Neuron

NeuroResource

Direct Targeted Quantitative Molecular Imaging

of Neurotransmitters in Brain Tissue Sections
Mohammadreza Shariatgorji,1 Anna Nilsson,1 Richard J.A. Goodwin,1,2 Patrik Källback,1 Nicoletta Schintu,3
Xiaoqun Zhang,3 Alan R. Crossman,4 Erwan Bezard,5 Per Svenningsson,3 and Per E. Andren1,*
1Biomolecular Imaging and Proteomics, National Center for Mass Spectrometry Imaging, Department of Pharmaceutical Biosciences,

Uppsala University, P.O. Box 591 BMC, 75124 Uppsala, Sweden

2AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire SK10 4TF, UK
3Center for Molecular Medicine, Department of Neurology and Clinical Neuroscience, Karolinska Institutet and Karolinska University Hospital,

17176 Stockholm, Sweden

4Faculty of Life Sciences, University of Manchester, Manchester M13 9PL, UK
5Université de Bordeaux, Institut des Maladies Neurodégénératives, UMR 5293 Bordeaux, France

*Correspondence: per.andren@farmbio.uu.se
http://dx.doi.org/10.1016/j.neuron.2014.10.011

SUMMARY Nuclear medicine imaging is a widely used tool for indirect

Current neuroimaging techniques have very limited
abilities to directly identify and quantify neurotrans-
mitters from brain sections. We have developed a
molecular-specific approach for the simultaneous
imaging and quantitation of multiple neurotransmit-
ters, precursors, and metabolites, such as tyrosine,
tryptamine, tyramine, phenethylamine, dopamine,
3-methoxytyramine, serotonin, GABA, glutamate,
acetylcholine, and L-alpha-glycerylphosphorylcho-
line, in histological tissue sections at high spatial
resolutions. The method is employed to directly
measure changes in the absolute and relative levels
of neurotransmitters in specific brain structures in
animal disease models and in response to drug treat-
ments, demonstrating the power of mass spectrom-
etry imaging in neuroscience.
INTRODUCTION
Small-molecule neurotransmitters such as the catecholamine
dopamine (DA), the amino acids g-amino butyric acid (GABA)
and glutamate (Glu), and acetylcholine (ACh) are critical chemi-
cal messengers that transmit signals between neurons. Changes
in their concentrations are associated with numerous normal
neuronal processes, such as sleep and aging, but also several
disease states, including Alzheimer’s disease, Parkinson’s
disease (PD), depression, drug addiction, and attention deficit
hyperactivity disorder. A better understanding of their relative
abundance and distribution would provide insights into these
complex neurological processes and disorders. At present, re-
searchers rely on indirect histochemical, immunohistochemical
(IHC), and ligand-based assays to detect these small-molecule
transmitter substances (de Jong et al., 2005; Falck et al., 1962;
Jones and Beaudet, 1987). The antibodies used in IHC often
have major limitations, such as an inability to distinguish be-
tween different transmitters (Keenan and Koopowitz, 1981).
visualization of the distribution, abundance, and activity of
neurotransmitters in the brain, relying on radiolabeled tracers
that emit gamma rays, which are detected using positron emis-
sion tomography (PET) or single photon emission computed
tomography scanners (Pimlott and Sutherland, 2011). Devel-
oping appropriate labeled compounds is often complicated,
particularly for studying endogenous chemical messengers
(Badgaiyan, 2011). There is often an inability to discriminate
between the labeled parent compound and a metabolite retain-
ing the label. Imaging techniques that can directly visualize the
localization of neurotransmitters and simultaneously quantitate
them without relying on a label would thus represent a scientific
breakthrough. Another significant advance in the molecular
analysis of neurological tissues would be the development of
a multiplex method that enables the simultaneous detection of
multiple endogenous compounds and pharmaceutical agents.
MALDI mass spectrometry (MS) imaging has been used
for direct molecule-specific compound detection, distribution
mapping, and identifying molecular species in tissue sections
(Caprioli et al., 1997; Cornett et al., 2007). It can generate multi-
plex full mass spectrum data at near-cellular spatial resolution.
Crucially, it provides a powerful tool for in situ visualization of
target compounds and for determining their abundance and
spatial distribution without requiring a great deal of a priori infor-
mation. Moreover, the data gathered during such experiments
can be used to simultaneously identify unknown endogenous
molecular changes due to disease or degeneration. MALDI-MS
can be used for the direct analysis of targets, ranging from small
molecules to large proteins (Schwamborn and Caprioli, 2010).
However, despite some notable successes, it is relatively insen-
sitive toward certain compound classes and sometimes suffers
from signal interference caused by matrix clusters/fragments
(Sugiura et al., 2012; Ye et al., 2013). To date, it has found only
limited applications in the study of neurotransmitters due to
their poor ionization efficiencies. As such, the development of
a reagent that would enable selective neurotransmitter derivati-
zation while also facilitating their detection by MS and acting
as an efficient matrix to assist their laser-induced desorption
and ionization would be a significant breakthrough.

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Here we describe a unique approach for the direct and
absolute quantitative imaging of classical and common neuro-
transmitters in histological brain tissue sections. We report
an example of the simultaneous mapping of biogenic amines
and amino acid chemical messengers following in situ derivati-
zation in brain tissue sections. The method is employed to
simultaneously measure changes in the levels of neurotransmit-
ters, including tyrosine, tryptamine, tyramine, phenethylamine,
DA, 3-methoxytyramine (3-MT), serotonin (5-HT), GABA, and
Glu, in specific brain structures of primates and rodents with
and without DA depletion and L-3,4-dihydroxyphenylalanine
(L-DOPA) treatment. In addition, we have developed a method
that can be used to image and quantitate ACh and an ACh
precursor, L-alpha-glycerylphosphoryl-choline (alpha-GPC) in
brain sections, as well as to study activity within cholinergic
pathways by measuring changes in ACh concentrations, imaged
at a 15 mm spatial resolution. Together, these experiments
demonstrate a significant advancement in the molecular imaging
arsenal available to neuroscientists.
RESULTS AND DISCUSSION
Without modification, most neurotransmitters are not directly
detectable by MALDI-MS due to their poor ionization efficiency
and the overlapping signals of isobaric compounds. To address
the limited ionization and desorption of neurotransmitters, we
developed a method for their in situ chemical derivatization.
Once modified, the neurotransmitters are readily ionized and de-
tected, enabling their identification and the quantitative mapping
of their distributions. Our strategy uses pyrylium salts such
as 2,4-diphenyl-pyranylium tetrafluoroborate (DPP-TFB), which
react selectively with primary amines to produce N-alkyl- or
N-aryl-pyridinium derivatives (Figure S1, available online). The
reaction proceeds under very mild conditions and occurs rapidly
at ambient temperature and pressure without any need for
stirring or agitation. Such conditions are required to preserve
the localization of endogenous compounds. The resulting
charged derivatives have laser desorption and ionization
efficiencies sufficient to enable the detection of endogenous
small-molecule primary amines (Johannesen et al., 2012; Quirke
et al., 1994), such as tyrosine, tryptamine, tyramine, phenethyl-
amine, DA, 3-MT, 5-HT, GABA, and Glu, simultaneously in a sin-
gle experiment. Importantly, the selective derivatization of the
primary amines separates their signals from those of overlapping
isobaric compounds. A MALDI-MS analysis of a representative
pyrylium derivatization reaction clearly showed that all of the
desired pyridinium ions were formed in high yields (Figure S1).
Furthermore, the DPP-TFB derivatives of endogenous primary
amines, including neurotransmitters and their metabolites and
precursors, undergo self-assisted laser desorption ionization.
As such, they are amenable to MALDI-MS imaging without
needing to be assisted by a matrix such as a-cyano-4-hydroxy-
cinnamic acid (CHCA).
The combination of pyrylium derivatization and multiplex data
acquisition generates hundreds of ion signals in each MALDI-MS
imaging experiment. This makes it possible to obtain images
showing the distribution of every individual ion within an m/z
window of 200 to 450 in the sample being studied (Figure 1A).
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Quantitative Molecular Imaging of Neurotransmitters
In addition to showing the distributions of known derivatives,
these images can be used to characterize unidentified ions
whose concentrations and localizations may be affected by
neurological events and diseases. We demonstrated this by
determining the localization of selected neurotransmitters in
brain tissue samples derivatized using DPP-TFB and also
2,4,6-trimethyl-pyranylium tetrafluoroborate (TMP-TFB) as a
confirmatory reagent. The identities of the signals for the neuro-
transmitter derivatives were verified by performing tandem
MS (MS/MS) experiments using derivatized synthetic standards
alongside the endogenous neurotransmitter. In all cases, the
product ions observed for the standards were identical to those
for the endogenous species. For example, both exogenous and
standard GABA generated a peak at m/z 208.1 and similar MS/
MS product ions when derivatized with TMP-TFB and m/z
318.1 and similar MS/MS product ions when derivatized with
DPP-TFB (Figure S1). Similarly, both endogenous and standard
DA yielded peaks at m/z 136 and m/z 232, respectively, after
derivatization with these reagents (Figure S1). In addition, the
elemental compositions obtained for the target ions from accu-
rate mass Fourier transform ion cyclotron resonance (FT-ICR)-
MS measurements were in good agreement with the expected
structures of the derivatized neurotransmitters (Tables S1 and
S2). Deuterated neurotransmitters were used as calibration stan-
dards to determine the absolute concentrations of the endoge-
nous neurotransmitters directly in brain tissue sections.
MALDI-MS Imaging of Neurotransmitters in Control
Rodent Brain Sections
The in situ neurotransmitter derivatization and imaging proce-
dure was initially demonstrated using coronal and sagittal mouse
and rat brain sections (Figures 1, S1, and S2). The distribution
of the pyrylium salt formed by derivatizing GABA with DPP-
TFB (m/z 318.1) in coronal rat brain tissue sections showed
that GABA is most abundant in the medial septum/diagonal
band region (MSDB) of the brain (Figure S2), which is consistent
with previous reports (Alreja et al., 2000). The MSDB provides a
major GABAergic input to the hippocampus (Alreja et al., 2000;
Qin and Luo, 2009). Imaging of sagittal rat brain sections (Fig-
ure 1B) revealed structures with high GABA concentrations,
including the hypothalamus, midbrain, and basal forebrain (Fig-
ure 1C). GABA is a dominant neurotransmitter in the hypothala-
mus and plays an important role in hypothalamic inhibitory
circuits (Decavel and Van den Pol, 1990). Moreover, the basal
forebrain and its substructures are reported to provide major
GABAergic inputs to the hippocampus (Alreja et al., 2000). The
importance of midbrain neurons in GABA recycling and the
corelease of GABA from the dopaminergic midbrain neurons
via plasma membrane uptake (Tritsch et al., 2014) may explain
the high GABA concentration in this region. Deuterated GABA
(D6-GABA) was used to determine absolute GABA concentra-
tions in rat brain tissue sections. The absolute concentration of
GABA in the coronal brain section as a whole was 5 nmol/mg,
while that in the MSDB region was 10 nmol/mg (Figure S2). These
values are consistent with previous results (Schaaf et al., 1985).
Other primary amine neurotransmitters, such as tyrosine,
tryptamine, tyramine, phenethylamine, DA, 3-MT, 5-HT, GABA,
and Glu, were detected and imaged in the same brain tissue
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sections that were treated with DPP-TFB to produce 2,4-diphe-
nylpyridinium derivatives. As before, this substantially increased
the neurotransmitter’s MALDI desorption/ionization efficiency
and generated a detectable signal at m/z 362.1 (Figure S1).
Glu was found in most structures of the brain but was mainly
localized in the striatal and cortex regions (Figure S2), which is
consistent with previous reports on the distribution of Glu trans-
porters (Bressan and Pilowsky, 2000; El Mestikawy et al., 2011;
Herzog et al., 2004). The identity of the Glu peak was verified by
MS/MS fragmentation of derivatized synthetic standards
(deuterated and nondeuterated) and the endogenous signal
(Figure S1).
Analyses of rat brain sagittal sections revealed high DA con-
centrations in the substantia nigra, striatum, and ventral pallidum
structures due to the nigrostriatal and mesolimbic DAergic path-
ways (Figure 1D). 3-MT, a methylated DA metabolite formed
by the action of the enzyme catechol-O-methyl transferase,
was also imaged and found to have the same distribution as
DA (Figure 1E). 3-MT was initially reported to be an inactive DA
Figure 1. MALDI-MS Imaging Data Display-
ing GABA, DA, and 3-MT and Distributions
in Rat Brain Tissue Sections
(A–I) The mass spectrum generated in a MALDI-
MS imaging experiment (A) contains hundreds of
ion signals associated with each individual pixel.
These can be used to create images showing the
distributions of each individual ion in the spectrum.
A sagittal tissue section from a rat brain (B) and the
associated relative abundances and distributions
of GABA (C), DA (D), and 3-MT (E). GABA was
found in all structures of the brain but was most
abundant in HY, BF and its substructures, and MB.
High DA concentrations were observed in SN,
CP, VS, and AON. The distribution of 3-MT
mirrored that of DA. DA imaging in a rat brain
coronal tissue section (F) revealed that it was most
abundant in the striatal region (G). By rescaling the
signal intensity of the latter image, it was possible
to detect DA in other parts of the brain, notably
in the A24, Pir, MS, and VDB. However, the
DA concentrations in these substructures were all
much lower than that in striatal region (H). The
intensities of the DA signals in each region of the
brain were compared to enable its relative quan-
titation. The inset bars show the rescaled DA signal
intensities for the Pir, MS and VDB, and A24
regions (I). MS data were acquired using either
a MALDI-TOF/TOF (A–E) or a MALDI-FT-ICR
mass spectrometer (G–I). Data are shown using a
rainbow scale, normalized against the total ion
count. Scale bar, 2 and 5 mm. Images were ac-
quired at spatial resolutions of 100 mm for sagittal
sections and 150 mm for coronal sections. Sub-
stantia nigra, SN; caudate-putamen, CP; ventral
striatum, STRv; anterior olfactory nucleus, AON;
cingulate cortex (area 24), A24; piriform cortex, Pir;
medial septal nucleus, MS; nucleus of the vertical
limb of the diagonal band, VDB; accumbens
nucleus, ACB; hypothalamus, HY; thalamus, TH;
hippocampus, HIPP; corpus callosum, cc; cere-
bral cortex, CTX; midbrain, MB; basal forebrain,
BF. See also Figure S1.
metabolite, but recent studies suggest that it is a neuromodula-
tor that acts as an agonist of the trace amine-associated recep-
tor 1 (Sotnikova et al., 2010). It is worth noting that distinguishing
between an endogenous compound and its metabolites is
very difficult when using techniques based on radiolabeled
compounds. In the coronal sections (Figure 1F), DA was mostly
localized in the striatal regions (Figure 1G), but it was also
present at lower concentrations in the cingulate cortex, piriform
cortex, medial septal nucleus, and the nucleus of the vertical
limb of the diagonal band (Figure 1H). The intensities of the DA
signals for selected regions (Figure 1F) were used to quantify
their DA levels in relative terms (Figure 1I), revealing that the
average DA concentration in the striatal region is about 40 times
higher than that in the cingulate cortex region.
Neurotransmitter MALDI-MS Imaging of Parkinson’s
Disease Models
To further demonstrate the potential of the derivatization/MALDI-
MS imaging methodology, it was used to analyze the distribution
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of neurotransmitters in experimental models of PD. When study-
ing neurodegeneration processes in PD models, it is desirable to
assess the integrity and function of the nigrostriatal DAergic
pathway. Various techniques have been used for this purpose,
including the quantitation of dopaminergic neurons and termi-
nals in the substantia nigra pars compacta and striatum by
immunostaining using an antibody against tyrosine hydroxylase
(TH), and the measurement of TH or DA transporters (DAT) in
striatal tissue extracts by immunoblotting (Ciliax et al., 1995;
Huot et al., 2007). Alternatively, the dopamine neurons can be
quantified by using autoradiography to measure the levels of
a radiolabeled DAT ligand (Boja et al., 1991). The best way to
quantitate DA is to use high-performance liquid chromatography
coupled with an electrochemical detector. However, this
approach provides no information on the neurotransmitter’s
anatomical localization. Dopaminergic terminals are heteroge-
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Quantitative Molecular Imaging of Neurotransmitters
Figure 2. MALDI-MS Images of Neurotrans-
mitters in Coronal Rat PD Model Tissue
Sections
(A–U) Imaging experiments were conducted
on brain tissue sections from unilateral sham-
lesioned-, unilateral 6-OHDA-lesioned, and uni-
lateral 6-OHDA-lesioned animals that were treated
with subchronic L-DOPA for 4 weeks, with the
final dose being given as deuterated (D3)-L-DOPA.
The images show the distributions of DA (A–F)
in sham-lesioned (A), 6-OHDA-lesioned (B),
and 6-OHDA-lesioned L-DOPA-treated animals.
Rescaling images (D–F) (with new max intensity
corresponding to 5% of the previous) made it
possible to determine the distribution of DA in
the 6-OHDA-lesioned side of the brain and in
structures with low DA concentrations, such as the
cortex. (D) Rescaled image from (A). (E) Rescaled
image from (B). (F) Rescaled image from (C).
Distribution of D3-DA (formed by the in vivo
breakdown of D3-L-DOPA is imaged in sham-
lesioned (G), 6-OHDA-lesioned (H), and 6-OHDA-
lesioned L-DOPA-treated animals (I). Distribution
of endogenous 3-MT is imaged in sham-lesioned
(J), 6-OHDA-lesioned (K), and 6-OHDA-lesioned
L-DOPA-treated animals (L), and distribution of
D3-3-MT (derived from D3-L-DOPA) is imaged
in sham-lesioned (M), 6-OHDA-lesioned (N), and
6-OHDA-lesioned L-DOPA-treated animals (O).
Distribution of GABA (P–R) is imaged in sham-
lesioned (P), 6-OHDA-lesioned (Q), and 6-OHDA-
lesioned L-DOPA-treated animals (R), and tyrosine
is imaged in sham-lesioned (S), 6-OHDA-lesioned
(T), and 6-OHDA-lesioned L-DOPA-treated ani-
mals (U). There are clear differences in the
concentrations of the studied amines in different
regions of the brain and between treatments. MS
images were acquired using a MALDI-FT-ICR
mass spectrometer. Data are shown using a
rainbow scale, normalized against the total ion
count. Scale bar, 2 mm; spatial resolution =
150 mm. See also Figure S2.
neously distributed in the striatum; in PD, it is mainly the
dorsolateral regions that exhibit reduced DA levels. Important
information might therefore be lost if the anatomical distribution
of DA is not determined. Our method allows the simultaneous
direct imaging and quantitation of primary amine neurotransmit-
ters in tissue sections from animal models of PD.
The PD models used in our experiments were generated
by injecting the neurotoxin 6-hydroxydopamine (6-OHDA) into
rodents’ medial forebrain bundles (Zhang et al., 2008) or by
intravenous administration of 1-methyl-4-phenyl-1,2,3,6-tetra-
hydropyridine (MPTP) to primates. Pyrylium derivatization
and MALDI-MS imaging were then used to quantify neurotrans-
mitter levels in rat brain sections by depositing known con-
centrations of deuterated standards onto vehicle control tissue
sections. The DA concentrations in the intact and 6-OHDA-
lesioned sides of rat striata were determined to be 124 pmol/mg
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Quantitative Molecular Imaging of Neurotransmitters
and 84 pmol/mg, respectively (Figure S2); these values are
consistent with previous results obtained by HPLC (Zhao and
Suo, 2008).
Pyrylium derivatization and MS imaging were used to evaluate
the neurochemical changes that occur in 6-OHDA PD models
with and without L-3,4-dihydroxyphenylalanine (L-DOPA) treat-
ment. Some of the model animals were treated with deuterated
L-DOPA (D3-L-DOPA), which is converted into deuterated
dopamine (D3-DA) in vivo. The derivatization/MALDI-MS imag-
ing protocol successfully distinguished endogenous DA from
both the administered D3-L-DOPA and D3-DA. Sham-lesioned
rat brain tissue sections had a normal DA distribution in their
striatal regions, but the intensity of the DA signal was signifi-
cantly lower in the lesioned side of the brain, with or without
D3-L-DOPA treatment (Figures 2A–2C). The distributions of
DA in the lesioned side of the brain and structures with low
DA levels, such as the cortex, were determined by rescaling
the intensities of the MS-derived images (Figures 2D–2F).
No interfering signal for D3-DA was detected in sham- or
6-OHDA-lesioned brain tissue sections (Figures 2G and 2H),
and the signal corresponding to D3-DA (derived from D3-L-
DOPA) was only observed in the nonlesioned sides of brains
treated with D3-L-DOPA (Figure 2I). This is presumably due to
the destruction of dopaminergic neurons, which are essential
for the uptake and conversion of L-DOPA to DA. The distribu-
tion images for 3-MT showed that its concentration was high
in the intact sides of the brain but lower in the lesioned sides
(Figures 2J–2L). The average concentration of 3-MT in the non-
lesioned sides of 6-OHDA-treated brains (Figures 2K and 2L)
was higher than that in sham-lesioned brains (Figure 2J). The
metabolic conversion of D3-L-DOPA to D3-3-MT was traced
by measuring the concentration of D3-3-MT, which was highest
in the nonlesioned side of the brain (Figure 2O); no interfering
signal was detected in untreated brains (Figures 2M and 2N).
This experiment also showed that D3-L-DOPA treatment
increased striatal GABA levels in the lesioned side of the brain
(Figure 2R) relative to those in sham- and 6-OHDA-lesioned
brain sections (Figures 2P and 2Q, respectively). Changes in
GABAergic signaling throughout the basal ganglia (including
the striatum and the substantia nigra pars reticulate) after the
destruction of dopaminergic neurons and L-DOPA treatment
have been extensively documented (Galeffi et al., 2003; Wang
et al., 2007). Tyrosine is the biosynthetic precursor of the
catecholamines and exhibited the same distribution as 3-MT
and DA (Figures 2S–2U).
Accurate mass FT-ICR-MS imaging was also performed on
selected regions of coronal brain sections, including the caudate
and putamen, of control and MPTP-lesioned primates (Figures
3A and 3B). The measured m/z values for selected neurotrans-
mitters matched the corresponding theoretical values with
sub-ppm deviations (Table S2). DA (Figure 3C) and 3-MT (Fig-
ure 3E) were localized in the caudate and putamen regions of
the brain. However, the concentrations of DA (Figure 3D) and
3-MT (Figure 3F) in the MPTP-lesioned brain sections were
around 30 and 100 times lower, respectively, than that in the
controls (Figure S3).
Several reports have discussed the varied roles of 5-HT in PD
(Fox et al., 2009). By reanalyzing the data from the experiments
used to study the distribution of DA and the effects of L-DOPA in
PD models, we were able to map the distribution of 5-HT in con-
trol (Figure 3G) and MPTP-lesioned (Figure 3H) brain tissue sec-
tions. 5-HT was localized to the nucleus basalis (Meynert), the
hypothalamic regions, and the substantia nigra. MPTP lesioning
increased its concentration in these regions by >40% (Figure S3).
Increases in 5-HT concentrations following MPTP-induced DA
denervation have previously been demonstrated by in vivo
microdialysis; it was suggested that they are due to the brain’s
compensatory processes and recovery mechanisms (Boulet
et al., 2008).
Increased levels of striatal GABA have been reported in
humans with PD, 6-OHDA-treated rats, and MPTP-treated pri-
mates. Striatal GABA levels increased by 70% in MPTP-lesioned
brains (Figures 3I, 3J, and S3) (Boulet et al., 2008); interestingly,
its concentration also rose in the hypothalamic regions. In addi-
tion to DA, 5-HT, and GABA, high mass accuracy FT-ICR imag-
ing maps were also generated for phenethylamine, tyramine, and
tryptamine (Table S2) in control and MPTP-treated primate
brains (Figure S3).
It is important to recall that while the derivatization/MALDI-MS
imaging technique makes it possible to simultaneously deter-
mine the distributions of multiple known species by interrogating
the data set from a single experiment in a targeted manner, it
can also provide information on as-yet unidentified molecular
species.
ACh Imaging and Analysis of the Distribution of
Cholinergic Neurons Using a Deuterated MALDI Matrix
While pyrylium derivatization provides a powerful tool for
studying primary amine neurotransmitters, some important
neurotransmitters, such as ACh and alpha-GPC do not contain
primary amines and so do not react with pyrylium cations. We
therefore developed an alternative method for their imaging.
MALDI-MS analysis generally requires the homogeneous appli-
cation of a matrix to the sample surface to facilitate the laser
desorption ionization process. One of the most widely used
matrices for MALDI-MS imaging of small molecules is CHCA.
Unfortunately, all matrices generate numerous readily detected
fragments and cluster ions in the low molecular weight region
of the mass spectrum, which can mask signals from target
analytes such as ACh (m/z 146.1). One way of avoiding such
target mass interference is to use a deuterated CHCA (D4-
CHCA) matrix (Shariatgorji et al., 2012), which shifts the inter-
fering signals by a minimum of 4 m/z units. This method was
used to facilitate the imaging of molecular ACh and alpha-GPC
(m/z 258.1) and thereby monitor changes in their abundance
in different parts of the brain during neurological events that
affect the cholinergic system. ACh and alpha-GPC were identi-
fied by MS/MS fragmentation (Figure S4).
Cholinergic neurons are anatomically ubiquitous. However, it
has been difficult to study their distribution in the CNS based
on the presence of ACh because of difficulties in detecting and
quantifying ACh in situ. Instead, cholinergic neuron distributions
have been studied by examining markers such as acetylcholin-
esterase (the enzyme that degrades ACh), muscarinic and nico-
tinic ACh receptors, and choline acetyltransferase (the enzyme
that catalyzes ACh synthesis). By using a deuterated CHCA
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matrix, we were able to directly investigate the cholinergic
neuron distribution in the brain based on the concentrations of
ACh in its various regions (Figure 4A). Imaging of rodent sagittal
sections taken from different levels of the brain revealed high
ACh concentrations in the frontal cortex, olfactory bulb, striatum,
hippocampus, medial habenula, tectum, thalamus, ventral
tegmental area, interpeduncular nucleus, pedunculopontine
tegmental nucleus, and the laterodorsal tegmental nucleus.
This is consistent with previous findings (Hammond et al., 1994).
High-Resolution MALDI-MS Imaging of ACh
High spatial resolution MALDI-MS imaging makes it possible to
map the distributions of neurotransmitters within substructures
of the brain with high spatial resolution. Using our methodology,
we determined the relative abundance of ACh in various sub-
structures of the hippocampus, cerebellum, and striatum at a
spatial resolution of 15 mm (Figures 4C–4E). The concentration
of ACh was greater in the gray matter of cerebellum than in the
white matter (Figure 4E). Certain subregions of the hippocampus
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Quantitative Molecular Imaging of Neurotransmitters
Figure 3. MALDI-MS Images of Neurotrans-
mitters in Coronal Primate PD Model Brain
Tissue Sections
(A–J) MALDI-MS images showing changes in
neurotransmitter levels in one hemisphere of a
coronal brain tissue sections from a control animal
(A) and an MPTP-lesioned animal (B). MALDI-FT-
ICR imaging of DA (C) and 3-MT (E) in control
brains showed that these compounds are most
abundant in the caudate and putamen structures.
Conversely, both DA (D) and 3-MT (F) are almost
absent in these regions after the destruction of
their dopaminergic neurons by MPTP-lesioning.
5-HT is mostly localized in the nucleus basalis
(Meynert) of the brains, and its concentration is
significantly lower in control brains (G) than in
MPTP-lesioned tissue sections (H). The 5-HT-rich
hypothalamic structure was also imaged in control
and MPTP-lesioned brain sections, revealing
that lesioning increased its 5-HT concentration.
The concentrations of GABA in the putamen and
caudate were significantly higher in the MPTP-
lesioned brain (J) than the control (I). All MS images
were acquired using a MALDI-FT-ICR mass
spectrometer. Data are shown using a rainbow
scale over a single range, normalized against the
total ion count. Scale bar, 10 mm; spatial resolu-
tion = 200 mm. Caudate, cd; putamen, Put. See
also Figure S3.
were also enriched in ACh, notably the
granule cell layer of the dentate gyrus
(DG-sg) and the pyramidal layer (sp) of
CA1 (Ammon’s horn) (Figures 4C and
4F). These subregions of the hippocam-
pus are delineated in Figure 4B, which
shows a MALDI-MS image generated
using data for a lipid with an m/z value
of 721.2.
The power of high spatial resolution MS
imaging was further demonstrated by
studying the distribution of ACh in the basal ganglia of the brain.
In the striatum, ACh serves as the transmitter of the large aspiny
interneurons, which have diameters of 20–60 mm (Kawaguchi,
1992) and comprise 1%–2% of the total striatal cell population
(Aosaki et al., 1998). While the cell bodies of the cholinergic inter-
neurons are relatively few in number, they densely innervate the
striatum with their extensive axonal arborizations (Holt et al.,
2005). As shown in Figure 4D, the striatal ACh concentrations
can be measured by MALDI-MS imaging with a spatial resolution
of 15 mm.
It is anticipated that the continuing development of MALDI-
MS imaging technologies will improve to a point that enables
the analysis of small sample areas of tissue sections at sub-
cellular resolution. Recently, it was demonstrated that MALDI-
MS instrumentation capable of imaging at resolutions < 1 mm
is technically feasible (Zavalin et al., 2012). Therefore, imaging
neurotransmitters at subcellular spatial resolution in the near
future would lead to further biological insights and make the
method even more compelling.
Neuron
Quantitative Molecular Imaging of Neurotransmitters
Caudate-putamen, CP; hippocampus, HIPP; cerebellum, CB; granule cell layer of the dentate gyrus, DG-sg; pyramidal layer of CA1, sp; cerebral cortex, CTX;
piriform area, PIR; striatum (ventral region), STRv; thalamus, TH; subiculum, SUB; stratum lacunosum-moleculare, Slm; stratum radiatum, Sr; stratum oriens, So;
cornu ammonis (field 2), CA2; cornu ammonis (field 3), CA3; polymorph layer of dentate gyrus, PO. See also Figure S4.
Imaging of ACh and Alpha-GPC in Brain Sections of
Tacrine-Administered Rodents
As a final illustration of the power of the deuterated matrix tech-
nique, it was used to investigate the distributions of ACh (Figures
5A and 5B) and alpha-GPC (Figures 5C and 5D) in sagittal sec-
tions of mouse brains from animals that had been treated with
10 mg/kg intraperitoneal tacrine (a centrally acting cholines-
terase inhibitor) and untreated control animals. The concentra-
tion of ACh in the tacrine-treated brains was approximately
seven times greater than that in the control brains, and the con-
centrations of alpha-GPC within brain structures decreased as
their ACh levels increased. In both the control (Figure 5A) and
tacrine-treated (Figure 5B) brain sections, the ACh concentration
was determined for selected brain structures using a standard
calibration curve created by spotting known quantities of deuter-
ated ACh (D9-ACh) onto consecutive control brain tissue sec-
tions (Figure S5). Absolute quantities of ACh (Figure 5E) and rela-
tive concentrations of alpha-GPC (Figure 5F) were determined
for the whole brain, hippocampus, caudate putamen, thalamus,
and cerebral cortex regions in both control and tacrine-treated
brains. The average measured concentration of ACh was consis-
tent with previous results (Stavinoha and Weintraub, 1974).
Conclusions
The methods presented herein represent powerful tools for the
quantitative imaging of neurotransmitters, including biogenic
amines, amino acids, and ACh in histological brain tissue sec-
tions. Pyrylium derivatization and deuterated CHCA matrices
Figure 4. High-Resolution MALDI-MS Im-
ages of ACh in a Sagittal Rat Brain Section
(A–F) MALDI-MS images of ACh in a rodent brain
sagittal tissue section. The relative distribution
and abundance of ACh determined by MS imaging
are consistent with existing data on the major
cholinergic pathways in the brain (A). ACh-rich
structures such as the HIPP, CP, CTX, and even
small structures such as the nuclei of the facial
nerves and pons are readily apparent in the
MALDI-MS image. High-resolution MALDI-MS
images (15 mm spatial resolution) of various
structures in consecutive brain tissue sections
were also acquired. Substructures of the hippo-
campus were delineated by high-resolution im-
aging of the distribution of a lipid with m/z 721.2
(B). The relative abundance and distribution of
ACh in the HIPP (C), a subregion of the CP indi-
cated by a dashed rectangular border in (A) (D),
and CB (E) were determined by high-resolution
imaging. The concentration of ACh in the gray
matter of CB is around five times greater than that
in the white matter. In the HIPP, high ACh con-
centrations are apparent in DG-sg and sp regions
(F). MS images were acquired using a MALDI-
TOF/TOF mass spectrometer. Data are shown
using a rainbow scale and are normalized against
the total ion counts; scale bars, 2 mm, 1 mm, and
500 mm; spatial resolutions = 100 mm and 15 mm.
extend the reach of MALDI-MS imaging, a technique with excel-
lent mass accuracy and resolving power, allowing it to be used in
the characterization of compounds that were previously prob-
lematic. Pyrylium derivatization converts low molecular weight
endogenous primary amines into readily ionized species of rela-
tively high molecular weight. This makes it possible to simulta-
neously image tyrosine, tryptamine, tyramine, phenethylamine,
DA, 3-MT, 5-HT, GABA, and Glu and other endogenous primary
amines in a single MALDI-MS experiment. The use of a deuter-
ated matrix allows one to sidestep problems caused by the
masking effect of matrix-derived ions in MALDI-MS analyses of
low molecular weight species, such as ACh, that lack a primary
amine group. Because MALDI-MS imaging can be performed at
a spatial resolution as low as 15 mm, these technologies make
it possible to measure the distributions of a wide range of neuro-
transmitters with high spatial resolution. The concentrations
of neurotransmitters in different subregions of the brain were
quantified by comparing the intensities of their signals to those
generated by known quantities of deuterated calibration stan-
dards. The techniques presented herein permit the simultaneous
quantitation and specific molecular imaging of multiple neuro-
transmitters in situ. As such, they should find diverse and imme-
diate applications in fields such as neuroscience, pharmacology,
drug discovery, neurochemistry, and pathology.
EXPERIMENTAL PROCEDURES
All chemicals and reagents were purchased from Sigma-Aldrich unless other-
wise stated and were used without further purification. Ketamine was obtained
Neuron 84, 697–707, November 19, 2014 ª2014 Elsevier Inc. 703

from Parke-Davis, and xylazine was obtained from Bayer. Deuterated analogs
of neurotransmitters and phenol-2,3,4,5,6-d5 (D5-phenol) were purchased
from CDN Isotopes. Water, methanol, and trifluoroacetic acid (TFA) were ob-
tained from Merck.
Animal Experiments
Male Sprague-Dawley rats (approximately 150 g; Scanbur) and C57BL/6J
male mice (3 months old, Charles River Laboratories) were housed in separate
air-conditioned rooms (with a 12 hr dark/light cycle) at 20 C and a humidity of
53%. Experiments were performed in agreement with the European Commu-
nities Council Directive of November 24, 1986 (86/609/EEC) on the ethical use
of animals and were approved by the local ethical committee at the Karolinska
Institute (N350/08).
Unilateral 6-OHDA lesioning of nigral dopaminergic axons was performed as
described previously (Zhang et al., 2008). Briefly, rats were anesthetized with
ketamine (80 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and pretreated with desi-
pramine (25 mg/kg, i.p.) and pargyline (5 mg/kg, i.p.). They were placed in a
stereotaxic instrument and injected with 6-OHDA (2.5 ml of a 5 mg/ml solution)
in the medial forebrain bundle (MFB) of the right hemisphere (AP = 2.8 mm,
ML = 2.0 mm, and V = 9.0 mm).
To determine the success of nigrostriatal denervation following unilateral
6-OHDA lesioning, a single injection of apomorphine (1 mg/kg, i.p.) was admin-
istered to the rodents 2 weeks after their treatment with 6-OHDA, and their
resulting contralateral rotations were measured over 30 min. Only rats that
performed more than 100 turns were used in subsequent studies. They were
killed by decapitation 4 weeks after the surgery, after which their brains
were removed, frozen in dry ice-cooled isopentane, and stored at 80 C until
needed.
704 Neuron 84, 697–707, November 19, 2014 ª2014 Elsevier Inc.
Neuron
Quantitative Molecular Imaging of Neurotransmitters
Figure 5. MALDI-MS Images of ACh and
Alpha-GPC Concentrations in Sagittal
Mouse Brain Sections from a Control Animal
and after Administration of 10 mg/kg of the
Cholinesterase Inhibitor Tacrine
(A–D) The absolute concentration of ACh in the
control brain section (A) is about seven times lower
than that observed following the injection of tacrine
(B). Conversely, the concentration of the ACh
precursor alpha-GPC is higher in the control
sample (C) than in the tacrine-treated brain (D). The
overall levels of alpha-GPC are reduced following
tacrine treatment. The absolute concentrations of
ACh in the control (A) and tacrine-treated (B)
mouse brains were quantified using a calibration
standard curve.
(E and F) Bar graphs showing the absolute con-
centration of ACh (E) and relative concentration of
alpha-GPC (F) with (black bars) and without (white
bars) administration of tacrine in whole brains and
selected structures. MS images were acquired
using a MALDI-TOF/TOF mass spectrometer. Data
are shown using a rainbow scale over a single
range, normalized against the total ion count. Scale
bar, 2 mm; spatial resolution = 100 mm. Caudate-
putamen, CP; hippocampus, HIPP; cerebral cor-
tex, CTX; thalamus, TH. See also Figure S5.
For D3-L-DOPA treatment experiments, 4 weeks
after the surgery, rats were treated with L-DOPA
(10 mg/kg, i.p.) and benserazide (7.5 mg/kg, i.p.)
once daily for 3 weeks. Animals were sacrificed
by decapitation 30 min after the last i.p. injection,
which was deuterated (D3)-L-DOPA (10 mg/kg)
and benserazide (7.5 mg/kg). Handling and storing
the brains was performed as described above.
Primate experiments were conducted using tissue from a previously pub-
lished brain bank (Fernagut et al., 2010; Porras et al., 2012; Santini et al.,
2010). Experiments were carried out in accordance with European Commu-
nities Council Directive of November 24, 1986 (86/609/EEC) for care of labora-
tory animals in an AAALAC-accredited facility following acceptance of study
design by the Institute of Lab Animal Science (Chinese Academy of Science,
Beijing, China) IACUC. Briefly, two female rhesus monkeys (Macaca mulatta,
Xierxing; mean age = 5 ± 1 years; mean weight = 5.3 ± 0.8 kg) were used.
One animal was kept untreated as control. The other animal received daily
MPTP (0.2 mg/kg, i.v.) according to previously published protocol (Bezard
et al., 1997, 2001a, 2001b). Following stabilization of the MPTP-induced syn-
drome, animal behavior was assessed as previously published (Ahmed et al.,
2010; Fasano et al., 2010; Fernagut et al., 2010; Muñoz et al., 2008; Porras
et al., 2012). The degree of parkinsonism was assessed using a validated
macaque clinical scale (Imbert et al., 2000). Animals were killed by sodium
pentobarbital overdose (150 mg/kg, i.v.), and brains were removed quickly
after death and immediately frozen by immersion in isopentane ( 45 C) and
stored at 80 C.
For the ACh experiments, mice that were 3 months old were injected with
saline or tacrine (10 mg/kg i.p.) and then sacrificed after 30 min according to
the procedure described above.
Tissue Section Preparation
The frozen brain tissues were cut using a cryostat-microtome (Leica
CM3050S, Leica Microsystems). Sagittal and coronal brain sections were
cut at a thickness of 14 mm. Tissue sections were transferred by thaw mounting
onto conductive indium tin oxide (ITO) glass slides (Bruker Daltonics) and
then stored at 80 C. Sections were desiccated at room temperature for
Neuron
Quantitative Molecular Imaging of Neurotransmitters
15 min prior to spotting of calibration standards, after which they were imaged
optically using a photo scanner (Epson perfection V500). The samples were
then coated with matrix for ACh experiments or derivatized with or without
matrix coating for primary amine imaging experiments. To enable the quanti-
tation of individual neurotransmitters, deuterated ACh, DA, or GABA dissolved
in 50% methanol was spotted onto the control tissue sections to serve as
calibration standards prior to derivatization and matrix application.
Derivatization of Calibration Standards and Endogenous
Primary Amines
DPP-TFB and TMP-TFB were dissolved in methanol to prepare 1 mg/ml stock
solutions. Derivatization was performed using solutions containing 150 ml of
the stock solution of DPP-TFB or TMP-TFB in 1.5 ml of 50% methanol,
buffered by 1 ml triethylamine (TEA). The derivatization solution was applied
to the tissue sections using an automatic sprayer (ImagePrep, Bruker Dalton-
ics). To facilitate the derivatization reaction and to avoid delocalization of the
endogenous compounds, reagents were added using a three-step process
involving spraying, incubation, and drying, which lasted for 2.5, 15, and 50 s,
respectively. The prepared slides were then incubated for 60 min in a chamber
saturated with the vapor arising from a 50% methanol solution. To acidify the
tissue sections, 1 ml of a solution containing acetic acid, water, and methanol
(10:45:45) was sprayed over the tissue using the same setup as above. The
treated tissue sections were then incubated once more in the methanol
vapor-saturated chamber for 30 min. Primate and D3-L-DOPA-treated rat
tissue sections were analyzed using a high concentration of DPP-TFB as reac-
tive matrix. DPP-TFB was dissolved in 1.2 ml of methanol to prepare 8 mg/ml
of the stock solution. This solution was diluted in 6 ml of 70% methanol con-
taining 3.5 ml of TEA. An automated pneumatic sprayer (TM-Sprayer, HTX
Technologies) was used to spray warm reagent over the dried samples. The
nozzle temperature was set at 80 C, and the reagent was sprayed (30 passes)
over the tissue sections at a linear velocity of 110 cm/min with a flow rate of
about 80 ml/min. Samples were then incubated for 15 min (dried by nitrogen
flow every 5 min) in a chamber saturated with the vapor arising from a 50%
methanol solution. Stock solutions of DPP-TFB and TMP-TFB (2 mg/ml in
methanol) and standards of DA, GABA, and Glu (1 mg/ml in methanol) were
used for in-solution derivatization experiments. Stock solutions of neuro-
transmitters (2 ml) and derivatization reagents (10 ml) were added to the solvent
containing 90 ml of 50% methanol, buffered by 0.1 ml TEA. The mixture was
incubated for 30 min at room temperature, followed by addition of 1 ml of acetic
acid. Samples were spotted (0.5 ml) to dry on stainless steel plates.
Matrix Coating and MALDI-MS Imaging Data Acquisition
CHCA (0.5 ml of 10 mg/ml in 50% ACN, 0.2% TFA) was spotted over dried
derivatized neurotransmitters on stainless steel plates. D4-CHCA was pre-
pared as reported previously (Shariatgorji et al., 2012). The MALDI-MS matrix
was applied to the tissue sections as follows. A 10 ml solution of 10 mg/ml
CHCA (for analysis of tissues with low concentration of derivatization re-
agent) or D4-CHCA (for analysis of ACh) in 50% ACN containing 0.2% TFA
was sprayed onto the tissues sections using a TLC sprayer. The sprayer
was maintained at a distance of 30 cm from the target during matrix coating,
and sections were allowed to dry between coating cycles. For high spatial
resolution (HSR) MALDI-MS imaging of ACh and low spatial resolution
(LSR) matrix-assisted imaging experiments, D4-CHCA and CHCA (5 mg/ml
dissolved in 50% ACN containing 0.2% TFA) were applied using an auto-
matic sprayer (TM-Sprayer, HTX Technologies). All parameters were opti-
mized in order to reach the best sensitivity and minimize delocalization of
target compounds. The nozzle temperature was set at 98 C (for HSR exper-
iments) and 90 C (for LSR experiments). The matrix was sprayed (eight
passes for HSR and six passes for LSR experiments) over the tissue sections
at a linear velocity of 110 cm/min with a flow rate of about 70 ml/min. The
quantity of matrix solution applied in this way was measured using an optical
sensor present in the automatic sprayer (ImagePrep, Bruker Daltonics) to
ensure that the same amount of matrix was applied to each slide. All
MALDI-MS imaging experiments were performed using either MALDI-TOF/
TOF (Ultraflextreme, Bruker Daltonics) or MALDI-FT-ICR (Solarix XR 12T,
Bruker Daltonics) mass spectrometer in positive ion mode using a Smart-
beam II 2 kHz laser. The mass spectrometer’s operating parameters were
set according to the manufacturer’s recommendations for optimal acquisi-
tion performance. The laser spot size was selected to yield intermediate
and sharp levels of focus (40 mm and 10 mm laser spot diameters) for
low and high spatial resolution analyses. The laser power was optimized at
the start of each run and then held constant during the MALDI-MS imaging
experiment. On-tissue and on-plate MS/MS fragmentation experiments
were performed in positive ion mode using the MALDI-TOF/TOF according
to the manufacturer’s recommended settings. Tissue sections were analyzed
in a random order to prevent any possible bias due to factors such as matrix
degradation or variation in mass spectrometer sensitivity. MS imaging
data were visualized using FlexImaging (Bruker Daltonics), version 3.0 for
TOF/TOF data and version 4.0 for FT-ICR data. Mass spectra analysis and
chemical formula calculations were performed in Data Analysis, version 4.2
(Bruker Daltonics). An in-house-developed software package was used for
data processing, normalization, and quantitation. Regions of interest were
manually defined in the analysis software using both the optical image and
MS imaging data (Källback et al., 2012). The concentration of the target
compounds in defined regions of interest were calculated by correlating their
signal intensities to a calibration curve derived from deuterated analogs of
the analytes spotted on control tissue sections considering the brain density
as 1.0 g/cm3 (Barber et al., 1970).
Autoradiographic Studies
Sections that were used to study the distribution of DAT (a dopamine trans-
porter) were preincubated in 50 mM Tris-HCl and 120 mM NaCl (pH 7.5) for
20 min and then incubated for 1 hr in the same buffer supplemented with
50 pM [125I] RTI-55 in the presence of 1 mM fluoxetine (a selective serotonin
reuptake inhibitor). The slides were washed twice for 10 s each in ice-cold
binding buffer, rapidly dipped in deionized water, and dried. The sections
were then exposed to Kodak BioMax MR films for 2 days.
SUPPLEMENTAL INFORMATION
Supplemental Information includes five figures and two tables and can be
found with this article online at http://dx.doi.org/10.1016/j.neuron.2014.10.011.
AUTHOR CONTRIBUTIONS
M.S. conceived the project and its essential ideas, assisted with the
MALDI-MS experiments and data evaluation, and contributed to discus-
sions. A.N. assisted with the MALDI-MS experiments and data evaluation
and contributed to discussions. N.S. conducted animal (mouse) and DAT
experiments and contributed to discussions. R.J.A.G. contributed to data
evaluation and discussions. X.Z. conducted animal (rat) experiments and
contributed to discussions. P.K. developed the quantitation software and
contributed to discussions. A.R.C and E.B. supervised the primate experi-
ments and contributed to discussions. P.S. supervised the rodent experi-
ments and contributed to discussions. P.E.A. supervised the overall
research, conceived the project and essential ideas, assisted with the
data evaluations, and contributed to discussions. All authors contributed
to the writing of the manuscript.
ACKNOWLEDGMENTS
The authors acknowledge Dr. Matthias Witt and Patrik Ek (Bruker Daltonics) for
assisting with FT-ICR-MS imaging and Dr. Andras Szabo and members of
Ubichem laboratory for synthesis of deuterated matrix. This work was
supported by the Swedish Research Council (Medicine and Health 2013-
3105, Natural and Engineering Science 2010-5421, and Research Infrastruc-
ture 2009-6050 to P.E.A.), AstraZeneca R&D, UK (to P.E.A. and A.N.),
Agence Nationale de la Recherche (ANR-12-BSV4-0001-01 to E.B.), and the
Michael J. Fox Foundation (to E.B.).
Accepted: September 29, 2014
Published: November 6, 2014
Neuron 84, 697–707, November 19, 2014 ª2014 Elsevier Inc. 705

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