Classical conditioning increases membrane-bound protein kinase C in rabbit cerebellum.

We examined membrane-bound protein kinase C (PKC) in the cerebellum of rabbits given paired presentations of a tone conditioned stimulus (CS) that co-terminated with a periocular electrical stimulation unconditioned stimulus (US) or unpaired presentations of the CS and US or restraint in the experimental context. PKC activation was measured by quantitative film autoradiography of [3H]phorbol 12,13-dibutyrate ([3H]PBt2) binding in the molecular and granule cells layers of lobule HVI, anterior vermis and Crus I, and in the dentate/interpositus nuclei. There was a statistically significant increase in [3H]PBt2 binding within the molecular layer of lobule HVI in rabbits given paired training relative to controls. The results indicate PKC activation in lobule HVI may be important in acquisition of conditioned eyeblink responses.

Secondary structure and Ca2+-induced conformational change of calexcitin, a learning-associated protein.

Calexcitin/cp20 is a low molecular weight GTP- and Ca2+-binding protein, which is phosphorylated by protein kinase C during associative learning, and reproduces many of the cellular effects of learning, such as the reduction of potassium currents in neurons. Here, the secondary structure of cloned squid calexcitin was determined by circular dichroism in aqueous solution and by Fourier transform infrared spectroscopy both in solution and on dried films. The results obtained with the two techniques are in agreement with each other and coincide with the secondary structure computed from the amino acid sequence. In solution, calexcitin is one-third in alpha-helix and one-fifth in beta-sheet. The conformation of the protein in solid state depends on the concentration of the starting solution, suggesting the occurrence of surface aggregation. The secondary structure also depends on the binding of calcium, which causes an increase in alpha-helix and a decrease in beta-sheet, as estimated by circular dichroism. The conformation of calexcitin is independent of ionic strength, and the calcium-induced structural transition is slightly inhibited by Mg2+ and low pH, while favored by high pH. The switch of calexcitin's secondary structure upon calcium binding, which was confirmed by intrinsic fluorescence spectroscopy and nondenaturing gel electrophoresis, is reversible and occurs in a physiologically meaningful range of Ca2+ concentration. The calcium-bound form is more globular than the apoprotein. Unlike other EF-hand proteins, calexcitin's overall lipophilicity is not affected by calcium binding, as assessed by hydrophobic liquid chromatography. Preliminary results from patch-clamp experiments indicated that calcium is necessary for calexcitin to inhibit potassium channels and thus to increase membrane excitability. Therefore the calcium-dependent conformational equilibrium of calexcitin could serve as a molecular switch for the short term modulation of neuronal activity following associative conditioning.

Time-resolved imaging of protein kinase C activation during sea urchin egg fertilization.

To study protein kinase C (PKC) activation during sea urchin egg fertilization we used three different fluorescent probes specific for PKC, namely, fim-1, which recognizes the catalytic site of the enzyme, and BODIPY- and NBD-phorbol esters interacting with the PKC regulatory domain. We were able to follow PKC activation during the early steps of fertilization, the three different probes giving the same fluorescent pattern. Within 120 s following insemination, the fluorescent signal increased and clustered in the cortical zone of the cell. The process was Ca2+ dependent and was inhibited in the presence of staurosporine, a PKC inhibitor. According to our in vitro probe characterization, this signal increase is due to PKC activation. These findings were further confirmed by Western blot analysis. This initial phase was followed by a rapid decrease which might be attributed to PKC hydrolysis by Ca2(+)-dependent proteases. The kinetics and the site distribution of PKC activation appear in complete agreement with the putative functions previously suggested for PKC during fertilization.

Imaging protein kinase C activation in living sea urchin eggs after fertilization.

The fluorescent dye NBD-phorbol acetate was used to visualize the activation of protein kinase C (PKC) in living Lytechinus pictus eggs during fertilization. The dye interacts directly with PKC as determined using a competitive binding assay. Quantitative image analysis of sequential images from laser-scanning confocal microscopy showed a significant reorganization of the signal in the vicinity of the cortical granules and the plasma membrane that began immediately following fertilization and persisted up to 1 hr (P<0.0001). At the concentrations employed, the NBD-phorbol dye was not capable of inducing a significant translocation of the fluorescent signal to the membrane, nor did it appear to interfere with the cell cycle. It therefore seems likely that the present in vivo results reflect the previously reported in vitro activation of protein kinase C immediately subsequent to fertilization. Such an interpretation is parsimonious with the results of parallel subcellular fractionation experiments using an N-terminal polyclonal antibody to sea urchin PKC which showed a significant (P<0.037) translocation of the enzyme from the cytosolic fraction to the membrane fraction 40 min subsequent to fertilization. This study supports and extends previous in vitro data suggesting that PKC activation subsequent to fertilization occurs at or near the egg plasma membrane, perhaps in association with arachadonic acid-rich cortical granules.

Protein kinase C in the hippocampus is altered by spatial but not cued discriminations: a component task analysis.

The exact role of the mammalian hippocampus in memory formation remains essentially as an unanswered question for cognitive neuroscience. Experiments with humans and with animals indicate that some types of mnemonic associative processes involve hippocampal function while others do not. Support for the spatial processing hypothesis of hippocampal function has stemmed from the impaired performance of rats with hippocampal lesions in tasks that require spatial discriminations, but not cued discriminations. Previous procedures, however, have confounded the interpretation of spatial versus cued discrimination learning with the number and kinds of irrelevant stimuli present in the discrimination. An empirical set of data describing a role of protein kinase C (PKC) in different mnemonic processes is similarly being developed. Recent work has implicated the activation of this serine-threonine kinase in a variety of learning paradigms, as well as long-term potentiation (LTP), a model system for synaptic plasticity which may subserve some types of learning. The present study employs the principles of component task analysis to examine the role of membrane-associated PKC (mPKC) in hippocampal-dependent memory when all factors other than the type of learning were equivalent. The results indicate that hippocampal mPKC is altered by performance in hippocampally-dependent spatial discriminations, but not hippocampally-independent cued discriminations and provide a general experimental procedure to relate neural changes to specific behavioral changes.

Alzheimer and beta-amyloid-treated fibroblasts demonstrate a decrease in a memory-associated GTP-binding protein, Cp20.

The two proteins most consistently identified in the brains of patients with Alzheimer disease (AD) have been beta-amyloid and tau, whose roles in the physiology or pathophysiology of brain cells are not fully understood. To identify other protein(s) involved in AD that have been implicated in physiological contexts, we undertook to analyze a specific memory-associated protein, Cp20, in fibroblasts from AD and control donors. Cp20, a GTP-binding protein that is a member of the ADP-ribosylation factor family, was significantly decreased in fibroblasts from AD patients. Normal control fibroblasts exposed to 10 nM beta-amyloid, the same concentration that induced AD-like K+ changes in control fibroblasts, showed a similar decrease in Cp20. Since it has been previously demonstrated that Cp20 is a potent regulator of K+ channels, these findings suggest that changes in this memory-associated protein may explain previously observed differences in AD K+ channels and suggest a pathophysiologic involvement linked to soluble beta-amyloid metabolism that could contribute to the characteristic memory loss of AD.

Incorporation of fluorescent lipids into living rabbit hippocampal and cerebellar slices.

Incorporation of exogenously applied fluorescent lipids into living cells was exploited to probe cellular structure and function in living hippocampal and cerebellar slices as assessed by fluorescent imaging techniques and intracellular recording. Nitrobenzoxadiole-phosphatidylcholine (NBD-PC) and BODIPY phorbol ester, in vitro substrates of phospholipase activity and protein kinase C, respectively, were incorporated and distributed into specific cell populations. In the hippocampal slice, both probes labeled the somata and proximal dendrites of pyramidal and granule cells but were hetrogeneously distributed across the different hippocampal fields. Changes in fluorescent properties of NBD-PC in individual pyramidal cell and granule cell somata were quantified upon challenge with a muscarinic agonist known to modulate phospholipase A2 activity. In the cerebellar slice, both probes labeled Purkinje cell bodies and dendrites but only NBD-PC labeled stellate and granule cells. The cellular and functional specificity of these fluorescent lipid probes shows great promise for monitoring biochemical events in complex neuronal systems with significant spatial and temporal resolution.