Corticotropin-releasing hormone (CRH) neurons in the paraventricular hypothalamic nucleus (PVH) are in the position to integrate stress-related information and initiate adaptive neuroendocrine-, autonomic-, metabolic- and behavioral responses. In addition to hypophysiotropic cells, CRH is widely expressed in the CNS, however their involvement in the organization of the stress response is not fully understood. In our experiments, we used the recently available Crh-IRES-Cre;Ai9 mouse line to study the recruitment of hypothalamic and extrahypothalamic CRH neurons in categorically distinct, acute stress reactions. To investigate the role of CRHPVH neurons in the initiation of stress reaction, we compared hormonal, behavioral and molecular responses of mice in which CRHPVH neurons were chemogenetically activated with those exposed to single or repeated restraint. Differences of acute-, chronic variable (CVS)- and chronic repeated restraint (CRS)-induced stress cascade features were also examined. 95 brain regions in the adult male mouse brain have been identified as containing putative CRH neurons with significant expression of tdTomato marker gene. With comparison of CRH mRNA and tdTomato distribution, we found match and mismatch areas. Reporter mice were then exposed to restraint, ether, high salt, lipopolysaccharide or predator odor stressors and neuronal activation was revealed by FOS immunohistochemistry. In addition to a core stress system, stressor-specific areas have been revealed to display activity marker FOS. A stressor-specific differential activation pattern was observed in CRH neurons in extrahypothalamic regions. Comparison of acute selective chemogenetic CRHPVH activation and acute restraint-induced stress cascade revealed several neurons, whose activation is CRHPVH-independent and/or are situated upstream to CRHPVH neurons. Hormonal and behavioral responses induced by repeated activation of CRHPVH differed from those, which were produced by repeted restraint stress, highlighting the importance of additional brain regions in organizing the stress response. In chronic stress (CRS and CVS) the expression of FOS was desensitized, while the mRNA of chronic markers (Fosl2 and Fosb) was differentially activated. Comprehensive description of stress-related CRH neurons in the mouse brain provides a starting point for a systematic functional analysis of the brain stress system and its relation to stress-induced psychopathologies.
