Methodological Approach for Immunohistochemical Analysis of Hypothalamic Orexin in Mice

Hyperphagia can develop secondary to hypercortisolism from stress or iatrogenic sources of glucocorticoids. Orexin (hypocretin) signaling in the brain plays a role in regulating adaptive behaviors including arousal, anxiety and feeding. Our aim was to localize hypothalamic orexin expression to explore its potential relationship with body mass index (BMI) changes following stress. Twenty 8-week-old C57BL/6 strain male mice were divided into four treatment groups of five mice. One group served as the non-stressed control. Each of the mice in the remaining three groups underwent a 1-hour conical tube restraint stress session once per day for five days. Following the fifth restraint stress period, all mice, including the controls, were euthanized. Weight, length, BMI and bioimpedance data including total body water, intracellular fluid, extracellular fluid, fat mass, free fat mass was recorded. Mice subjected to restraint stress showed reductions in BMI, likely due to decreased fat mass. We used immunohistochemistry to investigate the impact of restraint stress on orexin expression in the hypothalamus of mice and its potential implications for changes in BMI. Brain tissues from both control mice and stressed mice were extracted and then sectioned using a cryostat. Coronal brains sections were used to localize the hypothalamus. The primary antibody used was anti-orexin A antibody (ab6214 raised in rabbit). A fluorescently tagged secondary antibody (anti-rabbit; Alexa 488) binds to the primary antibody. Confocal microscope generated fluorescence emission indicates hypothalamic orexin expression, enabling the visualization and quantification of orexin within the brain tissue. Fluorescence specificity is verified with controls. This analysis provides a starting point to further the investigation of stress induced changes of orexin in mice exposed to restraint stress. This study provides the foundation for investigating stress-induced changes in orexin expression. Our aim is to further examine the potential link between orexin metabolic changes associated with stress. Grant received from UWL Undergrad Research and Creativity in Spring 2024. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.