Acute Intermittent Hypoxia Preconditioning Elicits Age and Sex-Dependent Changes in Molecules Known to Regulate Phrenic Motor Plasticity

Acute intermittent hypoxia (AIH) elicits a form of respiratory motor plasticity known as phrenic long-term facilitation (pLTF); preconditioning with daily AIH (dAIH) enhances pLTF, demonstrating metaplasticity. Two additional factors impacting pLTF expression are age and sex: whereas moderate AIH-induced pLTF decreases with age in males, it increases in middle aged females. Unfortunately, little is known concerning mechanisms of dAIH preconditioning or age-dependent sexual dimorphism in pLTF. Moderate AIH elicits 2 distinct signaling cascades in phrenic motor neurons initiated by 5HT2A/2B receptors (Q pathway) versus adenosine 2A or 5HT7 receptors (S pathway), respectively. The Q and S pathways interact via mutual cross-talk inhibition, a powerful regulator of pLTF expression. To begin exploring the hypotheses that dAIH preconditioning and age/sex effects on pLTF can be accounted for by shifts in molecules regulating the Q and S pathways, we assessed mRNA of key molecules known to regulate pLTF in ventral cervical spinal homogenates from segments containing the phrenic motor nucleus from young (3 month) and middle-aged (12 month) male and female Sprague-Dawley rats. Rats (n=8/group) were exposed to sham or dAIH for 14 days (15, 1 min hypoxic episodes/day), and sacrificed 24 hours after the last AIH exposure. Tissues were harvested, preserved in RNA stabilizing solution (<10 min), and stored (-80°C) until mRNA extraction. mRNA levels of molecules that regulate pLTF were assessed via RT PCR, including: serotonin 2A (Htr2a) & 2B (Htr2b); adenosine 2A (Adora2a); exchange protein activated by cAMP (Epac); PKA catalytic subunit (Prkaa1); PKA regulatory subunit (Prkar1a); fractalkine (Cx3cl1); phosphodiesterase type 4 (Pde4b); Gp91 and p47(NOX genes); and the Pkcδisoform. We also quantified protein expression in phrenic motor neurons (via immunofluorescence) for BDNF and adenosine-1A & -2A receptors in rats fixed with 4% paraformaldehyde (n=4/group; analysis via MATLAB codes to quantify optical density in labeled motor neurons). We found significantly more Pde4b, Adora2a, and Pkcδ mRNA in young and older females versus age matched males; Epacwas also elevated, but only in young females (p<0.001). p47 was increased in older rats of both sexes (p<0.01). p47, Cx3cl1, Adora2a and Pkcδ mRNA were reduced by dAIH in older females (p<0.05), but not other groups. There were age-dependent increases in adenosine 1A (p <0.0001) and 2A (p <0.0001) receptors, and BDNF (p<0.001) protein post-AIH in both sexes. Thus, there are age, sex and dAIH preconditioning effects on molecules known to regulate the Q and S pathways to phrenic motor facilitation. These novel findings advance our understanding of pLTF, and inform translational research concerning the therapeutic potential of dAIH to treat breathing deficits in individuals of different ages or sex. © FASEB.