Inhibitory and excitatory neurotransmission in the rat cerebellum

Extracellular recordings were made from Purkinje cells of the rat cerebellum in vitro and in vivo. Gamma-aminobutyric acid (GABA) and related amino acids were applied to the in vitro preparation by superfusion. GABA, muscimol, taurine, β-alanine and δ-aminolaevulinic acid had inhibitory actions on cell activity. Their potencies in terms of EC50 values (concentration to inhibit cell firing by 50%) were 0.29 ± 0.5mM, 0.22 ± 0.01μM, 0.69 ± 0.07mM, 0.66 ± 0.12mM and 1.03 ± 0.13mM respectively. Studies with the antagonists bicuculline methiodide (BMI), picrotoxin and pitrazepin indicated that the inhibitory effects of these drugs were by an action at the GABAA receptor site. Submaximal inhibitory responses to ionophoresed GABA (0.1M, 50nA) were potentiated by the RU32007 and flurazepam when applied by ionophoresis (0.1M, 50nA). This enhancement was blocked by the antagonist Ro15-1788 (10μM) applied by superfusion. Pressure application of L-glutamate and L-aspartate showed these compounds to have potent excitatory actions on Purkinje cell activity. Kainate and quisqualate produced pure excitatory actions which were antagonised by CNQX (10μM) applied by superfusion. NMDA was found to exert either a pure inhibitory action or a biphasic action of excitation followed by inhibition. Both actions could be blocked by (-)APV (10μM) applied by superfusion. The inhibitory actions of NMDA could also be blocked by BMI (1μM), indicating the involvement of GABA receptor in mediating this response. An endogenous inhibitory and excitatory mechanism was also demonstrated in the slice preparation by stimulation of the white matter (0.1-1.0mA, 0.1-1.0ms). Construction of post-stimulus time histograms revealed a short latency excitation followed by a period of inhibition of cell activity. The stimulus evoked inhibition was blocked by the BMI (1μM) and picrotoxin (10μM). The inhibitory period was also blocked by (-)APV (10μM), indicating the involvement of NMDA receptors in mediating the inhibition evoked by stimulation. Theexcitation was antagonised CNQX (10μM),indicating that the endogenous excitatory neurotransmitter, mediating the stimulus evoked excitation, works mainly through kainate/quisqualate receptors. The basal firing rate of Purkinje cells in vitro was not significantly affected by the benzodiazepine receptor agonists (RU32007 and Ro19-0528), antagonist (Ro15-1788) or inverse agonists (DMCM, β-CCE, Ro15-3505, Ro15-4513 and Ro19-4603) at 10μM. At these concentrations a bi-directional modulation of the stimulus evoked inhibition of cell activity was demonstrated. The agonists enhanced the inhibitory period and the inverse agonists reduced the period of inhibition. Both effects were reversed by Ro15-1788 (10μM). In vitro, the agonist RU32007 (1-4mg/Kg i.v.) was found to significantly reduce Purkinje cell firing whereas the inverse agonists DMCM, β-CCE, β-CCM and Ro15-4513 (1-5mg/kg i.v.) were found to increase cell firing in most cells studied. In addition, these ligands also had an effect on the period of inhibition caused by stimulation of the cerebellar cortex. The agonist RU32007 increased the period of inhibition and the inverse agonists decreased the period of inhibition. In most cases however, these changes were no more then would be expected as a result of increase or decrease in the firing rate, since it was shown that there was an exponential relationship between the firing rate and the period of inhibition. These changes in firing rate and period of inhibition are thought to be mediated by an action of benzodiazepines outside the cerebellum. Firing rate independent changes in the period of inhibition were observed in three experiments. These changes in inhibition are thought to be due to direct modulation of a GABAergic mechanism within the cerebellum.