CO2 exposure enhances Fos expression in hypothalamic neurons in rats during the light and dark phases of the diurnal cycle

Orexinergic (OX) neurons in the lateral hypothalamus (LH), perifornical area (PFA) and dorsomedial hypothalamus (DMH) play a role in the hypercapnic ventilatory response, presumably through direct inputs to central pattern generator sites and/or through interactions with other chemosensitive regions. OX neurons can produce and release orexins, excitatory neuropeptides involved in many functions, including physiological responses to changes in CO2/pH. Thus, in the present study, we tested the hypothesis that different nuclei (LH, PFA and DMH) where the orexinergic neurons are located, show distinct activation by CO2 during the light-dark cycle phases. For this purpose, we evaluated the Fos and OXA expression by immunohistochemistry to identify neurons that co-localize Fos+OXA in the LH, LPeF, MPeF and DMH in the light-inactive and dark-active phase in Wistar rats subjected to 3 hours of normocapnia or hypercapnia (7% CO2). Quantitative analyses of immunoreactive neurons show that hypercapnia caused an increase in the number of neurons expressing Fos in the LH, LPeF, MPeF and DMH in the light and dark phases. In addition, the number of Fos+OXA neurons increased in the LPeF and DMH independently of the phases of the diurnal cycle, whereas in the MPeF, this increase was observed exclusively in the light phase. Thus, we suggest that OX neurons are selectively activated by hypercapnia throughout the diurnal cycle, reinforcing the differential role of nuclei in the hypothalamus during central chemosensitivity.

位于外侧下丘脑（lateral hypothalamus, LH）、穹窿周区（perifornical area, PFA）以及背内侧下丘脑（dorsomedial hypothalamus, DMH）的食欲素能（Orexinergic, OX）神经元参与高碳酸血症通气反应，其作用机制推测为通过直接向中枢模式发生器（central pattern generator）位点输入信号，或是与其他化学感受区域产生相互作用。食欲素能神经元可合成并释放食欲素——一类参与多种生理功能的兴奋性神经肽，其功能涵盖对CO₂/pH变化的生理应答。因此，本研究旨在验证下述假说：分布于外侧下丘脑、穹窿周区与背内侧下丘脑的食欲素能神经元，其被CO₂激活的程度会随昼夜明暗周期的不同阶段存在差异。
为此，我们采用免疫组织化学技术检测Fos与食欲素A（Orexin A, OXA）的表达，以识别在Wistar大鼠的外侧下丘脑、外侧穹窿周区（lateral perifornical area, LPeF）、内侧穹窿周区（medial perifornical area, MPeF）以及背内侧下丘脑中共表达Fos与OXA的神经元。实验设置了静息光明期与活动黑暗期两个时间点，受试大鼠分别接受3小时的正常碳酸血症或高碳酸血症（7% CO₂）处理。
对免疫反应性神经元的定量分析显示：在静息光明期与活动黑暗期，高碳酸血症均可使外侧下丘脑、外侧穹窿周区、内侧穹窿周区以及背内侧下丘脑内表达Fos的神经元数量显著增加。此外，无论昼夜周期处于哪个阶段，外侧穹窿周区与背内侧下丘脑内的Fos+OXA双阳性神经元数量均有所上升；而在内侧穹窿周区，该现象仅见于静息光明期。
据此我们认为，食欲素能神经元在整个昼夜周期中可被高碳酸血症选择性激活，这进一步证实了下丘脑不同核团在中枢化学感受过程中发挥着差异化的作用。