Medullary 5-HT neurons: Switch from tonic respiratory drive to chemoreception during postnatal development.

Serotonin (5-HT) neurons contribute to respiratory chemoreception in adult mice, but it is unclear whether they play a similar role in neonatal mice. We studied breathing during development in Lmx1bf/f/p mice, which lack 5-HT neurons. From postnatal days 1-7 (P1-P7), ventilation of Lmx1bf/f/p mice breathing room air was 50% of WT mice (p<0.001). By P12, baseline ventilation increased to a level equal to WT mice. In contrast, the hypercapnic ventilatory response (HCVR) of neonatal Lmx1bf/f/p and WT mice was equal to each other, but were both much less than adult WT mice. By P21 the HCVR of WT mice increased to near adult levels, but the HCVR of Lmx1bf/f/p mice had not changed, and was 42% less than WT mice. Primary cell cultures were prepared from the ventromedial medulla of neonatal mice, and patch-clamp recordings were made from neurons identified as serotonergic by expression of a reporter gene. In parallel with developmental changes of the HCVR in vivo, 5-HT neurons had little chemosensitivity to acidosis until 12days in vitro (DIV), after which their response increased to reach a plateau around 25 DIV. Neonatal Lmx1bf/f/p mice displayed high mortality and decreased growth rate, and this worsened in hypoxia. Mortality was decreased in hyperoxia. These results indicate that maturation of 5-HT neurons contributes to development of respiratory CO2/pH chemoreception during the first few weeks of life in mice in vivo. A defect in the 5-HT system in early postnatal life decreases survival due in part to hypoxia.

The Alteration of Neonatal Raphe Neurons by Prenatal-Perinatal Nicotine. Meaning for Sudden Infant Death Syndrome.

Nicotine may link maternal cigarette smoking with respiratory dysfunctions in sudden infant death syndrome (SIDS). Prenatal-perinatal nicotine exposure blunts ventilatory responses to hypercapnia and reduces central respiratory chemoreception in mouse neonates at Postnatal Days 0 (P0) to P3. This suggests that raphe neurons, which are altered in SIDS and contribute to central respiratory chemoreception, may be affected by nicotine. We therefore investigated whether prenatal-perinatal nicotine exposure affects the activity, electrical properties, and chemosensitivity of raphe obscurus (ROb) neurons in mouse neonates. Osmotic minipumps, implanted subcutaneously in 5- to 7-day-pregnant CF1 mice, delivered nicotine bitartrate (60 mg kg(-1) d(-1)) or saline (control) for up to 28 days. In neonates, ventilation was recorded by head-out plethysmography, c-Fos (neuronal activity marker), or serotonin autoreceptors (5HT1AR) were immunodetected using light microscopy, and patch-clamp recordings were made from raphe neurons in brainstem slices under normocarbia and hypercarbia. Prenatal-perinatal nicotine exposure decreased the hypercarbia-induced ventilatory responses at P1-P5, reduced both the number of c-Fos-positive ROb neurons during eucapnic normoxia at P1-P3 and their hypercapnia-induced recruitment at P3, increased 5HT1AR immunolabeling of ROb neurons at P3-P5, and reduced the spontaneous firing frequency of ROb neurons at P3 without affecting their CO2 sensitivity or their passive and active electrical properties. These findings reveal that prenatal-perinatal nicotine reduces the activity of neonatal ROb neurons, likely as a consequence of increased expression of 5HT1ARs. This hypoactivity may change the functional state of the respiratory neural network leading to breathing vulnerability and chemosensory failure as seen in SIDS.

Cxs and Panx- hemichannels in peripheral and central chemosensing in mammals.

Connexins (Cxs) and Pannexins (Panx) form hemichannels at the plasma membrane of animals. Despite their low open probability under physiological conditions, these hemichannels release signaling molecules (i.e., ATP, Glutamate, PGE2) to the extracellular space, thus subserving several important physiological processes. Oxygen and CO2 sensing are fundamental to the normal functioning of vertebrate organisms. Fluctuations in blood PO2, PCO2 and pH are sensed at the carotid bifurcations of adult mammals by glomus cells of the carotid bodies. Likewise, changes in pH and/or PCO2 of cerebrospinal fluid are sensed by central chemoreceptors, a group of specialized neurones distributed in the ventrolateral medulla (VLM), raphe nuclei, and some other brainstem areas. After many years of research, the molecular mechanisms involved in chemosensing process are not completely understood. This manuscript will review data regarding relationships between chemosensitive cells and the expression of channels formed by Cxs and Panx, with special emphasis on hemichannels.

Prenatal to early postnatal nicotine exposure impairs central chemoreception and modifies breathing pattern in mouse neonates: a probable link to sudden infant death syndrome.

Nicotine is a neuroteratogen and is the likely link between maternal cigarette smoking during pregnancy and sudden infant death syndrome (SIDS). Osmotic minipumps were implanted in 5-7 d CF1 pregnant mice to deliver nicotine bitartrate (60 mg Kg(-1) day(-1)) or saline (control) solutions for up to 28 d. Prenatal to early postnatal nicotine exposure did not modify the number of newborns per litter or their postnatal growth; however, nicotine-exposed neonates hypoventilated and had reduced responses to hypercarbia (inhalation of air enriched with 10% CO(2) for 20 min) and hypoxia (inhalation of 100% N(2) for 20 s) at postnatal days 0-3 (P0-P3). In contrast, at postnatal day 8, nicotine-exposed neonates were indistinguishable from controls. Isolated brainstem-spinal cord preparations obtained from P0 to P3 nicotine-exposed neonates showed fictive respiration with respiratory cycles longer and more irregular than those of controls, as indicated by high short- and long-term variability in Poincaré plots. In addition, their responses to acidification were reduced, indicating compromise of central chemoreception. Furthermore, the cholinergic contribution to central chemosensory responses switched from muscarinic receptor to nicotinic receptor-based mechanisms. No significant astrogliosis was detectable in the ventral respiratory group of neurons with glial fibrillary acidic protein immunohistochemistry. These results indicate that nicotine exposure affects the respiratory rhythm pattern generator and causes a decline in central chemoreception during early postnatal life. Consequently, breathing would become highly vulnerable, failing to respond to chemosensory demands. Such impairment could be related to the ventilatory abnormalities observed in SIDS.

ATP- and ACh-induced responses in isolated cat petrosal ganglion neurons.

Chemoreceptor (glomus) cells of the carotid body are synaptically connected to the sensory nerve endings of petrosal ganglion (PG) neurons. In response to natural stimuli, the glomus cells release transmitters, which acting on the nerve terminals of petrosal neurons increases the chemosensory afferent discharge. Among several transmitter molecules present in glomus cells, acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) are considered to act as excitatory transmitter in this synapse. To test if ACh and ATP play a role as excitatory transmitters in the cat CB, we recorded the electrophysiological responses from PG neurons cultured in vitro. Under voltage clamp, ATP induces a concentration-dependent inward current that partially desensitizes during 20-30 s application pulses. The ATP-induced current has a threshold near 100 nM and saturates between 20-50 muM. ACh induces a fast, inactivating inward current, with a threshold between 10-50 muM, and saturates around 1 mM. A large part of the population of PG neurons (60%) respond to both ATP and ACh. Present results support the hypothesis that ACh and ATP act as excitatory transmitters between cat glomus cells and PG neurons.

Catecholamine release from isolated sensory neurons of cat petrosal ganglia in tissue culture.

The petrosal ganglion (PG) is entirely constituted by the perikarya of primary sensory neurons, part of which innervates the carotid body via the carotid sinus nerve (CSN). Application of acetylcholine (ACh) or nicotine (Nic) as well as adenosine 5'-triphosphate (ATP) to the PG in vitro increases the frequency of CSN discharges, an effect that is modified by the concomitant application of dopamine (DA). Since a population of PG neurons expresses tyrosine hydroxylase, and DA is released from the cat carotid body in response to electrical stimulation of C-fibers in the CSN, it is possible that DA may be released from the perikarya of PG neurons. Therefore, we studied whether ACh or Nic, ATP and high KCl could induce DA release from PG neurons in culture. Petrosal ganglia were excised from pentobarbitone-anesthetized adult cats, dissociated and their neurons maintained in culture for 7-21 days. Catecholamine release was measured by amperometry via carbon-fiber microelectrodes. In response to KCl, Nic, ACh or ATP application, about 25% of neurons exhibited electrochemical signals compatible with DA release. This percentage increased to 41% after loading the neurons with exogenous DA. The present results suggest that DA release may be induced from the perikarya of a population of PG neurons.

Dopamine inhibits ATP-induced responses in the cat petrosal ganglion in vitro.

The petrosal ganglion (PG) provides sensory innervation to the carotid sinus and carotid body through the carotid (sinus) nerve (CN). Application of either acetylcholine (ACh) or adenosine 5'-triphosphate (ATP) to the PG superfused in vitro activates CN fibers. Dopamine (DA) modulates the effects of ACh. We have previously shown that DA when applied to the PG modulates the effects of ACh on carotid sinus nerve fibers. We currently report the effects of DA on the ATP-induced responses in the isolated PG in vitro. While DA had no effect on the basal activity recorded from the CN, it reduced ATP-induced responses in a dose-dependent manner, when preceding ATP applications by 30 s. Our results suggest that DA-a transmitter present in a group of PG neurons and in carotid body cells-may act as an inhibitory modulator of ATP-evoked responses in PG neurons.