Developmental Expression of the GABAAReceptor a1 Subunit mRNA in the Rat Brain Carla Gambarana, Robin Pittman, and Ruth E. Siegel* Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 441 06

SUMMARY Recent studies have suggested that the GABAA, receptor complex, the site of action of the inhibitory neurotransmitter gamma amino-butyric acid (GABAA) and the anxiolytic benzodiazepines, is heterogeneous. Mareover, its composition may change during development. T o better understand the molecular basis of receptor heterogeneity, the levels and distribution of the mRNA encoding the a1 receptor subunit were examined in the developing and adult rat brain with quantitative in situ hybridization histochemistry. O u r studies demonstrate that a1 subunit mRNA expression changes during ontogeny. At late embryonic stages and in the first postnatal week, low levels of the mRNA were detected in the cortex, inferior colliculus, and hippocampus. T h e mRNA levels in these regions increased during the second and third postnatal weeks. Furthermore, a dramatic change in the distribution of the a1 subunit mRNA was

seen in the second postnatal week when the message first became detectable in the cerebellar cortex. During subsequent development and in the mature brain, the a1 subunit mRNA was most abundant in the cerebellum, olfactory bulb, and inferior colliculus, although the absolute levels of mRNA varied by as much as sixfold in selected brain regions. The mature distribution of a1 subunit mRNA, along with its temporal appearance in the cerebellum, suggests that this subunit is a constituent of the Type 1 benzodiazepine site of the GABAA receptor complex. Furthermore, the onset of a1 subunit mRNA expression in the cerebellar cortex coincides with a period of extensive synapse formation, raising the possibility that synaptic interactions modulate the appearance of this GABAA receptor subunit in the cerebellum.

INTRODUCTION

complex that mediates these multiple actions is composed of two polypeptide subunits, the a and the 0(Sigel, Stephenson, Mamalaki, and Barnard, 1983;Mamalaki, Stephenson, and Barnard, 1987), which preferentially bind the benzodiazepines and GABA, respectively (Deng, Ransom, and Olsen, 1986; Casalotti, Stephenson, and Barnard, 1986). Although biochemical studies have identified only two classes of subunits, several lines of evidence suggest that GABAAreceptor composition is heterogeneous and may vary in different brain regions. In pharmacological studies, two classes of benzodiazepine binding sites, Type 1 and Type 2, have been identified (Klepner, Lippa, Benson, Sano, and Beer, 1979). The two receptor types appear a1 different developmental stages and exhibit different spatial patterns of expression in rat and mouse brains (Lippa, Beer, Sano, Vogel, and Meyerson, 1981). An even greater diversity in re-

The GABAA receptor complex mediates the actions of garnma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain. Numerous studies have demonstrated that GABA-induced increases in chloride ion flux are modulated by several classes of clinically important compounds, including the anxiolytic benzodiazepines and the anticonvulsant barbiturates (Olsen, 1982; Tallman and Gallager, 1985; Stephenson, 1988). Receptor purification and photoaffinity labelling have indicated that the receptor Received June 6 , 1990; accepted June 8. 1990 Journal ofNeurobiology, Vol. 21. No. 8, pp. 1169-1 179 (1990) 0 1990 John Wiley ti Sons, Inc. CCC 0022-3034/90/080 1 169- 1 1$04.00 * To whom correspondence should be addressed.

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ceptor structure is suggested by molecular cloning. These studies have isolated multiple N and p subunit cDNAs indicating that the subunits are actually encoded by families of closely related genes (Levitan, Schofield, Burt, Rhee, Wisden, Kohler, Fujita, Rodriguez, Stephenson, Darlison, Barnard, and Seeburg, 1988; Ymer, Schofield, Draguhn, Werner, Kohler, and Seeburg. 1989a). Although it was initially believed that the entire receptor complex was composed of only these two subunits, later studies indicated that additional subunits were necessary, When cDNAs for the a and (Isubunits were coexpressed transiently in mammalian cells, the cells were sensitive to GABA but unresponsive to the benzodiazepines, a property that appears to be specified by the recently identified 72 subunit (Pritchett Sontheimer, Gorman, Kettenmann, Seeburg, and Schofield, 1988, Pritchett, Sontheimer. Shivers, Ymer, Kettenmann, Schofield, and Seeburg, 1989a). Finally, a cDNA encoding a 6 subunit has been identified (Shivers, Killisch, Sprengel, Sontheimer, Kohler, Schofield, and Seeburg, 1989), although the role of this subunit in the formation of a functional receptor complex has not yet been elucidated. To determine further the molecular basis for receptor heterogeneity, several groups have begun to examine the distribution of receptor subunit mRNAs in the central nervous system. Studies on the adult bovine brain have demonstrated that the cx subunit subtypes exhibit different and distinct distributions (Wisden, Morris, Darlison, Hunt, and Barnard, 1988, Wisden, Morris, Darlison, Hunt, and Barnard, 1989a). Although similar studies have been performed to examine a subunit expression in the rat brain, interpretation of these studies is complicated by the fact that probes detecting multiple species of mRNA have been used (Skquier, Richards, Malherbe, Price, Mathews, and Mohler, 1988; Montpied, Martin, Cottingham, Stubblefield, Ginns, an d Paul, 1988; Khrestchatisky MacLennan, Chiang. Xu, Jackson. Brecha, Sternini, Olsen, and Tobin, 1989). To understand better the structure of the GABAAreceptor complex in this species, we have examined the levels and distribution of the mRNA encoding the al receptor subunit in the developing rat brain with quantitative in situ hybridization histochemistry. This subtype appears to be the most abundant of all of the a subunits (Levitan et al., 1988; Wisden et al., 1988, Wisden, Morris. Darlison, Hunt, a nd Barnard, 1989b; Ymer, Draguhn, Kohler, Schofield, and Seeburg, 1989b) and is the most extensively characterized a subunit subtype

in the rat brain (Lolait, O’Carroll, Kusano, Muller, Brownstein, and Mahan, 1989; Khrestchatisky et al., 1989). Our studies demonstrate that crl subunit mRNA expression changes during ontogeny. Moreover, its developmental profile in the cerebellum raises the possibility that synaptic interactions play a role in regulating receptor gene expression. METHODS Tissue Sprague-Dawley rats (Zivic Miller) were killed at embryonic day 18 and postnatal days I , 4, 6, 8, 10, 14, 16, 21, 28, and 60. For each time point, brains were removed from four to six animals and immediately frozen on dry ice. Parasagittal sections (10 prn) were cut on a cryostat at the level of plates 80-82 (Paxinos and Watson. 1986) and mounted onto gelatin-coated slides. The sections were thcn stored at -80°C until processed for in situ hybridization histochemistry.

Probe Preparation Oligonucleotide probes 40 bases in length were made by solid-phase synthesis on an Applied Biosystems DNA synthesizer (courtesy of Dr. Pieter DeHaseth, Case Western Reserve University). The a 1 subunit probe was complementary to bases 809-848 of the bovine cDNA sequence (Schofield. Darlison, Fujita, Burt. Stephenson, Rodriguez, Rhee. Ramachandran, Reale, Glencorse, Seeburg, and Barnard, 1987), a putative extracellular domain that shares little homology with either the glycine or nicotinic acetylcholine receptors. This probe shares 87.5% nucleotide sequence homology with the rat a1 subunit cDNA that was published after these studies were begun (Khrestchatisky et al., 1989). A message sense probe complementary to the cul subunit probe was also prepared as a control for specificity. For in situ hybridization histochemistry, the probes were labelled to comparable specific activity on the 3‘ end using terminal deoxynucleotidyl transferase (Bethesda Research Labs) and [35S]deoxyadenosine5’-(a-thio)triphosphate (NEN) as previously described (Siegel, 1988).

In situ Hybridization Histochemistry In sitti hybridization histochemistry was performed essentially as previously described (Siegel, 1988). For each experiment, triplicatc sections for all time points were processed simultaneously. The sections were warmed to room temperature, fixed for 5 min with 4% formaldehyde in 0.1 M sodium phosphate (pH 7.2), and then rinsed three timcs in phosphate-buffered saline (PBS). The sections were incubated in 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0 (10 min), rinsed in 2 X SSC ( 1 X SSC = 0.15 M NaCI, 0.01 5 A4 sodium ci-

Ontogeny of GABAA Receptor mRNA trate. pH 7.0), and dehydrated sequentially, for 5 min each, in 70%, 80%, and 95% ethanol, chloroform, 95% ethanol, and then allowed to air dry. Following tissue processing, the sections were prehybridized with 50 yl of hybridization buffer containing 4 X SSC, 50% formamide, 10% dextran sulfate, 2 mg/mL single-stranded DNA for 1 h at room temperature. To prevent tissue drying, the sections were covered with parafilm coverslips during all incubations. The sections were incubated in 45 pl of the hybridization buffer containing 0.5-1 X lo6 counts per minute (cpm) of %-labelled probe and 100 mM dithiothreitol, for 2 1-22 h at room temperature. After incubation, the parafilm coverslips were floated off the slides in 2 X SSC. The slides were then washed four times for 15 min each in 2 X SSC containing 50% formamide at 3 5 T , dipped briefly in water, air dried, and processed for autoradiography. Two autoradiographic procedures were used. For more rapid signal detection. the slides were placed against Kodak X-AR film and exposed for 4 weeks at room temperature. This procedure yielded information concerning the regional distribution of the message. For cellular resolution of the signal, the slides were dipped in Kodak NTB 3 liquid emulsion diluted 1:1 with water and exposed at 4°C in a desiccated chamber for 8 weeks. The autoradiographs were developed in Kodak D I9 and stained with 0.4% cresyl violet to allow visualization of cells and overlying silver grains in phase-contrast optics. The slides were mounted in DPX (BDH) for observation.

Quantitation Film autoradiographs were quantitated by measuring optical densities using the Micro Computer Imaging Device (MCID) system (Imaging Research, Inc., Brock University. St. Catherine, Ontario). The optical density ofthe film (0.206 k 0.004, n = 7) was subtracted from all readings. Significant differences in relative hybridization signals were not observed when films were preexposed to increase sensitivity (Laskey and Mills, 1975), indicating that the exposures were in the linear response range of the film. Liquid emulsion autoradiographs were analyzed by determining grain densities over individual Purkinje neurons. These cells were identified by morphological criteria with phase-contrast optics. Total numbers of grains over at least 100 cells for each age were counted either manually or using the CUE 4 Image Analysis System (Olympus). Grains representing nonspecific binding of the probe were determined in two ways, both of which yielded similar results. In most cases, nonspecific grains were counted over areas of the emulsion-covered slide away from the tissue section. Second, the number of grains over Purkinje neurons in sections hybridized with the radioactively labelled sense probe were counted. Specific grain counts were obtained by subtracting the nonspecific grains from the total.

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RESULTS

Temporal and Spatial Distribution of the a1 Subunit mRNA The regional pattern of a1 subunit mRNA distribution in the developing rat brain was examined by hybridization histochemistry and film autoradiography. At embryonic day 18 and during the first postnatal week, only a weak hybridization signal was observed throughout the brain [Fig. l(A)]. During this time. the mRNA was most prominent in the developing cortex, hippocampus, inferior colliculus, and mitral cell layer of the olfactory bulb. In addition. the a1 subunit mRNA was detectable in the thalamic nuclei, striatum, superior colliculus, and the lower brain stem. By postnatal day 7-8, the mRNA was also observed in the deep cerebellar nuclei. Dramatic changes in the pattern of 01 subunit mRNA expression occurred during the second postnatal week [Fig. l(B,C)]. During this period, the mRYA was observed in the cerebellar cortex, an area previously lacking detectable hybridization. The a1 subunit mRNA was found in the molecular and granule cell layers by day 9 and rapidly increased in intensity during the following 2-3 days. At the same time, the intensity of the hybridization signal increased in other brain regions already exhibiting the al subunit mRNA at earlier developmental stages. The pattern of a1 subunit mRNA distribution did not change over the remainder of the developmental time course [Fig. l(D)]. In the adult brain, the most intense signals were observed in the cerebellum, olfactory bulb, inferior colliculus, and hippocampus, whereas signals of lower intensity were present in the cortex, thalamic nuclei, superior colljculus, and striatum. The specificity of the hybridization signal is supported by several lines of evidence. First, the specificity of the probe for the a1 subunit was confirmed by Northern blot analysis. With this approach, a broad band of approximately 4.5 kb was observed in the cerebellum and cortex of the adult rat brain (data not shown). An mRNA(s) of approximately the same size has previously been identified as that encoding the bovine and rat a1 subunit mRNAs (Siegel, 1988; Lolait et al., 1989; Khrestchatisky et al., 1989). Second, the specificity of the patterns generated by hybridization histochemistry were confirmed by probing adjacent sections with a message sense probe. Hybridizations with this probe yielded only background sig-

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Figure 1 Temporal and regional expression of a1 subunit mRNA in the rat brain. Tissue sections from animals at postnatal days 6 (A), 10 (B), 14 (C), and 28 (D) were hybridized with the 35S-labelledoligonucleotide probe and placed against Kodak X-AR film. IC = inferior colliculus; HI = hippocampus; CTX = cortex: MI = mitral cell layer of the olfactory bulb: G R = granule cell layer of the cerebellum; N = deep cerebellar nuclei: DG = dentate gyrus.

nals of uniform intensity over the entire tissue section (Fig. 2). Moreover, only background levels were observed over the white matter of the corpus callosum with either the sense or antisense probes. Quantitative Analysis of a1 Subunit mRNA Levels Temporal changes in a1 subunit mRNA levels in selected brain regions were examined further by densitometric analysis of the film autoradiographs. Two general patterns of expression were observed. In most areas, the hybridization signal increased gradually over the first 3 postnatal weeks, peaked by postnatal days 21 or 28, and then declined to the adult level (Tables 1,2). When this pattern was observed, the a1 subunit mRNA level in the adult was approximately 5 0 4 0 % of the maximal level of expression found at earlier developmental stages. A second and slightly different pattern was observed in the cerebellum (Table 3). In this region, where the mRNA was first detectable in the

second postnatal week, the hybridization signal increased very rapidly, was maximal by day 2 1, and then decreased to the adult level. In the granule cell layer of the cerebellar cortex, for example, the amount of a1 subunit mRNA increased approximately fourfold between postnatal days 8 and 14. Increases of similar magnitude were not observed in any other brain region over the same developmental period. The level of a 1 subunit mRNA expression varied greatly among different brain regions. At day 21, the peak of mRNA expression, a 6.3-fold difference was found between the region with the highest density of mRNA, the granule cell layer of the cerebellar cortex, and that with the lowest level of expression among the areas analyzed, the antenor olfactory bulb. In addition, the level of a1 subunit mRNA in the granule cell layer was 2.6and 1.2-foldgreater than that in the cerebral cortex and inferior colliculus. At postnatal day 4, when a1 subunit mRNA expression was lower in all brain regions, differences of the same magnitude

Ontogmy ofGABAA Receptor mRNA

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Table 1 Developmental Expression of a1 Subunit mRNA in Selected Brain Regions Age (Days)

Region

4

6

8

CTX Mi

4 9 + 13 191 + 13

6 5 + 13 233 f 35 43215 3 5 f 10 64k 5 46f 8

5 9 + 15

A0

Th IC

sc

-

31 + 16 92222 47f21

30214 33+ I1 130+ 10 5 3 + 13

10

14

16

21

28

60

8 2 k 12 136124 4 7 k 3 45 f 12 156k23 71 k 19

138+ 9 176t18 91f23 75 2 13 1 5 4 k 16 8 4 2 18

1 1 3 + 13 255+21 75523 7 5 5 12 2 4 9 + 15 91 t 14

1 2 8 t 19 220i16 84k28 121 + 2 7 282f30 109k 15

1095 3 272+18 61213 8 8 ? 12 249+21 9 3 + 12

7 2 5 12 238+17 4 1 t 6 72-+20 171 k 2 5 80t 16

Note: CTX = cortex; Mi = mitral cell layer of the olfactory bulb; A 0 = anterior olfactory nucleus; Th = thalamic nuclei; inferior colliculus; SC = superior colliculus. Brain sections from animals at the indicated postnatal ages were hybridiLed with JsS-labelleda1 subunit-specific probe and exposed 4 weeks for autoradiography. The optical density ofthe film image (O.D. X lo’) was analyzed as described in the Methods. Values from the cortex represent densities measurcd ovcr the soniatoscnsory cortex, and signals from the thalamus were measured over the entire thalamic rcgion at the level of plates 80-82 (Paxinos and Watson, 1986) for all experimental ages. All values represent thc mcan i- SEM of 4-6 separate experiments.

IC

=

were observed. The level of mRNA in the mitral cell layer of the olfactory bulb, the region cxhibiting the highest density, was 6. I - and 2. I-fold greater than that in the thalamic nuclei and inferior colliculus, respectively. Cerebellar Expression of the a1 Subunit mRNA As the most striking developmental changes in a1

subunit mRNA expression occurred in the cerebellum, m R N A levels in individual neurons were examined in greater detail in this region using high-resolution liquid emulsion autoradiography. During the first postnatal week, few positive cells could be detected. However, labelled cells were observed in the deep cerebellar nuclei by day 6-7, and grains were found over most large cells in this region at later developmental stages (Fig. 3). In the cerebellar cortex, positive Purkinje neurons were prominent by day 8 and a lower density of grains

was observed over the granule cell layer. Grains were observed in all layers of the cerebellar cortex by day 10 (Fig. 4). By day 14 and at later developmental stages, all Purkinje cells were positive, and a uniform distribution of grains was observed over the granule cell layer. Comparison of dark- and bright-field micrographs indicates that a majority of the granule cells were labelled. However, because granule cells are densely packed and the autoradiographic signal may spread beyond cellular boundaries, the possibility that some cells were unlabelled cannot be ruled out. To examine quantitative changes in LY 1 subunit mRNA expression in Purkinje neurons, the grain density over individual cells was determined. In a representative experiment (Fig. 5), the mean number of grains over Purkinje cells was 24.8 1.7 at postnatal day 8, the earliest time at which positive cells were reliably detected. By day 14, the average number of grains was 60.0 k 2.0, a 2.4-fold increase. The density of grains over individual Pur-

*

Table 2 Developmental Expression of a1 Subunit mKNA in the Hippocampus

Region CAIpy

4

6

8

10

14

16

21

28

60

89513 8 51e 19

87k19 66 -t 14 5 2 f 14

107-C20 75 + 17 75 k 2 7

127kX 97 f 12 9 0 + 14

159+15 127 16 1 2 4 2 19

156i19 117 t 2 2 105 k 15

167i15 1242 11 151 k 16

145kI2 108 12 1 2 6 t 15

121k11 95 f 10 167f38

c A 2 - 3 ~ ~ 50?

DGgr

+

*

Note: py = pyramidal layer; DG = dentate gyms; gr granule layer. Hybridization histochemistry and image analysis were performed as described in the legend for Table I . (O.D. X lo3)Only those hippocampal regions exhibiting significant signals are shown. In the molccular layers of CAI, CA2.3, and of the dentate gyms, areas which contain few neuronal cell bodies, only background signals wcre observcd. All values represent the mean f S.E.M. of 4-6 separate experimcnts.

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Table 3 Developmental Expression of a1 Subunit mRNA in the Cerebellum

Age (Days) Regon

8

10

14

16

21

28

60

Granule cell layer Purkinje cell layer Molecular layer Deep cerebellar nuclei

60 % 1 1

131 t 13

21 f 11

45+ 7

238 t 22 299222 56t_ 7

291 2 14 321 ? 2 4 55k 6

335 f 19 375k 9 106t 16

290 4 8 385k 4 9 5 % 13

263 +_ 23 260?36 9 9 k 12

62?

7 4 k 16

151 t 10

208 t 18

224t22

1602 14

l o o t 10

-

8

-

In situ hybridization histochemistry and image analysis was performed as described in Table 1. In the Purkinje cell layer. no signal could be routinely determined on the low-resolution film image (OD. X lo3) prior to day 14. All values represent the mean 2 S.E.M. of 4-6 separate experiments.

kinje cells reached a plateau by day 21 and remained constant throughout the rest of the experimental time course. Examination of the distribution of grain densities over individual cells suggests that the increases in a 1 subunit expression

occur throughout the entire Purkinje cell population. Only a single population of positive cells was observed, although these data were not subjected to a detailed statistical analysis (Fig. 6). Moreover, the increases in grain density were independent of increases in Purkinje cell size because the developmental profiles of grains/cell and grains/area were identical (data not shown).

DISCUSSION Numerous reports have examined the heterogeneity of the GABAAreceptor complex. In recent mo-

Figure 2 In yitu hybridization histochemistry using antisense and sense oligonucleotide probes. Adjacent tissue sections prepared from a p21 rat brain were hybridized with antisense (A) or sense (B) probes labelled to the same specific activity and exposed identically for autoradiography. Specific hybridization was only detected with the antisense probe.

Figure 3 Phase micrograph of a1 subunit mRNA in neurons of the deep cerebellar nuclei. Sections from a p28 rat brain were hybridized with 35S-labelledprobe, dipped in liquid emulsion, and exposed 8 weeks for autoradiography. Arrows indicate positive cell bodies. Scale bar = 150 urn.

Ontogmy of GABAAReceptor mRNA

lecular biological studies, cDNAs encoding multiple N and p, as well as 6 and y subunits, of the GABA,, receptor have been isolated (Olsen and Tobin, 1990). The existence of multiple receptors is supported by the recent purification of three a and three p subunit polypeptides from the rat brain (Fuchs and Sieghart, 1989) and the identification of multiple peptides using a subunit-specific antibodies (Stephenson, Duggan, and Casalotti, 1989; Fuchs, Adamiker. and Sieghart, 1990).

All of these findings support the possibility that different receptors, varying in composition, exist in different brain regions. To understand better receptor composition and the molecular basis of heterogeneity, we have examined the distribution of mRNA encoding the N I subunit in the developing and adult rat brain. Our studies demonstrate that the a1 subunit mRNA is widcly distributed throughout the rat brain. In early postnatal development, the highest

Figure 4 Autoradiographic localization of the N I subunit mRNA in the rat ccrcbellum. Dark-held (left) and phase (right) views of sections prepared from rats at postnatal days 10.2 1, and 28. Tissues were hybridized, dipped in liquid emulsion, and exposed 8 weeks for autoradiography. P = Purkinje cell: G = granule cell layer; N = deep cerebellar nuclei. Scale bar = 500 Pm.

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1176

= 0)

100

-

75

-

50

-

Y

u)

c ‘i 0

25 0 0

I

unit mRNA that we observed in the adult rat brain is similar to that reported in previous studies (Stquier et al., 1988; Montpied et al., 1988; Khrestchatisky et al., 1989), significant differences do exist. In the study by Montpied et al., (1988), 01 subunit mRNA appeared to be most abundant in the cerebellum, thalamus, and hypothalamus. Our quantitative studies similarly revealed that the highest levels of a 1 subunit mRNA existed in the cerebellum. In fact, the high resolution of our hybridizations demonstrated that the mRNA was most abundant i n the cerebellar Purkinje and granule cell layers, whereas lower levels existed in the molecular layer and in the deep cerebellar nuclei. However, in contrast to this previous study, significantly lower levels of the mRNA were observed in the thalamic nuclei. This discrepancy presumably reflects differences in the probes used for hybridization. While we used an oligonucleo-

:II:,a I

I

I

10

20

Adult

Age (days)

Figure 5 Developmental changes in the expression of the a1 subunit mRNA in Purkinje neurons o f the cerebellar cortex. The number of grains observed over individual Purkinje neurons following hybridization and liquid emulsion autoradiography was quantitated by image analysis. For days 10 through adult, each point represents the mean k S.E.M. of 100 cells on a single brain section. For day 8, where Purkinje neurons are more difficult to identify, grains were counted over 37 cells. Similar developmental patterns were observed in three separate experiments.

60

40

levels of hybridization were observed in the cerebral cortex, hippocampus, inferior colliculus, and mitral cell layer of the olfactory bulb. The mRNA was also abundant in the cerebellum at later developmental stages. In most brain regions, the level of mRNA increased gradually over the developmental time course. The increases in trl subunit expression, and the delay in its appearance in the cerebellum, parallel changes in GARA and benzodiazepine receptors detected by homogenate binding studies (Coyle and Enna, 1976; Candy and Martin, 1979; Aldinio, Balzano, and Toffano, 1980) and by receptor autoradiography (Palacios and Kuhar, 1982; C. Gambarana and R. E. Siegel, unpublished). In addition, whereas our report is the first to document developmental changes in a1 subunit mRNA levels in identified cell populations, studies on tissue homogenates also indicated that the expression of a subunit mRNAs changes in ontogeny (Levitan et al., 1988;Garrett, Duman, Saito, Blume, Vitek, and Tallman, 1988; Montpied, Ginns, Martin, Stetler, O’Carroll, Lolait, Mahan, and Paul, 1989). Such developmental changes do not appear to be unique to the 01 subunit as similar changes also occur in the expression of the mRNAs encoding the /3 and y subunits ( C . Gambarana and R.E. Siegel, unpublished). Although the regional distribution of a1 sub-

20

40

20

0

P2’

I

20

0

GRAINS/CELL

Figure 6 Distribution of grain density over Purkinje neurons. The number of grains over the entire Purkinje cell population increased with development.

Ontogcny of GA4B&Rerepior mRNA

tide probe that recognizes the a1 subunit mRNA exclusively, a cRNA probe that hybridized to three different transcripts was used in the previous study. Thus, while the subunit-specific oligonucleotide reveals only the a1 subunit mRNA, additional a subunit subtypes, which are presumably more abundant in the thalamus and hypothalamus, were detected with the cRNA probe. The spatial and temporal distribution of the rat a1 subunit mRNA suggests that this subunit 1s a constituent of the Type 1, but not the Type 2, benzodiazepine binding site. Several studies have demonstrated that these two receptors are pharmacologically distinct and exhibit different regional and temporal patterns of distribution (Klepner et al., 1979; Lippa et al., 1981; Chisholm, Kellogg, and Lippa, 1983). For example. the Type 1 receptor is enriched in the inferior colliculus and cerebellum of the adult rat brain, whereas the Type 2 receptor predominates in the spinal cord; a mixture of both receptors is found in the cortex and hippocampus. Moreover, the developmental pattern of the two receptors differs. The Type 2 receptor is present in the neonatal cortex and increases to adult levels over the next few weeks. In contrast. only low level5 of the Type 1 receptor are observed in the first postnatal week, and the receptor only becomes abundant during the second and third weeks of development. In fact, the cerebellar Type 1 receptor is first observed between postnatal days 7 and 16. Thus, the distribution of the Type 1 benzodiazepine receptor closely parallels the expression of thc a1 subunit mRNA in the rat brain. That the a1 subunit encodes a Type 1 receptor is also supported by two other studies. First, the properties of a , (I, and y subunits coexpressed in cultured mammalian cells have been examined. These studies indicate that cells containing the CY 1 subunit, but not the other a subunit subtypes, exhibited the pharmacological specificity of Type 1 G A BA,&xzodiazepine receptors (Pritchett, Luddens, and Seeburg, 1989b). Second, the distribution of a subunit mRNAs was examined in the bovine brain (Wisden et al., 1989a). This study revealed that the LY 1 subunit was most abundant in the cerebellum, inferior colliculus, olfactory bulb, and substantia nigra, regions enriched in the Type 1 receptor. In contrast, the a2 and 013 mRNAs were more abundant than the a1 mRNA in the striatum, The distribution of these two mRNAs does not correspond to that expected for the Type 2 receptor, and the properties of the encoded proteins are unknown. Moreover, recent studies indicate that the a2 subunit is not neuron-specific and

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is expressed in &a1 cells (Wisden, McNaughton, Dadison, Hunt, and Barnard, 198%). In the cerebellar Purkinje neurons, 011 subunit mRNA was not detected by hybridization histochemistry prior to the second postnatal week. In contrast, previous electrophysiological studies have demonstrated that Purkinje neurons are responsive to GABA at postnatal day 1 (Woodward, Hoffer, Siggins, and Bloom, 197 I). The reason for this discrepancy between the anatomical and physiological data is unclear. One possible explanation is that Purkinje cell sensitivity is mediated by a GABA,/benzodiazepine receptor encoded by a level of (Y 1 subunit mRNA too low to be detectable by hybndization. Alternatively, multiple genes encoding the receptor may exhibit differences in temporal expression. In this case, an 01 subunit mRNA other than a l may be expressed early in development, whereas the a 1 subunit may appear as the cerebellum matures. The events regulating receptor gene expression in the CNS are largely unknown. That the onset of LYI subunit mRNA is first observed in the cerebellum during the second postnatal week raises the possibility that synapse formation plays a role in regulating GABAAreceptor gene expression. The appearance of the trl subunit mRNA in the cerebellar cortcx is synchronous with the postnatal maturation of the cerebellum and with the formation of synaptic connections between the granule, basket, and stellate cells and their target Purkinje neurons and betwcen Golgi and granule cells. In addition, the a1 message is readily detectable in the deep cerebellar nuclei at day 7-8, by which time these cells have received afferent input from the Purkinje cells (Eccles, Ito, and Szentigothai, 1967; Altman, 1972a,b).Although synaptic events have not been implicated previously in the regulation of the GABA, receptor or other neurotransmitter receptor systems in the central nervous system, many properties of the nicotinic acetylcholine receptor in skeletal muscle cells are clearly modified by innervation (Salpeter and Loring, 1985: Schuetze, 1986). Furthermore, the molecular composition of the peripheral nicotinic receptor is altered by synaptogenesis (Gu and Hall, 1988) and denervation results in increased levels of receptor subunit mRNAs (Shieh. Ballivet. and Schmidt, lY88). I h e possibility that synaptic interactions similarly modulate GABAA receptor gene expression remains to be determined. We thank Dr. Gregory A. Ordway, Department of Psychiatry, Case Western Reserve University, for assis-

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tance with image analysis and Ms. Zoe Rodriguez for her excellent technical assistance. This work was supported by grants from the National Institute of Mental Health and the Mathers Foundation.

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