Developmental nicotine exposure alters cholinergic control of respiratory frequency in neonatal rats.

Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) alters the development of brainstem circuits that control breathing. Here, we test the hypothesis that DNE alters the respiratory motor response to endogenous and exogenous acetylcholine (ACh) in neonatal rats. We used the brainstem-spinal cord preparation in the split-bath configuration, and applied drugs to the brainstem compartment while measuring the burst frequency and amplitude of the fourth cervical ventral nerve roots (C4VR), which contain the axons of phrenic motoneurons. We applied ACh alone; the nicotinic acetylcholine receptor (nAChR) antagonist curare, either alone or in the presence of ACh; and the muscarinic acetylcholine receptor (mAChR) antagonist atropine, either alone or in the presence of ACh. The main findings include: (1) atropine reduced frequency similarly in controls and DNE animals, while curare caused modest slowing in controls but no consistent change in DNE animals; (2) DNE greatly attenuated the increase in C4VR frequency mediated by exogenous ACh; (3) stimulation of nAChRs with ACh in the presence of atropine increased frequency markedly in controls, but not DNE animals; (4) stimulation of mAChRs with ACh in the presence of curare caused a modest increase in frequency, with no treatment group differences. DNE blunts the response of the respiratory central pattern generator to exogenous ACh, consistent with reduced availability of functionally competent nAChRs; DNE did not alter the muscarinic control of respiratory motor output. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1138-1149, 2016.

Neonatal stress augments the hypoxic chemoreflex of adult male rats by increasing AMPA receptor-mediated modulation.

Neonatal stress disrupts the developmental trajectory of homeostatic systems. Adult (8- to 10-week-old) male rats exposed to maternal separation (a form of neonatal stress) display several traits reported in patients suffering from sleep-disordered breathing, including an augmented hypoxic chemoreflex. To understand the mechanisms behind this effect, we tested the hypothesis that neonatal stress augments glutamatergic neurotransmission in three regions involved in respiratory regulation, namely the nucleus of the solitary tract, the paraventricular nucleus of the hypothalamus and the phrenic motor nucleus. Maternal separation was performed for 3 h day(-1) from postnatal day 3 to 12. Control pups were undisturbed. Adult rats were instrumented for intracerebroventricular injection of the AMPA/kainate receptor antagonist CNQX (0-4.3 µm). Using plethysmography, ventilatory activity was measured at rest in awake animals during normoxia (fractional inspired O2 = 0.21) and during acute hypoxia (fractional inspired O2 = 0.12; 20 min). Following vehicle injection, the hypoxic ventilatory response of stressed rats was 35% greater than that of controls. Microinjection of CNQX attenuated the hypoxic ventilatory response, but the effect observed in stressed rats was greater than that in control animals. Autoradiography experiments showed that neonatal stress augments expression of AMPA receptors within the paraventricular nucleus of the hypothalamus and the phrenic motor nucleus. Quantification of brain-derived neurotrophic factor showed that neonatal stress augments brain-derived neurotrophic factor expression only within the paraventricular nucleus. We conclude that neonatal stress augments the hypoxic chemoreflex by increasing the efficacy of glutamatergic synaptic inputs projecting onto key respiratory structures, especially the paraventricular nucleus of the hypothalamus. These data provide new insight into the aetiology of sleep-disordered breathing.

Intermittent hypoxia and stem cell implants preserve breathing capacity in a rodent model of amyotrophic lateral sclerosis.

RATIONALE: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1(G93A) rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. OBJECTIVES: To preserve or restore phrenic nerve activity in SOD1(G93A) rats at disease end stage. METHODS: SOD1(G93A) rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage. MEASUREMENTS AND MAIN RESULTS: The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. CONCLUSIONS: AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS.

Testosterone restores respiratory long term facilitation in old male rats by an aromatase-dependent mechanism.

Steroidal sex hormones play an important role in the neural control of breathing. Previous studies in our laboratory have shown that gonadectomy in young male rats (3 months) eliminates a form of respiratory plasticity induced by intermittent hypoxia, known as long term facilitation (LTF). Testosterone replenishment restores LTF in gonadectomized male rats, and this is dependent on the conversion of testosterone to oestradiol by aromatase. By middle age (12 months), male rats no longer exhibit LTF of hypoglossal motor output; phrenic LTF is significantly reduced, and this persists into old age. We tested the hypothesis that LTF can be restored in old male rats by administration of testosterone. Intact Fischer 344 rats (>20 months) were implanted with Silastic tubing containing testosterone (T), T plus an aromatase inhibitor (T+ADT), or 5α-dihydrotestosterone (DHT), a form of testosterone not converted to oestradiol. One week post-surgery, LTF of hypoglossal and phrenic motor output was measured. By comparison with control rats, hypoglossal LTF was increased in testosterone-treated rats, with levels approaching that of normal young rats. LTF was not restored in T+ADT or DHT-treated rats. Aromatase levels in hypoglossal and phrenic nuclei did not change with age. As serum testosterone levels did not decline with age, local bioavailability of testosterone in old rats may be a limiting factor in the expression of this form of respiratory plasticity. Our findings suggest that testosterone supplementation could potentially be used to enhance upper airway control in the elderly.

Methazolamide does not impair respiratory work performance in anesthetized rabbits.

In human medicine, the carbonic anhydrase (CA) inhibitor acetazolamide is used to treat irregular breathing disorders. Previously, we demonstrated in the rabbit that this substance stabilized closed-loop gain properties of the respiratory control system, but concomitantly weakened respiratory muscles. Among others, the highly diffusible CA-inhibitor methazolamide differs from acetazolamide in that it fails to activate Ca(2+)-dependent potassium channels in skeletal muscles. Therefore, we aimed to find out, whether or not methazolamide may exert attenuating adverse effects on respiratory muscle performance as acetazolamide. In anesthetized spontaneously breathing rabbits (n = 7), we measured simultaneously the CO(2) responses of tidal phrenic nerve activity, tidal transpulmonary pressure changes, and tidal volume before and after intravenous application of methazolamide at two mean (+/- SE) cumulative doses of 3.5 +/- 0.1 and 20.8 +/- 0.4 mg/kg. Similar to acetazolamide, low- and high-dose methazolamide enhanced baseline ventilation by 52 +/- 10% and 166 +/- 30%, respectively (P < 0.01) and lowered the base excess in a dose-dependent manner by up to 8.3 +/- 0.9 mmol/l (P < 0.001). The transmission of a CO(2)-induced rise in phrenic nerve activity into volume and/or pressure and, hence, respiratory work performance was 0.27 +/- 0.05 ml x kg(-1) x kPa x unit(-1) under control conditions, but remained unchanged upon low- or high-dose methazolamide, at 0.30 +/- 0.06 and 0.28 +/- 0.07 ml x kg(-1) x kPa x unit(-1), respectively. We conclude that methazolamide does not cause respiratory muscle weakening at elevated levels of ventilatory drive. This substance (so far not used for medication of respiratory diseases) may thus exert stabilizing influences on breathing control without adverse effects on respiratory muscle function.

Sodium/myo-inositol cotransporter-1 is essential for the development and function of the peripheral nerves.

Sodium/myo-inositol cotransporter-1 (SMIT-1) is one of the transporters responsible for importing myo-inositol (MI) into the cells. MI is a precursor for a family of signal transduction molecules, phosphatidylinositol, and its derivatives that regulates many cellular functions. SMIT-1 null mice died soon after birth due to respiratory failure, but neonatal lethality was prevented by prenatal maternal MI supplement. Although the lung air sacs were closed, lung development was not significantly affected in the SMIT-1 null mice. The development of the peripheral nerves, including the brachial plexus, facial, vagus, and intercostal nerves, and the phrenic nerve that innervates the diaphragm was severely affected. All of these peripheral nerve abnormalities were corrected by prenatal MI supplement, indicating that MI is essential for the development of peripheral nerve and that neonatal lethality of the SMIT-1 knockout mice is most likely due to abnormal development of the nerves that control breathing. In the adult SMIT-1 deficient mice rescued by MI supplement, MI content in their brain, kidney, skeletal muscle, liver, and sciatic nerve was greatly reduced. The sciatic nerve, in particular, was most dependent on SMIT-1 for the accumulation of MI, and nerve conduction velocity and protein kinase C activity in this tissue were significantly reduced by SMIT-1 deficiency.

Abolishment of non-quantal release of acetylcholine from the mouse phrenic nerve endings by toosendanin.

The hyperpolarizing effect (H-effect) of d-tubocurarine on the end-plate of the isolated diaphragm pretreated with an anticholinesterase was irreversibly abolished by toosendanin (1 X 10(-5) g/ml), indicating the blockade of spontaneous non-quantal release of acetylcholine (ACh). The H-effect was also inhibited, but temporarily, when toosendanin (a dose of 0.6 LD50) was subcutaneously injected into the mouse and the diaphragm was isolated 40-120 min after injection. During such an inhibitory period, however, spontaneous release of ACh remained facilitated. It is concluded that the effect of toosendanin on non-quantal release of ACh was different from its effect on quantal release not only at the direction but also at the time course.