Autoradiographic localization of gamma-aminobutyric acid (GABA) receptors in the rat cerebellum.

Abstract

[3H]Muscimol was used in the light microscopic autoradiographic localization of 7-amninobutyrip acid (GABA) receptors in the rat cerebellum. The binding ofI3Hl muscimol to the slide-mounted tissue sections had all of the characteristics associated with a GABA receptor. It was saturable with appropriate kinetic constants and was blocked only by other GABAergic drugs. Autoradiographic studies revealed a very high density of receptors over the granule cell layer, a low level over'the molecular layer, and negligible receptor binding over white matter. This suggested a high localization of GABA receptors to granule 'cells, which is supported by other neurochemical studies. The autoradiographic procedure used here should be a useful method in further studies of receptors requiring high sensitivity and increased anatomical resolution.y-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system of mammals and elsewhere. One of the most intensively studied components of the GABAergic system has been the GABA receptor, Recently, conditions have been defined whereby one can utilize [3H]GABA to bind to a GABA receptor in membrane homogenates (1-6). More recently, muscimol (3-hydroxy-5-aminomethylisoxazole), a GABA receptor agonist, has also been used in its radiolabeled form to bind to GABA receptors (7). In binding studies, [3H]muscimol has the advantages that it has only a weak affinity for the GABA uptake system and is not an avid substrate for GABA-2-oxoglutarate aminotransferase (8, 9).Our laboratory has studied the light microscopic, autoradiographic localization of a number of neurotransmitter receptors, including cholinergic muscarinic (10-12), opiate (13-15), and dopaminergic receptors (16,17). We recently developed a method to localize receptors by autoradiographic methods at the light microscopic level that can be used with ligands that do not cross the blood/brain barrier or do not efficiently label receptors by in vivo injection (18). This method involves the in vitro labeling of receptors in slide-mounted tissue sections and the apposing of emulsion-coated cover slips to generate autoradiograms; it gives results identical to those found in earlier studies using in vivo labeling of receptors (18) Receptor Binding. Slides with mounted tissue sections were brought to room temperature and preincubated in 0.31 M Tris-citrate buffer, pH 7.1, at 2-4°C in an ice bath for 20 min. In extensive preliminary experiments, we found that this preincubation resulted in a marked increase in maximal specific binding of [3H]muscimol to the tissues. We suspect that this preincubation resulted in a depletion of endogenous GABA from the tissues, which would inhibit the binding of [3H]muscimol. A similar effect has been reported in published experiments with homogenates in which tissues are commonly washed or treated with Triton X-100 to enhance GABA binding (3,5). After preincubation, the slides were transferred to an incubation medium of 0.31 M Tris citrate (pH 7.1 at 2-40C) buffer contai...

Regional Distribution and Kinetics of Three Sites on the GABAA Receptor: Lack of Effect of Portacaval Shunting

“…The only exception was the Kd of muscimol, which was found to be 0.7-5.5 nM in our study, comparing reasonably well with �13 nM in whole brain sec tions in one study (Palacios et al, 1980), but not with 35-59 nM in two other studies (Penney et al, 1981;Pan et al, 1983). This discrepancy may be related to differences in technique; since muscimol binds to both the low-and high-affinity forms of the GABAA receptor, the conditions of the assay, es pecially the rinse time, can affect which form is predominantly reflected by the binding.…”

Autoradiographic Determination of Neurotransmitter Receptor Distributions in the Cerebral and Cerebellar Cortices,

“…Benzodiazepine receptor localization in the molecular layer of the cerebellar cortex (where the dendritic arborizations of the Purkinje cells reside) supports this hypothesis. Studies with human cerebellum demonstrated the same distribution of GABA receptors described here (18). However, while the rat cerebellum did not show any significant labeling of benzodiazepine receptors in the granule cell layer, the human cerebellum demonstrated equal concentrations of benzodiazepine receptors in the molecular layer and granule cell layer (34).…”

Differences between GABA Receptor Binding to Membranes from Cerebellum during Postnatal Development and from Cultured Cerebellar Granule Cells

“…In cerebellum the period of the largest in crease in the number of receptor sites coin cides with the development of the granule cells [34], in agreement with the suggestion by Simantov et al [38] and Olsen and Mikoshiba [31] that a major fraction of the GABA receptors in cerebellum resides on this cell type. Moreover, this is in line with autoradio graphic studies of GABA binding to cerebel lar slices [33] and cerebellar cultures [14][15][16] as well as with the finding that muscimol binding is enriched in cerebellar glomeruli [17]Although GABA or muscimol binding has been studied in cultured neurons from cere brum or spinal cord [7,8,15,39,41], no such studies have been performed on cerebellar cultures. Since it appears that granule cells possess a large fraction of the cerebellar GABA receptors and since this cell type can be cultured relatively easily [28] we decided to study GABA binding to such cells in cul ture.…”