Nongenomic Actions of 17-β Estradiol Restore Respiratory Neuroplasticity in Young Ovariectomized Female Rats

Abstract

Gonadal steroids modulate CNS plasticity, including phrenic long-term facilitation (pLTF), a form of spinal respiratory neuroplasticity resulting in increased phrenic nerve motor output following exposure to acute intermittent hypoxia (aIH; three 5 min episodes, 10.5% O₂). Despite the importance of respiratory system neuroplasticity, and its dependence on estrogen in males, little is known about pLTF expression or mechanisms of estrogen signaling in females. Here, we tested the hypotheses that (1) pLTF expression in young, gonadally intact female rats would be expressed during estrous cycle stages in which 17β-estradiol (E2) is naturally high (e.g., proestrus vs estrus), (2) pLTF would be absent in ovariectomized (OVX) rats and in physiological conditions in which serum progesterone, but not E2, is elevated (e.g., lactating rats, 3-10 d postpartum), and (3) acute E2 administration would be sufficient to restore pLTF in OVX rats. Recordings of phrenic nerve activity in female Sprague Dawley rats (3-4 months) revealed a direct correlation between serum E2 levels and pLTF expression in cycling female rats. pLTF was abolished with OVX, but was re-established by acute E2 replacement (3 h, intraperitoneal). To identify underlying E2 signaling mechanisms, we intrathecally applied BSA-conjugated E2 over the spinal phrenic motor nucleus and found that pLTF expression was restored within 15 min, suggesting nongenomic E2 effects at membrane estrogen receptors. These data are the first to investigate the role of ovarian E2 in young cycling females, and to identify a role for nongenomic estrogen signaling in any form of respiratory system neuroplasticity.SIGNIFICANCE STATEMENT Exposure to acute intermittent hypoxia induces phrenic long-term facilitation (pLTF), a form of spinal respiratory motor plasticity that improves breathing in models of spinal cord injury. Although pathways leading to pLTF are well studied in males and estradiol (E2) is known to be required, it has seldom been investigated in females, and underlying mechanisms of E2 signaling are unknown in either sex. We found that while ovariectomy abolished pLTF, it could be restored by acute systemic E2, or by intraspinal application of the membrane-impermeable E2 (BSA-conjugated E2; 15 min). The ability of nongenomic estrogen signaling within the cervical spinal cord to recover respiratory neuroplasticity in disorders of respiratory insufficiency suggests that membrane estrogen receptors may represent novel therapeutic targets to restore breathing in both sexes.

Keywords: estradiol; long-term facilitation; phrenic; plasticity.

Figure 1.

aIH induces pLTF in young proestrus, but not estrus rats, and systemic E2 restores pLTF to OVX rats. Representative traces of integrated phrenic neurograms over 90 min during and following aIH in young female rats. Baseline is indicated by the dashed line. All traces are identically scaled. A, Following exposure to aIH, females in proestrus (PRO; n = 8) exhibit a progressive increase in phrenic nerve burst amplitude above baseline (dashed line). B, Rats in estrus (EST; n = 8) do not express pLTF. C, OVX rats (n = 8) failed to express pLTF. D, E2 replacement (intraperitoneal) in OVX females (OVX+E; n = 8) rescued pLTF. E, In the absence of aIH, time-control (TC) rats (n = 5) displayed no time-dependent changes in phrenic nerve burst amplitudes over the same 90 min period. F, Quantification of pLTF magnitude in all treatment groups at 60 min following aIH, graphed as a percentage change from baseline. G, Phrenic nerve burst amplitudes in response to hypoxia, graphed as a percentage change from baseline. H, Phrenic nerve burst frequency in bursts per minutes (BPM) 60 min after aIH. ^†††p < 0.001, and ^†p < 0.05 versus baseline; ***p < 0.001 versus TC; ^‡‡‡p ≤ 0.001 versus PRO; ^###p ≤ 0.001 versus OVX+E.

Figure 2.

pLTF positively correlates with serum E2 levels, and inversely correlates with the P/E ratio. A, Serum E2 levels were significantly elevated in proestrus (PRO) and OVX+E rats relative to estrus (EST) and OVX rats. B, Regression analyses of serum E2 levels, which positively correlated with the magnitude of pLTF 60 min after aIH. C, Serum progesterone levels were reduced in OVX and OVX+E rats. D, Serum progesterone levels did not correlate with pLTF. E, The P/E ratios in the PRO and OVX+E groups were significantly lower than P/E ratios in the EST and OVX groups. F, The P/E ratio negatively correlated with pLTF. ***p < 0.001 and *p < 0.05 versus PRO; ^†††p ≤ 0.001 versus OVX+E; ^‡p < 0.05 versus EST. Overlaid box plots indicate the 25th and 75th percentile, and the median of each dataset; group means are denoted by bold lines.

Figure 3.

pLTF is absent in postpartum, lactating female rats despite elevated progesterone levels. A, Representative trace of an integrated phrenic neurogram over 90 min during and following aIH in postpartum, lactating rats. Baseline is indicated by the dashed line. B, Quantification of phrenic nerve burst amplitudes 60 min after aIH, graphed as percentage change from baseline; pLTF was not observed (n = 8). C, Phrenic nerve burst amplitudes in response to hypoxia, graphed as a percentage change from baseline, showed a robust response to hypoxia. D, E, Serum E2 levels were low (D) and serum progesterone levels approximated those in proestrus and estrus rats (E). Overlaid box plots indicate the 25th and 75th percentiles, and the median of each dataset; group means are denoted by bold lines.

Figure 4.

Activation of spinal membrane ERs with intrathecal E2-BSA restores pLTF in OVX rats. A, B, Representative traces of integrated phrenic neurograms over 90 min during and following aIH in OVX female rats receiving intrathecal (A) E2-BSA (n = 5) to activate membrane E2 receptors or (B) control BSA alone (n = 5). Baseline is indicated by the dashed line. E2-BSA given 15 min before aIH resulted in a progressive increase in phrenic nerve burst amplitude above baseline (dashed line). Rats receiving BSA alone did not express pLTF. C, In the absence of aIH, time-control (TC) rats (n = 6) receiving intrathecal injections displayed no time-dependent changes in phrenic nerve burst amplitudes over the same 90 min period. D, Quantification of pLTF magnitude following pretreatment with intrathecal E2-BSA or BSA alone reflect an E2-BSA-induced enhancement of phrenic burst amplitude 60 min following aIH relative to baseline, BSA-treated control rats, and time controls. The 50 nm E2-BSA dose was derived from dose–response studies (inset). E, F, Intrathecal E2-BSA led to enhanced phrenic amplitude in response to hypoxia (E), but had minimal impact on phrenic burst frequency (F).