GABAB receptor activation of Purkinje cells in cerebellar slices
Introduction
GABA (γ-aminobutyric acid), the main inhibitory neurotransmitter of the central nervous system, activates GABAA, the ligand-gated ion channel receptors and GABAB, the G protein coupled receptors (Alger and Nicoll, 1982, Bowery, 1989, Bowery, 1993, Misgeld et al., 1995, Kaupmann et al., 1997see Matsumoto, 1989). A GABAC type has also been described (Drew et al., 1984) but has been recently suggested to be a GABAA subgroup (Bowery and Brown, 1997). The distinct inhibitory action and mechanisms of these two receptor types as well as their localization have been widely studied in many brain structures. In the cerebellum, the presence of both types has been described (see Bowery et al., 1987). However, although it is well established by the radioautographic studies that the cerebellar cortex is particularly rich in GABAB receptors (Wilkin et al., 1981, Gehlert et al., 1985, Albin and Gilman, 1989, Turgeon and Albin, 1993see Bowery et al., 1987), only a few electrophysiological studies concern their inhibitory function on cerebellar Purkinje cells (PCs).
Before the two types of GABA receptors were distinguished, Curtis et al. (1974)demonstrated that Baclofen depressed the PC firing rate. This compound was later recognized as a GABAB agonist (Newberry and Nicoll, 1984a, Newberry and Nicoll, 1984b) and used in many other structures to activate these receptors. Recently, our study of rat PCs in vivo (Billard et al., 1992, Billard et al., 1993) have shown that the simple spikes evoked by the parallel fiber-PC activity, but not the complex spikes evoked by climbing fiber (CF)-PC activity, are dose dependently inhibited by electrophoretic application of Baclofen, and that this effect is reversed by 2-OH saclofen, the GABAB receptor antagonist. Schreurs et al. (1992)have shown in rabbit PCs recorded in slices, that inhibition is obtained when Baclofen is applied to the molecular layer where the presence of a high density of GABAB receptors was revealed by binding studies (see Bowery et al., 1987). The molecular layer of the cerebellar cortex contains the PC dendrites together with their excitatory (from parallel and climbing fibers) and inhibitory (from stellate cells) afferents.
GABAB receptors have pre- and post-synaptic localizations in several brain structures (see Misgeld et al., 1995). In the cerebellar cortex, a presynaptic inhibitory function localized on the excitatory terminals (heteroreceptors) has been described for the parallel fibers (Wojcik and Neff, 1984, Wojcik et al., 1990, Dittman and Regehr, 1996) and suggested for the climbing fibers (Kato and Fukuda, 1985). A postsynaptic localization of GABAB receptors on PC dendrites was described by Martinelli et al. (1992)but its inhibitory function is still unknown.
In the present work we show the electrophysiological effects of GABAB receptor activation in the PC and demonstrate their postsynaptic inhibitory function. The experiments were performed in cerebellar slices using conventional intracellular microelectrode recording and current clamp techniques while applying Baclofen and GABAB competitive antagonists.
Section snippets
Methods
The experiments were performed on 21–50 days old Sprague–Dawley rats, males and females. The animals were ether anesthetized and decapitated, the cerebellum rapidly dissected out and slices of 300 or 400 μm were cut in the parasagittal plane from a trimmed block containing the vermis, with a vibratome. Slices were incubated in the bathing medium at room temperature (22°C) for 1 h before being transferred to the recording chamber. The bathing medium contained in mM: NaCl, 124; KCl, 5; CaCl2,
Results
The results are based on recordings taken from 94 PCs. PCs were identified by the presence of both, fast Na dependent somatic spike and slow Ca dependent dendritic spikes (Llinas and Sugimori, 1980a, Llinas and Sugimori, 1980b). They were also identified by intracellular biocytine revealed histochemically. Fig. 1A shows one such PC which illustrates the large dendritic arborization occupying the entire width of the molecular layer. Since in many experiments the drug ejecting micropipette was
Discussion
The present results are based on the use of those agonists and antagonists which are generally recognized as specifically binding to particular types of receptors. Thus we demonstrate that the PCs of the cerebellar cortex are inhibited by activation of the GABAB receptors since inhibition is dose dependently induced by the specific agonist Baclofen and the responses were depressed by the specific antagonists 2-OH saclofen and CGP55845A.
The same conclusions were reached in our previous studies (
Acknowledgements
This work was partially funded by the Fondation pour la Recherche Medicale. The authors are indebted to Dr R.T. Kado for his helpful advice during this work and for reading and correcting the manuscript.
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