Plasticity Changes Of PV Neurons In Hippocampal CA1 Region Of Mice In Epilepsy Model

Epilepsy is a chronic brain dysfunctional disorder caused by sudden abnormal discharge of neurons.Abnormal discharge may be directly caused by the abnormal increase of excitability of pyramidal neurons,or indirectly caused by the decreased function of inhibitory interneurons（mainly containingγ-aminobutyric acid（GABA））.The hippocampus is the structural basis of learning and memory,and the stability of hippocampal neuron function is closely related to epilepsy.Pyramidal neurons（PNs）are regulated by interneurons（INs）to maintain homeostasis in the hippocampus.Most of the interneurons are GABAergic neurons,and parvalbumin（PV）neurons are one of the main subsets of GABAergic neurons.PV interneurons are activated to generate action potentials,target the soma,axon initial segment and proximal dendrites of pyramidal neurons,output inhibitory signals,and form a pericellular inhibitory effect on excitatory pyramidal neurons.Therefore,the alteration of GABAergic interneurons expressing PV is considered to be an important factor in the occurrence of epilepsy.It is known that PV neurons produce corresponding plastic changes to adapt to neural circuit reorganization in epilepsy.However,specific activation of PV neurons to study its mechanism in modulating hippocampal neural circuits is rarely reported.In this study,Nissl staining was used to observe the morphological and structural changes of neurons after epilepsy modeling.Furthermore,applying quantitative real-time PCR（q RT-PCR）and western blot（WB）,we first investigated the changes in gene and protein expression of PV neuron and GABAergic neuron marker GAD67 in the hippocampal CA1 region of the epilepsy models.Finally,combining optogenetics and whole-cell patch-clamp recordings,we provide direct evidence that presynapticγ-aminobutyric acid type B receptors（GABA_BRs）regulate the GABA release from PV interneurons onto CA1 pyramidal neurons.The experiment aims to provide new clues for the specific changes of PV neurons in the hippocampal CA1 region during epilepsy,and further explore new approaches for the treatment of epilepsy.Objective（1）To observe the morphological and structural features of neurons in epilepsy model.（2）To detect the expression of PV and GAD67 in hippocampal CA1 region in epilepsy model.（3）To explore the role and possible mechanism of PV neurons in hippocampal CA1 region in epilepsy.MethodsKainic acid（KA）was injected into the CA1 region of the mice hippocampus to induce an epilepsy model.A successful epilepsy model was defined with epileptic seizure behavior above gradeⅢand continuous epileptic seizure lasting more than 30minutes.The mice were executed at 3 days,1 week,2 weeks and 3 weeks after KA injection and tissue samples from the hippocampal CA1 region were collected.（1）Using Nissl staining to detect the morphological and structural changes of hippocampal neurons.（2）Using q RT-PCR to detect the m RNA expression level of PV and GAD67 in the hippocampal CA1 region of the mice epilepsy models.（3）Using WB to detect the protein expression of PV and GAD67 in the hippocampal CA1 region of the mice epilepsy models.（4）Using optogenetics and whole-cell patch-clamp recordings to specifically express and activate PV neurons and investigate the modulation of the GABA_BRs and voltage-gated calcium channels on the release of GABA in PV neurons terminal.Results（1）Nissl staining showed that the neurons in the hippocampal CA1 region of the control group were abundant and dense,while the neurons in the experimental group were reduced to a slightly different extent.The arrangement of neurons in the pyramidal layer of CA1 region was significantly disturbed,and some of them were morphologically deformed,showing cell shrinkage and reduction of Nissl bodies in the cytoplasm.（2）q RT-PCR showed that the m RNA expression of PV and GAD67 in the hippocampal CA1 region of mice was significantly reduced at all time points in the experimental group compared with the control group.（3）WB showed that the protein expression of PV and GAD67 in the hippocampal CA1 region of mice was significantly decreased at all time points in the experimental group compared with that in the control group.（4）Using optogenetics combined with whole-cell patch-clamp recordings,in the presence of the GABA_BRs agonist baclofen,it can significantly inhibited the amplitude of L-IPSCs（Light-evoked inhibitory postsynaptic currents）and reduced the release of GABA in PV neurons terminal.Based on the coupling of presynaptic membrane GABA_BRs to presynaptic membrane Ca²⁺channels,P/Q-type voltage-gated calcium channels are involved in the inhibition of GABA release from PV terminals in the presence ofω-agatoxin IVA,a specific P/Q-type calcium channel blocker.Conclusions（1）Nissl staining indicates that epilepsy induced differences in the morphological and structural characteristics of neurons in the CA1 region of the hippocampus and the plasticity changes may be related to the reorganization of neural network caused by epilepsy.（2）The low expression of PV and GAD67 in the hippocampus CA1 region of KA-induced epileptic mice may be related to the imbalance of excitation-inhibition neural circuit and the development of epilepsy.（3）The expression of GABA_BRs and calcium channels in PV neurons terminal and their function involved in GABA release from PV neurons may affect the occurrence and treatment of epilepsy.