Learning-induced alterations in hippocampal PKC-immunoreactivity: A review and hypothesis of its functional significance

To localize protein kinase C (PKC) in the hippocampus, PKC activity measures, mRNA in situ hybridization, and [³H]phorbol ester binding techniques were used until in the 1980s antibodies became available for in situ immunocytochemistry. In the late 1980s, PKC-isoform-specific antibodies were first used to map hippocampal PKC at the cellular and subcellular level. The mammalian hippocampus contains all four Ca²⁺-dependent PKC isoforms, but the (sub)cellular localization is both isoform- and species-specific. Hippocampally-dependent spatial and associative learning in rat, mice and rabbit induce an increase in PKC immunoreactivity (ir) in hippocampal principal cells studied 24 hours after the animals had learned the task. Among the four Ca²⁺-dependent PKC subtypes, this increase is selective for the γ-isoform. The presence of the γ-isoform in dendrite spines (the most likely site for synaptic plasticity and information storage), in contrast to PKCα, β1, and β2, may underlie the isoform-selectivity. Compared to fully trained animals, subjects halfway training showed intermediate levels of increased PKCγ-ir. Poor learners that were not able to learn the task showed considerably less enhanced PKCγ-ir as compared to good learners. Associative learning induced a decrease in astroglial PKCβ2 and γ-ir in those regions where a simultaneous increase in neuronal PKCγ-ir was observed. This decrease most likely reflects PKC down-regulation, enabling the astrocytes to maintain their K⁺ buffering capacity necassary to support neuronal activity such as accompanying learning and memory. Western blot analyses revealed that the increase in PKCγ-ir was not due to an increase in total amount of PKCγ, translocation, or the proteolytic generation of the fragment PKM. The increase in PKCγ-ir must therefore reflect a learning-induced conformational change in the PKCγ molecule that results in the exposure of the antigenic site(s). Although a large number of hippocampal pyramidal cells display learning-induced enhancement of PKCγ-ir at the 24 hours post-training time point, this does not indicate, however, that all synapses in these neurons are used, or that the maximal PKC signal transduction capacity per cell has been reached. The enhanced PKCγ-ir may reflect a form of activated PKC, since PKC stimulation by phorbol esters (both in hippocampal slices and mildly aldehyde fixed sections) mimicked the increase in PCKγ-ir similar as seen after learning. The most likely transmitter systems which may have induced the altered PKCγ-ir are acetylcholine and glutamate. Their contribution and interaction at the cellular level are depicted in a schematic circuit terminating on a CA1 pyramidal cell. Several functional roles for PKCγ in learning and memory are discussed, and a hypothetical model is proposed based on an endogeneous PKC inhibitor protein that may explain altered antibody-binding to PKCγ after learning. The immunocytochemical approach can contribute significantly to the ongoing attempts to decipher part of the cellular and biochemical mechanism of learning and memory. The development of ever more specific and better characterized antibodies reactive with different sites of proteins like PKCγ will offer the necessary tools for further immunocytochemical research to help unravel complex brain functions.