Safety Profile of Low-Intensity Pulsed Ultrasound-Induced Blood-Brain Barrier Opening in Non-epileptic Mice and in a Mouse Model of Mesial Temporal Lobe Epilepsy.

OBJECTIVE: It is unknown whether ultrasound-induced blood-brain barrier (BBB) disruption can promote epileptogenesis and how BBB integrity changes over time after sonication. METHODS: To gain more insight into the safety profile of ultrasound (US)-induced BBB opening, we determined BBB permeability as well as histological modifications in C57BL/6 adult control mice and in the kainate (KA) model for mesial temporal lobe epilepsy in mice after sonication with low-intensity pulsed ultrasound (LIPU). Microglial and astroglial changes in ipsilateral hippocampus were examined at different time points following BBB disruption by respectively analyzing Iba1 and glial fibrillary acidic protein immunoreactivity. Using intracerebral EEG recordings, we further studied the possible electrophysiological repercussions of a repeated disrupted BBB for seizure generation in nine non-epileptic mice. RESULTS: LIPU-induced BBB opening led to transient albumin extravasation and reversible mild astrogliosis, but not to microglial activation in the hippocampus of non-epileptic mice. In KA mice, the transient albumin extravasation into the hippocampus mediated by LIPU-induced BBB opening did not aggravate inflammatory processes and histologic changes that characterize the hippocampal sclerosis. Three LIPU-induced BBB opening did not induce epileptogenicity in non-epileptic mice implanted with depth EEG electrodes. CONCLUSION: Our experiments in mice provide persuasive evidence of the safety of LIPU-induced BBB opening as a therapeutic modality for neurological diseases.

In Vivo Injection of Anti-LGI1 Antibodies into the Rodent M1 Cortex and Hippocampus Is Ineffective in Inducing Seizures.

Autoimmune encephalitis (AIE) associated with antibodies directed against the leucine-rich glioma inactivated 1 (LGI1) protein is the second most common AIE and is responsible for deleterious neocortical and limbic epileptic seizures. Previous studies demonstrated a pathogenic role of anti-LGI1 antibodies via alterations in the expression and function of Kv1 channels and AMPA receptors. However, the causal link between antibodies and epileptic seizures has never been demonstrated. Here, we attempted to determine the role of human anti-LGI1 autoantibodies in the genesis of seizures by analyzing the impact of their intracerebral injection in rodents. Acute and chronic injections were performed in rats and mice in the hippocampus and primary motor cortex, the two main brain regions affected by the disease. Acute infusion of CSF or serum IgG of anti-LGI1 AIE patients did not lead to the emergence of epileptic activities, as assessed by multisite electrophysiological recordings over a 10 h period after injection. A chronic 14 d injection, coupled with continuous video-EEG monitoring, was not more effective. Overall, these results demonstrate that acute and chronic injections of CSF or purified IgG from LGI1 patients are not able to generate epileptic activity by themselves in the different animal models tested.

Kv1.1 channels inhibition in the rat motor cortex recapitulates seizures associated with anti-LGI1 encephalitis.

Autoimmune encephalitis associated with antibodies directed against the leucine-rich glioma inactivated 1 (LGI1) protein is responsible for specific tonic-dystonic motor seizures. Although dysfunctions in neuronal excitability have been associated with anti-LGI1 autoantibodies, their relation to seizures remain inconclusive. We developed a new in vivo experimental rat model to determine whether inhibition of Kv1.1 channels by dentrotoxin-K (DTX) in the primary motor cortex (M1) could recapitulate the human seizures and to elucidate their subtending cortical mechanisms. Comparing electro-clinical features of DTX-induced seizures in rats with those recorded from a cohort of anti-LGI1 encephalitis patients revealed striking similarities in their electroencephalographic (EEG) signature, frequency of recurrence and semiology. By combining multi-site extracellular and intracellular recordings of M1 pyramidal neurons in DTX rats, we demonstrated that the blockade of Kv1.1 channels induced a sequence of changes in neuronal excitability and synaptic activity, leading to massive suprathreshold membrane depolarizations underlying the paroxysmal EEG activity. Our results suggest the central role of Kv1.1 channels disruption in the emergence of anti-LGI1-associated seizures and suggest that this new rodent model could serve future investigations on ictogenesis in autoimmune encephalitis.

Depdc5 knockout rat: A novel model of mTORopathy.

DEP-domain containing 5 (DEPDC5), encoding a repressor of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, has recently emerged as a major gene mutated in familial focal epilepsies and focal cortical dysplasia. Here we established a global knockout rat using TALEN technology to investigate in vivo the impact of Depdc5-deficiency. Homozygous Depdc5(-/-) embryos died from embryonic day 14.5 due to a global growth delay. Constitutive mTORC1 hyperactivation was evidenced in the brains and in cultured fibroblasts of Depdc5(-/-) embryos, as reflected by enhanced phosphorylation of its downstream effectors S6K1 and rpS6. Consistently, prenatal treatment with mTORC1 inhibitor rapamycin rescued the phenotype of Depdc5(-/-) embryos. Heterozygous Depdc5(+/-) rats developed normally and exhibited no spontaneous electroclinical seizures, but had altered cortical neuron excitability and firing patterns. Depdc5(+/-) rats displayed cortical cytomegalic dysmorphic neurons and balloon-like cells strongly expressing phosphorylated rpS6, indicative of mTORC1 upregulation, and not observed after prenatal rapamycin treatment. These neuropathological abnormalities are reminiscent of the hallmark brain pathology of human focal cortical dysplasia. Altogether, Depdc5 knockout rats exhibit multiple features of rodent models of mTORopathies, and thus, stand as a relevant model to study their underlying pathogenic mechanisms.

Different mechanisms of ripple-like oscillations in the human epileptic subiculum.

OBJECTIVE: Transient high-frequency oscillations (HFOs; 150-600Hz) in local field potentials generated by human hippocampal and parahippocampal areas have been related to both physiological and pathological processes. The cellular basis and effects of normal and abnormal forms of HFOs have been controversial. This lack of agreement is clinically significant, because HFOs may be good markers of epileptogenic areas. Better defining the neuronal correlate of specific HFO frequency bands could improve electroencephalographic analyses made before epilepsy surgery. METHODS: Here, we recorded HFOs in slices of the subiculum prepared from human hippocampal tissue resected for treatment of pharmacoresistant epilepsy. With combined intra- or juxtacellular and extracellular recordings, we examined the cellular correlates of interictal and ictal HFO events. RESULTS: HFOs occurred spontaneously in extracellular field potentials during interictal discharges (IIDs) and also during pharmacologically induced preictal discharges (PIDs) preceding ictal-like events. Many of these events included frequencies >250Hz and so might be considered pathological, but a significant proportion were spectrally similar to physiological ripples (150-250Hz). We found that IID ripples were associated with rhythmic γ-aminobutyric acidergic and glutamatergic synaptic potentials with moderate neuronal firing. In contrast, PID ripples were associated with depolarizing synaptic inputs frequently reaching the threshold for bursting in most pyramidal cells. INTERPRETATION: Our data suggest that IID and PID ripple-like oscillations (150-250Hz) in human epileptic hippocampus are associated with 2 distinct population activities that rely on different cellular and synaptic mechanisms. Thus, the ripple band could not help to disambiguate the underlying cellular processes.

Synchrotron X-ray interlaced microbeams suppress paroxysmal oscillations in neuronal networks initiating generalized epilepsy.

Radiotherapy has shown some efficacy for epilepsies but the insufficient confinement of the radiation dose to the pathological target reduces its indications. Synchrotron-generated X-rays overcome this limitation and allow the delivery of focalized radiation doses to discrete brain volumes via interlaced arrays of microbeams (IntMRT). Here, we used IntMRT to target brain structures involved in seizure generation in a rat model of absence epilepsy (GAERS). We addressed the issue of whether and how synchrotron radiotherapeutic treatment suppresses epileptic activities in neuronal networks. IntMRT was used to target the somatosensory cortex (S1Cx), a region involved in seizure generation in the GAERS. The antiepileptic mechanisms were investigated by recording multisite local-field potentials and the intracellular activity of irradiated S1Cx pyramidal neurons in vivo. MRI and histopathological images displayed precise and sharp dose deposition and revealed no impairment of surrounding tissues. Local-field potentials from behaving animals demonstrated a quasi-total abolition of epileptiform activities within the target. The irradiated S1Cx was unable to initiate seizures, whereas neighboring non-irradiated cortical and thalamic regions could still produce pathological oscillations. In vivo intracellular recordings showed that irradiated pyramidal neurons were strongly hyperpolarized and displayed a decreased excitability and a reduction of spontaneous synaptic activities. These functional alterations explain the suppression of large-scale synchronization within irradiated cortical networks. Our work provides the first post-irradiation electrophysiological recordings of individual neurons. Altogether, our data are a critical step towards understanding how X-ray radiation impacts neuronal physiology and epileptogenic processes.