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Neuroscience 152 (2008) 1024–1031

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TEREOLOGICAL ESTIMATES OF DOPAMINERGIC, GABAERGIC ANDLUTAMATERGIC NEURONS IN THE VENTRAL TEGMENTAL AREA,

UBSTANTIA NIGRA AND RETRORUBRAL FIELD IN THE RAT

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. G. NAIR-ROBERTS,a,b S. D. CHATELAIN-BADIE,a1

. BENSON,a H. WHITE-COOPER,a J. P. BOLAMc

ND M. A. UNGLESSa,d*

Department of Zoology, University of Oxford, South Parks Rd, Oxford,X1 3PS, UK

Cardiff School of Biosciences, Cardiff University, Cardiff, CF10 3US,K

Medical Research Council Anatomical Neuropharmacology Unit, De-artment of Pharmacology, University of Oxford, Oxford, OX1 3TH, UK

Medical Research Council Clinical Sciences Centre, Faculty of Med-cine, Imperial College London, Hammersmith Hospital, Du Cane Rd,ondon, W12 0NN, UK

bstract—Midbrain dopamine neurons in the ventral tegmen-al area, substantia nigra and retrorubral field play key rolesn reward processing, learning and memory, and movement.

ithin these midbrain regions and admixed with the dopa-ine neurons, are also substantial populations of GABAergiceurons that regulate dopamine neuron activity and haverojection targets similar to those of dopamine neurons. Ad-itionally, there is a small group of putative glutamatergiceurons within the ventral tegmental area whose functionemains unclear. Although dopamine neurons have been in-ensively studied and quantified, there is little quantitativenformation regarding the GABAergic and glutamatergic neu-ons. We therefore used unbiased stereological methods tostimate the number of dopaminergic, GABAergic and gluta-atergic cells in these regions in the rat. Neurons were iden-

ified using a combination of immunohistochemistry (ty-osine hydroxylase) and in situ hybridization (glutamic acidecarboxylase mRNA and vesicular glutamate transporter 2RNA). In substantia nigra pars compacta 29% of cells werelutamic acid decarboxylase mRNA-positive, 58% in the ret-orubral field and 35% in the ventral tegmental area. There wereurther differences in the relative sizes of the GABAergic pop-lations in subnuclei of the ventral tegmental area. Thus, glu-amic acid decarboxylase mRNA-positive neurons repre-ented 12% of cells in the interfascicular nucleus, 30% in thearabrachial nucleus, and 45% in the parainterfascicular nu-

Present address: S. D. Chatelain-Badie, Medical Research Counciladiation & Genome Stability Unit, Harwell, Oxfordshire, OX11 ORD,K.

Corresponding author: Tel: �44-(0)-20-8383-8299; fax: �44-(0)-20-8383-337.-mail address: mark.ungless@imperial.ac.uk (M. A. Ungless).bbreviations: GAD, glutamic acid decarboxylase; HB, hybridizationuffer; IFN, interfascicular subnucleus of the ventral tegmental area;P, interpeduncular nucleus; lSN, lateral substantia nigra; PBP, para-rachial nucleus; PBS, phosphate-buffered saline; PBS-T, phosphate-uffered saline containing 0.1% Tween 20; PN, paranigral nucleus;Li, rostral linear nucleus; RRF, retrorubral field; RT-PCR, reverse

ranscriptase–polymerase chain reaction; SN, substantia nigra; SNpc,ubstantia nigra pars compacta; TH, tyrosine hydroxylase; VGluT,

hesicular glutamate transporter; vSN, ventral substantia nigra; VTA,entral tegmental area.

306-4522 © 2008 IBRO. Published by Elsevier Ltd.oi:10.1016/j.neuroscience.2008.01.046

1024

Open access under CC BY licens

leus. Vesicular glutamate transporter 2 mRNA-positive neu-ons were present in the ventral tegmental area, but notubstantia nigra or retrorubral field. They were mainly con-ned to the rostro-medial region of the ventral tegmentalrea, and represented approximately 2–3% of the total neu-ons counted (�1600 cells). These results demonstrate thatABAergic and glutamatergic neurons represent large pro-ortions of the neurons in what are traditionally considereds dopamine nuclei and that there are considerable hetero-eneities in the proportions of cell types in the differentopaminergic midbrain regions. © 2008 IBRO. Published bylsevier Ltd.

ey words: VGluT2, GAD, reward, midbrain, basal ganglia.

entral midbrain dopamine neurons play key roles in re-ard processing, learning and memory and movement

reviewed in Albin et al., 1989; Wise, 2004). In addition,heir dysfunction is implicated in a number of disorders,ncluding Parkinson’s disease (DeLong, 1990), schizo-hrenia (Goldstein and Deutch, 1992) and drug addictionHyman et al., 2006; Luscher and Ungless, 2006). Theyxert their influence through dense cortical and subcorticalrojections, most notably to the prefrontal cortex and basalanglia (Swanson, 1982; Oades and Halliday, 1987; Albint al., 1989). Ventral midbrain dopaminergic neurons arerganized into three cell groups: retrorubral field (RRF)A8), substantia nigra (SN) (A9), and ventral tegmentalrea (VTA) (A10), which, in addition to dopamine neurons,ontain significant populations of GABAergic neurons (Na-ai et al., 1983; Mugniani and Oertel, 1985; Kosaka et al.,987). The GABAergic neurons subserve roles, both in theegulation of dopamine neuron activity through their localxon collaterals, and in the regulation of the activity oftriatal and cortical neurons through their projecting axonsBayer and Pickel, 1991; Van Bockstaele and Pickel, 1995;arr and Sesack, 2000; Melis et al., 2002; Szabo et al.,002; Korotkova et al., 2004). The possibility of the pres-nce of GABA interneurons, however, cannot be excluded.ecent studies have also identified a population of gluta-atergic neurons in the VTA that project to prefrontal

ortex (Hur and Zaborszky, 2005). These glutamatergiceurons are a discrete population (i.e. they are negative forarkers of dopamine and GABA (Yamaguchi et al., 2007),nd are mainly located in rostro-medial VTA (Kawano etl., 2006; Yamaguchi et al., 2007)). Electrophysiologicaltudies have shown that dopamine neurons typically haveroad action potentials and slow firing rates (Grace andunney, 1983) compared with GABAergic neurons which

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ave narrow action potentials and fast firing rates (Stef-e.

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ensen et al., 1998). More recently, neurons that haveroad action potentials and slow firing rates but are non-opaminergic have been described both in vitro (Cameront al., 1997; Margolis et al., 2006) and in vivo (Ungless etl., 2004). One possibility is that these non-dopaminergiceurons are glutamatergic.

Although the numbers and distribution of dopamineeurons in the ventral midbrain have been quantified ex-ensively (e.g. Swanson, 1982; German and Manaye,993; Harris and Nestler, 1996; Pioli et al., 2004), little

nformation is known about the numbers and distribution ofhe populations of GABAergic and glutamatergic neurons.his type of quantitative data is critical for our understand-

ng of the functional roles of the GABAergic and glutama-ergic neurons and for our understanding of the functionaloles of the midbrain dopamine nuclei. We therefore un-ertook a stereological study of neurons expressing aABAergic marker (glutamic acid decarboxylase, GAD)nd a marker of glutamatergic neurons (vesicular gluta-ate transporter 2 (VGluT2)) within major midbrain dopa-inergic nuclei in the rat. Our approach was to perform in

itu hybridization to detect mRNA for GAD and VGluT2,ombined with immunohistochemistry for tyrosine hydrox-lase (TH) to reveal dopamine neurons. This allowed us tolearly and reliably resolve individual cell bodies of definedhenotypes and then quantify them using unbiased stere-logy.

EXPERIMENTAL PROCEDURES

nimals

en male Sprague–Dawley rats, weighing approximately 250–00 g, were used in this study. Procedures were conducted inccordance with the Animals (Scientific Procedures) Act of 1986United Kingdom) and the Society of Neuroscience policy on these of animals in neuroscience research. Every effort was madeo use the minimum number of animals and to minimize suffering.

reparation of brains for in situ hybridizationnd immunohistochemistry

nimals were deeply anesthetized using a combination of ket-mine (70 mg/kg) and medetomidine (0.5 mg/kg). They wereranscardially perfused with a phosphate-buffered paraformalde-yde fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH.4). Brains were removed and post-fixed for 12 h in the sameolution at 4 °C. They were sectioned in the coronal plane at0 �m using a vibrating microtome (Vibratome 1500; Vibratome,t. Louis, MO, USA). Free floating sections were rinsed in phos-hate-buffered saline (PBS), cryoprotected in a series of sucrose–thylene glycol solutions (15% followed by 30% sucrose–ethylenelycol) and stored frozen in 30% sucrose–ethylene glycol at20 °C until use. Solutions used for the preparation of brains and

ubsequent in situ hybridization steps were treated with an RNasenhibitor (0.1% DEPC).

iboprobes for in situ hybridization

ense and antisense digoxigenin-labeled RNA probes for VGluT2,AD 65 kDa isoform and GAD 67 kDa isoform were prepared using

everse transcriptase–polymerase chain reaction (RT-PCR) andn vitro transcription. cDNA transcript sequences were identified

nd downloaded from the Ensembl Rat Genome server (http:// s

ww.ensembl.org/Rattus_norvegicus/index.html). Clustal multi-equence alignment analysis (Chenna et al., 2003) and Blast2-CBI (http://www.ncbi.nlm.nih.gov/blast/) searches were used to

dentify 300–600 bp gene-specific regions within each transcript.rimers flanking target transcript regions were designed using theector NTI software package (Invitrogen). Details of primer se-uences are shown in Table 1.

T-PCR

wo male Sprague–Dawley rats were deeply anesthetized with anverdose of sodium pentobarbital. The brains were carefully re-oved and the VTA, SN, striatum, cortex and cerebellum were

apidly dissected out. Tissue samples were snap-frozen in liquiditrogen and stored at �80 °C until use. Frozen tissue samplesere thawed in Trizol for total RNA extraction using Invitrogen’srizol system.

cDNA transcripts were produced from total RNA extractssing a reverse transcriptase (Superscript First-strand synthesisystem, Invitrogen). Gene-specific cDNA sequences were selec-ively amplified from total cDNA preparations through PCR primedith the appropriate gene-specific primers (Taq polymerase PCRit, Qiagen). PCR products were analyzed on a 1% agarose gel.omparison of actual product size with predicted size was used tostablish that successful amplification of the desired target se-uence had occurred.

ynthesis of labeled riboprobes

CR products were used as templates for riboprobe synthesissing an in vitro transcription kit (Digoxigenin RNA Labeling kit;oche Diagnostics, UK). Labeled RNA products were subjected tolkaline hydrolysis in 0.1 M carbonate buffer (40 mM NaHCO3,

0 mM Na2CO3) to yield approximately 100 bp length riboprobes.

n situ hybridization

1:6 series of coronal sections (40 �m) through the midbrain wasybridized for GAD mRNA (both isoforms) (n�4) or VGluT2RNA (n�4). During sectioning, sections were collected in singleells. Eight to 10 free-floating sections encompassing an entire:6 series were combined in a single well for processing. Rostral–audal order was re-established for mounting after processing bynspection of dopaminergic groups. The sections were washedeveral times in phosphate-buffered saline containing 0.1%ween 20 (PBS-T) before transfer to hybridization buffer (HB,0% formamide, 5� SSC, 100 �g/ml denatured sonicated salmon

able 1. Primer sequences

soform Sequence

AD65Forward 5=-CTGTAATACGACTCACTATAGGGCACTTCTCCAC

GCAACAGACC-3=Reverse 5=-ACTGAATTAACCCTCACTAAAGGCGCTGTCTGTT

CCGATCCCC-3=AD67Forward 5=-ACTGTAATACGACTCACTATAGGGGCAGGCCTC

CAAGAACCT-3=Reverse 5=-ACTGAATTAACCCTCACTAAAGGAGCCCCATCAC

CGTAGCAACC-3=Glut 2Forward 5=-GCGCTAATACGACTCACTATAGGGGATTTGGTT

GCGTTAAGAC-3=Reverse 5=-GCGCAATTAACCCTCACTAAAGGCGGTTATCCT

GCTTCTTCTC-3=

perm DNA, 50 �g/ml heparin, 0.1% Tween 20) for preincubation

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R. G. Nair-Roberts et al. / Neuroscience 152 (2008) 1024–10311026

1 h at 65 °C). Denatured digoxigenin-labeled riboprobes (dilutedn HB) were added to sections, with hybridization for 12 h at 65 °C.fter a series of high to low stringency washes, sections were

ransferred to PBS for detection of DIG-labeled riboprobes using aouse monoclonal anti-DIG antibody conjugated to alkaline phos-hatase (Roche Diagnostics, UK; 1:2000 in PBS applied overnightt 4 °C). Sections were washed in PBS and transferred to a highH buffer (HP, 100 mM NaCl, 100 mM Tris pH 9.5, 0.1% Tween0, 50 mM MgCl2). A staining solution made up from NBT/BCIPnitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phos-hate) phosphatase substrate (Roche) diluted in HP was applied

or 2.5 h. The phosphatase reaction was terminated by washing inBS.

Optimal concentrations and incubation times were deter-ined for each riboprobe in preliminary dilution series and time

ourse experiments (results not shown). An incubation time of.5 h at room temperature was found to produce specific labelingf cell bodies in known GABAergic or glutamatergic cell regions,ithout significant background. In situ hybridization proceduresere also carried out using sense DIG-labeled riboprobes toontrol for the occurrence of non-specific signals. No cellularabeling was observed in sections hybridized with sense ribo-robes (results not shown).

H immunohistochemistry

ollowing in situ hybridization, sections were further processed toeveal TH immunoreactivity. The primary antibody was a sheepnti-TH from Chemicon (1:5000 in PBS-T with 3% normal rabbiterum, overnight at 4 °C), followed by a biotinylated secondaryntibody (diluted 1:200, biotinylated rabbit anti-sheep, Vector Lab-ratories; Burlingame, USA) overnight at 4 °C. An avidin–biotineroxidase enzyme complex was prepared and applied according

o manufacturer’s instructions (Vectastain Elite ABC kit). Finally,ections were incubated for 5 min in a DAB/hydrogen peroxideubstrate solution (prepared according to manufacturer’s instruc-ions, Vectastain DAB substrate kit, Vector Laboratories). Sec-ions were mounted in an aqueous mountant (Vectashield, Vectoraboratories) and coverslipped. Coverslips were fixed in placesing clear nail polish. This TH antibody is widely used (e.g. Albérit al., 2004; Schober et al., 2007) and only labels cells in dopa-inergic regions. In control experiments where the primary or

econdary antibody was omitted no labeling was observed. Inddition, we have observed complete overlap between TH immu-olabeling and in situ hybridization for TH mRNA (data nothown).

ell counting

ptical fractionator sampling was carried out on a Leica DMLBicroscope equipped with a motorized stage and Lucivid attach-ent (40� objective). Sampling was implemented using the Ste-

eoinvestigator software package (MicroBrightField Inc; Williston,SA). Midbrain dopaminergic groups were outlined on the basis ofH immunolabeling, with reference to a coronal atlas of the ratrain (Paxinos and Watson, 2005). Boundaries separating nucleind subdivisions were identified based on the location of anatom-

cal landmarks (e.g. fiber tracts), the neurochemical content ofells and regional variations in cell density, orientation and mor-hology (in line with descriptions in previous cytoarchitectonictudies, e.g. Phillipson, 1979a,b). For example, within the VTA theoundary separating dorsal PN (paranigral nucleus) from ventralBP (parabrachial nucleus) was established based on visiblehanges in the density of dopaminergic cell bodies and theirendritic orientation. Cell bodies in PN tended to be more denselyacked and with dendrites oriented in a ventromedial direction, inontrast with dopaminergic neurons in PBP, which were moreparsely distributed and appeared to lack any consistent dendritic

rientation. n

Total estimates of the number of GABAergic and dopaminer-ic neurons were obtained from four brains processed for GAD initu hybridization and TH immunohistochemistry. Estimates oflutamatergic neurons were obtained from a further four brainsrocessed for VGluT2 in situ hybridization and TH immunohisto-hemistry. In order for our counting to encompass the full rostro-audal extent of the relevant midbrain dopamine nuclei, eight to 10ections from a 1:6 series were analyzed for each brain. A randomtart was ensured by using a different well for the beginning ofach series combined with the fact that the beginning of theollection of sections from the vibratome varied from brain torain. Damaged/lost sections, of which there were few, wereccounted for by the Stereoinvestigator software. Prior to begin-ing counting a section, average thickness was measured on aumber of sections from different brains. There was no significanthrinkage from the original 38–40 �m thickness, as the sectionsere mounted in aqueous medium rather than dehydrated. Auard height of 8 �m was used with a sampling brick depth of0 �m. Pilot studies were used to determine suitable countingrame and sampling grid dimensions prior to counting. Countingrame information: counting frame width (X) was 68.2 �m, heightY) was 75 �m, depth was 20 �m. Guard regions of 8 �m thickere used for counting frame depth. Counting frame size was seto roughly 5 to 10 neurons were counted per frame and wasaintained across all regions sampled. Sampling grid sizes wereetermined for different major regions (e.g. SN, VTA, and RRF)uring pilot studies to allow approximately 15–20 sampling siteser region per section. Sampling grid size was equal for subnucleiithin each major region. The sizes were: SN ((X) 366 �m, (Y)76 �m); VTA ((X) 342 �m, (Y) 192 �m); RRF ((X) 351 �m, (Y)51 �m). The software carried out these computations. System-tic random sampling was implemented by the Stereoinvestigatoroftware until the designated region of interest was covered.

NBT-BCIP-positive in situ–labeled cells (blue) and DAB-pos-tive immunolabeled cells (brown) were counted simultaneouslyithin each counting frame position. A cell was only marked andounted if a nucleus surrounded by cytoplasm filled with a coloredrecipitate was clearly visible. Cells were counted only if theyame into focus when racking the focus down through the sam-ling brick.

mage capture and processing

olor digital images were acquired using a Leitz Dialux 22 micro-cope equipped with a Coolsnap color camera (Photometrics;ucson, USA) and Openlab software (Improvision; Coventry, UK).ontrast and color balance were adjusted using Adobe PhotoshopS in order to include information-containing pixels and to reflect

he true appearance of the tissue as far as possible. Line drawingsllustrating the distribution VGluT2-positive neurons in individualections were prepared from images exported from Stereoinves-igator, using Adobe Illustrator drawing software.

RESULTS

eneral appearance of labeling

oth in situ hybridization and immunohistochemical reac-ions produced colored precipitates. In the NBT-BCIP initu reaction, a blue–purple precipitate was deposited inhe cell body of positive neurons. This blue–purple precip-tate was clearly absent from the nucleus, with little or noroduct found in dendritic processes. Immunolabeling us-

ng the DAB-peroxidase system resulted in the formation ofbrown precipitate within the cell body, axonal and den-

ritic processes. Although some light brown staining of the

ucleus occurred, the intensity was always less than that

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een in the cytoplasm allowing clear visualization of nuclein labeled cells.

In a small number of VTA neurons, analysis at highagnification revealed the presence of both blue andrown precipitates, suggesting a positive signal for both initu and immunohistochemical reactions. Thus, an aver-ge of approximately 0.58% of neurons examined in theTA appeared to be double-labeled in GAD in situ/TH

mmunohistochemical preparations. This suggests that amall proportion of dopaminergic neurons in the VTA co-xpress GABAergic or glutamatergic markers as has beeneported previously (e.g. see Gonzalez-Hernandez et al.,001; Olson and Nestler, 2007 (GAD/TH colocalization)nd Kawano et al., 2006 (VGluT2/TH colocalization)).owever, the cell labeling methods used in this study areot suitable for an accurate quantitative analysis of double-

abeled neurons as both the in situ and immunohistochem-cal reactions produced opaque precipitates that interferedith the detection of the other. An accurate analysis of theossible colocalization of TH, GAD, and VGluT2 will re-uire the use of different cell labeling methods that utilizeifferent markers e.g. fluorescence or autoradiographic

abeling. For this reason, the data we present here dealnly with cells that we could confidently identify as singly-

abeled for GAD, TH or VGluT2. The small number ofutative double-labeled cells encountered was excludedrom the cell counting protocol.

opaminergic (TH-positive) neurons

s has been shown extensively on previous occasions,mmunohistochemical labeling for TH revealed significantopulations of dopaminergic neurons within the midbrainuclei (see Table 2). The total number of dopamine neu-ons contained within the three midbrain groups was ap-roximately 71,000 (bilateral count). There were clear dif-erences in the numbers of dopaminergic neurons amonghe different cell groups. The VTA contained approximately0,000 dopaminergic neurons (bilateral count), the SNcearly 25,000 (bilateral count) and the RRF approximately

able 2. Stereological estimates of the numbers of TH-immunoposi-ive neurons and GAD mRNA-positive neurons in the VTA, SN andRF (bilateral counts)

ucleus Subnucleus TH GAD TH/GADratio

RF 6163�1562 8541�1923 0.72�0.08TA 40174�5315 21011�2453 1.92�0.18

PBP 16435�4178 7047�2071 2.41�0.23IFN 3475�386 464�35 7.47�0.52Rli 3310�801 1556�560 3.24�1.22PN 6202�499 3167�254 2.01�0.25PIF 10752�1701 8777�1711 1.28�0.11

N 24906�4209 41214�4268 0.60�0.06Pc 15772�3160 6400�1545 2.60�0.28Pr 2430�728 21657�3544 0.11�0.02V 2941�670 4814�1370 0.73�0.14L 3763�539 8343�867 0.46�0.06

Data obtained from four animals; values are means�S.E.M. Eight to

V0 sections from a 1 in 6 series were analyzed per brain.

100 (bilateral count). There were also clear differences inhe number of TH-positive neurons in different subdivisionsithin each dopaminergic nucleus (see Table 2). WithinTA, the PBP (parabrachial) and PIF (parainterfascicular)ubnuclei contained the largest number of dopaminergicells (bilaterally approximately 16,700 and 10,700 dopami-ergic cells, respectively). In the SN, there were strikingifferences in the numbers of dopaminergic neurons in theubdivisions: the large majority of SN dopaminergic neu-ons were in the substantia nigra pars compacta (SNpc)ubdivision with fewer in the lateral (lSN), ventral (vSN)nd substantia nigra pars reticulata (SNpr) subdivisions.

ABAergic (mRNA GAD-positive) neurons

he in situ hybridization revealed a number of distinctopulations of GABAergic neurons spread across the mid-rain (see Table 2). GAD-positive labeling was particularlyrominent in the interpeduncular nucleus (IP) (Fig. 1A) andhe SNpr (Fig. 1B, C). In the VTA and SNpc, a smallerontingent of GAD-positive neurons was observed scat-ered throughout the dense TH-positive cell populationsFig. 1E, F). While GAD-positive cell bodies were observednfrequently in the interfascicular subnucleus of ventralegmental area (IFN), a higher density occurred in lateralNPc (SNl) and RRF (Fig. 1D). The estimates of absoluteumbers of GAD-positive neurons in the major midbrainopaminergic nuclei are shown in Table 2.

In addition to differences in absolute numbers of GAD-ositive neurons between different midbrain nuclei, be-ause of the differences in numbers of TH-positive neu-ons the relative proportions of different classes of neuronsaried (Table 2). The largest proportion of GAD-positiveeurons relative to TH-positive neurons was in the SNpr,here approximately 70% were GAD-positive and 30%H-positive. On the other hand, the smallest proportion ofABAergic cells was in the SNpc, where 29% of neuronsere positive for GAD.

Differences in the relative numbers of GAD-positiveeurons were also seen among the different subnucleiTable 2). Thus, in VTA the greatest proportion of GAD-ositive neurons (relative to TH neurons) was found in theIF subdivision, and the lowest in the IFN. As a general

rend, the proportion of GABAergic neurons relative toopaminergic neurons was greater in more caudal VTAubnuclei (PIF, PN), as compared with rostral VTA areasrostral linear nucleus (RLi), IFN). Variations in the relativeumbers of GABAergic neurons among the subnuclei ofhe SN were even more striking: GAD-positive neurons inNpc were outnumbered by TH-positive neurons by a

actor of about 2.5:1. In SNpr, vSN and lSN the situationas reversed: GAD-positive neurons in these areas out-umbered TH-positive neurons by factors of 9:1, 1.3:1 and:1 respectively.

lutamatergic (VGluT2-positive) neurons

he in situ hybridization data revealed that VGluT2-posi-ive cell bodies were far less abundant in the midbrain thanither TH-positive or GAD-positive neurons. Midbrain

GluT2-positive labeling was especially prominent in the

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ed nucleus (RN), located dorsal to VTA (Fig. 2A, B) andhe medial terminal nucleus of the optic tract (not shown).owever, few if any VGluT2-positive neurons were ob-erved in the SNpr, SNpc and RRF. On the other hand,

clearly-labeled, though relatively small, population ofGluT2-positive cells was apparent in VTA (Fig. 2C, D).ur quantitative analyses show that VGluT2-positive neu-

ons in the VTA account for 1600 neurons on average1599�94.4, mean�standard error of the mean; bilateralstimate obtained from four animals), which representsnly 2–3% of all neurons in the VTA. The VGluT2-positiveeurons displayed a far more restricted regional distribu-ion than TH-positive or GAD-positive neurons. Thus, mostf VGluT2-positive neurons were located in the rostralarts of the VTA and their numbers decreased sharply inaudal VTA (Fig. 3A, B). A higher density of VGluT2-ositive neurons was present in medial versus lateral por-ions of VTA: VGluT-positive cells were relatively enrichedn the medial subdivisions (rostral VTA, RLi, PN and medialBP; Fig. 3A, B).

DISCUSSION

uantitative aspects of the numbers and distribution ofopamine neurons in the VTA, SN and RRF have beenidely studied, however, it is becoming increasingly evi-ent that within these nuclei are populations of non-do-aminergic neurons that are of functional importance. We

ig. 1. In situ hybridization for GAD (blue reaction product) combinedentral midbrain. (A) GAD positive neurons in the IP ventral to theAD-positive neurons are present in the SNpr but also among the deigher-power image of the SNpr showing the clear differentiation betweote that the in situ labeling is confined to the perikaryon, whereas t

mage of the RRF showing GAD-positive neurons (black arrows) amonhe VTA (E: paranigral VTA; F: parabrachial). Some of the GAD-positiD, E); 50 �m (C, F).

ave therefore undertaken simultaneous quantitative anal- o

ses of dopaminergic, GABAergic and glutamatergic neu-onal populations in the VTA, SN and RRF using unbiasedtereology. This simultaneous analysis revealed consider-ble variation in the relative numbers of dopaminergic andABAergic neurons at regional and subregional levels. Ineneral, we found that there are more GABAergic neuronshan previously assumed. Our data also provide the firstnbiased stereological count of the glutamatergic neurons

n the VTA.

omparison of our data to previous estimates

ne other quantitative study of the total number of neuronsn the SN has been undertaken using unbiased stereologyOorschot, 1996). Their reported estimate (unilateralount) of the total number of neurons in the A9 group was3,500 which is in close agreement with the figure of3,060 that we estimate for the total cell population con-ained in all subdivisions of A9 (SNPc, SNPr, lSN, vSN).ther studies have concentrated almost exclusively onopamine neurons, and generally they report lower num-ers of dopaminergic neurons than we do (e.g. Swanson,982; German and Manaye, 1993; Harris and Nestler,996). For example, in the VTA using total cell counts (withbercrombie’s correction) Swanson (1982) found a total of4,000 (unilateral estimate) of which two-thirds were do-aminergic (TH-immunoreactive) and one-third non-dopa-inergic. In spite of the difference in absolute estimates,

unohistochemistry for TH (brown reaction product) in sections of the) Low-power micrograph of the nigral complex. Large numbers of

tributed TH-positive neurons in the SNpc and pars lateralis (lSN). (C)positive neurons (black arrows) and TH-positive neurons (gray arrow).munolabeling also extends into dendritic processes. (D) High-powerwn TH-positive neurons. (E, F) High-power images of subdivisions ofs are indicated by black arrows. Scale bars�200 �m (A, B); 100 �m

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ur estimate that 35% of all cells in VTA are GABAergic

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grees fairly well with Swanson’s estimate that at leastne-third of VTA cells are non-dopaminergic.

In a more recent, computer-assisted study dopaminergiceurons labeled by immunohistochemistry were counted in

he same midbrain cell groups as those studied here: A8,9 and A10 in the rat (German and Manaye, 1993). Bilat-ral cell counts obtained were: A8, 2600, A9, 21,000, A10,0,400. While our population estimate for the A9 groupSNPc) is similar to their estimate (25,000 bilateral), ouropulation estimates for VTA and RRF are higher (RRF:100 vs. 2600; VTA 40,000 vs. 20,400). These, and other,tudies did not typically use unbiased sampling methods,nd other features such as computer-assisted countingay have resulted in the lower numbers reported. Weelieve it is likely that our study provides a more accuratestimate because we used unbiased optical fractionatorampling (for discussion of the merits of unbiased stereol-gy see Howard and Reed, 1998).

ABAergic neurons in the VTA, SN and RRF

ew formal attempts have been made to quantify the pop-lations of GABAergic neurons within the VTA, SN andRF (also reviewed in Kalivas, 1993; e.g. Ford et al.,995). In general, they report lower figures than ours (1090or RRF, 1080 for SNPc and 1200 for VTA; Ford et al.,995), a difference that is likely to be related to the use of

mmunohistochemical methods with antibodies againstAD. Our choice of in situ hybridization for GAD mRNA to

ig. 2. In situ hybridization for VGluT2 (blue reaction product) combiositive neurons in the red nucleus located dorsal–medial to theGluT2-positive neurons in the red nucleus (note the occasional TH-ositive neuron (black arrow) in the RLi of VTA. (D) A VGluT2-positars�200 �m (A); 100 �m (C); 50 �m (B, D).

dentify GABAergic cells stemmed from previous difficul- p

ies we and others encountered in resolving significant num-ers of immunolabeled cell bodies in midbrain nuclei using aumber of commercially available antibodies against GADNair-Roberts, Chatelain-Badie, Bolam and Ungless, unpub-ished observations). We believe, therefore, that our resultsrovide a more accurate account of the GABAergic popula-

ion. In addition we have presented an analysis by subregionhat was previously lacking. Overall, our data suggest that theuantitative importance of GABAergic neurons relative to dopa-inergic neurons within the VTA, SN and RRF has been un-erestimated in previous studies. GABAergic neurons thusepresent a large proportion of neurons in dopaminergictructures in the ventral midbrain and are likely to be critically

nvolved in the regulation of the activity of dopaminergic neu-ons and the output of these nuclei.

lutamatergic neurons in the VTA

he existence of a distinct population of glutamatergic neu-ons identified on the basis of VGluT2 in situ hybridization hasecently been described (Kawano et al., 2006; Yamaguchi etl., 2007). Our identification of, and estimate of the totalumber of the glutamatergic neurons in VTA is thus in sup-ort of these findings. Furthermore, the regional distributionf the VGluT2 cells observed in our study (Fig. 3A, B) isonsistent with the findings of Yamaguchi et al. (2007).

We found that the VTA (bilateral) contains about 1600eurons (i.e. 2–3% of the total population of neurons in theTA). Although this is a small absolute number and a small

immunohistochemistry for TH (brown reaction product). (A) VGluT2-ich is heavily immunostained for TH. (B) Higher-power image ofprocess coursing among the glutamatergic neurons). (C) A VGluT2-n (black arrow) in the parabrachial region of the VTA (PBP). Scale

ned withVTA, whpositive

roportion of the total population, they were highly en-

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Fda((nVefigure legend, the reader is referred to the Web version of this article.

R. G. Nair-Roberts et al. / Neuroscience 152 (2008) 1024–10311030

iched in medial parts of rostral VTA, with a density thatailed off very rapidly in more caudal and lateral areas.ittle is known about these neurons, except that theyroject to medial prefrontal cortex and somatosensory cor-ex (Hur and Zaborszky, 2005). The existence of a neuro-hemically-distinct rostral VTA cell group may be relevanto the functional differences between rostral and caudalubdivisions of VTA (e.g. Carlezon et al., 2000; Olson etl., 2005). It is important to characterize this population oflutamatergic neurons and relate them to other identifiedopulations of non-dopaminergic neurons (Cameron et al.,997; Ungless et al., 2004).

In vitro slice studies have demonstrated that stimula-ion of the VTA evokes glutamatergic excitatory synapticurrents (EPSCs) in the nucleus accumbens (NAc) (Chu-ma et al., 2004). These experiments, and others, haveeen taken to support the idea that dopaminergic neuronso-release glutamate and dopamine (Sulzer et al., 1998;oyce and Rayport, 2000; Chuhma et al., 2004). An alter-ative possibility is that the separate population of non-opaminergic glutamatergic neurons in the VTA is respon-ible for fast excitatory transmission in this pathway. Fur-hermore, although a small proportion of dopamineeurons have been reported to express mRNA for VGluT2

n one study (Kawano et al., 2006), the findings ofamaguchi et al. (2007) suggest that dopaminergic neu-

ons in the SN/VTA do not express VGluT2. We also foundery few neurons that co-express TH and VGluT2, but asiscussed in the Results section our methods were notell-suited to assess co-localization.

CONCLUSION

ur detailed quantitative data relating to dopaminergic,ABAergic and glutamatergic neurons in the VTA, SN andRF lay an essential foundation for future functional in-ights into the principles of operation of the microcircuits ofhese regions and their interactions with other regions ofhe brain. Resolving a complete picture of local and globaleurochemical interactions in the midbrain dopamine nu-lei will require the investigation of the function of individual

dentified cell types, their interaction with other cell typesnd their projections.

cknowledgments—This research was supported by the Medicalesearch Council UK, The Royal Society, and The Parkinson’sisease Society. We thank Nina Bausek for advice on the man-facture of riboprobes and Juan Mena-Segovia for assistance withrain processing.

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(Accepted 11 January 2008)(Available online 7 February 2008)