulation of P2X7 receptors in primary mi-
launch microglial activation (the latter being assessed
croglia upregulated synthesis and induced release of CC-
morphologically and through expression of specific mark-
chemokine ligand 3 (CCL3)/macrophage inflammatory
ers isolectin GS-IB4 and CD68; Ref. 607). This microglial
protein-1␣; this release was blocked by P2X7 receptor
activation was sensitive to oxATP, thus confirming the
antagonists and selective inhibition of nuclear factor ac-
specific role for P2X7 receptors. Surprisingly, the initia-
tivated T cells (NFAT) (435). Similarly, P2X7 receptor
tion of the activation program required a P2X7 receptor-
activation increases expression and induces the release of
associated pore formation; in microglial cells transfected
CXCL2 chemokine from rat cultured microglia via NFAT
with a point mutant P2X7RG345Y (this mutant protein
retains the channel function but cannot form the pore), and MAPKs (p38, ERK, and JNK) signaling cascades
microglial activation was significantly reduced (607). The (830). P2X7 receptor-mediated Ca2⫹ influx was shown to
P2X7 receptors were also obligatory in mediating micro- suppress the synthesis of microglial response factor-1
glial activation in response to an intrahippocampal injec- gene (mrf-1; the latter being the part of microglial activa-
tion of A␤ protein (787); considering the pivotal role of tion induced by neuronal death in vitro and in vivo) at the
microglial activation in AD-associated neuroinflammation transcriptional level (440). Another group, however,
(115, 370, 767), the P2X7 receptors may be considered as found that P2X7-mediated Ca2⫹ influx significantly (⬃10
important therapeutical targets. times) increases the expression and release of MRF-1
The P2X7 receptors are linked to various intracellular protein from cultured rat microglia (904). Calcium influx
transcription factors. The activation of P2X7 receptors is through P2X7 receptors is instrumental in triggering the
linked to phosphorylation of another transcription factor, release of plasminogen from cultured rat microglia (413).
CREB (724). The P2X7 receptors play a central role in The release of plasminogen was inhibited either by clamp-
positive modulation of IFN-␥ induced upregulation of ing [Ca2⫹]i with BAPTA-AM or by removing extracellular
iNOS production and NO synthesis and RK1/2 phosphor- Ca2⫹ or by inhibiting P2X7 receptors with oxATP. The
ylation (314). The activation of P2X7 promotes release of specific P2X7 receptor agonist BzATP was much more
cytokines IL-1␣ and IL-1␤ from activated cultured micro- potent than ATP in triggering plasminogen release (413).
glial cells (90, 277), which may occur through membrane Sustained calcium influx through P2X7 receptors was also
vesicle formation and shedding (65). Similarly, in vivo in instrumental in increasing microglial production of endo-
P2X7 knockout (KO) mice, the systemic treatment with cannabinoid 2-arachidonoylglycerol (2-AG) through di-
LPS induced smaller increases in the brain IL-1␤ and rect Ca2⫹-dependent activation of diacylglycerol lipase
TNF-␣ compared with the wild-type controls (590). In and the simultaneous inhibition of monoacylglycerol
microglial cultures prepared from P2X7 KO animals, the lipase, the enzyme that degrades 2-AG (996). Importantly,
P2X7 receptors were demonstrated to be selectively cou- the stimulation of microglial P2X7 receptors can occur

following ATP release in astrocytes, with this release P2Y12 receptors (966). The activation of P2Y receptor-
being potentiated by IFN-␥ (953). mediated ER Ca2⫹ release often triggers secondary store-
The stimulation of microglial P2X7 receptors can in- operated Ca2⫹ entry; incidentally, the store-operated
duce neurotoxicity. In neuronal-microglial cocultures, Ca2⫹ influx is negatively regulated by concominant acti-
treatment with ATP and BzATP promoted neuronal cell vation of P2X receptors, because depolarization produced
death, which was prevented by P2X7 inhibition with ox- by stimulation of the latter reduces electro-driving force
ATP or Brilliant Blue G (841). This type of P2X7-depen- for Ca2⫹ (985). In ramified microglial cells from acute
dent neurotoxicity was mediated through microglial re- adult mice slices, activation of P2Y receptors triggers an
lease of superoxide and NO (694, 841), and treatment of activation of outward-rectifying K⫹ conductance (82).
cocultures with superoxide dismutase mimetic or with a The activation of P2Y receptors can also affect microglial
peroxynitrite decomposition catalyst was neuroprotec- secretion of cytokines and other biologically active sub-
tive. When microglia for cocultures was prepared from stances. The P2Y receptors, for example, were shown to
P2X7 KO mice, BzATP did not trigger neurotoxicity, fur- inhibit release of TNF-␣, IL-1␤, IL-6, and IL-12 in LPS-
ther corroborating the role for P2X7 receptors (841). The activated cultured rat and mice microglia (82, 669). In
P2X7-mediated IL-1 release from microglia was also im- contrast, the activation of P2Y1/P2Y11 receptors increased
plicated in neurotoxic effects in organotypic hippocampal IL-10 expression and release in LPS-activated rat micro-
slices (49). glia (820, 821). The activation of P2Y receptors in cultured
Under certain conditions, however, microglial P2X7 microglia also resulted in rapid accumulation of immedi-
receptors may acquire neuroprotective potential, as for ate early genes c-fos, junB, c-jun, and TIS11 (725). In the
example was suggested by studying ischemic insults fol- retina, stimulation of P2Y1 receptors induced morpholog-
lowing the middle cerebral artery occlusion in rats. In ical remodeling of microglia represented by the decrease
these experiments the activation of P2X7 receptors by in the cell soma size and retraction of processes, without
injection of BzATP alleviated, whereas the inhibition of affecting microglial cell density (938).
P2X7 receptors by oxATP exacerbated brain damage Cultured rat microglia were also shown to express
(1018). Incidentally, the microglial P2X7 receptors may UDP-preferring P2X6 receptors at both mRNA and pro-
also be somehow linked to the development of mood tein levels (467). These P2Y6 receptors are implicated
disorders. Behavioral analysis of P2X7 knockout mice in pyrimidine-dependent activation of microglial phago-
found that they were more resistant to induction of de- cytosis (467). Stimulation of microglia with UDP trig-
pression and more sensitive to antidepressant drugs (39). gered [Ca2⫹]i transients through activation of InsP3-
mediated Ca2⫹ release from the ER and induced phago-
8. P2Y receptors cytosis of fluorescent zymosan particles and latex
beads. The P2Y6 agonist reactive blue 2 inhibited both
Metabotropic P2Y purinoceptors belong to archetyp- Ca2⫹ signals and phagocytosis induced by UDP (467).
ical seven-transmembrane domain G protein-coupled re- The P2X6 receptors were also involved in regulation of
ceptors (5, 284). They are generally divided into the phagocytosis in vivo in kainate-lesioned CA1 and CA3
P2Y1,2,4,6,11 and P2Y12,13,14 groups based on phylogenetic hippocampal areas (467).
similarity and G protein preference (4). The P2Y1,2,4,6,11 The ADP-preferring P2Y12 receptors represent an im-
are linked with Gq/G11 proteins and regulate cytosolic portant target for purines in early microglial responses to
Ca2⫹ mobilization through PLC/InsP3/ER Ca2⫹ release injury in the CNS (365, 410). The P2Y12 receptors are
signaling cascade. The P2Y12,13,14 receptors are coupled to predominantly expressed in the brain, and within the
Gi/o proteins and inhibit adenylyl cyclase or modulate ion chan- brain they are predominantly expressed in microglial cells
nels activity (4). In addition, P2Y receptors are differentially as was shown by in situ hybridization and immunohisto-
activated by various nucleotides: the P2Y1,11,12,13 are sensi- chemistry (792). This specific expression was further cor-
tive to adenine nucleotides ATP/ADP, the P2Y4,6 are acti- roborated in experiments using a specific antibody
vated by uracil nucleotides UTP/UDP, the P2Y14 receptor against the COOH terminus of mouse P2Y12 receptors.
is specifically sensitive to UDP-glucose, and finally P2Y2 The labeling with this antibody (in both gray and white
receptors are activated by both adenine and uracil nucle- mater) colocalized with microglial-specific markers
otides (284). (EGFP expressed under control of microglia specific frac-
In cultured mouse microglia, the real-time semi-quan- talkine receptor promoter or with integrin CD11b anti-
titative RT-PCR analysis revealed the expression of P2Y6, sera; Ref. 365). The P2Y12 receptors were localized on the
P2Y12, P2Y13 receptors; the activation of these receptors processes and somatic membrane of resting microglial
participated in [Ca2⫹]i elevation evoked by various P2 cells (365). The activation of microglial cells following
receptors agonists (527, 568). In rat cultured microglia, brain injury induced a very significant decrease in P2Y12
nucleotide-induced Ca2⫹ signaling and [Ca2⫹]i oscillations receptors expression, which became almost absent 24 h
are the consequence of activation of P2Y2, P2Y6, and after the isolation of hippocampal slices (365). Similarly,

when microglia were activated in vivo by LPS injection B. Glutamate Receptors

into the striatum, the P2Y12 receptors expression de-
creased to nondetectable levels 4 days after the insult 1. Ionotropic receptors
(365).
At the very same time P2Y12 receptors expressed in A) AMPA RECEPTORS.In cultured rat microglia, the ad-
resting microglia appeared to be critical for inducing ministration of both glutamate and kainate triggered cat-
the morphological activation and chemotaxis following ionic currents (343, 652). These currents were completely
brain injury. ATP/ADP induced membrane raffling and inhibited by selective AMPA receptor blocker CNQX, and
microglial chemotaxis in vitro are sensitive to pharma- potentiated by allosteric modulators of AMPA receptors
cological inhibition of P2Y12 receptors with a specific 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxy-
antagonist AR-C69931MX (384) and are severely im- acetamide (PEPA) and cyclothiazide (CTZ). The AMPA-
paired in P2Y12 KO mice (365). Genetic deletion of the mediated currents in cultured microglia showed negligi-
P2Y12 gene severely affects the ability of microglial ble Ca2⫹ permeability (652). Transcription analysis using
cells to migrate, proliferate, and extend processes to- PCR revealed microglial expression of GluR2, GluR3, and
wards a mechanical lesion as was demonstrated both in GluR4 (652). In another study of rat cultured microglia,
vitro and in vivo (365). The P2Y12-mediated microglial the expression of GluR1, GluR2, and GluR3 mainly in flip
chemotaxis requires intracellular Ca2⫹ signaling (medi- form was reported (343). Activation of microglial AMPA
ated through PLC-InsP3-induced Ca2⫹ release) and the receptors by glutamate or kainate induced release of
activation of phosphatidylinositol 3=-kinase (PI3K) and TNF-␣ (343, 652), which was, rather surprisingly, blocked
Akt cascades (419, 673). The P2Y12 receptor activation by CTZ and PEPA, prompting authors (343) to suggest
that potentiation of AMPA receptors produces negative
engages integrin-␤1 signaling cascade, which controls
feedback to TNF-␣ release. The AMPA receptors are also
extension of microglial processes (674). There are also
implicated (together with unidentified metabotropic glu-
some indications that microglial chemotaxis involves
tamate receptors) in glutamate-induced microglial che-
activation of P2Y-regulated K⫹ channels (1004).
motaxis, which was found in microglial cultures and in
In the spinal cord, the P2Y12 receptors are also
the spinal cord slices (529).
confined exclusively to microglia and are critically in-
Activation of microglial AMPA receptors with 0.1–1
volved in the genesis of neuropathic pain (414). In
mM of kainate led to a rapid and substantial remodeling of
contrast to the CNS, however, spinal nerve injury trig-
the cytoskeleton manifested by condensation of cytoplas-
gers a significant upregulation of P2Y12 receptors at
mic actin filaments, rapid de- and repolymerization, and
both mRNA and protein levels (464, 920). The activa-
cytoplasmic redistribution of condensed actin bundles.
tion of these receptors (in parallel with activation of These changes may play a role in the regulation of motility
P2X4 receptors; see above) is instrumental in inducing and phagocytosis of activated microglial cells (156).
tactile allodynia and thermal hyperalgesia (464, 920). In Stimulation of cultured rat microglia with 500 ␮M
P2Y12 KO mice, nerve injury failed to produce allo- glutamate NMDA, AMPA, kainate, or mGluR agonist (RS)-
dynia, although the basic mechanosensitivity was not 3,5-dihydroxyphenylglycine (DHPG) markedly (23-fold)
affected (920). Furthermore, an intrathecal injection of and transiently upregulated c-fos gene expression (on
P2Y12 receptor blocker AR-C69931MX or oral adminis- both mRNA and protein levels); this stimulation required
tration of P2Y12 receptor antagonist clopidogrel mark- [Ca2⫹]i elevation (being blocked by BAPTA-AM) and in-
edly reduced allodynia in rats, which underwent surgi- volved the activation of glutamate receptors sensitive to
cal nerve injury (920). Direct injection of P2Y12 agonist specific antagonists; treatment of microglia with CNQX,
2-(methythio)adenosine 5=-diphosphate trisodium salt MK-801, or AIDA (group I mGluRs antagonist) completely
(2Me-SADP) mimicked nerve injury and increased neu- inhibited the increase in expression of immediately early
ropathic pain symptoms (464). Interestingly, the ge- genes (256). Such a broad sensitivity of gene expression
netic ablation of P2Y12 receptors did not affect micro- to very different glutamate receptors antagonists remains
glial activation, yet it definitely determined the devel- inexplicable, and these data were never independently
opment of neuropathic pain (920). Effects of P2Y12 confirmed.
receptor activation involve p38 mitogen-activated pro- B) KAINATE RECEPTORS. There is no direct evidence for
tein kinase (p38 MAPK) signaling pathway (464). the functional expression of kainate receptors in micro-
Recently, microglial cells were shown to express the glial cells. Despite the identification of mRNA for GluR5
new P2Y-like receptor GRP17, which is activated by UDP, kainate receptors in cultured rat microglial cells, electro-
UDP-glucose, and UDP-galactose as well as by cysteinyl- physiological analysis demonstrated that kainate-induced
leukotrienes LTD4 and LTC4; microglial expression of currents were carried through AMPA receptors (652).
this receptor appeared after brain injury (502). Treatment of primary cultured rat microglia with 100 ␮M

kainite was reported to initiate their activation and stim- (2S,2=R,3=R)-2-(2=3=-dicarboxycyclopropyl)glycine (agonist of
ulate release of TNF-␣ (1038). group II mGluRs) triggered the activation of microglial
C) NMDA RECEPTORS. The expression of NMDA receptors cells and induced neurotoxicity mediated through micro-
in microglia remains doubtful; only indirect evidence is glial release of TNF-␣ (437). The mGluR-dependent mi-
available. An injection of 50 nM NMDA into the sensori- croglial activation involved NF-␬B signaling. In contrast
motor cortex of 6-day-old rats triggered transient activa- to LPS-induced microglial activation, this mGluR-
tion of microglia with the appearance of pseudopodic dependent pathway does not involve MAPK signaling;
morphology (7), which however is not necessarily indic- neither does it promote NO production and release (437).
ative for microglial receptor. Treatment of rat microglial Further analysis indicated that cultured rat microglia ex-
primary cultures with MK-801 [a use-dependent (393) in- press mRNA and proteins of both group II (mGluR2 and
hibitor of NMDA receptors] induced cytotoxicity, which mGluR3 receptors; Ref. 906) and group III (mGluR4,
was decreased by the addition of glutamate, NMDA, and mGluR6, and mGluR8, but not mGluR7; Ref. 907). Activa-
(surprisingly) kainate, thus prompting authors to suggest tion of group II receptors triggered microglial activation
an expression of functional NMDA receptors (380). Both and neurotoxicity, promoted the release of TNF-␣ (908),
LPS- and neurotoxic Tat HIV protein-induced activation and was also suggested to participate in microglia activa-
of cultured mouse microglia were suppressed by MK-801 tion induced by chromogranin A, a secretory peptide
and another NMDA receptor inhibitor dextromethorphan present in neuritic plaques in Alzheimer’s disease (906). In
(910). Similarly, systemic administration of MK-801 pre- contrast, activation of group III receptors reduced micro-
vented rapid microglial activation in the hippocampus glial neurotoxicity following treatment with LPS or chro-
following ischemic insult (871). Finally, another set of mogranin A. On a molecular level, group III receptors
data suggested that the activation of NMDA receptors inhibited activity of adenylate cyclase (907).
induced MAPK phosphorylation in spinal cord microglia
from rats with streptozotocin-induced diabetes, which
C. GABA Receptors
may be somehow involved in diabetic hyperalgesia (189).
Thus all the evidence remains indirect.
The main CNS inhibitory neurotransmitter GABA has
a well-documented neuroprotective effect (273), and
2. Metabotropic glutamate receptors
GABA levels were reported to increase in patients with
In rat cultured microglial cells, expression of cerebral ischemia (395).
mGluR5a (group I members) receptors was identified at Functional GABAB receptors were identified in a sub-
mRNA level; subsequent [Ca2⫹]i recordings confirmed population of microglial cells in culture (Fig. 10) and in
their functional expression by demonstrating Ca2⫹ signals ⬃50% of tomato-lectin positive cells in brain slices (480).
in response to mGluR5 antagonist trans-(1S,3R)-1-amino- Stimulation of GABAB receptors in microglia activated an
1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD) (66). outwardly rectifying K⫹ current and triggered [Ca2⫹]i
There is some evidence that stimulation of group I transients. In addition, stimulation of GABAB receptors
mGluRs may regulate LPS-induced microglial activation reduced the release of IL-6 and IL-12p40 from LPS-stimu-
in primary cultures (269). The stimulation of group II lated cultured microglia (480). Both GABAB subunits 1
mGluRs by glutamate released from oxygen-glucose-de- and 2 were determined in a majority (⬎90%) of cultured
privation-stressed embryonic rat neurons cocultured with microglial cells by immunostaining, and both proteins
rat microglia or by endogenously applied glutamate or were detected by immunoprecipitation, with GABAB re-
FIG. 10. GABA triggered current in cul-

tured microglial cells. Left: the membrane was
clamped at ⫺20 mV and then repetitively to a
series of de- and hyperpolarizing potentials
(⫺120 to ⫹60 mV with 20-mV increment). GABA
was applied at 500 ␮M as indicated and trig-
gered an outward current at the holding poten-
tial and an increase in current amplitude. At this
time resolution, each series of voltage steps
appears as a single line. Right: by subtracting
the control currents from the currents at the
peak response, the I-V curve for GABA-induced
current was obtained. The extrapolated reversal
potential was at about ⫺80 mV and thus close to
the potassium equilibrium potential. [Modified
from Kuhn et al. (480), with permission from
Elsevier.]

ceptor subunit 1 being represented by three splice vari- matory protein-1␣ (MIP1-␣) from microglia (151). Inhibi-
ants GABAB(1a), GABAB(1b), and GABAB(1c) (142, 480). tion of GABA signaling by the GABAA receptors blocker
Immunostaining indicated also that GABAB receptors bicuculline increased the resting motility and volume
were preferentially concentrated in microglial lamellipo- sampling by resting microglia in vivo (645). This is most
dia (480). Facial axotomy increased the population of likely also an indirect effect mediated by neuronal or
GABAB receptors-expressing microglia in the facial nu- macroglial activity.
cleus (480).
At the same time, microglial cells may detect GABA-
ergic activity in their immediate environment indirectly, D. Cholinergic Receptors
as for example was found for amoeboid microglia in
postnatal (6 – 8 days) mouse corpus callosum slices (151). Cholinergic pathways control global anti-inflammatory
The application of GABAA agonist muscimol triggered a reactions in the brain, which are believed to be mainly
transient increase in IKIR in these amoeboid microglia; mediated through ␣7 nicotinic receptors, ␣7nAChRs (978).
this increase, however, completely disappeared when Importantly, cholinergic systems are impaired by numerous
cells were removed from the slice surface (Fig. 11). It neurodegenerative diseases, such as for example AD and
turned out that microglial responses to muscimol resulted Parkinson disease; the weakness of cholinergic transmis-
from the transient elevation of extracellular K⫹ concen- sion may facilitate the development of neuroinflammation
tration following stimulation of neuronal or macroglial particularly through decreasing the ACh input to micro-
GABAA receptors. This indirect mechanism had some glial cells (124).
functional consequences, however, because activation of The expression of neuronal ␣7 nicotinic receptors
IKIR stimulated release of chemokine macrophage inflam- was initially found in cultured mouse microglia through
FIG. 11. Muscimol-induced current responses

in microglia are only observed in the vicinity of
postnatal brain slice. A: illustration showing how
an amoeboid microglial cell could be lifted up from
the brain slice while still attached to the patch
pipette (left). Image of a cell lifted up 300 ␮m from
the slice (right). B: membrane currents were re-
corded while muscimol (100 ␮M; 30 s) was applied.
ATP (500 ␮M; 30 s) was applied as a positive con-
trol 4 min later. C: an illustration showing how the
brain slice was cut into half-hemispheres and one
was placed on a coverslip with cultured microglial
cells (left). Images showing the brain slice and a
cultured microglial cell approached with the patch
pipette at high and low resolutions (right). D: sim-
ilar to B, membrane currents of cultured microglial
cells were recorded during muscimol (100 ␮M; 30
s) application in the absence (left) and presence of
a brain slice (middle). The current-voltage plot was
made from muscimol-induced current. E: a ramified
microglial cell in the cortex close to the corpus
callosum in an acute brain slice prepared from an
adult (P35– 40) Iba1-EGFP transgenic mouse. EGFP
fluorescence and the same cell filled with Alexa
Fluor 594 (10 ␮g/ml) via the patch pipette are
shown (left). Membrane currents typical for a ram-
ified microglial cell were recorded while applying
muscimol (100 ␮M; 30 s) and ATP (1 mM; 30 s) 5
min apart. [From Cheung et al. (151).]

the combination of RT-PCR, western blot, immunofluo- tion of ␤1 receptors increased expression of IL-1␤ mRNA
rescence, and immunohistochemistry analyses (198, 833). (902). RT-PCR analysis of cultured rat microglial cells
In microglial cells from the retina of a rhesus monkey, revealed expression of mRNAs for ␣1A, ␣2A, ␤1, and ␤2
only ␣6 or ␤4 nAChR subunits were identified by immu- receptors (613, 902). Stimulation of ␤2 receptors with
nostaining (530). In human embryonic microglial cell cul- terbutaline and norepinephrine increased cytosolic cAMP
tures, RT-PCR revealed expression of mRNAs specific for levels (902). Norepinephrine also induced [Ca2⫹]i tran-
␣3, ␣5, ␣7, and ␤4 subunits (765). A rather unusual sig- sients originating from ER Ca2⫹ release in cultured rat
naling pathway associated with ␣7nAChRs was identified microglial cells (990). Norepinephrine attenuated the LPS-
in primary rat cultured microglia. The treatment of these induced release of NO, TNF-␣, and IL-6 (267). Selective
cells with nicotine induced [Ca2⫹]i transients, which were stimulation of ␣1 or ␤1 or ␤2 receptors suppressed the
blocked by the specific ␣7nAChRs blocker methyllycaco- expressions of IL-6 and TNF-␣ at transcriptional level
nitine (MLA; 10 nM), but they were also blocked by the (613). Activation of ␤2 ARs suppressed proliferation of
PLC inhibitor U73122 and the presumed InsP3 receptor microglia through cAMP-dependent mechanism (302).
blocker xestospongin C (884). Furthermore, nicotine-in- Norepinephrine, acting through ␤1/2 receptors, controlled
duced [Ca2⫹]i transients persisted in Ca2⫹-free extracel- p38 MAPK signaling cascades via both cAMP and PKA;
lular media, and nicotine failed to induce any currents in this inhibited ATP-induced release of TNF-␣ (620). Stim-
voltage-clamped microglial cells. These results suggested ulation of ␤ adrenoreceptors was shown to be involved in
that ␣7nAChRs in microglia could be linked to the PLC/ pro-inflammatory cytokine production in microglial cells
InsP3/Ca2⫹ release signaling pathway (884). The nicotine- following surgical trauma (979).
induced Ca2⫹ signals modulated the release of TNF-␣ in In mesencephalic neuronal-glial cultures, the selec-
response to either activation of P2X7 receptors (positive tive ␤2 receptor agonist salmeterol induced ERK phop-
modulation) or LPS (negative modulation) (884).
shorylation and microglial activation. Activated microglia
The activation of nACh receptors generally inhibits
upregulated the production of reactive oxygen species
the immune response of microglial cells, thus represent-
(ROS) by NADPH oxidase; the ROS in turn induced neu-
ing endogenous “cholinergic anti-inflammatory pathway”
rotoxicity (736).
(833). Nicotine was shown to inhibit ATP/P2X7-mediated
Adrenoreceptors can be also instrumental in regula-
ROS production in A␤1– 42 stimulated cultured microglial
tion of microglial migration and phagocytosis. Specifi-
cells, with this effect being antagonized by ␣-bungaro-
cally, this can be relevant for pathogenesis of AD, where
toxin, thus indicating specific involvement of ␣7nAChRs
early degeneration of locus ceruleus and depletion of
(608). Long-term (15 days) incubation of corticostriatal
adrenergic input to the brain affects the ability of micro-
organotypic slices with nicotine (3–30 ␮M) reduced the
thrombin-dependent activation of microglia (672). glia to provide for effective clearance of ␤-amyloid (371).
The activation of nAChRs in human embryonic mi-
croglial cultures by treatment with nicotine (15 min to 24 F. Dopamine Receptors
h in concentrations ranging between 3 and 300 ␮M) in-
creased the expression of human immunodeficiency virus
HIV-1 and modified the gene expression profile in HIV-1 Functional dopamine receptors have been identified
infected cells (765). At the same time, activation of in mouse and rat microglia, in culture, and in brain slices
nAChRs was reported to somewhat suppress microglial (267). With the use of subtype-specific ligands, D1- and
activation in response to IFN-␥ and the HIV-1 coat glyco- D2-like dopamine receptor-mediated membrane currents
protein gp120 (322). were recorded in voltage-clamp experiments. Dopamine
In cultured microglial cells from the human brain, triggered the inhibition of the constitutive potassium in-
application of carbachol triggered cytosolic [Ca2⫹]i tran- ward rectifier currents and activated potassium outward
sients, which were blocked by atropine and originated currents in a subpopulation of microglia (267). Chronic
from Ca2⫹ release from the ER, thus suggesting functional dopamine receptor stimulation enhanced migratory activ-
expression of muscarinic cholinoreceptors (990, 1031). ity and attenuated the LPS-induced NO release similar to
stimulation of adrenergic receptors. While, however, nor-
epinephrine attenuated the LPS-induced release of TNF-␣
E. Adrenergic Receptors and IL-6, dopamine was ineffective in modulating this
response. This indicates that dopamine does not mediate
Accumulation of intracellular cAMP following stimu- its effect by triggering adrenergic receptors, but provides
lation of ␤2 receptors provided the first evidence for the evidence for microglial dopamine receptors expression
expression of adrenergic receptors in microglia (727). (267). The dopamine-induced chemotaxis was confirmed
While ␤2 receptor stimulation suppressed LPS-induced also for cultured elderly human microglia (555). Indeed,
release of IL-12p40 in cultured microglia (727), stimula- the D1,2,3,4 (but not D5) receptors were identified on trans-

lational level in microglia in culture and visualized by B. Bradykinin Receptors

immunostaining in substantia nigra from Parkinsonian
brains (555), suggesting that activation of microglia in The bradykinin receptors, of which B1 and B2 sub-
Parkinson’s disease triggers expression of dopamine re- types are cloned and characterized, are seven-trans-
ceptors, which may explain an increase in microglia-re- membrane domain receptors coupled with G␣q and G␣i
lated toxicity towards dopamine-producing neurons proteins (373, 563, 752). These receptors are widely
(555). Incidentally, concentration of microglial cells in the expressed in the nervous system and are thought to
substantia nigra (the brain region primarily affected in mediate effects of bradykinin that are produced in the
PD) is the highest in the brain: in mouse microglia amount injured brain (649). Bradykinin receptors were initially
to ⬃12% of total cells number in this region, compared discovered in primary cultured rat microglia (648, 649).
with ⬃5% in the cortex and in the corpus callosum (498). In these mixed cerebrocortical cultures from neonatal
In Parkinson’s disease, microglial cells in substantia nigra (P3) Wistar rats, the exclusive expression of B2 recep-
are activated and concentrated around dystrophic dopa- tors at both transcriptional and protein levels was dem-
minergic neurons (768); the role of dopamine receptors in onstrated (648). The expression of B1 receptors in rest-
this specific activation and migration of microglia need to ing (or not fully activated) microglia was very low
be clarified further. The inhibition of D4 receptors by (648). The stimulation of cultured microglia with 100 –
200 nM bradykinin triggered an outward current in 14%
specific agonist L-745,870 suppressed microglial activa-
of cells studied; these currents were due to an activa-
tion in the spinal cord of SOD1 transgenic animal model
tion of KCa channels following an increase in [Ca2⫹]i
of amyotrophic lateral sclerosis (ALS); this reduced mi-
stimulated via B2/InsP3 cascade (648). Similar currents
croglial activation coincided with slowing down the dis-
were also observed in amoeboid microglia in situ in
ease progression (901).
forebrain slices of young mice (650). Activation of cul-
tured microglia with LPS or with 24 h treatment with
XI. RECEPTORS FOR NEUROHORMONES 300 nM of bradykinin induced a strong elevation in
AND NEUROMODULATORS expression of both B1 and B2 receptors (650). These
two types of receptors had a neuroprotective potential
by attenuating cytokines release from microglia (650).
A. PAF Receptors Bradykinin was also found to induce chemotaxis in
cultured microglia (397). This was mediated by activation of
Activation of PAF receptors triggers a strong che- B1 receptors (as judged by their agonist/antagonist sensitiv-
motaxic response of microglial cells; this response is ity and complete disappearance in cells from B1⫺/⫺ but not
B2⫺/⫺ mice). Action of bradykinin was mediated through
mediated through G protein and MAPK signaling path-
PKC and phosphoinositide 3-kinase, which activated the
ways (9). PAF can be produced by neurons following
Na⫹/Ca2⫹ exchanger (NCX) and intermediate-conductance
stimulation of NMDA receptors and [Ca2⫹]i elevation (9).
KCa channels (Fig. 12); in NCX⫹/⫺ heterozygotes, the effects
The expression of mRNA encoding PAF receptors (iden-
of bradykinin were substantially decreased (397).
tified by in situ hybridization and Northern blotting of rat
brains) was found to be the highest in microglia (614).
PAF triggered very rapid [Ca2⫹]i elevations in primary C. Histamine Receptors
cultured microglia from rats and humans; these Ca2⫹
signals were produced by both Ca2⫹ release and the ini- Histamine induced [Ca2⫹]i increase in ⬃30% of cul-
tiation of substantial store-operated Ca2⫹ influx (385, 614, tured rat microglia; this [Ca2⫹]i increase originated from
794, 982). The relative weight of Ca2⫹ release versus Ca2⫹ InsP3-induced Ca2⫹ release from the ER (29).
entry may vary considerably between species. In human
microglia, for example, the removal of extracellular Ca2⫹ D. Endothelin Receptors
reduced [Ca2⫹]i response by ⬃80%, indicating a predom-
inant role of Ca2⫹ influx (982). The PAF-induced Ca2⫹ Endothelins represent a family of peptides initially
influx affects microglial function. The long-lasting Ca2⫹ discovered as potent vasoactive compounds (1019). Two
entry following PAF stimulation produced release of ara- types of endothelin receptors, the ETA and ETB, were
chidonic acid from cultured microglia (614). Similarly, discovered in neural cells; the stimulation of these recep-
Ca2⫹ influx via a store-operated pathway increased ex- tors triggers Ca2⫹ mobilization via InsP3-induced intracel-
pression (but not secretion) of IL-6 in microglial cells lular Ca2⫹ release (621, 811, 937).
(794); the SOCE is also believed to be instrumental for Transcripts of ETB endothelin receptors were found
PAF-induced upregulation of expression and release of in purified cultured mouse microglial cells and in the
microglial response factor (MRF)-1 (904). individual cells (using single-cell PCR) from the same

FIG. 12. Schematic view of bradykinin receptor-induced microglial signaling. At the site of tissue injury, bradykinin and ATP are released or
produced. Bradykinin is rapidly degraded by peptidases known as kininase I, which generate Des-Arg 9-bradykinin, a B1 receptor agonist. Activation
of the B1 receptor coupled to Gq/11 results in the activation of PLC-␤ and PKC. PKC can phosphorylate Na⫹/Ca2⫹ exchanger (NCX), thus increasing
NCX activity in the reverse mode with subsequent increase in [Ca2⫹]i. Increase in [Ca2⫹]i can activate Ca2⫹-dependent K⫹ channels. Activation of
the B1 receptor is also coupled to Raf-dependent MAPK signaling, as well as activation of Ras and consecutive stimulation of PI3K. At the same time,
ATP and adenosine activate A1 adenosine and P2X/Y purinoceptors, which induce Ca2⫹ signaling through both InsP3-induced Ca2⫹ release and Ca2⫹
influx. Activation of P2Y12 and P2X4 contribute to the activation of PI3K signaling cascade. ATP/adenosine also activates a separate set of K⫹
channels, which is different from Ca2⫹-dependent K⫹ channels activated by bradykinin.
cultures. The functional expression of ETB receptors was endothelins and ETB receptors was reported in ischemic
further confirmed by Ca2⫹ imaging. The stimulation of cerebral cortex of adult rats subjected to MCAO (523).
mouse cultured microglia with ET-1 or ET-3 triggered
[Ca2⫹]i transients in a small (⬃13%) subpopulation of
E. Cannabinoid Receptors
cells. These Ca2⫹ responses were mimicked by specific
agonist BQ3020 and blocked by selective antagonist
BQ788. The ETB-mediated Ca2⫹ signals had a complex The cannabinoid signaling system comprises the
nature involving both Ca2⫹ release and store-operated endogenous cannabinoids, cannabinoid receptors (CB1,
CB2, orphan receptor GPR55 and 2 additional pharma-
plasmalemmal Ca2⫹ entry (603). In cultured human mi-
cologically identified receptors, which await cloning;
croglia, ET-1 and ET-3 induced [Ca2⫹]i transients in ⬃80%
Refs. 433, 858, 859), and cannabinoid degrading en-
of cells. These Ca2⫹ signals were also mediated by ETB
zymes. Cannabinoids may also modulate the activity of
endothelin receptors (as judged by their inhibition by
TRPV1, TRPA1, and TRPM8 channels by a yet unknown
selective antagonist BQ788) and were dependent on both mechanism (196). Rodent microglial cells in vitro ex-
intracellular Ca2⫹ release and plasmalemmal Ca2⫹ influx press CB1 and CB2 cannabinoid receptors as well as the
(573). third, not yet cloned, receptor (114, 259, 744, 766). The
Expression of both endothelins and ETB receptors constitutive expression of cannabinoid receptors in the
was detected in the amoeboid invading microglia in the resting microglia is very low, if at all existing. The
corpus callosum of neonatal rats (1002). This expression immunocytochemistry did not visualize CB1 receptors
diminished with age and almost completely disappeared in resting microglia; neither CB2 mRNA is detectable in
in the mature brains. the healthy brain tissue (114, 858, 859). The specific
Significant increase in ETB receptors expression in expression of CB2 receptors was found in perivascular
hippocampal microglia was found following brain ische- microglial cells in white matter of human cerebellum
mia induced by a 10-min bilateral carotid occlusion and (663), although this study relied solely on immunostain-
reperfusion (1017). Similarly, an increase in expression of ing with CB2-specific antibodies, which can have non-

specific binding (859). Microglial activation, however, F. Angiotensin II Receptors

results in rapid upregulation of cannabinoid receptors
expression (858). The functional expression of angiotensin II receptors
Microglial CB1 and CB2 receptors are coupled to Gi/o type 1 and 2 (AT1/AT2) was suggested based on RT-PCR
and Gi proteins, respectively (859). The activation of can- and pharmacological studies of cultured microglial cells
nabinoid receptors reduced NO production by fully acti- from embryonic rat brain. It was found that unstimulated
vated microglia and may inhibit production of cytokines microglia expressed mRNA for AT2 and angotensinogen
(114). The stimulation of CB2 receptors in cultured micro- but not for AT1. Treatments of cultures for 6 h with LPS
glia by selective agonist JWH-015 suppressed microglia induced additional expression of AT1 specific mRNA. The
activation by inhibiting CD40 signaling pathway, inhibited exposure of cultures to specific AT1 receptor antagonist
phosphorylation of JAK/STAT1 and production of NO and losartan (0.1–10 ␮M) suppressed morphological activa-
TNF-␣ (244). The treatment of rat cultured microglia with tion of microglia and reduced production of NO and IL-1␤.
endogenous and synthetic cannabinoids suppresses LPS- Furthermore, losartan almost completely inhibited LPS-
induced activation of NF-␬B (596).
induced TNF-␣ release (259); this action was mediated by
one of uncloned CB receptors.
A very high expression of CB2 receptors was found in G. Somatostatin Receptors
the CNS of mice with experimental autoimmune enceph-
alomyelitis (549). The upregulation of CB2 receptors was The RT-PCR of material obtained from cultured rat
due to synergistic action of IFN-␥ and GM-CSF, which microglial cells revealed an expression of sst2, sst3, and
both are expressed in experimental autoimmune enceph- sst4 (but not sst1 and sst5) subtypes of somatostatin
alomyelitis brains (549). Similarly, CB2 receptors are also receptor. The activation of these receptors by somatosta-
prominently expressed in microglial cells from the brains tin per se or by metabolically stable analog octreotide
of patients with AD, ALS, and AIDS-associated dementia (SMS 201–995, selective agonist of sst2, sst3, and sst5
(45– 47, 1022). Incidentally, acute treatment with 3,4- receptors) induced protein phosphorylation and inhibited
methylenedioxymethamphetamine (popularly known as microglial proliferation (272).
“ecstasy”) increased microglial expression of CB2 recep-
tors in the rat in vivo (917). H. Glucocorticoid and Mineralocorticoid Receptors
Generally, the activation of cannabinoid receptors
increases microglial proliferation (127) and reduces Binding studies suggested the functional expression
microglial neurotoxicity (859). The cannabinoid recep- of both glucocorticoid and mineralocorticoid receptors
tors are therefore potentially neuroprotective, and in- (GR and MR, respectively) in cultured microglia from the
deed, activation of CB2 receptors reduced neurotoxic- forebrain of newborn rats. The presence of GR was also
ity, neuroinflammation, brain edema, and death of stri- confirmed with immunoblotting using specific antibody.
atal neurons, simultaneously improving motor Activation of GR inhibited proliferation of cultured micro-
symptoms in the animal model of Huntington disease glia and enhanced lysosomal formation (900).
(682). The activation of CB2 receptors facilitated mi-
croglial uptake of A␤1– 42 peptide (244). The stimulation
I. Opioid Receptors
of cannabinoid receptors inhibited microglial activa-
tion by ␤-amyloid and reduced microglia-dependent
Kappa opioid receptors were identified in human
neurotoxicity (744). Massive release of anandamide,
fetal microglia using RT-PCR, immunohistochemistry,
observed upon brain lesions, was shown to exert neu-
and ligand binding assay (140); these receptors may reg-
roprotection through activating microglial CB1 and CB2 ulate microglial immune responses particularly in HIV-1-
receptors with subsequent induction of mitogen-acti- dependent encephalopathies and HIV-1-associated de-
vated protein kinase phosphatase-1 (MKP-1). The latter mentia (140). The activation of kappa-opioid receptors by
terminated the MAPK signaling transduction cascade, cocaine is somehow involved in potentiation of HIV-1
limiting thus TLR4-dependent activation of microglia expression and facilitation of HIV-1 associated dementia
(246, 760). Similarly, the activation of CB2 receptors (313). Similarly, the RT-PCR analysis demonstrated the
expressed in microglial cells in the spinal cord may constitutive expression of mu-opioid receptor mRNA in
limit inflammatory and pain reactions after transaction human microglia. The activation of these receptors in
of peripheral nerve (770). Finally, CB2 receptors reduce cultured microglial cells strongly inhibited their che-
HIV-1 expression in microglia possibly through down- motaxis towards C5a with an IC50 ⬃1 fM (141).
regulation of chemokine receptor CCR5, which is in- Exposure of feline cultured microglia to morphine
volved in virus entry into microglial cells (716). and endorphin triggered morphological activation, sup-

posedly mediated by mu3 (opiate alkaloid-selective) re- microglia in vitro, and stimulation of these receptors
ceptors, as suggested because of inhibitory effects of with VIP (0.1 ␮M) or with its functional/structural an-
naloxone (221). Microglial expression of mu3 receptors alog pituitary adenylyl cyclase-activating polypeptide
appears very early in evolution; the stimulation of mu3 (PACAP, 0.1 ␮M) strongly inhibited TNF-␣ production
receptors by morphine promotes NO release and induces in LPS-activated cultured rat microglia (455). These
activation of microglial cells in invertebrate, the mussel effects of VPAC1 receptor activation were mediated
Mytilus edulis (531). The mu-opioid receptors increase through cAMP-dependent pathway (455). The exposure
migration and upregulate expression of P2X4 purinocep- to VIP inhibited COX-2 expression and the production
tors in cultured rat microglia (388). On the contrary, in of prostaglandin E2 (PGE2) in activated cultured mouse
neuronal-glial cultures prepared from rat mesencephalon, microglia (330). Similarly, VIP and PACAP inhibited
morphine significantly reduced microglial neurotoxicity LPS-induced activation of and secretion of proinflam-
and LPS-induced microglial activation, albeit through an matory factors TNF-␣, IL-1␤, and NO from cultured
unknown pathway that, most likely, did not involve opioid microglial cells (212–214). In addition, VIP suppressed
receptors (737). Reportedly morphine can also cooperate microglial activation in vivo, after intraventricular LPS
with HIV-1 transactivating protein Tat in stimulating mi- injection or mechanical brain trauma (212).
croglial activation and secretion of proinflammatory fac- Stimulation of VPAC1 receptors was also reported to
tors (76). inhibit the activation and neurotoxic potential of cultured
microglial cells exposed to A␤1– 42. In this study, the ac-
tion of VPAC1 receptors was mediated through several
J. Neurokinin (Substance P) Receptors
intracellular signaling cascades that included p38 MAPK,
p42/p44 MAPK, and NF-␬B pathways (211, 215).
Neurokinin-1 (NK-1 or substance P) receptors were
detected in primary cultured murine and fetal human
microglia at mRNA and protein levels (491, 749). More- L. Neurotrophin Receptors
over, human microglia was shown to produce significant
amounts of substance P (491). Activation of NK-1 recep- The neurotrophins [nerve growth factor (NGF),
tors by nanomolar concentrations of substance P trig- brain-derived growth factor (BDNF), and neurotophins 3
gered the activation of transcriptional factor NF-␬B (749). and 4 (NT-3, -4)] exert their biological action through
Furthermore, the NK-1 receptors may regulate production activation of two classes of receptors, the Trk tyrosine
of cytokines in microglial cultures and in microglia in vivo kinase receptors and the p75 neurotrophin receptor
following stimulation with bacterial pathogens, Neisseria (p75NTR; for review, see Refs. 434, 695). The subclass of
meningitidis and Borrelia burgdorferi (144). Trk receptors, the truncated tropomyosin-related kinase
In rat cultured microglia, substance P, when admin- B-T1 (Trk-B1) receptors, were identified (at the mRNA
istered together with LPS, synergistically increased IL-1 level) in cultured rat microglia. Activation of these recep-
production (which was 4 times greater than after treat- tors by BDNF resulted in sustained [Ca2⫹]i elevation,
ment with LPS alone; substance P when added on its own which was mediated by an initial PLC/InsP3-driven Ca2⫹
did not affect IL-1 synthesis), with this action possibly release from the ER that followed by a long-lasting acti-
involving “nonclassical” NK-1 receptors (553). vation of the SOCE. Incubation with BDNF also de-
Activation of NK-1 receptors can enhance inflamma- creased release of NO from the activated microglia (597).
tory responses induced by bacterial infections of CNS LPS-induced activation of microglia can trigger the ex-
(750). At the same time, the pharmacological inhibition of pression of NGF in cultured microglial cells, which can
NK-1 receptors in cultured microglia by aprepitant (1 nM also be induced by stimulation of A2 adenosine receptors
to 10 ␮M) suppressed HIV-1 infection by reducing virus (366).
replication (983).
XII. CYTOKINE AND CHEMOKINE RECEPTORS

K. Vasoactive Intestinal Polypeptide Receptors
The vasoactive intestinal polypeptide (VIP) exerts A. Chemokine Receptors

its action through the activation of G protein-coupled
receptors of VPAC (VIP/pituitary adenylate cyclase- Chemokines comprise a growing family of chemoat-
activating peptide) receptors 1 and 2 (VPAC1/VPAC2; tractive cytokines with pivotal functions in cell migration
Ref. 496). The activation of these receptors is known to under both physiological and pathophysiological condi-
trigger anti-inflammatory and immunodepressive re- tions (25, 30, 310, 623, 783). Since the first description of
sponses in various peripheral tissues (e.g., Refs. 23, IL-8 as a neutrophil attractant in 1987, the list of identified
503). The VPAC1 receptor mRNA was identified in rat chemokines exceeded more than 50. Characterized by

presence of up to four conserved cysteine residues in is also another property of the chemokine system that is
their sequences, these small proteins of 8 –12 kDa fall into not shared by other cytokines. Ligands and receptors
four subgroups, i.e., the C, CC, CXC, and CX3C chemo- reveal some promiscuity in their interactions, allowing
kines (275, 492, 623). Most of the chemokines act upon individual ligands to bind more than a single receptor, and
release as soluble messengers that can create chemotac- vice versa. Considering the expression of several chemo-
tic gradients for cell migration, while especially CX3CL1 kine receptors by a given cell and the simultaneous pres-
(also known as fractalkine) occurs also in surface-at- ence of different chemokines in their environment, the
tached format. In addition, chemokines may associate movements and functions of cells could be orchestrated
with proteoglycans on the vascular endothelium or in the in a rather complex fashion. Not only the direct access to
extracellular matrix, thus providing also immobilized diverse signaling pathways including [Ca2⫹]i (30, 69, 74),
cues. A distinction as to inflammatory and homeostatic but also physical interaction with other receptor/effector
chemokines is based on the inducible versus constitutive systems, such as TLRs (927), further increase the options
expression, with overlap occurring as well (623). Initially of chemokine receptor-mediated control over cellular
the chemokines were believed to participate in the migra- functions.
tion and maturation of immune cell populations within The stimulation of cultured microglia with ␤-chemo-
and between immune organs as well as in the recruitment kines MCP-1/CCL2 (agonist of CCR2 receptors) or MIP-1␣/
and guidance of immune cells to inflammatory sites. Sub- CCL3 and RANTES/CCL5 (agonists of CCR5 receptors) trig-
sequent experiments also described chemokines as im- gered rapid [Ca2⫹]i transients in distinct subpopulations of
portant messengers in other tissues, which probably serve control and LPS-treated microglia; LPS-induced microglial
a multitude of functions beyond leukocyte attraction (62, activation resulted in an increase of Ca2⫹ responses. Both
69, 258). These other roles of chemokines may range from types of Ca2⫹ responses required a functional Ca2⫹ store (as
neovascularization, control of cell adhesion or regulation indicated by inhibition by thapsigargin), although MCP-1
of apoptosis and phagocytosis, to contribution to various responses were also dependent on extracellular Ca2⫹ and
signaling cascades (784, 927, 943). were blocked by ryanodine (74), thus indicating a combi-
The chemokines are expressed by neurons and mac- nation of initial Ca2⫹ influx and subsequent Ca2⫹-induced
roglial cells (69, 194) as well as by microglia in the human Ca2⫹ release. The CCR5-dependent MIP-1␣/CCL3 and
and mouse CNS (69, 195, 219, 323, 349, 351, 363, 454, 509, RANTES/CCL5-induced Ca2⫹ signals most likely involved
950). Basal and inducible levels may vary between species the InsP3-dependent Ca2⫹ release. Ultimately, both types
(74, 285), anatomical regions (193), ages (834), and even of [Ca2⫹]i transients were inhibited by PTX, reflecting the
between human individuals (579), with the latter variabil- role of G␣o and G␣i proteins (74). The role for PI3-K,
ity being likely a consequences of disease history. Micro- Bruton’s tyrosine kinase (Btk), and stimulation of cADPR
glial expression at transcriptional and/or protein level has production with possible activation of RyRs was also
been documented for rodent CCR1, CCR2, CCR7, and suggested (826).
CCR5 (74, 219), the latter being most abundant, as well as In human microglial cells from hippocampal slices,
for human CXCR1, CXCR3, and CCR3, whereas CCR4, 15-s application of MIP-1␣/CCL3 (2 mg/ml) trebled the
CCR5, CCR6, CXCR2, CXCR4, and CXCR5 were ex- amplitude of Ca2⫹-dependent K⫹ currents by a yet un-
pressed at much lower levels (285). Expression of induc- characterized mechanism (77), indicating possible role of
ible chemokines in microglia may be dynamically regu- KCa channels in regulation of microglia migratory activity.
lated in the course of an activation process. Feedback Activation of CXCR3 receptor by secondary lymphoid
signals from leukocyte populations attracted, in the first tissue chemokine CCL21 induced the activation of micro-
place, by microglial chemokines may subsequently reor- glial volume-regulated Cl⫺ channels, which was linked to
ganize the microglial chemoattractive cocktail. This can microglial migration (747).
result in a phenomenon of “self-limitation” as well as in a Microglial activation following neuropathological
shift of the chemoattractive call towards other cell pop- challenges affects the expression of chemokine receptors
ulations, as indicated for the Th1 cytokine IFN␥ and its (478). Recruitment of microglia to a site of infection,
impact on Th1 attraction (281, 363). lesion, or degeneration may include several factors in-
The cellular effects of chemokines are mediated cluding chemokines released from local cell populations.
through families of seven-transmembrane domain GPCRs, Monocyte/macrophage-attracting CCL2, for example, can
which are now classified in accordance to the systematic be produced by activated microglia (such as upon TLR
nomenclature of their ligands (e.g., CCR, CXCR, or stimulations), while representing itself a microglial che-
CX3CR). They are linked to numerous intracellular signal- moattractant. Thus microglia act as a source and a target
ing cascades (69), which include adenylate cyclase, phos- of chemokine actions, also in auto/paracrine fashion. The
pholipases, GTPases (Rho, Rac, and Cdc42), and some CCL2/CCR2 system seems to be crucial for the recruit-
kinases such as MAPK or phosphatidylinositol 3-kinase ment of myeloid cells to the CNS where they can acquire
(PI3-K) (30). Besides the link to G protein signaling, there microglia-like features (192, 586, 729). Distinct circulating

monocyte subsets have been discussed as giving rise to stages of multiple sclerosis; the CCR5 receptors were
populations replenishing tissue resident macrophages or suggested to be somehow linked to microglia-dependent
to comprise the infiltrates invading during pathological initiation of remyelination (924). Chemokine receptor
conditions. Expression of CCR identifies the respective functions in the CNS in general and in microglia in par-
subsets, and this is functionally underlined by its role as ticular reveal broad implications with remarkable mech-
the receptor for CCL2. The CX3CR1, the receptor for anisms and consequences. Activation of CCL21 receptors
CX3CL1 (fractalkine), represents another key molecule in was reported to mediate microglial activation in the thal-
this CNS-relevant monocyte subclassification (729). Mi- amus, which was related to neuropathic pain responses
croglial expression of CX3CR1 receptor mRNA was sup- following spinal cord injury (1036). CCL2, its receptor
pressed by treating cultures with LPS (73). The application of CCR2, and other chemokines in the hippocampi of pa-
0.1–1 nM of fractalkine to cultured microglia triggered tients and laboratory animals have been linked to seizures
[Ca2⫹]i transients; whereas at 10 nM it triggered [Ca2⫹]i and epilepsy (258).
oscillations (73). These fractalkine-induced Ca2⫹ signals CXCL10 and its receptor CXCR3 have been linked to
originated from ER Ca2⫹ release. The genetic deletion of various CNS pathologies (951). Studying the mechanisms
CX3CR1 enhanced microglial neurotoxicity in several by which this system mediates NMDA-induced neuronal
pathological models, including systemic inflammation in- toxicity in the hippocampus, the authors demonstrated
duced by an intraperitoneal injection of LPS, MPTP-in- that astrocytes and microglia cooperate to deliver the
duced model of Parkinson disease, and in a SOD1 over- effect and that the deficiency in either the ligand or the
expressing mouse used as a model for ALS (123). receptor diminished or enhanced cell death depending on
The expression of CCR5 receptors (on transcrip- the tissue subregion and that microglia was the responsi-
tional level) was increased following hypoxic-ischemic ble cellular element by which this difference in neuronal
insults and nerve injury (177, 307). LPS-induced expres- vulnerability is organized. This report thus documented
sion of mRNAs for inflammatory cytokines (IL-1␤, IL-6, not only regional diversity in signaling consequences for
TNF-␣) and iNOS in microglia were all suppressed by the CXCL10/CXCR3 system per se, but an intimate link of
RANTES/CCL5-induced activation of CCR5, and nerve in- this phenomenon to microglia, in this regard demonstrat-
jury-induced motor neuron death seen in wild-type ing the surprisingly refined organization of microglial
C56BL/6J mice was accelerated in CCR5 knock-out chemokine physiology within anatomically and function-
C57BL/6J, suggesting that CCR5-mediated neuron-glial ally circumscribed CNS divisions.
signaling protects neurons by suppressing microglia tox- A similarly intriguing mechanism involving microglia,
icity (307). astrocytes, and a chemokine system had been unraveled
The chemokines including CCL2, CCL21, or CX3CL1 earlier when studying astroglial contributions to gluta-
may serve as signals from endangered neurons to micro- mate toxicity (62). Binding of CXCL12 (also known as
glia (69). The CX3CL1 expressed in neurons may provide stromal cell-derived factor, SDF-1␣) to its receptor
a constitutive calming influence on CX3CR1-expressing CXCR4 in astrocytes causes InsP3 production, [Ca2⫹]i
microglia, thus representing a neuron-to-microglia signal- increases, and release/shedding of TNF-␣. The binding of
ing system similarly to already described for CD200/ TNF-␣ to its receptor triggers a second wave of signaling
CD200R or CD47/SIRP-1␣. Interruption of this influence (either in the same or in another astrocyte, through auto-
may release microglia from a control to allow activation crine or propagating mechanism) that causes PGE2 pro-
or enhanced responses to activating signals. Indeed, de- duction. The PGE2, in turn, induces the release of gluta-
ficiency in this chemokine signaling can lead to enhanced mate, which may serve in glial or glia-neuron communi-
severity of CNS damage in several disease models (123, cation, but which can also initiate neurotoxicity. In the
729). The CX3CR1/CX3CL1 system reveals some complex- latter situation, SDF-1␣ would also act on microglia, thus
ity as the protein expression may not be cell-specific, as driving enhanced TNF-␣ release from both glial popula-
surface-attached and soluble ligands may differ function- tions and ultimately causing massive glutamate release.
ally and as the system may have distinct importance in Chemokines may thus reveal even more important roles
different regions of the brain and spinal cord or impact in microglial activities as originally anticipated from their
differently depending on the disease scenario (75, 216, chemoattractive potential.
301).
There is increasing evidence for altered chemokine
signaling in diverse CNS diseases such as AD or multiple B. TNF-␣ Receptors
sclerosis (310, 924) which may involve microglia activa-
tion and “sterile inflammation” (864). Microglial cells from The action of TNF-␣ is mediated through two types of
AD brains may have elevated levels of CCR3 and CCR5 receptors, TNFR1 and TNFR2 (543). Stimulation of TNF-␣
receptors (310). Increased levels of CCR5 receptors were receptors enhanced microglial phagocytic activity (971).
also found in biopsic material from patients with early Activation of microglial cells induced by brain lesions was

significantly reduced in transgenic mice lacking TNF-␣ recognition receptors (PRRs) abundantly expressed in
receptors (95). Cultured mouse microglial cells release microglia had evolved to detect invading infectious agents
TNF-␣ following stimulation of TNF-␣ receptors 1, thus and to assist in the control of the adaptive immunity and
producing a positive autocrine loop, which can partici- govern the cooperative activities of effector cells (59, 350,
pate in microglial activation (483). In mice deficient in 681). In mammals, host defense is based on innate and
TNF-␣ receptors, microglial activation was very much adaptive immunity. Adaptive (also acquired or specific)
reduced in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine immunity occurred in jawed vertebrates and is carried by
(MPTP) model of Parkinson’s disease (854). specialized helper, effector, and regulatory T-cell popula-
tions as well as antibody-producing B lymphocytes. The
innate (also natural or native) immunity, the much older
C. Interleukin Receptors
defense system, is based on physical and chemical barri-
ers (e.g., epithelia), phagocytotic (e.g., macrophages) and
The IL-1 receptors are represented by IL-1 type I
natural killer (NK) cells, as well as a range of proteins
receptor (IL-1RI), IL-1 type II receptor (IL-1RII), and IL-1
with signal and effector functions, such as the comple-
receptor accessory protein (IL-1RAcP). A transcriptional
ment system.
analysis of purified cultured microglia showed weak ex-
The repertoire of germline-encoded nonclonal recep-
pression of IL-1RI and stronger expression of IL-1RII,
tors for detection of pathogen-associated molecular pat-
whereas the treatment with LPS significantly increased
terns (PAMPs) includes 1) lectin-type, mannose and
expression of IL-1RI, IL-1RII, and IL-1RacP. Cultured rat
␤-glucan receptors (e.g., dectin-1); 2) nucleotide binding
microglial cells also expressed mRNA for IL-10 receptor
and oligomerization domain (NOD)-like receptors; 3) re-
(IL-10R1), which was upregulated following LPS stimula-
ceptors characterized by a RNA helicase domain and two
tion; interestingly, IL-10 released by activated microglia or
caspase-recruitment domains (CARD), collectively known
cultured astrocytes inhibits microglial expression of IL-
now as RIG-I-like receptors (RLR); and 4) the Toll-like re-
10R1 by auto- or paracrine loops (504). In human micro-
ceptors (TLR) (see Refs. 299, 683, 786, 991 for review).
glial cells, mRNA transcripts for multiple interleukin re-
Reaching beyond pathogen detection, several PRRs,
ceptors (IL-1RI, IL-1RII, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R,
and specifically members of the TLR family claim a dual
IL-12R, IL-13R, and IL-15R) were detected (509).
function by also detecting endogenous molecules that are
It was suggested that IL-RII specifically regulates
generated, released, or modified upon tissue injury. These
effects of IL-1␤ on microglial cells by binding the excess
molecules are classified as damage- or danger-associated
levels of this cytokine (721). The stimulation of IL-1 re-
molecular patterns (DAMPs), or alarmins (63). Sharing
ceptors in cultured human microglial cells by IL-1␤ (2
receptors and inducing overlapping sets of genes, PAMPs
ng/ml) induced a slow increase in [Ca2⫹]i through an
identify strangers and DAMPs spell danger (470). Ulti-
uncharacterized mechanism, which potentially involved
mately, both inform about disturbed homeostasis and
both Ca2⫹ entry and intracellular Ca2⫹ release (326).
initiate appropriate reactions in dangerous situations
The activation or cultured mouse microglia with LPS
(561).
induced expression of IL-2 receptors, which were absent
in untreated cells. These newly acquired IL-2 receptors
mediated effects of IL-2 on activated microglia by enhanc- A. Toll-like Receptors
ing growth and viability. These effects were suppressed
The Toll receptors were initially discovered in Dro-
by treating cultures with anti-mouse IL-2 receptor beta-
sophila, where they are important for embryogenesis,
chain antibodies (797). The IL-2 receptors, coupled to
being involved in controlling dorsoventral polarity of the
Janus kinase 1 signaling pathway, were also shown to
fly (17).2 Further studies demonstrated the role of Toll
mediate effects of IL-15 on cultured microglia.
proteins in the immune defense of Drosophila (515). The
Spinal cord microglia showed sensitivity to IL-6, con-
mammalian analog, the Toll-like receptor, was first cloned
nected with a signal transducer and activator of transcrip-
in 1997 (578), and by now at least 11 human and 13 mouse
tion (STAT) pathway. This system was reported to be
TLRs have been described (519, 676). Toll-like receptors
activated after a peripheral nerve injury and may contrib-
are coupled to a complex signaling pathways (which, for
ute to development of mechanical allodynia and neuro-
most of TLRs except TRL3 involves adaptor protein
pathic pain (223).
MyD88) that end at the transcription factor AP-1 and/or in
the nuclear translocation of another transcription factor
XIII. PATTERN-RECOGNITION RECEPTORS NF-␬B (355, 426).
As the principal resident immune cells of the CNS, 2

The name “Toll receptors” was coined in the course of analyzing the
microglia serve at the first line of defense against exoge- data by the discoverer Christiane Nüsslein-Volhard, who exclaimed “Das
nous threats, such as viruses and bacteria. The pattern war ja toll!” (355).

TLRs are type I integral membrane glycoproteins. Incidentally, TLR4 receptors are also involved in micro-
TLR1/2, -6/2, -4, and -5 are located on the cell surface, glial activation following exposure to ethanol (274).
whereas TLR3, -7, -8, and -9 are confined to endosomal The Toll-like receptors directly control microglial
compartments. They relate to the interleukin-1 receptor activation. TLR2, for example, is important for micro-
(IL-1R) family, due to homology in cytoplasmic Toll/IL-1R glial activation following damage to sensory neurons,
(TIR) domains (11, 840). TLRs are characterized by leu- and TLR2⫺/⫺ mice showed reduced activation in the
cine-rich repeat motifs (for TLR4, e.g., within the extra- spinal cord following peripheral nerve injury (451).
cellularly portion), forming a horseshoe-like structure Intrathecal injection of antisense mRNA to TLR3 also
with its concave surface likely interacting with the ago- reduced spinal cord microglia activation and sup-
nists. TLRs are widely expressed in cells of the innate as pressed tactile allodynia following spinal nerve ligation
well as adaptive immune system, but also in nonimmune in rats (667). Similarly, tactile and thermal hypersensi-
cells and organs traditionally not considered as immuno- tivity following spinal nerve injury was attenuated in
logically active (799). TLR4⫺/⫺ mice and in mice and rats receiving TLR4
In the brain, Toll-like receptors are mainly expressed antisense oligodeoxynucleotides intrathecally (905).
in glia, although some of them can be detected in neurons Alzheimer’s disease is associated with significant ele-
(see Refs. 22, 125, 350, 469, 676 for review). The Toll-like vation in TLR expression in the brain (518, 974). In
receptors are widely expressed in microglia. Structural experiments in vitro in primary mouse microglial cul-
(mRNA and protein detection) and functional (responses tures, treatment with A␤1– 40 potentiated TLR2 and
to agonists) evidence was presented for TLR1 to 9 recep- TLR4-mediated responses, while inhibiting the TLR9
tors, for coreceptors, like CD14 and for TLR signaling (535). At the same time, all three receptors (TLR2,
components, such as MyD88 in the mouse and human TLR4, and TLR9) stimulated the uptake of amyloid
system by both in vitro and in vivo approaches (56, 96, ␤-protein by microglial cells (890). The role of TLR2 in
146, 226, 232, 233, 253, 340, 353, 422, 432, 508, 510, 677, A␤ uptake was mediated through the induction of ex-
pression of formyl peptide receptor mFPR2 (mouse
728, 738, 903, 918).
homolog of human GPCR formyl peptide receptor-like
TLRs are essential for mounting an immune re-
1, FPRL1) (146). The levels of Toll-like receptors in
sponse against infection, with different receptors being
CNS are generally upregulated in many other neurode-
sensitive to specific agents. TLR2 in heteromeric asso-
generative diseases, including multiple sclerosis, Par-
ciations with TLR1 or TLR6 conveys signals to bacterial
kinson’s disease, and ALS (see Ref. 676 for review),
tri- and diacyl lipopeptides, lipoteichoic acid, and pep-
however the specific role of microglial TLRs in these
tidoglycan. TLR3 recognizes virus-specific double-
forms of pathology remains unknown.
stranded RNA. TLR4 is activated by LPS, a major cell
Microglial activation, in turn, upregulates the synthe-
wall component of gram-negative bacterial strains. sis of Toll-like receptors (449, 566); similarly, the levels of
TLR5 responds to bacterial flagellin. TLR7 and TLR8 are TLRs in microglial cells are increased following hypoxia
sensors of single-stranded (viral) RNA, while TLR9 is (668) and focal cerebral ischemia (in the latter case the
sensitive to bacterial and viral unmethylated CpG DNA highest increase was found for TLR2; Ref. 1039). The
(11, 22, 125, 489, 677, 928). For several TLRs, such as expression of Toll-like receptors can also be regulated by
TLR10 or -11, ligand specifities have not clearly been inflammatory factors; for example, TNF-␣ (which may act
unravelled, and species differences exist for the func- in both para- and autocrine manner) stimulates expres-
tional expression of certain members, such as TLR7/8. sion of TLR2 in cultured mouse microglia (888).
How the recognition of structurally diverse ligands by a The Toll-like receptors also regulate microglial death
given TLR is organized cannot yet be explained in all following pathological activation. The TLR4 triggers mi-
cases, and surprising cross-recognition of apparently croglial apoptosis via autocrine production of IFN-␥,
unrelated motifs can be noticed (928). whereas TLR2 is coupled to caspase-8-dependent apopto-
Numerous reports demonstrated the importance of tic pathways (514). Similarly, TLR2 receptors were instru-
TLRs in various CNS diseases including infection, trauma, mental for microglial apoptosis following HIV-1 infection
stroke, neurodegeneration, and autoimmunity (28, 131, (20, 21). Conceptually, TLR-controlled microglial cell
511, 634, 641). The stimulation of TLRs triggers various death can represent an intrinsic mechanism preventing
programs of microglial activation and activates secretion microglial over-activation (676).
of cytokines and chemokines (22, 676). The stimulation of The TLRs are also involved in DAMP-induced sig-
TLR3, for example, induces the release of IL-6, IL-12, naling. Heat shock protein 60 (hsp60) was recently
chemokine ligand 10, TNF-␣, and IFN-␤ (13, 422); TLR2 shown to drive neurodegeneration (513). Released from
controls the secretion of IL-6 and IL-10 (422); TLR9 reg- CNS cells undergoing necrosis or apoptosis hsp60 was
ulates production of NO and TNF-␣ (398), whereas stim- found to drive a TLR4- and MyD88-dependent micro-
ulation of TLR4 triggers release of IL-6 and TNF-␣ (676). glial activation, with NO synthesis as well as associated

TABLE 5. Cytokines and chemokines receptors in microglia
Receptor Type Experimental Preparation/Technique Pharmacology Properties and Functional Relevance Reference Nos.
Chemokine receptors
CCR1, CCR2, CCR5 Rat/primary culture/RT-PCR, Agonists: Stimulation of CCR2 and CCR5 73, 74
Ca2⫹ imaging CCR2-MCP-1 triggered [Ca2⫹]i transients;
CCR5-MIP-1␣, RANTES activation of microglia by LPS
increased these Ca2⫹ responses.
CXCR1, CXCR3, and Human/primary culture/RT-PCR, Receptor expression was increased 285
CCR3 immunocytochemistry following treatments with TNF-␣
and IFN-␥.
CCR5 Neonatal rat forebrain/RT-PCR Hypoxia/ischemia increased CCR5 177
mRNA expression.
CCR5 Human hippocampus/acute Short application of MIP-1␣ 77
slices/whole cell increased the amplitude of K⫹
voltage-clamp currents 3-fold.
CXCR3 Mouse/primary cultures/whole Agonist: CCL21T Activation of CXCR3 receptors 747
cell voltage-clamp activated volume-regulated Cl⫺
channels.
TNF-␣ receptors
TNFR1 and TNFR2 Mouse/knockout and disease Agonist: TNF-␣ Reduced microglial activation 95, 353, 357, 888
models/primary culture, along with increased neuronal
isolated cells/protein damage in receptor-deficient
detection, also of soluble mice challenged by ischemia and
TNFR seizures, upregulation of TLR2.
Interferon (IFN) receptors
IFN␥R Mouse/primary culture, slice IFN-␥ Regulation and massive 110, 112, 349, 363, 585,
preparations reorganization of induced 866, 950
cytokine and chemokine
production, upregulation of MHC
II, induction of
immunoproteasome, induction of
neuronal markers and a
neuroprotective phenotype,
instruction of a M1-like reactive
phenotype.
IFNAR, type I IFN Mouse/primary culture, IFN-␤ Suppression of glutamate and 349, 730
receptor knockout in vivo superoxide production,
regulation of gene expression,
involvement in the control of
microglial functions in vivo and
ex vivo.
Interleukin receptors
IL-1R1/IL-1R2, and Human, mouse, rat/primary IL-1␤ Induction of inflammatory 19, 184, 509, 576, 721
related molecules cultures, tissues/mRNA mediators, like PGE2 and IL-6,
detection, antibody blockade activation of NF-␬B, p38, JNK,
and ERK1/2.
IL-2R␣/␤/␥ (␥c) Mouse, rat/primary cultures/ IL-2 as agonist, blocking Augmented NO production, 349, 352, 717, 782, 797
mRNA, partially protein antibodies against increased growth and viability,
detection receptor components indirect evidence for recruitment
of MHC II⫹ and CD11b⫹
microglia to lesions.
IL-4R Mouse, rat/primary cultures, IL-4 Induction of cytokines and 110, 112, 349, 585, 829,
brain and nerve tissue/gene chemokines with M2-like profile, 862, 950
profiling, detection of protein enhanced amyloid ␤ clearance,
regulation of scavenger receptor
expression, oligodendrocyte
markers and dendritic cell (DC)
marker, CD11c, downregulation
of proinflammatory cytokines,
induction of a M2-alternatively
activated phenotype.
IL-10R Human, rat/primary cultures, IL-10 Induction of a M2-deactivated 331, 349, 509, 585
brain tissue/mRNA, gene phenotype.
profiling, protein detection
Continued

Receptor Type Experimental Preparation/Technique Pharmacology Properties and Functional Relevance Reference Nos.
IL-13R Mouse primary or human IL-13 Induction of cytokines and 349, 509, 950
cultures chemokines with M2-like profile.
IL-15R␣ (sharing IL-2R␤ Mouse primary or human IL-15 as agonist Induction of intracellular signaling 352, 392, 509
and ␥ for signaling) cultures, mRNA, also at via JAK1 recruitment, support of
single-cell level, protein microglial survival, reduction of
detection, knockout animals NO production, indirect evidence
for recruitment of MHC II⫹
microglia to lesions.
IL-18R (IL-1 related) Mouse primary cultures, IL-18 Inducible signaling by IRAK and 726
TRAF6 recruitment, regulation
(attenuation) of induced IL-12
production.
axonal loss and neuronal death, consequences which XIV. OTHER RECEPTOR SYSTEMS
again would deliver hsp for a vicious cycle. Such a
mechanism could apply to various types of CNS dam-
age where innate immunity participates by TLR4-medi-
A. Calcium Receptors
ated recruitment and TLR4-organized release of toxic
compounds (513). Since it can bind LPS, hsp60 could
additionally lower the threshold for triggering re- The plasmalemmal calcium receptor (CaR), identi-
sponses to the PAMP in an infectious setting (680), with fied in 1993 by Brown et al. (91), is a member of a G
this probably being the beneficial facet of its TLR4 protein-coupled receptors of seven-transmembrane do-
interaction. TLR4 binding as well as cotrafficking prop- main topology, which is activated by relatively subtle
erties and roles in the initiation of an immune response (though physiological) changes in extracellular Ca2⫹ con-
have been assigned also to hsp22, hsp70, hsp72, hsp90, centration, and plays a critical role in the regulation of
gp96 (hsp90b1), chlamydial hsp60, and Francisella tu- serum Ca2⫹ homeostasis (92). The activation of CaR,
larensis hsp DnaK (24, 26, 43, 251, 470, 793, 925, 926). depending on cellular context, triggers multiple signaling
Similarly, the high-mobility group box 1 protein cascades, with the most common being InsP3 production
(HMGB1, HMG1, amphoterin) was found to inhibit amy- with subsequent InsP3-induced Ca2⫹ release or regulation
loid ␤ uptake (891) and to amplify inflammatory re- of plasmalemmal channels activity (987, 1020).
sponses in multiple sclerosis and its animal model, exper- The expression of CaR in cultured rat microglial cells
imental autoimmune encephalomyelitis (18). A direct link was demonstrated at both mRNA and protein levels (143).
of neurodegenerative processes in AD to microglial TLR4 The activation of these receptors either by an increase in
became obvious when amyloid ␤ fibrilles were found to [Ca2⫹]o from 0.75 to 3 mM or by CaR agonists (neomycin
bind to CD14, the prominent coreceptor for LPS signaling
or “CaR activator” R-467) increased the open probability
via TLR4 (270). CD14 and TLR-dependent mechanism
of 84 pS Ca2⫹-dependent K⫹ channels (143). The CaR
may promote phagocytotic clearance of A␤-containing
receptor may also be involved in the regulation of IKIR,
plaque material and/or participate in inflammatory re-
because fluctuations in [Ca2⫹]i are known to modulate the
sponses of microglia (270, 494, 751, 890). Pronounced
CD14 immunoreactivity was, indeed, observed for micro- activity of KIR channels in microglia (402). Incidentally,
glia in vicinity to AD lesion sites in sections from AD CaR in neurons may also be activated by ␤-amyloid pep-
brains (532). Importantly, a microglial CD36-TLR4-TLR6 tides (1021), whether this may also be the case for micro-
complex has now been identified to promote sterile in- glial CaRs remains unknown.
flammation in response to amyloid-␤ (864).
Finally, it has to be emphasized that TLR signaling
B. Leukotriene Receptors
can be neuroprotective, not only by driving clearance
of infectious agents, but by organizing CNS-intrinsic as
well as immune system-mediated (T cell-involving) sup- Cultured rat microglia expressed mRNAs for cystei-
port of neural cell survival, tissue preservation, and nyl leukotrienes (CysLTs) receptors of CysLT1 and
CNS functioning (325, 350). The critical question re- CysLT2 types, and stimulation of these cells by CysLT
gards the mechanisms by which TLRs could engage triggered a [Ca2⫹]i rise, which in turn triggered the release
detrimental or beneficial programs. of purines (mostly ATP) and CysLT (32).

TABLE 6. Toll-like receptors in microglia
Experimental
TLR1/2 Detection of mouse, primate Pam3Cys, Pam3CSK4, Evidence for expression, 28, 48, 96, 232, 268,
and human mRNAs in endogenous functional responses to 353, 363, 422,
vitro, ex vivo, protein agonists, chemically defined agonists 450, 677, 728,
detection by FACS, preparations of and microbial agents, 1044
immunoblotting, microbial cell wall regulation of and by other
immunocytochemical structures TLRs
localization in cells in
situ, cellular responses to
stimulation
TLR6/2 Detection of mouse and Peptidoglycan, Evidence for expression, 28, 96, 146, 232,
human mRNAs in vitro, zymosan, functional responses to 253, 268, 353,
ex vivo, protein lipoteichoic acid, chemically defined agonists 363, 422, 450,
detection by FACS, preparations of and microbial agents, 510, 677, 728
immunocytochemical microbial cell wall regulation of and by other
localization in cells in structures TLRs
situ, cellular responses to
stimulation
TLR3 Detection of mouse and Poly(I:C) Evidence for expression, 96, 422, 677, 918
human mRNA in vitro, ex function and regulation or
vivo, protein detection and by other TLRs
by FACS and
immunohistochemistry,
cellular responses to
stimulation
TLR4 Detection of mouse, primate LPS of various Evidence for expression, 49, 96, 232, 253,
and human mRNA in strains, functional responses to 353, 363, 382,
vitro, ex vivo, protein pneumolysin, chemically defined agonists 422, 511, 668,
detection by FACS and various and microbial agents, 677, 728, 739,
immunohistochemistry endogenous regulation of and by other 1044
(also for primate cells), factors TLRs, upregulation by
cellular responses to hypoxia
stimulation
TLR5 Detection of mouse, primate Flagellin, Evidence for expression and 48, 96, 422, 677,
and human mRNA in preparations of regulation by other TLRs, 881, 1044
vitro, ex vivo, protein microbial cell wall functional responses to
detection by structures microbial agents
immunoblotting,
responses of primate cells
to stimulation
TLR7/8 Detection of mouse, primate R848, imiquimod Evidence for expression and 96, 109, 422, 677,
and human mRNAs in (R837), regulation by other TLRs 1044
vitro, ex vivo, protein ssRNALyoVec,
detection by FACS,
responses of mouse and
primate cells to
stimulation
TLR9 Detection of mouse, primate CpG ODNs Evidence for expression, 48, 96, 109, 187,
and human mRNA in function responses by 232, 677
vitro, ex vivo, protein chemically defined agonists,
detection by FACS, regulation of and by other
cellular responses to TLRs
stimulation
CD14 (as a TLR coreceptor) Detection of mouse mRNA, Evidence for expression and 41, 56, 118, 146,
protein detection by functional roles as well as 450, 511, 728
immunoblot or FACS on regulation, e.g., by TLRs
cultured cells, localization
to human cells in situ
Approaches on isolated cells, rather than in vivo studies, are preferentially considered here as they detect mRNA and protein in defined material
and as they demonstrate more directly responses to agonistic compounds.
C. Notch Receptors expression was found in the corpus callosum, which con-
tains the highest concentration of invading microglia
Notch-1 receptors were identified in amoeboid mi- (119). Expression of Notch-1 receptor was downregulated
croglial cells in the early postnatal brain; the maximal with age and can be stimulated by LPS. The notch-1-

TABLE 7. Other receptor systems
Receptor Type Experimental Preparation/Technique Properties and Functional Relevance Reference Nos.
Calcium receptors, CaR Rat/primary cultures/RT-PCR, whole Stimulation of CaR by activators (neomycin 143
cell voltage clamp or a R-467) or by an increase of Ca2⫹o
from 0.75 to 3 mM increased the open
state probability of 84 pS Ca2⫹-activated
K⫹ channel.
Leukotriene receptors, Rat/primary cultures/Ca2⫹ imaging Stimulation of microglia with cysteinyl 32
CysLT1 and CysLT2 leukotrienes triggered [Ca2⫹]i rise.
Notch-1 receptor Rat/corpus callosum/ Notch-1 receptors were identified in 119, 335
immunocytochemistry amoeboid invading microglia in corpus
callosum. Notch-1 receptors are
suggested to regulate production and
secretion of NO and cytokines in
activated microglia.
Complement receptors, Human/mouse/primary cultures/RT- Activation of C5a and C3a receptors 486, 605, 653
C3a, C5a PCR, immunocytohemsitry/Ca2⫹ induced Ca2⫹ signaling via intracellular
imaging Ca2⫹ release. The C5a receptors also
control microglial motility.
Thrombin receptors, Rat, mouse/primary cultures/Ca2⫹ Thrombin triggered Ca2⫹ signals via 31, 602
PAR1,3,4 imaging activation of Ca2⫹ release from the ER.
Macrophage colony-stimulating Mouse/primary culture Activation of M-CSFR stimulates release of 593–595
factor receptors (M-CSFRs) NO and cytokines.
Epidermal growth factor Mouse/primary culture/whole cell Stimulation of microglial cells with EGF 401
receptors (EGFRs) voltage clamp increase the amplitude of IKDR via PTX-
sensitive G protein cascade.
CD200 receptors Rat, mouse/brain sections, primary CD200 receptors downregulate microglial 986a, 1000
culture/RT-PCR activation.
Lysophosphatidic acid Rat, mouse/primary culture/RT-PCR, Mouse microglia expressed LPA1 receptors; 303, 601
receptors, Ca2⫹ imaging rat LPA3 receptors. Exposure of
LPA1, LPA3 microglial cells to LPA triggered [Ca2⫹]i
transients associated with ER Ca2⫹
release.
Formyl peptide receptors, Mouse/primary culture/Ca2⫹ Activation of microglia increases 182, 913
FPR1, FPR2 imaging expression of FPR1/2; activation of
receptors triggers [Ca2⫹]i elevation.
Sigma-1 receptors Human, rat/primary culture/Ca2⫹ Activation of sigma-1 receptors inhibits 312, 346
imaging, migration assays, ATP-mediated Ca2⫹ signals and
pharmacological assays suppresses microglial inflammatory and
migratory responses.
dependent signaling was suggested to be involved in reg- tiated in the inflamed brain, although its expression in
ulation of cytokines and NO production (119). The stim- healthy CNS seems to be minimal (308). Expression of
ulation of Notch receptors in activated microglia de- C5a receptors in spinal cord microglia was significantly
creases NO production and secretion of inflammatory increased after peripheral nerve injury (337).
cytokines (335). The C5a receptors are implicated in the control of
microglial motility. In murine microglial cells in culture,
D. Complement Receptors the application of C5a rapidly (within seconds) induced a
ruffling of microglial membranes, which was followed by
The complement fragments C3a and C5a are anaphy- the extension of lamellipodia and the rearrangement of
lotoxins associated with the activation of complement the actin cytoskeleton (653). The motility reactions were
system, which represents an important part of the immu- independent from [Ca2⫹]i but were blocked by PTX and
nological response (328). Receptors for C5a and C3a frag- cytochalasin B, indicating a critical role for G proteins
ments are classic seven-transmembrane domain G protein- and cytoskeleton (653). Both C5a and C3a receptors trig-
coupled metabotropic receptors linked to several intracellu- gered Ca2⫹ signals in cultured mouse microglia (605);
lar signaling cascades, including PLC/InsP3-dependent these signals demonstrated biphasic kinetics, which rep-
pathway (315, 383). resented an initial InsP3-induced Ca2⫹ release (sensitive
C5a receptors were identified at both transcriptional to thapsigargin) from the ER store and the subsequent
and protein levels in cultured human microglia (486). The store-operated Ca2⫹ entry (sensitive to Ca2⫹ removal
expression of complement C5a receptors is greatly poten- from extracellular solution). The action of both receptors

involved PTX-sensitive G proteins (605). Microglial C5a equivalent of GRO␣ (CXCL1), monocyte chemoattractant
receptors are also coupled to K⫹ channels. The stimula- protein 1 (MCP-1, CCL2), macrophage inflammatory pro-
tion of C5a receptors triggered rapid (in ⬃20 s) and tein 1␣ and 1␤ (MIP-1␣/-1␤, CCL3/CCL4) or “regulated
transient upregulation of K⫹ conductance in primary cul- upon activation, normal T-cell expressed and secreted”
tured mouse microglia (401) that was blocked after incu- (RANTES, CCL5) (452, 602, 779, 878). Thrombin triggers
bation with 1 ␮g/ml PTX for 30 min prior to the recording. Ca2⫹ signals in microglial cells; these Ca2⫹ responses
The complement-induced stimulation of K⫹ currents, have PAR-typical features (fast kinetics, sensitivity to
however, was not linked to regulation of microglial mo- hirudin, and use-dependent destruction), suggesting func-
tility (401). tional expression of the respective receptors. All four
The C5a receptors participate in microglial-related PARs were also found in rodent microglia at both mRNA
pathogenesis of neuropathic pain. The complement frag- and protein levels (31, 602). Toxic consequences of
ment C5a applied intrathecally induced cold pain, thrombin administration in vivo and association with CNS
whereas an injection of C5a antagonist (synthetic cyclic lesions support the concept that the protease may ac-
AcF-[OPdChaWR] peptide) reduced cold allodynia in an- count for neuronal impairment and that microglial medi-
imals with peripheral nerve injury (337). ators could play a critical role (126, 224, 225, 317, 318, 348,
C1q, the recognition subcomponent of the classical 507, 675, 874, 1012). The Par-1 receptors activation was
complement activation pathway, in the CNS is produced suggested to be involved in microglial inflammatory re-
in rat brain microglia (but not in astrocytes or neurons) sponse in Parkinson’s disease. However, not all of the
within 24 h after an ischemic insult (798). C1q acts in an microglial activities assigned to thrombin may actually
autocrine fashion by triggering the release of IL-6, TNF-␣, relate to the proteolytic activation of PARs (354, 606, 989).
and NO as well as by inducing the oxidative burst in rat
primary microglial cells. These data indicate that 1) ex- F. Macrophage Colony-Stimulating
trinsic plasma C1q is involved in the initiation of micro- Factor Receptors
glial activation in the course of CNS diseases with blood-
brain barrier impairment and 2) C1q synthesized and The M-CSF is a hematopoietic cytokine, which is
released by activated microglia is likely to contribute in present in both neurons and glia. The corresponding re-
an autocrine/paracrine way to maintain and balance mi- ceptor, the M-CSF receptor (M-CSFR; encoded by proto-
croglial activation in the diseased CNS tissue (262). oncogene c-fms), is expressed in activated microglial
cells surrounding neuritic plaques in AD and also in mi-
E. Thrombin Receptors croglia after traumatic or ischemic brain insults (593–
595). The activation of M-CSFR stimulates release of NO
and proinflammatory cytokines and also promotes phago-
Thrombin (factor IIa) is best known for cleaving cytosis and the uptake of A␤ (593–595). Overexpression
fibrinogen as a central event in the coagulation cascade of M-CSFR in microglia had a significant neuroprotective
(495). Thrombin is suspected to cause severe damage to effect in organotypic hippocampal slices subjected to ex-
neural cells (318, 646, 977) and to act as a link between citotoxic stress (592).
injury, hemostasis, and inflammation (174, 646, 877).
Upon blood-brain barrier disruption, plasma components
can develop a significant impact on glial and neuronal G. Epidermal Growth Factor Receptors
cells (317, 318, 969).
Search for the direct molecular target of thrombin on Epidermal growth factor receptors (EGFRs) belong
the surface of platelets and other cells resulted in the to the ErbB family of receptor protein kinases (97). The
discovery of the proteinase-activated receptors (PARs). functional expression of these receptors was demon-
The protease cleaves the extracellular portion of a PAR strated in cultured mouse microglial cells; the exposure of
(832). The newly formed NH2-terminal sequence stretch these cells to EGF resulted in rapid increase in the am-
subsequently acts as an intramolecular (“tethered”) ligand plitude of K⫹ conductance (401). The EGF-induced mod-
that binds to an extracellular receptor domain to cause ulation of K⫹ channels was mediated through PTX-sensi-
cytosolic events, largely through G proteins (679, 857). tive Gi proteins; similar signaling cascades activated by
Four PARs have been identified thus far. Thrombin was EGF were reported for fibroblasts (181) and hepatocytes
shown to act on PAR1, PAR3, and PAR4, whereas PAR2 (567).
accepts trypsin or proteases with trypsin-like substrate
preference (85, 164, 173, 665). H. CD200 Receptors
Microglial cells respond to thrombin preparations
with the release of NO, various cytokines, and chemo- CD200 receptors (CD200Rs), activated by surface
kines, including TNF-␣, IL, IL-12(p40), KC, the mouse glycoprotein CD200 (or OX2), are expressed in various

types of myeloid cells and are involved in the regulation of embryonic human (312) and neonatal rat (346) brains.
phagocytic activity (1000). The CD200Rs were also iden- Activation of sigma-1 receptors suppresses ATP-induced
tified in rodent microglial cells and are generally involved Ca2⫹ signaling, membrane ruffling, as well as inflamma-
in downregulation of microglial activation (986a). tory and migratory responses in cultured microglial cells
(346).
I. Lysophosphatidic Acid Receptors
XV. MICROGLIAL PLASMALEMMAL
The actions of signaling phospholipids lysophospha- TRANSPORTERS
tidic acid (LPA), sphingosine-1-phosphate (S1P), and sph-
ingosylphosphorylcholine (SPC) are mediated through an
A. Xc Transporters
extended family of G protein-coupled receptors (158, 171,
693). The LPA receptors include three molecularly dis-
tinct types, LPA1/Edg2, LPA2/Edg4, and LPA3/Edg7. Rat Microglia, activated by various stimuli [e.g., by culture
primary cultured microglia expressed LPA3 mRNA recep- conditions (718), or by secreted amyloid precursor protein
tors, whereas cultured mouse cells expressed mRNA spe- (35) as well as by aggregated ␤-amyloid protein1– 40 (740), or
cific for LPA1 receptor (303, 601). Treatment of both by LPS (36)], release substantial amounts of glutamate,
cultures with LPA resulted in [Ca2⫹]i elevation, which was which may contribute to the excitotoxic damage of the
mostly produced by Ca2⫹ influx in rat microglia, whereas brain. Microglial release of glutamate occurs almost ex-
in mouse microglia, the dominant pathway was associ- clusively through glutamate-cystine antiporter Xc, which
ated with intracellular Ca2⫹ release (600, 601). Activation exchanges glutamate for cystine according to the respec-
of LPA3 receptors in rat cultured microglia also caused tive concentration gradients; as a consequence, removal
the release of ATP; the latter in turn induced membrane of extracellular cystine completely blocks microglial glu-
ruffling via activation of P2Y12 receptors (303). tamate release (35). The abnormal activation of Xc anti-
porter in reactive microglia may be linked to massive
demand for cystine/cysteine for replenishing gluthatione,
J. Formyl Peptide Receptors the latter being driven down by oxidative bursts accom-
panying microglial activation and brain insults (34, 36).
There are two subtypes of receptors to bacterial The release of glutamate from microglia via Xc trans-
N-formylpeptides: the high-affinity receptors FPR1 and porter was implicated in oligodendrocytes damage and
the low-affinity receptor FPR2 (500, 600). Both receptors death; the activation of microglia by LPS triggered prom-
belong to GPRC class, and the expression of both (albeit inent oligodendrocytes extinction in the whole-mount rat
at low levels) was found in primary mouse microglia optic nerve. This death was prevented by inhibition of Xc
(182). Treatment of microglial cells with LPS or A␤ mark- transporter or blockade of AMPA/KA receptors (222).
edly upregulated expression of FRP1/FRP2; in the stimu-
lated cells, activation of formyl peptide receptors trig-
B. Glutamate Transporters
gered [Ca2⫹]i elevation (182). The FPR2 expression is also
significantly upregulated by the exposure of primary mi-
croglial cultures to TNF-␣ and IL-10 (418). This was cor- The overall involvement of microglial glutamate
related with an increased chemotaxis to A␤1– 42 and fur- transport in glutamate homeostasis in the brain is rela-
ther FPR2-mediated endocytosis of amyloid ␤-protein tively minor as it accounts for ⬍10% of astroglial gluta-
(418). mate uptake (710). Baseline expression of GLT-1 and
The FPRs may also act as receptors to prion protein GLAST in naive animals is primarily localized in astro-
peptides (501). The microglial cells respond to PrP106 –126 cytes (824, 1010). At the same time, insults to the CNS
by generation of slow [Ca2⫹]i elevation (372, 836), which may upregulate microglial glutamate transporters, thus
was suggested to involve voltage-gated Ca2⫹ channels increasing their ability to accumulate glutamate in the
(see sect. VIII and Ref. 836), yet the role of FPRs has to regions of injury. In post mortem human tissue, microglia
be explored further. activated following stroke expressed EAAT-1/GLAST (as
determined by immunohystochemistry) during the first
week after the insult; this expression was detected in both
K. Sigma Receptors ramified and amoeboid microglial cells, and was down-
regulated at later stages (57). The expression of EAAT-1/
Sigma receptors of two subtypes, sigma-1 and sigma-2, are GLAST transporters was limited to microglia, as periph-
abundantly expressed in the brain; they are regulating eral macrophages did not have the glutamate transporter.
Ca2⫹ release from the ER (364). Sigma-1 receptors were Similarly, the expression of the EAAT-1/GLAST transport-
identified in primary microglial cultures prepared from ers was transiently upregulated in microglial cells at early

activated stages following traumatic brain injury in hu- inward current (456). Since glutamate release can be
mans, which may indicate their neuroprotective potential monitored electrophysiologically, a possible modulatory
(55). A rapid de novo expression of both GLAST and effect of a synthetic 11-amino-acid analog of amyloid-␤
GLT-1 transporters was identified in rat microglia follow- peptide (A␤25–35) on electrogenic glutamate transport in
ing controlled cortical impact injury; the expression starts primary cultured rat microglia was investigated in vitro
at 4 h and reaches the steady-state in 48 h after the injury (651). It was found that A␤ enhanced glutamate release
(948). The EAAT-1 transporters were also found in micro- from primary cultured rat microglia via the Na⫹-depen-
glial cells of HIV-1-positive patients (945). dent glutamate transporter, which was also activated by
An increase in EAAT-1 expression was also observed extracellular K⫹.
in microglial cells in samples from the cerebral cortex,
striatum, thalamus, and cerebellum from 14 patients with
C. Glucose Transporters
prion disease (Creutzfeldt-Jakob disease and fatal familial
insomnia), which may indicate a certain neuroprotective
adaptation of microglia in chronic prion infections (155). The exclusive expression of glucose transporter 5
The GLT-1/EAAT-2 receptors were found in activated (GLUT5) was demonstrated in microglial cells from sec-
microglia in cultures and in situ in facial nuclei of adult tions of human and rat brains (697). Expression of these
rats, which underwent unilateral facial nerve axotomy transporters was even suggested to be a specific marker
(533). Similarly, EAAT-2/GLT-1 (but not EAAT-1/GLAST) of microglial cells in situ (791). The primary isoforms in
transporters were identified in primary cultured rat mi- brain are GLUT1, detected at high concentrations as a
croglial cells, which also demonstrated the [14C]glutamate highly glycosylated form (55 kDa) in blood-brain barrier,
uptake (627), which was blocked by dihydrokainate. Mi- and also as a less glycosylated, 45-kDa form, present in
croglial [14C]glutamate uptake was stimulated by incuba- parenchyma, predominantly in the glia; GLUT3 in neu-
tion of cultured microglia with a neuronal conditioned rons; and GLUT5 in microglia (952). It was also shown
medium, suggesting some degree of neuronal control over that the 45-kDa form of glucose transporter 1 (GLUT1) is
glial glutamate transporters (628). Both EAAT-1/GLAST localized in oligodendrocytes and astrocytes but not in
and EAAT-2/GLT-1 were detected in rat cultured spinal microglia in the rat brain (1023).
microglia, and their activity was inhibited by activation of
P2X7 receptors (619). In brain autopsy tissue specimens D. Monocarboxylate Transporters
of HIV-1-infected patients, the expression of EAAT-2 by
activated microglia suggests a compensatory effect that An extended family (14 members) of monocarboxy-
protects neurons from glutamate neurotoxicity (1011). late transporters (MCTs) are represented by proton-de-
The glutamate uptake systems in microglial cells can pendent transporters, which provide for transmembrane
also have a self-defense function. The GLT-1 transporter transport of various energy substrates including lactate
is upregulated in microglial cells exposed to the Herpes (345). Three members of the MCT family, the MCT1,
simplex virus of both HSV-1 (encephalitis) and HSV-2 MCT2, and MCT4, are found in the central nervous sys-
(meningitis) types (708). Microglial cells also demon- tem: the MCT1 in endothelial cells and in astrocytes,
strated a higher resistance to HSV viruses compared with MCT2 in neurons, and MCT4 in astroglia (720). Microglia
neurons and astrocytes (708). This higher resistance can activated following compression-induced ischemia dem-
result from upregulated GLT-1 transporters, which in turn onstrated significant expression of MCT1 and MCT2, with
are instrumental in increasing microglial glutathione con- the latter possibly involved in supply of microglial cells
tent, as the microglial glutamate uptake was shown to be with energy substrates (609).
directly coupled to glutathione synthesis (710). The com-
plement-derived anaphylatoxin C5a also increases micro-
glial GLT-1 expression and glutamate uptake in a TNF-␣- E. ATP-Binding Cassette Transporters
independent manner (709).
The main functional role of the glutamate transport- The ATP-binding cassette (ABC) transporters belong
ers, especially in glial cells, is to maintain low glutamate to an extended superfamily, which comprises seven sub-
concentration in the extracellular space of the CNS (188, types (designated as ABCA to ABCG transporters; Ref.
773). However, the reverse glutamate transporter has 427). The expression of ABC transporters was reported in
been suggested as the mechanism of the neurotoxic rise cultured rat microglia, where they can be involved in the
in extracellular glutamate concentration during brain an- release of purines and cysteinyl leukotrienes (32). An
oxia (524). Transport of glutamate is driven by transmem- expression of ABCA1 transporters was found to be up-
brane ion gradients, and translocation of a single gluta- regulated in cultured microglia treated with mutant
mate molecule requires influx of three Na⫹ and one H⫹ (Swedish) amyloid precursor protein (468). High levels of
coupled with efflux of one K⫹ (1028), thus producing a net expression of ABC transporter G4 (ABCG4) protein was

found in microglial cells associated with neuritic plaques XVI. MIGRATION AND MOTILITY
in the AD human brains (939). The ABCG4 is responsible
for cholesterol transport and may be involved in AD- Cell migration plays a central role in many physio-
associated alterations of lipid metabolism. logical and pathophysiological processes, including im-
mune defense and wound healing. Microglial cells exhibit
F. Chloride Transporters two types of movement activity: in the ramified form, they
actively move their processes without translocation of the
Chloride channels and transporters were character- cell body. In the amoeboid form, they also move their
ized by whole cell recording and RT-PCR (1040). The processes, but in addition the entire cell can migrate
repertoire of chloride-conducting pathways in murine pri- within brain tissue including translocation of their soma.
mary microglial cells includes the K-Cl cotransporters, Migration either occurs during development, when cells
KCC1, KCC2, KCC3, and KCC4, as well as swelling-acti- of monocytic origin migrate into the brain or after a
vated chloride channels. KCl cotransporters induce K⫹ pathological insult when ramified microglia transform
and Cl⫺ efflux when swelling-activated chloride channels into the amoeboid form and migrate to the site of injury.
are activated. Most studies were performed in cell cultures from post-
natal mice and rats. These cells might therefore resemble
the amoeboid microglia early in postnatal development. It
G. Bicarbonate Transporters is not yet explored whether these forms of motility, mi-
gration, and process movement are distinctly regulated.
The presence of operational Na⫹/HCO⫺ 3 cotrans- Moreover, there is also not a systematic study comparing
porter and/or Na⫹-dependent Cl⫺/HCO⫺ 3 exchanger was the migratory behavior of microglia during development
found in cultured microglia (260). Anion exchange-medi- and in response to pathology. There are many candidate
ated chloride/bicarbonate transporter is a major compo- molecules that serve as signals for pathological events to
nent in the regulation of intracellular pH. The functional microglia including ATP (190, 384), cannabinoids (973),
consequences of changes in anion exchange structure morphine (892), the chemokine CCL21 (747), lysophos-
may range from acidosis, disturbance of cytoskeleton phatidic acid (806), and bradykinin (397). In addition, ion
integrity, and untimely or impaired recognition of cells by channels and transporters play an important role in cell
components of the immune system, such as microglia. migration (Fig. 13), such as K⫹ channels, Cl⫺ channels,
Microglial cells express a distinct set of pH regulatory Na⫹/H⫹ exchanger, Cl⫺/HCO⫺ 3 exchanger, and Na⫹/
carriers that control for a defined level of intracellular ⫺
HCO3 cotransporter, which all are linked to actin cyto-
pH (80). skeleton (813, 814). Although the roles of these mecha-
nisms in microglial migration were not analyzed in detail,
H. Hⴙ/Kⴙ pump there is evidence for the contribution of Cl⫺ and K⫹
channels, as well as the Na⫹/Ca2⫹ exchanger and Ca2⫹-
When monitoring K⫹ concentration in cultured rat permeable stretch-activated cation channels (397).
microglia with ion-selective microelectrodes, Shirihai et
al. (831) found that the H⫹-K⫹-ATPase plays the leading A. Control of Processes Motility In Vivo
role in regulation of potassium and proton gradients. The
Na⫹-K⫹-ATPase, in contrast, was not functionally active.
Microglial processes are not static, but rapidly move
The KD for microglial H⫹/K⫹ pump was ⬃3.7 mM, thus
within the brain parenchyma as shown by real-time imag-
being in the physiological range of extracellular K⫹ con-
ing using two-photon laser microscopy. Process move-
centrations (831).
ments are highly random in direction. At rest, however,
these process movements do not result in overt cellular
I. Proton Pump migration (190, 645). Processes rapidly move towards a
lesion as experimentally triggered by a laser. This rapid
The proton pump was suggested to participate in chemotactic response can be mimicked by local injection
volume regulation in microglia, macrophages, and T lym- of ATP and can be inhibited by the ATP-hydrolyzing en-
phocytes (538). Proton pump inhibitors possess anti-in- zyme apyrase or by blockers of G protein-coupled puri-
flammatory effects and can decrease human microglial nergic receptors and connexin channels, which are highly
and monocytic neurotoxicity. In addition, proton pump expressed in astrocytes. It was speculated that ATP acts
inhibition combined with nonsteroidal anti-inflammatory first on astrocytes, which then release ATP and trigger the
drugs may be effective in the treatment of a broad spec- microglial response (190). The responsible receptor sub-
trum of neurodegenerative diseases associated with acti- type is P2Y12 (see sect. X). Microglia in mice deficient for
vation of microglia (361). P2Y12 show significantly diminished directional branch

FIG. 13. Model summarizing the role

of ion channels and transporters in con-
trolling microglial migration. The cytoso-
lic Ca2⫹ signals induced by activation of
metabotropic receptors and InsP3 cas-
cade and/or by Ca2⫹ entry through iono-
tropic receptors or reverse mode of Na⫹/
Ca2⫹ exchanger induces the retraction of
the rear part of a migrating cell, which is
paralleled by massive K⫹ efflux via Ca2⫹-
dependent K⫹ channels and shrinkage of
the cell at the rear (retraction site).
Transporters such as Na⫹/H⫹ and Cl⫺/
HCO⫺ 3 exchangers at the front of migrat-
ing cells (protrusion site) are reported to
contribute to the extension of the actin
projection (lamellipodium) by mediating
salt and osmotically obliged water up-
take. [Based on Schwab (814).]
extension toward sites of damage. Moreover, P2Y12 ex- P2Y12 receptors control both chemotaxis of amoeboid
pression is robust in the “resting” state, but reduced after microglia and process motility of ramified microglia as
microglial activation. described above. P2Y12 receptor-mediated activation of
the PI3K pathway and increased Akt phosphorylation are
B. Control of Microglial Migration required for microglial chemotaxis in response to ATP
(419). Akt phosphorylation was reduced upon chelation
Under pathological conditions such as lesion, stroke, of extracellular calcium, suggesting that ionotropic P2X
neurodegenerative disorders, and tumor invasion, acti- receptors are also involved in microglial chemotaxis act-
vated microglia migrate to the site of injury. Using time- ing through the PI3K pathway. Pharmacological blockade
lapse confocal microscopy in acutely isolated living slices or knockdown of the P2X4 receptor in microglia by RNA
from adult brain-injured mice, extensive migration of peri- interference suppressed the microglial chemotaxis, sug-
lesional microglia was apparent by 24 h after injury and gesting that P2X4 as well as P2Y12 receptors are involved
peaked at 3 days with an average migration speed of ⬃5 in ATP-induced microglial chemotaxis (673). As for ADP-
␮m/min and peak speeds ⬎10 ␮m/min. This was not a induced chemotaxis of microglia, the role for P2Y12/13
collective, directed migration towards the lesion edge as receptors and the ␤-integrin was reported (633). Activa-
might be expected in the presence of chemoattractive tion of purinergic receptors triggers a K⫹ outward con-
gradients, but rather a random walk migration (121, 122). ductance (82). The induction of P2Y-associated outward
In the ex vivo retina, focal laser injury leads to a transition potassium current in microglia is required for the ATP-
of microglia from a fixed to a migratory phenotype while induced chemotactic response and baseline motility
retaining their ramified morphology (505). There are a (1004).
number of receptor systems that control the migration of The CD39 and CD73 are the dominant cellular ecto-
microglial cells. Most studies were performed in culture nucleotidases of microglial cells that degrade nucleotides
using migration assays based on twin chambers separated to nucleosides, including adenosine. These ectonucleoti-
by a membrane. It is important also to distinguish be- dases play an important role in the ATP-induced che-
tween random migration and chemotaxis, i.e., directed motaxis. ATP failed to stimulate P2 receptor-mediated
migration along a chemical gradient. migration in CD39-deficient microglia. However, the ef-
fects of ATP on migration in CD39-deficient microglia
1. Neurotransmitters were restored by costimulation with adenosine or by
ATP had been reported to be a chemoattractant for addition of a soluble ectonucleotidase. This indicates that
microglial cells (384, 419, 673). It was reported that ATP- costimulation of purinergic and adenosine receptors is a
induced microglial migration was inhibited by PTX, sug- requirement for microglial migration and that the expres-
gesting that activation of Gi/o proteins induced by P2Y sion of CD39 controls the ATP/adenosine balance. CD39
receptors is part of the intracellular signaling cascade deletion has also an impact in a pathological context,
controlling microglial migration (384). Extracellular ATP since the accumulation of microglia/macrophages in a
induces membrane ruffling and chemotaxis of microglia model of ischemia was markedly decreased in CD39-
mediated by the Gi/o protein-coupled P2Y12 receptor. Thus deficient animals (266).

As described above, microglial cells express AMPA- 3. Cannabinoids and related compounds
type and metabotropic glutamate receptors. It was re-
Microglial cannabinoid receptor of CB2 type and ab-
cently demonstrated that glutamate triggers microglial
normal cannabidiol-sensitive receptors stimulate micro-
membrane ruffling and migration to a source of glutamate
glial migration (973). Cannabinoid stimulation triggers the
in cell culture and in spinal cord slices. This chemotaxis
activation of the extracellular signal-regulated kinase 1/2
event is meditated by both AMPA and metabotropic glu-
signal transduction pathway. Cannabinoids are released
tamate receptors (529). Dopamine and epinephrine also
in a pathological condition, since excessive stimulation of
enhanced migratory activity (267).
neurons by glutamate together with carbachol dramati-
cally increases the production of the endocannabinoid
2. Chemokines 2-arachidonylglycerol. Similarly, pathological stimulation
of microglial cells with ATP also increases the production
As mentioned previously, chemokines are expressed
of 2-arachidonylglycerol (973). In LPS-stimulated primary
by neurons and macroglia as well as microglia and, when
cultured microglial cells, a CB2 agonist inhibits microglial
released after cellular damage, may act as major chemoat-
migration (771).
tractants for microglia. Damaged neurons express the
Focal cerebral ischemia induces rapid neuronal
chemokine CCL21, which acts as a chemotactic substance
death in the ischemic core, which gradually expands to-
for microglial cells (68). A local application of CCL21 for
ward the penumbra, partly as the result of a neuroinflam-
30 s to microglial cells in culture and in brain slices
matory response. It is known that propagation of neuro-
triggered a Cl⫺ conductance with lasted for tens of min-
inflammation involves migration of microglial cells. En-
utes, and the chemotaxis response was sensitive to Cl⫺
docannabinoids are an important factor that increased
channel blockers. The response is mediated by CXCR3
microglial cell motility after the ischemic insult (296). It
receptors as revealed by studies involving CXCR3 and
was found that in mouse cerebral cortex focal ischemia
CCR7 receptor-deficient animals. Therefore, the CCL21-
greatly increases palmitoylethanolamide (PEA), moder-
induced Cl⫺ current is a prerequisite for the chemotaxis
ately increases anandamide (arachidonylethanolamide,
response mediated by the activation of CXCR3 but not
AEA), and does not affect the level of 2-arachidonylglyc-
CCR7 receptors, indicating that in brain CCL21 acts via a
erol. It was further demonstrated that PEA potentiates
different receptor system than in lymphoid organs (747).
AEA-induced microglial cell migration, without affecting
Fractalkine or CX3CL1 is released by both neurons
other parameters of microglial activation, such as prolif-
and astrocytes, while the CX3CL1 receptor (CX3CR1) is
eration, particle engulfment, and NO production. This
predominantly expressed in microglial cells. Both the
potentiation of microglial cell migration by PEA involves
movement of processes under resting conditions and in
reduction in cAMP levels. In line with this, evidence was
response to tissue injury as well as the migration of
provided that PEA acts through Gi/o-coupled receptors.
microglia in response to injury was slowed down in
Interestingly, the receptors engaged by PEA are pharma-
CX3CR1-deficient retinal explants (525).
cologically distinct from CB1 and CB2 cannabinoid recep-
Microglial migration can be induced by stromal cell-
tors, as well as from the WIN and abn-CBD (abnormal-
derived factor-1␣ (SDF-1␣), acting through CXC chemo-
cannabidiol) receptors. The results show that PEA and
kine receptor 4 (CXCR4). Migration triggered by CXCR4-
AEA increase after focal cerebral ischemia and synergis-
specific ligands is accelerated by hypoxia. This effect is
tically enhance microglial cell motility. Because such a
due to the activation of hypoxia inducible factor-1␣ (HIF-
response could participate in the propagation of the focal
1␣) and PI3K/Akt signaling pathway, which leads to the
cerebral ischemia-induced neuroinflammation within the
enhancement of CXCR4 expression (986). Pharmacologi-
CNS, and because PEA is likely to act through its own
cal blockade of the cysteine-cysteine chemokine receptor
receptor, a better understanding of the receptor engaged
5 reduced migration velocity in acutely isolated living
by PEA may help the search for improved therapies
slices from adult brain-injured mice (121). MCP-1, a mem-
against neuroinflammation.
ber of ␤-chemokine subfamily, regulates the migration of
microglia, monocytes, and lymphocytes to the inflamma-
4. Lysophosphatidic acid
tory site in the CNS (839). Following a hypoxic insult,
amoeboid microglial cells in the neonatal rats increase Lysophosphatidic acid (LPA) enhances chemokinetic
MCP-1 production via NF-␬B signaling pathway. This in- migration of murine microglial cells. In the presence of 1
duces further migration and accumulation of amoeboid ␮M LPA, the mean migration rate of microglial cells was
microglial cells from the neighboring areas to the periven- increased 3.8-fold. In patch-clamp studies, LPA induces
tricular white matter. Amoeboid microglial cells are the activation of a Ca2⫹-activated K⫹ current. The LPA-stim-
main source of MCP-1, and this may offer an explanation ulated migration of microglial cells was inhibited by spe-
for the periventricular white matter being susceptible to cific inhibitor of IKCa1 Ca2⫹-activated K⫹ channels charyb-
hypoxic damage in neonatal brain (217). dotoxin, which decreased the migration rate by ⬃60%.

Therefore, it was concluded that IKCa1 Ca2⫹-activated K⫹ (0.2 ng/ml). It was concluded that both NGF and TGF-␤
channels are required for LPA-stimulated migration of contribute to microglial recruitment (199).
microglial cells (806). Hepatocyte growth factor-like protein (HLP)/macro-
phage stimulating protein (MSP), which was reported to
5. Morphine be involved in the regulation of peripheral macrophage
activation, promoted microglial migration without affect-
Morphine, in a relatively high concentration (1 ␮M), ing cell survival and proliferation. Ron, the receptor for
induced morphological changes and ruffling in cultured HLP, is expressed in primary microglia, and HLP greatly
microglia and triggered chemotaxis as assayed by Boyden increased the mRNA levels of inflammatory cytokines,
chamber analysis (892). Addition of morphine to micro- including IL-6 and GM-CSF, and iNOS (885).
glia stimulated gene expression of BDNF as early as in 1
h, which persisted for ⬎12 h; in parallel, ERK1/2 (extra- 8. ␤-Amyloid
cellular signal-regulated kinase 1/2) phosphorylation was
It is well established that microglial cells accumulate
also induced. Activation of Gi/o, PI3-K gamma, and Rac
at senile plaques in AD (370, 767). The pattern recognition
are involved in chemotaxis, whereas indirect pathways
receptor CD36 initiates a signaling cascade that promotes
through ERK1/2 phosphorylation induced by unknown
microglial activation and chemotaxis to ␤-amyloid depos-
growth factors generated through a metalloprotease acti-
its in the brain (875). Downstream of CD36 are the focal
vation are linked to the enhanced BDNF gene expression
adhesion-associated proteins p130Cas, Pyk2, and paxillin
(892).
representing novel members of the tyrosine kinase signal-
Morphine increased microglial migration via a spe-
ing pathway; assembly of this complex is essential for
cific interaction between ␮-opioid and P2X4 receptors
microglial migration (875). Chemokines may be involved
mediated through PI3-K/Akt pathway activation (388).
in the recruitment of microglia to senile plaques, because
The action of morphine is accomplished in two phases.
neutralizating antibodies against chemokines significantly
The initial phase develops in minutes, involves PI3-K/Akt
attenuated A␤-induced microglial clustering and the en-
pathway activation, and leads to acutely enhanced migra-
largement of A␤ aggregates (391). A recent study, how-
tion. The longer-term phase occurs within hours and in-
ever, did not find that the presence of microglia in a
volves increased expression of Iba1 and P2X4 receptors
mouse model of AD has an impact on plaque load (336).
that facilitates a promigratory phenotype (388).
6. Bradykinin C. Inhibitors of Microglial Migration
Bradykinin can attract microglia to a lesion or inflam- 1. LPS/IFN-␥

mation sites where bradykinin is upregulated. This in-
Normally, cultured microglia are nonmigratory and
volves inducible bradykinin receptors of B1 type. Brady-
have amoeboid cell morphology, no polarity, many short
kinin-induced microglial migration is PTX insensitive,
processes that extend into lamellipodia in opposing direc-
suggesting that the intracellular signaling is Gi/o indepen-
tions, and undulating cell membrane projections. After
dent and distinct from ATP-induced microglial migration
addition of LPS, some cells acquire polarity by forming a
or chemotaxis (397). Downstream signaling cascade in-
large lamellipodium and begin to migrate, although this
cludes activation of PI3-K and PKC, followed by subse-
migration ceases in 2 h (6). Proinflammatory stimulation
quent phosphorylation and activation of NCX in reverse
with LPS/IFN-␥ induces an IL-10-mediated downregula-
mode as well as activation of KCa channels. From phar-
tion of cell surface antigen expression and loss of migra-
macological study, intermediate-conductance Ca2⫹-de-
tory and phagocytic activity (88). In retinal explants, mi-
pendent K⫹ channels are supposed to be activated (397);
croglia readily migrate and are negative for MHC class II,
these channels are distinct from those activated by pu-
and iNOS, while producing IL-12. In response to LPS/
rines.
IFN-␥, microglia produce IL-10, which inhibits both their
migration and activation (129).
7. Growth factors
2. Minocycline
NGF induced chemotaxis of microglial cells through
the activation of TrkA receptor. In addition, the NGF- Minocycline, a semisynthetic tetracycline derivative
induced chemotactic activity was increased in the pres- with anti-inflammatory and neuroprotective effects unre-
ence of low concentrations (0.2 ng/ml) of TGF-␤, which at lated to its antimicrobial action, significantly reduced mi-
this concentration is also chemotactic per se. On the croglial migration to cellular debris, astrocyte-condi-
contrary, NGF-induced microglial migration was reduced tioned medium, ADP, and algesic mediators and inhibited
in the presence of chemokinetic concentration of TGF-␤ the expression of CD29 (␤1-integrin) but not CD18 (␤2-

integrin). Minocycline reduced the effect of extracellular went caspase-3-mediated cell death. In the absence of
potassium and later decreased microglial Kv1.3 expres- microglia, these neurons can survive, indicating that ap-
sion (664). optotic cells deliver an “eat me” signal to the microglial
cells (550). Microglial cells are also considered to be
3. Wogonin involved in synapse removal during development (863)
and potentially in pruning synapses in the postnatal brain.
Microglial migration toward MCP-1 was inhibited by
The role of microglia phagocytosis in neurodegenera-
wogonin, an active component from the root of Scutel-
tion is established in many experimental paradigms. For
laria baicalensis Georgi and had neuroprotective effect,
example, microglial cells are instrumental for removing
via suppression of NF-␬B activity (719).
the dendritic trees of the parvalbumin-positive interneu-
rons in the dentate gyrus following entorhinal cortex
4. Dexamethasone
lesion (746). In response to the lesion, microglial cells
MCP-1 production is suppressed by dexamethasone, accumulate at the molecular layer in the dentate gyrus,
an anti-inflammatory and immunosuppressive drug, re- the location of the synaptic contacts of entorhinal fibers
sulting in the inhibition of subsequent microglial cell mi- and dendrites of the interneurons. This accumulation is
gration to the inflammatory site by regulating MKP-1 ex- mediated by signaling through CXCR3 receptors. When
pression and the p38 and JNK MAPK pathways (1037). microglial attraction to the lesion site in the molecular
layer is impaired by deletion of the chemokine receptor
CXCR3, the dendritic trees of the interneurons are pre-
D. Microglial Residence in the Brain
served. Thus microglial cells also recognize signals which
tell them to remove parts of a cell.
Exogenous microglia enter the brain and migrate into
In addition to cells and parts of cells, microglia are
ischemic hippocampal lesions (403– 405, 882). The entry
also known to phagocytose molecules and debris such as
of intra-arterially injected microglia and macrophages
myelin or amyloid deposits. There are several studies that
into the brain using a rat muscle graft model was tested to
have established that ␤-amyloid is taken up by microglia
compare their respective abilities to invade the brain
in culture. For example, fibrous A␤ in vitro induces
parenchyma. Isolated microglia without any activation
phagocytosis through a recently characterized A␤ cell
treatment entered into the brain with or without the mus-
surface receptor complex comprising the B-class scaven-
cle graft, while macrophages activated by phorbol 12-
ger receptor CD36, ␣6␤1 integrin, and CD47 (integrin-
myristate 13-acetate entered the brain only in the pres-
associated protein), this complex therefore being distinct
ence of the muscle graft, suggesting that microglia have a
from that used by the classical phagocytic receptors, the
higher affinity for the brain than macrophages (403). Mi-
immunoglobulin, or complement receptors (465). In con-
croglia isolated from a mixed glial culture drawn from
trast, soluble A␤ is taken up by fluid-phase macropinocy-
neonatal Mongolian gerbils produced high amounts of
tosis, a mechanism distinct from phagocytosis and recep-
BDNF and glial cell line-derived neurotrophic factor
tor-mediated endocytosis (548). Recently, the role of sig-
(GDNF). The gerbil microglia retained the capability to
nal regulatory protein-beta1 (SIRP␤1) in regulation of A␤
migrate into the brain parenchyma after intra-arterial in-
phagocytosis was identified in microglial cells isolated
jection. After invasion, microglia retained their BDNF and
from amyloid precursor protein J20 transgenic mice and
GDNF productive ability and expressed large amounts of
in AD patients (305).
BDNF and GDNF in damaged brain areas, which suggests
While microbes and related particles are recognized
that microglia protect damaged neurons (882). In addi-
by the family of Toll-like receptors, the apoptotic neurons
tion, GDNF was reported to activate transmembrane re-
are recognized by different receptor systems. These in-
ceptor tyrosine kinase Ret in microglial cells in CA1/CA3
clude asialoglycoprotein-like-, vitronectin-, and phospha-
hippocampal slices (78).
tidylserine-mediated receptors (995).
There are multiple factors, which can regulate the
XVII. PHAGOCYTOSIS phagocytic activity. The intracellular chloride channel
(ClIC1) seems to be important for microglial phagocytic
Microglial cells are the professional phagocytes of activity. Pharmacological inhibition of this channel or
the CNS tissue (Fig. 14 and supplementary video 2). This downregulation of its expression by small interfering
function is important for the normal brain, during brain RNA impairs phagocytic activity. The growth factor cili-
development, and in pathology and regeneration (for re- ary neurotrophic factor increased microglial phagocytosis
view, see Refs. 630, 637). through a Ca2⫹-mediated pathway (509a). Similarly,
During development, microglial cells play a specific GDNF and M-CSF increased the phagocytotic capability
role in removing apoptotic cells. In the developing cere- of the microglia (139, 594). Substrate-bound complement
bellum they phagocytose Purkinje neurons, which under- component C1q was shown to enhance both FcR-and

FIG. 14. Phagocytosis of dead/damaged cells. Dead/

damaged cells can be labeled by incubation of the slice
with a solution containing 0.1% trypane blue. These cells
were probably damaged by the slicing or dissection pro-
cedure. The sequence follows the ingestion of a trypane
blue-filled dead or damaged cell (black arrow) by an
amoeboid microglial cell and the subsequent shrinkage of
the phagosome. In the first picture, the microglial cell has
attached to the trypane blue-filled cell and surrounds it
with its pseudopods (small white arrowheads). Six min-
utes later, the cell is ingested. Finally, the dark phagosome
containing the trypane blue cell is condensed to roughly
half of its diameter within less than half an hour. Note the
increase in the concentration of the dye during this pro-
cess. Bar ⫽ 10 ␮m. [From Brockhaus et al. (87), with
permission from John Wiley and Sons.]
CR1-mediated phagocytosis two- to fourfold (988). Con- mentally important, remains largely without an answer.
versely, the prostanoid receptor subtype 2 (EP2), down- Without doubt microglial cells have the capacity to sense
regulates phagocytosis, because ablation of EP2 en- synaptic activity through their extended complement of
hanced microglial A␤ accumulation in cell culture (526, neurotransmitter receptors. Can this interaction bear
827, 828). As mentioned above, ATP is an important sig- functional consequences? Recent in vivo imaging experi-
naling molecule for microglia. The metabotropic P2Y6 ments have demonstrated that microglia in their surveil-
receptor controls microglial phagocytosis (412). In- lance state constantly scans the synaptic contacts dwell-
creased levels of mRNA encoding P2Y6 receptors were ing in its territorial domain (972) through establishing
correlated to the activation state. Thus the P2Y6 receptor brief (4 –5 min) contacts with synaptic structures. Impor-
is upregulated when neurons are damaged and could tantly, in the conditions of ischemia, these contacts were
function as a sensor/trigger for phagocytosis (467). At the much more persisting; they lasted for an hour or more
same time, activation of P2X7 receptors in cultured rat (972). Even more importantly, these events of prolonged
microglia suppressed phagocytosis in a Ca2⫹-independent liaisons between microglial processes and synapses often
manner; inhibition of P2X7 expression with lentiviral-me- caused the disappearance of the latter. This behavior may
diated shRNA interference or with oxATP/BBG restored indicate the constant monitoring of the synaptic status by
the phagocytic activity (261). resting microglia. A question remaining is the molecular
instruction by which microglia understands the (activity)
XVIII. NEURONAL-MICROGLIAL INTERACTION: status of a synaptic structure. It could be the very local
POSSIBLE ROLE IN NEURAL level of neurotransmitter, which would allow for the eval-
PLASTICITY? uation of an individual synapse; rather than an averaged
transmitter concentration. On the other hand, short inter-
Do microglial cells play any physiological functions action via adhesion molecules could help the transient
in the undisturbed brain? This question, although funda- contact establishment between neuronal and microglial

processes. NCAM, integrins, preferentially structures with teases, substrates, and receptors in the hemostatic, im-
a signaling component in both directions could be candi- mune, and nervous systems upon injury, but some still not
dates. fully understood interactions between molecular and cel-
Any abnormalities in synaptic strength/performance lular players and targets in the control of neuronal cir-
may trigger local responses of microglial cells, which cuits. Dynamic neuronal-microglial interactions were also
although not inducing the full activation process can ini- reported to participate in postnatal remodeling of the
tiate remodeling of synaptic architecture. Indeed, the role visual cortex. Importantly, the microglial phenotype was
of microglia in removing synapses was shown long ago, regulated by visual experience with a faster ramification
when it was shown that microglia are responsible for in animals that were rendered monocular from the mo-
removing the synapses from axotomized motoneurons ment of birth (764).
(71), the process that became known as “synaptic strip- Microglia appear as an important player in the resto-
ping.” Interestingly, the actual removal of synapses pre- ration of neuronal connectivity by controlling the reactive
ceded by decrease in the synaptic strength, which could synaptogenesis following the insult (58). The regenerative
be mediated by ATP/adenosine release from microglial potential of microglia is well documented; the importance
cells at the early activation stages (1016). A similar pro- of microglia for recovery of neuronal connectivity and
cess of synaptic stripping was observed in the cortex in synaptic repair has been repeatedly demonstrated (40, 94,
response to focal inflammation (923). The “striping” pro- 599). Furthermore, transplantation of microglia/macro-
cess is specific, as it predominantly removes excitatory phages into lesioned optic nerve or spinal cord signifi-
glutamatergic synapses leaving inhibitory inputs opera- cantly improved posttraumatic regeneration (499, 742).
tional, thus limiting neuronal excitability and glutamate Contributions of microglia, in particular certain pheno-
toxicity (528). This specificity of action is associated with types, to the plasticity of the CNS may include support of
the MHC class F receptors, which are present in both neurogenesis as well (113, 577). Recent studies have
neurons and microglia (183). Of course, these examples found T cells and microglia to be important to maintain
are coming from pathological realm, yet they indicate that both neurogenesis in the hippocampus and spatial learn-
microglial cells in principle are able to remove or remodel ing abilities. Apparently, contributions of the adaptive
synapses operating in their territorial domains. An initial immune system and the local innate immunity are re-
drop in synaptic efficacy could trigger the “microsurgical” quired to keep the capacity for cellular renewal as well as
elimination by microglia, which would thereby interfere to support higher CNS functions (1042). It was also sug-
with neuronal communication. This microglia-to-neuron gested that TLRs modulate adult hippocampal neurogen-
interaction would reach much further than the previous esis. TLR2 and TLR4 were identified in neural stem/pro-
modes of neurotrophin supply or the passive listening to genitor cells and were found to exert opposite effects on
broadcasted signals. their proliferation and differentiation (769). Agonists of
At the same time, microglia can potentially be in- these TLRs would simultaneously affect microglia. The
volved in the opposite process of creating new synapses. importance of a normal microglial function has been dem-
Several lines of evidence indicate the involvement of mi- onstrated in mice with a homozygous loss of function
croglia in regulation of synaptogenesis in the early post- mutation in Hoxb8 (149). These animals suffer from com-
natal brain. Microglia can stimulate synaptogenesis by pulsive grooming and hair removal, a syndrome resem-
secreting thrombospondins (TSPs) (137, 599); the latter bling an obsessive-compulsive disorder in humans. The
belong to extracellular matrix proteins critically impor- Hoxb8 lineage was found to associate in the CNS with a
tant for synaptic formation and are also produced by microglial subpopulation, and wild-type bone marrow
astrocytes (157). TSP1 interacts with the integrin-associ- transplantation was sufficient to rescue the mutant-carry-
ated protein CD47, the latter accepting also signal regu- ing animals from their abnormal behavior (149). So, both
latory protein (SIRP)␣, a transmembrane protein ex- synaptic contacts and neuronal elements themselves
pressed in neurons and macrophages (558). The SIRP␣- could depend on or benefit from the active microglia
CD47 system is involved in the regulation of migration engagement. Furthermore, recent studies revealed that
and phagocytosis, immune homeostasis, and neuronal microglia may play an active (or at least supportive) role
networks. CD47 and its two ligands, SIRP␣ and TSP1, may in the functional integrity of the CNS and its normal
thus play not only homeostatic roles in the immune sys- physiological performance even at the level of learning
tem, but participate in synaptic patterning involving the and behavior. Links between immunological and neuro-
innate immune cell of the CNS, i.e., the microglia (941). psychiatric disorders could thus also involve disrupted
Indeed, thrombin, the blood coagulation factor II(a) and microglia activity.
name-giving inducer of TSP in platelets, which has been Microglial-derived factors may also affect synaptic
associated with synapse elimination and plasticity (936, transmission directly. This, for example, was observed
1043), has its receptors expressed on microglia (31). in the spinal cord, where stimulation of microglia with
There is probably not only shared use of critical pro- ATP triggers release of BDNF, which in turn affects Cl⫺

distribution in neurons, thus turning GABA- and glycine- CNS, the very same cytokine as produced by microglia
mediated postsynaptic responses from inhibitory to excit- then drives astroglial proliferation in response to injury.
atory (175). This type of microglia-neuronal interactions Microglial cells are also intimately involved in the
has been discussed already in the context of neuropathic development of the nervous system, which relies upon
pain (see section IX), although similar mechanisms may tightly controlled balance between neurogenesis and neu-
be in operation in the absence of pathological insults. ronal death. Microglia have dual action on neurogenesis,
Direct action of microglia on synaptic plasticity was also as both detrimental and supportive effects were reported
observed in hippocampal slices treated with A␤1– 42. This (245). The underlying mechanisms of this dichotomy are
treatment inhibited induction of LTP, which in turn was still obscure, although most likely they depend on the
mediated through NO released by activated microglia differences in the activation state of microglia, and hence
(981). Microglial cells have the potential to influence ho- differential secretion of cytotoxic or protective/instruc-
meostatic synaptic scaling, which entails uniform adjust- tive factors (816). Differentiation of neural precursors
ments in the strength of all synapses on a cell mechanism isolated from supraventricular zone in culture, for exam-
distinct from long-term synaptic potentiation or depres- ple, required the presence of microglia or microglia-con-
sion. Stellwagen and Malenka (860) found that synaptic ditioned media (629, 975). Furthermore, microglia-derived
scaling in response to prolonged blockade of activity is factors are involved in directing migration and final in-
mediated by the proinflammatory cytokine TNF-␣. They struction of newly born neural cells (2). As to whether
demonstrated that glia are the source of the TNF-␣, while microglial cells can ever give rise to other cells, including
they did not distinguish between astrocytes and micro- those with neuronal phenotype, is an intriguing question
glia. They conclude that glia actively participate in the (110). Similar reports have been published even earlier,
homeostatic activity-dependent regulation of synaptic but remained of little resonance (351). Even though mi-
connectivity by modulating TNF-␣ levels (860). At the croglia have been shown to express some stem cell mark-
ers and have therefore been considered as a kind of
same time however, induction of LTP by strong tetanic
immature cell (see sect. III), trans-differentiations of that
stimulation of Schaffer collaterals did not affect motility
kind, i.e., shift towards neuronal orientation, would chal-
of microglial processes in acute hippocampal slices; nei-
lenge much of the concept of a mesodermal origin of
ther did it induce any current responses from neighboring
microglia.
microglial cells (1005).
The role of microglia in controlling neuronal extinc-
NO is not only the poisonous gas used by immune
tion during periods of developmentally associated pro-
cells for cytotoxic attack. In the CNS it is, first of all, a
grammed cell death was demonstrated in several brain
neuromodulatory factor. The NO contribution for the neu-
regions. In the retina, for example, the massive neuronal
roendocrine axis lies in the control of releasing factors
programmed death follows the early invasion of macro-
and hormones (756); this ability may, at least partially, phages (135). Dissection of the retina prior to macro-
allow microglia to exert certain influences, especially un- phages invasion prevents neuronal demise, whereas fur-
der immune-activating conditions. Microglial NO release ther addition of microglia to retinal preparation induces
could thus integrate activities of the endocrine system in cell death. The pro-apoptotic action of microglia is medi-
responses to infection and injury, when the NOS is in- ated through NGF, exemplifying the other side of a neu-
duced as part of a respective reactive phenotype profile. rotrophic factor supply (290). Similarly microglial cells
Microglial cells can also have a trophic role, indi- induce apoptotic death of Purkinje neurons in organo-
cated by distinct neurotrophin expression (247). There is typic slices mediated through microglia-produced super-
some early evidence that secretory proteases secreted by oxide ions (550). In the embryonic (E12-E13) spinal cord,
microglia may provide for trophic regulation of neuronal invading microglia control the apoptotic death of mo-
circuits (466), affecting their growth, differentiation, and toneurons via secretion of TNF-␣ (818); incidentally, mo-
circuitry formation. Conceptually, depending on the sta- toneurons transiently express TNF-␣ receptor 1 (TNFR1)
tus, microglial cells are able to release a multitude of precisely at that stage (818).
factors with powerful neurotrophic action (611). It would To conclude, microglial cells do have the potential to
still require a systematic approach, however, to determine regulate the development, structuring, and function of
the regional and temporal patterns of neurotrophin pro- neuronal networks. They constantly monitor the status of
duction by microglia. This production may occur during synaptic contacts and receive information from neuronal
development versus induction upon request by endan- networks. Potentially, at least, microglial cells are also
gered neurons. Keeping a broader perspective on devel- able to remodel neuronal connectivity and thus partici-
opmental potential, a number of cytokines, as being more pate in physiological processes within neural networks.
typically associated with the inflammatory response, may Multiple activation states of microglial cells may allow the
have roles in the maturating CNS. The astroglial growth existence of “resting active” microglia, or even “active”
factor capacity of IL-1 is one example (321). In the adult compartments in the processes of surveillant microglia,

which dynamically interact with neural circuitry and pro- prevent and dampen or facilitate the progression of a
vide it with additional plastic capabilities. given brain disease.
Commitment to distinct reactive phenotypes as in-
structed by specific activating signals may then have a ACKNOWLEDGMENTS
rather variable impact on the neur(on)al community in We thank the reviewers for their meticulous revision of the
the affected (infected, injured, degenerating) tissue re- manuscript and for very constructive comments.
gion (351, 816). While such distinct and overlapping func- Address for reprint requests and other correspondence: H.
tional orientations have been studied intensively in extra- Kettenmann, Max-Delbrück-Center for Molecular Medicine,
neural macrophages, still much has to be addressed with Robert-Rössle-Straße 10, 13092 Berlin-Buch, Germany (e-mail:
regard to microglia (166, 622). It will be important to kettenmann@mdc-berlin.de); or A. Verkhratsky, The University
of Manchester, Oxford Road, Manchester, M13 9PT, UK (e-mail:
identify the nature and sources of such instructing signals
Alexej.Verkhratsky@manchester.ac.uk).
as they govern functional orientations of microglia and
their shifts along an activation episode. Equally impor- GRANTS
tant, little is known about the heterogeneity of microglia,
H. Kettenmann and U. Hanisch were supported by Deutsche
i.e., the differences in functional capacities of individual
Forschungsgemeinschaft (Grant SFB/TRR43) and Bundesministe-
microglial populations between and within CNS regions. rium für Bildung und Forschung. M. Noda was supported by
Finally, in pathological situations with blood-derived Grants-in-Aid for Scientific Research from the Japan Society for
monocytes/macrophages infiltrating the CNS, features Promotion of Science. A. Verkhratsky was supported by Biotech-
and functions of resident microglia and the invading cells nology and Biological Science Research Council UK, Royal Society,
may overlap or complement each other, with both detri- Alzheimer Research Trust (UK), and International Association for
mental and beneficial consequences (823, 837). Why is the Promotion of Cooperation with Scientists from the Indepen-
there a need for recruiting peripheral macrophages when dent States of the Former Soviet Union.
the microglia could fulfill all tasks? Answers are awaited
for understanding microglia in health and disease and to DISCLOSURES
evaluate the therapeutical potential of correcting their No conflicts of interest, financial or otherwise, are declared
dysregulation. by the authors.
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Physiology of Microglia

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Physiology of Microglia

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Physiol Rev 91: 461–553, 2011;

Max-Delbrück-Center for Molecular Medicine, Berlin-Buch; University of Göttingen, Institute of

I. The Discovery and Definition of Microglia 462

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C. Interleukin receptors 511

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FIG. 3. Activated glial cells associ-

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FIG. 5. Morphology of resting and activated mi-

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TABLE 1. Markers of microglia

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Calcium signaling/homeostatic system, which is opera- A. Intracellular Ca2ⴙ Stores

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TABLE 2. Ion channels in microglia

Experimental Biophysical Properties and

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Biophysical Properties and

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Biophysical Properties and

Aquaporins Rat/RT-PCR/Western blotting ? Expression of AQP4 was 915

See text for definitions.

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