Using electrophysiological, biochemical, and autoradiographic techniques, changes in protein kinase C (PKC) activity in specific regions of the hippocampus have been previously implicated in classical conditioning of the nictitating membrane response of the rabbit. Here we report that activation of PKC is potentiated 2-to 3-fold in synaptosomes of the hippocampal CAl and CA2 to -3 regions in rabbits that have undergone classical conditioning of the nictitating membrane response. This potentiation is apparently due to a change in the biochemical properties of PKC within the synaptosomes and is not a result of an increase in total PKC activity. This observation correlates a subcellular biochemical change with classical conditioning of a mammal.Several studies have suggested that the activation of protein kinase C (PKC) influences normal synaptic functioning (1, 2) and plays a role in long-term potentiation (3, 4) and associative learning (1,(5)(6)(7)(8). Although PKC activity has been measured in synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) of the cerebral cortex (9) and SPMs of the hippocampus (10), there are no reports documenting learning-induced changes in PKC activity in synaptosomes from specific brain regions. Our laboratory has previously shown that there are changes in PKC location and function in the hippocampus of rabbits that have undergone a classical conditioning paradigm (5,7,8). In the present report, we further investigate the nature of leaming-specific changes in PKC by monitoring synaptosomal PKC activity from specific microdissected regions of the hippocampus of rabbits that have demonstrated associative learning. Using this approach, we provide evidence that there is a change in the activation properties ofPKC in the synaptosomal fraction obtained from classically conditioned animals and not in samples from control animals. This change in activation properties was indicative of a change intrinsic to the SPMs, although it does not rule out postsynaptic involvement. MATERIALS AND METHODSAssociative Learning. Adult male albino rabbits (Oryctolagus cuniculus) weighing 1.5-3.0 kg were individually housed, given free access to food and water, and maintained on a 12-hr light/12-hr dark cycle. Animals were allocated randomly to one of three groups, which consisted of (i) paired stimulus presentations, (ii) unpaired stimulus presentations, or (iii) no stimulus presentations (naive). Paired and unpaired subjects received 1 day of preparation and 3 consecutive days of stimulus presentation. The procedure was identical to that of Bank et al. (8). Naive subjects received 1 day of adaptation and 3 consecutive days of restraint. On adaptation day, the rabbits were prepared for periorbital electrical stimulation and recording of nictitating membrane movement and then adapted to the training chambers for the length of time of subsequent training sessions (80 min). Training sessions for paired subjects consisted of 80 presentations of a 400-ms, 100-Hz, 82-decibel tone conditioned s...
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