Optogenetic Dissection Of Epilepsy-related Neural Circuits In Mice

Among human diseases,brain disorders account for up to 13% of all pathologies.Epilepsy is a prevalent neurological disorder associated with significant mortality risk,and principally known for harmful seizures.However,the mechanisms underlying epileptic seizures are still not fully understood,resulting the limited effect of clinical treatment.Temporal lobe epilepsy(TLE)is one of the most common epilepsy syndromes in adults.TLE profoundly alters hippocampus and entorhinal cortex structures in patients,and is most likely associated with many epilepsy symptoms such as epileptogenesis,seizures propagation,and epilepsy-induced brain dysfunctions.Previous work shows that neuronal excitation/inhibition imbalances are linked with seizures,while epileptogenesis could be due to hyper-synchronization of the hippocampal or parahippocampal excitatory neurons.It is generally accepted that epilepsy is a neural circuit-level phenomenon which may influence multiple brain regions including the hippocampus,entorhinal cortex,thalamus,amygdala,and motor cortex.Recent research suggests that seizures may propagate from temporal lobe to extensive regions of the cerebral cortex,and impairs consciousness consequently.Studies in brain slices imply that seizures propagate via an entorhinal cortex –hippocampal-entorhinal cortex(EC-HPC-EC)re-entrant loop,while studies in isolated brains demonstrate that seizures propagated within a HPC-EC-HPC re-entrant loop.Those discrepancies should be due to limitations in current research methodologies.Benefited from the recent advances of novel technologies in neuroscience research,especially the development of optogenetics,we now have the opportunity to identify the characteristics of neural circuitry of ictal propagation and epilepsy-induced brain dysfunctions.Combined these two aspects together,this work may shed light on future therapeutic treatments of TLE.The main findings are as follows:(1)In order to dissect the epilepsy related neural circuits,we developed electrophysiological techniques and methods,including: 1)electrochemically co-depositied poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate)/nerve growth factor/ dexamethasone phosphate/poly(vinyl alcohol)/poly(acrylic acid)interpenetrating polymer networks for improving neural/tissue interface for chronic studies in vivo;2)multi-functional optrode array combining KA injection with electrophysiological recording and optogenetic stimulation in vivo;3)improved method for multi-region electrophysiological recording(up to 8 brain regions)and optogenetic stimulation in vivo.(2)We applied optogenetic and electrophysiological methodologies to dissect the propagating direction of ictal seizures in the hippocampus-entorhinal cortex structures of KA-treated mice in vivo.We recorded spikes and LFPs in the dentate gyrus/hilus(DGH),medial entorhinal cortex(MEC),and primary motor cortex(M1)while simultaneously delivering optogenetic stimulation of DGH or MEC interneurons.By investigating the cross-area spike-triggered-average of LFPs(st LFPs),and the cross-correlation of instantaneous amplitudes of LFPs,we found a feed-forward propagation pathway of ictal discharges from the DGH to MEC,instead of a re-entrant loop.We demonstrated that selectively activating DGH GABAergic interneurons significantly inhibited ictal discharges in the DGH,MEC and M1,while optical stimulation in the MEC suppressed seizures only in local networks,not in the DGH or M1.Our data provide strong,direct evidence that the DGH dominates the propagation of ictal discharges.Furthermore,we also found that cyclic light delivery to the DGH shows a long-lasting inhibitory effect on ictal seizures,significantly reducing the frequency of behavioural seizures in freely moving animals.Our finding implies that DGH GABAergic interneurons can directly influence the propagation of seizures in the HP–EC circuitry,which may be a potential therapeutic target for TLE.(3)We found that the hippocampal structures of mice were notably altered after chronic TLE,and the animals exhibit significant reduced fear to predator.C-Fos mapping after predator fear test reveals that the hippocampal neurons are highly involved in innate fear response,implying that the epileptic hippocampal neurons may be attributed to the abnormal fear response.By using electrophysiological recording,optogenetic stimulation we found that there is a circuit from d HPC to hypothalamus via septum.And results from multi-region LFP recording during predator fear test suggested that hippocampus-hypothalamus circuit theta oscillations may contribute to predator fear behavior modulation,which were significantly suppressed in chronic TLE mice.Moreover,the c-Fos expression also showed that the neural activity of brain areas in hippocampus-hypothalamus circuit is significantly decreased compared with normal animal,which is consistent with electrophysiological results.Therefore,the hippocampus-hypothalamus circuit could be involved in epilepsy-related abnormal innate fear response.In summary,our improvement of electrophysiological and optogenetic techniques may facilitate in vivo dissection of neural circuits.The circuit-level study on the propagation of ictal seizures and the mechanisms of abnormal innate fear response may provide insight into the improved treatment for epilepsy,and contribute to understanding other neuropsychiatric disorders.