gamma Isoform-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 Ca(2+)-dependent protein kinase C (PKC) isoforms (PKC alpha, -beta I, -beta II, or -gamma) 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 PKC gamma 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 PKC alpha, -beta I, or -beta II in in hippocampal neurons. The increase in PKC gamma immunoreactivity (ir) was significantly less (P < 0.05) in poor learners than in good learners. The correlation between the degree of PKC gamma-ir and the total number of conditioned responses across training sessions was both positive and significant. These results suggest that PKC gamma is the major Ca(2+)-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 PKC gamma 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 gamma-isoform must therefore be due to an increased access to the antigenic site(s) as a result of alteration in the tertiary structure of PKC gamma or in quaternary interactions of PKC gamma in situ.