γIsoform-Selective Changes in PKC Immunoreactivity after Trace Eyeblink Conditioning in the Rabbit Hippocampus

An immunocytochemical examination of the rabbit hippocampus was done to determine which of the Ca2+-dependent protein kinase C (PKC) isoforms (PKCα, -βI, -βII, or -γ) are involved in associative learning. The hippocampally dependent trace eyeblink conditioning task was used for behavioral training, and pseudoconditioned and naive animals served as controls. Significant increases (P < 0.05) in staining intensity were found with antibodies reactive with the catalytic or the regulatory domain of PKCγ in conditioned animals compared with naive and pseudoconditioned subjects at a 24-h post-conditioning time point. The increase was found in CA1 and CA3 pyramidal cell bodies, in apical dendrites and the proximal part of the basilar dendrites, and in cell bodies of dentate granule cells. In contrast, no conditioning-specific changes were found for PKCα, -βI, or -βII in hippocampal neurons. The increase in PKCγ immunoreactivity (ir) was significantly less (P < 0.05) in poor learners than in good learners. The correlation between the degree of PKCγ-ir and the total number of conditioned responses across training sessions was both positive and significant. These results suggest that PKCγ is the major Ca2+-dependent PKC isoform involved in hippocampal neurons during acquisition of associative memories. Immunoblots revealed no conditioning-induced increase in the total amount or translocation of PKCγ at the 24-h time point, and no proteolytic PKC fragments were observed. In agreement with the Western blot data, PKC activity did not differ among naive, pseudoconditioned, and trace conditioned animals. The conditioning-induced increase in antibody binding to the γ-isoform must therefore be due to an increased access to the antigenic site(s) as a result of alteration in the tertiary structure of PKCγ or in quaternary interactions of PKCγ in situ