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Galanin receptors type 1 (GalR1) and/or type 2 (GalR2) represent unique pharmacological targets for treatment of seizures and epilepsy. Previous studies have shown that the endogenous peptide ligand galanin exerts powerful anticonvulsant effect through activation of these two G protein-coupled receptors, which are highly expressed in the temporal lobe of rodent brain. Here we report the characterization of a putative GalR2-positive allosteric modulator CYM2503. CYM2503 potentiated the galanin-stimulated IP1 accumulation in HEK293 cells stably expressing GalR2 receptor, whereas it exhibited no detectable affinity for the [sup.125]I galanin--binding site of GalR2 receptor, an effect consistent with that of a positive allosteric modulator. In the rat Li-pilocarpine status epilepticus model, CYM2503, injected intraperitoneally, increased the latency to first electrographic seizure and the latency to first stage 3 behavioral seizure, decreased the latency to the establishment of status epilepticus, and dramatically decreased the mortality. In a Li-pilocarpine seizure model in mice, CYM2503 increased the latency to first electrographic seizure and decreased the total time in seizure. CYM2503 also attenuated electroshock-induced seizures in mice. Thus, CYM2503 provides a starting point for the development of anticonvulsant therapy using the galanin R2 receptor as target. galanin | seizure | G protein-coupled receptor | status epilepticus doi/ 10.1073/pnas.1008986107

The optogenetic approach to gain control over neuronal excitability both in vitro and in vivo has emerged as a fascinating scientific tool to explore neuronal networks, but it also opens possibilities for developing novel treatment strategies for neurologic conditions. We have explored whether such an optogenetic approach using the light-driven halorhodopsin chloride pump from Natronomonas pharaonis (NpHR), modified for mammalian CNS expression to hyperpolarize central neurons, may inhibit excessive hyperexcitability and epileptiform activity. We show that a lentiviral vector containing the NpHR gene under the calcium/calmodulin-dependent protein kinase II[alpha] promoter transduces principal cells of the hippocampus and cortex and hyperpolarizes these cells, preventing generation of action potentials and epileptiform activity during optical stimulation. This study proves a principle, that selective hyperpolarization of principal cortical neurons by NpHR is sufficient to curtail paroxysmal activity in transduced neurons and can inhibit stimulation train-induced bursting in hippocampal organotypic slice cultures, which represents a model tissue of pharmacoresistant epilepsy. This study demonstrates that the optogenetic approach may prove useful for controlling epileptiform activity and opens a future perspective to develop it into a strategy to treat epilepsy. epilepsy | hippocampus | NpHR | paroxysmal depolarizing shift (PDS) | stimulation train-induced bursting (STIB)

Members of the SLC4 bicarbonate transporter family are involved in solute transport and pH homeostasis. Here we report that disrupting the Sic4a10 gene, which encodes the [Na.sup.+]-coupled [Cl.sup.-]-HC[O.sub.3]- exchanger Slc4al0 (NCBE), drastically reduces brain ventricle volume and protects against fatal epileptic seizures in mice. In choroid plexus epithelial cells, Slc4al0 localizes to the basolateral membrane. These cells displayed a diminished recovery from an acid load in KO mice. Slc4al0 also was expressed in neurons. Within the hippocampus, the Slc4al0 protein was abundant in CA3 pyramidal cells. In the CA3 area, propionate-induced intracellular acidification and attenuation of 4-aminopyridine-induced network activity were prolonged in KO mice. Our data indicate that Slc4a10 is involved in the control of neuronal pH and excitability and may contribute to the secretion of cerebrospinal fluid. Hence, Slc4a10 is a promising pharmacological target for the therapy of epilepsy or elevated intracranial pressure. cerebrospinal fluid | epilepsy | ion transport | knockout mouse | pH regulation

Gap junctions have been postulated to exist between the axons of excitatory cortical neurons based on electrophysiological, modeling, and dye-coupling data. Here, we provide ultrastructural evidence for axoaxonic gap junctions in dentate granule cells. Using combined confocal laser scanning microscopy, thin-section transmission electron microscopy, and grid-mapped freeze-fracture replica immunogold labeling, 10 close appositions revealing axoaxonic gap junctions ([approximately equal to] 30-70 nm in diameter) were found between pairs of mossy fiber axons ([approximately equal to] 100-200 nm in diameter) in the stratum lucidum of the CA3b field of the rat ventral hippocampus, and one axonal gap junction ([approximately equal to] 100 connexons) was found on a mossy fiber axon in the CA3c field of the rat dorsal hippocampus. Immunogold labeling with two sizes of gold beads revealed that connexin36 was present in that axonal gap junction. These ultrastructural data support computer modeling and in vitro electrophysiological data suggesting that axoaxonic gap junctions play an important role in the generation of very fast (>70 Hz) network oscillations and in the hypersynchronous electrical activity of epilepsy. axoaxonic | connexin | electrical synapse | epilepsy | synchronization

Differential composition of GABAA receptor (GABAAR) subunits underlies the variability of fast inhibitory synaptic transmission; alteration of specific GABAAR subunits in localized brain regions may contribute to abnormal brain states such as absence epilepsy. We combined immunocytochemistry and high-resolution ImmunoGold electron microscopy to study cellular and subcellular localization of [GABA.sub.A]R [alpha]l, [alpha]3, and [beta]2/[beta]3 subunits in ventral posterior nucleus (VP) and reticular nucleus (RTN) of control rats and WAG/Rij rats, a genetic model of absence epilepsy. In control rats, [alpha]1 subunits were prominent at inhibitory synapses in VP and much less prominent in RTN; in contrast, the [alpha]3 subunit was highly evident at inhibitory synapses in RTN. [beta]2/[beta]3 subunits were evenly distributed at inhibitory synapses in both VP and RTN. ImmunoGold particles representing all subunits were concentrated at postsynaptic densities with no extrasynaptic localization. Calculated mean number of particles for al subunit per postsynaptic density in nonepileptic VP was 6.1 [+ or -] 3.7, for a3 subunit in RTN it was 6.6 [+ or -] 3.4, and for [beta]2/[beta]3 subunits in VP and RTN the mean numbers were 3.7 [+ or -] 1.3 and 3.5 [+ or -] 1.2, respectively. In WAG/Rij rats, there was a specific loss of [alpha]3 subunit immunoreactivity at inhibitory synapses in RTN, without reduction in [alpha]3 subunit mRNA or significant change in immunostaining for other markers of RTN cell identity such as GABA or parvalbumin, [alpha]3 immunostaining in cortex was unchanged. Subtle, localized changes in GABAAR expression acting at highly specific points in the interconnected thalamocortical network lie at the heart of idiopathic generalized epilepsy. absence epilepsy | inhibition | quantitative electron microscopy

Our objective was to evaluate the brain regions showing increased and decreased metabolism in patients at the time of generalized bursts of epileptic discharges in order to understand their mechanism of generation and effect on brain function. By recording the electroencephalogram during the functional MRI, changes in the blood oxygenation level-dependent signal were obtained in response to epileptic discharges observed in the electroencephalogram of 15 patients with idiopathic generalized epilepsy. A group analysis was performed to determine the regions of positive (activation) and negative (deactivation) blood oxygenation level-dependent responses that were common to the patients. Activations were found bilaterally and symmetrically in the thalamus, mesial midfrontal region, insulae, and midline and bilateral cerebellum and on the borders of the lateral ventricles. Deactivations were bilateral and symmetrical in the anterior frontal and parietal regions and in the posterior cingulate gyri and were seen in the left posterior temporal region. Activations in thalamus and midfrontal regions confirm known involvement of these regions in the generation or spread of generalized epileptic discharges. Involvement of the insulae in generalized discharges had not previously been described. Cerebellar activation is not believed to reflect the generation of discharges. Deactivations in frontal and parietal regions remarkably followed the pattern of the default state of brain function. Thalamocortical activation and suspension of the default state may combine to cause the actual state of reduced responsiveness observed in patients during spike-and-wave discharges. This brief lapse of responsiveness may therefore not result only from the epileptic discharge but also from its effect on normal brain function. absence | epilepsy | thalamus

Multifunctional Ca2+/ calmodulin-dependant protein kinase II (CaMK) appears to play a large role in the control of neuron excitability in mammalian nervous systems, with a possible role in epilepsy. Studies on the affects of CaMK alpha-subunit null mutations in transgenic mice found that such mutations led to high excitability and and epileptic seizures. Similar mutation of the gamma-subunit had no affects on excitability.

High-resolution recordings and fluctuation analysis of miniature inhibitory postsynaptic currents reveal that an increase in functional receptor number in kindled granule cells supports increased GABA(A) receptor-mediated inhibition. This lasts for at least 48 hours after induction. The increased number of receptors does not affect single-channel conductance or kinetics. Increasing the number of functional receptors is an effective way to increase inhibition in mammalian brain.

Adenosine is an inhibitor of neuronal activity in the brain. The local release of adenosine from grafted cells was evaluated as an ex vivo gene therapy approach to suppress synchronous discharges and epileptic seizures. Fibroblasts were engineered to release adenosine by inactivating the adenosine-metabolizing enzymes adenosine kinase and adenosine deaminase. After encapsulation into semipermeable polymers, the cells were grafted into the brain ventricles of electrically kindled rats, a model of partial epilepsy. Grafted rats provided a nearly complete protection from behavioral seizures and a near-complete suppression of afterdischarges in electroencephalogram recordings, whereas the full tonic-clonic convulsions in control rats remained unaltered. Thus, the local release of adenosine resulting in adenosine concentrations [is less than] 25 nM at the site of action is sufficient to suppress seizure activity and, therefore, provides a potential therapeutic principle for the treatment of drug-resistant partial epilepsies.

The regulated expression of type A [Gamma]-aminobutyric acid receptor ([GABA.sub.A]R) subunit genes is postulated to play a role in neuronal maturation, synaptogenesis, and predisposition to neurological disease. Increases in GABA levels and changes in [GABA.sub.A]R subunit gene expression, including decreased [Beta]1 mRNA levels, have been observed in animal models of epilepsy. Persistent exposure to GABA down-regulates [GABA.sub.A]R number in primary cultures of neocortical neurons, but the regulatory mechanisms remain unknown. Here, we report the identification of a TATA-less minimal promoter of 296 bp for the human [GABA.sub.A]R [Beta]1 subunit gene that is neuron specific and autologously down-regulated by GABA. [Beta]1 promoter activity, mRNA levels, and subunit protein are decreased by persistent [GABA.sub.A]R activation. The core promoter, 270 bp, contains an initiator element (Inr) at the major transcriptional start site. Three concatenated copies of the 10-bp Inr and its immediate 3' flanking sequence produce full neural specific activity that is down-regulated by GABA in transiently transfected neocortical neurons. Taking these results together with those of DNase I footprinting, electrophoretic mobility shift analysis, and 2-bp mutagenesis, we conclude that GABA-induced down-regulation of [Beta]1 subunit mRNAs involves the differential binding of a sequence-specific basal transcription factor(s) to the Inr. The results support a transcriptional mechanism for the down-regulation of [Beta]1 subunit [GABA.sub.A]R gene expression and raises the possibility that altered levels of sequence-specific basal transcription factors may contribute to neurological disorders such as epilepsy. transcription | neural specific | epilepsy

Several models that develop epileptiform discharges and epilepsy have been associated with a decrease in the activity of calmodulin-dependent kinase II. However, none of these studies has demonstrated a causal relationship between a decrease in calcium/calmodulin kinase II activity and the development of seizure activity. The present study was conducted to determine the effect of directly reducing calcium/calmodulin-dependent kinase activity on the development of epileptiform discharges in hippocampal neurons in culture. Complimentary oligonucleotides specific for the subunit of the calcium/calmodulin kinase were used to decrease the expression of the enzyme. Reduction in kinase expression was confirmed by Western analysis, immunocytochemistry, and exogenous substrate phosphorylation. Increased neuronal excitability and frank epileptiform discharges were observed after a significant reduction in calmodulin kinase II expression. The epileptiform activity was a synchronous event and was not caused by random neuronal firing. Furthermore, the magnitude of decreased kinase expression correlated with the increased neuronal excitability. The data suggest that decreased calmodulin kinase II activity may play a role in epileptogenesis and the long-term plasticity changes associated with the development of pathological seizure activity and epilepsy.

Kainic acid (KA)-induced status epilepticus in adult rats leads to delayed, selective death of pyramidal neurons in the hippocampal CA1 and CA3. Death is preceded by down-regulation of glutamate receptor 2 (GluR2) mRNA and protein [the subunit that limits [Ca.sup.2+] permeability of [Alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors] in CA1 and CA3, as indicated by in situ hybridization, immunolabeling, and quantitative Western blotting. GluR1 mRNA and protein are unchanged or slightly increased before cell death. These changes could lead to formation of GluR2-lacking, [Ca.sup.2+]-permeable AMPA receptors and increased toxicity of endogenous glutamate. GluR2 immunolabeling is unchanged in granule cells of the dentate gyrus, which are resistant to seizure-induced death. Thus, formation of [Ca.sup.2+]-permeable AMPA receptors may be a critical mediator of delayed neurodegeneration after status epilepticus. epilepsy | seizures | [Alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors | neurodegeneration | kainate

Epileptic seizures are associated with increases in hippocampal excitability, but the mechanisms that render the hippocampus hyperexcitable chronically (in epilepsy) or acutely (in status epilepticus) are poorly understood. Recent evidence suggests that substance P (SP), a peptide that has been implicated in cardiovascular function, inflammatory responses, and nociception, also contributes to hippocampal excitability and status epilepticus, in part by enhancing glutamate release. Here we report that mice with disruption of the preprotachykinin A gene, which encodes SP and neurokinin A, are resistant to kainate excitoxicity. The mice show a reduction in the duration and severity of seizures induced by kainate or pentylenetetrazole, and both necrosis and apoptosis of hippocampal neurons are prevented. Although kainate induced the expression of bax and caspase 3 in the hippocampus of wild-type mice, these critical intracellular mediators of cell death pathways were not altered by kainate injection in the mutant mice. These results indicate that the reduction of seizure activity and the neuroprotection observed in preprotachykinin A null mice are caused by the extinction of a SP/neurokinin A-mediated signaling pathway that is activated by seizures. They suggest that these neurokinins are critical to the control of hippocampal excitability, hippocampal seizures, and hippocampal vulnerability.