Role And Mechanisms Of MSDB-vSub Cholinergic Circuit In Depression-like Behaviors Of Mice

Background:Major depressive disorder(MDD)is one of the most severe mental disorders characterized by persistent sadness and emotional numbness,usually comorbid with cognitive dysfunction and physical symptoms,and affects>10%of the worldwide population during their lifetime.According to the structural neuroimaging and postmortem studies,the frontal lobe and hippocampus are the most common regions manifesting a total volume decrease in MDD patients.Subiculum is the main subregion that controls output connections of the hippocampus.However,much less is known about the mechanisms of action of the subiculum in the brain network of MDD patients.Accumulating evidence suggests that the cholinergic system in the basal forebrain is critical in mood regulation.Both human and animal studies have shown that acetylcholine(ACh)is increased in the brain during MDD.Differing from the nucleus basalis magnocellularis in the forebrain targeting the cortex,the cholinergic neurons in the medial septum and diagonal band of Broca(MSDB)densely project to the hippocampus.However,the connection and function of MSDB cholinergic and vSub are poorly understood.Objective:In this study,we aimed to assess the activation of ventral subiculum(vSub)pyramidal neurons in the mouse depression model of chronic unpredictable mild stress(CUMS).To explore the role of MSDB-vSub cholinergic circuit in MDD and the underlying pathophysiological mechanisms.To provide insight into a new strategy to treat MDD through modulating ACh neurotransmitters for the treatment of depression.Methods:CUMS was used to construct a mouse depression model.Depression-like and anxiety-like behaviors were evaluated by sucrose preference test,tail suspension test,forced swimming test and open field test.Immunofluorescence staining,whole cell patch clamp electrophysiology and in vivo electrophysiology recording were used to test the neural activity and synaptic transmission of pyramidal neurons in vSub.The fluorescent ACh or GABA sensor was applied to monitor the change of cholinergic or GABAergic transmission in vSub.Retrograde and anterograde tracing to confirm the direct innervation of vSub by MSDB cholinergic neurons.Following the activation/inhibition of MSDB-vSub cholinergic circuits through virus injection along with chemogenetic manipulation,we investigated the role and mechanism of the circuit in depression-like behaviors.Results:(1)Chronic unpredictable mild stress could induce typical depression-like behaviors.(2)We found CUMS induced the vSub pyramidal neurons hyperactivation and excitation/inhibition imbalance by c-fos co-staining and whole-cell patch-clamp recording.(3)Chemogenetic stimulating vSub excitability neurons induce depression-like behaviors.(4)By retrograde and anterograde tracing,we confirmed the dense MSDB cholinergic innervation of the vSub.(5)Immunofluorescence staining was used to confirm MSDB cholinergic neurons could co-release GABA.(6)CUMS increased the ACh signal in CUMS mice by ACh sensor.(7)Chemogenetic stimulation of this MSDB-vSub innervation induced hyperactivation of vSub pyramidal neurons along with depression-like behaviors.(8)The depression-like behaviors induced by chemogenetic stimulation of this pathway and CUMS were reversed by local infusion of atropine(a muscarinic receptor antagonist)into the vSub.(9)CUMS decreased the GABA signal in CUMS mice by GABA sensor.(10)Chemogenetic inhibiting MSDB-vSub cholinergic pathway induces vSub glutamatergic hyperactivation and depression-like behaviors.(11)Downregulating GABA co-release by expressing Cre-dependent AAV-sh-v GAT in MSDB cholinergic neurons of Ch AT-Cre mice induced depression-like behaviors with vSub hyperactivation.(12)Infusion of a GABA_A receptor agonist in vSub attenuated the depression-like behaviors of mice.Conclusions:CUMS induces hyperactivation of vSub neurons.MSDB-vSub cholinergic pathway induces vSub pyramidal neurons hyperactivation and depression-like behaviors through excessive ACh or insufficient GABA release in vSub.Atropine or muscimol reverses the hyperactivation of vSub pyramidal neurons induced by activating or inhibiting the MSDB-vSub cholinergic pathway and reduces depression-like behaviors.