Roles Of Piriform Cortex’s Epileptic State-related Neuronal Ensembles In Temporal Lobe Epilepsy

Epilepsy is a common chronic central nervous system disorder with poor clinical management and prognosis.Temporal lobe epilepsy(TLE),in which the hippocampus is the main focus,is the most common type of refractory epilepsy.Epilepsy is characterized as two states:seizures and interictal periods.Seizures are caused by abnormal synchronous neuronal discharges in one or more areas of the brain and are unpredictable,recurrent,and transient in nature;the interictal period,the period between seizures,occupies most of the time as a pathological state for people with epilepsy.Previous studies have shown that seizures and interictal periods may be generated by different neural circuits and cellular mechanisms.However,it is not clear how interictal and ictal phases are interconverted and influenced by each other.The piriform cortex(Pir)plays a key role in the generation and propagation of seizures and is also the brain region where interictal discharges originate.In this study,we will focus on exploring the roles and mechanisms of neuronal populations in the Pir associated with ictal and interictal states in TLE.We first used an early gene-driven cell labelling technique,the enhanced synaptic activity-responsive element E-SARE,to label focal seizure-related ensemble(FS-Ens)and interictal period-related ensemble(IP-Ens)in the Pir in the mouse hippocampal kindling model.Immunohistochemical experiments showed that FS-Ens were more numerous and mainly distributed in the deeper layers of Pir,while IP-Ens were less numerous and mainly distributed in the superficial layers of Pir,and both had similar neuronal composition types,mostly glutamatergic neurons.Reverse trans-monosynaptic viral tracing showed no significant differences in the upstream inputs of the two ensembles.These results suggest that FS-Ens and IP-Ens are two non-overlapping neuronal subpopulations distributed in different subregions of Pir.To observe the function of these two ensembles in seizures,we used in vivo calcium fiber recording and found that FS-Ens showed a significant increase in calcium activity during FS,while IP-Ens showed a decreasing trend in calcium activity during FS,suggesting that IP-Ens and FS-Ens have different functional responses during FS.Further,we explored the effects of these two ensembles on seizures by optogenetic manipulations,and found that direct photoactivation of FS-Ens induced seizures,whereas photoactivation of IP-Ens did not,suggesting that these two ensembles have different epileptogenic capacities.Interestingly,activation of a small group of FS-Ens had a stronger epileptogenic effect than activation of all glutamatergic neurons in the Pir,suggesting that FS-Ens may be the actual causative neurons responsible for the seizure onset and other endogenous antiepileptic subpopulations may exist in Pir.Further,we found that prior photoactivation of IP-Ens during the interictal period significantly retarded seizure development,while photoinhibition of IP-Ens promoted it,suggesting that IP-Ens can bidirectionally regulate the process of seizure formation.However,bidirectional modulation of IP-Ens had no effect on generalized seizure(GS)secondary to TLE formation,suggesting that IP-Ens plays a key regulatory role in the early FS phase of epilepsy rather than the later GS phase,and that IP-Ens has a "time window" of intervention.To explore the functional evolution of IP-Ens during the progression of epilepsy formation from FS to GS,we used calcium fiber recording and immediate early gene(IEG)staining,and the results showed that there is functional activation of IP-Ens in GS;by means of reverse trans-monosynaptic viral tracing,IP-Ens received more upstream cellular inputs when it moved from FS to GS,suggesting that there is neural circuit reorganization of IP-Ens in the later GS phase.Next,we analyzed the functional connectivity between the Pir and hippocampus during FS and GS,and found that prior activation of IP-Ens decreased coherence between Pir and hippocampus during FS but not GS.,which partly explains why IP-Ens lost its antiepileptic effect during GS.Finally,to explore the generalizability of the therapeutic implications of targeting IPEns,we introduced the kainic acid(KA)-induced seizure model to further validate the function of these two ensembles and found that activation of Seizure-Ens,but not IP-Ens,induced seizures;meanwhile,pharmacogenetic activation of IP-Ens attenuated KAinduced acute seizures.These results suggest that IP-Ens can be applied as an antiepileptic target in other animal models of epilepsy.Taken together,this study reveals for the first time the role of the Pir’s neuronal ensembles associated with epileptic states in epilepsy.Our results show that activation of Seizure-Ens but not IP-Ens can induce seizures,and prior activation of IP-Ens can produce anti-epileptic effects through desynchronization,suggesting that IP-Ens may be an effective target for clinical precision intervention in TLE formation.