Adaptation of Respiratory-Related Brain Regions to Long-Term Hypercapnia: Focus on Neuropeptides in the RTN.

Long-term hypercapnia is associated with respiratory conditions including obstructive sleep apnea, chronic obstructive pulmonary disease and obesity hypoventilation syndrome. Animal studies have demonstrated an initial (within hours) increase in ventilatory drive followed by a decrease in this response over the long-term (days-weeks) in response hypercapnia. Little is known about whether changes in the central respiratory chemoreflex are involved. Here we investigated whether central respiratory chemoreceptor neurons of the retrotrapezoid nucleus (RTN), which project to the respiratory pattern generator within the ventral respiratory column (VRC) have a role in the mechanism of neuroplasticity associated with long-term hypercapnia. Adult male C57BL/6 mice (n = 5/group) were used. Our aims were (1) to determine if galanin, neuromedin B and gastrin-releasing peptide gene expression is altered in the RTN after long-term hypercapnia. This was achieved using qPCR to measure mRNA expression changes of neuropeptides in the RTN after short-term hypercapnia (6 or 8 h, 5 or 8% CO2) or long-term hypercapnia exposure (10 day, 5 or 8% CO2), (2) in the mouse brainstem, to determine the distribution of preprogalanin in chemoreceptors, and the co-occurrence of the galanin receptor 1 (GalR1:Gi-coupled receptor) with inhibitory GlyT2 ventral respiratory column neurons using in situ hybridization (ISH) to better characterize galaninergic RTN-VRC circuitry, (3) to investigate whether long-term hypercapnia causes changes to recruitment (detected by cFos immunohistochemistry) of respiratory related neural populations including the RTN neurons and their galaninergic subset, in vivo. Collectively, we found that hypercapnia decreases neuropeptide expression in the RTN in the short-term and has the opposite effect over the long-term. Following long term hypercapnia, the number of RTN galanin neurons remains unchanged, and their responsiveness to acute chemoreflex is sustained; in contrast, we identified multiple respiratory related sites that exhibit blunted chemoreflex activation. GalR1 was distributed in 11% of preBötC and 30% of BötC glycinergic neurons. Our working hypothesis is that during long-term hypercapnia, galanin co-release from RTN neurons may counterbalance glutamatergic inputs to respiratory centers to downscale energetically wasteful hyperventilation, thereby having a role in neuroplasticity by contributing to a decrease in ventilation, through the inhibitory effects of galanin.