Lalah Niane

Ventilatory and carotid body chemoreceptor responses to purinergic P2X receptor antagonists in newborn rats

Adenosine triphosphate, acting through purinergic P2X receptors, has been shown to stimulate ventilation and increase carotid body chemoreceptor activity in adult rats. However, its role during postnatal development of the ventilatory response to hypoxia is yet unknown. Using whole body plethysmography, we measured ventilation in normoxia and in moderate hypoxia (12% fraction of inspired O₂, 20 min) before and after intraperitoneal injection of suramin (P2X₂ and P2X₃ receptor antagonist, 40 mg/kg) in 4-, 7-, 12-, and 21-day-old rats. Suramin reduced baseline breathing (∼20%) and the response to hypoxia (∼30%) in all rats, with a relatively constant effect across ages. We then tested the effect of the specific P2X₃ antagonist, A-317491 (150 mg/kg), in rats aged 4, 7, and 21 days. As with suramin, A-317491 reduced baseline ventilation (∼55%) and the hypoxic response (∼40%) at all ages studied. Single-unit carotid body chemoreceptor activity was recorded in vitro in 4-, 7-, and 21-day-old rats. Suramin (100 μM) and A-317491 (10 μM) significantly depressed the sinus nerve chemosensory discharge rate (∼80%) in normoxia (Po₂ ∼150 Torr) and hypoxia (Po₂ ∼60 Torr), and this decrease was constant across ages. We conclude that, in newborn rats, P2X purinergic receptors are involved in the regulation of breathing under basal and hypoxic condition, and P2X₃-containing receptors play a major role in carotid body function. However, these effects are not age dependent within the age range studied.

Role of cholinergic-nicotinic receptors on hypoxic chemoreflex during postnatal development in rats

We tested the hypothesis that the function of cholinergic-nicotinic receptors on respiration is age dependent. To this end, we used whole body plethysmography to measure breathing frequency (fR), tidal volume (V(T)) and minute ventilation (V (E)) under normoxia (21% O(2)) in rats at 1, 4, 7, 12 and 21 postnatal days before and after administration of epibatidine (nicotinic agonist 5 microg/kg, i.p.). In normoxia, epibatidine increased fR and V (E) in a proportionally age-dependent manner (p for age <0.001), without affecting V(T). We then focused on P4 and P12 rats, as representative of this developmental pattern, to measure ventilatory response to moderate hypoxia (12% O(2), 20 min) after i.p. injection of saline (control), epibatidine (5 microg/kg), or (nicotinic antagonist, 1mg/kg). At these doses, both drugs selectively target peripheral nicotinic receptors. Epibatidine enhanced the hypoxic ventilatory response while hexamethonium significantly reduced it. These effects were significantly greater in P12 than in P4 rats (p for age <0.001). In P12 rats, in vitro recordings of carotid sinus nerve activity showed that superfusion with nicotine enhanced chemosensory discharge rate in normoxia and hexamethonium reduced the discharge rate in hypoxia. We also identified the nicotinic receptor alpha7 subunit by immunohistochemistry in carotid bodies from P12 rat. These data show that the role of cholinergic-nicotinic receptor on hypoxic chemoreflex is age dependent and this effect likely implicates carotid body nicotinic receptor activation.

Carotid sinus nerve stimulation, but not intermittent hypoxia, induces respiratory LTF in adult rats exposed to neonatal intermittent hypoxia

We tested the hypothesis that exposure to neonatal intermittent hypoxia (n-IH) in rat pups alters central integrative processes following acute and intermittent peripheral chemoreceptor activation in adults. Newborn male rats were exposed to n-IH or normoxia for 10 consecutive days after birth. We then used both awake and anesthetized 3- to 4-mo-old rats to record ventilation, blood pressure, and phrenic and splanchnic nerve activities to assess responses to peripheral chemoreflex activation (acute hypoxic response) and long-term facilitation (LTF, long-term response after intermittent hypoxia). In anesthetized rats, phrenic and splanchnic nerve activities and hypoxic responses were also recorded with or without intact carotid body afferent signal (bilateral chemodenervation) or in response to electrical stimulations of the carotid sinus nerve. In awake rats, n-IH alters the respiratory pattern (higher frequency and lower tidal volume) and increased arterial blood pressure in normoxia, but the ventilatory response to repeated hypoxic cycles was not altered. In anesthetized rats, phrenic nerve responses to repeated hypoxic cycles or carotid sinus nerve stimulation were not altered by n-IH; however, the splanchnic nerve response was suppressed by n-IH compared with control. In control rats, respiratory LTF was apparent in anesthetized but not in awake animals. In n-IH rats, respiratory LTF was not apparent in awake and anesthetized animals. Following intermittent electrical stimulation, however, phrenic LTF was clearly present in n-IH rats, being similar in magnitude to controls. We conclude that, in adult n-IH rats: 1) arterial blood pressure is elevated, 2) peripheral chemoreceptor responses to hypoxia and its central integration are not altered, but splanchnic nerve response is suppressed, 3) LTF is suppressed, and 4) the mechanisms involved in the generation of LTF are still present but are masked most probably as the result of an augmented inhibitory response to hypoxia in the central nervous system.

Antagonism of progesterone receptor suppresses carotid body responses to hypoxia and nicotine in rat pups

We tested the hypothesis that antagonism of progesterone receptor (PR) in newborn rats alters carotid body and respiratory responses to hypoxia and nicotinic receptor agonists. Rats were treated with the PR antagonist mifepristone (daily oral gavage 40 μg/g/d) or vehicle between postnatal days 3 and 15. In 11-14-day-old rats, we used in vitro carotid body/carotid sinus nerve preparation and whole body plethysmography to assess the carotid body and ventilatory responses to hypoxia (65 mmHg in vitro, 10% O2 in vivo) and to nicotinic receptor agonists (as an excitatory modulator of carotid body activity-nicotine 100 μM for in vitro studies, and epibatidine 5 μg/kg, i.p., which mainly acts on peripheral nicotinic receptors, for in vivo studies). The carotid body responses to hypoxia and nicotine were drastically reduced by mifepristone. Compared with vehicle, mifepristone-treated rats had a reduced body weight. The ventilatory response to epibatidine was attenuated; however, the hypoxic ventilatory response was similar between vehicle and mifepristone-treated pups. Immunohistochemical staining revealed that mifepristone treatment did not change carotid body morphology. We conclude that PR activity is a critical factor ensuring proper carotid body function in newborn rats.

Systemic blockade of nicotinic and purinergic receptors inhibits ventilation and increases apnoea frequency in newborn rats.

We hypothesized that the combined blockade of peripheral cholinergic and purinergic receptors alters the baseline breathing pattern and respiratory responses to carotid body stimuli (hypoxia, hyperoxia and hypercapnia). Rat pups at 4 (P4) and 12 days of postnatal age (P12) received an intraperitoneal injection of either saline vehicle or hexamethonium + suramin (Hex, 1 mg kg−1, nicotinic receptor antagonist; Sur, 40 mg kg−1, P2X receptor antagonist; both of which act mainly on peripheral receptors). Compared with the control animals (saline‐injected rats), the Hex + Sur‐treated rats demonstrated the following features: (1) decreased baseline ventilation and increased frequency of apnoea and breath‐by‐breath irregularities, with a larger effect in the P4 than in the P12 rats; (2) a decreased peak minute ventilation and respiratory frequency response to hypoxia (fractional inspired oxygen 12%), with a greater effect in the P12 than in the P4 rats; (3) an attenuated decline of the respiratory frequency during hyperoxia (fractional inspired oxygen 50%) to a similar magnitude in rats of both ages; and (4) a decreased hypercapnic ventilatory response (fractional inspired carbon dioxide 5%) to a similar magnitude in rats of both ages. We conclude that the cholinergic nicotinic and purinergic P2X receptors are essential to maintain an adequate baseline pattern in normoxia. They also contribute, albeit not exclusively, to the hypoxic ventilatory response, with an age‐specific effect, most probably linked to the cholinergic component, which might partly underlie the postnatal maturation of peripheral chemoreceptors.

Age-Dependent Changes in Breathing Stability in Rats

The respiratory control system rapidly develops during the perinatal period in mammalian species. Therefore, premature birth prevents the completion of important neurological maturation processes, which can cause periodic breathing and apneas. The ventilatory response to hypoxia (HVR) is among the test that have been used to study the immature respiratory control system in human and animal subjects (Carroll 2003; Cohen and Katz-Salamon 2005). This response is biphasic because the early increase in the HVR of newborn mammals is not sustained as in adults but decreases to below baseline values (Bissonnette 2000; Cohen and Katz-Salamon 2005; Niane and Bairam 2011). In a preliminary report, we have suggested that rats at postnatal days 1, 4, 7 and 12 (P1, P4, P7 and P12) may be selected to represent the postnatal changes in the biphasic HVR pattern (peak and steady state) and apnea frequency (Niane and Bairam 2011). To better understand the relationship between the maturation of respiratory control and the decreased apnea frequency with age (Niane and Bairam 2011), we re-analyzed our data in P1-, P4-, P7-, P12-, P21- and P90-day-old rats. The following two parameters were studied as an index of respiratory stability during development: the coefficient of variation of minute ventilation and the apnea types (spontaneous vs. post-sigh). The correlation between apnea types and the coefficient of variation of minute ventilation under baseline and steady-state conditions of HVR were assessed.