Neonatal maternal separation and sex-specific plasticity of the hypoxic ventilatory response in awake rat

Abstract

We tested the hypothesis that neonatal maternal separation (NMS), a form of stress that affects hypothalamo-pituitary-adrenal axis (HPA) function in adult rats, alters development of the respiratory control system. Pups subjected to NMS were placed in a temperature and humidity controlled incubator 3 h per day for 10 consecutive days (P3 to P12). Control pups were undisturbed. Once they reached adulthood (8-10 weeks old), rats were placed in a plethysmography chamber for measurement of ventilatory and cardiovascular parameters under normoxic and hypoxic conditions. Measurement of c-fos mRNA expression in the paraventricular nucleus of the hypothalamus (PVH) combined with plasma ACTH and corticosterone levels confirmed that NMS effectively disrupted HPA axis function in males. In males, baseline minute ventilation was not affected by NMS. In contrast, NMS females show a greater resting minute ventilation due to a larger tidal volume. The hypoxic ventilatory response of male NMS rats was 25% greater than controls, owing mainly to an increase in tidal volume response. This augmentation of the hypoxic ventilatory response was sex-specific also because NMS females show an attenuated minute ventilation increase. Baseline mean arterial blood pressure of male NMS rats was 20% higher than controls. NMS-related hypertension was not significant in females. The mechanisms underlying sex-specific disruption of cardio-respiratory control in NMS rats are unknown but may be a consequence of the neuroendocrine disruption associated with NMS. These data indicate that exposure to a non-respiratory stress during early life elicits significant plasticity of these homeostatic functions which persists until adulthood.

Figure 1. Male/female comparisons of the effects of neonatal maternal separation (NMS) on basal c-fos mRNA signal in the PVH and endocrine indicators of HPA axis activation in adult rats

A, radiographs of PVH section for each group; B, corresponding bar graphs showing mean optical density for controls (filled bars) and rats previously subjected to NMS (open bars). Between group and between sex comparisons of plasma ACTH concentrations (C) and plasma corticosterone concentrations (D). Data are expressed as means ± s.e.m.†Statistically different from corresponding control value (P < 0.05) and *statistically different from females (P < 0.05).

Figure 2. Effects of neonatal maternal separation (NMS) on time-course of breathing frequency response to hypoxia in adult females (A) and males (B)

Graphics show frequency change (in breaths min⁻¹) from baseline in controls (filled circles; males: n = 25 and females: n = 15) and rats previously subjected to NMS (open triangles; males: n = 15 and females: n = 18). Data are expressed as means ± s.e.m.†Statistically different from corresponding control value (P < 0.05). Note that all values are different from baseline (P < 0.05); however, no symbols are shown for simplicity.

Figure 3. Effects of neonatal maternal separation (NMS) on selected ventilatory variables in adult female (left hand panels) and male (right hand panels) rats

A and D, minute ventilation; B and E, breathing frequency; C and F, tidal volume. Measurements were taken under baseline condition (normoxia) and after 20 min of exposure to moderate hypoxia (F_IO2 = 0.12). Data are compared between controls (filled circles; males: n = 25 and females: n = 15) and rats previously subjected to NMS (open triangles; males: n = 15 and females: n = 18). Values are expressed as means ± s.E.M.†Statistically different from corresponding control value (P < 0.05) and *statistically different from baseline (P < 0.05).

Figure 4. Effects of neonatal maternal separation (NMS) on hypoxic ventilatory response of females (A) and male rats (B)

For each group, selected ventilatory variables (minute ventilation, tidal volume, breathing frequency and inspiratory flow) were measured after 20 min exposure to moderate hypoxia (F_IO2 = 0.12) and expressed as a percentage changes from normoxic baseline values. Filled bars represent control animals (males: n = 25 and females: n = 15) and open bars represent rats subjected to NMS (males: n = 15 and females: n = 18). Data are expressed as means ± s.e.m.^†Statistically different from corresponding control value (P < 0.05); ^‡statistically different from corresponding control value (P = 0.06).

Figure 5. Effects of neonatal maternal separation (NMS) on arterial blood pressure (A and B) and heart rate (C and D) in adult male (right hand panels) and female rats (left hand panels) under baseline (room air) condition and after 20 min exposure to moderate hypoxia (F_IO2 = 0.12)

Controls: filled circles (males: n = 8 and females: n = 8), and NMS rats: open triangles (males: n = 7 and females: n = 7). Values are expressed as means ± s.e.m.†Statistically different from corresponding control value (P < 0.05); *statistically different from baseline at P < 0.05; + statistically different from baseline at P < 0.1.