Glutamate decarboxylase (GAD65) gene expression is increased by dopamine receptor agonists in a subpopulation of rat striatal neurons

Molecular Brain Research 48 Ž1997. 333–345

Research report

Glutamate decarboxylase ž GAD65/ gene expression is increased by dopamine receptor agonists in a subpopulation of rat striatal neurons Nathalie Laprade, Jean-Jacques Soghomonian

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Centre de Recherche en Neurobiologie and Departement d’Anatomie, UniÕersite´ LaÕal, Quebec, Que., ´ ´ ´ Canada Accepted 4 March 1997

Abstract The mRNA levels encoding for the two isoforms of glutamate decarboxylase ŽGAD65 and GAD67. were measured in the adult rat striatum following systemic administration of dopamine receptor agonists. Double-labeling in situ hybridization histochemistry was used to measure GAD65 or GAD67 mRNA levels in neurons labeled or not with a preproenkephalin ŽPPE. cRNA probe. Chronic treatment with the D1rD2 dopamine receptor agonist apomorphine or with the D1 dopamine receptor agonist SKF-38393 induced an increase in GAD65 but not GAD67 mRNA levels in different sectors of the striatum. These effects were abolished by pre-administration of the D1 dopamine receptor antagonist SCH-23390. On double-labeled sections, GAD65 mRNA labeling was distributed in neurons labeled and unlabeled with the PPE cRNA probe. About half of all neuronal profiles labeled with the GAD65 cRNA probe were also labeled with the PPE cRNA probe. Quantification of labeling at cellular level demonstrated a significant increase of GAD65 mRNA levels in PPE-unlabeled neurons. On the other hand, no significant changes of GAD65 mRNA levels were detected in PPE-labeled neurons. Our results demonstrate a differential effect of dopamine receptor agonists on striatal GAD65 and GAD67 gene expression. In particular, we show that GAD65 mRNA levels are selectively increased in presumed striato-nigral neurons following treatments with dopamine receptor agonists. These data provide evidence that the GAD65 isoform is preferentially involved in the regulation of GABAergic neurotransmission in striato-nigral neurons. q 1997 Elsevier Science B.V. Keywords: Glutamic acid decarboxylase; Glutamate decarboxylase ŽGAD.; g-Aminobutyric acid ŽGABA.; Striato-nigral; Dopamine; D1 receptor; mRNA; Hybridization histochemistry, in situ

1. Introduction Striatal projection neurons use g-aminobutyric acid ŽGABA. as their neurotransmitter and contain the GABAsynthesizing enzyme, glutamate decarboxylase ŽGAD. w6,31,44,47,51x. The enzyme GAD exists at least as two isoforms ŽGAD65 and GAD67. of distinct molecular weight that are encoded by different genes w12,29x. Anatomical studies have shown that the mRNAs encoding for the two GADs are co-localized in many neurons of the Abbreviations: GAD, glutamate decarboxylase; PPE, preproenkephalin; 6-OHDA, 6-hydroxydopamine; MPTP, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine; mRNA, messenger ribonucleic acid; SKF-38393, 1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine-7,8-diolhydrochloride; SCH-23390, Ž R q .-7-chloro-8-hydroxy-3-methyl-1phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine hydrochloride. ) Corresponding author. Centre de Recherche en Neurobiologie, Hopital ˆ de l’Enfant-Jesus, 1401 18eme Que. ´ ` rue, Quebec, ´ ´ G1J 1Z4, Canada. Fax: q1 Ž418. 649-5910; E-mail: [email protected] 0169-328Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 1 1 2 - 5

basal ganglia including the striatum w13,43x. Striatal GABAergic projection neurons in the rat can be subdivided into two distinct subpopulations. The striato-pallidal neurons preferentially express the peptide enkephalin and the D2 dopamine receptor and the striato-nigralrentopeduncular neurons preferentially express the peptides substance P and dynorphin and the D1 dopamine receptor w18,19,21,24,25,35,36,65x. There is extensive evidence that dopamine receptors are involved in the regulation of striatal GABAergic neurons. For instance, dopamine or D1 receptor agonists are able to increase striatal GABA release as measured in vitro on brain slices w4,17,60x or in vivo by push-pull cannula or microdialysis techniques w23,64x. The stimulatory effect of D1 dopamine receptors on striatal GABA release appears to involve GABAergic neurons of the striato-nigralrentopeduncular pathway since application of SKF-38393 in the striatum increases concomitantly the release of GABA in the striatum, the substantia nigra and the entopeduncular

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N. Laprade, J.-J. Soghomonianr Molecular Brain Research 48 (1997) 333–345

nucleus w16,64x. On the other hand, D2 dopamine receptors appear to exert an inhibition w4,23,50x or have no effect w64x on striatal GABA release. The regulation of striatal GABAergic neurons by dopamine receptors can also be evidenced through measurements of GAD activity or GAD gene expression. For instance, rats with 6-OHDA lesions of dopamine neurons exhibit increased GAD activity w52x, GAD67 mRNA levels and GAD immunoreactivity w40,52,54,59x. Similar increases are observed following blockade of D2 dopamine receptors in adult rats w8,32,40,49,54x which suggests that dopamine through D2 receptors is involved in a tonic inhibition of striatal GAD activity and GAD67 gene expression. Striatal GAD65 gene expression, on the other hand, is not altered following D2 dopamine receptor blockade or lesion of dopamine neurons in adult rats w32,54x. However, a recent study has shown that the administration of D1 dopamine receptor agonists to adult rats induces an increase in striatal GAD65 but not GAD67 mRNA levels w32x. In addition to demonstrating a differential regulation of the two GAD isoforms by dopamine receptor subtypes, this result suggests that the GAD65 isoform is preferentially up-regulated in response to D1 receptor stimulation in striato-nigral neurons of the adult rat. Such a possibility would provide further evidence for the still debated stimulatory role of D1 dopamine receptors on GABAergic striato-nigral neurons w26x. The goal of the present study was first to provide further evidence that chronic administration of dopamine D1 receptor agonists to adult rats induce a selective increase in GAD65 mRNA levels and second to determine whether this increase occurs in presumed striato-pallidal, striato-nigral or both subpopulations of neurons. The effects of the dopamine receptor agonists apomorphine or SKF-38393 on the levels of the GAD65 mRNA were measured in striatal neurons labeled or not with a preproenkephalin ŽPPE. cRNA probe which was used as a preferential marker of striato-pallidal neurons.

2. Materials and methods 2.1. Animals and treatment Adult male Sprague–Dawley rats ŽCharles River, Montreal. weighing 230–250 g at the beginning of the experiments were used. They were kept under a 12 h lightrdark cycle, with food and water available ad libitum. In a first experiment, three groups of 6 rats were injected s.c. for 10 days twice daily with one of the dopamine receptor agonists apomorphine Ž5 mgrkg. or SKF-38393 Ž12.5 mgrkg.. SKF-38393 was dissolved in 0.2% acetic acid and apomorphine in 0.2% ascorbic acid. Control rats received 0.2% acetic acid in saline. In a second experiment aimed at confirming the specificity of the effects of dopamine receptor agonists, five groups of 6 rats each were injected

s.c. for 10 days twice daily with either apomorphine Ž5 mgrkg., SKF-38393 Ž12.5 mgrkg., apomorphine and SCH-233390 Ž0.2 mgrkg. or SKF-38393 and SCH-23390 Ž0.2 mgrkg.. The antagonist SCH-23390 was injected 1r2 h before the injection of the agonists. Drugs were dissolved as in the previous experiment. Control rats were injected with 0.2% acetic acid in saline. In all cases, the first injection was performed between 09:00 and 10:00 h and the second injection between 17:00 and 18:00 h. The last day of the treatment, the rats were injected in the morning only. All drugs were purchased from Research Biochemicals Incorporated ŽRBI; Natick, MA.. Animals were killed 3 h after the last injection by decapitation and their brains were quickly removed and stored at y808C. Tissue sections Ž10 m m-thick. were cut at the striatal level in the coronal plane on a cryostat and thaw-mounted onto slides coated with gelatin and stored at y808C until further processing. 2.2. RadioactiÕe and digoxigenin-labeled probes Radiolabeled complementary RNA ŽcRNA. probes were produced after in vitro transcription from cDNA clones encoding either for the feline GAD67 or the rat GAD65 w12,29x. The feline GAD67 cDNA shows 97% sequence identity with the more recently cloned rat cDNA w12,29x. The digoxigenin-labeled probe was produced after transcription of a cRNA probe encoding for the rat PPE w63x. The cDNAs inserted into bluescript ŽGAD65. or PSP64r65 ŽGAD67 and PPE. vectors were linearized with HindIII ŽGAD65., BamH1 ŽGAD67. or Sac1 ŽPPE. restriction enzymes. Transcription of the cRNAs from the cDNAs was then performed using a riboprobe kit ŽPromega. in presence of 2.5 m M of w 35 SxUTP Ž1000 Cirmmol; New England Nuclear. and 10 m M unlabeled UTP Žfor radioactive probe. or 0.166 mM of DIG-UTP Ždigoxigenin-labeled UTP; Boehringher Mannheim. and 0.33 mM unlabeled UTP Žfor digoxigenin-labeled probe. with ATP, CTP and GTP in excess and SP6 Žfor PPE and GAD67. or T3 Žfor GAD65. RNA polymerases. The reaction was carried out for 2 h at 378C and then the template was digested with DNase I. The labeled cRNA was purified by phenolrchloroform extraction and ethanol precipitation. The length of the cRNAs was reduced to 100–150 nucleotides by partial alkaline hydrolysis to improve accessibility of the probe w9x. 2.3. In situ hybridization and radioautography All solutions and buffer were prepared with distilled water treated with diethylpyrocarbonate ŽDEPC; Sigma. to inhibit RNase activity and autoclaved. Brain sections were quickly dried at room temperature and fixed for 5 min in a solution of 3% paraformaldehyde in phosphate buffer Ž1 M; pH 7.2. containing 0.02% DEPC. Sections were treated for 10 min with triethanolamine Ž0.1 M; pH 8.0. contain-

N. Laprade, J.-J. Soghomonianr Molecular Brain Research 48 (1997) 333–345

ing 0.25% acetic anhydride and then for 30 min with Tris-glycine Ž1 M; pH 7.0. before being dehydrated and air-dried. Each section was then covered with 3–3.5 ng of radiolabeled cRNA probe and 4 ng of the digoxigeninlabeled probe diluted in 20 m l of hybridization solution containing 40% formamide, 10% dextran sulfate, 4 = SSC, 10 mM dithiothreitol, 1% sheared salmon sperm DNA, 1% yeast tRNA, 1 = Denhardt’s solution containing 1% RNase-free bovine serum albumin. The sections were covered with parafilm, placed in humidified boxes and the hybridization reaction was carried out for 4 h at a temperature of 508C. Sections were then washed in 50% formamide and 2 = SSC at 528C for 5 min and 20 min, in RNase A Ž100 m grml; Sigma. and 2 = SSC for 30 min at 378C, in 50% formamide and 2 = SSC at 528C for 5 min, in 2 = SSC and 0.05% Triton X-100 at room temperature for 3 = 5 min and in Tris buffer Ž0.1 M; pH 7.5; buffer 1. containing 0.15 M NaCl, 0.3% Triton X-100 and 2% normal sheep serum for 30 min at room temperature under mild agitation. Sections were then covered with 100 m l of the anti-digoxigenin Fab fragment conjugated with alkaline phosphatase ŽBoehringher Mannheim. diluted 1 : 500 in buffer 1 and left overnight at 48C. Then, sections were rinced for 3 = 7 min in buffer 1 and for 2 = 5 min in Tris buffer Ž1 M; pH 9,5; buffer 2. containing 0.1 M NaCl and 0.05 M MgCl. Slides were then incubated in buffer 2 containing 0.24 mgrml levamisole, 75 mgrml of 4Nitroblue tetrazolium ŽNTB. and 50 mgrml 5-Bromo-4chloro-3 indolyl phosphate ŽBoehringher Mannheim. and kept in the dark Žreactives are light-sensitive. until detection of the non-radioactive labeling Ž1–2 h.. The reaction was stopped by dipping the slides in a Tris buffer Ž10 mM; pH 8.0. containing 1 mM EDTA. Sections were then washed in SSC Ž2 = . for 15 min, quickly dipped in ammonium acetate Ž300 mM. and dehydrated in 70% ethanol before being exposed to Kodak X-OMAT-AR X-ray films for 10 days. For emulsion radioautography, sections were then dipped in an Amersham LM-1 nuclear emulsion, air-dried and stored at 48C in light-tight boxes in the presence of dessicant. Following 4–8 days of exposure, the sections were developped in Kodak D-19 for 3.5 min at 148C and mounted with Aquaperm mounting media ŽFisher Scientific.. 2.4. Analysis of labeling Level of GAD65 or GAD67 mRNA labeling in the striatum was quantified by densitometry on X-ray films with a MacIntosh computer and an Ultimage image analysis software ŽGraftek, France.. The optical density of labeling in each striatal sector was calculated after subtracting the optical density of the film and standardization against emulsion-coated filters ŽKodak.. Internal 14 C standards ŽAmersham. were used to insure that measurements were made in the linear portion of the film. Two sectionsranimal were analyzed for each probe. The aver-

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age level of labeling was calculated for each rat and in each striatal region sampled. Overall statistical significant differences in GAD65 or GAD67 radioautographic labeling between the experimental groups of rats was determined for each striatal sector using a one-way analysis of variance ŽANOVA. while post-hoc paired comparisons were performed with the Protected Least Significant Difference Fisher’s test. Statistical significance was defined as P - 0.05. The cellular distribution of labeling with the GAD65 or GAD67 and PPE cRNA probes was examined on emulsion radioautographs by light microscopy. First, camera lucida drawings of all single- or double-labeled neurons were made at =25 magnification in a representative area of the dorsolateral striatum. One sectionrrat was analyzed. The numbers of neurons labeled with the radioactive GAD65 or GAD67 cRNA probe alone or double-labeled with one of the GADs and the digoxigenin PPE cRNA probes were then determined. Radioautographic level of labeling in those sampled neurons was measured by computerized image analysis ŽNIH Image 1.55.. In that case, emulsion radioautographs were observed under dark-field Žto quantify GAD mRNA level in PPE-positive neurons. or brightfield Žto quantify GAD mRNA levels in PPE-negative neurons. illumination at =40 magnification. The area covered by silver grains in each neuron was measured and expressed as a number of pixelsrneuron. Because the number of silver grains is linearly proportional to the area they occupy, this later measure was used as an index of the relative intensity of radioautographic labeling. For each rat, a sample of f 50 PPE-labeled and 50 PPE-unlabeled neurons was analyzed. We have previously demonstrated that such a sample allows a reproducible estimate of levels of GAD mRNA labeling in individual striatal neurons w54x. Comparison of labeling between control and drug-injected animals was made on sections processed in parallel. Sections adjacent to those double-labeled with the GAD65 and PPE cRNA probes were Nissl-stained in order to provide an estimate of the total number of neurons present in the sampled area of the striatum. Statistical significant differences in GAD65 or GAD67 mRNA labeling between the five experimental groups of rats was calculated using a one-way analysis of variance ŽANOVA. for each striatal sector. Post-hoc pair-wise comparison of GAD65 or GAD67 mRNA labeling between each of the experimental group was then performed for each striatal sector with a Fisher’s test with P - 0.05 considered significant.

3. Results 3.1. Effects of chronic treatments with dopamine receptor agonists on GAD65 and GAD67 mRNA leÕels The effects of dopamine receptor agonists on GAD gene expression were first analyzed on X-ray films ra-

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Fig. 1. Negative images of X-ray films from frontal brain sections at a stereotaxic level A s 10.7 according to the stereotaxic atlas of Paxinos and Watson w45x. Sections were processed for in situ hybridization with a 35 S-labeled GAD65 cRNA Žleft column; exposure time: 10 days. or GAD67 cRNA probe Žright column; exposure time: 14 days.. Sections are from adult control rats ŽA,B., rats chronically treated with either apomorphine ŽC,D., apomorphine combined with SCH-23390 ŽE,F. SKF-38393 ŽG,H. or SKF-38393 combined with SCH-23390 ŽI,J.. Note the increase in labeling for the GAD65 mRNA in rats treated with apomorphine or SKF-38393. Scale bar s 300 m m.

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dioautographs at a rostral striatal level ŽA s 10.7 according to Paxinos and Watson’s stereotaxic atlas w45x.. Chronic apomorphine or SKF-38393 administration to adult rats induced a marked increase in striatal GAD65 mRNA levels ŽFig. 1A,C,G, Fig. 3A.. These effects were abolished by the co-administration of the D1 dopamine receptor antagonist, SCH-23390 ŽFig. 1A,C,E,G,I, Fig. 3A.. Quantitative analysis in different sectors of the striatum revealed highly significant differences in GAD65 mRNA

Fig. 3. Levels of GAD65 ŽA. and GAD67 ŽB. mRNA in four different striatal sectors ŽDM, dorsomedial; VM, ventromedial; DL, dorsolateral; VL, ventrolateral. and over the whole striatal surface in control adult rats and adult rats chronically treated with apomorphine, SKF-38393, apomorphineqSCH-23390 or SKF-38393qSCH-23390 at the stereotaxic level A s10.7. The values represent the mean intensity of labeling measured on X-ray films by computerized densitometry and expressed as a percentage of the controls. The data Žmean"S.E.M.. were obtained from 5 rats in each experimental group. Statistical differences in labeling in each striatal sector were determined after a single-way ANOVA. Pair-wise comparisons between different experimental conditions were made according to Fisher’s test. ) P - 0.05 or ) ) P - 0.01 when compared to controls; ¶ P - 0.05 or ¶¶ P - 0.01 when compared to apomorphine and a P - 0.05 or aa P - 0.01 when compared to SKF-38393-treated rats.

Fig. 2. Negative images of X-ray films from frontal brain sections at stereotaxic level A s9.2 according to the atlas of Paxinos and Watson. Sections have been processed for in situ hybridization with a 35 S-labeled GAD65 and a digoxigenin-labeled PPE cRNA probe Žexposure time: 10 days.. Sections are from an adult control rat ŽA., and from adult rats chronically treated with either apomorphine ŽB. or SKF-38393 ŽC.. Note the increase in labeling for the GAD65 mRNA in rats treated with apomorphine or SKF-38393. Scale bar s 300 m m.

labeling between groups in the dorsomedial Ž F4,18 s 4.9; P s 0.0078., the ventromedial Ž F4,18 s 8.0; P s 0.0007., the dorsolateral Ž F4,18 s 4.3; P s 0.0134., the ventrolateral Ž F4,18 s 6.5; P s 0.0020. striatal sectors and over the whole striatal surface Ž F4,18 s 5.8; P s 0.0035.. Increased labeling ranged from 26 to 36% following apomorphine and from 34 to 53% following SKF-38393 ŽFig. 3A.. Quantitative analysis of labeling in adjacent sections demonstrated that neither apomorphine nor SKF-38393 administration induced significant changes in striatal GAD67 mRNA levels ŽFig. 1B,D,H, Fig. 3B.. Chronic administration of SCH-23390 combined with either apomorphine or SKF38393 did not induce significant changes in GAD67 mRNA levels ŽFig. 1B,D,F,H,J, Fig. 3B..

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Fig. 4. Levels of GAD65 mRNA in the striatum of control adult rats and adult rats chronically treated with apomorphine or SKF-38393 at the stereotaxic level A s9.2. Brain sections were processed for double-labeling in situ hybridization with a GAD65 radioactive probe and a digoxigenin-labeled PPE cRNA probe. The values represent the mean intensity of labeling measured on X-ray films by computerized densitometry and expressed as a percentage of the controls. The data Žmean"S.E.M.. were obtained from 5 rats in each experimental group. Labeling was measured in four different striatal sectors ŽDM, dorsomedial; VM, ventromedial; DL, dorsolateral; VL, ventrolateral; total, whole striatal surface.. Statistical differences in labeling in each striatal sector were determined after a single-way ANOVA. Post-hoc comparisons between different experimental conditions were made according to Fisher’s test. ) P - 0.01 when compared to control rats in striatal sectors that showed significant differences after the ANOVA.

alkaline-phosphatase reaction and clusters of silver grains ŽFig. 6.. Neurons radioactively labeled with the GAD65 cRNA probe but unlabeled with the digoxigenin PPE cRNA probe were identified as clusters of silver grains Ž) 10 grains. that clearly stood-out from the scattered distribution of background grains. In most cases, each cluster of silver grains was dense enough to evoke the characteristic shape of a profile of neuronal somata. Clusters of silver grains that did not satisfy to these criteria Ži.e. with - 10 grains or that did not evoke a cellular shape. were not included in our subsequent quantitative analysis. Observation of eosin-hematoxylin-stained sections demonstrates that dense clusters of silver grains in the striatum labeled with a GAD65 cRNA probe invariably indicate the presence of labeled profiles of neuronal somata Žnot illustrated; see also w46,54x.. Light microscopic observation of double-labeled sections revealed that all digoxigenin-labeled neurons in the striatum were also labeled with the GAD65 cRNA probe. However, a significant number of neurons labeled with the GAD65 cRNA probe was not labeled with the PPE cRNA

The effects of apomorphine or SKF-38393 on GAD65 mRNA levels were reproduced in another group of adult rats and were examined at a more caudal striatal level ŽA s 9.2 according to Paxinos and Watson’s atlas w45x. ŽFig. 2, Fig. 4.. Again, highly significant differences in GAD65 mRNA levels between the experimental groups were measured in the dorsomedial Ž F2,15 s 5.8; P s 0.0134., the ventromedial Ž F2,15 s 4.5; P s 0.0298., the dorsolateral Ž F2,15 s 4.9, P s 0.0233., the ventrolateral Ž F2,14 s 6.0; P s 0.0135. sectors and over the whole striatal surface Ž F2,14 s 4.1; P s 0.0389.. Increased labeling ranged from 15 to 34% following apomorphine and from 16 to 31% following SKF-38393 administration ŽFig. 4.. The effects of SKF-38393 or apomorphine on GAD65 mRNA levels were observed on sections processed with a GAD65 cRNA probe only ŽFig. 1, Fig. 3. or simultaneously processed with a GAD65 radioactively labeled and a PPE digoxigenin-labeled cRNA probe ŽFig. 2, Fig. 4.. 3.2. Cellular distribution of striatal GAD65 mRNA labeling in control and agonist-treated rats The cellular distribution of GAD65 mRNA labeling was first analyzed on sections simultaneously processed with a radioactively labeled GAD65 and a digoxigenin-labeled PPE cRNA probe. Sections exhibited single- or doublelabeled neuronal profiles throughout the striatum ŽFig. 6B,D,F.. Double-labeled neurons were easily detected by the combination of a dense deposit resulting from the

Fig. 5. The circle on the frontal section illustrates the location and size of the dorsolateral striatal area in which labeling was quantified at single cell level. The large circle illustrates an example of distribution of singleand double-labeled neurons in the selected area. All labeled neurons in that area were analyzed. Filled circles: neurons double-labeled with the PPE and GAD65 cRNA probes. Empty circles: neurons labeled with the GAD65 cRNA probe only.

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Table 1 Mean " S.E.M. numbers Žper mm2 of tissue section. of GAD65 or PPE mRNA-containing and Nissl-stained neuronal profiles in a comparable dorsolateral striatal sector at stereotaxic level A s 10.7 or 9.2 A s 10.7

Control Apomorphine SKF-38393

A s 9.2

Nissl

PPE

GAD65

Nissl

PPE

GAD65

616 " 60 656 " 54 732 " 34

225 " 10 230 " 12 253 " 19

514 " 39 633 " 08 682 " 44

602 " 15 562 " 13 582 " 17

287 " 14 283 " 13 292 " 12

553 " 15 541 " 12 558 " 14

Mean number of neurons sampled in a microscopic field corresponding to 1 mm2 . The total number of neurons labeled with the GAD65 mRNA probe, doubled-labeled with the GAD65 and the PPE cRNA probes or Nissl-stained in the dorsolateral portion of the striatum was calculated at stereotaxic level A s 10.7 and 9.2. Data are from adult control rats and from rats chronically treated with apomorphine or SKF-38393. The values are mean " S.E.M. from 5 rats per condition.

Fig. 6. Bright-field photomicrographs of brain sections processed for in situ hybridization histochemistry with a 35 S-labeled GAD65 cRNA probe and a digoxigenin-labeled PPE cRNA probe in a representative lateral striatal sector at =40 ŽA,C,E. and =25 magnification ŽB,D,F.. Labeling is from adult control rats ŽA,B. and from adult rats treated with apomorphine ŽC,D. or SKF-38393 ŽE,F.. Neurons labeled with the GAD65 cRNA probe only are indicated by the arrowheads and neurons labeled with both the PPE and the GAD65 probes are indicated by arrows. Note the increase in GAD65 mRNA labeling in PPE-unlabeled neurons in apomorphine- or SKF-38393- treated rats. Scale bar s 10 m m Žin A,C,E.; scale bar s 30 m m Žin B,D,F..

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probe ŽFig. 6.. The numbers of labeled neurons were determined in a representative dorsolateral sector of the striatum Žsuch as illustrated on Fig. 5.. In that case, all single- or double-labeled neurons found in a striatal area corresponding to 0.212 mm2 of tissue were mapped ŽFig. 5.. At the two rostro-caudal levels examined ŽA s 10.7 and 9.2., the numbers of profiles labeled with the GAD65 probe were comparable in control, apomorphine- and SKF38393-treated rats ŽTable 1.. These numbers were also comparable to the numbers of Nissl-stained neuronal profiles that were detected on adjacent sections. In control rats, the proportion of GAD65-labeled neurons that were double-labeled with the PPE cRNA probe reached 52% at level A s 9.2 and 44% at level A s 10.7. These proportions were comparable in the apomorphine- or SKF38393-treated groups of rats and at the two rostro-caudal levels examined ŽTable 1..

Fig. 8. Histograms of frequency distributions of GAD65 mRNA labeling in PPE-labeled and PPE-unlabeled neurons of the lateral striatum at stereotaxic level A s10.7. Data are from adult control rats ŽA,D. and from adult rats treated with apomorphine ŽB,E. or SKF-38393 ŽC,F., Quantification of silver grains over individual striatal neurons was performed by computerized image analysis Žsee Section 2 for details.. The area covered by silver grains is expressed in number of pixelsrneuron. A sample of 50 neuronsrrat from 4 rats in each experimental condition was analyzed.

3.3. LeÕels of GAD65 mRNA labeling in PPE-labeled and PPE-unlabeled neurons

Fig. 7. Levels of GAD65 mRNA labeling in single PPE-labeled and PPE-unlabeled neurons in a representative lateral sector of the striatum at stereotaxic level A s10.7 ŽA. and A s9.2 ŽB.. Radioautographic labeling was measured by computerized image analysis Žsee Section 2 for details.. The values are mean"S.E.M. of the mean number of pixelsrneuron and are expressed as a percentage of the controls. Data are from adult control rats and adult rats chronically treated with apomorphine or SKF-38393. A sample of 50 neuronsrrat from 5 rats per experimental condition was analyzed. Pair-wise comparisons between experimental groups were made with a Fisher’s test in the sectors that showed statistically significant differences after the ANOVA. ) P - 0.05 or ) ) P - 0.01 when compared to controls.

Observation of the emulsion radioautographs revealed that GAD65 mRNA labeling in striatal neurons was higher in rats treated with apomorphine and SKF-38393 than in controls ŽFig. 6.. Increased labeling was apparent in PPEunlabeled but not in PPE-labeled neurons ŽFig. 6A,C,E.. These differences were confirmed by quantitative analysis of GAD65 mRNA labeling in those PPE-labeled and PPEunlabeled neurons that were previously mapped in the dorsolateral sector of the striatum Žsee Section 2 for details.. At the two rostro-caudal levels examined, the intensity of GAD65 mRNA labeling in PPE-labeled neurons was not significantly different in apomorphine or SKF38393-treated rats than in the controls ŽFig. 6A,C,E, Figs. 7–9.. In PPE-unlabeled neurons, however, ANOVAs revealed highly significant differences in GAD65 mRNA

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phine or SKF-38393 induces an increase in GAD65, but not GAD67, mRNA levels in the adult rat striatum. Double-labeling in situ hybridization experiments further revealed that the GAD65 mRNA was distributed in a large proportion of striatal neurons but its increased levels following apomorphine or SKF-38393 administration occurred only in a subpopulation of neurons that were not labeled with a PPE cRNA probe. 4.1. Dopamine receptor subtypes differentially regulate striatal GAD65 and GAD67 gene expression

Fig. 9. Histograms of frequency distributions of GAD65 mRNA labeling in PPE-labeled and PPE-unlabeled neurons of the lateral striatum at stereotaxic level A s9.2. Data are from adult control rats ŽA,D. and from adult rats treated with apomorphine ŽB,E. or SKF-38393 ŽC,F., Quantification of silver grains over individual striatal neurons was performed by computerized image analysis Žsee Section 2 for details.. The area covered by silver grains is expressed in number of pixelsrneuron. A sample of 50 neuronsrrat from 4 rats in each experimental condition was analyzed.

labeling between the experimental groups Ž F2,9 s 6.3; P s 0.0195 at level 10.7 and F2,9 s 35.8; P - 0.0001 at level 9.2.. At the rostral-most level ŽA s 10.7., the increase in GAD65 mRNA labeling in PPE-unlabeled neurons was of 64% after apomorphine and 51% after SKF-38393 ŽFig. 7.. At the caudal-most level ŽA s 9.2., the effects were slightly lower reaching 44% after apomorphine and 36% after SKF-38393 treatment ŽFig. 7.. The histograms of frequency distribution of GAD65 mRNA labeling in individual striatal neurons are shown on Figs. 8 and 9 and illustrate the shift of labeling in the population of PPE-unlabeled ŽDIG y . but not PPE-labeled ŽDIG q . neurons at the two rostro-caudal levels analyzed.

4. Discussion The present study demonstrates that chronic systemic administration of the dopamine receptor agonists apomor-

In agreement with a previous report w32x, the present results demonstrate that chronic treatment with apomorphine or with SKF-38393 induce an increase in GAD65 mRNA levels in the adult rat striatum. Since these effects were abolished by co-administration of SCH-23390, they are likely to involve a selective action on D1 dopamine receptors. On the other hand, apomorphine or SKF-38393 failed to alter significantly the mRNA levels encoding for GAD67. In keeping with previous demonstrations that chronic blockade of D2 dopamine receptors or 6-OHDA lesions of dopamine neurons increase striatal GAD67 but not GAD65 mRNA levels w32,54x, the present results confirm our original observations w32x that the gene expression of the GAD65 and GAD67 isoforms is differentially regulated in the adult rat striatum by D1 and D2 dopamine receptors. Increases in GAD65 mRNA levels following apomorphine- or SKF-38393 administration were significant only in the subpopulation of striatal neurons that were unlabeled with the PPE probe. Most of these neurons can be considered as striato-nigral neurons that contain the mRNAs encoding for the peptides dynorphin and substance P but not enkephalin w18x. On the other hand, we have produced preliminary evidence that increased GAD67 mRNA levels following D2 receptor blockade is restricted to presumed striato-pallidal neurons w33x. The likelihood that the D1 and D2 dopamine receptors, respectively, stimulate GAD65 and inhibit GAD67 gene expression in different subpopulations of striatal neurons is consistent with the differential distribution of these receptors in striato-nigral and striatopallidal neurons w19,24,25,36,65x. Although GAD65 gene expression appears to be preferentially regulated by D1 dopamine receptors in striatonigral neurons in the adult rat, increases in both GAD65 and GAD67 mRNA levels in those neurons have been recently reported in adult rats lesioned with 6-OHDA as neonates w34x. The likelihood that the two GAD isoforms can be regulated at gene level in a single neuron is also supported by earlier studies in which parallel changes in GAD67 and GAD65 mRNA levels were measured in the rat or monkey pallidum following experimental lesions of dopamine neurons w46,53,55x. The significance of these different patterns of regulation of GAD65 and GAD67

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gene expression is presently unknown. On the basis of anatomical observations in the rat brain, it has previously been proposed that each GAD isoform might be differentially associated with different populations of GABAergic neurons exhibiting phasic or tonic patterns of electrical activity w15x. The selective changes in GAD65 andror GAD67 gene expression might thus reflect specific modulations in the firing pattern of striatal neurons. 4.2. Role of D1 receptor subtypes on the regulation of striato-nigral GABAergic neurons The finding that D1 dopamine receptor agonists induce an increase in GAD65 gene expression in presumed striato-nigral neurons can be taken as further evidence that D1 receptors stimulate GABAergic activity in these neurons. Indeed, local application of a D1 agonist in the striatum is able to increase GABA release in the striatum w16,23,60,64x, the substantia nigra w64x and the entopeduncular nucleus w16x. When directly delivered into the substantia nigra, D1 dopamine receptor agonists are also able to increase the release w17,50,64x or the synthesis w1x of GABA. Because D1 dopamine receptors are present on striato-nigral terminals and on the neuronal somata of striatal efferent neurons w2,24x, it is likely that they exert a positive control on GABA release at both pre- and postsynaptic level. A stimulatory effect of D1 receptors on striatal GABAergic neurons is, however, not supported by a recent study showing that the in vivo rate of GABA synthesis following systemic administration of SKF-38393 is not increased w26x. In addition, it has also been reported that different doses of the full D1 receptor agonist SKF81297 can stimulate or inhibit striatal GABA release w7x. This biphasic effect rises the interesting possibility that different populations of striatal D1 receptors might exert opposite effects on the regulation of striatal GABAergic neurons. Because dopamine receptor agonists were systemically administered, the mechanisms involved in increased GAD65 gene expression in our experimental conditions are unclear. Indeed, in addition to a direct action on striato-nigral neurons, it cannot be excluded that their effects might involve an indirect action through polysynaptic pathways. Direct application of apomorphine or SKF38393 in the substantia nigra decreases the release of dopamine in the striatum w50x. Therefore, systemic apomorphine or SKF-38393 administration could exert a direct excitatory effect on striato-nigral neurons and an indirect inhibitory effect that would be secondary to a decrease of dopamine release. The involvement of dopamine neurons on the regulation of GABAergic striato-nigral neurons following systemically administered SKF-38393 can also be suspected because of the opposite effect of the agonist on the electrophysiological activity of nigral neurons in normal and in 6-OHDA-lesioned rats w27,42,61,62x. Although there is no evidence in support of

it, other pathways such as the cortico-striatal might also be involved in the effects of systemically administered dopamine receptor agonists. The hypothesis that systemically administered D1 dopamine receptor agonists exert an excitatory effect on the activity of GABAergic striato-nigral neurons is also supported by indirect evidence. For instance, systemic L-DOPA administration induces a D1- but not D2-dependent increase in glucose utilization in the two targets of the striato-nigral pathway, the entopeduncular nucleus and the substantia nigra pars reticulata w57,58x. In addition, the levels of substance Prdynorphin andror their mRNAs, which are preferentially distributed in striato-nigral neurons, are up-regulated following chronic administration of either apomorphine or SKF-38393 in normal w20,37,38x or dopamine-depleted rats w11,19,20,28,39x. Finally, a recent study has shown that systemically injected D1 dopamine receptor agonists are able to increase the expression of the early gene zifr268 in presumed striato-nigral but not striato-pallidal neurons w22x. 4.3. Significance of differential regulation of GADs In addition to differences in their molecular weight, the two GAD enzymes differ in their affinity for the co-factor pyridoxal-phosphate. Whereas about half of brain GAD65 is bound with the co-factor, almost all GAD67 is found as an holo-enzyme, i.e. associated with its co-factor w12,41x. These data combined with earlier studies showing that GAD67 is regulated at gene level has led to the speculation that GAD65 activity was mainly regulated by co-factor availability w30,41x whereas GAD67 activity was regulated through changes in GAD67 gene expression. Our results thus provide evidence that striatal GAD65 can also be regulated at gene level and that such a regulation might be involved in the modulation of activity of subpopulations of GABAergic neurons. Another feature of the brain GAD65 enzyme is its preferential distribution in axon terminals w12,14,30x. Moreover, in vitro studies have shown that GAD65 is associated with the surface of vesicle-like structures w56x. In contrast, GAD67 appears preferentially distributed in the cytoplasm of cell bodies w12,14x. In view of this specific association with vesicles and because GABA might be physiologically released in a vesicular as well as a non-vesicular manner w3,5,10,48x, it is tempting to speculate that the two GAD isoforms are differentially involved in vesicular vs. non-vesicular GABA release. In this case, our results would tend to indicate that D1 dopamine receptor agonists preferentially induce an increase in vesicular vs. non-vesicular GABA release by striato-nigral neurons. 4.4. Conclusions The present study provides original evidence that GAD65 but not GAD67 gene expression is increased in

N. Laprade, J.-J. Soghomonianr Molecular Brain Research 48 (1997) 333–345

presumed striato-nigral neurons following chronic systemic administration of D1 or D1rD2 dopamine receptor agonists to adult rats. These effects are consistent with an excitatory role of D1 dopamine receptors on GABAergic neurotransmission in striato-nigral neurons. Although its functional significance remains to be determined, the preferential involvement of the GAD65 isoform in the regulation of D1-mediated stimulation of striato-nigral neurons in adult rats could have critical consequences on the release of GABA andror on its post-synaptic action on nigral and pallidal neurons. In particular, we suggest that the regulation of GAD65 gene expression by dopamine receptor agonists might reflect a modulation of vesicular GABA release in striato-nigral neurons.

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Acknowledgements We wish to thank Dr. Allan J. Tobin ŽUCLA. for the gift of the GAD67 and GAD65 cDNAs. The technical assistance of Ms. I. Deaudelin is also acknowledged. This research has been supported by the ‘Fonds de la Recherche en Sante´ du Quebec ´ ŽFRSQ.’, the Parkinson Foundation of Canada and the Natural Sciences and Engineering Research Council of Canada ŽNSERC..

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