Sex differences in the developing brain impact stress-induced epileptogenicity following hyperthermia-induced seizures.

Febrile seizures (FS) are common, affecting 2-5% of children between the ages of 3 months and 6 years. Complex FS occur in 10% of patients with FS and are strongly associated with mesial temporal lobe epilepsy. Current research suggests that predisposing factors, such as genetic and anatomic abnormalities, may be necessary for complex FS to translate to mesial temporal lobe epilepsy. Sex hormones are known to influence seizure susceptibility and epileptogenesis, but whether sex-specific effects of early life stress play a role in epileptogenesis is unclear. Here, we investigate sex differences in the activity of the hypothalamic-pituitary-adrenal (HPA) axis following chronic stress and the underlying contributions of gonadal hormones to the susceptibility of hyperthermia-induced seizures (HS) in rat pups. Chronic stress consisted of daily injections of 40 mg/kg of corticosterone (CORT) subcutaneously from postnatal day (P) 1 to P9 in male and female rat pups followed by HS at P10. Body mass, plasma CORT levels, temperature threshold to HS, seizure characteristics, and electroencephalographic in vivo recordings were compared between CORT- and vehicle (VEH)-injected littermates during and after HS at P10. In juvenile rats (P18-P22), in vitro CA1 pyramidal cell recordings were recorded in males to investigate excitatory and inhibitory neuronal circuits. Results show that daily CORT injections increased basal plasma CORT levels before HS and significantly reduced weight gain and body temperature threshold of HS in both males and females. CORT also significantly lowered the generalized convulsions (GC) latency while increasing recovery time and the number of electrographic seizures (>10s), which had longer duration. Furthermore, sex-specific differences were found in response to chronic CORT injections. Compared to females, male pups had increased basal plasma CORT levels after HS, longer recovery time and a higher number of electrographic seizures (>10s), which also had longer duration. Sex-specific differences were also found at baseline conditions with lower latency to generalized convulsions and longer duration of electrographic seizures in males but not in females. In juvenile male rats, the amplitude of evoked excitatory postsynaptic potentials, as well as the amplitude of inhibitory postsynaptic currents, were significantly greater in CORT rats when compared to VEH littermates. These findings not only validate CORT injections as a stress model, but also show a sex difference in baseline conditions as well as a response to chronic CORT and an impact on seizure susceptibility, supporting a potential link between sustained early-life stress and complex FS. Overall, these effects also indicate a putatively less severe phenotype in female than male pups. Ultimately, studies investigating the biological underpinnings of sex differences as a determining factor in mental and neurologic problems are necessary to develop better diagnostic, preventative, and therapeutic approaches for all patients regardless of their sex.

Favorable adverse effect profile of brivaracetam vs levetiracetam in a preclinical model.

Levetiracetam (LEV), and its newer selective analog brivaracetam (BRV), are two seizure medications that share an innovative mechanism of action targeting the Synaptic Vesicle Protein 2A (SV2A), altering neurotransmitter release and decreasing seizure frequency. Behavioral changes are the most significant adverse effects reported by patients taking LEV. We hypothesize that BRV, the more potent SV2A analog, could exert less behavioral side effects, as it requires lower doses than LEV. Using Kainic Acid (KA)-treated and control rats, we measured adverse behavioral effect profiles of LEV, BRV, or Saline, on social and nonsocial behaviors. Our data indicate that both tested drugs had no effect on locomotion, anxiety levels, fear learning, depression-like behavior, and memory retention in rats. However, when considering social interactions, we first confirmed the epilepsy-induced strong increase in aggressive behaviors and specific hippocampal neuronal loss. We furthermore observed, in Sham rats, that LEV-treated animals were 2 times faster to attack at first encounter, had 5 times more aggressive behaviors, and had significantly less social behaviors than control rats. In all circumstances, BRV rats behaved like Saline rats, suggesting that BRV treatment in rats leads to significantly less aggressive behaviors than LEV treatment at the doses used, while there are limited differential effects between these two drugs on other types of behaviors. Since increased aggressiveness has been reported in patients well controlled on LEV, this study indicates based on our findings, that BRV could represent an effective alternative to LEV to limit aggressiveness problems due to this antiepileptic drug (AED) therapy.

Reducing premature KCC2 expression rescues seizure susceptibility and spine morphology in atypical febrile seizures.

Atypical febrile seizures are considered a risk factor for epilepsy onset and cognitive impairments later in life. Patients with temporal lobe epilepsy and a history of atypical febrile seizures often carry a cortical malformation. This association has led to the hypothesis that the presence of a cortical dysplasia exacerbates febrile seizures in infancy, in turn increasing the risk for neurological sequelae. The mechanisms linking these events are currently poorly understood. Potassium-chloride cotransporter KCC2 affects several aspects of neuronal circuit development and function, by modulating GABAergic transmission and excitatory synapse formation. Recent data suggest that KCC2 downregulation contributes to seizure generation in the epileptic adult brain, but its role in the developing brain is still controversial. In a rodent model of atypical febrile seizures, combining a cortical dysplasia and hyperthermia-induced seizures (LHS rats), we found a premature and sustained increase in KCC2 protein levels, accompanied by a negative shift of the reversal potential of GABA. In parallel, we observed a significant reduction in dendritic spine size and mEPSC amplitude in CA1 pyramidal neurons, accompanied by spatial memory deficits. To investigate whether KCC2 premature overexpression plays a role in seizure susceptibility and synaptic alterations, we reduced KCC2 expression selectively in hippocampal pyramidal neurons by in utero electroporation of shRNA. Remarkably, KCC2 shRNA-electroporated LHS rats show reduced hyperthermia-induced seizure susceptibility, while dendritic spine size deficits were rescued. Our findings demonstrate that KCC2 overexpression in a compromised developing brain increases febrile seizure susceptibility and contribute to dendritic spine alterations.

Cortical GABAergic interneurons in cross-modal plasticity following early blindness.

Early loss of a given sensory input in mammals causes anatomical and functional modifications in the brain via a process called cross-modal plasticity. In the past four decades, several animal models have illuminated our understanding of the biological substrates involved in cross-modal plasticity. Progressively, studies are now starting to emphasise on cell-specific mechanisms that may be responsible for this intermodal sensory plasticity. Inhibitory interneurons expressing γ-aminobutyric acid (GABA) play an important role in maintaining the appropriate dynamic range of cortical excitation, in critical periods of developmental plasticity, in receptive field refinement, and in treatment of sensory information reaching the cerebral cortex. The diverse interneuron population is very sensitive to sensory experience during development. GABAergic neurons are therefore well suited to act as a gate for mediating cross-modal plasticity. This paper attempts to highlight the links between early sensory deprivation, cortical GABAergic interneuron alterations, and cross-modal plasticity, discuss its implications, and further provide insights for future research in the field.

Long-term consequences of a prolonged febrile seizure in a dual pathology model.

Clinical evidence suggests that febrile status epilepticus (SE) in children can lead to acute hippocampal injury and subsequent temporal lobe epilepsy. The contribution of febrile SE to the mechanisms underlying temporal lobe epilepsy are however poorly understood. A rat model of temporal lobe epilepsy following hyperthermic SE was previously established in our laboratory, wherein a focal cortical lesion induced at postnatal day 1 (P1), followed by a hyperthermic SE (more than 30 min) at P10, leads to hippocampal atrophy at P22 (dual pathology model) and spontaneous recurrent seizures (SRS) with mild visuospatial memory deficits in adult rats. The goal of this study was to identify the long term electrophysiological, anatomical and molecular changes in this model. Following hyperthermic SE, all cortically lesioned pups developed progressive SRS as adults, characterized by the onset of highly rhythmic activity in the hippocampus. A reduction of hippocampal volume on the side of the lesion preceded the SRS and was associated with a loss of hippocampal neurons, a marked decrease in pyramidal cell spine density, an increase in the hippocampal levels of NMDA receptor NR2A subunit, but no significant change in GABA receptors. These findings suggest that febrile SE in the abnormal brain leads to hippocampal injury that is followed by progressive network reorganization and molecular changes that contribute to the epileptogenesis as well as the observed memory deficits.

A 3D model to evaluate retinal nerve fiber layer thickness deviations caused by the displacement of optical coherence tomography circular scans in cynomolgus monkeys (Macaca fascicularis).

The main objective of the study was to analyze deviations in retinal nerve fiber layer (RNFL) thickness measurements caused by the displacement of circular optic disc optical coherence tomography scans. High-density radial scans of the optic nerve heads of cynomolgus monkeys were acquired. The retinal nerve fiber layer was manually segmented, and a surface plot of the discrete coordinates was generated. From this plot, the RNFL thicknesses were calculated and compared between accurately centered and intentionally displaced circle scans. Circle scan displacement caused circumpapillary retinal nerve fiber layer thickness deviations of increasing magnitude with increasing center offset. As opposed to the human eye, horizontal displacement resulted in larger RNFL thickness deviations than vertical displacement in cynomolgus monkeys. Acquisition of high-density radial scans allowed for the mathematical reconstruction and modelling of the nerve fiber layer and extrapolation of its thickness. Accurate and strictly repeatable circle scan placement is critical to obtain reproducible values, which is essential for longitudinal studies.

Humanized mouse model of Rasmussen's encephalitis supports the immune-mediated hypothesis.

Rasmussen's encephalitis (RE) is a chronic inflammatory brain disorder that causes frequent seizures and unilateral hemispheric atrophy with progressive neurological deficits. Hemispherectomy remains the only treatment that leads to seizure freedom for this refractory epileptic syndrome. The absence of an animal model of disease has been a major obstacle hampering the development of effective therapies. Here, we describe an experimental mouse model that shares several clinical and pathological features with the human disease. Immunodeficient mice injected with peripheral blood mononuclear cells from RE patients and monitored by video electroencephalography developed severe seizures of cortical origin and showed intense astrogliosis and accumulation of human IFN-γ- and granzyme B-expressing T lymphocytes in the brain compared with mice injected with immune cells from control subjects. We also provide evidence for the efficacy of α4 integrin blockade, an approved therapy for the treatment of multiple sclerosis and Crohn's disease, in reducing inflammatory markers associated with RE in the CNS. This model holds promise as a valuable tool for understanding the pathology of RE and for developing patient-tailored experimental therapeutics.

Regional analysis of neurofilament protein immunoreactivity in the hamster's cortex.

The laminar distribution of several distinct populations of neurofilament protein containing neurons has been used as a criterion for the delineation of cortical areas in hamsters. SMI-32 is a monoclonal antibody that recognizes a non-phosphorylated epitope on the medium- and high-molecular weight subunits of neurofilament proteins. As in carnivores and primates, SMI-32 immunoreactivity in the hamster neocortex was present in cell bodies, proximal dendrites and axons of some medium and large pyramidal neurons located in cortical layers III, V and VI. A small population of labeled multipolar cells was also found in layer IV. Neurofilament protein immunoreactive neurons were found throughout isocortical areas. Very few labeled cells were encountered in supplemental motor area, insular cortex, medial portion of associative visual cortex and in parietal association cortex. Our data indicate that SMI-32 immunoreactive cells can be efficiently used to trace boundaries between neocortical areas in the hamster's brain. The regional distribution SMI-32 immunoreactivity in the hamster cortex corresponds quite closely with cortical areas as defined by their cytoarchitecture and myeloarchitecture. The primary sensory cortical areas contain the most intense of SMI-32 immunoreactivity and are also those with the highest density of myelinated axons. Very low SMI-32 immunoreactivity was found in orbital, insular, perirhinal, cingulate and infralimbic cortices, which are also poor in myelinated axons. This supports the association between SMI-32 immunoreactivity and myelin contents.

Role of GPR55 during Axon Growth and Target Innervation.

Guidance molecules regulate the navigation of retinal ganglion cell (RGC) projections toward targets in the visual thalamus. In this study, we demonstrate that the G-protein-coupled receptor 55 (GPR55) is expressed in the retina during development, and regulates growth cone (GC) morphology and axon growth. In vitro, neurons obtained from gpr55 knock-out (gpr55(-/-) ) mouse embryos have smaller GCs, less GC filopodia, and have a decreased outgrowth compared with gpr55(+/+) neurons. When gpr55(+/+) neurons were treated with GPR55 agonists, lysophosphatidylinositol (LPI) and O-1602, we observed a chemo-attractive effect and an increase in GC size and filopodia number. In contrast, cannabidiol (CBD) decreased the GC size and filopodia number inducing chemo-repulsion. In absence of the receptor (gpr55(-/-) ), no pharmacologic effects of the GPR55 ligands were observed. In vivo, compared to their wild-type (WT) littermates, gpr55(-/-) mice revealed a decreased branching in the dorsal terminal nucleus (DTN) and a lower level of eye-specific segregation of retinal projections in the superior colliculus (SC) and in the dorsal lateral geniculate nucleus (dLGN). Moreover, a single intraocular injection of LPI increased branching in the DTN, whereas treatment with CBD, an antagonist of GPR55, decreased it. These results indicate that GPR55 modulates the growth rate and the targets innervation of retinal projections and highlight, for the first time, an important role of GPR55 in axon refinement during development.

Subdural porous and notched mini-grid electrodes for wireless intracranial electroencephalographic recordings.

BACKGROUND: Intracranial electroencephalography (EEG) studies are widely used in the presurgical evaluation of drug-refractory patients with partial epilepsy. Because chronic implantation of intracranial electrodes carries a risk of infection, hemorrhage, and edema, it is best to limit the number of electrodes used without compromising the ability to localize the epileptogenic zone (EZ). There is always a risk that an intracranial study may fail to identify the EZ because of suboptimal coverage. We present a new subdural electrode design that will allow better sampling of suspected areas of epileptogenicity with lower risk to patients. METHOD: Impedance of the proposed electrodes was characterized in vitro using electrochemical impedance spectroscopy. The appearance of the novel electrodes on magnetic resonance imaging (MRI) was tested by placing the electrodes into a gel solution (0.9% NaCl with 14 g gelatin). In vivo neural recordings were performed in male Sprague Dawley rats. Performance comparisons were made using microelectrode recordings from rat cortex and subdural/depth recordings from epileptic patients. Histological examinations of rat brain after 3-week icEEG intracerebral electroencephalography (icEEG) recordings were performed. RESULTS: The in vitro results showed minimum impedances for optimum choice of pure gold materials for electrode contacts and wire. Different attributes of the new electrodes were identified on MRI. The results of in vivo recordings demonstrated signal stability, 50% noise reduction, and up to 6 dB signal-to-noise ratio (SNR) improvement as compared to commercial electrodes. The wireless icEEG recording system demonstrated on average a 2% normalized root-mean-square (RMS) deviation. Following the long-term icEEG recording, brain histological results showed no abnormal tissue reaction in the underlying cortex. CONCLUSION: The proposed subdural electrode system features attributes that could potentially translate into better icEEG recordings and allow sampling of large of areas of epileptogenicity at lower risk to patients. Further validation for use in humans is required.

Atypical febrile seizures, mesial temporal lobe epilepsy, and dual pathology.

Febrile seizures occurring in the neonatal period, especially when prolonged, are thought to be involved in the later development of mesial temporal lobe epilepsy (mTLE) in children. The presence of an often undetected, underlying cortical malformation has also been reported to be implicated in the epileptogenesis process following febrile seizures. This paper highlights some of the various animal models of febrile seizures and of cortical malformation and portrays a two-hit model that efficiently mimics these two insults and leads to spontaneous recurrent seizures in adult rats. Potential mechanisms are further proposed to explain how these two insults may each, or together, contribute to network hyperexcitability and epileptogenesis. Finally the clinical relevance of the two-hit model is briefly discussed in light of a therapeutic and preventive approach to mTLE.

Electroencephalographic and physiologic changes after tricaine methanesulfonate immersion of African clawed frogs (Xenopus laevis).

The objective of this study was to determine electroencephalographic and complementary physiologic changes in Xenopus leavis frogs after bath immersion in MS222. We also evaluated the addition of sodium pentobarbital injected intracoelomi- cally 2 h after MS222 immersion to achieve euthanasia. Frogs (n = 9) weighing 105.5 ± 8.4 g (mean ± 1 SD) were immersed in MS222 at either 1 or 3 g/L until anesthesia was achieved; a conductive stainless steel screw then was implanted in the skull on top of the outer pial surface of the brain. Frogs were immersed again in MS222 at the same concentration as previously, and electroencephalograms, heart rate, oxygen saturation, and respiratory movements were recorded. Amplitude and mean frequency of the electroencephalographic signal were evaluated at 15-min intervals until a flat-line signal was achieved. At 2 h after induction, frogs were injected intracoelomically with sodium pentobarbital (0.5 mL; 240 mg/mL) to accelerate euthanasia. Immersion of frogs in 1 or 3 g/L of MS222 depressed cerebral activity within 30 min without a significant effect on cardiac function. Intracoelomic injection of sodium pentobarbital at 2 h after MS222 administration rapidly (3.2 ± 1.7 min) induced cardiac arrest. In conclusion, immersion in MS222 can be used for the collection of organs from X. laevis frogs, but the addition of pentobarbital is required to achieve euthanasia.

Early-life stress is associated with gender-based vulnerability to epileptogenesis in rat pups.

During development, the risk of developing mesial temporal lobe epilepsy (MTLE) increases when the developing brain is exposed to more than one insult in early life. Early life insults include abnormalities of cortical development, hypoxic-ischemic injury and prolonged febrile seizures. To study epileptogenesis, we have developed a two-hit model of MTLE characterized by two early-life insults: a freeze lesion-induced cortical malformation at post-natal day 1 (P1), and a prolonged hyperthermic seizure (HS) at P10. As early life stressors lead to sexual dimorphism in both acute response and long-term outcome, we hypothesized that our model could lead to gender-based differences in acute stress response and long-term risk of developing MTLE. Male and female pups underwent a freeze-lesion induced cortical microgyrus at P1 and were exposed to HS at P10. Animals were monitored by video-EEG from P90 to P120. Pre and post-procedure plasma corticosterone levels were used to measure stress response at P1 and P10. To confirm the role of sex steroids, androgenized female pups received daily testosterone injections to the mother pre-natally and post-natally for nine days while undergoing both insults. We demonstrated that after both insults females did not develop MTLE while all males did. This correlated with a rise in corticosterone levels at P1 following the lesion in males only. Interestingly, all androgenized females showed a similar rise in corticosterone at P1, and also developed MTLE. Moreover, we found that the cortical lesion significantly decreased the latency to generalized convulsion during hyperthermia at P10 in both genders. The cortical dysplasia volumes at adulthood were also similar between male and female individuals. Our data demonstrate sexual dimorphism in long-term vulnerability to develop epilepsy in the lesion + hyperthermia animal model of MTLE and suggest that the response to early-life stress at P1 contributes significantly to epileptogenesis in a gender-specific manner.

Distribution of calcium binding proteins in visual and auditory cortices of hamsters.

The morphology and distribution of neurons immunoreactive (ir) to parvalbumin (PV), calretinin (CR) and calbindin (CB) were studied in the primary visual (V1) and auditory (A1) cortices of hamsters. Cortical cell populations were labelled immunohistochemically using a glucose oxidase-diaminobenzidine-nickel combined revelation method. Quantitative analysis revealed significant differences between V1 and A1 in the density and distribution of their neuronal population. CBir cells exhibited several typologies in both cortical regions. Most cells were multipolar even though many of them had bitufted or bipolar morphologies. These cells were distributed in layers II/III and in layer V of both A1 and V1, but were more numerous in layer V of V1. CRir cells were of the fusiform type with long bipolar dendritic arbours. These were similarly distributed in both cortices with a peak in superficial layers II/III. PVir cells were also found in both cortices and had round or oval-shaped somata with multipolar processes. They were mostly located in layer V for V1 and in layers III/IV for A1. Visual and auditory primary cortices can thus be differentiated on the basis of their immunoreactivity to specific calcium binding proteins.