Dopaminergic neurons in the ventral tegmental area : role of L-type calcium channels in firing regulation

Liu, Yudan (2009) Dopaminergic neurons in the ventral tegmental area : role of L-type calcium channels in firing regulation. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Dopaminergic projections from the ventral tegmental area constitute the mesolimbocortical system that underlies drug abuse and schizophrenia, primarily as the result of increased dopamine transmission. Essentially, more intense spiking releases more dopamine at the terminal, such as fast single spike firing or burst firing. L-type calcium channels are expressed in dopaminergic neurons, however their role in regulating firing frequency and modes remains unknown. In this thesis, I combined patch clamp recording, western blotting and L-type calcium channel transgenic mice to examine the effects of L-type calcium channels on the firing behavior of dopaminergic neurons in brain slices. Results revealed that calcium influx through L-type calcium channels following FPL64176 or (S)-(-)-Bay K8644 application induced burst firing independent of dopamine, glutamate or calcium from internal stores. The burst firing induced as such was completely blocked by the substrate site protein kinase C (PKC) inhibitor chelerythrine but not by the diacylglycerol site inhibitor calphostin C. Western blotting analysis showed that FPL64176 and (S)-(-)-Bay K8644 increased the cleavage of PKC to generate protein kinase M (PKM) and the specific calpain inhibitor MDL28170 blocked this increase. Prevention of PKM production by inhibiting calpain or depleting PKC blocked burst firing induction whereas direct loading of purified PKM into cells induced burst firing. Activation of the NMDA type glutamate or cholinergie receptors known to induce burst firing increased PKM expression. These results indicate that calcium influx through L-type calcium channels activates a calcium-dependent protease that cleaves PKC to generate constitutively active and labile PKM resulting in burst firing of dopaminergic cells (Chapter 2). Next, I examined the role of the L-type calcium channel and PKC in firing responses to carbachol, NMDA or AMPA. All three ligands induced a reversible increase in firing, however, only the carbachol-induced increase was attenuated by the PKC inhibitors chelerythrine and GF 109203X. The L-type calcium channel blocker nifedipine partially blocked carbachol-induced excitation similar to the PKC inhibitors. PKC inhibition and L-type calcium channel blockade did not significantly alter NMDA- or AMPA-induced excitation. Concurrent blockade of PKC and L-type calcium channels with chelerythrine and nifedipine did not additionally suppress carbachol-induced excitation indicating they were sequential events in the same signaling pathway. Furthermore, preincubation with the PKC inhibitor GP 109203X reduced the carbachol-induced increase in nifedipine-sensitive high-voltage gated calcium currents. These results indicate that cholinergic activation enhances PKC activity, which in turn facilitates L-type calcium channel opening to excite dopaminergic cells (Chapter 3). Then, I examined which subtype of L-type calcium channels was involved in firing activities in L-type transgenic mice that have a mutant dihydropyridine (DHP) site. Single spike firing was inhibited to the same extent by the DHP site blocker nifedipine in both Caᵥ1.2DHP⁽⁺/⁺⁾ and Caᵥ1.2DHP⁽⁻/⁻⁾ mice. The DHP site opener (S)-(-)-Bay K8644 and the non-DHP opener FPL64176 induced bursting of dopaminergic cells in Caᵥ1.2DHP⁽⁻/⁻⁾ mice similarly and the DHP blocker nifedipine blocked bursting induced by either agent. Since Cav1.2 and Cav1.3 are the only subtypes expressed in dopaminergic cells, these results underscore the importance of Caᵥ1.3 L-type calcium channels in single spike and burst firing of these cells (Chapter 4).

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