Date of Award

2009

Degree Type

Thesis

Degree Name

Master of Science

Program

Physiology and Pharmacology

Supervisor

Dr. Michael O. Poulter

Second Advisor

Cezar Gavrilovici

Third Advisor

Vladimir Zhurov

Abstract

Aim It was previously reported that the efficacy of the neuroactive steroid (THDOC) was reduced after the induction of stage 5 amygdala kindled seizures. As the phosphorylation has some implications on THDOC pharmacology such as altered desensitization rate of GABAa receptors, we hypothesized that kindling induced (long term) changes in the phosphorylation of GABAa receptors. Methods In order to test this hypothesis we manipulated phosphorylation state of GABAa receptors by employing agents that either activated PKC (PMA, 100 nM), inhibited phosphatase (FK-506,100 nM) or activated phosphatase (Li+ Palmitate, 100 nM). Through patch clamp recordings in layers II of the piriform cortex prepared from kindled and non-kindled male Sprague Dawley rats (200-250 gm), GABAa synaptic responses were measured. We also carefully monitored the extra-synaptic currents to determine whether the extra-synaptic transmission was affected. We applied THDOC (100 nM) for a minimum of 10 minutes, a protocol that was shown previously to enhance currents by as much as 100 %. Findings We found that PMA and FK-506 prolonged the deactivation of the mIPSCs. The subsequent application of THDOC reduced the amplitude of mIPSCs, but THDOC prolonged

mIPSCs decay time further. However, the net affect was that THDOC did not enhance charge transfer, an outcome that was identical to those found in kindled tissue. Both the inactive phorbol (a-PMA, 100 nM) and the PKC antagonist (BIS I, 100 nM) had no effect on the THDOC synaptic modulation. Interestingly, Li+ palmitate reversed the effect of kindling on THDOC modulation in synaptic transmission, but it showed no effect on extrasynaptic transmission. Conclusion taken together these data indicated that phosphorylation had profound effects on THDOC modulation and kindling reduced the THDOC efficacy by potentially inducing the phosphorylation of GABAa receptors.

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