Cell-type Specific Neural Circuit Mapping And Functional Identification In The Anterior-dorsal Bed Nucleus Of The Stria Terminalis

The bed nucleus of the stria terminalis(BNST)is a highly heterogeneous limbic forebrain region that processes stress and reward.BNST is characterized by its extensive functional connections with multiple brain structures to associate the external stimuli with the behavioral feedback,which contributes to the expression and regulation of various emotional states,such as anxiety.Anxiety disorder is a common mental disease with mood disorder,and harms the individual physical and mental health in recent years.Rodent studies have found that pathological changes in different subregions of BNST and its neural circuits are the basis for the formation and development of anxiety disorders.Notably,specific anxiety-related behaviors have been demonstrated to mitigate by lesions of the anterior-dorsal BNST(Ad BNST).Previous neuroanatomical work has shown that there are various neuronal subpopulations of neurotransmitters and neuropeptides in the Ad BNST,such as the corticotropin-releasing hormone-positive(CRH+)and the protein kinase C-?-positive(PKC-?+).Specifically,the existence of CRH+ neurons in the Ad BNST is thought to be tightly linked with the regulation of anxiety.However,the underlying neural circuit mechanism remains elusive.Therefore,comprehensively understanding the specific cell types that functioned in regulating anxiety state and their connectivities that exist in the Ad BNST would help to extend the neural circuit mechanisms involved in the formation and development of anxiety disorders,and also provide new theoretical targets for its clinical treatment.In this study,we combine transgenic mice,neural circuit tracing,ex vivo electrophysiology,in vivo calcium imaging and chemogenetics to investigate the specific cell types and the local and long-range connections from Ad BNST,as well as the roles of the Ad BNST in regulating anxiety-like behaviors.The main conclusions are summarized as follows:(1)Two distinct neuronal subpopulations(CRH+ and PKC-?+)were identified in the Ad BNST by immunofluorescent staining.Paired recordings,as well as electrophysiological recordings via optogenetic stimulation,were performed to examine the local networks of the CRH and PKC-? neurons in Ad BNST.The results showed that the synaptic strength was significantly different between the two cell subpopulations,which indicates the pathway of functional regulation by the CRH and PKC-? neurons in Ad BNST.(2)By using the rabies-virus-based(RV)trans-synaptic retrograde tracing and Credependent adeno-associated(AAV)virus anterograde tracing strategy,we elucidated the whole-brain input and output mapping of the CRH+ and PKC-?+ neurons in Ad BNST.The results showed that the two cell types had similar input patterns,and exhibited significant differences in the projection density of some efferent brain regions.These results provided important anatomical basis for the research on the specific neural mechanism of regulating anxiety.(3)It is clear that the critical roles of Ad BNST CRH+ and PKC-?+ neurons in regulating anxiety.In vivo calcium imaging revealed the two cell types were both activated by the anxiety-related aversive stimuli,thus demonstrating that they are closely related to the regulation of anxiety.Chemogenetic manipulation of CRH+ and PKC-?+neurons had opposing effects on anxiety-like behaviors,further suggesting the functional heterogeneity within the two specific Ad BNST cell types.In summary,by investigation of the structure and function of two specific cell types(CRH+ and PKC-?+)in the Ad BNST,we concluded that,the two types of neurons play significant but independent roles in regulating anxiety-like behaviors.Our research may provide new insights in clinical treatment of anxiety disorder,and also shed light on the future studies of neural circuit mechanism.