Classical conditioning induces long-term translocation of protein kinase C in rabbit hippocampal CA1 cells.

Abstract

The role of the Ca2 /phospholipid-dependent, diacylglycerol-activated enzyme protein kinase C (PKC) in rabbit eyelid conditioning was examined. PKC was partially purified from the CAl region of hippocampal slices from naive, pseudoconditioned, and conditioned rabbits 24 hr after the rabbits were well conditioned. Crude membrane and cytosol fractions were prepared. In conditioned rabbits, significantly more PKC activity (63.3%) was associated with the membrane fraction (and significantly less with the cytosol fraction) compared to naive (42.0%) and pseudoconditioned (44.7%) animals. These differences in distribution of enzyme activity were paralleled by differences in stimulation of enzyme activity by Ca2", phospholipid, and diacylglycerol.There were no between-group differences in basal protein kinase activity. These results suggest that there is a long-term translocation of PKC from cytosol to membrane as a result of conditioning. Autoradiographic binding of radioactive phorbol 12,13-dibutyrate to PKC demonstrated that almost all specific binding was in the stratum radiatum, a region containing the proximal apical dendrites of CAl pyramidal neurons. Therefore, this may be the site of the conditioningspecific PKC translocation, a locus well-suited to underlie the biophysical effects of conditioning.Recent work in our laboratory has established that classical conditioning of the nictitating membrane/eyelid response causes a biophysical record similar to that of Hermissenda classical conditioning-both records involving persistent reductions of a Ca2+-dependent outward K+ current, IK(Ca)(1-4). A long-lasting decrease in IK(Ca) was measured as a decrease in the late afterhyperpolarization (AHP) following impulses of hippocampal CA1 cells (1, 2). In addition, it was found that conditioning induces an enhancement in the summation of postsynaptic potentials (5). Both the AHP reduction and the enhanced postsynaptic potential summation can be mimicked by bath application of phorbol esters, a class of pharmacological agents that activate Ca2+/phospholipid-dependent protein kinase C (PKC) (5-7). Similarly, in Hermissenda, injection of purified PKC into type B photoreceptors reduces IA (an early, rapidly inactivated outward K+ current) and IK(Ca)' but only when paired with a "calcium load" (8). We therefore examined the role of PKC activation as a possible biochemical mechanism underlying the biophysical changes induced by classical conditioning. We now report a molecular record of nictitating membrane/eyelid conditioning: a long-lasting (24 hr), conditioning-specific translocation of PKC activity from the cytosol to the membrane compartment of CA1 cells isolated from the hippocampal slice. This finding is novel because the translocation is of long duration, is learning-specific, and is intrinsic to a population of vertebrate neurons known to undergo biophysical alterations as a result of this type of learning. In addition, the present finding provides a possible point of convergence between mechanisms of learning a...

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