Single-cell Reconstruction Reveals Input Patterns And Pathways Into Corticotropin-releasing Factor Neurons In The Central Amygdala

Corticotropin-releasing factor(CRF)is a peptide consisting of 41 amino acids mainly produced by the hypothalamus.It not only acts as an endocrine factor to regulate stress,digestion,reproduction,immunity in peripheral target organs through the classic hypothalamic-pituitary-adrenal axis.It also acts as a neuromodulator to regulate feeding,energy metabolism and emotional responses in the central nervous system.The basis behind functional diversity is morphological heterogeneity.The diversity of regulatory functions involved in CRF suggests that there may be heterogeneity in morphological features and neural circuits,but there are still few relevant systematic researches.In this study,by crossing CRF transgenic mice with Ai9/Ai14 red fluorescent reporter mice,the distribution and morphology of CRF neurons in the whole brain were specifically displayed at single-cell resolution,meanwhile,the differences in fluorescent protein expression between Ai9 and Ai14 were also systematically compared.The results showed that CRF neurons were widely distributed in the central nervous system,especially in the hypothalamus and striatum,where they were more densely distributed and showed morphological heterogeneity.In addition,it was found that the neurons labeled by Ai9 had higher brightness and the neurons labeled by Ai14 had higher signal-to-noise ratio.This finding provides an experimental basis for the strategy of selecting different reporter mice when studying the morphology of neurons.In the limbic system,apart from the hypothalamus,CRF neurons are also densely distributed in the central amygdala(CeA).The amygdala is the central nucleus for processing negative emotions such as fear and anxiety,and the CeA,as the main output nucleus of the amygdala,has complex neuronal composition and connectivity.However,little is known about the function of these densely distributed CRF neurons.Due to the limitation of experimental technology at that time,previous studies on neuronal input to the amygdala could not accurately trace a single type of neuron in the nucleus at the monosynaptic level.In this paper,by employing CRF-Cre transgenic mice,combined with a novel deficient rabies virus approach,the starter neurons were restricted to the CRF-expressing neurons.And all neurons,which monosynaptic input to CeA-CRF neurons at the whole-brain scale,could be specifically displayed at single-cell resolution through CLARITY technology,light-sheet imaging and three-dimensional reconstruction.For the first time,the monosynaptic input atlas to CeA-CRF neurons were quantitatively mapped in this work.The CeA-CRF neurons received inputs from 44 brain regions,which were clustered into 6 groups based on the correlation between each other.In addition to the inputs from amygdala,CeA-CRF neurons mainly received input from the piriform cortex,insular cortex,and caudoputamen.By calculating the input strengths of each nucleus,it was found that the top-down projections were mostly inputs in the form of convergence,while the bottom-up projections were mostly inputs in the form of diffusion,and by fluorescence in situ hybridization,it was found that the top-down inputs were mostly neurons expressing the excitatory neuron marker type ⅡCa2+/calmodulin-dependent protein kinase,while bottom-up inputs were mostly inhibitory neurons with the marker of glutamate decarboxylase.Through threedimensional imaging and single-cell reconstruction techniques,it was found that the morphological complexity of input neurons in the cortex was inversely proportional to their depth in the cortex,and the input neurons in the somatosensory cortex were mostly distributed in the fourth layer.Finally,by employing whole-brain-scale light-sheet imaging and in situ reconstruction techniques,the complete morphology of a single input neuron in the somatosensory cortex,dorsomedial thalamus,and periaqueductal gray were respectively demonstrated in a modified 3D-reference atlas of a mouse brain.Among them,a fiber pathway of a single input neuron was highlighted in the periaqueductal gray passing through the midbrain reticular nucleus,the parvicellular part of subparafascicular nucleus,the medial lemniscus,the zona incerta of thalamus,the subthalamic nucleus,the globus pallidus,the internal capsule,the caudate putamen,and finally reaching the CeA.The colocalization of the axons with the postsynaptic protein PSD-95 suggested the possibility of a new neural signaling of transmitting information through these nuclei to these en passant fibers.In conclusion,this study delineates the diversity of CRF neurons at the level of cell morphology and circuit connectivity and provides a structural basis for further studies on the functions and mechanisms involved in CeA-CRF neurons.In addition,the amygdala receives multimodal input,which provides a biological model for neuromorphic computing,and the in-situ display of en passant fiber and nucleus also provides a reference for craniocerebral surgery.