| ObjectiveThe mechanism of the development of temporal lobe epilepsy(TLE)is complex,current anti-epileptic drugs,based on traditional theory "excitation-inhibition imbalance",are only effective in seizure control,but infective in epileptogenesis.Previous studies demonstrated that basal forebrain is the main source of cholinergic input to the hippocampus,which is closed involved in epileptogenesis.However,as cholinergic neurons are largely heterogeneous based on their molecular and structural phenotype,the previous mechanism in TLE is still needed further clarified.As a cholinesterase inhibitor,Huperzine A(Hup A)has good antiepileptic activity in specific types of epilepsy,but whether its antiepileptic effect is related to the regulation of the function of the cholinergic neurons circuit in the brain is unclear.Thus,our project mainly to clarify the role and mechanisms of the subiculum-projecting cholinergic subgroup in the development of TLE and to evaluate the antiepileptic activity of Hup A and its correlation with the regulation of cholinergic system.MethodsIn the mouse hippocampal kindling model,using optogenetics,chemical genetics,in vivo optical fiber recording and classical pharmacological intervention firstly to investigate the role and characteristics of the cholinergic subgroup projected from the subiculum in the development of TLE.Further,the downstream neural circuits and molecular mechanism in the cholinergic subgroup projected from the subiculum in the development of TLE were elucidated by anterograde or retrograde trans-monosynaptic virus tracing labeling,molecular biology and cell-specific gene intervention.Finally,using several epileptic models combined with pharmacological intervention,electrophysiological and behavioral evaluation to study the role and mechanism of Hup A in TLE.ResultsFirst,we found that subiculum-projecting cholinergic subgroup is structural separative compared with the cholinergic subgroup projected to other hippocampal subregions by retrograde viral tracing.Then we found the release of acetylcholine transmitter in subiculum presents an inhibited state in epilepsy by fiber photometry recording.Further,we chose optogenetic methods selective active the dorsal subiculum(dSub)-projecting cholinergic subgroup promoted the formation and development of TLE from early stage,while selective active the ventral subiculum(vSub)-projecting cholinergic subgroup promoted the formation and development of TLE from the late stage.Combined with pharmacological methods,it was found that the development of TLE accelerated by the subiculum-projecting cholinergic subgroup is mediated by cholinergic receptors(mainly muscarinic).Then,we found MS/VDB cholinergic neurons have little project to dSub,and much more project to vSub,we chose immumohistochemical staining found that cholinergic neurons project to subicular mainly is glutamatergic neurons but not GABAergic neurons.Selective optogenetic stimulation of MS/VDB-vSub cholinergic circuit could activate vSub glutamatergic neurons,and chemo-genetic inhibition of vSub glutamatergic neuron reversed the effect of promoting TLE induced by activation of MS/VDB-vSub cholinergic circuit.Similarly,selective optogenetic stimulation of MS/VDB-dSub cholinergic circuit could activate dSub the Ca2+activity of astrocytes,and inhibition of the activity of astrocytes in dSub reversed the effect of promoting TLE induced by activation of MS/VDB-dSub cholinergic circuit.Finally,we evaluated the pharmacodynamics of Hup A in different epilepsy models,and found that Hup A both alleviates acute seizures and TLE,Furthermore,selective pharmacological interventions in different hippocampal subregions showed that Hup A may exert antiepileptic effects by acting on α7 nicotinic acetylcholine receptors of dCAl.ConclusionOur study found that the cholinergic subgroups projecting from the basal forebrain to different subregions of the hippocampus are heterogeneous in structural regulation and it also heterogeneous in functional regulation during epileptogenesis.The subiculum-projecting cholinergic circuit promoted the occurrence of TLE,while dSub mainly activated astrocytes and vSub mainly through glutaminergic neurons to mediated the mechanism of pro-seizure effects.The antiepileptic effect of Hup A is related to the regulation of cholinergic activity,but the antiepileptic effect is different in different brain regions.This study plays an important role in enriching the theoretical analysis of the disease mechanism of cholinergic involvement in the occurrence of TLE,and is expected to provide an important experimental basis for the clinical study of the target of precision medicine therapy for TLE. |
