Neonatal maternal separation and enhancement of the hypoxic ventilatory response in rat: the role of GABAergic modulation within the paraventricular nucleus of the hypothalamus

Abstract

Neonatal maternal separation (NMS) affects respiratory control development as adult male (but not female) rats previously subjected to NMS show a hypoxic ventilatory response 25% greater than controls. The paraventricular nucleus of the hypothalamus (PVN) is an important modulator of respiratory activity. In the present study, we hypothesized that in awake rats, altered GABAergic inhibition within the PVN contributes to the enhancement of hypoxic ventilatory response observed in rats previously subjected to NMS. During normoxia, the increase in minute ventilation following microinjection of bicuculline (1 mm) within the PVN is greater in NMS versus control rats. These data show that regulation of ventilatory activity related to tonic inhibition of the PVN is more important in NMS than control rats. Microinjection of GABA or muscimol (1 mM) attenuated the ventilatory response to hypoxia (12% O2) in NMS rats only. The higher efficiency of microinjections in NMS rats is supported by results from GABAA receptor autoradiography which revealed a 22% increase in GABAA receptor binding sites within the PVN of NMS rats versus controls. Despite this increase, however, NMS rats still show a larger hypoxic ventilatory response than controls, suggesting that within the PVN the larger number of GABAA receptors either compensate for (1) a deficient GABAergic modulation, (2) an increase in the efficacy of excitatory inputs converging onto this structure, or (3) both. Together, these results show that the life-long consequences of NMS are far reaching as they can compromise the development of vital homeostatic function in a way that may predispose to respiratory disorders.

Figure 1

Comparison of the time course of breathing frequency and minute ventilation responses to hypoxia Comparison of the time course of breathing frequency (A) and minute ventilation (C) responses to hypoxia (12% O₂) following microinjection of PBS in PVN between rats previously subjected to neonatal maternal separation (NMS; •) and controls (▵). Preliminary experiments comparing the effects of different doses of GABA on the mean breathing frequency (B) and mean minute ventilation (D) measured over the 5 first minutes of hypoxia between NMS and control rats. Values are expressed as mean ±s.e.m.†Statistically different from corresponding control value and *statistically different from PBS (P < 0.05).

Figure 2

Microinjection sites and effects of GABAergic agents on resting ventilatory activity in NMS and control rats A, anatomical location of the 34 microinjection sites in the paraventricular nucleus of the hypothalamus (PVN; Bregma −1.8) where phosphate-buffered saline (PBS; vehicle), GABA, bicuculline or muscimol were injected. The bicuculline panel also shows three injection sites outside the PVN (○, control; ▵, neonatal maternal separation (NMS)) for which breathing frequency data are shown in C. B, comparison of the change in basal (normoxic) ventilatory activity (expressed as percentage change from preinjection values) measured 10 min after PBS, GABA, bicuculline or muscimol microinjection (10 ng each) in the PVN between rats subjected to NMS (open bars) and controls (filled bars). Measured ventilatory variables include breathing frequency (f), tidal volume (V_t) and minute ventilation (V˙_E). C, comparison of the change in ‘resting’ breathing frequency (expressed in absolute data) following bicuculline microinjection according to cannula placement within the PVN. The symbols with crosses inside show data from three rats (two controls, one NMS) for which the cannula was outside. In this graph, data for the two control rats were averaged. These data show that the effects of the injection are site-specific. Data are expressed as means ±s.e.m.†Statistically different from corresponding control value and *statistically different from PBS (P < 0.05). PaDC, paraventricular hypothalamic nucleus, dorsal cap; PaLM, paraventricular hypothalamic nucleus, lateral magnocellular part; PaMP, paraventricular hypothalamic nucleus, medial parvicellular part; PaV, paraventricular hypothalamic nucleus, ventral part; f, fornix; 3V, third ventricle; AHP, anterior hypothalamic area, posterior; AHC, anterior hypothalamic area, central; Pe, periventricular hypothalamic nucleus; Opt, Optic tract; sox, supraoptic decussation.

Figure 3

Neonatel maternal seperation (NMS) augments the effects of GABA agonists on the breathing frequency response to hypoxia Comparison of the effects of microinjections of GABA (A) muscimol (B) or bicuculline (C) within the PVN on the breathing frequency response to moderate hypoxia (12% O₂) between rats previously subjected to neonatal maternal separation (NMS; ▵) and controls (•). Note that the light grey symbols show the data obtained from PBS-treated rats (Figure 1A) for comparison. Data are expressed as means ±s.e.m.†Statistically different from corresponding control value and bars with asterisk indicate a series of values statistically different from corresponding PBS value (P < 0.05).

Figure 4

Comparison of the effects of microinjection of PBS (100 nl), GABA (1 mm), bicuculline (1 mm) or muscimol (1 mm) in the PVN on the ‘late phase’ (last 5 min) of the ventilatory response to moderate hypoxia (12% O₂). Ventilatory variables shown are breathing frequency (A), tidal volume (B), minute ventilation (C) and inspiratory flow (D). Results are expressed as a percentage of normoxic (baseline) values obtained 1 min before the injections. Data are shown as means ±s.e.m.†Statistically different from corresponding control value and *statistically different from PBS (P < 0.05). ‡Indicates a value statistically different from corresponding control value (P = 0.06).

Figure 5

Comparison of the [³H]muscimol and [³H]flunitrazepam binding within the PVN between rats previously subjected to neonatal maternal separation (NMS) and controls Representative autoradiographs of [³H]muscimol (A) and [³H]flunitrazepam (B) binding in PVN section for each group of rats. In these panels, the scale bar is 250 μm.C, binding curves for [³H]muscimol obtained in NMS (•) and control rats (▵) showing that NMS augments the number of GABA_A recptors within the PVN. D, binding curves for [³H]flunitrazepam obtained in NMS and control rats showing that NMS decreased the number of benzodiazepine binding sites within the PVN. For each concentration, data are expressed as means ±s.e.m.†statistically different from corresponding control value (P < 0.05).

Figure 6

Neonatel maternal seperation (NMS) decreases GABA_A receptor α₁ subunit mRNA signal in the PVN A, comparison of representative autoradiographs showing basal GABA_A receptor α₁ subunit mRNA signal in the PVN of NMS and control rats. The scale bar is 250 μm.B, mean optical density of the α₁ subunit mRNA signal for NMS (open bars) and controls (filled bars). These data show that, within the PVN, NMS decreased α₁ subunit mRNA expression level; this subunit is the most widespread GABA binding site in the CNS. Data are expressed as means ±s.e.m.†Statistically different from corresponding control value (P < 0.05).