A study of ion channels modulating synaptic transmission using a cerebellar Purkinje cell nerve-bouton preparation

In this thesis a nerve-bouton preparation of Purkinje cells has been characterised. Mechanically isolated Purkinje cells are shown to retain active afferent nerve terminals. This provides a simplified system where the effects of manipulating the ion channels in nerve boutons can be studied without the potentially confounding influences of the rest of the presynaptic cell or surrounding tissue. Isolated Purkinje cells were initially identified for whole cell patch-clamp recordings by their distinctive size and shape. Vesicular release of neurotransmitter was evident by spontaneous inward synaptic currents with a characteristic time course. Antagonist application established that isolated Purkinje cells receive a mixture of inhibitory GABAergic and excitatory glutamatergic inputs. Changes in the frequency, amplitude, and burst behaviour of these spontaneously occurring synaptic currents were used to infer properties of the afferent boutons. Because rat Purkinje cells can be distinguished by their lack of postsynaptic NMDA receptors the presynaptic effects of NMDA application could be readily investigated. NMDA caused an increase in the frequency of postsynaptic events. The NMDA-induced increase was found to be sensitive to external magnesium and TTX application. NMDA application was found to increase the frequency of both GABAergic events and glutamatergic events. Physiologically, NMDA receptors in afferent inhibitory terminals are thought to be activated by the retrograde release of glutamate. So experiments were performed to determine if retrograde release of glutamate could also increase the frequency of glutamatergic events, however it was found that this process has a much more pronounced influence on the GABAergic events. Properties of afferent boutons were also probed with a range of potassium channel blockers. The relevant topics covered are pharmacology, synaptic transmission, and the role of NMDA receptors in the cerebellum and the main technique used is whole-cell patch clamp recording.