GABA Forebrain Subunit mRNA Distribution

8/14/2019 GABA Forebrain Subunit mRNA Distribution

1/23

The Journal of Neuroscience, March 1992, f2(3) : 1040-l 0

The Distribution of 13 GABA, Receptor Subunit mRNAs in the RatBrain. I. Telencephalon, Diencephalon, Mesencephalon

W. Wisden, D. J. Laurie, H. Monyer, and P. H. SeeburgLaboratory of Molecular Neuroendocrinology, Zentrum fijr Molekulare Biologie, University of Heidelberg, D-6900Heidelberg, Germany

The expression patterns of 13 GABA, receptor subunit en-coding genes (q-as, &-@,, y,--r3, 6) were determined in adultrat brain by in situ hybridization. Each mRNA displayed aunique distribution, ranging from ubiquitous (a, mRNA) tonarrowly confined (a6 mRNA was present only in cerebellargranule cells). Some neuronal populations coexpressed largenumbers of subunit mRNAs, whereas in others only a fewGABA, receptor-specific mRNAs were found. Neocortex,hippocampus, and caudate-putamen displayed complex ex-pression patterns, and these areas probably contain a largediversity of GABA, receptors. In many areas, a consistentcoexpression was observed for a, and b2 mRNAs, whichoften colocalized with -r2 mRNA. The a,& combination wasabundant in olfac tory bulb, globus pallidus, inferior collicu-Ius, substantia nigra pars reticulata, globus pallidus, zonaincerta, subthalamic nucleus, medial septum, and cerebel-lum. Colocalization was also apparent for the (Y* and fi3mRNAs, and these predominated in areas such as amygdalaand hypothalamus. The a3 mRNA occurred in layers V and

VI of neocortex and in the reticular thalamic nucleus. In muchof the forebrain, with the exception of hippocampal pyra-midal cells, the ar, and 6 transcripts appeared to codistribute.In thalamic nuclei, the only abundant GABA, receptor mRNAswere those of a,, a4, &, and 6. In the medial geniculate tha-lamic nucleus, a,, a,, &, 6, and yJ mRNAs were the principalGABA, receptor transcripts. The a5 and 8, mRNAs generallycolocalized and may encode predominantly hippocampalforms of the GABA, receptor. These anatomical observa-tions support the hypothesis that a,&y2 receptors are re-sponsible for benzodiazepine I (BZ I) binding, whereas re-ceptors containing a*, a3, and a, contribute to subtypes ofthe BZ II site. Based on significant mismatches between a,/6and y mRNAs, we suggest that in viv o, the CY~ ubunit con-tributes to GABA, receptors that lack BZ modulation.

GABA is the principal inhibitory transmitter in vertebrate brain.GABA produces its inhibitory effect by interacting with two

Received Aug. 8, 1991; revised Oct. 21, 1991; accepted Oct. 23, 1991.We gratefully acknowledge Ulla Keller for expert and dedicated technical help

and Jutta Rami for efficient secretarial skills. D.J.L. was a recipient of a EuropeanScience Exchange Program fellowship awarded by the Royal Society (London).W.W. he ld an EMBO long-term fellowship. This work was supported by theBundesministerium Blr Forschung und Technolog ie, Grant BCT 364 AZ 231/7291, the Deutsche Forschungsgemeinschaft (SFB 3 17, B9), and the Fonds derChemischen Industrie (P.H.S.).

Correspondence should be addressed to W. Wisden, Laboratory of MolecularNeuroend ocrinology, Zentrum fur Molekulare Bio logie , University of Heidelb erg,Im Neuenhe imer Feld 282, D-6900 Heidelb erg, Germany.

Copyright 0 1992 Society for Neuro science 0270-6474/92/121040-23$ 05.00/O

classes f molecules on the target cell: (1) GABA, receptors,which are igand-gated anion channels hat exhibit a diverse andclinically important pharmacology, being the locus of action forbarbiturates, benzodiazepines BZs), and steroids, which all actalosterically to modify the efficacy of GABA (Haefely and Pole,1986; Lambert et al., 1987; Puia et al., 1990); ethanol alsoappears to mediate some of its effects through this receptor(Wafford et al., 1990, 199 1); and (2) GABA, receptors, whichare coupled to G-protein-mediated cellular responses Bowery,1989). These two GABA receptor classes ct on different timescales, often in the same synapse Dutar and Nicoll, 1988).

Our knowledge regarding the molecular composition of theGABA, receptor has ncreased considerably over recent years.Once thought to be a single molecular species Haring et al.,1985) this receptor now presents a staggering molecular diver-sity revealed by cDNA cloning of GABA, receptor subunits. Inkeeping with other members of the ligand-gated ion channelsuperfamily (Unwin, 1989; Cooper et al., 1991), the GABA,receptor is probably assembled s a pentameric structure from

a number of possible subunit classes. he subunit stoichiometryin any given GABA, receptor complex is unknown. In the ro-dent there are currently six a-subunits ((Y,*~), three @-subunits(p&3,), three y-subunits (7,-y,), and a &subunit (Olsen andTobin, 1990; Seeburg et al., 1990; Herb et al., 1992; Ltiddensand Wisden, 1991; Wilson-Shaw et al., 1991). Additionally ap-subunit cDNA has recently been isolated from human retina(Cutting et al., 199 1).Molecular diversity rather than uniformityprobably accounts for the pharmacological heterogeneity ofGABA, receptors seen by numerous aboratories (Unnerstall etal., 198 1; Young et al., 1981; Sieghart, 1989; Olsen et al., 1990).

Different subunit combinations confer disparate pharmacol-ogies on GABA, receptors expressed from combinations ofcDNAs. For example, the y-subunit class s required to confera generally robust BZ responsiveness n any cul/3 ubunit com-bination (Pritchett et al., 1989b), such hat a minimum require-ment for conventional GABA, receptor pharmacology wouldbe an LYJ~~T~ ombination (where x is any variant). However,studies on alxpXyz eceptors reveal that it is the members of thee-subunit class hat dictate which type of BZ ligand binds to,and allosterically modulates, the receptor complex (Pritchett etal., 1989a,b; Ltiddens et al., 1990; Pritchett and Seeburg, 1990;Seeburg et al., 1990; Ltiddens and Wisden, 1991). The a,-con-taining complexes exhibit high affinity for CL 218-872 and/3-carbolines, whereas he complexes containing cu,, LYE, nd LYEshow ower affinity for these compounds Pritchett et al., 1989a;Pritchett and Seeburg, 1990). However, all display high affinity

for the BZ antagonist Ro 15-1788 and, particularly, for the


2/23

The Journal of Neuroscience, March 1992, f2(3) 1041

alcohol antagonist Ro 15-45 13. The cyqand (Ye subunits incombination with a &y2 subunit pair bind Ro 15-45 13 at a siteinsensitive to diazepam (Ltiddens et al., 1990; Wisden et al.,1991 b), and hence CY,&, and a&y, receptors do not bind BZagonists. The p-subunits have been reported to modulate currentamplitudes (Sigel et al., 1990) and appear to differ in bindingof GABA analogs and pentobarbital (Bureau and Olsen, 1990).

The role of the &subunit remains unclear, but this subunit formshomomeric channel complexes gated by GABA (Shivers et al.,1989). The p-subunit appears to be quantitatively unimportantin rodent CNS, with its mRNA restricted to retina (Cutting etal., 1991).

Clearly, in order to proceed with electrophysiological andpharmacological analyses, it is important to know which subunitcombinations occur in vivo. Two approaches are available: map-ping the site of protein expression using subunit-specific anti-bodies (immunocytochemistry), or tracing sites of gene expres-sion using in situ hybridization. The immunocytochemicalapproach, although providing useful information about the cel-lular distribution of GABA, receptors, s severely hindered bythe dif ficulty of raising unique antibodies. Previously used an-tibodies, fo r example, monoclonal antibody (mAb) bd- 17 andmAb 62-361 (Richards et al., 1986, 1987; de Blas et al., 1988),react with both & and & subunits (Fuchs et al., 1988; Ewert etal., 1990, 199 1). However, one recent report using syntheticpeptide-derived antibodies has demonstrated different ial lo-calization of immunoreactivity specif ic for (Y, , o(~, and LYE ub-units in rat brain (Zimprich et al., 199 1). The yz and &subunitshave also been detected immunohistochemically using peptide-specif ic antisera (Benke et al., 199 1a,b).

The sites of gene expression of some o f the subunits havebeen partially mapped by in situ hybridization. In rodent brainthese are the mRNAs for (Y, Sequier et al., 1988; Khrestchatiskyet al., 1989; Lolait et al., 1989; Hironaka et al., 1990; Malherbe

et al., 1990b; Seeburg et al., 1990; MacLennan et al., 1991), (Y*(Seeburg et al., 1990; MacLennan et al., 1991; Persohn et al.,1991; Wisden et al., 199 a), (Y) Seeburg et al., 1990; Persohnet al., 1991; Wisden et al., 1991a), LYEWisden et al., 1991b), Ye(Khrestchatisky et al., 1989; MacLennan et al., 1991, termeda14 by these authors), LYE Kato, 1990; Lilddens et al., 1990), p,(SCquier et al., 1988; Malherbe et al., 1990b; Seeburg et al.,1990; Zhang etal., 1990) p2 and & (Lolait et al., 1989; Seeburget al., 1990; Zhang et al., 1990), y, (Ymer et al., 1990), yz (Shiverset al., 1989; Malherbe et al., 1990b; Persohn et al., 199 1; Wisdenet al., 1991a), and 6 (Shivers et al., 1989). In bovine brain, theffl, a2, and (Ye mRNAs (Wisden et al., 1988, 1989a,b) and p,mRNAs (Siegel, 1988), and in chicken brain the (Y, mRNA(Bateson et al., 199 la), have also been studied.

However, no systematic comparison is currently possible. First,studies have selectively focused on different brain regions and/or different species, and second, the mRNA abundance has beenassessed by oligonucleotides, cRNA, or DNA restriction frag-ment probes. The combined results make a systematic com-parison between data diff icul t. In addition, studies employingextended cRNA or DNA probes may produce misleading re-sults, since such probes may cross-hybridize to closely relatedgene family members.

In this and the accompanying article (Laurie et al., 1992), wehave undertaken a systematic comparison of the brain distri-bution ofthe 13 currently known rat GABA, receptor transcriptsby in situ hybridization using unique oligonucleotide probes

specif ic for each subunit mRNA.

Materials and MethodsFor detection of GABA, receptor subunit transcripts, 45base antisenseoligonucleotides were synthesized, each of a unique sequence often takenfrom the region encoding the divergent intracellular area between pu-tat ive transmembrane domains M3 and M4. The oligonucleotides wereconstructed complementary to rat cDNA encoding subunit residues asfollows: 01~,342-356 (Khrestchatisky et al., 1989); 01~,340-344 (Khre-stcha tisky et al., 1991); (Ye, 61-375 (Malherbe et al., 1990a); (Ye,15-30ofthe signal peptide (Ymer et al., 1989a; Wisden et al., 1991b); (Ye, 55-369 (Khrestchatisky et al., 1989; Malherbe et al., 1990a; the subunittermed 01~by us is termed LYE y Khrestchatisky et al., 1989); (Ye,342-356 (Lilddens et al., 1990); @,, 382-396 (Ymer et al., 1989b); &, 382-396 Ymer et al., 1989b); &, 380-394 (Ymer et al., 1989b); y ,, 341-354 (Ymer et al., 1990); yz, 338-352 (Shivers et al., 1989); y3, 343-358(Herb et al., 1992; see also Wilson-Shaw et al., 1991, for the mousesequence); 6, 335-349 (Shivers et al., 1989).

The procedures used (Monyer et al., 199 1; Wisden et al., 199 1c) werea modification of those by Young et al. (1986). Probes were 3 endlabeled using a 30: 1 molar ratio o f &S-dATP (1200 Ci/mmol; Amer-sham) to oligonucleotide, and terminal deoxynucleotidyl transferase(Boehringer Mannheim). Unincorporated nucleotides were removed byBio-Span 6 chromatography columns (Bio-Rad). Nonperfused brainswere removed and frozen on dry ice. Sections (14 wrn) were cut on acryos tat, mounted onto poly-L-lysine-coated slides, and dried at roomtemperature. Sections were fixed in 4% paraformaldehyde, washed inphosphate-buffered saline, and dehydrated into 95% ethanol for storageat 4C until required. Prior to hybridization, sections were removedfrom ethanol and allowed to air dry. Labeled probe dissolved in hy-bridization buffer (0.06 fmol, 1000 dpm/pl) was then applied to sections.Hvbridization buffer contained 50% formamide/ x SSC (1 x SSC:O. 15~Nacl, 0.0 15M Na-citrate)/lO% dextran sulfate. Hybridization was at42C overnight. Sections were washed to a final stringency of 1 x SSCat 60C before alcohol dehydration and exposure to Kodak XARS film.Anatomy of sections and autoradiographs was determined using theatlas of Paxinos and Watson (1986), and fo r thalamus the monographof Jones (1985) was consulted. Signal spec ifici ty was assessed by use ofcompetition experiments in which radiolabeled probes were hybridizedto sections in the presence of an excess (50-fold) unlabeled probe. Thisresulted in blank autoradiographs. Specificity was also confirmed byreference to previous reports of the distribution of GABA, transcriptsin rat performed by other laboratories or other methods (e.g., Northern

blot analysis): the distribution of (Y, (Northern blot and insitu hybrid-ization


3/23

Table 1. Distribution of q-ah, &-&, y,--y3 , and 6 mRNAs of GABA, receptors in the CNS

Olfactory bulbPeriglomerularTufted cellsh4itral cellsGram& cells

al a2 a3 a4 as a6 & ?/j

0 (+I++ $1 +

;f8 8 i

(;I ;I

+++ 0 0 ++ 00 +++ (1) ++ ++ i 0 & +

Neocortexlayer n/mlayer IVlayer V/VI

Pyriform cortex

++ ++ + ++ + ++ + + + g 8 )I; +

++ + ++ + + 0 + + g;+++ +++ ++ ++ 0 0 + + +

HippocampusCA1 s tr. pyramidalis ++ +++ (+I ++ +++ 0 i-i+CA3 str. pywnidak + +++ (+I ++ +++ +++ {iiDG granule cells ++ +++ + +++ + i +++

ri+ ++

Teuia recta + +++ 0 +++ ++ 0 +++ 0 0

Basal nucleiCaudak -putamen ++ ++ + +Nucleus accumbens :z; ++ ):I ++ i 8 ii; + +Globus pallidus ii+

(:I

0i

0

Endopeduncular n. +++ 0 0 :

0:

0

Claustnun ++ ++ ii+ + + ++Subthalamic nucleus ++ 0 0 0 0 0 0 &i

(%I

Amygdalacentral amygdaloid I ++ (+j +med. amygdaloid n. :; +++

=I+ 8 ii +

(;I8

lateral amygdaloid n ++ +++ + + (+I 0 + (+I 0

Septumbed nucleus s. tlahalsep~medial septumdiagonal band

Medial Habeoula

+ +++ ii + $38

+++ +++ + t+i

0+

+++ 0+++ +

;:;(:I 8

0 (:I 0 80 (+I + 0

(+I ++ (+I 0 0 0 (+I (+I 0

&

++++-I-+++

0

+++ii

+++

++

++

0

$;ii+

ii+++++

+it+

;+I+++ii+

(+I

ii+++++++++++++++++++(+I

++++++

(;I

:

++

it

G-i

(+I+++

0+++

83

++++++ii+

+++iiii+

+++ii+++++++

iii$

i--G+++++

+++0+

ii

(+"I$1

8

t+;

8

ii+++

++

(+I

0

(+I+

0+++

0+++

yl

$1

A

::;(+I+

+++0

[I;++

({I

t++++(+I

+++++

(+"I

+

8+

r :;

f;+

8

+++

&+

(+I

(+I

(:I

8++0

P

+++

+++(+)

+++ii+++

+++++++++

0

+++

++

++

++

++

++++++

++

(+I++

:ij++

(:I+

+++++

ii

++

++

+++++

0++

6

TbalamusMcdio dorsal ii 0 0 +++ +Paraventicular n. + ++ + +++ i (+I 3Rhomboid nucleus ++ ++ t+ +++

({I0 + :

Dorsolat. geniculate ++Ventnht. geuiculatf t+ 8 :

++++++ 8 8 8

ri; +(+I ++

Medial geniculate ++ 0 +++ 0 0Parafascicular n. +++ (+I +++ (:I ;+;

+t0

Reticular nucl. + (:I + (+I 8 8 0Ventr. posterior n. +Zouaincuta ++ : A

+++8 8

8 [I; tt0 0 (+) 0

HypothalamusMedialpreoptiCisea

::;

+++ + (;I + 0 +Arcuate nucl. ++

(:I+ 0 0

(;I8

Dorsomedial nucl. + 0 0 0 0Ventmmedial nucl. (+I ii +

(A+ 0 + 8 0

MidbrainRed nucleus it+ (+I 0 0 0 0 0 (+I 0

Inferior colliculiCenhal nucleus +++ + (+I 0 0 0 0 (+I 0

Substantia nigraPars reticulata +++ $1 (+I 0 (+IPars compacta (+I + + ii 8 (:I (+I i

Cerebellumstellate/Basketcells +++Purkinje +++ 8

;fi z i 8

0i

Beqgmaun glia 0 +Granule cells +++ 0 8 (:I :

0A

8 0+++ (+I ttt

In situ hybridization signals obtained with Y Glabeled oligonucleotide probes on serial sections were assessed as intense, + + f; strongly positive, + +weakly positive , (+); or not detectab le, 0.


4/23

The Journal of Neuroscience, March 1992, C (3) 10

broad range of structures. The results compiled from the figuresand also unpublished data are summarized in Table 1. Resultsfor the olfactory bulb and cerebellum are described and dis-cussed in detai l in the accompanying article (Laurie et al., 1992).

Telencephalon (cortex, hippocampal formation, septum,striatum)

NeocortexIn the neocortex and hippocampus, 12 of the 13 subunits arepresent (Figs. 1-14). The (YemRNA is restricted completely tothe cerebellum (Fig. l), and its expression will not be furtherdescribed here (see accompanying article, Laurie et al., 1992).Considering first the a-subunit class, (Y, mRNA is present incortex in a laminated pattern, with layers II/III and V/VI ex-pressing higher levels than layer IV (Figs. 1, 5, 11). In contrast,the c+ mRNA is most predominant in layer II, although it ispresent in deeper layers (Figs. 1, 3, 5, 7, 9). The (YemRNAoccurs in a gradient reciprocal to that of (Ye,with layer VI ex-pressing most of this transcript (Figs. 1, 5, 7, 9). The q mRNAappears to be highest in layers II and III, although significantlevels are present in deeper layers (Figs. 1,3,7,9). The (YemRNAis rare in cortex, but the expression pattern appears weaklydelineated in layer VI (Figs. 1, 3, 5, 7, 9).

For the p-subunit mRNAs in cortex (Figs. 2,4, 6,8, lo), thatof p, resembles (YemRNA in that it is present at overall uni-formly low levels, but layer VI has slightly higher levels. The& and 6, transcripts are present in similar amounts in the samepattern of lamination, with layers II/III, V, and VI having higherlevels than layer IV (Figs. 2, 4, 6, 8, 10).

All three y-subunit genes are expressed in cortex (Figs. 2, 4,6, 8, lo), although only the pattern of yZ expression appearsstrongly laminated, similar to that of the LY,,&, and & mRNAs.The y, mRNA is present at uniformly low levels throughout alllayers of the cortex. Interestingly, parts of the corpus callosum

appear to contain targets hybridizing with the y, probe, as wit-nessed by the lack of a signal boundary between cortex andcaudate putamen for the y, autoradiographs (e.g., Figs. 2, 4, 6).In addi tion, white matter tracts in the hippocampus also appearto be weakly labeled. This effect seems specific because (1) twoindependent y, oligonucleotides recognizing different parts ofthe mRNA (Ymer et al., 1990) give the same result, (2) thesignal can be competed by competition with unlabeled probe,and (3) y, probes do not label white matter tracts in the cere-bellum or olfactory bulb (Fig. 2; Laurie et al., 1992). No othersubunit mRNAs could be detected in corpus callosum, and thedemarcation between cortex and caudate-putamen was alwayspronounced for these subunit mRNAs.

The d-subunit mRNA pattern resembles that of the (Yeand CX~mRNAs, with layer II showing moderate levels (Figs. 3, 5, 7,9, 11).

Piriform cortex

The piriform cortex expresses every subunit mRNA, to a greateror lesser extent, except (Ye data not shown). The most abundanttranscripts are (Y,-(Y~, & &, y2, and 6 (Figs. 3-6). The autora-diographic images obtained over this area are often extremelyintense.

HippocampusExamining the hippocampal expression of the a-subunit genes,the CQmRNA is consistently the most abundant product and is

expressed at high levels in the CA 1, CA3, and dentate gyrus ce ll

layers (Figs. 1, 7, 9, 12). The 01,and q mRNAs are also foundin all sectors of the hippocampus (Figs. 1, 7, 9, 11, 12). The LYEmRNA occurs mainly in the dentate granule cells, with somepyramidal cell expression also. The (YemRNA appears as abun-dant as the (Y* ranscript in the CA1 and CA3 areas, but is lessprominent in the dentate gyms (Figs. 1, 7, 9, 12). In fact, theLYEmRNA appears to encode a predominantly hippocampal

subunit, since it is virtually absent from most other areas of thebrain (see also Khrestchatisky et al. , 1989).Regarding P-subunit mRNAs, all three are present in CAl,

CA3, and dentate gyrus cell layers (Figs. 2, 8, 10, 13), with p,and ps being more abundant than & mRNA. Reminiscent ofthe 01~mRNA, the p, mRNA is expressed at its highest levelsin the hippocampus but is rare elsewhere. All three y-subunitmRNAs are detectable in the hippocampus (Figs. 2, 8, 10, 13)with the yZ mRNA being most abundant. The ys mRNA is ratherrare, cortical levels being higher than those in the hippocampus.The 6 mRNA is restricted to dentate granule cells at the reso-lution of x-ray film autoradiographs (Figs. 7, 9, 11, 12).

Tenia tecta

The tenia tecta, a structure embryologically related to hippo-campus, expresses a number of subunit genes at very high levels(Figs. 3, 4). These abundant mRNAs are CQ,q, q, /I,, and &.No y-subunit or a-subunit mRNAs are detected in this structure.

SeptumThe lateral septum contains a variety of subunit mRNAs, themost abundant of which are LY*,OIL,&, and y, transcripts (Figs.3,4). Some subunit mRNAs such as (Ye, 2, and ys are completelyabsent from the lateral septum. The medial septal nucleus (datanot shown; Wisden et al. , 199 1b) and the nucleus of the diagonalband contain very high levels of cy,, &, and y2 mRNAs (Table1; Figs. 5, 6). This is consistent with these two nuclei being

anatomically linked and containing the same cell types (Bleierand Byne, 1985). In the ventral septum, a different profile ofsubunit transcripts is found. The bed nucleus of the stria ter-minalis contains very high levels of (Yeand y, transcripts (sat-urating autoradiographic signals), moderate levels of a3, p,, andps mRNAs and low levels of LY,,q, &, and y2 mRNAs (Table1; Figs. 5, 6).

Basal ganglia: caudate-putamen, nucleus accumbens, andglobus pallidus

In the caudate nucleus the most prevalent a-subunit mRNAsare CQ and q (Figs. 1, 3, 5). However, longer exposure timesalso reveal the presence of (Y, and C+ mRNAs. The LYEmRNAwas undetectable. The predominant P-subunit in the caudate is/I, (Figs. 2, 4, 6) followed in order of abundance by & and p,mRNAs. All three y-subunit mRNAs are present at low levelsin caudate, with the y3 mRNA slightly elevated relative to theothers (Figs. 2, 4, 6). The &subunit mRNA is moderately ex-pressed in the caudate nucleus (Figs. 3, 5, 11).

Levels of subunit transcripts in the nucleus accumbens par-allel their respective levels in caudate. Subunit mRNAs abun-dant in the caudate are also abundant in the nucleus accumbens,while those mRNAs rare in nucleus accumbens are also rare incaudate. The most abundant accumbens transcripts are thoseof Ly2, , and p,. However, the y3 mRNA appears to be elevatedin the medial parts of the nucleus accumbens (Fig. 4).

In the globus pallidus, the major a-subunit mRNA is that of

(Y, (Fig. 5). There are also smaller but significant amounts of LYE


5/23

1044 Wisden et al. - GABA , Receptor mRNA Distribution

Figure 1.4.4 mm.

Distribution of GABA, receptor a-subunit mRNAs (q-0~~) in horizontal rat brain sections. See Appendix for abbreviations. Scale bar,

and a3 mRNAs in this area (Fig. 5). The & mRNA is the only@mRNA found in the globus pallidus (Fig. 6). Regarding they-subunit class, y, mRNA is marginally the more abundanty-transcript in this region (Fig. 6). The 6 and y3 mRNAs are notexpressed n the globus pallidus (Figs. 5, 6).

Subthalamic nucleusIn the subthalamic nucleus, he (Y, and & mRNAs are the most

significant GABA, receptor transcripts present (Figs. 9, 10). y2

mRNA is also n this area, but at lower levels (Fig. 10). Longerexposure times also indicate the presence of ys mRNA (notshown).

AmygdalaIn the amygdaloid complex, CQ mRNA is found at very highlevels and is the predominant a-subunit mRNA in the medialamygdaloid nucleus (Fig. 9), lateral amygdaloid nucleus (Fig.

7), and posterior medial cortical nucleus not shown). All other


6/23

The Journal of Neuros cience , March 1992, Q(3) 10

Figure 2. Distribution of GABA, receptor B-subunitmRNAs j3,-&) and -subunit mRNAs n-/J in horizontal at brain sections. ee Appendixfor abbreviations. cale ar, 4.4 mm.

a-subunit mRNAs, except (Ye, are also present in these nucleibut at differing degrees. The rarest is cr5 mRNA. Similarly, allP-genes are expressed n these nuclei, with & generally beingthe best expressed p-subunit mRNA (Figs. 8, 10).

The differential expression of the y-subunit genes n the amyg-daloid nuclei s noteworthy. For example, y, mRNA is expressedat striking levels in the medial amygdaloid nuclei (Fig. lo), anarea where there is relatively little of the y2 and yp mRNAs.Indeed, this nucleus contains some of the highest amounts of

y, mRNA in the brain. However, yZ mRNA is the main rep-

resentative of the y-class n the general amygdaloid area (Figs.8, 10). The y3 mRNA is present n diffuse, low levels throughoutthe complex. The &transcript is absent rom the amygdala Figs.7, 9).

Diencephalon (epithalamus, thalamus, and hypothalamus)The medial habenula expresses ignificant quantities of CQ nd& mRNAs and some y, and y2 mRNAs (Figs. 7, 8). All other

subunit mRNAs are rare or undetectable.


7/23

1048 W isden et al.l GABA, Receptor mRNA Distribution

Figure 3. Distribution of (Y,-cY~nd 6 GABA, receptor subunit mRNAs in coronal sections at the level of caudate nucleus and nucleus accumbens.See Appendix for abbreviations. Scale bar, 2.8 mm.

ThalamusThe IX, and LY., ranscripts are the most prominent a-subunitmRNAs throughout the whole thalamus. On sections hroughcaudal portions of the thalamus, the dorsal lateral geniculate,ventral lateral geniculate, and the ventral posterior nuclei areclearly positive with the CY, nd CQ robes Figs. 7, 9, 11, 14) asis the lateral dorsal complex (Fig. 1). The q and a3 mRNAs arealso present n the thalamus, but in more restricted subpopu-lations (Figs. 7, 9, 14). The q mRNA was absent from all

thalamic nuclei examined (Figs. 7, 9).

Examining the thalamic distribution ofthe a-subunit mRNAsin more detail, several eatures become apparent. The q mRNAis very abundant in most nuclei of the thalamus, with someexceptions. For example n the zona incerta, a part of the ventralthalamus, (Y, mRNA is the only a-variant present Fig. 9), whilein the reticular thalamus q mRNA is absent but q mRNA ispresent (Figs. 7, 9). The CQ nd (Ye mRNAs are absent rom theventral posterior nucleus where the (Y, and aa mRNAs appear(Figs. 7, 9). In very caudal parts of the thalamus, such as themedial geniculate nucleus, the (Y, and CQ mRNAs predominate,

with a4 mRNA being the most abundant (Fig. 12). The CY* nd


8/23

The Journal of Neuroscience, March 1992, 72(3)1

Figure 4. Distribution of &+3, and y,-y3 GABA, receptor subunit mRNAs in coronal sections t the level of caudate nucleus nd nucleusaecumhens. eeAppendix for abbreviations. cale ar, 2.8 mm.

(Y, mRNAs appear to be restricted to predominantly midlinenuclei (e.g., paraventricular nuclei, rhomboid nuclei) and alsothe central lateral nucleus, and seem o colocalize in these struc-tures. Both the (Ye nd (Y) mRNAs are absent from the medio-

dorsal nucleus Fig. 7).

Certain areas of the thalamus show a surprising degree ofmicroheterogeneity with regard to a-subunit expression. This isillustrated by the parafascicular thalamic nucleus Fig. 14, PF),which encircles the fasciculus etroflexus (fr) fiber tract (Jones,1985; Paxinos and Watson, 1986). The LYE ranscript is the high-


9/23

1048 W&den et al. - GABA , Receptor mRNA Distribution

Figure 5. Distribution ofLY,-C+ nd 6 GABA, receptor subunit mRNAs in coronal sections at the level of globus pallidus and medial preoptichypothalamic area. See Appendix for abbreviations. Scale bar, 2.8 mm.

est overall in this nucleus and is more abundant in the lateraland ventral parts than in the most medial part (Fig. 14). Incontrast, (Y, mRNA is largely restricted to the lateral portion(Fig. 14 (Y,, arrowheads), and the CQ mRNA is present in themost medial midline portion (Fig. 14, CY~, rrowhead). The (YemRNA is present at diffuse low levels throughout the parafas-cicular nucleus Fig. 9).

A pronounced feature of @-subunit mRNA expression n thal-

amus s the diffuse low-level expression of B, and & mRNAs.

Levels for these mRNAs increase n midline nuclei, for example,paraventricular, rhomboid, and central lateral nuclei. By con-trast, p2 mRNA is ubiquitous in thalamus (Figs. 2, 8, 10, 13,14), and its pattern resembles n detail that of (Y, mRNA, bothin spatial distribution and relative abundance.

All members of the y-subunit mRNA class are poorly ex-pressed n the thalamus (Figs. 2, 8, 10, 13). Of these, the yzmRNA is the most abundant, with the y, and y, probes giving

weaker, diffuse signals. The yS mRNA is present at moderate


10/23

The Journal o f Neuro scienc e, March 1992, 12(3) 104

Figure 6. Distribution of @,+3, nd 7,~~ GABA, receptor ubunit mRNAs n coronal ections t the evel of globus allidus nd medial preoptichypothalamic rea. SeeAppendix for abbreviations. cale ar, 2.8 mm.

levels in the medial geniculate nucleus (Fig. 13), being moreabundant than y, and y2 mRNAs in this nucleus.

The b-subunit mRNA is observed n a broad range ofthalamicnuclei and colocalizes with the a., mRNA in the medial genic-ulate, ventral posterior, ventral lateral geniculate, and dorso-lateral geniculate nuclei (Figs. 5, 7, 9, 11, 12). However, unlikethe a4 mRNA, 6 mRNA is absent rom the parafascicular ha-lamic nucleus Fig. 9). The 6 mRNA also appears o be absent

from midline nuclei such as the paraventricular nucleus andrhomboid nucleus, and is also absent rom the reticular nucleus(Fig. 7). However, it is found in the mediodorsal nucleus Fig.7). In this respect, b-gene expression s reciprocal to the thalamicnuclei expressing he a2 and a, subunit genes. For example, theautoradiographic signals for a2 and aj mRNAs result in theformation of a trident-like pattern (comprising he centrolateral,

paraventricular, and rhomboid nuclei) in the middle of the thal-


11/23

1050 W&den et al.l GABA, R eceptor mRNA Distribution

Figure 7. Distribution of GABA, receptor or-subunit mRNAs (q-q and 6) in coronal sections of rat brain at the level of medial habenula. SeeAppendix for abbreviations. Scale bar, 3 mm.


12/23

The Journal of Neuroscience, March 199% 733)1051

Figure 8. Distribution of GABA, receptor &subunit mRNAs 8,~&) and r-subunit mRNAs (y,-y3) in coronal sections t the level of medialhabenula. ee Appendix or abbreviations. cale ar, 3 mm.

amus Fig. 7), and the 6 mRNA distribution traces he negativeimage of this pattern (Fig. 7).

HypothalamusIn the hypothalamus, the most prominent mRNA is that of cx2

(Figs. 5, 7, 9, 14). This mRNA is abundantly present in the

medial preoptic, dorsomedial, ventromedial, and arcuate nucleiand in the dorsal hypothalamic area. The CY,, J, and (Ye ubunitmRNAs are also found in these nuclei, but in lower amounts.The q mRNA appears to be absent from the hypothalamus(Fig. 14).

Regarding B-subunits, he P3 mRNA predominates n the me-


13/23

1052 Wisden et al. - GABA, Receptor mRNA Distribution

Figure 9. Distribution of q-a, and 6 GABA, receptor subunit mRNAs in coronal sections at the level of the parafascicular nucleus. See Appendixfor abbreviations. Scale bar, 3 mm.


14/23

The Journal of Neuroscience, March 1992, 7273) 105

I

Figure 10. Distribution of/3,-& and 7,-y, GABA, receptor subunitmRNAs n coronal ections t level of the parafascicular ucleus. ee Appendixfor abbreviations. Scale bar, 3 mm.

dial preoptic, dorsomedial, ventromedial, and arcuate nuclei y, and y2 mRNAs are found in dorsomedial, ventromedial, and(Figs. 6,8, 10). A low amount of& mRNA is seen n the arcuate arcuate nuclei (Figs. 8, 10). The y, mRNA conspicuously pre-and the ventromedial nuclei, whereas & mRNA is rare in all dominates in the medial preoptic area (Fig. 6). The &subunit

hypothalamic nuclei examined. Considering he y-subunit class, mRNA is undetectable n hypothalamus.


15/23

1054 Wisden et al.l GABA, Receptor mRNA Distribution

Figure II. Comparison of the distribution of q, LX.,, , and d-subunit mRNAs in horizontal sections. See Appendix for abbreviations. scale3.1 mm.

bar.


16/23

The Journal of Neuroscience, March 1992, 12(3) 10

Fimr Fe 2. Coronal sections t the level of the medial geniculate ucleus nd substantia igra llustrating differential patterns of q-a,mkNAs. See Appendix for abbreviations. Scale bar, 1.6 mm.

Midbrain (colliculi, substantia nigra, red nucleus)Throughout the general midbrain area, the most noticeablemRNAs are (Y,, q, & Pa, and y2 (Figs. 12, 13). However, theautoradiographic signals obtained with the (Y,, &, and to someextent the yz probes are very punctate, suggesting xpression nlarge cells. The patterns obtained with the LYE nd p3 probes aremore uniform and diffuse (Figs. 12, 13). Some subunit mRNAs(LYE,3,, and 6) are entirely absent rom any midbrain structuresexamined, whereas others are generally absent but very prom-inent in certain nuclei, for example, (Ye n substantia nigra com-pacta (Fig. 12).

Substantia nigra

The substantia nigra pars reticulata contains high levels of (Y,(see also Hironaka et al., 1990) and p2 mRNAs with y1 and yZ

and6

transcripts also present (Figs. 12, 13). The substantia nigra parscompacta contains oj, (Ye, &, and y2 mRNAs (Figs. 12, 13).

Red nucleusThe red nucleus see lso Hironaka et al., 1990, for LY,) xpressesthe same GABA, receptor subunit genes s hat of the substantian&a reticula@ that is, a,, &, and y2 mRNAs, with only bor-derline to zero degrees of expression for the others (Figs. 12,13).

Superior colliculusAll levels of the superior colliculus contain cq, p2,and /z mRNAs,although deeper ayers contain larger amounts (Figs. 12, 13). Incontrast, the q., q, and a)5 ranscripts are largely found in the

superficial ayer. The (Ye mRNA occurs mainly in the optic nervelayer. The p, mRNA is restricted to the optic layer, and the &


17/23

1056 Wisden et al.l GABA, Receptor mRNA Distribution

Figure 13. Coronal sections at the level of the medial geniculate nucleuskubstantia nigra illustrating patternsof @,-j3,mRNAs and y,yJ mRNAs.SeeAppendix for abbreviations. cale ar, 1.6 mm.

mRNA is more highly expressed hroughout the superior col-liculi.

Inferior colliculusIn the inferior colliculus (central nucleus), the main GABA,receptor transcripts are (Y,, &, and yZ (Figs. 1, 2, 11).

patterns can be analyzed to deduce plausible in vivo subunitcombinations that may constitute molecularly and functionallydistinct receptor subtypes. In the following discussion, we list

DiscussionIn this study we have documented he regional brain distributionof 13 rat GABA, receptor subunit mRNAs. Their expression

such combinations and endeavor to match these suggested ub-

types with previously published pharmacological characteristicsin both brain membranes and engineered expression systems.

antibodies will be required to address his problem. However,

To derive plausible combinations, brain regions n which alimited subset of subunit genes was expressed were naturally

there is evidence in favor of more than one GABA, receptor

more amenable than regions with highly complex expression

on different parts of hippocampal neurons. GABA, receptors

patterns. For example, dentate granule cells pose an extreme

on pyramidal cell soma differ from those on dendrites in terms

problem, since hey seem o contain every subunit mRNA withthe exception of that encoding Ye. hus, either a arge complexityof GABA, receptors exists on one cell type or there are sub-populations of granule cells, each expressing particular subsetsof receptors. Sophisticated multiple-labeling experiments with


18/23

The Journal of Neuroscience, March 1992, L!?(3) 1

Fi.gz re 14. Differential distribution of CX,, Y?,d, and B, mRNAs in the thalamus (enlargements from Figs. 9, 10). Arrowheads defined in text.Appendix for abbreviations. Scale bar, 1.8 mm.-. -

of agonist preference (Alger and Nicoll, 1982; Nicoll and Dutar,1989).Similarly, the neocortex seems refractory to analysis owing

to the large repertoire of different cell populations. However,there are certain shared regional patterns of subunit mRNAswith regard to different laminae. For example, the cr,, Q, and 6mRNAs appear higher in layers II and III, relative to otherlaminae (Table 1). The (Ye,c+, and @, transcripts are highest inlayer VI relative to their abundance in other layers. Althoughthe a,, &, &, and yz mRNAs are in every layer, they appearhighest in layers II/III and V/VI. These correlations may besignificant. For the (Y,, (Ye, and (Y) subunits, the cortical poly-pept ide distribution correlates with the mRNA pattern (Zim-prich et al., 199 l), and the same mRNA pattern is observed inbovine cortex (Wisden et al., 1988). Moreover, these broadgroupings of mRNAs are consistent with groupings in otherbrain regions (thalamus, colliculi, caudate nucleus). For example(Yeand 6 mRNAs often codistribute, the (Yeand @, mRNA pat-terns look very similar, and the a,&y, combination appearsfrequently (see below). This is suggestive of at least three corticalreceptor subtypes containing LYJ, Q3,, and (~,&y~. Other sub-units, most obviously -r-variants, would presumably also be apart of these cores.

Deduced GABA, receptor subunit combinations

c~,/3JyJ. The (Y, and & mRNAs are most widely codistributedin the brain (Table 1). In addition, the -yz mRNA often colo-

calizes with this pair. In areas such as the central nucleus of the

See

inferior colliculi and the red nucleus, only (Y,, &, and yz mRNAsare found. This core combination is also found in mitral cellsof the olfactory bulb and in cerebellar Purkinje cells (accom-panying article , Laurie et al., 1992). In coexpression experi-ments, the cy,&y, receptor embodies the complete profile ofclassical GABA, electrophysiological responses (Sigel et al.,1990; Verdoom et al., 1990). This subunit combination is alsosupported by immunoprecipitation studies (Benke et al., 199 la).However, it appears that other y-variants can be used. For ex-ample, in the globus pallidus, both (~,&y, and cu,&y, combi-nations may occur. Some regions, such as the zona incerta,islands of Calleja, and the subthalamic nucleus, express veryhigh levels of cy, and p2 mRNAs, with no other GABA, receptormRNAs present except for moderate levels of yz mRNA.

LYJ~&J, aJ,(rJ. Another frequently occurring combinationis that of CQ and & mRNAs. For example, in the nucleus ac-cumbens, caudate nucleus, medial habenula, numerous amyg-daloid nuclei, and in many hypothalamic nuclei, the CQ& pairoccurs in combinat ion with various y-variant mRNAs. In thespinal cord, the &&-pair rule also seems to hold, with probablea&y, complexes occurring on motor neurons (Persohn et al.,1991; Wisden et al., 1991a). The (Y* and & mRNAs also colo-calize in the olfactory bulb granule cell layer (Laurie et al., 1992).It is interesting to note that the (1~~nd & together with CQandB, are the most abundant hippocampal mRNAs. The distri-bution and intensity of the LYEnd p, probe hybridization signalsappear to be identical throughout most of the brain (with the

exception of the olfactory bulb), suggesting that they could well


19/23

1058 Widen et al. - GABA, Receptor mRNA Distribution

be partners. Such an q/I,-containing receptor would occur main-ly in the hippocampus. The properties of both a,P,y, and cu,&y,recombinant receptors have been studied in Xenopus oocytesand transfected kidney cells (Sigel et al., 1990; Puia et al., 199 l),with receptors containing the 8, subunit being less sensitive todiazepam potentiation of GABA responses (Sigel et al., 1990).

cr,~u,/3+thalamic receptors.This set of mRNAs is highly ex-

pressed in overlapping areas of the thalamus. With the exceptionof ys mRNA in the medial geniculate nucleus, y mRNAs areexiguous in thalamus. This data may be suggestive of anin vivoreceptor complex containing ayz, LYE, , and &subunits of un-known stoichiometry. Such a receptor in the absence of a y-sub-unit might be expected to bind GABA, ligands but not BZs(Pritchett et al., 1989a,b; Shivers et al., 1989), and indeed, thediencephalic distribution of high-affinity 3H-muscimol andHGABA, sites (Palacios et al., 198 1; Bowery et al., 1987; Olsenet al., 1990) strikingly resembles the distribution of the CX~ RNA.For example, high-affinity 3H-muscimol sites are prevalent inthalamus and rare in the hypothalamus. Thalamic BZ sites asassessed by 3H-flunitrazepam binding are fivefold less abundantthan 3H-muscimol sites (Olsen et al. , 1990), suggesting that themajority of thalamic GABA, receptors do not bind BZs (Un-nerstall et al. , 1981). The a-subunit is probably present in asubset of the high-affinity muscimol binding receptors since itis present in a more limited number of thalamic nuclei than thecy,, q, and & mRNAs. Thus, it is possible that many thalamicreceptors may be a,cw.,& receptors. In certain parts of the thal-amus, such as the reticular nucleus, cr3 mRNA appears to re-place that of ad, a result consistent with immunocytochemicalstudies using qspecific antibodies (Zimprich et al., 199 1).

With regard to cu,cu,&G eceptors, the evidence for the occur-rence of two different a-variants in the same complex is uncer-tain. An antibody specific for the CX, ubunit coprecipitates undernondenaturing conditions other photolabeled subunits in ad-

dition to that of a, (tagged by either 3H-flunitrazepam or 3HRo15-45 13), whereas only the labeled (Y, subunit is precipitatedunder denaturing conditions (Ltiddens et al., 199 1). On the otherhand, other immunoprecipitation studies on bovine brain sug-gest that the (Y, and (Y*subunits are in largely distinct complexes(Duggan and Stephenson, 1990), in agreement with our in situhybridization data. InXenopus oocytes, addition of cr, or (Y~ ocr,p,y, combinations made little difference to the receptor prop-erties (Sigel et al., 1990).

Correlation of mRNA levels with protein levelsOur inferences of subunit combinations from differential mRNAdistributions is critically dependent on the hypothesis that mRNAlevels reflect protein levels. Unfortunately, there is currently noproof that this assumption holds. Additionally, the protein mightbe located in processes (dendrites, axon terminals) far from thesoma where the mRNA resides. Nevertheless, the rela tive sub-unit mRNA abundances correlate well with the immunocyto-chemical results obtained with the small number of specificantibodies tested so far (Benke et al., 199 1a,b; Zimprich et al.,1991). For example, using (Y,, q, and a3 subunit-specific anti-bodies on globus pallidus, an anti-,-subunit antibody gives avery strong signal, (Ye s present in a small number of cells, and(Ye s absent (Zimprich et al., 1991). Of the three a-subunitantibodies, that of (Ye produces the most intense reaction indentate gyrus (Zimprich et al., 1991), in line with ourin situhybridization results. The protein levels of the d-subunit appear

to follow mRNA levels and distribution faithfully (Shivers etal., 1989; Benke et al., 1991b). There are, however, some dis-

crepancies with the yz results. Some of the strongest -y2-im-munoreactive labeling appeared in the islands of Calleja and inthe substantia nigra (Benke et al. , 199 la), areas that, althoughpositive for yz mRNA, are not the most marked areas of yzmRNA abundance (Shivers et al., 1989; Malherbe et al., 1990b;present results). The hippocampus, while containing high levelsof yz mRNA (considerably higher than substantia nigra), con-

tains only moderate levels of yz immunoreactivity. Althougharguments could be made regarding the relative specificites ofimmune sera, it is possible that some degree of distortion maybe present in our inferences resulting from differences in mRNAturnover rates in different cell types.

GABA, receptor mRNA distribution and pharmacologyThe a-subunits. Expression studies on recombinant receptorsshow that it is the a-subunit class that confers major pharma-cological differences with respect to BZs on the receptor com-plexes cyIp;uz. The (Y, &yz complexes display BZ I-type binding,whereas (~,p,y, and a3@,yz combinations display indistinguish-able BZ II binding (Pritchett et al., 1989a). GABA, receptorscontaining the CX~ubunit also display a BZ II-like pharmacology(Pritchett and Seeburg, 1990) whereas those conta ining aq donot appear to bind BZ agonists (Wisden et al. , 199 1b). The brainareas expressing the highest level of a, mRNA, that is, olfactorybulb, medial septum (Wisden et al., 1991b), globus pallidus,zona incerta, central nucleus of the inferior colliculi, red nucleus,substantia nigra pars reticulata, and cerebellum are preciselythose regions that are mainly of the BZ I type (Young et al.,198 1; Niddam et al., 1987; Sieghart, 1989). These areas containrelatively low levels of mRNA for the other BZ agonist-bindinga-subunit variants (+, (Ye, YJ. One potentia l diff iculty with theassignment of the cu,&y, combination as a BZ I subtype is thatBZ I binding is enriched in cortical layer IV (Young et al., 198 1;Niddam et al., 1987; Olsen et al., 1990), while the mRNAs are

not. However, this could be due to spatial mismatches betweenreceptor protein present in dendrites and mRNA in the soma.Conversely, the spinal cord (Persohn et al., 199 1; Wisden et

al., 199 1a), the nucleus accumbens, caudate nucleus, and partsof the amygdala have relatively litt le (Y, mRNA but expresshighly the (Y* and/or a3 mRNAs. These areas contain predom-inantly BZ II sites (Young et al., 198 1; Niddam et al., 1987).These data are concordant with in vitro expression binding dataon the a-subunits. Additionally, areas that have mixed popu-lations of BZ I and BZ II binding sites (e.g., cortex, hippocam-pus) have mixed populations of mRNAs encoding BZ agonist-binding subunits.

A puzzling observation is that, in receptor autoradiography,the GABA agonists 3H-muscimol and 3H-GABA fail to decoratehypothalamic and amygdaloid areas (Bowery et al., 1987; Olsenet al., 1990), even though in both of these regions CQ and p3mRNAs are well expressed. It is possible that a2/@, subunit-containing receptors have a high affinity for certain GABA an-tagonists, since the distribution of the GABA, antagonist 3H-SR-9553 1 matches the distribution of a2 mRNA (Bristow andMartin, 1988; Olsen et al., 1990); its binding in cortex decreasesfrom superficial to deep layers, and the highest density ofbindingis in the hippocampus and nucleus accumbens, with interme-diate levels present in caudate nucleus. Low densities of sitesare observed in thalamic nuclei, substantia nigra, and both layersof the cerebellum.

It appears that forebrain also contains an unusual type of

GABA, receptor, constructed in part from the (Yesubunit. Therecombinant (a&,rJ is characterized by 3H-Ro 15-45 13 binding


20/23


not displaceable by diazepam (Wisden et al., 199 1b). The prop-erties of this cu,/3,yz subtype are reminiscent of cerebellar 01~subunit-containing GABA, receptors (Liiddens et al., 1990).Although (YemRNA is abundant in many forebrain areas, Ro15-45 13 binding that is resistant to BZ agonist displacementdoes not seem very common except in the cerebellum (Sieghartet al., 1987; Turner et al., 1991). However, a low amount of

diazepam-insensitive Ro 15-45 13 binding has been detected incortex, hippocampus, and striatum, although not in the thala-mus (Turner et al., 1991). This may suggest that a fraction ofthe (Ye ubunits combines with y-subunits in some brain regions(cortex, hippocampus, striatum), but not in other areas. Forexample, the thalamus is the principal region for aq gene ex-pression, but because ofthe relative scarcity ofy-subunit mRNAs,thalamic (Yecontaining receptors may exhibit binding of mus-cimol but not BZs (see below).

The P-subunits. In recombinant receptors assembled from(Y-, p-, and y-subunits, the three P-subunits appear to be func-tionally interchangeable isoforms (Pritchett et al., 1989a; Ymeret al., 1989b), yet clearly their marked differential distributionsof mRNAs (see also Lolait et al., 1989; Zhang et al., 1990; Laurieet al., 1992) would suggest some functional significance forp-variants. Recently, a fourth p cDNA variant has been isolatedfrom avian brain cDNA libraries (Bateson et al., 199 1b), but itis not at present clear if a rat fi4 homolog exists or whether itrepresents an avian idiosyncrasy. So far, the only differencesreported for recombinant p-subunits (/3, vs. p,) are those ofcurrent amplitudes in receptors expressed in Xenopus oocytes(Sigel et al., 1990). This could simply be due to different effi-ciencies of protein expression in the oocyte system. However,other subtle differences may emerge if the cloned P-subunits aretested with their most likely in vivo partners. For example, ithas been suggested that natural receptors containing & and &subunits differ in their affinity to GABA analogs and pentobar-

bital (Bureau and Olsen, 1990).The y-subunits. The y-subunits are required for allosteric po-tentiation by BZs of (Y- and @subunit-containing complexes(Pritchett et al., 1989b; Ymer et al., 1990; Herb et al., 1992).Of the three known members, the yz mRNA is the most uni-versal and abundant. It is also the most studied in terms offunction (Pritchett et al., 1989a,b; Sige l et al., 1990; Verdoomet al., 1990). Additional complexity has recently arisen in thatthe y2 mRNA exists in two splice versions, resulting from anexonic insertion into the cytoplasmic loop between transmem-brane segments M3 and M4 (Whiting et al., 1990; Kofuji et al.,1991; Wafford et al., 1991). This splicing event is predicted togenerate a yz polypeptide with the addition of a target sequencefor prote in kinase C. As assessed by PCR analysis, the relativeabundances of the yz splice variants depend on the brain region(Whiting et al., 1990). However, our y2 probe would hybridizeto both of these mRNA versions.

In certain limbic areas of the brain such as amygdala, hy-pothalamus, and septum, yz mRNA appears to be replaced byconsiderable amounts of y, mRNA. Thus, future compoundsselective for y, subunit-containing GABA, receptors might beexpected selectively to modulate neurons of affective circuits.The y, subunit would appear to contribute to only a minorityof CNS GABA, receptors, as assessed by its low mRNA abun-dance. Yet other regions, for example, the tenia tecta and thethalamus, contain none or very litt le, respectively, of any of theknown y-subunit mRNAs, even though high levels of non-y-

subunit transcripts are detected in these regions. How the dif-ferent y-subunits affect the functional properties of the (Y- and

P-subunits is a largely unexplored area, and many of the com-binations we have suggested here have not yet been subjectedto binding or electrophysiological analysis. However, differenty-subunits can differentially modulate responsiveness of a-sub-units to BZs and related compounds (Ymer et al., 1990; Herbet al., 1992). Specifically, the y, subunit confers positive mod-ulation by /3-carbolines and DMCM (6,7-dimethoxy-4-ethyl-p-carboline-3-carbolic acid methyl ester) on LVJ~, omplexes (Puiaet al., 199 l), and the replacement of y2 with either y, or ys leadsto a general lowering of the response to BZs. Consequently, they2 subunit may not always be the appropriate y-subunit to usewhen testing a-subunit pharmacology in vitro.

The&subunit. This subunits role in GABA, receptor functionhas remained an enigma. In a large number of regions (princi-pally thalamic nuclei) it colocalizes with the CX,, d, and pz mRNAs,although its distribution is more restricted (see Fig. 11). Theseresults suggest that in vitro experiments should be designed withrecombinant receptors containing both CY~ nd &subunits. Asoriginally suggested for the d-subunit alone (Shivers et al., 1989),ol,a,@-containing receptors may have high affinity to muscimolbut lack BZ binding sites. In the cerebellum, a,a,@-containingreceptors would correspond to high-affinity muscimol sites inthe granule cel l layer (Laurie et al., 1992). Thus, &subunits maypreferentially associate with a-subunits that do not bind BZagonists.

ConclusionsA number of plausible GABA, receptor combinations have beeninferred based on mRNA distribution. Immunoprecipitationand immunocytochemical studies in addition to modem patch-clamping methodology on brain slices (Edwards et al., 1989)could be used to test the validity of these predictions, usingrecombinant receptors as a reference. Although the original clas-sification of BZ I and BZ II receptors could be criticized for

being too reductionistic, it seems that a systematic comparisonof GABA, receptor subunit distributions in the brain still en-ables this distinction to be maintained with qualifications. Thus,based on their patterns of gene expression and recombinantexpression studies, we would propose that the a,&y, receptorscorrespond to the BZ I subtype, and receptors containing c&y,,(~$~y~, and c@,y, are three subtypes of BZ II receptor. Addi-tional as yet unclassified subtypes of receptor would emerge ifthese subunits occur with the y, or y3 subunits. The receptorscontaining (Yeand cygwould diverge from the main family of BZreceptors because of their very restricted BZ ligand bindingprofile in vitro. Indeed, in some brain regions the (Yesubunitmay contribute to BZ-insensitive GABA, receptors because itsmRNA fails to colocalize with that of any known y-subunit.

The physiological significance of such GABA, receptor com-plexity remains to be determined. Low-affinity receptors onpresynaptic terminals could serve as autoreceptors mediatingnegative feedback of transmitter release. Additionally, differentGABA, receptor complexes could differ in mean channel opentime or desensitization rate, with constraints of neuronal ge-ometry dictating the type of desensitization rate or channel con-ductance that is required to produce a given hyperpolarizationper unit time. Alternatively, neurons receiving a strong excit-atory input may require GABA, receptors of greater conduc-tance and slower densensitization rates than neurons in whichsuch input is less. Analogous situations for receptor diversityalso extend to other ligand-gated ion channels in brain, most

notably the neuronal nicotinic receptors (Wada et al., 1989;Morris et al., 1990), glycine receptors (Betz, 1991; Malosio et


21/23

1060 Wisden et al.l GABA , Receptor mRNA Distribution

al., 199 l), and glutamate receptors (Bettler et al., 1990; Sommeret al., 1990; Monyer et al., 199 1; Werner et al., 1991). Futureexperimental directions could involve homologous recombi-nation experiments to see if it is possible to dissect out thefunction of the different receptor subtypes.

AppendixList of anatomical abbreviationsacaAcbArcAVBS TCAl-4cb

ELCGD

E:.CPUCtX

ctx11DA

EsDLGDMEPfrGPHY

feeICj

i!i

E&C

LsMDMeMGMHbMPOOBGPPFPirPVRRhRt

&CSNRSPFSThSUGTSttVLGVMHVPVPMZI

anterior commissure, anterioraccumbens nucleusarcuate hypothalamic nucleusanteroventral thalamic nucleusbed nucleus, stria terminalisfields l-4 of Ammons horncerebellumcentral amygdaloid nucleuscentral graycentral gray, dorsalclaustrumcentrolateral thalamic nucleuscaudate putamenneocortexneocortex, layer 2dorsal hypothalamic areadiagonal banddentate gyrusdorsal lateral geniculate thalamic nucleusdorsomedial hypothalamusendopeduncular nucleusfasciculus retroflexusglobus pallidushypothalamusinternal capsuleinferior colliculusislands of Callejainfmlimbic cortexlateral amygdaloid nucleuslaterodorsal thalamic nucleuslateral posterior thalamic nucleus, mediocaudal

lateral septummediodorsal thalamic nucleusmedial amygdaloid nucleusmedial geniculate nucleusmedial habenular nucleusmedial preoptic nucleusolfactory bulboptic nerve layer, superior colliculusparafascicular thalamic nucleuspiriform cortexpamventricular thalamic nucleusred nucleusrhomboid thalamic nucleusreticular thalamic nucleussuperior colliculussubstantia nigra pars compactasubstantia nigra pars reticulatasubparafascicular thalamic nucleussubthalamic nucleussuperficial gray layer, superior colliculustriangular septal nucleustenia tectaventral lateral geniculate nucleusventromedial hypothalamic nucleusventral posterior thalamic nucleusventral posteromedial thalamic nucleuszona incerta

ReferencesAlger BE, Nicoll RA (1982) Pharmacological evidence for two kinds

of GABA receptor on rat hippocampal pyramidal cells studiedinvitro. J Physiol (Lond) 328: 125-14 1.

Bateson AN, Harvey RJ, Wisden W, Glencorse TA, Hicks AA, HuntSP, Barnard EA, Darlison MG ( 199 1a) The chicken GABA, recep-tor (Y,subunit cDNA sequence and localization of the correspondingmRNA. Mol Brain Res 9:333-339.

Bateson AN, Lasham A, Darlison MG (199 1b) y-Aminobutyric acid,receptor heterogeneity is increased by alternative splicing of a novel@-subunit gene transcript. J Neurochem 56:1437-1440.

Benke D, Mertens S, Trzeciak A, Gillessen D, Mijhler H (199 la) GA-BA, receptors display association of yZ subunit with (Y,- and j32,3-subunits. J Biol Chem 266:4478-1483.

Benke D, Mertens S, Trzeciak A, Gillessen D, Mohler H( 199 1b) Iden-tification and immunohistochemical mapping of GABA, receptorsubtypes containing the b subunit in rat brain. FEBS Lett 283:145-149.

Bettler B, Boulter J, Hermans-Borgmeyer I, OShea-Greenfield A, De-neris ES, Moll C, Borgmeyer U, Hollmann M, Heinemann S (1990)Cloning of a novel glutamate receptor subunit GluR 5: expression inthe nervous system during development. Neuron 5: 583-595.

Betz H (199 1) Glycine receptors: heterogeneous and widespread ex-pression in the mammalian brain. Trends Neurosci 14:458-46 1.

Bleier R, Byne W (1985) Septum and hypothalamus. In: The rat ner-vous system, Vol 1 (Paxinos G, ed), pp 87-l 18. Sydney: Academic.

Bowery NG ( 1989) GABA, receptors and their sianificance in mam-malian pharmacology. Trends Pharmacol Sci lo:20 l-407.

Bowery NG, Hudson AL, Price GW (1987) GABA, and GABA,

receptor site distribution in the rat central nervous system. Neurosci-ence 20:365-383.Bristow DR, Martin IL (1988) Light microscopic autoradiographic

localization in rat brain o f the bindina sites for the GABA, receptorantagonist [HI SR 9553 1: comparison-with [HI GABA, distribution.Eur J Pharmacol 148:283-288.

Bureau M, Olsen RW (1990) Multiple distinct subunits of ther-aminobutyric acid-A receptor protein show different ligand-bindingaffinities . Mol Pharmacol 37:497-502.

Cooper E, Couturier S, Ballivet M (1991) Pentameric structure andsubunit stoichiometry of a neuronal nicotinic acetylcholine receptor.Nature 350:235-238.

Cutting GR, Luo L, OHara BF, Kasch LM, Montrose-Rafizadeh C,Donovan DM, Shimada S, Antonarakis SE, Guggino WB, Uhl GR,Kazazian HH (199 1) Cloning of the GABA p, cDNA: a novel GA-BA, receptor subunit highly expressed in retina. Proc Nat1 Acad SciUSA 88:2673-2677.

de Blas AL, Vitorica J, Friedrich P (1988) Localizaton of the GABA,receptor in rat brain with a monoclonal antibody to the 57,000 M,peptide of the GABA, receptor/benzodiazepine receptor/Cl- channelcomplex. J Neurosci 8:602-6 14.

Duggan MJ, Stephenson FA (1990) Biochemical evidence for the ex-istence of y-aminobutyrate, receptor isooligomers. J Biol Chem 265:383 l-3835.

Dutar P, Nicoll RA (1988) A physiological role for GABA, receptorsin the central nervous system. Nature 322: 156-l 58.

Edwards FA, Konnerth A, Sakmann B, Takahashi T (1989) A thinslice preparation for patch clamp recordinn from neurones in themammalian central nervous system. Pfluegers Arch 4 14:600-6 12.

Ewert M, Shivers BD, Liiddens H, Miihler H, Seeburg PH (1990)Subunit selectivi ty and epitope characterization of mAbs directedagainst the GABA,/benzodiazepine receptor. J Cell Biol 110:2043-2048.

Ewert M, de Blas AL, Mohler H, Seeburg PH (1991) A prominentepitope on GABA, receptors is recognized by two different mono-clonal antibodies. Brain Res, in press.

Fuchs K, Mohler H, Sieghart W (1988) Various proteins from ratbrain, specifical ly and irreversibly labelled by [3H]flunitrazepam, aredistinct (Ysubunits of the GABA-benzodiazepine receptor complex.Neurosci Lett 90~3 14-3 19.

Garrett KM, Saito N, Duman RS, Abel MS, Ashton RA, Fujimori S,Beer B, Tallmann JF, Vitek MP, Blume AJ (1990) Differential ex-pression of y-aminobutyric acid,, receptor subunits. Mol Pharmacol37:652-657.

Haefely W, Pole P (1986) Physiology of GABA enhancement by ben-zodiazepines and barbiturates. In: Benzodiazepine/GABA, receptorsand chloride channels: structural and functional properties (OlsenRW, Venter JC, eds), pp 97-133. New York: Liss.

HIring P, Stahli C, Schoch P, Takacs B, Staehelin T, Mijhler H (1985)Monoclonal antibodies reveal structural homogeneity of y-amino-


22/23


butyric acid/benzodiazepine receptors in different brain areas. ProcNat1 Acad Sci USA 82:48374841.

Herb A, Wisden W, Lilddens H, Puia G, Vicini S, Seeburg PH (1992)A third y subunit of the GABA, receptor fa mily. Proc Nat1 Acad SciUSA, in press.

Hironaka T, Morita Y, Hagihira S, Tateno E, Kita H, Tohyama M(1990) Localization of GABA, receptor a, subunit mRNA-contain-ing neurons in the lower brainstem of the rat. Mol Brain Res 7:335-345.

Jones EG (1985) The thalamus. New York: Plenum.Kato K (1990) Novel GABA, receptor LY ubunit is expressed only in

cerebellar granule cells. J Mol Biol 2 14:6 19-624.Khrestchatisky M, MacLennan AJ, Chiang M-Y, Xu W, Jackson MB,

Brecha N, Stemini C, Olsen RW, Tobin AJ (1989) A novel (Ysubunitin rat brain GABA, receptors. Neuron 3:745-753.

Khrestchatisky M, MacLennan AJ, Tillakaratne NJK, Chiang M-Y,Tobin AJ (199 1) Sequence and regional distribution of the mRNAencoding the a2 polypeptide of rat y-aminobutyric acid, receptors. JNeurochem 56:1717-1722.

Kofuji P, Wang JB, Moss SJ, Huganir RL, Burt DR (199 1) Generationof two forms o f the y-aminobutyric acid, receptor y2 subunit in miceby alternative splicing. J Neurochem 56:7 13-7 15.

Lambert JJ, Peters JA,Cottrell GA (1987) Actions o f synthetic andendogenous steroids on the GABA, receptor. Trends Pharmacol Sci8~224-227.

Laurie DJ, Seeburg PH, Wisden W (1992) The distribution of 13GABA, receotor subunit mRNAs in the rat brain. I I. Olfactorv bulband cerebellum. J Neurosci 12: 1063-1076.

Lolait SJ, OCarroll AM, Kusano K, Mahan LC (1989) Pharmaco-logical characterization and region specific expression in brain of the& and &-subunit of the rat GABA, receptor. FEBS Lett 258: 17-2 1.

Liiddens H, Wisden W (199 1) Function and pharmacology of multipleGABA, receptor subunits. Trends Pharmacol Sci 12:49-5 1.

Liiddens H, Pritchett DB, Kiihler M, Killisch I, Keinlnen K, MonyerH, Sprengel R, Seeburg PH (1990) Cerebellar GABA, receptor se-lective for a behavioural alcohol antagonist. Nature 346648-65 1.

Lilddens H. Killisch I. Seebura PH (1991) More than one (Yvariantmay exist in a GABA,/benzidiazepine receptor complex. J ReceptorRes 11:535-551.

MacLennan AJ, Brecha N, Khrestchatisky M, Stemini C, TillakaratneNJK, Chiang M-Y, Anderson K, Lai M, Tobin AJ (199 1) Indepen-dent cellular and ontogenetic expression o f mRNAs encoding three(Ypolypeptides ofthe rat GABA, receptor. Neuroscience 43:369-380.

Malherbe P, Sigel E, Baur R, Pershon E, Richards JG, MGhler H ( 199Oa)Functional expression sites of gene transcription of a novel LY ubunitof the GABA, receptor in rat brain. FEBS Lett 260:261-265.

Malherbe P, Sigel E, Baur R, Pershon E, Richards JG, Mbhler H(1990b) Functional characteristics and sites of gene expression o f theo;;~$,, y,-isoform of the rat GABA, receptor. J Neurosci 10:2330-

Malosio M-L, Marqueze-Pouey B, Kuhse J, Betz H (1991) Wide-spread expression of glycine receptor subunit mRNAs in the adultand develooina rat brain. EMBO J 10:2401-2409.

Monyer H, .Seeb&g PH, Wisden W (199 1) Glutamate-operated chan-nels: developmentally early and mature forms arise by alternativesplicing. Neuron 6:799-8 10.

Morris BJ, Hicks AA, Wisden W, Darlison MG, Hunt SP, Barnard EA(1990) Distinct regional expression of nicotinic acetylcholine recep-

tor genes in chick brain. Mol Brain Res 7:305-3 15.Nicoll RA, Dutar P (1989) Physiological roles of GAB% and GABA,receptors in synaptic transmission in the hippocampus. In: Allostericmodulation of amino acid receptors: therapeutic implications (Bar-nard EA, Costa E, eds), pp 195-205. New York: Raven.

Niddam R, Dubois A, Scatton B, Arbilla S, Langer SZ (1987) Auto-radiographic localization of [3H]zolpidem binding in the rat CNS.Comparison with the distribution of [3H]flunitrazepam binding sites.J Neurochem 49:890-899.

Olsen RW, Tobin AJ (1990) Molecular biology of GABA, receptors.FASEB J 4: 1469-l 480.

Olsen RW, McCabe RT, Wamsley JK (1990) GABA, receptor sub-types: Autoradiographic comparison o f GABA, benzodiazepine, andconvulsant binding sites in the rat central nervous system. J ChemNeuroanat 3159-76.

Palacios JM, Wamsley JK, Kuhar MJ (1981) High affin ity GABAreceptors-autoradiographic localization. Brain Res 222:285-307.

Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates,2d ed. Sydney: Academic.

Persohn E, Malherbe P, Richards JG (1991) In situ hybridizationhistochemistry reveals a diversity of GABA, receptor subunit mRNAsin neurons of the rat spinal cord and dorsal root ganglia. Neuroscience42~497-507.

Pritchett DB, Seeburg PH (1990) y-Aminobutyric acid, receptor QL~subunit creates novel type II benzodazepine receptor pharmacology.J Neurochem 54: 1802-1804.

Pritchett DB, Ltlddens H, Seeburg PH (1989a) Type I and type IIGABA, benzodiazepine receptors produced in transfected cells. Sci-ence 246:1389-1392.

Pritchett DB, Sontheimer H, Shivers BD, Ymer S, Kettenmann H,Schofield PR, Seeburg PH (1989b) Importance of a novel GABA,receptor subunit for benzodiazepine pharmacology. Nature 338:582-585.

Puia G, Santi M, Vicini S, Pritchett DB, Purdy RH, Paul SM, SeeburgPH, Costa E (1990) Neurosteroids act on recombinant human GA-BA, receptors. Neuron 4~759-765.

Puia G, Vicini S, Seeburg PH, Costa E ( 199 1) Influence of recombinanty-aminobutyric acid, receptor subunit composition on the action ofallosteric modulators of y-aminobutyric acid-gated Cl- currents. MolPharmacol39:69 l-696.

Richards JG, Mijhler H, Haefely W (1986) Mapping benzodiazepinereceptors in the CNS by radiohistochemistry and immunohistochem-

istry. In: Neurohistochemistry: modem methods and applications(Panula P, Pairannta H, Soinita S, eds), pp 629-677. New York: Liss.Richards JG. Schoch P. Hiring P. Takacs B. Miihler H f 1987) Re-

solving GABA,lbenzodiaze&re receptors: cellular and subckllularlocalization in the CNS with monoclonal antibodies. J Neurosci 7:1866-1886.

Seeburg PH, Wisden W, Verdoom TA, Pritchett DB, Werner P, HerbA, Ltlddens H, Sprengel R, Sakmann B (1990) The GABA, reeeptorfamily: molecular and functional diversity . Cold Spring Harb Symp@ant Biol 55:29-40.

Siquier JM, Richards JG, Malherbe P, Price GW, Mathews S, MiihlerH (1988) Mapping of brain areas containing RNA homologous tocDNAs encoding the LY nd @ subunits of the rat GABA, y-amino-butyrate receptor. Proc Nat1 Acad Sci USA 85:78 15-78 19.

Shivers BD, Killisch I, Sprengel R, Sontheimer H, Kiihler M, SchofieldPR, Seeburg PH (1989) Two novel GABA, receotor subunits existin distinct neuronal s&populations. Neuron3:32?-337.

Siegel RE (1988) The mRNAs encoding GABA,/benzodiazepine re-ceptor subunits are localized in different cell populations ofthe bovinecerebellum. Neuron 1:579-584.

Sieghart W (1989) Multiplicity of GABA,-benzodiazepine receptors.Trends Pharmacol Sci lo:4074 11.

Sieghart W, Eichinger A, Richards JG, Miihler H (1987) Photoaffinitylabelling of benzodiazepine receptor proteins with the partial agonist[H]Ro 15-45 13: a biochemical and autoradiographic study. J Neu-rochem 48:46-52.

Sigel E, Baur R, Trube G, Miihler H, Malherbe P (1990) The effectof subunit composition of rat brain GABA, receptors on channelfunction. Neuron 5:703-7 11.

Sommer B, Kainiinen K, Verdoom TA, Wisden W, Bumashev N, HerbA, Kiihler M, Takagi T, Sakmann B, Seeburg PH (1990) Flip andflop: a cell specif ic functional switch in glutamate operated channelsof the CNS. Science 249:158&1585.

Turner DM, Sapp DW, Olsen RW (199 1) The benzodiazepine/alcoholantagonist Ro 15-45 13: binding to a GABA, receptor subtype thatis insensitive to diazepam. J Pharmacol Exo Ther 257:1236-1242.

Unnerstall JR, Kuhar %IJ, Niehoff DL, Palacios JM (1981) Benzo-diazepine receptors are coupled to a subpopulation of GABA recep-tors: evidence from a quantitative autoradiographic study. J Phar-macol Exp Ther 218:797-804.

Unwin N (1989) The structure of ion channels in membranes of ex-citable cells. Neuron 3:665-676.

Verdoom TA, Draguhn A, Ymer S, Seeburg PH, Sakmann B (1990)Functional properties of recombinant GABA, receptors depend uponsubunit composition. Neuron 4:919-928.

Wada E, Wada K, Boulter J, Deneris E, Heinemann S, Patrick J, Swan-son LW (1989) Distribution of alpha2, alpha3, alpha4, and beta2neuronal nicotinic receptor subunit mRNAs in the central nervoussystem: a hybridization histochemical study in the rat. J Comp Neurol284:314-335.


23/23

1062 Wisden et al. - GABA , Receptor mRNA Distribution

Wafford KA, Burnett DM, Dunwiddie TV, Harris RA (1990) Geneticdifferences in the ethanol sensitivity o f GABA, receptors expressedin Xenopus oocytes. Science 249:29 l-293.

Wafford KA, Burnett DM, Leidenheimer NJ, Burt DR, Wang JB, KofujiP, Dunwiddie TV, Harris RA, Sikela JM (199 1) Ethanol sensit ivi tyof the GABA, receptor expressed in Xenopus oocytes requires 8 aminoacids contained in the y2L subunit. Neuron 7~27-33.

Werner P. Voiat M. Keiniinen K. Wisden W. Seebera PH (199 1) Clon-ing of a p&i& high-affinity kainate receptor ixpresged piedomi-nantly in hippocampal CA3 cells. Nature 35 1:742-744.

Whiting P, McKeman RM, Iversen LL (1990) Another mechanismfor creating diversity in y-aminobutyric type A receptors: RNA splic-ing directs expression of two forms o f y2 subunit, one of which con-tains a protein kinase C phosphorylation site. Proc Nat1 Acad SciUSA 87:9966-9970.

Wilson-Shaw D, Robinson M, Gambarana C, Siegel RE, Sikela JM(199 1) A novel y subunit of the GABA, receptor identified usingthe polymerase chain reaction. FEBS Lett 2842 1 -2 15.

Wisden W, Morris BJ, Darlison MG, Hunt SP, Barnard EA (1988)Distinct GABA, receptor (Ysubunit mRNAs show differentiated pat-terns of expression in bovine brain. Neuron 1:933-947.

Wisden W. McNauahton LA. Darlison MG. Hunt SP. Barnard EA(1989a) Differentyal distribbtion of GABA, receptor in bovine cer-ebellum-localization of az mRNA in Bergmann glia layer. NeurosciLett 106:7-12.

Wisden W, Morris BJ, Darlison MG, Hunt SP, Barnard EA (1989b)Localization of GABA, receptor a subunit mRNAs in relation toreceptor subtypes. Mol &a&Res 5:305-3 10.

Wisden W, Gundlach AL, Barnard EA, Seeburg PH, Hunt $P(199 la)Distribution ofGABA, receptor subunit mRNAs in rat lumbar spinalcord. Mol Brain Res 10:179-183.

Wisden W, Herb A, Wieland H, KeinHnen K, Liiddens H, Seeburg PH(199 1b) Cloning, pharmacological characteristics and expression pat-tern of the rat GABA, receptor a4 subunit. FEBS Lett 289:227-230.

Wisden W, Morris BJ, Hunt SP (1991~) In situ hybridization withsynthetic DNA probes. In: Molecular neurobiology-a practical ap-proach, Vo12 (Chad J, Wheal H, eds), pp 205-225. Oxford: OxfordUP/IRL.

Ymer S, Draguhn A, Kiihler M, Schofield PR, Seeburg PH (1989a)Sequence and expression of GABA, receptor (r4 subunit. FEBS Lett258:119-122.

Ymer S, Schofield PR, Draguhn A, Werner P, Kiihler M, Seeburg PH(198913) GABA, receptor j3 subunit heterogeneity: functional ex-pression of cloned cDNAs. EMBO J 8:1665-1670.

Ymer S, Draguhn A, Wisden W, Werner P, Keiniinen K, Schofield PR,Sprengel R, Pritchett DB, Seeburg PH (1990) Structural and func-tional characterization of the y, subunit of GABA,ibenzodiazepinereceptors. EMBO J 9:3261-3267.

Young WS III, Niehoff DL, Kuhar MJ, Beer B, Lippa AS (198 1) Mul-tiple benzodiazepine receptor localization by light microscopic ra-diohistochemistrv. J Pharmacol EXD Ther 216:425436.

Young WS III, Mezey E, Siegel RE -( 1986) Quantitative in situ hy-bridization histochemistry reveals increased levels of corticotropinreleasing factor mRNA after adrenalectomy in rats. Neurosci Lett 70:198-203.

Zhang JH, Sato M, Noguchi K, Tohyama M (1990) The differentialexpression patterns of the mRNAs encoding @ subunits (j3,, & and&) of GABA, receptors in the olfac tory bulb and its related areas inthe rat brain. Neurosci L&t 119:257-260.

Zimprich F, Zezula J, Sieghart W, Lassmann H (1991) Immunohis-tochemical localization of the (Y, , (Yeand (Y) subunits of the GABA,receptor in the rat brain. Neurosci Lett 127: 125-128.

Documents

GABA Forebrain Subunit mRNA Distribution