Abstract
Enhanced activity of the dopaminergic system originating in the ventral tegmental area is implicated in addictive and psychiatric disorders. Burst firing increases dopamine levels at the synapse to signal novelty and salience. We have previously reported a calcium-dependent burst firing of dopamine cells mediated by L-type channels following cholinergic stimulation; this paper describes a cellular mechanism resulting in burst firing following L-type channel activation. Calcium influx through L-type channels following FPL 64176 or (S)-(-)-Bay K8644 induced burst firing independent of dopamine, glutamate, or calcium from the internal stores. 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 FPL 64176 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 N-methyl-D-aspartic acid type glutamate or cholinergic receptors known to induce burst firing increased PKM expression. These results indicate that calcium influx through L-type channels activates a calcium-dependent protease that cleaves PKC to generate constitutively active and labile PKM resulting in burst firing of dopamine cells, a pathway that is involved in glutamatergic or cholinergic modulation of the central dopamine system.
Highlights
Dopaminergic (DA)2 projections from the ventral tegmental area (VTA) constitute the mesolimbocortical system that underlies drug abuse and schizophrenia, primarily as the result of increased DA transmission [1,2,3]
We present results that Ca2ϩ influx through L-type channels activates a Ca2ϩ-dependent protease, which in turn cleaves protein kinase C (PKC) to generate a labile fragment that is constitutively active to induce burst firing in DA neurons
Activation of NMDA and Cholinergic Receptors Increases protein kinase M (PKM) Expression—To test whether PKM is involved in the physiological regulation of burst firing in DA neurons, glutamate, or cholinergic agonists known to induce burst firing in DA cells were used to stimulate VTA slices and the amount of PKM was detected by Western blotting
Summary
Dopaminergic (DA)2 projections from the ventral tegmental area (VTA) constitute the mesolimbocortical system that underlies drug abuse and schizophrenia, primarily as the result of increased DA transmission [1,2,3]. To further validate the role of PKM in L-type channel-induced burst firing, we compared the levels of phospho-PKC and PKM by Western blotting total protein lysates from slices that were treated with (S)-(Ϫ)-Bay K8644 or FPL 64176 alone and with MDL 28170 followed by FPL 64176.
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