Organotypic rat cerebellar slice culture as a model to analyze the molecular pharmacology of GABA A receptors

Abstract

The preservation of the neuronal circuitry in rat cerebellar slice cultures provides an advantage in monitoring the development and characterizing the pharmacology of GABA A receptor subtypes. Sprague–Dawley rats, 8–11 days of age, were decapitated, their cerebella were cut into 400-μm slices and transferred into culture dishes. Cell viability and organotypic cerebellar organization of the culture remained well preserved up to 3 weeks. Autoradiographic procedures were introduced in these advanced culture technique and employed [ 3H]Ro 15-4513 in the absence and presence of 10 μM diazepam to visualize all benzodiazepine (BZD) and diazepam-insensitive (DIS) binding sites, respectively. Since expression of the α6 subunit variant of the GABA A/BZD receptor is restricted to the cerebellar granule cells and the BZD receptor agonist diazepam has very low affinity for this subunit, changes in DIS [ 3H]Ro 15-4513 binding sites during cultivation time can be attributed to changes in α6 subunit expression. A time-dependent development of total and DIS [ 3H]Ro 15-4513 binding sites were observed in the culture with a trend towards an increase in GABA A receptor α6 subunit levels during the first week. These findings suggest that explant preparations can be used to examine morphological changes in rat cerebellar slices. In addition, these preparations can be utilized to study the pharmacological effects of GABA A/BZD selective drugs on postnatal development of GABA A receptors in rat cerebellum.