One of the most challenging problems in the field of neuroscience is understanding how the CNS orchestrates motor coordination. Lesion studies have shown that the cerebellum is necessary for several types of motor activity including its coordination. Anatomical and physiological studies have demonstrated that cerebellar Purkinje cells (PCs) are central elements in this process. PCs integrate the excitatory and inhibitory inputs arriving in the cerebellar cortex, and are its sole output. Many factors influence this integrative process including the cellular and molecular properties of PCs. One of the more unique properties of PCs is the ability to generate rhythmic electrical activity in vivo as well as in vitro. It is unclear whether this rhythmic activity results from endogenous cellular properties, or is an emergent network property. Several lines of evidence suggest that it is endogenous, resulting from the types and distribution of ion channels along the cell membrane. However the evidence to date has been indirect since synaptic contribution are difficult to rule out either in vivo or in cerebellar slices. In preliminary experiments we have found that embryonic rat PCs survived and differentiated in dispersed cell culture. After one month in culture, PCs were identified using cell-specific immunostaining and displayed electrical properties similar to PCs in cerebellar slices. In particular they generated complex firing patterns (including bursts of action potentials) and dendritic localization of Ca2+ fluxes, the latter monitored using Ca-imaging techniques. Thus, this culture preparation appears ideally-suited for examining the source of the rhythmic electrical activity in cerebellar PCs. In this project we plan to explore whether cultured PCS display endogenous pacemaker activity under conditions that will eliminate synaptic considerations. We will also use single-channel methods to characterize the types of Ca channels present in cultured PCs, and assess whether dendritic Ca2+ influx is necessary for pacemaker activity. We will also determine whether adult rat PCs display endogenous pacemaker activity following acute isolation. The role of cyclic nucleotides and calcium ions in pacemaker activity will also be examined. These experiments will allow us to assess the role of synaptic transmission in the generation of rhythmic electrical activity in PCs. The results of these studies will provide important information regarding the cellular and molecular properties of PCs, and may offer insight into how PC excitability contributes to cerebellar integration.
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