Nair, J.; Marciante, A. B.; Lurk, C.; Kelly, M. N.; Mitchell, G. S.

Acute intermittent hypoxia (AIH) elicits a form of respiratory motor plasticity known as phrenic long-term facilitation (LTF). Repetitive daily AIH (dAIH) exposure enhances phrenic LTF, demonstrating a form of metaplasticity. Two additional factors impacting phrenic LTF are age and sex. For example, moderate AIH-induced phrenic LTF decreases with age in males, but increases in middle-aged females. However, little is known concerning cellular mechanisms of dAIH effects or age-dependent sexual dimorphism in phrenic LTF. Moderate AIH elicits distinct signaling cascades within phrenic motor neurons initiated by 5HT2 receptors (Q pathway) versus adenosine 2A or 5HT7 receptors (S pathway), respectively. The Q and S pathways interact via mutual crosstalk inhibition, a powerful regulator of phrenic LTF. To test the hypothesis that dAIH, age and sex effects on phrenic LTF are associated with differential expression of molecules known to regulate the Q and S pathways, we assessed mRNA of key regulatory molecules in ventral cervical homogenates from spinal segments containing the phrenic motor nucleus from young (3 month) and middle-aged (12 month) male and female Sprague-Dawley rats. Since CNS estrogen levels impact molecules regulating the Q and/or S pathways, mRNA was correlated with serum estradiol. Rats (n=8/group) were exposed to sham (21% O2) or dAIH (15, 1 min episodes of 10.5% inspired O2 per day) for 14 days, and sacrificed 24 hours post-dAIH. mRNA for molecules known to regulate phrenic LTF were assessed via RT PCR, including: brain derived neurotrophic factor (Bdnf); serotonin 2A (Htr2a); 2B (Htr2b); and (Htr7) receptors; adenosine 2a (Adora2a) receptors; exchange protein activated by cAMP (Epac1); p38 MAP kinase [Mapk14 () & Mapk11 ({beta})]; PKA catalytic subunit (Prkaa1); PKA regulatory subunit (Prkar1a); fractalkine (Cx3cl1); phosphodiesterase type 4 (Pde4b); NAPDH-gp91 (Cybb) and p47 (ncf1); and the PKC{delta} isoform (Prkcd). Significantly higher Pde4b, Adora2a, and Prkcd mRNA were found in young and middle-aged females versus age-matched males; Epac1 was elevated, but only in young females (p<0.001). Ncf1 was increased in middle-aged versus young adult rats of both sexes (p<0.01). Ncf1, Cx3cl1, Adora2a and Prkcd mRNA were reduced by dAIH in middle-aged females (p<0.01), but not other groups. Serum estradiol levels positively correlated with Epac1 (r2=0.29, p=0.002), Mapk14 (r2=0.31, p=0.001), Mapk11 (r2=0.20, p=0.014), and Prkar1a (r2=0.20 p=0.013) mRNA. With higher serum estradiol levels, dAIH decreased Mapk14 mRNA (slope difference p=0.001). Thus, age, sex and dAIH preconditioning influence molecules known to regulate the Q and S pathways to phrenic motor facilitation. These novel findings advance our understanding of phrenic LTF, and inform translational research concerning the therapeutic potential of dAIH to treat breathing deficits in individuals of different ages or sex.