Neuroscience Vol. 45, No. 2, pp. 423-432, 1991 F’rintcd in Great Britain 0306-4522/91 53.00 + 0.00 Pergamon Press plc 0 1991IBRO zyxwvu REGION-SPECIFIC EXPRESSION OF MESSENGER RNAs ENCODING GABA, RECEPTOR SUBUNITS IN THE DEVELOPING RAT BRAIN C. GAMBARANA, C. E. BEATIIE, Z. R. RODRIGUEZ and R. E. SIEGEL* Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, U.S.A. Ahatraet-The distribution and levels of messenger RNAs encoding the zyxwvutsrqponmlkjihgfedcbaZYXWVU K 1, j 1, 82, 83, and y2 subunits of the GABA, receptor in the developing and adult rat brain were investigated using quantitative in zyxwvutsrqponmlk situ hybridization histochemistry and subunit-specific probes. Regional localization of the subunit messenger RNAs was determined with film autoradiography and expression in identified neuronal cell populations was examined using higher resolution techniques. Each of the GABA, receptor subunit messenger RNAs exhibits a distinct pattern of localization in the developing and adult brain. Of the subunits examined, the 0: 1, 82, and y2 are the most abundant and are found in many brain regions, including the olfactory bulb, cortex, hippocampus, thalamic nuclei, and inferior colliculus. In addition, these subunit messenger RNAs are prominent in the cerebellum where virtually all cells of the deep cerebellar nuclei and Purkinje cell layer are labeled. The levels of most of the subunit messenger RNAs, with the exception of that encoding the /?l subunit, increase during postnatal development. While the ul, 82, and y2 subunit messenger RNAs rise in parallel in many regions and identified cell populations, different subsets of receptor subunit messenger RNAs are co-ordinately expressed at other sites. The greatest increases in subunit messenger RNA levels occur in the cerebellar cortex during the second postnatal week, a period coincident with cerebellar maturation. The codistribution of different GABA, receptor subunit messenger RNAs in various regions of the developing and adult nervous systems supports the hypothesis that multiple receptor compositions exist. Moreover, that different subunit messenger RNAs exhibit coordinate changes in expression in different regions and cell populations suggests that receptor gene expression is modulated by cell type-specific signals. The temporal changes in subunit messenger RNA levels in the cerebellum raise the possibility that synaptogenesis may play a role in receptor gene regulation in this brain region. The GABA, receptor complex is a Iigand-gated ion channel that mediates the actions of GABA, the major inhibitory neurotransmitter in the central nervous system. It is also the site of action for a number of clinically important compounds, including the benzodiazepines and barbiturates (reviewed by Olsen and Tobin23). Initial biochemical studies suggested that the receptor was assembled from two subunit types a and /34~‘“~33 and existed as an a2#l2 heterotetramer.‘* However, recent molecular biologi- cal studies indicate that receptor composition is far more complex. Two novel cDNAs encoding y and 6 subunits have been identified,26,M and multiple isoforms of the a, /I, and y subunits have been characterixed 9,10.14,15.27.37,35-1 While numerous GABAA receptor subunits have been identified, the actual composition of the func- tional receptor in vivo remains unclear. As an approach to this issue, several groups have begun to investigate the properties of receptors produced by the assembly of different subunits expressed in Xeno- pus oocytes or mammalian cells.9~12~1~‘7~2s~26~30~~36~~ *To whom correspondence should be addressed. Abbreviutims: PBS, phosphate-buffered saline; SSC, sodium chloride-sodium citrate buffer. These studies have indicated that several subunit combinations confer sensitivity to GABA. Ex- pression of single subunit mRNAs, as well as combi- nations of different subunit mRNAs, all produce GABA-gated channels. However, the conductance and gating properties of the resulting channels differ.L5~23~35~36 In addition, the allosteric modulation of GABA sensitivity by the benzodiazepines requires the expression of the 72 subumt.26 While electrophysio- logical studies have demonstrated that different subunit combinations expressed in vitro produce channels having distinct functional properties, the relationship between these complexes and the compo- sition of the GABAA receptor in vivo is unknown. To approach this issue, several recent studies have examined the distribution of GABA, receptor sub- unit mRNAs in the central nervous system by in situ hybridization histochemistry and Northern blot analysis. Studies performed on adult brains indicate that the subunits and their isoforms exhibit different, but partially overlapping, patterns of distri- bution.12*‘426,M*38*42 In contrast, much less is known about receptor gene expression in the developing nervous system. Studies using Northern blot analysis demonstrated that the levels of the mRNAs encoding the a 1 and /31 receptor subunits in the rat cortex 423