A QUANTITATIVE AUTORADIOGRAPHIC STUDY OF GABA-A AND BENZODIAZEPINE RECEPTORS IN THE BRAIN OF THE FROG, RANA-ESCULENTA

Specific binding sites for GABA(A) and benzodiazepines were detected in the brain of the frog Rana esculenta after the in vitro incubation of tissue sections with their respective specific agonists [H-3] muscimol and [H-3] flunitrazepam. Conditions for the binding assay were optimized and as a result binding was saturable and specific. Quantitative autoradiographic receptor measurements in the different brain sections showed that elevated levels of [H-3] muscimol binding sites were found in the two layers of the cerebellum (periventricular and external) with the highest binding densities being detected in the periventricular layer. Relatively high densities of [H-3] muscimol binding sites were also observed in the torus semicircularis of the mesencephalon and in the thalamic nucleus rotundus and posterolateral nucleus, plus the mitral cell layer of the olfactory bulb, the amygdala pars lateralis and the striatum of the telencephalon. Intermediate to low binding levels were obtained in the remaining brain areas such as the external layer of the optic tectum, the dorsomedial and dorsolateral anterior thalamic nuclei, the medial and lateral pallium, the medial septal nucleus, the preoptic nucleus, the dorsal and ventral infundibular nuclei of the hypothalamus and the interpeduncular nucleus. Autoradiographic evaluation of benzodiazepine receptors revealed that binding levels of [H-3] flunitrazepam were overall lower than those of the GABA(A) sites. In fact the highest [H-3] flunitrazepam binding levels were observed in the striatum, external layer of the optic tectum and the torus semicircularis. Intermediate receptor levels were encountered in the mitral cell and glomerular layers of the olfactory bulb, the nucleus rotundus and the lateral and medial pallium, whereas low levels were found in the remaining brain areas. It is interesting to note that the lowest binding densities of [H-3] flunitrazepam plus an inverse binding pattern to that shown for the GABA(A) receptors were encountered in the two layers of the cerebellum, Consequently, when the binding densities of the two ligands were expressed as a ratio ([H-3] MUS/[H-3] FLU), we noticed that the highest ratio values were observed in the periventricular (17.1) and external (8) layers of the cerebellum, while a ratio of 1 was obtained in the external layer of the optic tectum. The heterogeneous binding relationship of these two GABA(A) molecular components encountered in key brain areas, such as the cerebellum and the optic tectum, of the frog Rana esculenta could provide interesting insights concerning the role of GABA in these brain sites.