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After the discovery of calretinin, a protein with high sequence homology to calbindin D-28k, the validity of immunohistochemical results obtained using polyclonal antibodies for this protein, was in question. In order to validate the previous results on the localization of calbindin D-28k in the brain, we localized the protein by highly specific monoclonal antibodies and revealed its mRNA histochemically by in situ hybridization. In general there was good agreement between the results obtained using these two different techniques and those reported in previous publications. The concordance was particularly impressive for the cerebral cortex, basal ganglia, basal nucleus of Meynert, hippocampus, thalamus, cerebellum and superior colliculus. In the amygdala and hypothalamus the low spatial resolution of in situ hybridization did not allow precise definition of some nuclei displaying a positive reaction for the protein. In the rhombencephalon, cells of the parabrachial nuclei and the dorsal raphe nucleus expressed calbindin D-28k. Neurons in the dorsal horn of the spinal cord and some horizontal cells of the retina were tagged with both methods. The only discrepancy was the presence of immunoreactive ependymal cells, whereas mRNA never occurred in cells lining the ventricles. Thus, the combined approach has established the widespread distribution of cells expressing calbindin D-28k in the rat brain.

An in situ hybridization technique was used to determine the distribution in rat brain of RNA homologous to cDNA clones encoding the a and .8 subunits of the rat brain GABAA yaminobutyrate receptor. The subunit proteins were mapped in adjacent sections autoradiographically and immmunohistochemically. Many brain areas containing high densities of GABAA receptors showed strong hybridization signals with both the a-and the 1subunit antisense RNA probe-e.g., cerebral cortex, hippocampus, and cerebellum. On a cellular level, a dense dendritic localization of GABAA receptors was correlated with a strong hybridization in the corresponding somata-e.g., in mitral cells of the olfactory bulb, pyramidal cells of hippocampus, granule cells of the dentate gyrus, and Purkinje and granule cells of the cerebellum. In some brain areas-e.g., substantia nigra--the intensity of the hybridization signal with the ,&subunit probe was much weaker than that with the a-subunit probe, whereas the inverse ratio of hybridization intensity was found in others-e.g., in bed nucleus. This regional heterogeneity in the hybridization pattern may reflect regional differences in RNA stability, transcription rate, or subunit composition. The results open the way for studies on the regulation of GABAA-receptor gene expression in normal and pathological brain in situ.The most abundant inhibitory neurotransmitter in the brain, ytaminobutyrate (GABA), facilitates chloride conductance of neuronal membranes by means ofGABAA receptors (1, 2). The function of GABAA receptors can be allosterically modulated, in particular by drugs acting at the benzodiazepine receptor (BZR) (3, 4); these drugs are widely used in the treatment of anxiety, insomnia, and epilepsy. The GABAA receptor complex is a heterooligomer consisting of a and p subunits (5-7) that not only contain the binding sites for GABA, BZR ligands, and other drugs (5-9) but also form the chloride channel (10).The identification of neurons that express the GABAA receptor is of therapeutic interest, since these cells propagate the drug effects elicited at the BZR. Such neurons have been identified electrophysiologically by their response to GABA and BZR ligands (1, 2). In addition, the distribution of the receptor protein helped to identify some GABA-recipient neurons (11, 12). However, an extensive mapping of neurons that express the GABAA receptor complex has not been possible. In situ hybridization histochemistry was therefore used to study the expression of mRNA homologous to the aand p-subunit cDNAs of the GABAA receptor in rat brain. The hybridization pattern was compared to the known regional, cellular, and subcellular distribution of the GABAA receptor protein in the rat central nervous system (11)(12)(13)(14).MATERIALS AND METHODS Preparation of Tissue. Male rats [specific pathogen-free (SPF) albino, Fullinsdorf, Switzerland] weighing 120-130 g were killed by transcardiac perfusion for 20-30 min with ice-cold phosphate-buffered saline containing as fixative either 4% (wt/vol) paraformal...

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