Jana Velíšková

Sex and hormonal influences on seizures and epilepsy

Epilepsy is the third most common chronic neurological disorder. Clinical and experimental evidence supports the role of sex and influence of sex hormones on seizures and epilepsy as well as alterations of the endocrine system and levels of sex hormones by epileptiform activity. Conversely, seizures are sensitive to changes in sex hormone levels, which in turn may affect the seizure-induced neuronal damage. The effects of reproductive hormones on neuronal excitability and seizure-induced damage are complex to contradictory and depend on different mechanisms, which have to be accounted for in data interpretation. Both estradiol and progesterone/allopregnanolone may have beneficial effects for patients with epilepsy. Individualized hormonal therapy should be considered as adjunctive treatment in patients with epilepsy to improve seizure control as well as quality of life.

Altered neurotransmitter release, vesicle recycling and presynaptic structure in the pilocarpine model of temporal lobe epilepsy

In searching for persistent seizure-induced alterations in brain function that might be causally related to epilepsy, presynaptic transmitter release has relatively been neglected. To measure directly the long-term effects of pilocarpine-induced status epilepticus on vesicular release and recycling in hippocampal mossy fibre presynaptic boutons, we used (i) two-photon imaging of FM1-43 vesicular release in rat hippocampal slices; and (ii) transgenic mice expressing the genetically encoded pH-sensitive fluorescent reporter synaptopHluorin preferentially at glutamatergic synapses. In this study we found that, 1-2 months after pilocarpine-induced status epilepticus, there were significant increases in mossy fibre bouton size, faster rates of action potential-driven vesicular release and endocytosis. We also analysed the ultrastructure of rat mossy fibre boutons using transmission electron microscopy. Pilocarpine-induced status epilepticus led to a significant increase in the number of release sites, active zone length, postsynaptic density area and number of vesicles in the readily releasable and recycling pools, all correlated with increased release probability. Our data show that presynaptic release machinery is persistently altered in structure and function by status epilepticus, which could contribute to the development of the chronic epileptic state and may represent a potential new target for antiepileptic therapies.

Differential antagonism of tetramethylenedisulfotetramine-induced seizures by agents acting at NMDA and GABAA receptors

Tetramethylenedisulfotetramine (TMDT) is a highly lethal neuroactive rodenticide responsible for many accidental and intentional poisonings in mainland China. Ease of synthesis, water solubility, potency, and difficulty to treat make TMDT a potential weapon for terrorist activity. We characterized TMDT-induced convulsions and mortality in male C57BL/6 mice. TMDT (ip) produced a continuum of twitches, clonic, and tonic-clonic seizures decreasing in onset latency and increasing in severity with increasing dose; 0.4mg/kg was 100% lethal. The NMDA antagonist, ketamine (35mg/kg) injected ip immediately after the first TMDT-induced seizure, did not change number of tonic-clonic seizures or lethality, but increased the number of clonic seizures. Doubling the ketamine dose decreased tonic-clonic seizures and eliminated lethality through a 60min observation period. Treating mice with another NMDA antagonist, MK-801, 0.5 or 1mg/kg ip, showed similar effects as low and high doses of ketamine, respectively, and prevented lethality, converting status epilepticus EEG activity to isolated interictal discharges. Treatment with these agents 15min prior to TMDT administration did not increase their effectiveness. Post-treatment with the GABA(A) receptor allosteric enhancer diazepam (5mg/kg) greatly reduced seizure manifestations and prevented lethality 60min post-TMDT, but ictal events were evident in EEG recordings and, hours post-treatment, mice experienced status epilepticus and died. Thus, TMDT is a highly potent and lethal convulsant for which single-dose benzodiazepine treatment is inadequate in managing electrographic seizures or lethality. Repeated benzodiazepine dosing or combined application of benzodiazepines and NMDA receptor antagonists is more likely to be effective in treating TMDT poisoning.

Prenatal Corticosteroids Modify Glutamatergic and GABAergic Synapse Genomic Fabric: Insights from a Novel Animal Model of Infantile Spasms

Prenatal exposure to corticosteroids has long‐term postnatal somatic and neurodevelopmental consequences. Animal studies indicate that corticosteroid exposure‐associated alterations in the nervous system include hypothalamic function. Infants with infantile spasms, a devastating epileptic syndrome of infancy with characteristic spastic seizures, chaotic irregular waves on interictal electroencephalogram (hypsarhythmia) and mental deterioration, have decreased concentrations of adrenocorticotrophic hormone (ACTH) and cortisol in cerebrospinal fluid, strongly suggesting hypothalamic dysfunction. We have exploited this feature to develop a model of human infantile spasms by using repeated prenatal exposure to betamethasone and a postnatal trigger of developmentally relevant spasms with NMDA. The spasms triggered in prenatally primed rats are more severe compared to prenatally saline‐injected ones and respond to ACTH, a treatment of choice for infantile spasms in humans. Using autoradiography and immunohistochemistry, we have identified a link between the spasms in our model and the hypothalamus, especially the arcuate nucleus. Transcriptomic analysis of the arcuate nucleus after prenatal priming with betamethasone but before trigger of spasms indicates that prenatal betamethasone exposure down‐regulates genes encoding several important proteins participating in glutamatergic and GABAergic transmission. Interestingly, there were significant sex‐specific alterations after prenatal betamethasone in synapse‐related gene expression but no such sex differences were found in prenatally saline‐injected controls. A pairwise relevance analysis revealed that, although the synapse gene expression in controls was independent of sex, these genes form topologically distinct gene fabrics in males and females and these fabrics are altered by betamethasone in a sex‐specific manner. These findings may explain the sex differences with respect to both normal behaviour and the occurrence and severity of infantile spasms. Changes in transcript expression and their coordination may contribute to a molecular substrate of permanent neurodevelopmental changes (including infantile spasms) found after prenatal exposure to corticosteroids.

Sex-specific behavioral traits in the Brd2 mouse model of juvenile myoclonic epilepsy

Idiopathic generalized epilepsy represents about 30–35% of all epilepsies in humans. The bromodomain BRD2 gene has been repeatedly associated with the subsyndrome of juvenile myoclonic epilepsy (JME). Our previous work determined that mice haploinsufficient in Brd2 (Brd2+/−) have increased susceptibility to provoked seizures, develop spontaneous seizures and have significantly decreased gamma‐aminobutyric acid (GABA) markers in the direct basal ganglia pathway as well as in the neocortex and superior colliculus. Here, we tested male and female Brd2+/− and wild‐type littermate mice in a battery of behavioral tests (open field, tube dominance test, elevated plus maze, Morris water maze and Barnes maze) to identify whether Brd2 haploinsufficiency is associated with the human behavioral patterns, the so‐called JME personality. Brd2+/− females but not males consistently displayed decreased anxiety. Furthermore, we found a highly significant dominance trait (aggression) in the Brd2+/− mice compared with the wild type, more pronounced in females. Brd2+/− mice of either sex did not differ from wild‐type mice in spatial learning and memory tests. Compared with wild‐type littermates, we found decreased numbers of GABA neurons in the basolateral amygdala, which is consistent with the increase in aggressive behavior. Our results indicate that Brd2+/− haploinsufficient mice show no cognitive impairment but have behavioral traits similar to those found in patients with JME (recklessness, aggression). This suggests that either the BRD2 gene is directly responsible for influencing many traits of JME or it controls upstream regulators of individual phenotypes.

Combined diazepam and MK-801 therapy provides synergistic protection from tetramethylenedisulfotetramine-induced tonic-clonic seizures and lethality in mice.

The synthetic rodenticide, tetramethylenedisulfotetramine (TMDT), is a persistent and highly lethal GABA-gated Cl(-) channel blocker. TMDT is clandestinely produced, remains popular in mainland China, and causes numerous unintentional and deliberate poisonings worldwide. TMDT is odorless, tasteless, and easy to manufacture, features that make it a potential weapon of terrorism. There is no effective treatment. We previously characterized the effects of TMDT in C57BL/6 mice and surveyed efficacies of GABAergic and glutamatergic anticonvulsant treatments. At 0.4 mg/kg i.p., TMDT produced neurotoxic symptomatology consisting of twitches, clonic and tonic-clonic seizures, often progressing to status epilepticus and death. If administered immediately after the occurrence of the first clonic seizure, the benzodiazepine diazepam (DZP) effectively prevented all subsequent seizure symptoms, whereas the NMDA receptor antagonist dizocilpine (MK-801) primarily prevented tonic-clonic seizures. The latter agent, however, appeared to be more effective at preventing delayed death. The present study further explored these phenomena, and characterized the therapeutic actions of DZP and MK-801 as combinations. Joint treatment with both DZP and MK-801 displayed synergistic protection against tonic-clonic seizures and 24 h lethality as determined by isobolographic analysis. Clonic seizures, however, remained poorly controlled. A modification of the treatment regimen, where DZP was followed 10 min later by MK-801, yielded a reduction in both types of seizures and improved overall outcome. Simultaneous monitoring of subjects via EEG and videography confirmed effectiveness of this sequential regimen. We conclude that TMDT blockage at GABAA receptors involves early activation of NMDA receptors, which contribute to persistent ictogenic activity. Our data predict that a sequential combination treatment with DZP followed by MK-801 will be superior to either individual therapy with, or simultaneous administration of, these two agents in treating TMDT poisoning.

Oestradiol Regulates Neuropeptide Y Release and Gene Coupling with the GABAergic and Glutamatergic Synapses in the Adult Female Rat Dentate Gyrus

Neuropeptide Y (NPY) is an endogenous modulator of neuronal activity affecting both GABAergic and glutamatergic transmission. Previously, we found that oestradiol modifies the number of NPY immunoreactive neurones in the hippocampal dentate gyrus. In the present study, we investigated which oestrogen receptor type is responsible for these changes in the number of NPY‐positive neurones. Furthermore, we determined the effects of oestrogen receptor activation on NPY release. Finally, we examined the contribution of oestrogen toward the remodelling of the GABAergic and glutamatergic gene networks in terms of coupling with Npy gene expression in ovariectomised rats. We found that activation of either oestrogen receptor type (ERα or ERβ) increases the number of NPY‐immunopositive neurones and enhances NPY release in the dentate gyrus. We also found that, compared to oestrogen‐lacking ovariectomised rats, oestrogen replacement increases the probability of synergistic/antagonistic coupling between the Npy and GABAergic synapse genes, whereas the glutamatergic synapse genes are less likely to be coupled with Npy under similar conditions. The data together suggest that oestrogens play a critical role in the regulation of NPY system activity and are also involved in the coupling/uncoupling of the Npy gene with the GABAergic and glutamatergic synapses in the female rat dentate gyrus.

β-Estradiol unmasks metabotropic receptor-mediated metaplasticity of NMDA receptor transmission in the female rat dentate gyrus.

Loss of estrogen in women following menopause is associated with increased risk for cognitive decline, dementia and depression, all of which can be prevented by estradiol replacement. The dentate gyrus plays an important role in cognition, learning and memory. The gatekeeping function of the dentate gyrus to filter incoming activity into the hippocampus is modulated by estradiol in a frequency-dependent manner and involves activation of metabotropic glutamate receptors (mGluR). In the present study, we investigated whether estradiol (EB) modulates the metaplastic effect of inducing synaptic long-term potentiation (LTP) on subsequent propensity for expression of LTP in the dentate gyrus. At medial perforant path-dentate granule cell synapses in hippocampal slices of ovariectomized female rats, EB replacement was critical for an initial induction of LTP to enhance the magnitude of subsequent LTP elicited by a second high-frequency stimulation, metaplasticity, which was not present in slices from oil-treated control animals. EB enhanced expression of group I mGluRs, and the metaplastic effect of EB on LTP required activation of group I mGluRs that led to Src-family tyrosine kinase-mediated phosphorylation of NR2B subunits of N-methyl-d-aspartate receptors (NMDAR) that enhanced the magnitude of NMDAR-dependent LTP. Our data show that EB effects on LTP in the hippocampal dentate gyrus require activation of group I mGluRs, which in turn leads to functional metaplastic regulation of NR2B subunit-containing NMDARs, as opposed to direct effects of EB on NMDARs.

Estradiol does not affect spasms in the betamethasone-NMDA rat model of infantile spasms

This study attempted to validate the effects of neonatal estradiol in ameliorating the spasms in the prenatally betamethasone‐primed N‐methyl‐d‐aspartate (NMDA) model of infantile spasms in rats as shown previously in a mouse Arx gene knock‐in expansion model of infantile spasms.

Gonadal status-dependent effects of in vivo β-estradiol administration to female rats on in vitro epileptiform activity induced by low [Mg2+]o in combined hippocampus-entorhinal cortex slices

There are controversial data regarding estrogen effects on neuronal excitability. We investigated whether β-estradiol (EB) administration to ovariectomized (OVX) or gonadally intact female rats alters epileptiform activity within the dentate gyrus network induced in vitro by removing [Mg2+]o in combined hippocampus-entorhinal cortex slices. In vivo EB administration significantly influenced the epileptiform activity in gonadal status-dependent manner. The onset of epileptiform discharges was modestly delayed in slices from OVX rats replaced with physiologically relevant doses of EB but the number of discharges was not affected. In contrast, EB administration to gonadally intact rats had robust effects such that: EB delayed the onset of discharges but significantly increased their number within the dentate gyrus network. Our data suggest that EB in physiologically relevant concentrations does not seem to negatively affect hippocampal neuronal excitability, nevertheless supraphysiological EB levels may enhance seizure severity.