Metabotropic-mediated kainate receptor regulation of IsAHP and excitability in pyramidal cells.

Kainate receptors (KARs) on CA1 pyramidal cells make no detectable contribution to EPSCs. We report that these receptors have a metabotropic function, as shown previously for CA1 interneurons. Brief kainate exposure caused long-lasting inhibition of a postspike potassium current (I(sAHP)) in CA1 pyramidal cells. The pharmacological profile was independent of AMPA receptors or the GluR5 subunit, indicating a possible role for the GluR6 subunit. KAR inhibition of I(sAHP) did not require ionotropic action or network activity, but was blocked by the inhibitor of pertussis toxin-sensitive G proteins, N-ethylmaleimide (NEM), or the PKC inhibitor calphostin C. These data suggest how KARs, putatively containing GluR6, directly increase excitability of CA1 pyramidal cells and help explain the propensity for seizure activity following KAR activation.

Metabotropic regulation of intrinsic excitability by synaptic activation of kainate receptors.

Prolonged modification of intrinsic neuronal excitability is gaining prominence as an activity-dependent form of plasticity. Here we describe a potential synaptic initiation mechanism for these changes in which release of the transmitter glutamate acts on kainate receptors to regulate the postspike slow afterhyperpolarization (sAHP). This action of synaptically released glutamate was occluded by previous kainate application. Furthermore, inhibition of glutamate uptake enhanced the effects of synaptic activation. Glutamate-mediated kainate receptor inhibition of sAHP current (I(sAHP)) was blocked by the PKC inhibitor calphostin C, confirming the requirement for a metabotropic signaling cascade. These data describe a new physiological function for glutamate release: activation of metabotropic kainate receptors, which control directly the excitability of pyramidal cells and probably contribute to prolonged excitability changes.

Oxadiazolylindazole sodium channel modulators are neuroprotective toward hippocampal neurones.

We report the discovery of a new class of neuroprotective voltage-dependent sodium channel modulators exemplified by (5-(1-benzyl-1H-indazol-3-yl)-1,2,4-oxadiazol-3-yl)methanamine 11 (CFM1178). The compounds were inhibitors of [(14)C]guanidinium ion flux in rat forebrain synaptosomes and displaced binding of the sodium channel ligand [(3)H]BW202W92. 11 and the corresponding N(2)-benzyl isomer, 38 (CFM6058), demonstrated neuroprotective activity in hippocampal slices comparable to sipatrigine. CYP450 enzyme inhibition observed with 11 was reduced with 38. In electrophysiological experiments on dissociated hippocampal neurons, these two compounds caused use- and voltage-dependent block of sodium currents. Sodium channel isoform profiling against Na(v)1.1-1.8 demonstrated that the standard sodium channel blocker lamotrigine had modest activity against Na(v)1.1, while sipatrigine was generally more potent and less selective. 11 and 38 showed potent activity against Na(v)1.6, pointing to pharmacological block of this isoform being consistent with the neuroprotective effect. 38 also showed use dependent block of Na(v)1.6 in HEK cells.

Protein kinase signalling requirements for metabotropic action of kainate receptors in rat CA1 pyramidal neurones.

Hippocampal pyramidal neurones display a Ca(2+)-dependent K(+) current responsible for the slow afterhyperpolarization (I(sAHP)), a prominent regulator of excitability. There is considerable transmitter convergence onto I(sAHP) but little information about the interplay between the kinase-based transduction mechanisms underlying transmitter action. We have added to existing information about the role of protein kinase C (PKC) in kainate receptor actions by demonstrating that direct postsynaptic activation of PKC with either 1-oleoyl-2-acethylsn-glycerol (OAG) or indolactam is sufficient to inhibit I(sAHP). The physiological correlate of this action - activation of PKC by kainate receptors - requires G alpha(i/o) proteins. The cAMP/PKA system is well documented to subserve the actions of monoamine transmitters. We have found an additional role for the cAMP/PKA system as a requirement for kainate receptor-mediated inhibition of I(sAHP). Inhibition of adenylyl cyclase with dideoxyadenosine or PKA with either H89 or RpcAMPs blocked kainate receptor-mediated actions but did not prevent the actions of direct PKC activation with either OAG or indolactam. We therefore propose that the PKA requirement is upstream from the actions of PKC. We additionally report a downstream link in the form of increased mitogen-activated protein (MAP) kinase activity, which may explain the long duration of metabotropic actions of kainate receptors on I(sAHP).

Hippocalcin: a new solution to an old puzzle.

In hippocampal pyramidal neurons, calcium entry following an action potential burst results in a slow afterhyperpolarization (sAHP) that critically regulates subsequent excitability. Although this potassium current was described two decades ago, the mechanism whereby the rise in intracellular calcium generates the sAHP was, until now, not known. In this issue of Neuron, Tzingounis et al. now show that calcium binding to hippocalcin, a member of the NCS family, is one of the necessary steps involved in production of the sAHP.

Kinetics of ion channel modulation by cAMP in rat hippocampal neurones.

Ion channel regulation by cyclic AMP and protein kinase A is a major effector mechanism for monoamine transmitters and neuromodulators in the CNS. Surprisingly, there is little information about the speed and kinetic limits of cAMP-PKA-dependent excitability changes in the brain. To explore these questions, we used flash photolysis of caged-cAMP (DMNB-cAMP) to provide high temporal resolution. The resultant free cAMP concentration was calculated from separate experiments in which this technique was used, in excised patches, to activate cAMP-sensitive cyclic nucleotide-gated (CNG) channels expressed in Xenopus oocytes. In hippocampal pyramidal neurones we studied the modulation of a potassium current (slow AHP current, I(sAHP)) known to be targeted by multiple transmitter systems that use cAMP-PKA. Rapid cAMP elevation by flash photolyis of 200 microm DMNB-cAMP completely inhibited the K(+) current. The estimated yield (1.3-3%) suggests that photolysis of 200 microm caged precursor is sufficient for full PKA activation. By contrast, extended gradual photolysis of 200 microm DMNB-cAMP caused stable but only partial inhibition. The kinetics of rapid cAMP inhibition of the K(+) conductance (time constant 1.5-2 s) were mirrored by changes in firing patterns commencing within 500 ms of rapid cAMP elevation. Maximal increases in firing were short-lasting (< 60 s) and gave way to moderately enhanced levels of spiking. The results demonstrate how the fidelity of phasic monoamine signalling can be preserved by the cAMP-PKA pathway.

On the regulation of NMDA receptors by nitric oxide.

Nitric oxide (NO) is generated in central synapses on activation of N-methyl-D-aspartate (NMDA) receptors and exerts physiological effects by changing cGMP levels. NO has frequently also been claimed to engage a different mechanism, namely the covalent modification of thiol residues (S-nitrosation), and thereby exert a negative feedback on NMDA receptors. Tests of this hypothesis were conducted by recording NMDA receptor-mediated synaptic potentials in the CA1 area of rat hippocampal slices. Manipulations designed to increase or decrease endogenous NO levels had no effect. Addition of exogenous NO using a NONOate donor in concentrations up to 30-fold higher than those needed to evoke maximal cGMP accumulation also had no effect. Nevertheless, in agreement with previous findings, photolysis of a caged NO derivative with UV light led to an enduring block of synaptic NMDA receptors. To address these contradictory results, NMDA receptor-mediated currents were recorded from HEK-293 cells transfected with NR1 and NR2A subunits. As found in slices, photolysis of caged NO inhibited the currents whereas perfusion of NO (up to 5 microM) was ineffective. However, when NO was supplied at a concentration found to be effective when released photolytically (5 microM) and the cells simultaneously exposed to the UV light used for photolysis, NMDA receptor-mediated currents were inhibited. This effect was not observed at more physiological NO concentrations (10 nM range). The results indicate that neither endogenous NO nor exogenous NO in supra-physiological concentration inhibits synaptic NMDA receptors; the combination of high NO concentration and UV light can give an artifactual result.

SK channels and the varieties of slow after-hyperpolarizations in neurons.

Action potentials and associated Ca2+ influx can be followed by slow after-hyperpolarizations (sAHPs) caused by a voltage-insensitive, Ca2+-dependent K+ current. Slow AHPs are a widespread phenomenon in mammalian (including human) neurons and are present in both peripheral and central nervous systems. Although, the molecular identity of ion channels responsible for common membrane potential mechanisms has been largely determined, the nature of the channels that underlie the sAHPs in neurons, both in the brain and in the periphery, remains unresolved. This short review discusses why there is no clear molecular candidate for sAHPs.