Expression of N-Methyl-D-AspartateReceptor Subunit mRNA in theHuman Brain: Mesencephalic

Dopaminergic Neurons

TIMOTHY J. COUNIHAN,1 G. BERNHARD LANDWEHRMEYER,2

DAVID G. STANDAERT,1 CHRISTOPH M. KOSINSKI,1 CLEMENS R. SCHERZER,1

LORRIE P. DAGGETT,3 GONUL VELICELEBI,3 ANNE B. YOUNG,1

AND JOHN B. PENNEY, JR.1*1Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts 02114

2Department of Neurology, Albert-Ludwigs-University, Freiburg, Germany3SIBIA Neurosciences, Inc., La Jolla, California 92037

ABSTRACTEvidence is accumulating that glutamate-mediated excitotoxicity plays an important role

in neuronal degeneration in Parkinson’s disease (PD). In addition, alterations in excitatoryamino acid neurotransmission in the basal ganglia contribute to the clinical manifestations ofmotor dysfunction. However, detailed knowledge of the anatomical distribution and subtypespecificity of glutamate receptors in the dopamine neurons of human substantia nigra (SN)has been lacking. In order to test the hypothesis that selective expression of particularN-methyl-D-aspartate receptor (NR) subunit mRNA contributes to the differential vulnerabil-ity of specific neuronal populations to excitotoxic injury in PD, we have used a quantitativedual label, in situ hybridization technique with ribonucleotide probes to examine the cellulardistribution of NR subunit mRNA in postmortem human mesencephalic dopaminergicneurons from subjects with no known neurological disorder. Analysis of both film autoradio-grams and emulsion-dipped sections demonstrated significant labeling of nigral neurons foreach NR subunit. Neuronal labeling was most intense for the NR1 and NR2D subunits, withlow level labeling for the remaining subunits. In addition, we examined four subregions of theventral mesencephalon for differential expression of NR subunit mRNA. For all NR subunits,the pars lateralis (PL) exhibited the most intense signal, while neurons of the ventral tiersubstantia nigra pars compacta (SNpc) failed to demonstrate a preponderance of a particularsubunit. These results demonstrate that NRs are expressed to a significant degree indopaminergic neurons of the SN and that their distribution does not correlate with thecharacteristic pattern of neuronal degeneration in PD. J. Comp. Neurol. 390:91–101, 1998.r 1998 Wiley-Liss, Inc.

Indexing terms: NMDA; receptor subtypes; Parkinson’s disease; in situ hybridization;

excitotoxicity

There is increasing evidence for involvement of theglutamatergic system in the pathogenesis of Parkinson’sdisease (PD; Albin and Greenamyre, 1992; Beal, 1995).Particular interest has centered on the ionotropic N-methyl-D-aspartate receptor (NR), in part because of its unusualphysiologic properties and complex pharmacology but alsobecause of its role in mediating excitotoxic cell damage(Choi and Rothman, 1990). NRs are hetero-oligomers ofproteins derived from two gene families: NR1, which isessential for channel activity, and NR2, which modulatesthe properties of channels (Nakanishi, 1992; Hollmann

and Heinemann, 1994). NR1 subunits are encoded by asingle gene, which contains three variably spliced regions,yielding eight distinct NR1 protein isoforms (Karp et al.,

Grant sponsor: USPHS; Grant numbers: NS 31579, AG 11337; Grantsponsor: DFG; Grant numbers: Ko 1696/1-1, SFB 505.

*Correspondence to: Dr. John B. Penney, Jr., Department of Neurology,Massachusetts General Hospital, Fruit Street, Boston MA 02114.E-mail: penney@helix.mgh.harvard.edu

Received 17 March 1997; Revised 31 July 1997; Accepted 5 August 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 390:91–101 (1998)

r 1998 WILEY-LISS, INC.

1993; Planells-Cases et al., 1993; Foldes, 1994; Zukin andBennett, 1995; Hess et al., 1996). NR2 subunits areencoded by four different genes, NR2A-D (Ishii et al., 1993;Monyer et al., 1994). In vivo, NRs are thought to becomposed of an NR1 subunit, together with differentcombinations of NR2 subunits (Buller et al., 1994). Thefunctional properties of NR channels, such as bindingaffinities for agonists and antagonists and the threshold ofvoltage-dependent Mg11 blockade, are determined by theconstituent NR2 subunits (Kutsuwada et al., 1992; Ishii etal., 1993; Buller et al., 1994; Laurie and Seeburg, 1994).

There is evidence that NRs are present on dopaminergicneurons of the substantia nigra pars compacta (SNpc)(Ball et al., 1994; Standaert et al., 1994; Ulas et al., 1994;Christoffersen and Meltzer, 1995) and that they are lost asthe dopamine neurons are destroyed in PD (Difazio et al.,1992). Furthermore, NR activation is involved in stimulat-ing the electrical bursting activity, which regulates striataldopamine release (Krebs et al., 1991; Johnson et al., 1992;Overton and Clarke, 1992) as well as the expression oftyrosine hydroxylase (TH; Arias et al., 1992).

In addition to the important role of glutamate in thephysiologic regulation of movement, it has been suggestedthat excitotoxic neuronal injury, mediated by glutamate,may be partially responsible for the loss of dopaminergicneurons in PD (Albin and Greenamyre, 1992). NR chan-nels regulate the transmembrane flux of calcium ions,which can trigger a number of intracellular processes thatmay contribute to excitotoxic death (Choi and Rothman,1990). Moreover, activation of NRs is toxic to dopamineneurons in culture (Marey-Semper et al., 1995), and NRantagonists have been shown to be protective in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) models of PD(Turski et al., 1991; Brouillet and Beal, 1993).

In the rodent brain, NR1 and NR2 subunit mRNA’sexhibit differential regional expression (Standaert et al.,1994; Landwehrmeyer et al., 1995; Standaert et al., 1996),but the cellular localization of these receptors in humanSN is unknown. In view of current anticipation that NRantagonists may have a promising neuroprotective role inPD, we have determined the NR phenotype of groups oftyrosine hydroxylase-containing neurons from the ventralmesencephalon of normal human brains by using duallabel in situ hybridization histochemistry.

MATERIALS AND METHODS

Postmortem specimens

Tissue blocks from the brains of seven donors with nohistory of neurological illness were obtained from theBrain Tissue Resource Center, McLean Hospital, Belmont,MA, and the Michigan Brain Disease Program, Ann Arbor,MI. The subjects consisted of four females and three males,age 58623 years (mean 6 standard deviation); postmor-tem delay time of 1766 hours. At autopsy, the brain wasdivided in the mid-sagittal plane and the brainstem wascut transversely at the level of the exit of the oculomotornerve. A single 5 mm block from one side was quick-frozenin chilled isopentane and stored at 270°C until use.

Generation of probes

All of the NR probes were constructed from complemen-tary DNA (cDNA) subclones of human NR subunits iso-lated at SIBIA Neurosciences, Inc. (La Jolla, CA). The NR1probe was derived from a 4297 base pair cDNA (Hess et al.,

1996), spanning the entire coding sequence of NR1, butwhich did not contain the alternatively spliced exon 5(Zukin and Bennett, 1995). The NR2A probe was derivedfrom a cDNA spanning nucleotides (nt) -1159-2134 of thecoding region of the NR2A subunit (Hess et al., 1996). BothNR2B (Hess et al., 1996) (nt -209-5258) and NR2C (L.P.Daggett, unpublished observation; Genbank accessionnumber U77782; nt -24-4153) probes were derived fromconstructs containing the entire open reading frame ofthese genes. For NR2D, a probe spanning approximately95% of the open reading frame (Genbank accession num-ber U77783) was used. Antisense and sense probes weregenerated from opposite strands of the linearized plasmidconstructs by run-off transcription using RNA polymeraseT3 or T7, respectively.

A DNA template for synthesizing an RNA probe for TH(Grima et al., 1987) was constructed using a polymerasechain reaction (PCR) strategy that is a modification of themethod of Sitzmann and LeMotte (1993). ComplementaryDNA was prepared from whole human brain poly A1mRNA (Clontech, Palo Alto, CA) by using a ReverseTranscriptase Reaction Kit and oligodeoxythymidine prim-ers (Promega, Madison, WI) as directed by the manufac-turer. Software analysis by using Oligo 4.0 (NationalBiosciences, Inc., Plymouth, MN) was used to optimizeprimer design. Outer primers consisting of 20mers (Table1) specific for the targeted gene segment and amplifying asegment of 1090 base pairs were used to amplify DNAobtained by reverse transcription of human brain poly AmRNA (Promega). The product of this amplification servedas starting material for amplification with an inner primerpair, consisting of 43mers (Table 1), 20 bases that werespecific for the target, and 23 bases that encoded promot-ers for the RNA polymerases SP6 or T7. The final templatesize was 456 base pairs, which recognized all three formsof the human TH mRNA (Grima et al., 1987). All primerswere prepared by using an Applied Biosystems (FosterCity, CA) 392 DNA synthesizer. Amplification with outerprimer pairs was conducted by using 10 ng cDNA, 0.5 pmolof each primer, 200 µM deoxynucleotide triphosphates,and 2.0 units of Taq DNA polymerase in assay buffer B(Fisher Scientific, Pittsburgh, PA) in a total volume of 50µl. Magnesium ion concentrations of 1.5 µM were used.After 36 cycles of amplification, the products were studiedby electrophoresis on 1.5% agarose gels stained withethidium bromide. Amplification with inner primer pairswas conducted in a similar manner, using 1 µl of the outerprimer product in place of the human brain cDNA. Theinner primer product was purified by using the WizardPCR Prep system (Promega), diluted to 0.3 µg/µl in 10 mMTris-HCl, pH 8.0, plus 1 mM ethylenediamine tetraaceticacid (EDTA; TE) buffer and stored at 220°C.

TABLE 1. Primers Used for Generation of TyrosineHydroxylase (TH) DNA Template

OUTER PRIMERS (58-38) Bases 257–1327

Upper primer GCC GTG CTA AAC CTG CTC TTLower primer TGT CCT TGG CGT CAC TGA AG

INNER PRIMERS (58-38) Bases 308–698

Upper primer CTG TAA TAC GAC TCA CTA TAG GGTCCC GAG CTG TGA AGG TGT T

Lower primer GGG ATT TAG GTG ACA CTA TAG AATTCC AGG TGG CAA TCT CCT C

92 T.J. COUNIHAN ET AL.

Labeling of RNA probes

Radioactively labeled RNA probes to each NR subunitwere synthesized by in vitro transcription of 0.75 µg ofeach template by using 160 units SP6, T3 or T7 RNApolymerase (Promega) in a reaction mixture containing330 µCi of [35S]CTP (. 1,000 Ci/mmol, Dupont-NEN,Boston, MA), 100 pmoles unlabeled CTP, 10 nmoles each ofATP, GTP, and UTP, and 20 units RNasin (Promega) inTranscription Buffer (Promega, 40 mM Tris-HCl, pH 7.9, 6mM MgCl2, 2 mM spermidine, and 10 mM NaCl) with 10mM dithiothreitol (DTT; total volume 21 µl). This reactionmixture was incubated for 1 hour at 37°C, and then thetemplate was digested by addition of 1 unit RQ1 DNase(Promega) and incubated at 37°C for an additional 15minutes. The RNA product was subsequently isolated byprecipitation in ethanol and resuspended in 75 µl of TEbuffer with 100 mM DTT and 80 units RNasin. Incorpora-tion of the radioactive label was assessed by liquid scintil-lation counting. Radiolabeled probes were stored at 270°C and used within one week.

The digoxygenin-labeled RNA probe for human TH wassynthesized in a similar manner by using 0.6 µg oftemplate DNA, 160 units of SP6 or T7 polymerase, 7.0nmoles digoxygenin 11-UTP, 13.0 nmoles of unlabeledUTP, 20 nmoles each of ATP, CTP, and GTP (DIG RNALabeling Mix, Boehringer-Mannheim, Indianapolis, IN),and 40 units RNasin in a total volume of 20 µl ofTranscription Buffer. This mixture was incubated for 2hours at 37°C, the template was digested with 1 unit RQ1DNase at 37°C for 15 minutes, EDTA was added to a finalconcentration of 20 mM, and the labeled probe was puri-fied by precipitation from ethanolic solution. The resultingpellet was resuspended in 50 µl of TE buffer with 40 unitsRNAsin, and stored at 220°C.

Northern blot analysis

Northern blot analysis was used to evaluate the specific-ity of the TH RNA probe (Fig. 1). Probes for Northernanalysis were prepared from PCR products by randomprimer extension, by using the Prime-a-Gene system (Pro-mega) and [32P]dCTP. Incorporation of radioactive probewas assessed by liquid scintillation counting. A commer-cially available human brain Northern blot (MTN Blot III,Clontech), containing approximately 2 µg of polyA1 RNAper lane from eight different brain regions, was hybridizedwith labeled probe in ExpressHyb hybridization solution(Clontech) for 1 hour. Blots were washed four times in 23SSC (13 5 0.15 M NaCl, 0.015 M sodium citrate), 0.05%sodium dodecyl-sulfate (SDS) for 10 minutes each at roomtemperature, followed by two washes in 0.13 SSC, 0.1%SDS at 50°C for 20 minutes, and then exposed to X-ray film(Hyperfilm-MP, Amersham, Arlington Heights, IL) at270°C with an intensifying screen for 3 days (Fig. 1).

In Situ Hybridization

Sections (12 µm) of human midbrain were cut by using acryostat and stored at 270°C. The slides were warmed toroom temperature, fixed in 4% paraformaldehyde in 0.1 Mphosphate buffer, pH 7.4 (10 minutes), washed in threechanges of 0.1 M phosphate-buffered saline containing 0.9g per liter NaCl, pH 7.4 (PBS, 5 minutes each), acetylatedin 0.1 M triethanolamine, pH 8.0, with 0.25% acetic

anhydride (10 minutes), rinsed in PBS (5 minutes), dehy-drated through graded ethanol solutions (2 minutes each),and delipidated in chloroform. Combinations of 35S-labeledand digoxygenin-labeled probes were hybridized in a buffercontaining 50% formamide, 0.3 M NaCl, 10 mM Tris, pH8.0, 5 mM EDTA, 10% dextran sulfate, 13 Denhart’ssolution, 100 mM DTT, 0.1% SDS, 0.1% sodium thiosul-fate, 100 µg/ml salmon sperm DNA, 250 µg/ml yeast tRNA,and 250 µg/ml yeast total RNA at 50°C for 4 hours. Thequantity of radioactive probe included was adjusted toachieve 150,000 cpm/µl hybridization buffer, whereas forthe digoxygenin labeled probe, 1 µl probe stock was addedto each 100 µl of hybridization buffer. After hybridization,sections were washed in 23 SSC at room temperaturebriefly, and then in 0.13 SSC at 70°C for 1 hour. They werethen treated with RNase A, 100 µg/ml (in 0.5 M NaCl, 10mM Tris-HCl, and 1 mM EDTA, pH 7.2) for 30 minutes at37°C, rinsed in RNase buffer for 15 minutes, and washedtwo more times in 0.13 SSC at 70°C for 30 minutes each.Detection of the digoxygenin label was performed by usingrabbit anti-digoxygenin antisera coupled to alkaline phos-phatase (1:1,000 dilution, 5 hours at room temperature,Boehringer Mannheim), followed by incubation in a sub-strate solution containing nitro blue tetrazolium (0.34mg/ml) and bromo-chloro-indole-phosphate (0.18 mg/ml)in 100 mM Tris-HCl, 100 mM NaCl, and 50 mM MgCl2, pH9.5 for 9 hours at room temperature. After completion ofthe digoxygenin detection, slides were rinsed briefly inwater and 70% ethanol and dried. Film autoradiogramswere prepared by apposing the slides to Hyperfilm b-Max(Amersham) for 3 to 7 days. Slides were then dipped inIlford K5 emulsion (Polysciences, Inc., Warrington, PA),diluted 1:1 with distilled water, stored at 4°C, and devel-

Fig. 1. Northern blot analysis of human tyrosine hydroxylase (TH),by using human brain multiple tissue poly A1 mRNA (Clontech),probed with the PCR amplified product labeled with 32P, as describedin the text. Numbers indicate the position of RNA size standards inkilobases (kb). An intense band is noted, about 2 kb in size, in the lanecorresponding to substantia nigra. Two very faint bands of similar sizeare also visible in the lanes corresponding to caudate and subthala-mus.

NR SUBUNITS IN MIDBRAIN DOPAMINE NEURONS 93

oped at 8 weeks. Specificity of the hybridizations wasmonitored by the use of sense strand probes and RNAasedigestion of samples.

Analysis of in situ hybridization

Emulsion autoradiograms were examined under bright-field illumination. Two sections from each of the sevenbrains were included in the analyses. Neurons from fourcell groups were analyzed: the medial paranigral nucleus(PN, analogous to the ventral tegmental area of therodent), dorsal and ventral tiers of the SNpc, and parslateralis (PL). Regional boundaries of the SN were inter-preted according to the schemes of Olszewski and Baxter(1954), with additional reference to Bogerts (1981) andGibb and Lees (1991).

Quantitative analysis of NR subunit mRNA expressionwas performed by using a computer-assisted image analy-sis system (M1, Imaging Research, St. Catharine’s, On-tario; Landwehrmeyer et al., 1995). The image analysissystem was used first to outline the soma of each labeledneuron and then was focused on the overlying grains. Boththe area of the neuronal profile, in µm2, as well as thenumber of grains present were digitized and used tocompute the intensity of the labeling of each neuron, ingrains per 1,000 µm2. The grains could be differentiatedfrom the underlying melanin by their different plane offocus, as well as by using the program filter settings. Thecenter of each region of interest was identified at lowmagnification; neurons were then analyzed in adjacentfields using a 1003 water immersion lens (Leitz, Deerfield,IL), counting from the center toward the periphery of eachregion, until 30 digoxigenin-labeled cells had been counted.The intensity of the autoradiographic background wasmeasured by using a circular sample of 315 µm2, whosesize was chosen to correspond closely to the overall meanarea of the neuronal profiles. Data from 30 near-adjacentregions of this size overlying the crus cerebri were ob-tained from each section studied.

The intensity of labeling in each of the populations ofneurons were compared to each other and to backgroundlabeling by using a 2-way analysis of variance with re-peated measures for brains and regions, with a signifi-cance level of 5%. The number of neurons counted wastreated as the repeated measure (Leuba et al., 1989).Post-hoc tests (Fisher’s Predicted LSD) were used tocalculate main effects for regions. The analyses wereperformed on a commercially available statistics softwarepackage (SuperANOVA, Abacus Concepts, Inc., Berkeley,CA). For figure presentation purposes, photographic im-ages were digitized, arranged as a montage, and labeledusing standard image editing software (Adobe Photoshop3.0, Mountain View, CA).

RESULTS

Northern blot analysis

Northern blot analysis of the TH probe demonstrated anintense band in the lane corresponding to the SN (Fig. 1),of approximately 2.0 kilobases in size, which is consistentwith the known size of human TH (Grima et al., 1987) andrat TH mRNAs (Grima et al., 1985). Two faint bands of thesame size are also visible in the lanes corresponding tocaudate and subthalamic nucleus. The band in the lanethat corresponds to the caudate is probably a reflection ofthe population of TH-positive neurons known to be present

in the striatum (Dubach et al., 1987). The reason for theband in the subthalamus is uncertain but may be due todissection contamination of the subthalamus by nigraltissue.

Regional distribution of NR subunitmRNA expression

Inspection of film autoradiograms showed different dis-tributions of labeling with the NR subunit probes acrossthe midbrain (Fig. 2). Hybridization with each of the NRprobes produced discernible signal in neurons of the SNpc.Intense signal was also seen in the PL. Low level signalwas detected in the red nucleus and retrorubral areas.Hybridization with the NR1 subunit probe produced in-tense signal in all neuronal subpopulations of the SNpc(Fig. 2B). The apparent discrepancy in labeling intensitybetween the dorsal and ventral tier neurons on autoradio-grams is due to the lower cell density of the dorsal tierneurons than is the case in the ventral tier. In somesections where the central gray substance was identifiable,a modest signal was noted. Hybridization with the probefor NR2A produced only a faint signal throughout themidbrain (Fig. 2C). The NR2B (Fig. 2D) and NR2C (Fig.2E) specific probes produced only a modest signal in theSNpc, while hybridization with the NR2D specific probeproduced an intense signal within all nigral areas (Fig.2F). There was minimal labelling of the SN pars reticulata(SNpr) regions with the NR2C and NR2D subunit specificprobes. Control studies using hybridization with senseprobes to each NR subunit, in addition to pretreatment ofsections with RNase, failed to produce a signal signifi-cantly above background.

Cellular distribution of NR subunitmRNA expression

Inspection of emulsion autoradiograms following duallabel hybridization with each NR subunit probe, in combi-nation with a digoxigenin-labeled mRNA probe for TH,showed that variable clusters of silver grains were seenoverlying the neurons labeled with TH (Figs. 4, 5). Withinthe neuronal subpopulations of the SNpc, there was a widevariation in the extent of TH-staining. Neurons of theventral tier showed prominent cytoplasmic staining,whereas many neurons of the dorsal tier and PL tended tobe more heavily melanized with relatively little cytoplas-mic TH-staining. Occasional scattered TH-labeled neu-rons were seen that were not labeled with the isotopicprobe. In addition to the SNpc neurons, TH staining wasseen in the smaller neurons of the retrorubral area andperiaqueductal gray substance. The majority of theseneurons were also labeled with the isotopic probes, espe-cially NR1 and NR2D subunits. Within the regions of theSNpc, no neurons were identified that were labeled withsilver grains but which were TH-negative.

For quantitative analysis, four regions of the SN wereanalyzed (Fig. 2A): PN, dorsal and ventral tier SNpcneurons, and PL. Significant labeling of TH-positive neu-rons compared to background (P,.05 by ANOVA) wasdetected for all regions for the NR1 and NR2D 35S-labeledprobes. In the case of the NR2A and NR2B probes, labelingof only the PL reached statistical significance over back-ground, while with the NR2C probe, all regions exceptneurons of the dorsal tier SNpc demonstrated statisticallysignificant signal, compared to background (Table 2).There was some variability in the extent of labeling among

94 T.J. COUNIHAN ET AL.

brains for each of the 35S-labelled probes, although therewas no correlation with either the age of the patient orpostmortem delay time (data not shown). In addition, twobrains were analysed at different levels (at the level of theemergence of the oculomotor nerve and at the level of thedecussation of the superior cerebellar peduncle) to exam-

ine for rostro-caudal differences in NR subunit mRNA geneexpression. No differences were observed in signal be-tween rostral and caudal sections for any of the 35S-labelled probes (data not shown).

NR1 probe. Hybridization with a probe common to allisoforms of NR1 resulted in significant signal over allregions examined (Table 2, Figs. 3, 4). Quantitative analy-sis of each region showed intense labeling of PL andventral tier neurons, followed by the PN and dorsal tier(F[4,6] 5 10.08, P,.0001). Post-hoc tests (Fisher’s Pro-tected LSD) showed no significant differences in signalbetween dorsal and ventral tier neurons, although thenumber of grains overlying neurons of the PL was signifi-cantly higher than over the dorsal tier SNpc neurons.

NR2A probe. A weak hybridization signal with theprobe for NR2A was seen over all regions (F[4,6] 5 3.80,P,.02). Only neurons of the PL demonstrated labeling

Fig. 2. Low magnification (1.253) photomicrograph of a transversesection through human mesencephalon at the level of the exit of theoculomotor nerve, probed with digoxygenin-labeled TH (A), and filmautoradiograms hybridized with 35S-labeled NR-specific antisense

RNA probes for NR1, NR2A, NR2B, NR2C, and NR2D (B–F). Regionsanalysed are indicated: Paranigral nucleus (PN), dorsal (Dor), andventral (Ven) tier neurons of the substantia nigra pars compacta(SNpc), and the pars lateralis (PL). Scale bar 5 4 mm for A–F.

TABLE 2. Mean Grain Density/1,000 µm2 (Mean 6 standard error of themean) for Each NR Subunit in Different Subregions of the Substantia

Nigra Pars Compacta

NR1 NR2A NR2B NR2C NR2D

PN1 118 6 6* 24 6 2 14 6 1 16.2 6 1.1* 105.7 6 6.9*DORSAL 105 6 4* 12 6 1 14 6 1 9.3 6 0.8 78.5 6 2.2*VENTRAL 130 6 5* 12 6 1 16 6 1 10.3 6 0.6* 75.9 6 2*PL1 168 6 11* 34 6 2* 41 6 2.5* 15 6 1.5* 137 6 5.7*BKGD1 14 6 1 8 6 0.4 4.8 6 0.5 4 6 0.3 18.7 6 0.7

*P , 0.05, compared to background.1PN, paranigral nucleus; PL, pars lateralis; BKGD, background.

NR SUBUNITS IN MIDBRAIN DOPAMINE NEURONS 95

Fig. 3. Quantitative analysis of NR subunit mRNA hybridizationsignals in neuronal subgroups of the human ventral mesencephalon.Hybridization signal intensity is expressed as grain density (silvergrains/1,000 µm2 of neuronal profile), with error bars indicatingstandard error of the mean. Four regions were analyzed: paranigralnucleus (PN), dorsal (Dor) and ventral (Ven) tier neurons of the

substantia nigra pars compacta (SNpc), and the pars lateralis (PL).Signal intensities that were not statistically different (P,.05) frombackground are designated by white bars. Neuronal groups that differsignificantly from PL are marked with an asterisk, and those thatdiffer significantly from PN are designated with a cross.

96 T.J. COUNIHAN ET AL.

that was statistically significant compared to background.Post-hoc tests showed significant differences in signalintensity between PL and SNpc neurons.

NR2B probe. Hybridization with the NR2B proberesulted in a weak signal across all regions with theexception of the PL (F[4,6] 5 6.04, P,.002). As with theNR2Aprobe, quantitative analysis revealed labeling signifi-cantly above background only for PL neurons. Regionalcomparisons showed significant differences between PLand the other neuronal groups examined.

NR2C probe. The number of grains overlying neuronswas low for all nigral regions (although greater thanbackground except for dorsal tier SNpc neurons) followinghybridization with the NR2C probe (F[4,6] 5 7.04, P,.001).There were significant differences in NR2C mRNA geneexpression between neurons of the PN and SNpc usingpost-hoc tests.

NR2D probe. Hybridization with the NR2D proberevealed intense labeling in all midbrain neurons exam-ined (F[4,6] 5 7.21, P,.001; Fig. 5). As with the other

probes, neurons with the highest grain density werelocated in the PL, followed by the PN, dorsal and ventraltier neurons. There were significant inter-regional differ-ences in signal between the PL and neurons of the SNpc.

DISCUSSION

By using a dual label in situ hybridization technique, wehave observed that the genes encoding NR subunits ex-hibit differential patterns of expression in dopamine neu-rons of the human midbrain. For regional analysis of NRsubunit mRNA expression, we examined four well-definedareas of the SN: the PN, the ventral and dorsal tierneurons of the SNpc, referred to by Olszweski and Baxter(1954) as the pars a and pars b, respectively, and the PL. Inthe normal SN at the level of the oculomotor nerve, theseneuronal cell groups are relatively easily defined by boththeir morphology and melanin content, as seen by lightmicroscopy (Fig. 2; Gibb et al., 1990). In these studies, wehave found a significant NR subunit mRNA expression in

Fig. 4. Expression of NR1 mRNA in dopaminergic neurons of thehuman ventral mesencephalon. Each panel shows autoradiographicsilver grains produced by a 35S-labeled probe for NR1, as well asalkaline phosphatase reaction product produced by hybridization of

digoxygenin-labeled TH probe. Four regions are shown (A–D): para-nigral nucleus (PN), dorsal (Dor), and ventral (Ven) tier neurons of thesubstantia nigra pars compacta (SNpc), and the pars lateralis (PL).Scale bar 5 30 µm.

NR SUBUNITS IN MIDBRAIN DOPAMINE NEURONS 97

dopaminergic neurons of the SN. Analysis of inter-regionaldifferences showed that for each NR subunit, the PL wasthe most intensely labeled, while the dorsal neurons of theSNpc had low grain density for all subunits. No significantdifference in signal intensity was found between dorsaland ventral tier neurons for any of the NR subunits.

Attempting to compare the abundance of mRNA be-tween probes for a given region can be difficult, as theintensity of the hybridization signal is not only affected bythe specific activity of the probe and the abundance of thetarget, but also by the relative preservation of a particularmRNA in the histologic process, and the efficiency ofhybridization of the probe to the target. (Young and Kuhar,1986; Tecott et al., 1994). The NR probes varied little intheir number of cytosines (<1500), with the exception theNR2A probe (227); this could result in underrepresenta-tion of the radioactive signal. The intensity of hybridiza-tion signal may be variable when using human tissue, inwhich patient age, antemortem factors, and postmortemdelay time may influence mRNA stability. A number ofprecautions were taken to ensure as much uniformity ofhybridization as possible between sections: a single batchof label was used for each probe and all sections for a givenprobe were processed in parallel. For the mRNAs ofinterest, we used RNAprobes rather than oligodeoxynucleo-tides, as the former confer a greater sensitivity of hybrid-ization. Keeping the limitations outlined above in mind,one may compare the relative signal intensities obtainedwith the five NR probes. Neurons of the SN expressedmRNA of all NR subunits to a varying degree, with theNR1 probe resulting in intense labeling throughout thedopaminergic neurons of the SN. The NR2D probe alsoresulted in dense labeling of TH-positive cells in allsubregions but showed maximal expression in the PL andPN; NR2A, NR2B, and NR2C subunits are expressed inlow levels across all regions studied. A potential concern isthe degree of cross-hybridization between probes, particu-larly in view of the 40–50% sequence homology betweenmembers of the NR2 family (Mori and Mishina, 1995). Byusing Northern blot analysis, Kosinski et al. found nosignificant cross hybridization between any of the sameprobes used in this study with the exception of minimalcross-hybridization of the NR2C probe with the NR2DmRNA (see Kosinski et al., 1997).

The dual label in situ hybridization method employedhere allows for the simultaneous visualization of twomRNAs within the same neuron and permits quantitativeanalysis of the hybridization signal produced by the 35S-labelled probes. Although the majority of midbrain dopa-minergic neurons are readily identified microscopically bythe presence of neuromelanin, not all dopamine neuronsare melanized (Hirsch et al., 1988). While single label insitu hybridization only allows for quantitative determina-tion of silver grains overlying the neuromelanin, whichmay underestimate the total grain count per neuron, theuse of nonisotopic digoxigenin labeled probe for human THidentifies the full perikarial profile and allows for a morerepresentative quantitative analysis. TH is the rate-limiting enzyme in the synthesis of all catecholamines,and as such is not a specific marker for dopaminergicneurons. However, since mesencephalic TH immunoreac-tive neurons lack immunohistochemical staining for dopa-mine β-hydroxylase (required for the conversion of dopa-mine to norepinephrine), they are assumed to bedopaminergic (Pearson et al., 1983).

Overall, the data presented here are similar to previousfindings in the rodent (Standaert et al., 1994). In the rat,the NR1 subunit is expressed throughout the brain includ-ing the SN; of the NR2 subunits, only NR2D, and to alesser extent NR2C, showed high levels of expression inthe SN. The NR1 probe used in the present study wasselected to hybridize to all splice variants; hence the highlevel of expression does not take into account the differentsplice forms, which are known to have a more restrictedpattern of expression in the rat SN (Standaert et al., 1993).

The abundant signal for NR2D in the SN is of particularinterest because of its unusual expression profile andfunctional properties. In contrast to the other NR sub-units, the 2D transcript is present in embryonic structuresof the rodent central nervous system (Monyer et al., 1994)and declines to low levels in the adult, where expression isrestricted principally to hindbrain structures. In addition,these channels exhibit extremely long offset decay times(agonist specific deactivation time) as well as weak Mg11

blockade and insensitivity to blockade by polyamines(Williams, 1995). These unique physiological and pharma-cological properties may confer an integrative function onthese receptors, particularly in view of their prominentexpression on interneurons (Standaert et al., 1996) andduring development. Interestingly, recombinant NRs con-taining the NR2D subunit are known to have a loweraffinity for MK801 than do other NR receptors (Laurie andSeeburg, 1994). Involvement of NR2D-containing recep-tors in the regulation of motor function in vivo has beenconfirmed by recent investigators by using mice deficientin the e4 gene, the murine homologue of NR2D, whichexhibit reduced spontaneous behavioral activity in anopen field test (Ikeda et al., 1995). Recent work usingtransfected HEK-cell lines has shown that heteromeric NRcalcium responses are subunit dependent (Grant et al.,1996); in cells transfected with NR1/2C or NR1/2D subunitcompositions, no intracellular calcium responses to NRagonists were observed.

Relevance to selective vulnerability ofneurons in Parkinson’s disease

Excitotoxic effects mediated by NRs have been sug-gested to have a role in neurodegenerative disordersincluding PD (Greenamyre and O’Brien, 1991; Beal, 1995).So-called ‘‘weak’’ excitotoxicity may occur as a consequenceof defective energy metabolism, leading to persistent acti-vation of NR channels and excessive calcium influx, thelatter being a potent mediator of cell death. A characteris-tic feature of PD is that the severity of injury to dopamineneurons varies greatly in different subregions of the SN(Fearnley and Lees, 1991). In PD, neuronal loss is mostsevere in the caudal and ventrolateral SN, and thisregional selectivity of PD is relatively specific, contrastingwith predominant loss of dorsal tier neurons in ageing.Kish et al. (1988) found more severe dopamine loss in themid and caudal putamen of PD brains than in other basalganglia areas. Hirsch and Agid (1988) reported that mela-nized midbrain neurons are more likely to degenerate inPD than non-melanized neurons, and that within theSNpc, the ventral tier of neurons is more likely to degener-ate than the dorsal tier, even though the latter containmore melanin. Neurons of the PN and PL are relativelyspared until late in the disease. Recent immunohistochemi-cal and in situ hybridization studies in the squirrel mon-

98 T.J. COUNIHAN ET AL.

key demonstrated that NR1 receptors are more dense onthe SNpc ventral tier neurons than in the neurons of thedorsal tier or VTA (Paquet et al., 1997). Further studieshave suggested that dorsal tier SNpc neurons may berelatively resistant to the degenerative process becausethese neurons express the calcium-buffering protein calbin-din-D28k (Yamada et al., 1990), although the distribution ofthese proteins has been disputed (McRitchie and Halliday,1995).

We hypothesized that selective expression of particularNR subunits might contribute to the differential vulnerabil-ity of specific neuronal populations to excitotoxic injury inPD. The present study demonstrates selective expressionof NR subunits in human SN, both in terms of the NRphenotype (predominantly NR1 and NR2D) as well asdifferential regional expression (the PL exhibits the mostabundant signal for each subunit). Furthermore, the datademonstrate that the neurons that are most vulnerable todegeneration in PD do not appear to contain either aspecific NR phenotype, or an excess of NRs that wouldaccount for their selective vulnerability.

There is an increasing body of evidence to suggest thatNR antagonists might play a significant role in the treat-ment of PD (Greenamyre and O’Brien, 1991; Uitti et al.,1996). NR antagonists protect nigral neurons in MPTPmodels of PD (Turski et al., 1991; Brouillet and Beal,1993), suggesting a possible clinical application as neuro-protective agents in PD. In addition, they may have a rolein the symptomatic therapy of PD by pharmacologicalantagonism of the overactive subthalamic nucleus, whoseprojections are glutamatergic (Bergman et al., 1990;Brotchie et al., 1991). Thus, pharmacological modulationof glutamatergic neurotransmission in PD may representan important therapeutic strategy both in terms of retard-ing pathological progression and improving clinical fea-tures of motor dysfunction. Treatment of patients withnonspecific NR antagonists, however, can lead to unaccept-able side effects, in part on account of the NR’s importancein learning and memory. The possibility exists of usingselective NR antagonists therapeutically without encoun-tering significant side effects. Recent pre-clinical studiesusing NR2B antagonists show some promise (Fischer et

Fig. 5. Expression of NR2D mRNA in dopaminergic neurons of thehuman ventral mesencephalon. Each section shows autoradiographicsilver grains produced by a 35S-labeled probe for NR2D, as well asalkaline phosphatase reaction product produced by hybridization of

digoxygenin-labelled TH probe. Four regions are shown (A–D): para-nigral nucleus (PN), dorsal (Dor) and ventral (Ven) tier neurons of thesubstantia nigra pars compacta (SNpc), and the pars lateralis (PL).Scale bar 5 30 µm.

NR SUBUNITS IN MIDBRAIN DOPAMINE NEURONS 99

al., 1996; Loeschmann et al., 1996; Vartanian and Boxer,1996), although their clinical usefulness as neuroprotec-tive agents may be limited by the relative low density ofNR2B receptors present on nigral neurons. With theemerging understanding of the physiological and pharma-cological properties of NRs, it may become possible toexploit the unique receptor subtype localization in the SNto provide a potent therapeutic agent to treat PD.

ACKNOWLEDGMENTS

The authors thank the Brain Tissue Resource Center,McLean Hospital, and the Michigan Brain Disease Pro-gram for providing the brain tissue. We also thank Z.Hollingsworth and J. Kerner for excellent technical assis-tance. This work was supported by USPHS grants NS31579 and AG 11337, and a Cotzias fellowship from theAmerican Parkinson Disease Association to D.G.S.. C.M.K.and G.B.L. are the recipients of DFG grants Ko 1696/1-1and SFB 505, respectively. T.J.C. is the recipient of aNational Parkinson Foundation fellowship.

LITERATURE CITED

Albin, R.L., and J.T. Greenamyre (1992) Alternative excitotoxic hypothesis.Neurology 42:733–738.

Arias, M.J., F.D. Martinez, and J. Aceves (1992) Glutamate stimulation oftyrosine hydroxylase is mediated by NMDA receptors in the ratstriatum. Brain Res. 569:317–322.

Ball, E., P. Shaw, P. Ince, and M. Johnson (1994) The distribution ofexcitatory amino acid receptors in the normal human midbrain andbasal ganglia with implications for Parkinson’s disease: A quantitativeautoradiographic study using [3H]MK-801, [3H]glycine, [3H]CNQXand [3H]kainate. Brain Res. 658:209–218.

Beal, M.F. (1995) Aging, energy, and oxidative stress in neurodegenerativediseases. Ann. Neurol. 38:357–366.

Bergman, H., T. Wichmann, and M. DeLong (1990) Reversal of experimen-tal parkinsonism by lesions of the subthalamic nucleus. Science 249:1436–1438.

Bogerts, B. (1981) A brainstem atlas of catecholaminergic neurons in man,using melanin as a natural marker. J. Comp. Neurol. 197:63–80.

Brotchie, J., I. Mitchell, M. Sambrook, and A. Crossman (1991) Alleviationof parkinsonism by antagonism of excitatory amino acid transmissionin the medial segment of the globus pallidus in rat and primate. Mov.Disord. 6:133–138.

Brouillet, E., and M. Beal (1993) NMDA antagonists partially protectagainst MPTP induced neurotoxicity in mice. Neuroreport 4:387–390.

Buller, A.L., H.C. Larson, B.E. Schneider, J.A. Beaton, R.A. Morrisett, andD.T. Monaghan (1994) The molecular basis of NMDA receptor subtypes:Native receptor diversity is predicted by subunit composition. J.Neurosci. 14:5471–5484.

Choi, D.W., and S.M. Rothman (1990) The role of glutamate neurotoxicity inhypoxic-ischemic neuronal death. Annu. Rev. Neurosci. 13:171–182.

Christoffersen, C., and L. Meltzer (1995) Evidence for N-methyl-D-aspartate and AMPA subtypes of the glutamate receptor on substantianigra dopamine neurons: Possible preferential role for N-methyl-D-aspartate receptors. Neuroscience 67:373–381.

Difazio, M.C., Z. Hollingsworth, A.B. Young, and J.B. Penney (1992)Glutamate receptors in the substantia nigra of Parkinson’s diseasebrains. Neurology 42:402–406.

Dubach, M., R. Schmidt, D. Kunkel, D.M. Bowden, R. Martin, and D.C.German (1987) Primate neostriatal neurons containing tyrosine hy-droxylase: Immunohistochemical evidence. Neurosci. Lett. 75:205–210.

Fearnley, J., and A. Lees (1991) Ageing and Parkinson’s disease; substantianigra regional selectivity. Brain 114:2283–2301.

Fischer, G., A. Bourson, J. Kemp, and H. Lorez (1996) The neuroprotectiveactivity of RO 25-6981, a NMDA receptor NR2B subtype selectiveblocker. Soc. Neurosci. Abstr. 22:1760.

Foldes, R.L. (1994) Cloning and sequence analysis of additional splicevariants encoding human N-methyl-D-aspartate receptor (NR1) sub-units. Gene 147:303–304.

Gibb, W.R.G., and A.J. Lees (1991) Anatomy, pigmentation, ventral anddorsal subpopulations of the substantia nigra, and differential celldeath in Parkinson’s disease. J. Neurol. Neurosurg. Psych. 54:388–396.

Grant, E., B. Backsai, L. Kricka, D. Pleasure, and D. Lynch (1996)Heteromeric NMDA receptor calcium responses are subunit dependentin transfected HEK-293 cells. Soc. Neurosci. Abstr. 22:68.

Greenamyre, J.T., and C.F. O’Brien (1991) N-methyl-D-aspartate antago-nists in the treatment of Parkinson’s disease. Arch. Neurol. 48:977–981.

Grima, B., L. A., F. Blanot, N. Biguet, and J. Mallet (1985) Complete codingsequence of rat tyrosine hydroxylase mRNA. Proc. Natl. Acad. Sci. USA82:617–621.

Grima, B., A. Lamouroux, C. Boni, J.-F. Julien, F. Javoy-Agid, and J. Mallet(1987) A single human gene encoding multiple tyrosine hydroxylaseswith different predicted functional characteristics. Nature 326:707–711.

Hess, S.D., L.P. Daggett, J. Crona, C. Deal, C.-C. Lu, A. Urrutia, L.Chavez-Noriega, S.B. Ellis, E.C. Johnson, and G. Velicelebi (1996)Cloning and functional characterization of human heteromeric N-methyl-D-aspartate receptors. J. Pharmacol. Exp. Ther. 278:808–816.

Hirsch, E., A. Graybiel, and Y. Agid (1988) Melanized dopaminergic neuronsare differentially susceptable to degeneration in Parkinson’s disease.Nature 334:345–348.

Hollmann, M., and S. Heinemann (1994) Cloned glutamate receptors.Annu. Rev. Neurosci. 17:31–108.

Ikeda, K., K. Araki, C. Takayama, Y. Inoue, T. Yagi, S. Aizawa, and M.Mishina (1995) Reduced spontaneous activity of mice defective in the e4subunit of the NMDA receptor channel. Mol. Brain Res. 33:61–71.

Ishii, T., K. Moriyoshi, H. Sugihara, K. Sakurada, H. Kadotani, M. Yokoi, C.Akazawa, R. Shigemoto, N. Mizuno, M. Masu, and S. Nakanishi (1993)Molecular characterization of the family of N-methyl-D-aspartate recep-tor subunits. J. Biol. Chem. 268:2836–2843.

Johnson, S., V. Seutin, and R. North (1992) Burst-firing in dopamineneurons induced by N-methyl-D-aspartate: Role of the electrogenicsodium pump. Science 258:665–667.

Karp, S.A., M. Masu, T. Eki, K. Ozawa, and S. Nakanishi (1993) Molecularcloning and chromosomal localization of the key subunit of the humanN-methyl-D-aspartate receptor. J. Biol. Chem. 268:3728–3733.

Kish, S.J., K. Shannak, and O. Hornykiewicz (1988) Uneven pattern ofdopamine loss in the striatum of patients with idiopathic Parkinson’sdisease: Pathophysiologic and clinical implications. N. Engl. J. Med.318:876–880.

Kosinski, C.M., D.G. Stomdaert, T.J. Counihan, C.R. Scherzer, J.A. Kerner,L.P. Daggett, G. Velicelebi, J.B. Penney, Jr., A.B. Young, and G.B.Landwehrmeyer (1997) Expression of N-Methyl-D-Aspartate ReceptorSubunit mRNAs in the Human Brain: Striatum and Globus Pallidus. J.Comp. Neuro. 390:63–74.

Krebs, M.-O., F. Trovero, M. Desban, C. Gauchy, J. Glowinsky, and M.-L.Kemel (1991) Distinct presynaptic regulation of dopamine releasethrough NMDA receptors in striosome and matrix-enriched areas of therat striatum. J. Neurosci.11:1256–1262.

Kutsuwada, T., N. Kashiwabuchi, H. Mori, K. Sakimura, E. Kushiya, K.Araki, H. Meguro, H. Masaki, T. Kumanishi, and M. Arakawa (1992)Molecular diversity of the NMDA receptor channel. Nature 358:36–41.

Landwehrmeyer, G.B., D.G. Standaert, C.M. Testa, J.B. Penney, and A.B.Young (1995) NMDA receptor subunit mRNA expression by projectionneurons and interneurons in rat striatum. J. Neurosci. 15:5297–5307.

Laurie, D.J., and P.H. Seeburg (1994) Ligand affinities at recombinantN-methyl-D-aspartate receptors depend on subunit composition. Eur. J.Pharmacol. 268:335–345.

Leuba, G., N. Jeanpretre, R. Kraftsik, and J.-M. Fritschy (1989) Samplesize and statistical power in the hierarchical analysis of variance:Applications in morphometry of the nervous system. J. Neurosci. Meth.29:231–239.

Loeschmann, P.-A., U. Wullner, J. Schulz, G. Fischer, J. Kemp, and T.Klockgether (1996) Antiparkinsonian activity of the NMDA2B antago-nist RO 25-6981 in rats. Soc. Neurosci. Abstr. 22:721.

Marey-Semper, I., M. Gelman, and M. Levi-Strauss (1995) A selectivetoxicity toward cultured mesencephalic dopaminergic neurons is in-duced by the synergistic effects of energetic metabolism impairmentand NMDA receptor activation. J. Neurosci. 15:5912–5918.

McRitchie, D.A. and G.M. Halliday (1995) Calbindin D28-containing neu-rons are restricted to the medial substantia nigra in humans. Neurosci-ence 65:87–91.

Monyer, H., N. Burnashev, D.J. Laurie, B. Sakmann, and P.H. Seeburg(1994) Developmental and regional expression in the rat brain andfunctional properties of four NMDA receptors. Neuron 12:529–540.

100 T.J. COUNIHAN ET AL.

Mori, H., and M. Mishina (1995) Structure and function of the NMDAreceptor channel. Neuropharmacology 34:1219–1237.

Nakanishi, S. (1992) Molecular diversity of glutamate receptors andimplications for brain function. Science 258:597–603.

Olszewski, J., and D. Baxter (1954) Cytoarchitecture of the HumanBrainstem. Basel: S. Karger, pp. 52–58.

Overton, P., and D. Clarke (1992) Iontophoretically administered drugsacting at the N-methyl-D-aspartate receptor modulate burst-firing inA9 dopamine neurons in the rat. Synapse 10:131–140.

Paquet, M., M. Tremblay, J.-J. Soghomonian, and Y. Smith (1997) AMPAand NMDA glutamate receptor subunits in midbrain dopaminergicneurons in the squirrel monkey: An immunohistochemical and in situhybridization study. J. Neurosci. 17:1377–1396.

Pearson, J., M. Goldstein, K. Markey, and L. Brandeis (1983) Humanbrainstem catecholamine neuronal anatomy as indicated by immunocy-tochemistry with antibodies to tyrosine hydroxylase. Neuroscience8:3–32.

Planells-Cases, R., W. Sun, A.V. Ferrer-Montiel, and M. Montal (1993)Molecular cloning, functional expression, and pharmacological charac-terization of an N-methyl-D-aspartate receptor subunit from humanbrain. Proc. Natl. Acad. Sci. USA 90:5057–5061.

Sitzmann, J.H., and P.K. LeMotte (1993) Rapid and efficient generation ofPCR-derived riboprobe templates for in situ hybridization histochemis-try. J. Histochem. Cytochem. 41:773–776.

Standaert, D.G., G.B. Landwehrmeyer, J.A. Kerner, J.B. Penney, and A.B.Young (1996) Expression of NMDAR2D glutamate receptor subunitmRNA in neurochemically identified interneurons in the rat neostria-tum, neocortex, and hippocampus. Mol. Brain Res. 42:89–102.

Standaert, D.G., C.M. Testa, J.B. Penney, and A.B. Young (1993) Alterna-tively spliced isoforms of the NMDAR1 glutamate receptor: differentialexpression in the basal ganglia of the rat. Neurosci. Lett. 152:161–164.

Standaert, D.G., C.M. Testa, J.B. Penney, and A.B. Young (1994) Organiza-tion of N-methyl-D-aspartate glutamate receptor gene expression in thebasal ganglia of the rat. J. Comp. Neurol. 343:1–16.

Tecott, L.H., J.H. Eberwine, J. Barchas, and K.L. Valentino (1994) Method-ological considerations in the utilization of in situ hybridization. In J. H.Eberwine, K. L. Valentino and J. Barchas (eds): In Situ Hybridization inNeurobiology. Oxford: Oxford University Press, pp. 3–23.

Turski, L., K. Bressler, K.-J. Rettig, P.-A. Loeschmann, and H. Wachtel(1991) NMDA antagonists protect nigral neurons in MPTP models ofPD. Nature 349:414–418.

Uitti, R., A. Rajput, J. Ahlskog, K. Offord, D. Schroeder, M. Ho, M. Prasad,A. Rajput, and P. Basran (1996) Amantadine treatment is an indepen-dent predictor of improved survival in Parkinson’s disease. Neurology46:1551–1556.

Ulas, J., F.B. Weihmuller, L.C. Brunner, J.N. Joyce, J.F. Marshall, and C.W.Cotman (1994) Selective increase of NMDA-sensitive glutamate bind-ing in the striatum of Parkinson’s disease, Alzheimer’s disease, andmixed Parkinson’s/Alzheimer disease patients: An autoradiographicstudy. J. Neurosci. 14:6317–6324.

Vartanian, M., and P. Boxer (1996) Prevention of NMDA-induced braininjury in neonatal rats by subtype selective NMDA (NR2B) antagonists.Soc. Neurosci. Abstr. 22:1279.

Williams, K. (1995) Pharmacological properties of recombinant N-methyl-D-aspartate receptors containing the epsilon-4 (NR2D) subunit. Neurosci.Lett. 184:181–184.

Yamada,T., P.L. Mc Geer, K.G. Baimbridge, and E.G. McGeer (1990)Relative sparing in Parkinson’s disease of substantia nigra dopamineneurons containing calbindin-D28k. Brain Res. 526:303–307.

Young, W.S., III, and M.J. Kuhar (1986) Quantitative in situ hybridizationand determination of mRNA content. In G. R. Uhl (ed): In SituHybridization in Brain. New York: Plenum Press, pp. 243–248.

Zukin, R.S., and M.V.L. Bennett (1995) Alternatively spliced isoforms of theNMDAR1 receptor subunit. Trends Neurosci. 18:306–313.

NR SUBUNITS IN MIDBRAIN DOPAMINE NEURONS 101