Date of Award

Spring 2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Michelle Mynlieff

Second Advisor

Edward M. Blumenthal

Third Advisor

James T. Buchanan, Thomas J. Eddinger, David A. Wagner

Abstract

During the early postnatal period, the inhibitory neurotransmitter γ-aminobutyric acid (GABA) facilitates current through voltage-dependent L-type calcium channels by activating metabotropic GABAB receptors in the rat hippocampus. In the present study, the effects of the GABAB receptor agonist baclofen on L-type currents were tested using whole-cell voltage clamp recording on neurons isolated from the superior region of hippocampi obtained from pups of various ages to determine the exact time course of L-type current facilitation. The facilitation of L-type current by GABAB receptors is more prominent during the second week of development.

One developmental process that L-type current may be involved in is changes in the expression of the K+Cl- co-transporter (KCC2) and N+K+2Cl- co-transporter (NKCC1), which are necessary in the maturation of the GABAergic system. To investigate whether calcium influx through L-type channels and GABAB receptor activation affects the expression of chloride transporters during the early neonatal period, hippocampal cultures isolated from day 0 pups were treated with a GABAB agonist or an L-type channel antagonist for one week. Steady state KCC2 and NKCC1 levels were determined by Western blot analysis. Blockade of L-type channels drastically reduced KCC2 expression but not NKCC1 expression, suggesting that the upregulation of KCC2 in the first postnatal week is dependent on calcium influx through L-type channels.

The involvement of protein kinase C (PKC) and A (PKA) in the signaling pathway of L-type current modulation by GABAB receptors was also investigated using electrophysiological experiments. The facilitatory response of baclofen was blocked in the presence of PKC inhibitors, but not PKA inhibitors. Direct activation of PKC using a phorbol ester mimicked the facilitation of L-type current seen with baclofen, whereas facilitation was not seen with direct activation of PKA with a cAMP analogue. Together, these experiments have demonstrated that the facilitation of L-type current by GABAB receptor activation is maximal during the second postnatal week in development and is mediated by PKC. In addition, calcium influx through L-type channels also contributes to the maturation of the GABAergic system.

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