A Cholinergic Medial Septum-hippocampal Pathway Mediates The Development Of Temporal Lobe Epilepsy

Epilepsy is a prevalent neurological disorder with recurrent unprovoked seizures.Insight into the neural mechanisms of TLE has previously focused mainly on the canonical balance of excitatory glutamate and inhibitory gamma-aminobutyric acid(GABA).The main anti-epileptic drugs also act on inhibiting glutamatergic transmission and promoting GABAergic transmission.However,current anti-epileptic drugs based on above mechanisms often fail to control seizures and even result side effects.Moreover.temp oral lobe epilepsy(TLE)is the most common type of refractory epilepsy with 75%resistant to anti-epileptic drugs.Therefore,it is urgent to explore non-traditional mechanisms and related cirucits to find more effective interventions and drug targests.Here,combine optogenetics,chemogenetics,calcium signaling recording,in vivo single unit recording,in vitro electrophysisology recording,and retrograde trans-monosynatpic tracing,we revealed a meidial septum(MS)cholinergic circuit that potently attenuates seizure in TLE.Data from magnetic resonance imaging(MRI)and diffusion tensor imaging(DTI)verified that TLE patients with hippocampal sclerosis had a smaller volume of MS compared to healthy controls,and the connectivity between MS and pathological ipsilateral hippocampus reduced compared with that between MS and contralateral hippocampus.Above results indicated that MS might have a correlation with human TLE.Then,in the mouse TLE model,we electrically destroyed the MS of WT mice and found that it promoted the progression of behavioral seizure stage and prolonged after-discharge duration(ADD),which indicated that MS might have endogenous anti-epiletic componentTo investigate the endogenous anti-epileptic component in MS,we optogenetically activated cholinergic,glutamatergic and GABAergic neurons respectively,and found that only activation of cholinergic neurons retarded the development of epilepsy.To further define the role of MS cholinergic neurons in epilepsy,we used calcium fiber photometry and single-unit recordings,and found that MS cholinergic neurons ceased firing during hippocampal seizures.In the hippocampal kindling TLE model,optogenetic activation of MS cholinergic neurons significantly retarded the development of behavioral stages and shortened the ADD.In the intra-hippocanpal kainic acid(KA)-induced chronic TLE model,chemogenetic prolonged activation of MS cholinergic neurons significantly decreased the total number and duration of spontaneous recurrent seizures.While specific inhibition or kill MS cholinergic neurons prior to kindling both promoted progression of behavioral seizure and prolonged ADD.These results demonstrated that MS cholinergic neurons bidirectionally modulate hippocampal seizures and show sufficient and nessary effect on TLEWe further explore the MS cholinergic related anti-seizure ciruit.MS cholinergic neurons could directly project to hippocampus and also give rise to local intra-MS connections to GABAergic and glutamatergic neurons which further providing projections to the hippocampus.We found that direct photo-stimulation in the CA1 showed an inhibitory effect on the development of hippocampal seizures and the effect can be reversed by a prior focal injection of blocker cocktails of acetylcholine receptor antagonists.Further,we found that both M and N cholinergic receptors were involved in the anti-seizure effect.However,blocker cocktails did not reverse the anti-seizure effect of optogenetic activation of MS cholinergic neurons.Above experiments indicated that the direct,but not the indirect MS-hippocampal cholinergic projections,mediated the anti-seizure effect,which also depended on both M and N receptors.To investigate how the activation of MS cholinergic neurons modulate local hippocampal neurons.In vivo/in vitro electrophysiology combined with modified rabies virus tracing studies showed that direct MS-hippocampal cholinergic projections preferentially targeted hippocampal GABAergic neurons and functionally activated them which mediated the anti-seizure effect.Moreover,we found that chemogenetic inhibition of hippocampal GABAergic neurons abrogated the anti-seizure effect of optogenetic activation of cholinergic projections in the dorsal hippocampal CA1.Above results indicated that hippocampal GABAergic neurons are downstream effector of anti-seizure MS-hippocampal cholinergic circuit.Moreover,due to the large diversity of GABAergic neurons in the hippocampus,we further investigated whether parvalbumin(PV)and somatostatin(SST)positive neurons,two major subtypes of GABAergic neurons,were differently targeted by the MS cholinergic input.We found that the chemogenetic inhibition of hippocampal SST rather than PV neurons reversed the anti-seizure effect of MS-hippocapus cholinergic circuit,which was mimicked by activating SST neurons.Together,these findings underscore the notable anti-seizure role of the direct cholinergic MS-hippocampus circuit in TLE through driving downstream SST effector.Collectively,our results revealed a neural circuit,originating from MS cholinergic neurons to hippocampal SST positive GABAergic neurons,involved in TLE.This may provide a better understanding of pathological network changes and the precise target for anti-epileptic drugs and interventions.