Canonical transient receptor potential channels (TRPCs) are receptor-operated cation channels that are activated in response to phospholipase C signaling. Although TRPC1 is ubiquitously expressed in the brain, TRPC4 expression is the most restrictive, with the highest expression level limited to the lateral septum. The subunit composition of neuronal TRPC channels remains uncertain because of conflicting data from recombinant expression systems. Here we report that the large depolarizing plateau potential that underlies the epileptiform burst firing induced by metabotropic glutamate receptor agonists in lateral septal neurons was completely abolished in TRPC1/4 double-knockout mice, and was abolished in 74% of lateral septal neurons in TRPC1 knockout mice. Furthermore, neuronal cell death in the lateral septum and the cornu ammonis 1 region of hippocampus after pilocarpine-induced severe seizures was significantly ameliorated in TRPC1/4 double-knockout mice. Our data suggest that both TRPC1 and TRPC4 are essential for an intrinsic membrane conductance mediating the plateau potential in lateral septal neurons, possibly as heteromeric channels. Moreover, excitotoxic neuronal cell death, an underlying process for many neurological diseases, is not mediated merely by ionotropic glutamate receptors but also by heteromeric TRPC channels activated by metabotropic glutamate receptors. TRPC channels could be an unsuspected but critical molecular target for clinical intervention for excitotoxicity.

Rhodamine 123 staining and electron microscopy were used to reveal a correlation between the ultrastructural and functional state of cultured cerebellar granule cells after short glutamate treatment. Glutamate exposure (15 min, 100 lxM) in Mg2+-free solution caused considerable ultrastructural alterations in a granule cell: clumping of the chromatin, swelling of the endoplasmic reticulum and mitochondria, and disruption of the mitochondrial cristae. After glutamate treatment, the mitochondria of the neurons lost their ability to sequester rhodamine 123. Both the N-methyl-D-nspartate receptor channel blocker MK-801 (30 ItM) and cobalt chloride (2 mM) prevented the deteriorative effects of glutamate. These data suggest that glutaumte-induced Ca 2+ overload of the neurons can lead to nonspecific permeability of the inner mitochondrial membrane, resulting in neuronal death.

SUMMARYStatus epilepticus (SE) is associated with a dynamic plasticity of postsynaptic neurotransmitter receptors. The plasticity of AMPA receptor (AMPAR)-mediated glutamatergic transmission during established SE (ESE), after development of benzodiazepine resistance, was evaluated. There was increased frequency and inward rectification of AMPAR-mediated excitatory postsynaptic currents at Schaffer collateral -CA1 pyramidal neuron synapses during ESE. Surface expression of the GluA1 subunit increased, and this was a consequence of N-methyl-D-aspartate receptor activation. Further, diminishing glutamate release by activation of somatostatin receptors prevented SE. These studies suggest that AMPAR-mediated glutamatergic transmission is strengthened during ESE. KEY WORDS: Receptor trafficking, AMPA receptor, Glutamate, Status epilepticus, Plasticity, CA1 pyramidal neurons.Status epilepticus (SE), characterized by continual, self-sustained seizures, is a dynamic and rapidly evolving neurologic condition. As SE progresses, electrographic seizures become continuous, grade V behavioral seizures are observed in rats, and benzodiazepines (BDZs) fail to terminate seizures (e.g., Walton & Treiman, 1988;Kapur & Macdonald, 1997). This is an animal model of established SE (ESE). Understanding synaptic plasticity during ESE will help discover newer targets to treat BDZ-refractory SE. We investigated AMPA receptor (AMPAR)-mediated neurotransmission during ESE. We previously found that the expression of the GluA2 subunit of AMPARs in hippocampal principal neurons is dynamically reduced during ESE, leading to the expression of calcium-permeable AMPARs (Rajasekaran et al., 2012). Extending these findings, herein we hypothesize that AMPAR-mediated excitatory conductance is progressively enhanced during ESE. To test this hypothesis, SE was induced in lithium-pretreated adult male rats using pilocarpine, and the animals were studied either 10 min (early ESE) or 60 min (late ESE) after the onset of the first grade V behavioral seizure using a combination of electrophysiological and biochemical studies as described previously (Rajasekaran et al., 2012;Kozhemyakin et al., 2013).AMPAR-mediated excitatory postsynaptic currents (EPSCs) were recorded from CA1 pyramidal neurons (CA1-PNs) and dentate granule cells (DGCs) by voltageclamp technique. Analysis of recordings obtained from CA1-PNs revealed that the frequency of action-potential independent EPSCs (m-EPSCs) increased with increasing seizure duration (p < 0.0001, one-way analysis of variance [ANOVA]). The mean m-EPSC frequency in CA1-PNs of the control group was 0.39 AE 0.05 Hz (n = 14 cells/6 animals), whereas in the late ESE group, it was 1.59 AE 0.4 Hz (n = 12 cells/7 animals, p < 0.05, Tukey's test). In contrast, the mean m-EPSC frequency in CA1-PNs of the early ESE group was similar to controls (0.22 AE 0.04 Hz, n = 8 cells/6 animals). The amplitude of m-EPSCs in CA1 PNs from both early ESE (11.74 AE 0.8 pA) and late ESE (11.97 AE 0.8 pA) groups were similar to that in the control...

Central cholinergic overstimulation results in prolonged seizures of status epilepticus in humans and experimental animals. Cellular mechanisms of underlying seizures caused by cholinergic stimulation remain uncertain, but enhanced glutamatergic transmission is a potential mechanism. Paraoxon, an organophosphate cholinesterase inhibitor, enhanced glutamatergic transmission on hippocampal granule cells synapses by increasing the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in a concentration-dependent fashion. The amplitude of mEPSCs was not increased, which suggested the possibility of enhanced action potential-dependent release. Analysis of EPSCs evoked by minimal stimulation revealed reduced failures and increased amplitude of evoked responses. The ratio of amplitudes of EPSCs evoked by paired stimuli was also altered. The effect of paraoxon on glutamatergic transmission was blocked by the muscarinic antagonist atropine and partially mimicked by carbachol. The nicotinic receptor antagonist α -bungarotoxin did not block the effects of paraoxon; however, nicotine enhanced glutamatergic transmission. These studies suggested that cholinergic overstimulation enhances glutamatergic transmission by enhancing neurotransmitter release from presynaptic terminals.

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