Autonomic Neuroscience: Basic and Clinical 153 (2010) 69–78

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Autonomic Neuroscience: Basic and Clinical

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u

Review

Visceral afferents — Determinants and modulation of excitability

Michael J. Beyak ⁎
Gastrointestinal Diseases Research Unit (GIDRU), Queen's University, Kingston, Ontario, Canada

a r t i c l e i n f o a b s t r a c t

Article history: An essential property of visceral sensory afferents is to be able to alter their firing properties in response to
Received 25 June 2009 changes in the microenvironment at the level of the sensory ending. Significant progress has been made in
Received in revised form 13 July 2009 recent years in understanding the ionic mechanisms of the regulation of afferent neuronal excitability, and in
Accepted 20 July 2009
identifying the mechanisms by which this can be altered. This article will review some of the recent
developments in the state of knowledge regarding mechanisms of increased excitability after inflammation,
Keywords:
and pharmacological modulation of excitability, concentrating on afferent nerves innervating the GI tract and
Visceral afferent
Ion channels
urinary bladder.
Pharmacology © 2009 Elsevier B.V. All rights reserved.
Excitability

Contents

1. Techniques — recording from afferent nerves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

1.1. Afferent nerve fibre recording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
1.2. Isolated cell recording — intracellular/patch clamp recording. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
2. Ion channel determinants of excitability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
2.1. Voltage gated sodium (NaV) channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
2.2. Voltage gated potassium (KV) channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3. Pharmacological modulation of excitability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4. Ligand gated ion channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.1. TRPV channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.2. P2X receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4.3. Serotonin — 5HT3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4.4. GPCRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
5. Protease activated receptors — PARs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
6. Metabotropic glutamate receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
7. Somatostatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
8. GABAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
9. Satiety hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
10. Gasotransmitters NO/H2S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
11. Chronic alteration in visceral excitability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
11.1. Inflammation and afferent excitability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
11.2. Mechanisms of inflammation induced hyperexcitability — alterations in receptor and ion channel function . . . . . . . . . . . . . . . . 75
12. Visceral hyposensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
13. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

At the heart of the function of the sensory neuron is its ability

to change its firing pattern in response to alterations in the micro-
⁎ Departments of Medicine and Physiology, Queen's University, GIDRU Wing, Kingston
General Hospital, 76 Stuart St, Kingston, ON, Canada K7L 2V7. Fax: +1 613 548 2426. environment of the nerve ending. This enables detection of physiolo-
E-mail address: beyakm@queensu.ca. gical stimuli, as well as noxious ones. In addition, afferent neurons,

1566-0702/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.autneu.2009.07.019
70 M.J. Beyak / Autonomic Neuroscience: Basic and Clinical 153 (2010) 69–78
including visceral afferents exhibit a great degree of plasticity in their
ability to respond to a given stimulus. That is to say the response to the
same stimulus is context dependent, and is influenced by what is
happening in the microenvironment at the level of the sensory ending.
For the purposes of this review “excitability” will be considered to refer
to the degree of response of a neuron in terms of action potential
output to a given stimulus. An increase in excitability is an action
potential in response to a lesser stimulus, or a greater number of action
potentials fired in response to a similar stimulus. This review will
concentrate on selected recent advances in the study of the regulation
of visceral afferent excitability, focusing on afferents innervating the
gastrointestinal tract and urinary bladder.
1. Techniques — recording from afferent nerves
There are a number of experimental approaches that can be taken
to assess visceral afferent excitability, and in broad categories they can
be divided into recordings from nerve fibres (teased single axons or
multiunit recordings) or recordings from single neuronal cell bodies.
1.1. Afferent nerve fibre recording
This technique involves extracellular recording of afferent nerve
activity using a wire or suction electrode, and then this signal is
differentially amplified, giving a readout of individual action potentials
or “spikes”. These types of recordings can be performed in vitro or
in vivo. The advantage of this approach is that the response of the
nerve ending to quasi physiological stimuli can be easily assessed (e.g.
stretch of a hollow viscus, application of mediators to the nerve end-
ing). In addition, the events recorded are reflective of the behaviour of
the specialized sensory ending lying in the wall of the viscus of interest.
The disadvantage of this technique is that the only variable that can be
assessed is firing frequency. Analysis of the waveform obtained has
little usefulness in gaining insight into the biophysical mechanisms of
changes in excitability.
1.2. Isolated cell recording — intracellular/patch clamp recording
For the reasons indicated above, there needs to be alternate ways of
examining electrical events in more detail at the level of the afferent
neuronal membrane. For this reason a number of investigators have
developed techniques for recording from isolated neuronal cell bodies
(typically in vitro). Most commonly whole cell patch clamp techniques
have been used, however some have used sharp microelectrode
intracellular recordings. As sensory neuronal cell bodies lie in ganglia,
these ganglia are often isolated and cells dispersed and maintained in
short term culture for electrophysiological recording. Thus isolated
neuronal cells (e.g. Nodose or dorsal root ganglion neurons) can be
individually recorded from, and recordings can be made in current
clamp mode (to measure membrane potential responses) or voltage
clamp mode (to measure flux of ions across the cell membrane). The
advantage of these techniques is that very detailed measurements of
individual ionic currents underlying excitability can be made and the
response of an individual neuron to depolarizing stimuli can be as-
sessed. The disadvantages are twofold. First, when recording from
randomly selected sensory ganglion neurons, one cannot be comple-
tely sure what organs they project to. Many investigators have tackled
this problem through the use of retrogradely transported fluorescent
dyes, which are applied to the organ of interest prior to cell harvest,
allowing the identification of a specific population of neurons
projecting to an organ of interest (see Moore et al., 2002; Yoshimura
et al., 1996 as examples). The second disadvantage is harder to address.
That is the assumption of these types of recordings is that the events
occurring at the membrane of the cell body are similar to that at the
level of the nerve terminal in situ. This may not be entirely correct, as
there is likely directed transport of receptors and channels to areas in
which they will ultimately be used, such as the specialized afferent
ending. Nonetheless, these techniques have given tremendous insight
into the regulation of sensory neuronal excitability and when com-
pared with results obtained from afferent nerve fibre recording ex-
periments, have fared reasonably well.
2. Ion channel determinants of excitability
2.1. Voltage gated sodium (NaV) channels
Voltage gated sodium channels are essential in mediating the rapid
upstroke of the action potential . The regenerative depolarization,
then further channel activation, and subsequent further depolariza-
tion result in the rapid upstroke of the action potential (Hille, 2001).
The threshold for activation of the sodium channel is the key in setting
the action potential voltage threshold. Equally important is the time
dependent inactivation of these channels, terminating the upstroke,
and permitting repolarization of the membrane. Sensory neurons are
relatively unique in that the voltage gated channels present are
limited to few subunits (Waxman et al., 1999b) In visceral sensory
neurons, many of these are unmyelinated a delta or C fibres, and thus
also carry a unique repertoire of sodium channel alpha subunits.
Voltage gated sodium channels in visceral sensory neurons can be
broadly categorized into 2 categories based on their sensitivities to the
puffer fish toxin tetrodotoxin (TTX) (Waxman et al., 1999a). Most
visceral sensory neurons have both TTX sensitive and TTX resistant
sodium currents (Beyak et al., 2004; Bielefeldt et al., 2002a,b; Stewart
et al., 2003; Yoshimura & de Groat, 1997). The alpha subunits
underlying these currents are primarily NaV 1.7 (TTX sensitive) and
NaV 1.8 and 1.9 (TTX resistant). NaV 1.7 mediates a TTX sensitive
current with relatively low threshold for activation, rapid inactivation,
slow repriming kinetics and a hyperpolarized steady state inactivation
curve. NaV 1.8 is responsible for a current with a higher threshold for
activation, slower inactivation kinetics, rapid repriming kinetics and a
more depolarized steady state inactivation curve (see Fig. 1A). NaV1.8
channels have been the focus of intense study in visceral sensation
because of their relatively selective expression in sensory neurons, and
their modulation by inflammatory states. NaV 1.9 results in a relatively
persistent current with a very hyperpolarized availability curve, and is
likely involved in setting the membrane potential and threshold for
action potential. Relatively few colon projecting afferents exhibit
currents similar to those mediated by NaV 1.9, and this may reflect the
fact that most colon afferents are peptidergic, rather than IB4 positive
(Beyak et al., 2004; Roberts et al., 1993) — and the latter have been
shown to express NaV 1.9 (Fang et al., 2006) preferentially. Until
recently the presence of these voltage gated sodium channel subunits
in visceral afferents was assumed, based on the expression profiles in
nonselected DRG and vagal neurons, however a recent paper by King
et al. has demonstrated the presence of transcript for NaV 1.7, 1.8 and
1.9 in colon projecting DRG neurons, using laser capture microdissec-
tion techniques (King et al., 2009). In addition, in bladder projecting
DRG neurons, the presence of NaV 1.8 and to a lesser degree 1.9 have
been demonstrated, using in situ hybridization techniques (Black et al.,
2003).
2.2. Voltage gated potassium (KV) channels
Voltage gated potassium channels are also critical in setting the
excitability of the visceral afferent neuron. They serve to repolarize the
cell membrane, and limit repetitive firing. They can be broadly clas-
sified based on their inactivation kinetics into inactivating (A-type)
currents or noninactivating IK (or delayed rectifier) currents. IA currents
tend to have a more depolarized availability curve, and lower thresholds
for inactivation while IK currents have a more depolarized availability
curve and increased thresholds for activation (see Fig. 1B). Reduction
in A currents results in repetitive firing in visceral sensory neurons,
 M.J. Beyak / Autonomic Neuroscience: Basic and Clinical 153 (2010) 69–78 71

Fig. 1. Voltage gated channels involved in determining excitability. A) Different voltage gated sodium currents in colon projecting DRG neurons. Left — fast inactivating TTX Sensitive
current. Middle — more slowly inactivating TTX resistant current. Right — ultra slowly inactivating TTX resistant current. Inset — prolonged time course showing little inactivation of
this current over 200 s (modified from Beyak et al., 2004 with permission). B) Typical voltage gated potassium currents in ileum projecting DRG neurons. Top left — total potassium
currents elicited by stepwise depolarizing voltage commands from a holding potential of − 100 mV. Top middle — holding potential of − 60 mV isolates the sustained IK current. Top
right — digital subtraction of the two previous currents reveals IA an inactivating potassium current. Bottom left — total potassium currents from holding potential of − 100 mV.
Middle — currents in the presence of 1 mM 4-aminopyridine, a blocker of IA currents. Bottom right — digital subtraction reveals the presence of IA. Note similarity of these currents to
those obtained in the above 3 panels (modified from Stewart et al., 2003 with permission).

highlighting the importance of this channel in regulating visceral sen-
sory neuronal excitability (Moore et al., 2002). Decreases in IK current
would be expected to broaden the action potential, and possibly to
depolarize the membrane, as there is likely some of this current active at
around resting potentials. The molecular determinants of these currents
are likely complex, as studies in unselected DRG neurons indicate
expression of numerous KV subunits, many of which underlie these
types of currents (Kim et al., 2002; Park et al., 2003; Yang et al., 2004).
In addition to IA and IK, calcium activated potassium currents also
likely play a role in visceral afferent excitability. These currents are
likely involved in the afterhyperpolarization and in responses to a
variety of mediators. The presence of these currents has not been
demonstrated in DRG or neurons projecting to the viscera, however
nodose neurons appear to have functional calcium activated potas-
sium channels (Hay & Kunze, 1994a,b).
3. Pharmacological modulation of excitability
Visceral afferents display responsiveness to a wide range of endog-
enous and exogenous ligands. This property has been termed by
Kirkup et al. as “promiscuous chemosensitivity. (Kirkup et al., 2001)”
In simple categories, the receptors mediating these actions can be
classed into ligand gated ion channels and membrane receptors that
activate downstream signaling cascades (e.g. GPCRs), that ultimately
impact on the ion channel determinants of excitability discussed
above (e.g. via channel phosphorylation). As the list of mediators
potentially impacting on the function of visceral afferents is immense,
I will limit the discussion to those examples from the recent liter-
ature, and those receptors that may be important in chronic alter-
ations in afferent sensitivity. The reader is otherwise directed to other
reviews for a more comprehensive list (Beyak et al., 2006a; Blackshaw
& Gebhart, 2002; Blackshaw et al., 2007).
4. Ligand gated ion channels
Ligand gated channels, as the name suggests, consist of a membrane
receptor — channel complex, that when activated by ligand binding,
results in the flux of ions across the cell membrane, thus resulting in
alterations in membrane potential. This can depolarize the membrane
resulting in a so called generator potential, and thus activate voltage
gated sodium channels and an action potential.
4.1. TRPV channels
TRPV channels (transient receptor potential vanilloid) are a family
of ligand gated channels and are a member of the TRP family of
channels including the TRPC (canonical TRPs), TRPA (ankyrin), TRPM
(melastatin) and TRPPs (polycystin) members (for recent review see
Patapoutian et al., 2009). The TRPVs, in particular TRPV1 and TRPV4
have been the focus of a number of interesting studies of visceral
afferents in recent years. Activation of these channels results in an
inward nonselective cation current. With regard to TRPV1, this is the
receptor for capsaicin, the pungent ingredient in chili pepper. How-
ever it is activated by a number of other physical and chemical stimuli
such as acid, heat and likely mechanical forces. Deletion of TRPV1
results in diminished afferent responses to mechanical stimuli and
acid in the intestine (Rong et al., 2004), stomach (Bielefeldt & Davis,
2008), colon (Sugiura et al., 2007; Jones et al., 2005) and urinary
bladder (Daly et al., 2007) (see Fig. 2). In isolated colon projecting
DRG neurons TRPV mediated acid induced currents as well (Sugiuar
72 M.J. Beyak / Autonomic Neuroscience: Basic and Clinical 153 (2010) 69–78
Fig. 2. TRPV1 is involved in afferent mechanosensitivity in the jejunum, colon and urinary bladder. A) Example of jejunal afferent nerve responses to ramp distention in wildtype and
TRPV1−/− mice. Mechanosensitivity is attenuated in the TRPV1−/− animals (modified from Rong et al., 2004 with permission). B) Example of responses of bladder afferents to
ramp distention in wildtype and TRPV1 −/− mice. Similar to the jejunum, there is a significant attenuation of responses to distention in the knockout animals (modified from Daly et
al., 2007 with permission). C) In the colon, deletion of TRPV1 or ASIC3 genes results in attenuation of responses of muscular mucosal afferents to circular stretch (Modified from Jones
et al., 2005 with permission).
et al., 2004). TRPV4 is also a mechanosensitive channel, that is sen-
sitive to heat (noxious). It also plays a critical role in mechanosensa-
tion in the colon and upper GI tract (Brierley et al., 2008). In addition
these channels can be modulated by activation of other receptors such
as PARs (Sipe et al., 2008) (see below) and their function altered by
chronic inflammation.
4.2. P2X receptors
ATP, in addition to being the intracellular energy molecule, also
is known to function as an extracellular signaling molecule. It acts on
two classes of receptors, P2Y (which are GPCRs) and P2X (ligand gated
channels. P2X receptors have been shown to be present in colonic (Xu
et al., 2008), gastric (Castelucci et al., 2003; Dang et al., 2005; McIlwrath
et al., 2009) intestinal (Kirkup et al., 1999) and bladder afferent neurons
(Dang et al., 2008). ATP is a potent stimulus to afferent firing in all types
of afferents tested, including esophageal, gastric, jejunal, colonic, lung,
carotid body, ureteric and urinary bladder afferents (Dang et al., 2008,
2005; Cockayne et al., 2005; Wang & Neuhuber, 2003; Rong et al., 2002;
Brouns et al., 2000; Kirkup et al., 1999).
In isolated visceral sensory neuronal cell bodies ATP and P2X
agonists activate inward currents (Xu et al., 2008; Dang et al., 2008).
The most important P2X subtypes in sensory neurons are thought to be
P2X2 and P2X3 which can form functional homomeric or heteromeric
receptors. Thus experiments examining the role of P2X receptors in
visceral sensory function have had to utilize double knockout animals.
In the urinary bladder, P2X2/3 double knockout animals had reduced
ATP responses as well as reduced mechanosensation (Cockayne et al.,
2005). In contrast in the jejunum, mechanosensation was preserved in
knockout mice, and was unaffected by P2X antagonism, but the
enhanced excitability induced by inflammation was blunted by P2X
antagonists, revealing a role for P2X 2/3 receptors in post inflammatory
states (Rong et al., 2009).
4.3. Serotonin — 5HT3
As greater than 90% of the body's 5-HT is found in the GI tract, it is
not surprising that there exists a serotonergic signaling system in
visceral afferents. 5HT3 receptors are known to be present on both
colonic (Hicks et al., 2002) and small intestinal afferent nerves
(Keating et al., 2008; Hillsley & Grundy, 1998; Hillsley et al., 1998; Li
et al., 2004). 5HT results in an increase in afferent firing in this
population that is nearly completely attenuated by selective antago-
nists of the 5-HT3 receptor. Furthermore in isolated abdominally
projecting vagal afferent neurons, 5-HT results in inward currents
characteristic of the 5-HT3 receptor (Keating et al., 2008). The
importance of this receptor in visceral afferent function is highlighted
by the use of 5-HT3 antagonists in the treatment of both visceral pain
states (e.g. alosetron for IBS) and emesis (ondansetron and granisetron
for chemotherapy and postoperative emesis). Furthermore these
actions of 5-HT, particularly those on vagal afferents likely underlie
some of the nutrient sensing functions of these afferent nerves (Li
et al., 2004; Zhu et al., 2001).
4.4. GPCRs
There are literally dozens of G-protein coupled receptor systems that
can increase or decrease afferent excitability. As a detailed review of all
of these is beyond the scope of this article, and is handled in a variety of
previous reviews, we will focus here on advances made in recent years
on protease activated receptors (PARs), metabotropic glutamate
receptors (mGluRs) GABAB receptors and somatostatin receptors, as
well as mediators involved in satiety that act via vagal afferents.
5. Protease activated receptors — PARs
The protease activated receptors (PARs 1–4) are a recently described
class of G-protein coupled, seven transmembrane receptors that are
expressed in a variety of tissues (Coelho et al., 2003). The best
characterized on visceral afferents is PAR2. Its endogenous ligands
include tryptase and trypsin. Sources of these proteases include mast
cells and other inflammatory cells, digestive enzymes and possibly
bacteria themselves (Cottrell et al., 2003). In both nerve recording
experiments and experiments on isolated cells, PAR-2 activation results
in increased excitability, depolarization and afferent neuronal firing
(Kayssi et al., 2007; Kirkup et al., 2003) (see Fig. 3A). Recent studies have
made significant progress in identifying the ionic mechanisms of action
of PAR-2, and its ion channel targets appear to be diverse. Firstly, PAR-2
is capable of inhibiting voltage gated potassium channels, in particular IK
currents, which are inhibited by more than 50% by PAR-2 activation
(Kayssi et al., 2007). There was no effect on voltage gated sodium
currents, or A-type potassium currents (see Fig. 3B). These effects on
excitability were dependent on the PKC and ERK pathways. In addition,
PAR-2 is also capable of sensitizing both TRPV1 (Amadesi et al., 2006)
and TRPV4 (Sipe et al., 2008) currents in colonic DRG neurons.
Furthermore, the ability of PAR-2 to evoke action potential discharge
from DRG neurons was significantly inhibited in mice deficient in
TRPV4, indicating an essential role in the actions of PAR-2, on visceral
afferents. Consistent with this, PAR-2 induced hyperalgesia was absent
in TRPV4 knockout animals as well (Sipe et al., 2008).
 M.J. Beyak / Autonomic Neuroscience: Basic and Clinical 153 (2010) 69–78
Fig. 3. PAR-2 excites visceral afferents by inhibiting potassium currents. A) PAR-2 agonists
excite rat mesenteric afferents in vivo. Intravenous injection of SLIGRL, a PAR-2 agonist
results in an increase in mesenteric afferent firing. Whereas the reverse peptide LRGILS has
no effect (modified from Kirkup et al., 2003 with permission). B) Effect of PAR-2 on
potassium currents in colon projecting DRG neurons. Application of the PAR-2 agonist
inhibits IK currents, but not IA (modified from Kayssi et al., 2007 with permission).
6. Metabotropic glutamate receptors
Recent work from the Blackshaw lab has pointed to a role for
metabotropic glutamate receptors in modulation of afferent sensitiv-
ity. Receptor mRNA and protein for multiple mGluR subtypes are
present in vagal afferents (Page et al., 2005). Agonists of group II and
III receptors inhibit mechanosensitivity, and antagonists of these
73
receptors enhance mechanosensitivity, suggesting the presence of
endogenous glutamatergic tone (Page et al., 2005). The source of
endogenous glutamate is not known, however afferent terminals
themselves do express glutamate transporters (Berthoud & Neuhuber,
2000) and glutamate may also be released from the enteric nervous
system (Wiley et al., 1991). Two separate reports suggest an
excitatory effect of a group I mGluR, mGluR5 on mechanosensitivity
in both gastroesophageal vagal afferents and colonic afferents (Page
et al., 2005; Lindstrom et al., 2008), with the latter paper providing
evidence for an effect on visceral pain. Interestingly, mGlur5 appears
to have a role as well in bladder afferent function, however at a
central, rather than peripheral level, as antagonism of this receptor
had no effect on bladder afferent discharge, but that systemic
administration inhibited mictruition. (Hu et al., 2009). Taken together
this line of work confirms the existence of both excitatory and
inhibitory mGluRs on gastrointestinal afferents. What remains to be
seen is how these competing influences interact in various patho-
physiological and physiological states. However they remain inter-
esting targets for drug targeting for disorders of GI motility and pain.
7. Somatostatin
Somatostatin is an endogenous peptide present in enteroendo-
crine D cells in the GI tract. Clinical studies suggest that somatostatin
agonists inhibit visceral sensation. A number of recent papers point to
a role for SST2 receptors in modulating visceral afferent excitability. In
the anaesthetized rat, SST2 agonists octreotide and BIM23027 have
effects on both vagal and spinal afferents (Booth et al., 2001). The
inhibitory effect on baseline discharge was inhibited by chronic
vagotomy, after which vagal afferent fibres projecting to the intestine
would be degenerated, suggesting a vagal site of action. However SST2
mediated inhibition of responses to distension and bradykinin were
not altered by vagotomy suggesting an effect on spinal afferents.
Subsequent experiments using animals deficient in the SST2 gene
revealed spontaneous hyperexcitability, and enhanced responses to
mechanical and chemical (acid) stimuli (Rong et al., 2007). This
suggests that endogenous somatostatin may act as a “brake” on the
excitability of intestinal afferents. Interestingly this system seems to
be downregulated in the post inflammatory state, and may contribute
to post inflammatory hyperexcitability (Aerssens et al., 2007).
8. GABAB
Gamma amino butyric acid type B receptors (GABAB) have also
been recently demonstrated to play a role in visceral afferent sen-
sitivity. Given the inhibitory effects of GABAB agonists on the transient
lower esophageal relaxations (TLESRs) that mediate gastroesophageal
reflux (Blackshaw et al., 2000; Partosoedarso et al., 2001), attention
has been directed to the afferent limb of this reflex, namely the vagal
afferent innervation of the proximal stomach and esophagus. GABAB
agonists inhibit mechanosensitive responses in vagal afferents
innervating the proximal stomach and esophagus (Page & Blackshaw,
1999) of the ferret. In the guinea pig, functional GABA receptors are
present in the nodose ganglion, however not in the afferent ending,
indicating significant species differences with regard to this receptor.
The peripheral source of GABA is unknown. Nonetheless, GABAB re-
mains an interesting target to reduce afferent input from the proximal
stomach to reduce the incidence of TLESRs.
9. Satiety hormones
Given the emerging public heath problem of obesity, there is
increasing attention being focused on gastrointestinal derived satiety
hormones and their action on afferents innervating the GI tract. Many
of these hormones exert satiety effects that at least partially depend
on gastrointestinal (vagal) afferents. Among the mediators studied,
74 M.J. Beyak / Autonomic Neuroscience: Basic and Clinical 153 (2010) 69–78

glucagon like peptide-1 (GLP-1), cholecystokinin (CCK) and leptin endings (IGLEs) raise the tantalizing possibility of nitrergic crosstalk
have been directly shown to augment vagal afferent activity. In the between the enteric nervous system and extrinsic sensory afferents.
case of CCK, the receptor involved is known to be the CCK-1 (pre- With regard to H2S, there is an emerging yet conflicting body of
viously known as CCK-A) subtype. CCK increases firing in gastro- literature supporting its role in altering visceral afferent activity. The
intestinal afferent fibres innervating the stomach (Blackshaw & GI tract is unique in that H2S can both be derived from endogenous
Grundy, 1990), intestine (Li et al., 1999) and liver (Cox & Randich, sources, via the action of H2S producing enzyme systems, or from
1997) and activates isolated nodose ganglion cells (Peters et al., “exogenous sources” namely the product of bacterial metabolism by
2006b). Recent studies have also provided insight into some of the resident colonic bacteria (Moore et al., 2003). Initial reports suggested
potential ionic mechanisms of action. For example Ritter et al. have an antinociceptive role for H2S in the colon (Distrutti et al., 2006), as
shown that a nonselective cation conductance is activated by CCK in exogenous H2S inhibited visceromotor reflexes induced by colonic
nodose neurons, and potassium conductances are inhibited (Peters distention. These effects appeared to involve the activation of KATP
et al., 2006a). Preliminary work suggests that the cation conduc- channels, which would argue against involvement of primary af-
tance is pharmacologically similar to the TRPC channels (Beyak et al., ferents, as activation of potassium channels would tend to inhibit
2006b). Similarly the leptin receptor is expressed in human and afferent excitability. However a recent report suggests a pro-nociceptive
rodent nodose ganglion neurons, and leptin is known to activate role for luminal H2S, by evidence that pain related behaviours are
mesenteric afferents, as well as isolated nodose ganglion neurons enhanced by luminal H2S (Matsunami et al., 2009). The reasons for the
(Peters et al., 2006b; Buyse et al., 2001). Leptin appears to also both discrepancies in these findings are unclear, however possibilities
inhibit an outward current and activate an inward current, however include, secondary effects of H2S on smooth muscle tone, as H2S is a
the former action seems more prominent. known relaxer of GI smooth muscle (Gallego et al., 2008), or differences
With regard to GLP-1, there is emerging literature pointing to the in the concentrations of mediators involved. Importantly there are no
importance of visceral afferents of the vagus in its satiety and glucose published studies demonstrating the effects of H2S on visceral afferent
homeostatic effects (Abbott et al., 2005; Vahl et al., 2007), however, activity, however it appears to exert effects on bladder smooth muscle
the evidence demonstrating GLP-1 activating visceral afferents is still
sparse. There are direct electrophysiological recordings from hepatic
afferents showing an increase in firing rate in response to GLP-1
(Nishizawa et al., 2000), and nodose ganglia express the GLP-1
receptor mRNA (Nakagawa et al., 2004), and are depolarized by GLP-1
(Kakei et al., 2002). Preliminary work suggests an excitatory effect of
GLP-1 of intestinal afferents (Gaisano & Beyak, 2007), and a recent
report indicates that gastric vagal afferents are sensitive to GLP-1
in vivo (Bucinskaite et al., 2009). The mechanisms of action of GLP-1
are as yet not clear, however preliminary results from our lab suggest
an important role for inhibition of voltage gated potassium currents,
both IA and IK type currents (Gaisano & Beyak, 2007). PYY, another
distally released enteroendocrine hormone, appears to exert actions
on satiety and gastric emptying via vagal afferents however there are
no reports of it directly activating vagal afferents to date (Abbott et al.,
2005). On the topic of “orexigenic” i.e. appetite stimulating hormones,
the only ones to date studied are ghrelin and orexin. Ghrelin seems to
diminish vagal afferent firing (Date et al., 2002), and inhibit
mechanosensitivity (Page et al., 2007) which would tend to decrease
vagal satiety input to the brain. Orexin itself has no direct effect on
jejunal afferent activity, but inhibits the excitatory action of the satiety
hormone CCK (Burdyga et al., 2003).

10. Gasotransmitters NO/H2S

The original gaseous neurotransmitter described was nitric oxide

(NO) and in subsequent years both carbon monoxide (CO) and more
recently hydrogen sulfide (H2S) have been described. The literature for
these mediators affecting the discharge properties of afferent nerves
innervating the viscera is sparse, but has begun to emerge in recent
years.
A recent report suggests a role for peripheral nitric oxide in mod-
ulating the activity of gastrointestinal vagal afferent endings. Page
et al. (2009) have shown that neuronal nitric oxide synthase (nNOS)
inhibition enhanced mechanosensitivity, but only of those mechan-
osensitive mucosal afferents, not those presumed to be present in the
smooth muscle layer. In addition, exogenous NO donors inhibited Fig. 4. Endogenous nitric oxide inhibits visceral afferent mechanosensitivity. A) Inhibition
mechanosensitivity, as did exogenous stimulators of the cyclic GMP of endogenous NO production with L-NAME (open symbols) enhances responses of
pathway — the second messenger pathway acted upon by nitric oxide mucosal afferents to stroking with calibrated von Frey hairs. B) L-NAME has no effect on
(see Fig. 4). The source of endogenous NO mediating these actions responses on tension units (which respond to circumferential stretch). C) Raw nerve
tracing of a mucosal unit and its responses to stroking of the receptive field with 50 mg von
is thought to be mucosal enteroendocrine cells expressing nNOS, Frey hairs. i) control trace ii) note enhancement of responses after L-NAME iii) L-arginine,
though the presence of specialized afferent endings terminating in the the natural substrate of nitric oxide synthase reverses the effects of L-NAME (modified
myenteric ganglia themselves, the so called intraganglionic laminar from Page et al., 2009 with permission).
 M.J. Beyak / Autonomic Neuroscience: Basic and Clinical 153 (2010) 69–78 75

contraction and intestinal secretion via an action on capsaicin sensitive
visceral afferents (Patacchini et al., 2004; Schicho et al., 2006). There is
however one preliminary study demonstrating an excitatory effect of
H2S on mesenteric afferent firing (Rong et al., 5 A.D.). Studies of the
direct effects of hydrogen sulfide on afferent excitability will be critical
in unraveling this potentially important novel signaling pathway.
11. Chronic alteration in visceral excitability
The above sections have focused on mechanisms by which the
firing properties of visceral afferents can be altered on an “acute” or
minute to minute basis. However there are clearly situations where
the function of these nerves is altered on a more long term basis,
causing relative “hypersensitivity” or “hyposensitivity.” These con-
cepts are relevant in disease states such as visceral pain, where normal
visceral functions often result in painful sensations (visceral allody-
nia) or exaggerated responses to normally painful stimuli (visceral
hyperalgesia). Indeed observations of altered sensory thresholds exist
in a variety of visceral pain disorders such as noncardiac chest pain,
functional dyspepsia, irritable bowel syndrome and interstitial
cystitis. With this in mind, many investigators have developed models
of visceral disease, typically inflammation, to study the medium and
long term impact on the function of visceral afferents. The review by
Vergnolle in this issue will discuss in detail numerous aspects of the
effects of inflammation on visceral afferent function.
inflammatory changes had resolved. Recordings from colonic afferent
nerve fibres after the resolution of inflammation also revealed increased
mechanosensitive responses (Ibeakanma et al., 2009). Recent papers
have also identified that after resolution of inflammation induced by
TNBS, a model of inflammatory bowel disease, that hyperexcitiability of
colonic afferents is present both during acute inflammation and after
recovery, though hypersensitivity was only observed in the acute phase
in splanchnic afferents, and not pelvic fibres, suggesting differences
between subtypes of fibres. (Hughes et al., 2009)
11.2. Mechanisms of inflammation induced hyperexcitability — alterations
in receptor and ion channel function
Inflammation clearly causes alteration in the membrane properties
of visceral afferent neurons, and this suggests a fundamental alter-
ation in the major determinants of neuronal excitability, namely vol-
tage gated potassium and sodium channels. With regard to voltage
gated potassium currents a variety of models of acute inflammation in

11.1. Inflammation and afferent excitability

As it is intuitive that inflammation results in pain, it follows that a

number of models of inflammation have been used to study the
impact of visceral afferent function. Studies using recordings from
isolated sensory neurons from the dorsal root ganglion have provided
a number of insights. Yoshimura and DeGroat first characterized
changes in excitability of bladder projecting DRG neurons in a model
of cyclophosphamide induced cystitis (Yoshimura & de Groat, 1999).
Neurons projecting to the inflamed bladder (identified by retrograde
labeling) exhibited lower current thresholds, fired more action
potentials at suprathreshold stimulus strengths, and had greater
input resistances. Subsequently studies using a model of inflamma-
tory bowel disease (trinitrobenzene sulphonic acid — TNBS) in the
ileum (Stewart et al., 2003) and colon (Beyak et al., 2004), and models
of ulcer disease (Dang et al., 2004) in the stomach have also revealed
similar effects on current threshold (rheobase) and repetitive firing. It
is important that these experiments were performed on cells removed
from the inflammatory in vivo milieu for several hours, suggesting the
presence of longer term changes in excitability, eliminating the types
of influences discussed in the previous sections.
The above important studies examined the effects of active inflam-
mation, but as many human conditions of visceral hypersensitivity
such as IBS occur in the absence of obvious inflammation, the question
remained whether these changes can persist after the resolution of
inflammation. Two recent studies have addressed this issue using
differing models of self limited gastrointestinal infection. Keating et al.
demonstrated that after resolution of the acute inflammatory phase of
intestinal infection with Trichinella spiralis that there were increased
responses to mechanical and chemical stimuli at the level of the afferent
terminal, and that hyperexcitability was present in isolated labeled gut
projecting sensory neurons at this time point as well — similar to
changes previously seen on neurons recorded from during the acute
Fig. 5. Increased excitability of visceral afferents in the post inflammatory state.
phase of inflammation. (Keating et al., 2008) (see Fig. 5) A more recent Transient inflammation was induced with infection with Trichinella spiralis. A) jejunal
study by Ibeakanma et al. used a mouse model of Citrobacter rodentium afferent responses to distension in the control, acute inflammatory period (day 14–16
infection, a self limited bacterial infectious colitis, similar to that seen post infection) and post inflammatory periods (after resolution of inflammation).
with pathogenic E. coli infection in humans. Again similar changes of During acute inflammation, there is a reduction in mechanosensitive responses,
however after resolution, there is evidence of increased response of jejunal afferent
decreased rheobase, and increased repetitive firing were seen in the nerves to distention. B) In isolated nodose ganglion neurons, there is evidence of
acute inflammatory period, and the increased repetitive DRG neuronal decreased rheobase and increased repetitive firing at twice rheobase in the post
firing persisted in post infections state, at time points when the acute inflammatory period (modified from Keating et al., 2008 with permission).
76 M.J. Beyak / Autonomic Neuroscience: Basic and Clinical 153 (2010) 69–78
the stomach (Dang et al., 2004), ileum (Stewart et al., 2003), colon
(Ibeakanma et al., 2009) and urinary bladder (Yoshimura & de Groat,
1999) have suggested that voltage gated currents are suppressed, and
their steady state inactivation properties altered such that fewer
channels are available for activation. Indeed the effects of TNBS ileitis
on labeled DRG neurons was very similar to that of administration of
the IA blocker 4-aminopyridine (Moore et al., 2002). Whether or not
only IA or IA and IK type currents are suppressed vary between
studies; however in most studies the suppression of IA is a consistent
finding. Following the resolution of inflammation, these suppressive
effects on potassium currents, at least IA, seem to persist (Ibeakanma
et al., 2009).
Important changes in voltage gated sodium channels have also been
demonstrated in inflammation. In TNBS colitis and ileitis, TTX resistant
sodium currents are increased, and their activation thresholds reduced
(Beyak et al., 2004; Stewart et al., 2003). Other investigators examining
bladder, and stomach afferents (Bielefeldt et al., 2002a,b) have made
similar findings. The effect on NaV currents is relatively selective. In
addition the kinetics of the channels are also altered, with the speed of
recovery increasing after inflammation, and the threshold for activation
is lowered (Beyak et al., 2004). The increase in this rapidly repriming
TTX-R current, combined with the decreased activation threshold would
tend to result in increased ability to fire repetitively with a lower
threshold for action potential electrogenesis. The critical role for the NaV
1.8 TTX-R current is illustrated using elegant studies by Hillsley et al.
(2006), in which transient jejunitis due to infection with Nippostron-
gylus brasiliensis induced hyperexcitability of intestine projecting DRG
neurons, and increased TTX-R currents. These investigators used NaV 1.8
knockout mice, and showed that post infectious hyperexcitability was
absent in these animals. Whether the observed changes in TTX-R
currents are due to alterations in channel expression or to post-
translational modeling is an important question. A recent paper has
attempted to address this issue, examining protein expression of im-
portant channel subunits NaV 1.7, 1.8 and 1.9 with western blot tech-
niques as well as examining mRNA levels using quantitative rtPCR
performed on labeled colon projecting neurons by laser capture micro-
dissection in animals with and without TNBS colitis. At time points
corresponding to previous studies showing enhanced excitability,
expression of NaV1.8 protein was increased, however there was no
change in mRNA, suggesting that changes are occurring at the post-
translational level (King et al., 2009).
In addition to the aforementioned changes in voltage gated
channels, there is also evidence for changes in the number and or
function of ligand gated channels as well. With regard to TRPV1, a
variety of models of inflammation in the viscera up regulate its
expression, and in a number of models is necessary for pain related
behaviours as well as afferent nerve mechanohypersensitivity (De
Schepper et al., 2008; Phillis et al., 2009). In addition, a recent
interesting report suggested that down regulation of TRPA1 atten-
uates colitis induced hyperalgesia (Yang et al., 2008). P2X currents
also seem to be unregulated by gastric (Dang et al., 2005) and
bladder (Dang et al., 2008) (see Fig. 6) inflammations as are afferent
neural responses to ATP. The situation with 5-HT may however be
different. Keating et al. (2008) showed that despite increased
responses to exogenous 5-HT, and increased 5-HT availability, that
5-HT mediated currents were actually decreased in the post inflam-
matory state. This suggested that in some instances, receptors may
down regulate in the face of increased availability of the ligand.
Taken together, the evidence to date is that visceral inflammation
results in multiple changes in ion channel function, including aug-
menting of sodium channel function, while at the same time inhibiting
potassium channels that normally allow a neuron to accommodate a
depolarizing stimulus, thus leading to decreased action potential
thresholds and repetitive firing. The enhancement of function of a
number of ligand gated channels further increases the effect that ligands
will have on the nerve terminal.
Fig. 6. Enhanced P2X receptor function in bladder projecting DRG neurons following
inflammation. Left panels are currents evoked by superfusion of ATP, right panels are
currents evoked by superfusion of the P2X agonist αβ-methylene ATP. B represents
currents from control neurons, B⁎ represents currents in neurons from animals with
cystitis. After inflammation currents are significantly increased (modified from Dang
et al., 2008 with permission).
12. Visceral hyposensitivity
While the investigation of the pathogenesis of visceral pain has
driven the study of visceral afferent hypersensitivity, the topic of
visceral hyposensitivity has been relatively neglected by all but a few
researchers. In humans hyposensitivity to visceral stimuli may
underlie constipation and defacatory problems. (Gladman et al.,
2009) Certainly there is impairment of somatic and probably visceral
afferent function in disorders such as diabetes mellitus. In incontinent
patients with diabetes, there is significantly impaired detection of
normal rectal filling (Caruana et al., 1991; Sun et al., 1996; Wald &
Tunuguntla, 1984). A recent paper examining rectal afferent function
in a rat model of diabetes suggests that there is significant impairment
of fibres that detect low threshold stretch, with relative preservation
of responses to near noxious levels of mechanical stimulation (Beyak
et al., 2009). The mechanisms for this are unclear, and this is an open
field for investigation. In addition there is ample evidence for
impaired satiety related afferent input to the CNS in diet induced
obesity in humans and animals (Covasa et al., 2000a,b), however there
has been little to no direct investigation of responses of satiety related
vagal afferents in models of obesity. Perhaps the impaired within meal
feedback signaling that appears to be present in both obese animals
and humans could be due to hyposensitivity of satiety related vagal
afferents innervating the stomach, liver and intestine. However this
hypothesis remains to be tested.
13. Summary and conclusions
Visceral afferents display a wide range of responsiveness to
mechanical and chemical stimuli. The basic determinants of excit-
ability are the membrane voltage gated sodium and potassium
channels, and the multitudes of mediators that act on them impact
the function of these channels directly or indirectly. Furthermore the
ability to change in the long term the properties of the determinants
of excitability results in states of hyper- and possibly hypo-excitability
and have important implications for a variety of disease states such as
visceral pain, diabetes and obesity.
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