Daily Acute Intermittent Hypoxia Elicits Age & Sex-Dependent Changes in Molecules Regulating Phrenic Motor Plasticity

Abstract

ABSTRACTAcute 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)versusadenosine 2A or 5HT7 receptors (S pathway), respectively. The Q and S pathways interactviamutual 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 O2per day) for 14 days, and sacrificed 24 hours post-dAIH. mRNA for molecules known to regulate phrenic LTF were assessedviaRT 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(α) &Mapk11(β)]; PKA catalytic subunit (Prkaa1); PKA regulatory subunit (Prkar1a); fractalkine (Cx3cl1); phosphodiesterase type 4 (Pde4b); NAPDH–gp91 (Cybb) and p47 (ncf1); and the PKCδ isoform (Prkcd). Significantly higherPde4b,Adora2a, andPrkcdmRNA were found in young and middle-aged femalesversusage-matched males;Epac1was elevated, but only in young females (p<0.001).Ncf1was increased in middle-aged versus young adult rats of both sexes (p<0.01).Ncf1, Cx3cl1,Adora2aandPrkcdmRNA were reduced by dAIH in middle-aged females (p<0.01), but not other groups. Serum estradiol levels positively correlated withEpac1(r2=0.29, p=0.002),Mapk14(r2=0.31, p=0.001),Mapk11(r2=0.20, p=0.014), andPrkar1a(r2=0.20 p=0.013) mRNA. With higher serum estradiol levels, dAIH decreasedMapk14mRNA (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.