Serum Estradiol Level Correlates With Molecules Regulating Phrenic Motoneuron Plasticity

Abstract

Acute Intermittent Hypoxia (AIH) elicits phrenic long‐term facilitation (pLTF), an important model of respiratory motor plasticity. Although moderate AIH‐induced pLTF exhibits profound age‐dependent sexual dimorphism, little is known concerning the age or sex‐dependent changes in molecules regulating the Q or S pathways to phrenic motor facilitation. AIH elicits pLTF via distinct signaling pathways in phrenic motor neurons initiated by 5HT2A/2B(Q pathway) or 5HT7 & Adenosine 2A receptors (S pathway); these pathways interact via powerful crosstalk inhibition. Moderate AIH (mAIH)‐induced pLTF declines ~50% from 3 to 12 months of age in male rats (Zabka et al., 2001). Gonadectomy abolishes pLTF in young adult males, and it is restored by supplemental testosterone; pLTF is restored due to testosterone conversion into estrogen in the CNS (Zabka et al., 2006). Thus, CNS estrogen must impact keys molecules regulating the Q and/or S pathways to phrenic motor facilitation. Here, we tested the hypothesis that serum estradiol is correlated with keys molecules known to regulate the Q and S pathways in rats. We assessed the mRNA of molecules known to regulate pLTF in ventral cervical spinal homogenates containing the phrenic motor nucleus from young (3 month) and middle‐aged (12 month) male and female Sprague‐Dawley rats; mRNA levels were correlated with serum estradiol levels assessed via ELISA. Spinal cord tissues were preserved in “RNA Later” solution <10 min from harvest, and stored at ‐80°C until mRNA extraction. RT PCR was used to quantify mRNA levels for: serotonin 2A (Htr2a) & 2B (Htr2b) and adenosine 2A receptors (Adora2a); exchange protein activated by cAMP (EPAC); PKA catalytic subunit (Prkaa1); PKA regulatory subunit (Prkar1a); fractalkine (CX3CL1); phosphodiesterase type 4 (pde4b); NOX genes (gp91 and p47); and PKCδ. We found that serum estradiol was positively correlated with mRNA expression of EPAC (r2=0.28, p=0.002), pde4b mRNA (r2=0.15, p=0.029) and adora2a (r2=0.15, p=0.030), but not the other molecules studied. We conclude that serum estradiol may shift the Q‐S pathway balance, although we have not yet demonstrated a causal relationship. An important limitation of this study is that serum estradiol concentration may differ from that in the ventral cervical spinal cord. In future studies, it is important to test the significance in these neurochemical shifts in mechanisms giving rise to the age‐dependent sexual dimorphism in AIH‐induced pLTF.