Long-term hypercapnia does not alter Galanin immunoreactivity in mouse brain respiratory and chemoreceptor regions.

Long-term hypercapnia has been implicated in respiratory disorders such as sleep apnea and chronic obstructive pulmonary disease (COPD). However, the mechanisms by which long-term hypercapnia contributes to central respiratory dysfunction in these conditions are unclear. The brainstem respiratory circuit, including the ventral respiratory column (VRC), comprises distinct neuronal populations that govern expiratory and inspiratory drive. The neuropeptide galanin is extensively distributed in brainstem chemoreceptor regions and the VRC, and microinjection of galanin into the VRC inhibits breathing and attenuates ventilatory reflex responses to acute hypercapnia. Nevertheless, the normal pattern of regional expression of galanin in the mouse brain is not known and the impact of long-term hypercapnia on galanin immunoreactivity (Gal-ir) in the vicinity of brainstem respiratory and chemoreceptor regions has not yet been studied. Therefore, we aimed to determine Gal-ir across the mouse brain and investigate the impact of prolonged hypercapnia exposure on Gal-ir in brainstem respiratory and chemoreceptor regions (pons and medulla). Adult male Phox2b-EGFP (MGI ID: 5776545) mice (31±2 g, n=5 mice/group) were exposed to hypercapnia (8% CO ; balance room air) or room air (control) for 10 or 14 days. To determine Gal-ir, brain slices were collected and processed for immunohistochemistry. Quantitative measurement of Gal-ir was performed using ImageJ 1.53a (a QuPath-integrated version of ImageJ) where a positive area fraction, reflecting the proportion of positively labeled pixels, was calculated for each region of interest [locus coeruleus, the retrotrapezoid nucleus, caudal-nucleus of the solitary tract (cNTS), rostral-NTS (rNTS), VRC, lateral reticular nucleus, and caudoventrolateral reticular nucleus]. Under control conditions, we found that Gal-ir was broadly present across the medulla oblongata, and abundant in the caudal aspects when compared with the rostral levels. The cNTS had a higher average percentage of Gal-ir area (p = 0.043, p = 0.05, 2-way ANOVA) compared to rNTS following 10- and 14-days of room air exposure, respectively. A significant difference in Gal-ir (p = 0.011, 2-way ANOVA) was observed between the cNTS and rNTS following 14-days of hypercapnia exposure. Overall, there was no significant difference of Gal-ir in all regions assessed after long-term hypercapnia compared to controls. Collectively, we found that long-term hypercapnia does not induce changes in galanin neuropeptide levels within respiratory and chemoreceptor regions in the mouse brain. Under control and hypercapnia conditions, cNTS contains higher Gal-ir compared to rNTS. Further investigation is required to elucidate the involvement of NTS-galaninergic subsets in respiratory homeostasis.