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Access Type

WSU Access

Date of Award

1-1-2010

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Pharmacology

First Advisor

RODRIGO ANDRADE

Abstract

One of the most characteristic features of pyramidal cells in the prefrontal cortex (PFC) is that they present a slow afterhyperpolarizing current (IsAHP) that plays a critical role in the regulation of neuronal excitability. This current is modulated by receptors acting via GΑq/11 G proteins, thus it is thought that neurotransmitters regulate neuronal excitability through the inhibition of this current. IsAHP is known to be mediated by calcium-activated potassium channels, however, neither the identity of the channel underlying this current nor its mechanism of activation are yet well understood. Recent reports have questioned a direct role of calcium in the activation of the channels underlying the IsAHP in hippocampus, suggesting the neuronal calcium sensor (NCS) protein hippocalcin as one of the plausible proteins involved in the triggering of IsAHP; therefore, one of the aims of this work will be to examine the role of hippocalcin and other NCS proteins in the development of IsAHP in pyramidal neurons of the PFC.

In the present study we used loss of function and overexpression techniques to demonstrate that hippocalcin is an important component in the development of the IsAHP in pyramidal neurons of the prefrontal cortex. Furthermore, we also established that neurocalcin-Δ, a close related member of the visinin-like protein subfamily, but not VILIP-2 produced similar effects on IsAHP. Transfection with either hippocalcin or neurocalcin- also altered the kinetic of IsAHP reducing its rate of decay.

Another characteristic feature of IsAHP is that it runs down upon prolonged whole-cell recordings. This rundown was reduced when a precursor of PtdIns(4,5)P2 was added to the recording pipette. Furthermore, blocking the resynthesis of PtdIns(4,5)P2, greatly increased the rate of IsAHP rundown. Reducing PtdIns(4,5)P2 levels or its availability at the plasma membrane greatly reduced IsAHP amplitude highlighting the relevance of this phospholipids in the developing of IsAHP. On the other hand, increasing PtdIns(4,5)P2 levels at the plasma membrane, by expressing its synthetic enzyme (PIPK5), did not significantly increased IsAHP amplitude, but strongly increased its sensibility to activation by calcium, thus ruling out a direct role of PtdIns(4,5)P2 on activating IsAHP in pyramidal neurons of the prefrontal cortex. These results propose the idea of an concerted mechanism of IsAHP activation between calcium influx and PtdIns(4,5)P2 availability at the plasma membrane and present IsAHP not as a single unitary current, but rather as the embodiment of a biochemical gating mode.