Whole-brain Mapping Of Direct Inputs To Corticotropin Releasing Hormone Neurons In Anterior Cingulate Area

Corticotropin-releasing hormone(CRH),having neuroendocrine and neurotransmitter properties,is a critical neuromodulator of CNS stress response in mammalian,and is essential for several brain functions,including appetite control,immune function,cognitive and motor behavior.CRH neurons play a crucial role in the regulation of the hypothalamic–pituitary–adrenal(HPA)axis.The anterior cingulate area(ACA)is especially crucial for the performance of executive functions,error monitoring and emotional processing.Therefore,characterizing the whole brain afferent pathways of CRH neurons in the ACA can expand the field of knowledge about molecular functions and neural circuit mechanism of CRH neurons.However,current traditional neural network tracers have not provided comprehensive description of the input and output projections of specific neuronal types in the ACA,whereas conditional replication of the expressed neurotropic virus can achieve that.Here,we utilized rabies virus-based monosynaptic retrograde tracing system and CRH-IRES-Cre mice to map the whole-brain afferent presynaptic partners of the CRH neurons in the ACA.The study found that the ACA CRH neurons integrate information from the cortex,thalamus,basal forebrain,amygdala,midbrain,hindbrain,and several other brain regions.In addition,we performed immunochemical staining with choline acetyltransferase(ChAT)to identify cholinergic neurons in the NDB,and the results showed that about 30% rabies-labeled neurons in the NDB were ChAT positive(cholinergic cells).At the same time,we used tyrosine hydroxylase(TH)and tryptophan hydroxylase 2(Tph2)to characterize the DA neurons and the 5-HT neurons in the ventral tegmental area(VTA),median raphe(MR)and dorsal raphe(DR),respectively.The results showed that:(1)None of the rabies-labeled cells are TH positive in the VTA;(2)Approximately 21% and 73% rabies-labeled neurons in the MR and DR were Tph2 positive,respectively.Taken together,our viral tracing results efficiently mapped a comprehensive list of monosynaptic inputs to the ACA CRH neurons,and revealed a comprehensive atlas of the presynaptic partners of the ACA CRH neurons.Furthermore,we showed that the cholinergic system and serotoninergic system in the basal forebrain and raphe nuclei,respectively,provide neuromodulatory inputs to the ACA CRH neurons.These findings together expand our knowledge about the connectivity of the cortical GABAergic neurons and provide a foundation for future exploration of the circuit function of cortical CRH neurons.