| Abstract No.: | B-B2043 |
| Country: | Canada |
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| Title: | T-TYPE CALCIUM CHANNELS ARE ACTIVATED BY SYNAPTIC INPUTS TO TRIGGER AN AHP IN CEREBELLAR PURKINJE CELLS |
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| Authors/Affiliations: | 2 Jordan Engbers*; 1 Bruce McKay; 2 Renata Rehak; 2 Gerald Zamponi; 2 Ray Turner;
1 Baylor College of Medicine, Houston, TX, USA ; 2 University of Calgary, AB, Canada
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| Content: | Objectives: Spike output in cerebellar Purkinje cells is under the control of distinct synaptic responses evoked by parallel fiber (PF) and climbing (CF) inputs. Both PF- and CF-evoked depolarizations evoke an AHP and pause in spike firing that has been suggested to involve the activation of postsynaptic calcium dependent potassium (KCa) channels. We recently reported an extensive distribution of T type calcium channel isoforms over the Purkinje cell soma-dendritic axis that could mediate subthreshold increases in postsynaptic calcium. In the present study we tested the hypothesis that PF and CF synaptic inputs to Purkinje cells activate calcium influx through postsynaptic T-type channels to trigger a KCa-mediated AHP.
Materials and Methods: Cerebellar slices were prepared from P18-25 rats to be maintained at 32-35°C in vitro and patch clamp recordings obtained from Purkinje cell somata through DIC-IR visualization. To selectively test the role of postsynaptic ion channels we injected simulated EPSCs (sEPSC) to evoke PF or CF-equivalent sEPSPs at the soma in the presence or absence of TTX. The amplitude of sEPSCs were varied from 0.1 - 1.0 nA in accordance with previous recordings under voltage clamp and direct synaptic activation of either pathway. Drugs were either bath applied or focally ejected from a pressure electrode.
Results: Both PF and CF sEPSCs evoked a graded EPSP and spike output pattern equivalent to that evoked by direct synaptic activation, followed by a graded postsynaptic AHP of up to 150 ms duration. Blocking calcium current with general calcium channel blockers promoted a larger membrane depolarization by sEPSCs and prolonged the CF-evoked complex spike depolarization. The AHP following either PF or CF sEPSPs was selectively reduced by blockers of T-type calcium channels (100 µM nickel, 0.5 µM Mibefradil), but not blockers of high voltage activated P-type calcium channels (200 nM Agatoxin IVA, 30 µM cadmium). Synaptically evoked AHPs were reduced by BK potassium channel blockers (200 nM iberiotoxin / 100 nM charybdotoxin) but not the SK potassium channel blocker apamin (200 nM). Western Blot analysis failed to report a coimmunoprecipitation between T-type calcium and BK potassium channels in homogenates of either brain tissue or HEK cells expressing the channels, revealing primarily a functional coupling between these channel subtypes. Synaptically evoked AHPs were directly involved in controlling Purkinje cell excitability in that blocking T-type calcium channels enhanced spike output and increased the capability of both PF and CF sEPSPs to toggle Purkinje cells to an up-state of prolonged spike discharge.
Conclusions: These results reveal a functional coupling between low voltage activated T type calcium channels and BK potassium channels that allow even subthreshold EPSPs to generate an AHP capable of modulating cell excitability.
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