Theta oscillations (4-10 Hz) are a prominent electrophysiological feature of the hippocampus and are highly dependent on the activity of interneurons and GABAergic signaling. In mesial temporal lobe epilepsy (MTLE), theta oscillations are disrupted in the hippocampus and extra-hippocampal structures. We studied here the time-sensitive changes of hippocampal theta oscillations during epileptogenesis and whether the ictogenic effects induced by the optogenetic stimulation of hippocampal CaMKII-positive principal cells are mirrored by changes in theta oscillations. CaMKII-ChR2 mice (n = 11) were treated with kainic acid (15 mg/kg, i.p.) and recorded from one day before status epilepticus (SE) to 25 days after. In a subgroup of mice (n = 6), optogenetic stimulation (1 Hz, 180 s ON, 220 s OFF) of CaMKII-positive principal cells was performed for 15 days starting 3 h after SE. In non-stimulated, control mice (n = 5), we found that the power of theta oscillations significantly increased during the latent phase and was then followed by a gradual decrease that marked the transition between the latent and chronic epileptic phases. In stimulated animals (n = 6), power of theta oscillations followed a similar time-sensitive pattern. Our findings suggest that time-sensitive changes in theta power are reliable biomarkers of inhibitory interneuron GABAergic function during epileptogenesis.

Catamenial epilepsy is characterized by increased seizure frequency or severity during specific phases of the menstrual cycle, presumably driven by changes in the balance between excitation and inhibition. GABAA receptor-mediated inhibition, which is involved in focal epileptic disorders rests on the presynaptic release of GABA by interneurons. Work performed in the 1980s identified loss of interneuron function in seizure onset zones and the ability of GABAA receptor antagonists to induce epileptiform synchronization thus indicating that decreased inhibition leads to seizures. However, in vitro and in vivo findings obtained during the last four decades from animal models and epileptic patients have challenged this view. Here, we will first review such active, though unexpected, contribution of interneurons (and thus of inhibition) in seizure initiation and maintenance. We will then address the blood level changes in the sex hormones progesterone and estrogen occurring in humans and rodents during the ovarian cycles, and their potential involvement in specific types of catamenial epilepsy. Finally, we will discuss the active contribution of parvalbumin (PV)-positive GABAergic interneurons to seizure activity. Due to high estrogen blood levels, these cells become hyperexcitable during periovulation and when ovariectomized females are treated with 17β-estradiol; this estrogen β receptor-mediated mechanism makes seizures last longer during periovulation. Such novel role of PV-positive interneurons in increasing focal seizures during proestrus/estrus may lead to formulate new therapeutic interventions in controlling seizure exacerbation during periovulation.

Background: Catamenial epilepsy, which is defined as a periodicity of seizure exacerbation occurring during the menstrual cycle, has been reported in up to 70% of epileptic women. These seizures are often non-responsive to medication and our understanding of the relation between menstrual cycle and seizure generation (i.e. ictogenesis) remains limited. Methods: Here, we employed the in vitro 4-aminopyridine model of epileptiform synchronization, to analyze the effects induced by optogenetic activation of parvalbumin (PV)-positive interneurons at 8 Hz during estrous and non-estrous phases in female PV-ChR2 mice. Results: We found that: (i) optogenetic stimulation of PV-positive interneurons induced an initial interictal spike followed by field oscillations occurring more often in estrous (59%) than in non-estrous slices (17%); (ii) these oscillations showed significantly higher power in estrous compared to nonestrous slices (p < 0.001); (iii) significantly higher rates of interictal spikes and ictal discharges were identified in both estrous and non-estrous slices during optogenetic stimulation of PV-positive interneurons compared to periods of no stimulation (p < 0.05); and (iv) ictal events appeared to occur more frequently during optogenetic stimulation in estrous compared to non-estrous slices. Conclusion: Our findings show that optogenetic activation of PV-interneurons leads to more powerful network oscillations and more frequent ictal discharges in estrous than in non-estrous slices. We conclude that during the rodent estrous cycle, PV-interneuron hyperexcitability may play a role in epileptiform synchronization and thus in catamenial seizures.

Catamenial epilepsy is characterized by seizure exacerbation during specific phases of the menstrual cycle and affects up to 70 % of epileptic women. These seizures are often non-responsive to medication and our understanding of the relationship between menstrual cycle and seizure generation remains limited. Previous experiments using the in vitro 4-aminopyridine model of epileptiform synchronization demonstrated that the proestrus and estrus, which are associated to high blood estrogen levels, favor seizure generation; this effect presumably results from enhanced parvalbumin (PV)-positive interneuron excitability caused by estrogen receptor β-mediated activity. Here, we used the in vivo kainic acid (KA) model of mesial temporal lobe epilepsy in PV-ChR2 mice to establish the influence of the estrous cycle on: (i) the induction of status epilepticus (SE), and (ii) the severity of spontaneous seizures and interictal spikes in female mice that were treated with systemic KA (i.p) injection. We found that SE induced during proestrus results in irregular estrous cycles without affecting SE severity, the occurrence/duration of spontaneous seizures and of interictal spike rates during the chronic phase. Moreover, chronic seizures were more frequent and significantly longer during the estrous phases compared to the non-estrous phases. Overall, our findings demonstrate that the hormonal state at the time of SE induction influences the estrous cycle regularity without affecting seizure or interictal spike burden during the chronic epileptic period. However, during the chronic period, seizure severity is promoted through the 4-5 days fluctuation in sex hormones coinciding with the estrous cycle, and specifically during the estrous phases.

Spreading depolarization (SD) is a transient disruption of electrographic activity that slowly propagates through the gray matter by chemical contiguity, and it is characterized by a large depolarization of neurons and glial cells. SD, which is associated with massive changes in ion homeostasis, including extreme increases in [K+]o, was shown to occur in various neurological diseases such as migraine and traumatic brain injury. It is hypothesized to also occur in epilepsy. We review here the cellular and pharmacological features of SD that was mainly induced in vitro by different pharmacological manipulations as well as its relationship to focal seizures that can concomitantly occur in these in vitro and in vivo preparations. Recent experimental evidence points to SD playing a role in controlling seizure generation, but other studies have reported that SD facilitates ictogenesis. We conclude that further work is needed to firmly identify the role of SD in modulating focal seizure generation. These future experiments should also help in clearly defining the role played by SD in the manifestation of sudden unexpected death in epilepsy.

Time-frequency analysis of focal seizure electroencephalographic signals performed with depth electrodes in human temporal lobe structures has revealed the occurrence at onset of oscillations at approximately 30-100 Hz that feature a monotonic rapid decay in frequency content. This seizure onset pattern, referred to as chirp, has been identified as a highly specific and sensitive marker of focal seizures that are characterized by low-voltage fast activity. We report that this chirp pattern is also observed in animal models of temporal lobe epilepsy in both in vivo and in vitro preparations. We propose here that chirps mirror the involvement of synchronous interneuron firing that is known to represent a specific cellular mechanism leading to the initiation of focal seizures, in particular those characterized by low-voltage fast activity.

Mesial temporal lobe epilepsy (MTLE) is characterized by recurring focal seizures that arise from limbic areas and are often refractory to pharmacological interventions. We have reported that optogenetic stimulation of PV-positive cells in the medial septum at 0.5 Hz exerts seizure-suppressive effects. Therefore, we compared here these results with those obtained by optogenetic stimulation of medial septum PV-positive neurons at 8 Hz in male PV-ChR2 mice (P60-P100) undergoing an initial, pilocarpine-induced status epilepticus (SE). Optogenetic stimulation (5 min ON, 10 min OFF) was performed from day 8 to day 12 after SE at a frequency of 8 Hz (n = 6 animals) or 0.5 Hz (n = 8 animals). Surprisingly, in both groups, no effects were observed on the occurrence of interictal spikes and interictal high frequency oscillations (HFOs). However, 0.5 Hz stimulation induced a significant decrease of seizure occurrence (p < 0.05). Such anti-ictogenic effect was not observed in the 8 Hz protocol that instead triggered seizures (p < 0.05); these seizures were significantly longer under optogenetic stimulation compared to when optogenetic stimulation was not implemented (p < 0.05). Analysis of ictal HFOs revealed that in the 0.5 Hz group, but not in the 8 Hz group, seizures occurring under optogenetic stimulation were associated with significantly lower rates of fast ripples compared to when optogenetic stimulation was not performed (p < 0.05). Our results indicate that activation of GABAergic PV-positive neurons in the medial septum exerts seizure-suppressing effects that are frequency-dependent and associated with low rates of fast ripples. Optogenetic activation of medial septum PV-positive neurons at 0.5 Hz is efficient in blocking seizures in the pilocarpine model of MTLE, an effect that did not occur with 8 Hz stimulation.

Abstract Optogenetics rests on the activation of light-gated ion channels or pumps in transgenic animals, and it is used to reversibly excite or silence specific neuronal populations, with millisecond precision and with a higher spatial accuracy than electrical stimulation. To date, epilepsy researchers have used optogenetics to study this neurological disorder, and in particular focal seizures that originate from a discrete brain area as in mesial temporal lobe epilepsy. Here, we will review findings obtained by employing optogenetics in in vitro and in vivo models of focal epileptiform synchronization. We will also address the use of this technique to control acute and chronic spontaneous seizures induced by the administration of chemoconvulsants in animal models of mesial temporal lobe epilepsy, such as the kainic acid and the pilocarpine model. These findings reveal that optogenetic activation or inhibition of excitatory or inhibitory cells can exert paradoxical effects on seizure occurrence depending on the frequency and timing of light stimulation, as well as on the targeted neuronal populations. We also consider the limitations associated with the use of optogenetics and the potential issues that must be solved before this approach is translated to human application. We conclude that optogenetics, despite some limitations, represents an innovative and promising approach to improve our understanding of brain function in health and disease.

Catamenial epilepsy, defined as a periodicity of seizure exacerbation during the menstrual cycle, affects up to 70 % of epileptic women. Seizures in these patients are often non-responsive to medication; however, our understanding of the relation between menstrual cycle and seizure generation (i.e. ictogenesis) remains limited. We employed here field potential recordings in the in vitro 4-aminopyridine model of epileptiform synchronization in female mice (P60-P130) and found that: (i) the estrous phase favors ictal activity in the entorhinal cortex; (ii) these ictal discharges display an onset pattern characterised by the presence of chirps that are thought to mirror synchronous interneuron firing; and (iii) blocking estrogen receptor β-mediated signaling reduces ictal discharge duration. Our findings indicate that the duration of 4AP-induced ictal discharges, in vitro, increases during the estrous phase, which corresponds to the human peri-ovulatory period. We propose that these effects are caused by the presumptive enhancement of interneuron excitability due to increased estrogen receptor β-mediated signaling.

Abstract The subiculum has for a long time been neglected and solely considered as the output region of the hippocampus, conducting or relaying inputs to the entorhinal cortex. However, significant evidence does not support such a passive role. The subiculum is a three-layer cortex structured associated with highly organized substructures. Preeminent bursting neuronal behavior and local microcircuits favor the production of strong neuronal activities. All hippocampal rhythms such as theta, gamma, sharp-wave ripples, and ripples can be generated by the subiculum. As a result, the subiculum is an important limbic structure involved in spatial location and memory. The role of the subiculum in epilepsy was highlighted by studies on human postoperative tissues, showing that it is a major and autonomous site of genesis of epileptic interictal and ictal activities. In these studies, a leading role of interneurons in the buildup of epileptic activities was suggested, and chloride dysregulation leading to depolarizing and potentially excitatory effects of GABAA signaling was unraveled. Evidence indicates that the subiculum not only anatomically “supports” hippocampal outputs, but that it also actively participates to the generation of epileptic activities in the temporal lobe.

Dimethyl sulfoxide (DMSO) is commonly used to dissolve water-insoluble drugs due to its dipolar and aprotic properties. It also serves as a vehicle in many pharmacological studies. However, it has been reported that DMSO can induce seizures in human patients, lower seizure threshold in vivo, and modulate ion receptors activities in vitro. Therefore, we investigated here the effect of 0.03% and 0.06% DMSO, which are 10 to 50 times lower than what usually employed in previous studies, in the 4-aminopyridine (4AP) model of epileptiform synchronization in male mouse brain slices. We found that 0.03% and 0.06% DMSO increase 4AP-induced ictal discharge rate, while 0.06% DMSO decreases ictal discharge duration. Our results suggest that the effects of DMSO on neuronal excitability deserve further analysis and that investigators need to be aware of its confounding effect as a solvent, even at very low concentrations.

Developmental and epileptic encephalopathies (DEEs) feature altered brain development, developmental delay and seizures, with seizures exacerbating developmental delay. Here we identify a cohort with biallelic variants in DENND5A, encoding a membrane trafficking protein, and develop animal models with phenotypes like the human syndrome. We demonstrate that DENND5A interacts with Pals1/MUPP1, components of the Crumbs apical polarity complex required for symmetrical division of neural progenitor cells. Human induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division with an inherent propensity to differentiate into neurons. These phenotypes result from misalignment of the mitotic spindle in apical neural progenitors. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state, ultimately shortening the period of neurogenesis. This study provides a mechanism for DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families. Developmental and epileptic encephalopathies are devastating neurological disorders. Here, the authors establish a cohort of patients with variants in the gene DENND5A and use human stem cells to discover a disease mechanism involving altered cell division.

Abstract The neurophysiological features of focal seizures have been extensively studied in humans and in animal models. Electroencephalogram (EEG), local field potentials, and cellular recordings have identified the prevalent characteristics of focal epileptic activities and highlighted the possible underlying mechanisms. In this chapter we will examine how focal seizures develop over time: (1) by describing EEG seizure patterns identified with intracerebral recordings in patients suffering from focal epilepsies during presurgical monitoring; (2) by reviewing the experimental findings obtained from animal models that reproduce the focal seizure patterns described in humans; and (3) by summarizing the most common hypotheses on the network mechanisms involved in the initiation, development, and termination of focal seizure activity. These findings suggest that regardless of the mechanisms involved in the onset of focal seizures, a quite reproducible sequence of excitability changes contribute to set the conditions for seizure progression and termination.

We review here the neuronal mechanisms that cause seizures in focal epileptic disorders and, specifically, those involving limbic structures that are known to be implicated in human mesial temporal lobe epilepsy. In both epileptic patients and animal models, the initiation of focal seizures - which are most often characterized by a low-voltage fast onset EEG pattern - is presumably dependent on the synchronous firing of GABA-releasing interneurons that, by activating post-synaptic GABAA receptors, cause large increases in extracellular [K+] through the activation of the co-transporter KCC2. A similar mechanism may contribute to seizure maintenance; accordingly, inhibiting KCC2 activity transforms seizure activity into a continuous pattern of short-lasting epileptiform discharges. It has also been found that interactions between different areas of the limbic system modulate seizure occurrence by controlling extracellular [K+] homeostasis. In line with this view, low-frequency electrical or optogenetic activation of limbic networks restrain seizure generation, an effect that may also involve the activation of GABAB receptors and activity-dependent changes in epileptiform synchronization. Overall, these findings highlight the paradoxical role of GABAA signaling in both focal seizure generation and maintenance, emphasize the efficacy of low-frequency activation in abating seizures, and provide experimental evidence explaining the poor efficacy of antiepileptic drugs designed to augment GABAergic function in controlling seizures in focal epileptic disorders.

Emerging evidence suggests that the medial septum can control seizures occurring in focal epileptic disorders, thus representing a therapeutic target. Therefore, we investigated whether continuous optogenetic activation of inhibitory parvalbumin (PV)-positive interneurons in the medial septum can reduce the occurrence of spontaneous seizures in the pilocarpine model of mesial temporal lobe epilepsy (MTLE). Light pulses (450 nm, 25 mW, 20-ms pulse duration) were delivered at 0.5 Hz (5 min ON, 10 min OFF) with a laser diode fiber light source between day 8 and day 12 after status epilepticus (SE) in PV-ChR2 mice (n = 8). Seizure rates were significantly lower during time periods of optogenetic stimulation (days 8-12) compared with before implementation of optogenetics (days 4-7) (P < 0.05). Moreover, between day 13 and day 21 after SE seizure rates were still significantly lower compared with before optogenetic stimulation (i.e., between day 4 and day 7) (P < 0.05). No seizures were recorded between day 10 and day 12 in all animals, and no seizures occurred up to 3 days after the end of optogenetic stimulation (days 13-15). Our findings indicate that activation of PV interneurons in the medial septum abates seizures in the pilocarpine model of MTLE. Moreover, the persisting anti-ictogenic effects suggest that stimulation of the medial septum could alter the progression of MTLE.NEW & NOTEWORTHY The medial septum could represent a therapeutic target to treat patients with focal epilepsy. In this study, we show that optogenetic activation of inhibitory parvalbumin-positive interneurons in the medial septum can block spontaneous seizures and prevents their reoccurrence for ∼5 days after the end of stimulation. Our findings suggest that the anti-ictogenic effects induced by stimulation of the medial septum could also alter the progression of mesial temporal lobe epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most common form of focal epilepsy and it is characterized by seizures that are often refractory to medications. Seizures in MTLE have two main patterns of onset that have been termed hypersynchronous (HYP) and low-voltage fast (LVF) and are believed to mainly depend on the activity of excitatory principal cells and inhibitory interneurons, respectively. In this study, we investigated whether unilateral open-loop optogenetic activation of CaMKII-positive principal cells in the hippocampus CA3 region favors the generation of spontaneous HYP seizures in kainic acid-treated (KA) CaMKII-ChR2 mice. Optogenetic activation of CA3 principal cells (1 Hz, 180 s ON, 220 s OFF) was implemented for 15 days after KA-induced status epilepticus. We found that both LVF and HYP seizures occurred in nonstimulated CaMKII-ChR2 (n = 6) and stimulated CaMKII-Cre (n = 5) mice. In contrast, optogenetic activation of principal cells in CaMKII-ChR2 mice (n = 5) triggered only HYP seizures that were characterized by high fast ripple (250-500 Hz) rates during the pre-ictal and ictal periods. These results provide firm evidence that in MTLE spontaneous seizures with different onset patterns depend on distinct neuronal network mechanisms of generation. They also demonstrate that HYP seizures occurring in vivo along with their associated fast ripples depend on the activity of principal cells in the CA3 region.NEW & NOTEWORTHY Previous evidence suggested that different seizure onset patterns rely on the activity of distinct neuronal populations. In this study, we show for the first time that in vivo optogenetic stimulation of CaMKII principal cells in kainic acid-treated mice triggers hypersynchronous-onset seizures that are associated with fast ripples. Our findings indicate that in patients with predominant HYP-onset seizures, anticonvulsant treatments should be aimed at limiting the firing of principal neurons in the seizure onset zone.

Dysregulation of protein synthesis is one of the key mechanisms underlying autism spectrum disorder (ASD). However, the role of a major pathway controlling protein synthesis, the integrated stress response (ISR), in ASD remains poorly understood. Here, we demonstrate that the main arm of the ISR, eIF2a phosphorylation (p-eIF2a), is suppressed in excitatory, but not inhibitory, neurons in a mouse model of fragile X syndrome (FXS; Fmr1-/y). We further show that the decrease in p-eIF2a is mediated via activation of mTORC1. Genetic reduction of p-eIF2a only in excitatory neurons is sufficient to increase general protein synthesis and cause autism-like behavior. In Fmr1-/y mice, restoration of p-eIF2a solely in excitatory neurons reverses elevated protein synthesis and rescues autism-related phenotypes. Thus, we reveal a previously unknown causal relationship between excitatory neuron-specific translational control via the ISR pathway, general protein synthesis, and core phenotypes reminiscent of autism in a mouse model of FXS.

Biological aging can be described as accumulative, prolonged metabolic stress and is the major risk factor for cognitive decline and Alzheimer's disease (AD). Recently, we identified and described a quinone reductase 2 (QR2) pathway in the brain, in which QR2 acts as a removable memory constraint and metabolic buffer within neurons. QR2 becomes overexpressed with age, and it is possibly a novel contributing factor to age-related metabolic stress and cognitive deficit. We found that, in human cells, genetic removal of QR2 produced a shift in the proteome opposing that found in AD brains while simultaneously reducing oxidative stress. We therefore created highly specific QR2 inhibitors (QR2is) to enable evaluation of chronic QR2 inhibition as a means to reduce biological age-related metabolic stress and cognitive decline. QR2is replicated results obtained by genetic removal of QR2, while local QR2i microinjection improved hippocampal and cortical-dependent learning in rats and mice. Continuous consumption of QR2is in drinking water improved cognition and reduced pathology in the brains of AD-model mice (5xFAD), with a noticeable between-sex effect on treatment duration. These results demonstrate the importance of QR2 activity and pathway function in the healthy and neurodegenerative brain and what we believe to be the great therapeutic potential of QR2is as first-in-class drugs.