Single-cell reconstruction reveals input patterns and pathways into corticotropin-releasing factor neurons in the central amygdala in mice

ARTICLE https://doi.org/10.1038/s42003-022-03260-9 OPEN Single-cell reconstruction reveals input patterns and pathways into corticotropin-releasing factor neurons in the central amygdala in mice 1234567890():,; Chuan Huang Guo-Qiang Bi 1,2 ✉, Yu Wang1,2, Peng Chen1,2, Qing-Hong Shan1,2, Hao Wang3,4, Lu-Feng Ding1,2, 1,2 & Jiang-Ning Zhou 1,2 ✉ Corticotropin-releasing factor (CRF) neurons are one of the most densely distributed cell types in the central amygdala (CeA), and are involved in a wide range of behaviors including anxiety and learning. However, the fundamental input circuits and patterns of CeA-CRF neurons are still unclear. Here, we generate a monosynaptic-input map onto CeA-CRF neurons at single-cell resolution via a retrograde rabies-virus system. We find all inputs are located in 44 nested subregions that directly innervate CeA-CRF neurons; most of them are top-down convergent inputs expressing Ca2+/calmodulin-dependent protein kinase II, and are centralized in cortex, especially in the layer 4 of the somatosensory cortex, which may directly relay information from the thalamus. While the bottom-up divergent inputs have the highest proportion of glutamate decarboxylase expression. Finally, en passant structures of single input neuron are revealed by in-situ reconstruction in a modified 3D-reference atlas, represented by a Periaqueductal gray-Subparafascicular nucleus-Subthalamic nucleus-Globus pallidus-Caudoputamen-CeA pathway. Taken together, our findings provide morphological and connectivity properties of inputs onto CeA-CRF neurons, which may provide insights for future studies interrogating circuit mechanisms of CeA-CRF neurons in mediating various functions. 1 Hefei National Laboratory for Physical Sciences at the Microscale, Chinese Academy of Sciences Key Laboratory of Brain Function and Diseases, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, PR China. 2 Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China. 3 National Engineering Laboratory for Brain-inspired Intelligence Technology and Application, University of Science and Technology of China, Hefei, China. 4 Institute of Artificial Intelligence, Hefei Comprehensive National Science Center, Hefei, China. ✉email: ; COMMUNICATIONS BIOLOGY | (2022)5:322 | https://doi.org/10.1038/s42003-022-03260-9 | www.nature.com/commsbio 1 ARTICLE T COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-022-03260-9 he amygdala has been demonstrated to play a critical role in a range of brain functions—including emotions, learning, memory, attention, and perception—especially in terms of processing environmental stimuli associated with fear and reward1. It has been posited that the complex anatomy of the amygdala and its diverse neuronal subtypes confer a wide variety of important functions2. Furthermore, increased attention has recently been devoted to elucidating specific amygdalar circuits and their corresponding functions1. However, different regions of the amygdala have unique connections with other brain structures3, and different molecularly defined neurons in the amygdala undertake distinct functions in various behaviors. The refined but basic neural circuits and input patterns to the amygdala remain unknown, and their identification may provide guidance for a better understanding of the behaviors in which the amygdala is involved. The basolateral amygdala (BLA) and central amygdala (CeA) are two major nuclei that play essential roles in various behaviors. Substantial information processing occurs between the BLA and CeA. As the main integrated input nucleus into the amygdala, the BLA receives inputs from upstream loci and transmits this information to the CeA. In contrast, the CeA acts as the output nucleus of the amygdala, such that it innervates several downstream brain regions that enable the body to adaptively respond to external stimuli4. However, in addition to receiving information from the BLA, the CeA also receives direct inputs from both the thalamus and cortex, and contributes to the expression of innate behaviors and associated physiological responses5. The CeA comprises a wide array of molecularly distinct cell types, which play different roles in amygdala-mediated behaviors6. In addition to neuronal subtypes expressing either somatostatin (SST) or protein kinase C-δ (PKC-δ) in inhibitory circuits encoding fear7, peptide-expressing neurons in the CeA have recently been investigated8. Corticotropin-releasing factor (CRF) is a stress-related peptide that is expressed in a large subpopulation of CeA neurons9. CRF acts as an endocrine factor to regulate stress responses via the hypothalamic–pituitary–adrenal axis, and acts as a neuromodulator in the central nervous system to regulate food intake, energy metabolism, and emotional responses10–12. Interestingly, CeA-CRF neurons represent one of the most densely distributed populations of CRF neurons throughout the brain13, which has attracted the attention of many research groups14,15. By employing CRF-Cre mice or rats, it has been discovered that they are involved in mediating stress16, pain17, alcohol addiction18, and fear19,20. In addition, by injecting the rabies virus, a fraction of putative excitatory input brain regions were retrogradely traced in previous research, which is consistent with the current data set21. However, so far there is no study that systematically elaborates the input of CeA-CRF neurons at the whole-brain scale. To elucidate the functions of CeA-CRF neurons, it is necessary to comprehensively dissect their connectivity. The amygdala sends projections from the CeA to the stria terminalis, basal forebrain, various hypothalamic nuclei, midline thalamic nuclei, and the brainstem22. Major efferent pathways of the amygdala to subcortical destinations of the limbic system include the stria terminalis, which travels along the lateral aspect of the fornix and through the caudothalamic groove and terminates in the bed nucleus of the stria terminalis. The ventral amygdalofugal pathway is another important efferent pathway from the amygdala that originates from the BLA and CeA and connects to the striatum, namely to the nucleus accumbens, as well as to the basal forebrain, medial dorsal nucleus of the thalamus, and lateral hypothalamus23. As for the afferent pathways of the amygdala, fibers carrying inputs into the amygdala exhibit a considerable correspondence with efferent fibers carrying outputs from the amygdala. The amygdala receives information from all sensory 2 inputs—which originate from the olfactory bulb and temporal/ anterior cingulate cortices—and also receives visceral inputs, which are transmitted from the hypothalamus, septal area, orbital area, and parabrachial nucleus22. Here, we employed a restricted rabies virus system to provide a systematic dissection of whole-brain monosynaptic inputs onto CeA-CRF n (...truncated)