Molecular, physiological and behavioral characterization of the heterozygous Df[h15q13]/+ mouse model associated with the human 15q13.3 microdeletion syndrome.

The human 15q13.3 microdeletion syndrome (DS) is caused by a heterozygous microdeletion (MD) affecting six genes: FAN1; MTMR10; TRPM1; KLF13; OTUD7A; and CHRNA7. Carriers are at risk for intellectual disability, epilepsy, autism spectrum disorder, and schizophrenia. Here we used the Df[h15q13]/+ mouse model with an orthologous deletion to further characterize molecular, neurophysiological, and behavioral parameters that are relevant to the 15q13.3 DS. First, we verified the expression and distribution of the α7 nicotinic acetylcholine receptor (nAChR), a gene product of the CHRNA7, in cortical and subcortical areas. Results revealed similar mRNA distribution pattern in wildtype (WT) and heterozygous (Het) mice, with about half the number of α7 nAChR binding sites in mutants. Hippocampal recordings showed similar input/output responses of field excitatory post-synaptic potentials and theta-burst induced long-term potentiation in WT and Het mice. Het males exhibited impaired spatial learning acquisition in the Barnes Maze. Indicative of increased seizure susceptibility, Het mice developed secondary seizures after 6-Hz corneal stimulation, and had significantly increased sensitivity to the chemoconvulsant pentylenetetrazol resulting in increased spiking in hippocampal EEG recordings. Basal mRNA expression of brain derived neurotrophic factor and activity regulated immediate early genes (c-fos, Arc, Erg-1 and Npas4) during adolescence, a critical period of brain maturation, was unaffected by genotype. Thus, the MD did not show gross neuroanatomical, molecular, and neurophysiological abnormalities despite deficits in spatial learning and increased susceptibility to seizures. Altogether, our results verify the phenotypic profile of the heterozygous Df[h15q13]/+ mouse model and underscore its translational relevance for human 15q13.3 DS.

Epigenetic Histone Deacetylation Inhibition Prevents the Development and Persistence of Temporal Lobe Epilepsy.

Epilepsy is a chronic brain disease characterized by repeated unprovoked seizures. Currently, no drug therapy exists for curing epilepsy or disease modification in people at risk. Despite several emerging mechanisms, there have been few studies of epigenetic signaling in epileptogenesis, the process whereby a normal brain becomes progressively epileptic because of precipitating factors. Here, we report a novel role of histone deacetylation as a critical epigenetic mechanism in epileptogenesis. Experiments were conducted using the histone deacetylase (HDAC) inhibitor sodium butyrate in the hippocampus kindling model of temporal lobe epilepsy (TLE), a classic model heavily used to approve drugs for treatment of epilepsy. Daily treatment with butyrate significantly inhibited HDAC activity and retarded the development of limbic epileptogenesis without affecting after-discharge signal. HDAC inhibition markedly impaired the persistence of seizure expression many weeks after epilepsy development. Moreover, subchronic HDAC inhibition for 2 weeks resulted in a striking retardation of epileptogenesis. HDAC inhibition, unexpectedly, also showed erasure of the epileptogenic state in epileptic animals. Finally, butyrate-treated animals exhibited a powerful reduction in mossy fiber sprouting, a morphologic index of epileptogenesis. Together these results underscore that HDAC inhibition prevents the development of TLE, indicating HDAC's critical signaling role in epileptogenesis. These findings, therefore, envisage a unique novel therapy for preventing or curing epilepsy by targeting the epigenetic HDAC pathway.

Novel therapeutic approaches for disease-modification of epileptogenesis for curing epilepsy.

This article describes the recent advances in epileptogenesis and novel therapeutic approaches for the prevention of epilepsy, with a special emphasis on the pharmacological basis of disease-modification of epileptogenesis for curing epilepsy. Here we assess animal studies and human clinical trials of epilepsy spanning 1982-2016. Epilepsy arises from a number of neuronal factors that trigger epileptogenesis, which is the process by which a brain shifts from a normal physiologic state to an epileptic condition. The events precipitating these changes can be of diverse origin, including traumatic brain injury, cerebrovascular damage, infections, chemical neurotoxicity, and emergency seizure conditions such as status epilepticus. Expectedly, the molecular and system mechanisms responsible for epileptogenesis are not well defined or understood. To date, there is no approved therapy for the prevention of epilepsy. Epigenetic dysregulation, neuroinflammation, and neurodegeneration appear to trigger epileptogenesis. Targeted drugs are being identified that can truly prevent the development of epilepsy in at-risk people. The promising agents include rapamycin, COX-2 inhibitors, TRK inhibitors, epigenetic modulators, JAK-STAT inhibitors, and neurosteroids. Recent evidence suggests that neurosteroids may play a role in modulating epileptogenesis. A number of promising drugs are under investigation for the prevention or modification of epileptogenesis to halt the development of epilepsy. Some drugs in development appear rational for preventing epilepsy because they target the initial trigger or related signaling pathways as the brain becomes progressively more prone to seizures. Additional research into the target validity and clinical investigation is essential to make new frontiers in curing epilepsy.

Catamenial-like seizure exacerbation in mice with targeted ablation of extrasynaptic δGABA-a receptors in the brain.

Neurosteroids play a key role in catamenial epilepsy, a menstrual cycle-related seizure clustering in women with epilepsy. While neurosteroids act on all GABA-A receptor isoforms, they cause greater effects on extrasynaptic δGABA-A receptors that mediate tonic inhibition in the brain. Previously, we identified a potential GABA-A receptor mechanism for catamenial epilepsy. However, the precise functional role of extrasynaptic δGABA-A receptors in the pathophysiology of catamenial epilepsy remains unclear. In this study, we utilized mice lacking extrasynaptic δGABA-A receptors (δKO) to investigate whether reduction of tonic inhibition affects catamenial seizure susceptibility or intensity. Intact female wildtype (WT) and δKO mice were subjected to hippocampus kindling until they exhibited stage 5 seizures. Elevated gonadal hormone-based neurosteroid levels were induced by standard gonadotropin regimen and neurosteroid withdrawal (NSW) was triggered by finasteride. NSW increased susceptibility to, as well the intensity of evoked catamenial-like seizures in WT and δKO mice. However, fully kindled δKO mice exhibited an accelerated and augmented response to NSW, with a more rapid increase in seizure susceptibility and intensity than WT mice undergoing the NSW paradigm. Moreover, δKO mice in NSW showed reduced benzodiazepine sensitivity, but in stark contrast to the increased neurosteroid sensitivity observed in WT animals, δKO mice displayed no change in neurosteroid sensitivity in response to NSW. The increased catamenial seizure exacerbation and alterations in antiseizure drug responses are consistent with NSW-induced changes in the abundance of δGABA-A receptors. Collectively, these findings provide evidence of a potential protective role for extrasynaptic δGABA-A receptors in catamenial-like seizures. © 2017 Wiley Periodicals, Inc.

Antiseizure Activity of Midazolam in Mice Lacking δ-Subunit Extrasynaptic GABA(A) Receptors.

Midazolam is a benzodiazepine anticonvulsant with rapid onset and short duration of action. Midazolam is the current drug of choice for acute seizures and status epilepticus, including those caused by organophosphate nerve agents. The antiseizure activity of midazolam is thought to result from its allosteric potentiation of synaptic GABA(A) receptors in the brain. However, there are indications that benzodiazepines promote neurosteroid synthesis via the 18-kDa cholesterol transporter protein (TSPO). Therefore, we investigated the role of neurosteroids and their extrasynaptic GABA(A) receptor targets in the antiseizure activity of midazolam. Here, we used δ-subunit knockout (DKO) mice bearing a targeted deletion of the extrasynaptic receptors to investigate the contribution of the extrasynaptic receptors to the antiseizure activity of midazolam using the 6-Hz and hippocampus kindling seizure models. In both models, midazolam produced rapid and dose-dependent protection against seizures (ED50, 0.4 mg/kg). Moreover, the antiseizure potency of midazolam was undiminished in DKO mice compared with control mice. Pretreatment with PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide], a TSPO blocker, or finasteride, a 5α-reductase neurosteroid inhibitor, did not affect the antiseizure effect of midazolam. The antiseizure activity of midazolam was significantly reversed by pretreatment with flumazenil, a benzodiazepine antagonist. Plasma and brain levels of the neurosteroid allopregnanolone were not significantly greater in midazolam-treated animals. These studies therefore provide strong evidence that neurosteroids and extrasynaptic GABA(A) receptors are not involved in the antiseizure activity of midazolam, which mainly occurs through synaptic GABA(A) receptors via direct binding to benzodiazepine sites. This study reaffirms midazolam's use for controlling acute seizures and status epilepticus.

Female-biased anorexia and anxiety in the Syrian hamster.

Anorexia and anxiety cause significant mortality and disability with female biases and frequent comorbidity after puberty, but the scarcity of suitable animal models impedes understanding of their biological underpinnings. It is reported here that in adult or weanling Syrian hamsters, relative to social housing (SH), social separation (SS) induced anorexia characterized as hypophagia, weight loss, reduced adiposity, and hypermetabolism. Following anorexia, SS increased reluctance to feed, and thigmotaxis, in anxiogenic environments. Importantly, anorexia and anxiety were induced post-puberty with female biases. SS also reduced hypothalamic corticotrophin-releasing factor mRNA and serum corticosteroid levels assessed by RT-PCR and RIA, respectively. Consistent with the view that sex differences in adrenal suppression contributed to female biases in anorexia and anxiety by disinhibiting neuroimmune activity, SS elevated hypothalamic interleukin-6 and toll-like receptor 4 mRNA levels. Although corticosteroids were highest during SH, they were within the physiological range and associated with juvenile-like growth of white adipose, bone, and skeletal muscle. These results suggest that hamsters exhibit plasticity in bioenergetic and emotional phenotypes across puberty without an increase in stress responsiveness. Thus, social separation of hamsters provides a model of sex differences in anorexia and anxiety during adulthood and their pathogeneses during adolescence.