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Kindling is a use-dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca(2+)-independent) and Ca(2+)-stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling-associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.
The release of somatostatin (somatostatin-like immunoreactivity) from hippocampal slices during the development of hippocampal kindling in rats was measured under resting and depolarizing conditions. Preliminary experiments in naive rats showed that the spontaneous efflux of somatostatin (4.0 +/- 0.3 fmol/ml every 10 min) was independent of external Ca2+ but was reduced to 71.5 +/- 6% of baseline (P < 0.05) during 20 min incubation with 5 microM tetrodotoxin. Neuronal depolarization with 25, 50 and 100 mM KCl induced a Ca(2+)-dependent somatostatin release, respectively 4.3 +/- 0.4, 16.7 +/- 1.6 and 22.0 +/- 1.3 times baseline (P < 0.01). Veratridine caused a dose-dependent Ca2+ and tetrodotoxin (5 microM) sensitive release ranging from 6.5 +/- 0.1 to 13.0 +/- 1.4 times baseline at 1.4 microM and 50 microM respectively (P < 0.01). One week after the last of three consecutive stage 5 seizures (full seizure expression) or 48 h after the last stage 2 stimulation (preconvulsive stage), 50 mM KCl-induced somatostatin release was significantly higher (1.8 +/- 0.1, P < 0.01) than in shams (animals implanted with electrodes but not stimulated) in the stimulated and contralateral hippocampus. Somatostatin release measured under resting conditions was increased by 1.5 times in the stimulated hippocampus at stage 2 (P < 0.05) and by 2.2 and 1.7 times in both hippocampi at stage 5 (P < 0.01). Forty-eight hours after the induction of a single afterdischarge no significant changes were found in either spontaneous or 50 mM KCl-induced release of somatostatin.(ABSTRACT TRUNCATED AT 250 WORDS)
The selective metabotropic glutamate receptor agonist, trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD), stimulates phosphoinositide hydrolysis and elicits a number of electrophysiological responses in the hippocampus. If these effects are mediated by the same receptor subtype, they should undergo parallel developmental regulation. Therefore, we compared the phosphoinositide hydrolysis response and the electrophysiological responses to trans-ACPD at two different developmental stages. Trans-ACPD-stimulated phosphoinositide hydrolysis was significantly greater in hippocampal slices from immature (6-11-day-old) rats than from adults. In contrast, trans-ACPD elicited decreases in spike frequency adaptation and in the amplitude of the slow afterhyperpolarization in roughly equal percentages of immature and adult CA1 pyramidal cells. Similar results were obtained using the putative endogenous agonist, glutamate. These data support the hypothesis that certain electrophysiological effects of trans-ACPD are mediated by a metabotropic glutamate receptor that is distinct from the phosphoinositide hydrolysis-linked glutamate receptor.
The selective metabotropic glutamate receptor agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) stimulates phosphoinositide hydrolysis and elicits several physiological responses in rat hippocampal slices. However, recent studies suggest that the physiological effects of trans-ACPD in the hippocampus are mediated by activation of a receptor that is distinct from the phosphoinositide hydrolysis-linked receptor. Previous experiments indicate that cyclic AMP mimics many of the physiological effects of trans-ACPD in hippocampal slices. Furthermore, recent cloning and biochemistry experiments indicate that multiple metabotropic glutamate receptor subtypes exist, some of which are coupled to yet unidentified effector systems. Thus, we performed a series of experiments to test the hypothesis that ACPD increases cyclic AMP levels in hippocampal slices. We report that 1S,3R- and 1S,3S-ACPD (but not 1R,3S-ACPD) induce a concentration-dependent increase in cyclic AMP accumulation in hippocampal slices. This effect was blocked by the metabotropic glutamate receptor antagonist L-2-amino-3-phosphonoproprionic acid but not by selective antagonists of ionotropic glutamate receptors. Furthermore, our results suggest that 1S,3R-ACPD-stimulated increases in cyclic AMP accumulation are not secondary to increases in cell firing or to activation of phosphoinositide hydrolysis.
Protein kinase C (PKC) is thought to play an important role in neuronal function by mediating changes in synaptic strength. Specifically, it has been argued that persistent PKC activation underlies the maintenance of long-term potentiation (LTP) of synaptic transmission in the hippocampus, a model widely used to study mammalian learning and memory. Because the induction of LTP is known to be dependent upon Ca2+ influx into the postsynaptic neuron, we investigated Ca(2+)-dependent mechanisms that operate to elicit persistent PKC activation in the hippocampus. Hippocampal homogenates were incubated with Ca2+ for a brief period and subsequently assayed for persistent changes in basal (Ca(2+)-independent) PKC activity, using the selective PKC substrate neurogranin(28-43) (NG(28-43)). After Ca2+ incubation, basal PKC phosphorylation of NG(28-43) was increased and expression of the increased activity could be inhibited by PKC(19-36), a selective peptide inhibitor of PKC. These data indicate the presence of a persistently activated form of PKC in Ca(2+)-pretreated hippocampal homogenates. The persistently activated PKC was localized to the soluble fraction of homogenates. Generation of the soluble, persistently activated form of PKC was blocked by the calpain inhibitor, leupeptin, suggesting a proteolytic activation of PKC. Column chromatography and Western blots indicated the presence of PKM, a proteolytic fragment of PKC that is active in the absence of calcium, diacylglycerols, or phospholipid cofactors. Thus, Ca2+ induces proteolytic activation of PKC in hippocampal homogenates. This suggests that proteolytic activation is a plausible candidate as a mechanism underlying the persistent activation of PKC associated with LTP.
Selective activation of metabotropic glutamate receptors with trans-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) stimulates phosphoinositide hydrolysis and elicits three major physiological responses in area CA1 of the hippocampus. These include direct excitation of pyramidal cells, blockade of synaptic inhibition, and decreased transmission at the Schaffer collateral-CA1 pyramidal cell synapse. Physiological effects of trans-ACPD are thought to be mediated by activation of phosphoinositide hydrolysis. However, it is now clear that multiple metabotropic glutamate receptor subtypes exist, some of which are not coupled to phosphoinositide hydrolysis. Thus, we performed a series of studies aimed at determining whether the physiological effects of trans-ACPD in the hippocampus are mediated by activation of the predominant phosphoinositide hydrolysis-linked glutamate-receptor. We report that L-2-amino-3-phosphonopropionic acid (L-AP3), an antagonist of trans-ACPD-stimulated phosphoinositide hydrolysis, does not inhibit the physiological effects of trans-ACPD in area CA1 at concentrations that maximally inhibit trans-ACPD-stimulated phosphoinositide hydrolysis in this region. Furthermore, 1S,3S-ACPD activates the phosphoinositide hydrolysis-linked glutamate receptor but does not reduce evoked field excitatory postsynaptic potentials (EPSPs) in area CA1. However, we report that the physiological effects of 1R,3S- and 1S,3R-ACPD are consistent with the hypothesis that these effects are mediated by activation of a metabotropic glutamate receptor. Thus, our data are consistent with the hypothesis that the physiological effects of trans-ACPD in area CA1 of the hippocampus are mediated by metabotropic glutamate receptors that are distinct from the AP3-sensitive phosphoinositide hydrolysis-linked glutamate receptor.