Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

We aimed to delineate sex-based differences in neuroplasticity that may be associated with previously reported sex-based differences in physiological alterations caused by repetitive succession of hypoxemia-reoxygenation encountered during obstructive sleep apnea (OSA). We examined long-term changes in the activity of brainstem and diencephalic cardiorespiratory neuronal populations induced by chronic intermittent hypoxia (CIH) in male and female mice by analyzing Fosb expression. Whereas the overall baseline and CIH-induced Fosb expression in females was higher than in males, possibly reflecting different neuroplastic dynamics, in contrast, structures responded to CIH by Fosb upregulation in males only. There was a sex-based difference at the level of the rostral ventrolateral reticular nucleus of the medulla, with an increase in the number of FOSB/ΔFOSB-positive cells induced by CIH in males but not females. This structure contains neurons that generate the sympathetic tone and which are involved in CIH-induced sustained hypertension during waking hours. We suggest that the sex-based difference in neuroplasticity of this structure contributes to the reported sex-based difference in CIH-induced hypertension. Moreover, we highlighted a sex-based dimorphic phenomenon in serotoninergic systems induced by CIH, with increased serotoninergic immunoreactivity in the hypoglossal nucleus and a decreased number of serotoninergic cells in the dorsal raphe nucleus in male but not female mice. We suggest that this dimorphism in the neuroplasticity of serotoninergic systems predisposes males to a greater alteration of neuronal control of the upper respiratory tract associated with the greater collapsibility of upper airways described in male OSA subjects.