Overexpression of KCNN4 channels in principal neurons produces an anti-seizure effect without reducing their coding ability.

Gene therapy offers a potential alternative to the surgical treatment of epilepsy, which affects millions of people and is pharmacoresistant in ~30% of cases. Aimed at reducing the excitability of principal neurons, the engineered expression of K+ channels has been proposed as a treatment due to the outstanding ability of K+ channels to hyperpolarize neurons. However, the effects of K+ channel overexpression on cell physiology remain to be investigated. Here we report an adeno-associated virus (AAV) vector designed to reduce epileptiform activity specifically in excitatory pyramidal neurons by expressing the human Ca2+-gated K+ channel KCNN4 (KCa3.1). Electrophysiological and pharmacological experiments in acute brain slices showed that KCNN4-transduced cells exhibited a Ca2+-dependent slow afterhyperpolarization that significantly decreased the ability of KCNN4-positive neurons to generate high-frequency spike trains without affecting their lower-frequency coding ability and action potential shapes. Antiepileptic activity tests showed potent suppression of pharmacologically induced seizures in vitro at both single cell and local field potential levels with decreased spiking during ictal discharges. Taken together, our findings strongly suggest that the AAV-based expression of the KCNN4 channel in excitatory neurons is a promising therapeutic intervention as gene therapy for epilepsy.

Antiepileptogenic Effects of Anakinra, Lamotrigine and Their Combination in a Lithium-Pilocarpine Model of Temporal Lobe Epilepsy in Rats.

Temporal lobe epilepsy is a common, chronic disorder with spontaneous seizures that is often refractory to drug therapy. A potential cause of temporal lobe epilepsy is primary brain injury, making prevention of epileptogenesis after the initial event an optimal method of treatment. Despite this, no preventive therapy for epilepsy is currently available. The purpose of this study was to evaluate the effects of anakinra, lamotrigine, and their combination on epileptogenesis using the rat lithium-pilocarpine model of temporal lobe epilepsy. The study showed that there was no significant difference in the number and duration of seizures between treated and untreated animals. However, the severity of seizures was significantly reduced after treatment. Anakinra and lamotrigine, alone or in combination, significantly reduced neuronal loss in the CA1 hippocampus compared to the control group. However, the drugs administered alone were found to be more effective in preventing neuron loss in the hippocampal CA3 field compared to combination treatment. The treatment alleviated the impairments in activity level, exploratory behavior, and anxiety but had a relatively weak effect on TLE-induced impairments in social behavior and memory. The efficacy of the combination treatment did not differ from that of anakinra and lamotrigine monotherapy. These findings suggest that anakinra and lamotrigine, either alone or in combination, may be clinically useful in preventing the development of histopathological and behavioral abnormalities associated with epilepsy.

Alterations in mRNA and Protein Expression of Glutamate Receptor Subunits Following Pentylenetetrazole-induced Acute Seizures in Young Rats.

Acute seizures can severely affect brain function and development. However, the underlying pathophysiological mechanisms are still poorly understood. Disturbances of the glutamatergic system are considered one of the critical mechanisms of neurological abnormalities. In the present study, we analyzed changes in the expression of NMDA and AMPA receptor subunits in the different brain regions (dorsal hippocampus, amygdala, and the medial prefrontal, temporal, and entorhinal cortex) using a pentylenetetrazole (PTZ) model of seizures in 3-week-old rats. A distinctive feature of this model is that the administration of PTZ causes severe acute seizures, which are not followed by the development of spontaneous recurrent seizures later on. Subunit expression was analyzed using qRT-PCR and Western blotting during the first week after seizures. The most pronounced alterations of mRNA and protein levels were observed in the dorsal hippocampus. We found decreased expression of the GluA2 mRNA 7 days after seizures (PSE7), as well as reduced GluN2a protein levels on PSE7. Significant alterations in the expression of different receptor subunits in the mRNA but not protein levels were observed in the entorhinal cortex and amygdala. In contrast, in the medial prefrontal and temporal cortex, we found almost no changes in the expression of the studied genes. The identified changes deepen our understanding of post-seizure disturbances in the developing brain and confirm that although various brain structures are involved in seizures, the hippocampus is the most vulnerable.

Reference Gene Validation in the Brain Regions of Young Rats after Pentylenetetrazole-Induced Seizures.

Reverse transcription followed by quantitative polymerase chain reaction (qRT-PCR) is a powerful and commonly used tool for gene expression analysis. It requires the right choice of stably expressed reference genes for accurate normalization. In this work, we aimed to select the optimal reference genes for qRT-PCR normalization within different brain areas during the first week following pentylenetetrazole-induced seizures in immature (P20-22) Wistar rats. We have tested the expression stability of a panel of nine housekeeping genes: Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1, and Ywhaz. Based on geometric averaging of ranks obtained by four common algorithms (geNorm, NormFinder, BestKeeper, Comparative Delta-Ct), we found that the stability of tested reference genes varied significantly between different brain regions. The expression of the tested panel of genes was very stable within the medial prefrontal and temporal cortex, and the dorsal hippocampus. However, within the ventral hippocampus, the entorhinal cortex and amygdala expression levels of most of the tested genes were not steady. The data revealed that in the pentylenetetrazole-induced seizure model in juvenile rats, Pgk1, Ppia, and B2m expression are the most stable within the medial prefrontal cortex; Ppia, Rpl13a, and Sdha within the temporal cortex; Pgk1, Ppia, and Rpl13a within the entorhinal cortex; Gapdh, Ppia, and Pgk1 within the dorsal hippocampus; Rpl13a, Sdha, and Ppia within the ventral hippocampus; and Sdha, Pgk1, and Ppia within the amygdala. Our data indicate the need for a differential selection of reference genes across brain regions, including the dorsal and ventral hippocampus.

Astrocytic Atrophy Following Status Epilepticus Parallels Reduced Ca2+ Activity and Impaired Synaptic Plasticity in the Rat Hippocampus.

Epilepsy is a group of neurological disorders commonly associated with the neuronal malfunction leading to generation of seizures. Recent reports point to a possible contribution of astrocytes into this pathology. We used the lithium-pilocarpine model of status epilepticus (SE) in rats to monitor changes in astrocytes. Experiments were performed in acute hippocampal slices 2-4 weeks after SE induction. Nissl staining revealed significant neurodegeneration in the pyramidal cell layers of hippocampal CA1, CA3 areas, and the hilus, but not in the granular cell layer of the dentate gyrus. A significant increase in the density of astrocytes stained with an astrocyte-specific marker, sulforhodamine 101, was observed in CA1 stratum (str.) radiatum. Astrocytes in this area were also whole-cell loaded with a morphological tracer, Alexa Fluor 594, for two-photon excitation imaging. Sholl analyses showed no changes in the size of the astrocytic domain or in the number of primary astrocytic branches, but a significant reduction in the number of distal branches that are resolved with diffraction-limited light microscopy (and are thought to contain Ca2+ stores, such as mitochondria and endoplasmic reticulum). The atrophy of astrocytic branches correlated with the reduced size, but not overall frequency of Ca2+ events. The volume tissue fraction of nanoscopic (beyond the diffraction limit) astrocytic leaflets showed no difference between control and SE animals. The results of spatial entropy-complexity spectrum analysis were also consistent with changes in ratio of astrocytic branches vs. leaflets. In addition, we observed uncoupling of astrocytes through the gap-junctions, which was suggested as a mechanism for reduced K+ buffering. However, no significant difference in time-course of synaptically induced K+ currents in patch-clamped astrocytes argued against possible alterations in K+ clearance by astrocytes. The magnitude of long-term-potentiation (LTP) was reduced after SE. Exogenous D-serine, a co-agonist of NMDA receptors, has rescued the initial phase of LTP. This suggests that the reduced Ca2+-dependent release of D-serine by astrocytes impairs initiation of synaptic plasticity. However, it does not explain the failure of LTP maintenance which may be responsible for cognitive decline associated with epilepsy.