Sex Differences in the Epilepsies and Associated Comorbidities: Implications for Use and Development of Pharmacotherapies.

The epilepsies are common neurologic disorders characterized by spontaneous recurrent seizures. Boys, girls, men, and women of all ages are affected by epilepsy and, in many cases, by associated comorbidities as well. The primary courses of treatment are pharmacological, dietary, and/or surgical, depending on several factors, including the areas of the brain affected and the severity of the epilepsy. There is a growing appreciation that sex differences in underlying brain function and in the neurobiology of epilepsy are important factors that should be accounted for in the design and development of new therapies. In this review, we discuss the current knowledge on sex differences in epilepsy and associated comorbidities, with emphasis on those aspects most informative for the development of new pharmacotherapies. Particular focus is placed on sex differences in the prevalence and presentation of various focal and generalized epilepsies; psychiatric, cognitive, and physiologic comorbidities; catamenial epilepsy in women; sex differences in brain development; the neural actions of sex and stress hormones and their metabolites; and cellular mechanisms, including brain-derived neurotrophic factor signaling and neuronal-glial interactions. Further attention placed on potential sex differences in epilepsies, comorbidities, and drug effects will enhance therapeutic options and efficacy for all patients with epilepsy. SIGNIFICANCE STATEMENT: Epilepsy is a common neurological disorder that often presents together with various comorbidities. The features of epilepsy and seizure activity as well as comorbid afflictions can vary between men and women. In this review, we discuss sex differences in types of epilepsies, associated comorbidities, pathophysiological mechanisms, and antiepileptic drug efficacy in both clinical patient populations and preclinical animal models.

Changes in Both Neuron Intrinsic Properties and Neurotransmission Are Needed to Drive the Increase in GnRH Neuron Firing Rate during Estradiol-Positive Feedback.

Central output of gonadotropin-releasing hormone (GnRH) neurons controls fertility and is sculpted by sex-steroid feedback. A switch of estradiol action from negative to positive feedback initiates a surge of GnRH release, culminating in ovulation. In ovariectomized mice bearing constant-release estradiol implants (OVX+E), GnRH neuron firing is suppressed in the morning (AM) by negative feedback and activated in the afternoon (PM) by positive feedback; no time-of-day-dependent changes occur in OVX mice. In this daily surge model, GnRH neuron intrinsic properties are shifted to favor increased firing during positive feedback. It is unclear whether this shift and the observed concomitant increase in GABAergic transmission, which typically excites GnRH neurons, are independently sufficient for increasing GnRH neuron firing rate during positive feedback or whether both are needed. To test this, we used dynamic clamp to inject selected previously recorded trains of GABAergic postsynaptic conductances (PSgs) collected during the different feedback states of the daily surge model into GnRH neurons from OVX, OVX+E AM, and OVX+E PM mice. PSg trains mimicking positive feedback initiated more action potentials in cells from OVX+E PM mice than negative feedback or OVX (open feedback loop) trains in all three animal models, but the positive-feedback train was most effective when applied to cells during positive feedback. In silico studies of model GnRH neurons in which >1000 PSg trains were tested exhibited the same results. These observations support the hypothesis that GnRH neurons integrate fast-synaptic and intrinsic changes to increase firing rates during positive feedback.SIGNIFICANCE STATEMENT Infertility affects 15%-20% of couples; failure to ovulate is a common cause. Understanding how the brain controls ovulation is critical for new developments in both infertility treatment and contraception. Ovarian estradiol alters both the intrinsic properties of gonadotropin-releasing hormone (GnRH) neurons and synaptic inputs to these cells coincident with production of sustained GnRH release that ultimately triggers ovulation. We demonstrate here using dynamic clamp and mathematical modeling that estradiol-induced shifts in synaptic transmission alone can increase firing output, but that the intrinsic properties of GnRH neurons during positive feedback further poise these cells for increased response to higher frequency synaptic transmission. These data suggest that GnRH neurons integrate fast-synaptic and intrinsic changes to increase firing rates during the preovulatory GnRH surge.

Nucleus-specific modulation of phasic and tonic inhibition by endogenous neurosteroidogenesis in the murine thalamus.

Neurosteroids are potent allosteric modulators of GABAA receptors (GABAA Rs). Although the effects of exogenous neurosteroids on GABAA R function are well documented, less is known about effects of neurosteroids produced by local endogenous biosynthesis. The neurosteroidogenic enzymes 5α-reductase and 3α-hydroxysteroid dehydrogenase are expressed in two nuclei of somatosensory thalamus, the thalamic reticular nucleus (nRT) and ventrobasal nucleus (VB). Here, the effects of acute blockade of neurosteroidogenesis by the 5α-reductase inhibitor finasteride on phasic and tonic GABAA R-mediated currents were examined in nRT and VB of mice. In nRT, finasteride altered the decay and amplitude, but not the frequency, of phasic currents, with no effect on tonic inhibition. In VB neurons, by contrast, finasteride reduced both the size and frequency of phasic currents, and also reduced the degree of tonic inhibition. These studies thus provide novel evidence for endogenous modulation of GABAA R function by 5α-reduced neurosteroids in the mature thalamus.

Differential impacts on multiple forms of spatial and contextual memory in diazepam binding inhibitor knockout mice.

Learning and memory are fundamental processes that are disrupted in many neurological disorders including Alzheimer's disease and epilepsy. The hippocampus plays an integral role in these functions, and modulation of synaptic transmission mediated by γ-aminobutyric acid (GABA) type-A receptors (GABAA Rs) impacts hippocampus-dependent learning and memory. The protein diazepam binding inhibitor (DBI) differentially modulates GABAA Rs in various brain regions, including hippocampus, and changes in DBI levels may be linked to altered learning and memory. The effects of genetic loss of DBI signaling on these processes, however, have not been determined. In these studies, we examined male and female constitutive DBI knockout mice and wild-type littermates to investigate the role of DBI signaling in modulating multiple forms of hippocampus-dependent spatial learning and memory. DBI knockout mice did not show impaired discrimination of objects in familiar and novel locations in an object location memory test, but did exhibit reduced time spent exploring the objects. Multiple parameters of Barnes maze performance, testing the capability to utilize spatial reference cues, were disrupted in DBI knockout mice. Furthermore, whereas most wild-type mice adopted a direct search strategy upon learning the location of the target hole, knockout mice showed higher rates of using an inefficient random strategy. In addition, DBI knockout mice displayed typical levels of contextual fear conditioning, but lacked a sex difference observed in wild-type mice. Together, these data suggest that DBI selectively influences certain forms of spatial learning and memory, indicating novel roles for DBI signaling in modulating hippocampus-dependent behavior in a task-specific manner.

Astrocytes potentiate GABAergic transmission in the thalamic reticular nucleus via endozepine signaling.

Emerging evidence indicates that diazepam-binding inhibitor (DBI) mediates an endogenous benzodiazepine-mimicking (endozepine) effect on synaptic inhibition in the thalamic reticular nucleus (nRT). Here we demonstrate that DBI peptide colocalizes with both astrocytic and neuronal markers in mouse nRT, and investigate the role of astrocytic function in endozepine modulation in this nucleus by testing the effects of the gliotoxin fluorocitrate (FC) on synaptic inhibition and endozepine signaling in the nRT using patch-clamp recordings. FC treatment reduced the effective inhibitory charge of GABAA receptor (GABAAR)-mediated spontaneous inhibitory postsynaptic currents in WT mice, indicating that astrocytes enhance GABAAR responses in the nRT. This effect was abolished by both a point mutation that inhibits classical benzodiazepine binding to GABAARs containing the α3 subunit (predominant in the nRT) and a chromosomal deletion that removes the Dbi gene. Thus, astrocytes are required for positive allosteric modulation via the α3 subunit benzodiazepine-binding site by DBI peptide family endozepines. Outside-out sniffer patches pulled from neurons in the adjacent ventrobasal nucleus, which does not contain endozepines, show a potentiated response to laser photostimulation of caged GABA when placed in the nRT. FC treatment blocked the nRT-dependent potentiation of this response, as did the benzodiazepine site antagonist flumazenil. When sniffer patches were placed in the ventrobasal nucleus, however, subsequent treatment with FC led to potentiation of the uncaged GABA response, suggesting nucleus-specific roles for thalamic astrocytes in regulating inhibition. Taken together, these results suggest that astrocytes are required for endozepine actions in the nRT, and as such can be positive modulators of synaptic inhibition.

Sniffer patch laser uncaging response (SPLURgE): an assay of regional differences in allosteric receptor modulation and neurotransmitter clearance.

Allosteric modulators exert actions on neurotransmitter receptors by positively or negatively altering the effective response of these receptors to their respective neurotransmitter. γ-Aminobutyric acid (GABA) type A ionotropic receptors (GABAARs) are major targets for allosteric modulators such as benzodiazepines, neurosteroids, and barbiturates. Analysis of substances that produce similar effects has been hampered by the lack of techniques to assess the localization and function of such agents in brain slices. Here we describe measurement of the sniffer patch laser uncaging response (SPLURgE), which combines the sniffer patch recording configuration with laser photolysis of caged GABA. This methodology enables the detection of allosteric GABAAR modulators endogenously present in discrete areas of the brain slice and allows for the application of exogenous GABA with spatiotemporal control without altering the release and localization of endogenous modulators within the slice. Here we demonstrate the development and use of this technique for the measurement of allosteric modulation in different areas of the thalamus. Application of this technique will be useful in determining whether a lack of modulatory effect on a particular category of neurons or receptors is due to insensitivity to allosteric modulation or a lack of local release of endogenous ligand. We also demonstrate that this technique can be used to investigate GABA diffusion and uptake. This method thus provides a biosensor assay for rapid detection of endogenous GABAAR modulators and has the potential to aid studies of allosteric modulators that exert effects on other classes of neurotransmitter receptors, such as glutamate, acetylcholine, or glycine receptors.

Focal cortical infarcts alter intrinsic excitability and synaptic excitation in the reticular thalamic nucleus.

Focal cortical injuries result in death of cortical neurons and their efferents and ultimately in death or damage of thalamocortical relay (TCR) neurons that project to the affected cortical area. Neurons of the inhibitory reticular thalamic nucleus (nRT) receive excitatory inputs from corticothalamic and thalamocortical axons and are thus denervated by such injuries, yet nRT cells generally survive these insults to a greater degree than TCR cells. nRT cells inhibit TCR cells, regulate thalamocortical transmission, and generate cerebral rhythms including those involved in thalamocortical epilepsies. The survival and reorganization of nRT after cortical injury would determine recovery of thalamocortical circuits after injury. However, the physiological properties and connectivity of the survivors remain unknown. To study possible alterations in nRT neurons, we used the rat photothrombosis model of cortical stroke. Using in vitro patch-clamp recordings at various times after the photothrombotic injury, we show that localized strokes in the somatosensory cortex induce long-term reductions in intrinsic excitability and evoked synaptic excitation of nRT cells by the end of the first week after the injury. We find that nRT neurons in injured rats show (1) decreased membrane input resistance, (2) reduced low-threshold calcium burst responses, and (3) weaker evoked excitatory synaptic responses. Such alterations in nRT cellular excitability could lead to loss of nRT-mediated inhibition in relay nuclei, increased output of surviving TCR cells, and enhanced thalamocortical excitation, which may facilitate recovery of thalamic and cortical sensory circuits. In addition, such changes could be maladaptive, leading to injury-induced epilepsy.

Show Me the Meaning of Being Lonely (and Its Effects on Seizure Burden and Comorbidities).

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A Single Test to Study Social Behavior and Repetitive Self-grooming in Mice.

The ability to recognize and interact with members of the same species is essential for social communication. Investigating the neural substrates of social interest and recognition may offer insights into the behavioral differences present in disorders affecting social behavior. Assays used to study social interest in rodents include the 3-chamber test, a partition test, and a social interaction test. Here, we present a single protocol that can be used to quantify the level of social interest displayed by mice, the ability to distinguish between different individual mice (social recognition), and the level of repetitive self-grooming displayed. In the first part of the protocol, a social habituation/dishabituation test, the time spent by a test mouse sniffing a stimulus mouse is quantified over 9 trials. In the first 8 interactions, the same stimulus mouse is used repeatedly; on the ninth trial, a novel stimulus mouse is presented. Intact social recognition is indicated by a progressive decrease in the investigation time over trials 1-8, and an increase in trial 9. The interval between each social trial is used to quantify self-grooming, a stereotyped repetitive behavior in mice. We also present a method for randomized, blinded analysis of these behaviors to increase rigor and reproducibility of results. Therefore, this single behavioral test enables ready assessment of phenotypes of both social and repetitive behaviors in an integrated manner in the same animals. This feature can be advantageous in understanding interactions between these behaviors and phenotypes in mouse models with genetic variants associated with autism and other neurodevelopmental or neuropsychiatric disorders, which are often characterized by these behavioral differences.

Inhibition Gets a New KAR Smell.

Ionotropic and metabotropic kainate receptor signaling regulates Cl- homeostasis and GABAergic inhibition Garand D, Mahadevan V, Woodin MA. J Physiol. 2018. doi:10.1113/JP276901 Potassium chloride cotransporter 2 (KCC2) plays a critical role in the regulation of chloride (Cl-) homeostasis within mature neurons. The KCC2 is a secondarily active transporter that extrudes Cl- from the neuron, which maintains a low intracellular Cl-concentration [Cl-]. This results in a hyperpolarized reversal potential of GABA ( EGABA), which is required for fast synaptic inhibition in the mature central nervous system. Potassium chloride cotransporter 2 also plays a structural role in dendritic spines and at excitatory synapses and interacts with "excitatory" proteins, including the GluK2 subunit of kainate receptors (KARs). Kainate receptors are glutamate receptors that display both ionotropic and metabotropic signaling. We show that activating KARs in the hippocampus hyperpolarizes EGABA, thus strengthening inhibition. This hyperpolarization occurs via both ionotropic and metabotropic KAR signaling in the CA3 region, whereas it is absent in the GluK1/2-/- mouse, and is independent of zinc release from mossy fiber terminals. The metabotropic signaling mechanism is dependent on KCC2, although the ionotropic signaling mechanism produces a hyperpolarization of EGABA even in the absence of KCC2 transporter function. These results demonstrate a novel functional interaction between a glutamate receptor and KCC2, a transporter critical for maintaining inhibition, suggesting that the KAR:KCC2 complex may play an important role in excitatory:inhibitory balance in the hippocampus. Additionally, the ability of KARs to regulate chloride homeostasis independently of KCC2 suggests that KAR signaling can regulate inhibition via multiple mechanisms. Activation of kainate-type glutamate receptors could serve as an important mechanism for increasing the strength of inhibition during periods of strong glutamatergic activity.

Estrous Cycle Monitoring in Mice with Rapid Data Visualization and Analysis.

The estrous cycle provides a readout of reproductive health in female laboratory rodents, and estrous cycle stage can be an important physiological variable. Accurate assessment of estrous cycle stage is also important in producing timed pregnancies for developmental studies. Here, we provide a protocol for evaluation of estrous cycle stage through a minimally invasive procedure of acquiring cells lining the vaginal cavity and immediate microscopic visual assessment of these cells without drying or staining. When performed over several consecutive days, the pattern of progression through the four main stages of the estrous cycle, and disruptions to this pattern, can be determined. We also present software that enables more efficient cycle stage data analysis and pattern visualization. These protocols and tools will thus facilitate the incorporation of female animals in laboratory experiments and enhance the assessment of relationships between the reproductive cycle and overall physiology and behavior.

Estradiol induces diurnal shifts in GABA transmission to gonadotropin-releasing hormone neurons to provide a neural signal for ovulation.

Ovulation is initiated by a surge of gonadotropin-releasing hormone (GnRH) secretion by the brain. GnRH is normally under negative feedback control by ovarian steroids. During sustained exposure to estradiol in the late follicular phase of the reproductive cycle, however, the feedback action of this steroid switches to positive, inducing the surge. Here, we used an established ovariectomized, estradiol-treated (OVX+E) mouse model exhibiting daily surges to investigate the neurobiological mechanisms underlying this switch. Specifically, we examined changes in GABA transmission to GnRH neurons, which can be excited by GABA(A) receptor activation. Spontaneous GABAergic postsynaptic currents (PSCs) were recorded in GnRH neurons from OVX+E and OVX mice in coronal and sagittal slices. There were no diurnal changes in PSC frequency in cells from OVX mice in either slice orientation. In OVX+E cells in both orientations, PSC frequency was low during negative feedback but increased at surge onset. During the surge peak, this increase subsided in coronal slices but persisted in sagittal slices. Comparison of PSCs before and during tetrodotoxin (TTX) treatment showed TTX decreased PSC frequency in OVX+E cells in sagittal slices, but not coronal slices. This indicates estradiol acts on multiple GABAergic afferent populations to increase transmission through both activity-dependent and -independent mechanisms. Estradiol also increased PSC amplitude during the surge. Estradiol and the diurnal cycle thus interact to induce shifts in both GABA transmission and postsynaptic response that would produce appropriate changes in GnRH neuron firing activity and hormone release.

Vasoactive intestinal polypeptide can excite gonadotropin-releasing hormone neurons in a manner dependent on estradiol and gated by time of day.

A surge of GnRH release signals the LH surge that triggers ovulation. The GnRH surge is dependent on a switch in estradiol feedback from negative to positive and, in rodents, a daily neural signal, likely from the suprachiasmatic nuclei. Vasoactive intestinal polypeptide (VIP) may be involved in suprachiasmatic nuclei-GnRH neuron communication. Here we assessed the effects of acute VIP (5 min treatment) on GnRH neuron function using targeted extracellular recordings of firing activity of GnRH neurons in brain slices. We examined the effect of VIP on firing rate at different times of day using an established ovariectomized, estradiol-treated (OVX+E) mouse model that exhibits daily LH surges timed to the late afternoon. Cells from OVX animals (no estradiol) did not respond to VIP, regardless of time of day. With estradiol, the effect of VIP on GnRH neurons was dependent on the time of recording. During negative feedback, OVX+E cells did not respond. VIP increased firing in cells recorded during surge onset, but this excitatory response was reduced at surge peak. Acute treatment of OVX+E cells during surge peak with a VIP receptor antagonist decreased GnRH neuron firing. This suggests endogenous VIP may both increase GnRH neuron firing during the surge and occlude response to exogenous VIP. These data provide functional evidence for VIP effects on GnRH neurons and indicate that both estradiol and time of day gate the GnRH neuron response to this peptide. VIP may provide an excitatory signal from the circadian clock that helps time the GnRH surge.

Critical roles for fast synaptic transmission in mediating estradiol negative and positive feedback in the neural control of ovulation.

A switch in the balance of estradiol feedback actions from negative to positive initiates the GnRH surge, triggering the LH surge that causes ovulation. Using an ovariectomized, estradiol-treated (OVX+E) mouse model that exhibits daily switches between negative in the morning and positive feedback in the evening, we investigated the roles of fast synaptic transmission in regulating GnRH neuron firing during negative and positive feedback. Targeted extracellular recordings were used to monitor activity of GnRH neurons from OVX+E and OVX mice in control solution or solution with antagonists to both ionotropic glutamate and gamma-aminobutyric acid receptors (blockade). Blockade had no effect on activity of OVX cells. In contrast, in OVX+E cells in the morning, blockade increased activity compared with control cells, whereas in the evening, blockade decreased activity. In vivo barbiturate sedation of OVX+E mice that blocked LH surge induction prevented the in vitro evening changes in firing rate and response to blockade. These observations suggest at least partial inversion of the negative-to-positive switch in estradiol feedback action and indicate that changes in fast synaptic transmission to GnRH neurons and within the network of cells presynaptic to GnRH neurons are critical for mediating estradiol negative and positive feedback actions on GnRH neurons. Fast synaptic transmission may also affect GnRH neuron activity indirectly through altering release of excitatory and inhibitory neuromodulators onto GnRH neurons at specific times of day. Fast synaptic transmission is thus critical for proper generation and timing of the GnRH surge.

Classical estrogen receptor alpha signaling mediates negative and positive feedback on gonadotropin-releasing hormone neuron firing.

During the female reproductive cycle, the neuroendocrine action of estradiol switches from negative feedback to positive feedback to initiate the preovulatory GnRH and subsequent LH surges. Estrogen receptor-alpha (ERalpha) is required for both estradiol negative and positive feedback regulation of LH. ERalpha may signal through estrogen response elements (EREs) in DNA and/or via ERE-independent pathways. Previously, a knock-in mutant allele (ERalpha-/AA) that selectively restores ERE-independent signaling onto the ERalpha-/- background was shown to confer partial negative but not positive estradiol feedback on serum LH. The current study investigated the roles of the ERE-dependent and ERE-independent ERalpha pathways for estradiol feedback at the level of GnRH neuron firing activity. The above ERalpha genetic models were crossed with GnRH-green fluorescent protein mice to enable identification of GnRH neurons in brain slices. Targeted extracellular recordings were used to monitor GnRH neuron firing activity using an ovariectomized, estradiol-treated mouse model that exhibits diurnal switches between negative and positive feedback. In wild-type mice, GnRH neuron firing decreased in response to estradiol during negative feedback and increased during positive feedback. In contrast, both positive and negative responses to estradiol were absent in GnRH neurons from ERalpha-/- and ERalpha-/AA mice. ERE-dependent signaling is thus required to increase GnRH neuron firing to generate a GnRH/LH surge. Furthermore, ERE-dependent and -independent ERalpha signaling pathways both appear necessary to mediate estradiol negative feedback on serum LH levels, suggesting central and pituitary estradiol feedback may use different combinations of ERalpha signaling pathways.

Subregion-Specific Impacts of Genetic Loss of Diazepam Binding Inhibitor on Synaptic Inhibition in the Murine Hippocampus.

Benzodiazepines are commonly prescribed to treat neurological conditions including epilepsy, insomnia, and anxiety. The discovery of benzodiazepine-specific binding sites on γ-aminobutyric acid type-A receptors (GABAARs) led to the hypothesis that the brain may produce endogenous benzodiazepine-binding site ligands. An endogenous peptide, diazepam binding inhibitor (DBI), which can bind these sites, is thought to be capable of both enhancing and attenuating GABAergic transmission in different brain regions. However, the role that DBI plays in modulating GABAARs in the hippocampus remains unclear. Here, we investigated the role of DBI in modulating synaptic inhibition in the hippocampus using a constitutive DBI knockout mouse. Miniature and evoked inhibitory postsynaptic currents (mIPSCs, eIPSCs) were recorded from CA1 pyramidal cells and dentate gyrus (DG) granule cells. Loss of DBI signaling increased mIPSC frequency and amplitude in CA1 pyramidal cells from DBI knockout mice compared to wild-types. In DG granule cells, conversely, the loss of DBI decreased mIPSC amplitude and increased mIPSC decay time, indicating bidirectional modulation of GABAAR-mediated transmission in specific subregions of the hippocampus. eIPSC paired-pulse ratios were consistent across genotypes, suggesting that alterations in mIPSC frequency were not due to changes in presynaptic release probability. Furthermore, cells from DBI knockout mice did not display altered responsiveness to pharmacological applications of diazepam, a benzodiazepine, nor flumazenil, a benzodiazepine-binding site antagonist. These results provide evidence that genetic loss of DBI alters synaptic inhibition in the adult hippocampus, and that the direction of DBI-mediated modulation can vary discretely between specific subregions of the same brain structure.

Dynamic and Sex-Specific Changes in Gonadotropin-Releasing Hormone Neuron Activity and Excitability in a Mouse Model of Temporal Lobe Epilepsy.

Reproductive endocrine disorders are prominent comorbidities of temporal lobe epilepsy (TLE) in both men and women. The neural mechanisms underlying these comorbidities remain unclear, but hypothalamic gonadotropin-releasing hormone (GnRH) neurons may be involved. Here, we report the first direct demonstrations of aberrant GnRH neuron function in an animal model of epilepsy. Recordings of GnRH neuron firing and excitability were made in acute mouse brain slices prepared two months after intrahippocampal injection of kainate (KA) or control saline, a well-established TLE model in which most females develop comorbid estrous cycle disruption. GnRH neurons from control females showed elevated firing and excitability on estrus compared with diestrus. By contrast, cells from KA-injected females that developed prolonged, disrupted estrous cycles (KA-long) showed the reverse pattern. Firing rates of cells from KA-injected females that maintained regular cycles (KA-regular) were not different from controls on diestrus, but were reduced on estrus. In KA-injected males, only GnRH neurons in the medial septum displayed elevated firing. In contrast to the diestrus versus estrus and sex-specific changes in firing, GnRH neuron intrinsic excitability was elevated in all KA-injected groups, indicating a role for afferent synaptic and neuromodulatory inputs in shaping overall changes in firing activity. Furthermore, KA-injected females showed cycle-stage-specific changes in circulating sex steroids on diestrus and estrus that also differed between KA-long and KA-regular groups. Together, these findings reveal that the effects of epilepsy on the neural control of reproduction are dynamic across the estrous cycle, distinct in association with comorbid estrous cycle disruption severity, and sex-specific.

Disrupted female estrous cyclicity in the intrahippocampal kainic acid mouse model of temporal lobe epilepsy.

OBJECTIVE: Reproductive dysfunction is a comorbidity that commonly occurs with temporal lobe epilepsy (TLE). Characterization of this comorbidity in various models of TLE in mice will greatly facilitate mechanistic investigations of the relationship between reproductive disorders and seizures initiated in the hippocampus. Here we investigate the impact on female reproductive estrous cyclicity in the intrahippocampal kainic acid mouse model of TLE and demonstrate the utility of using this model for future mechanistic studies. METHODS: Kainic acid (KA) or saline vehicle was stereotaxically injected in the right dorsal hippocampus of adult female C57BL/6J mice. Development of epilepsy was assessed by video monitoring for behavioral seizures. Reproductive function was assessed by daily estrous cycle monitoring and ovarian morphology. Estrous cycles were monitored for up to 2 months after injection. Ovarian morphology was examined by histological staining and assessment of follicular and luteal development. RESULTS: We observed spontaneous behavioral seizures in 82% of kainic-acid-treated mice. Irregular estrous cycles developed within 2 months after kainic acid injection. Sixty-seven percent of KA-treated mice showed disrupted estrous cycles, typically characterized by increased estrous cycle length, increased time spent in diestrus (nonfertile stage), and decreased time spent in estrus by 42 days post-KA injection. The estrous cycle disruption, however, was not accompanied by major changes in ovarian morphology or follicular development. KA-treated mice also displayed increased weight gain compared to control mice. SIGNIFICANCE: These data indicate that comorbid female irregular estrous cyclicity arises in the intrahippocampal kainic acid mouse model of TLE. This is the first demonstration of disrupted reproductive endocrine function in a mouse model of TLE initially produced by an insult specifically targeted to the hippocampus. This model should thus be useful for basic studies investigating the neural mechanisms driving comorbid reproductive dysfunction in epilepsy in women.

Regulation of Thalamic and Cortical Network Synchrony by Scn8a.

Voltage-gated sodium channel (VGSC) mutations cause severe epilepsies marked by intermittent, pathological hypersynchronous brain states. Here we present two mechanisms that help to explain how mutations in one VGSC gene, Scn8a, contribute to two distinct seizure phenotypes: (1) hypoexcitation of cortical circuits leading to convulsive seizure resistance, and (2) hyperexcitation of thalamocortical circuits leading to non-convulsive absence epilepsy. We found that loss of Scn8a leads to altered RT cell intrinsic excitability and a failure in recurrent RT synaptic inhibition. We propose that these deficits cooperate to enhance thalamocortical network synchrony and generate pathological oscillations. To our knowledge, this finding is the first clear demonstration of a pathological state tied to disruption of the RT-RT synapse. Our observation that loss of a single gene in the thalamus of an adult wild-type animal is sufficient to cause spike-wave discharges is striking and represents an example of absence epilepsy of thalamic origin.

Feedback modulation of neural network synchrony and seizure susceptibility by Mdm2-p53-Nedd4-2 signaling.

BACKGROUND: Neural network synchrony is a critical factor in regulating information transmission through the nervous system. Improperly regulated neural network synchrony is implicated in pathophysiological conditions such as epilepsy. Despite the awareness of its importance, the molecular signaling underlying the regulation of neural network synchrony, especially after stimulation, remains largely unknown. RESULTS: In this study, we show that elevation of neuronal activity by the GABA(A) receptor antagonist, Picrotoxin, increases neural network synchrony in primary mouse cortical neuron cultures. The elevation of neuronal activity triggers Mdm2-dependent degradation of the tumor suppressor p53. We show here that blocking the degradation of p53 further enhances Picrotoxin-induced neural network synchrony, while promoting the inhibition of p53 with a p53 inhibitor reduces Picrotoxin-induced neural network synchrony. These data suggest that Mdm2-p53 signaling mediates a feedback mechanism to fine-tune neural network synchrony after activity stimulation. Furthermore, genetically reducing the expression of a direct target gene of p53, Nedd4-2, elevates neural network synchrony basally and occludes the effect of Picrotoxin. Finally, using a kainic acid-induced seizure model in mice, we show that alterations of Mdm2-p53-Nedd4-2 signaling affect seizure susceptibility. CONCLUSION: Together, our findings elucidate a critical role of Mdm2-p53-Nedd4-2 signaling underlying the regulation of neural network synchrony and seizure susceptibility and reveal potential therapeutic targets for hyperexcitability-associated neurological disorders.