At least 20% of pregnant women smoke, and their offspring have a higher than normal incidence of impaired cardiac function, autonomic nervous system disorders, sleep disorders, delayed speech and central and obstructive apneas. Importantly, recent studies document that the main respiratory phenotype in nicotine-exposed human neonates is a higher incidence of obstructive sleep apnea. It is widely accepted that obstructive apnea is caused largely by abnormal activation of tongue muscles, which are in turn controlled by hypoglossal motoneurons. Work in our laboratory beginning in 2003-2004 shows that in utero and early neonatal nicotine exposure (developmental nicotine exposure, DNE) leads to complex changes in breathing and hypoglossal motoneuron structure and function, including: a) desensitization of nAChRs;b) reduced excitatory synaptic input;c) increased input resistance, suggesting that the neurons are smaller;d) altered neuronal responses to inhibitory and excitatory agonists, including nicotine;e) altered ventilatory control in vivo, including increased apnea duration, with the entire apneic period associated with the loss of tongue muscle activity. Here we propose a series of studies designed to systematically examine the effects of DNE on both presynaptic and postsynaptic regulation of hypoglossal motoneuron function, motoneuron morphology, including estimates of the distribution of glutamatergic and GABAergic synapses upon motoneurons, and control of the tongue musculature in vivo.
Specific Aim 1 tests the hypothesis that DNE reduces the release of both excitatory and inhibitory neurotransmitters from glutamatergic, GABAergic and glycinergic neurons in the vicinity of the hypoglossal motoneurons, using whole cell voltage clamp techniques.
Aim 2 is designed to determine how DNE exaggerates the post-synaptic response of hypoglossal motoneurons to agonists of GABAA, glycine, NMDA and AMPA receptors. These post-synaptic effects will be evaluated by blocking presynaptic input to hypoglossal motoneurons, and studying postsynaptic effects by injecting small volumes of receptor agonists, while measuring changes in whole cell current and conductance under voltage clamp.
Aim 3 tests the hypothesis that DNE disrupts the normal signals that regulate dendritic growth and synapse formation, leading to a reduction in the number of glutamatergic and GABAergic synapses formed upon the hypoglossal motoneurons. This hypothesis will be tested by filling motoneurons with dyes, and using 3-dimensional confocal microscopy to reconstruct the motoneuron cell body and dendritic tree, followed by detailed measures of somatic and dendritic anatomy. These data will be coupled with immunohistochemistry to examine the distribution of glutamatergic and GABAergic synapses that impinge upon the motoneurons, and how the number, position and density of these synapses change with DNE.
Aim 4 examines the very real consequences of DNE by testing the hypothesis that DNE leads to an increased frequency and duration of obstructive, central and mixed apneas in vivo, due to reduced tongue muscle activation and diminished neuromuscular responses to changes in upper airway pressure. For these studies we will use lightly anesthetized neonatal rat pups wherein measurements of rib cage expansion and the EMG activity of inspiratory intercostal and tongue muscles are recorded. We will measure the frequency and duration of central, obstructive and mixed apneas, and the genioglossus EMG before, during and after each apneic episode. Reflex control of tongue muscles evoked by changing upper airway pressure will also be measured and quantified. All experiments will be done in neonatal rat pups exposed to either nicotine (experimental group) or saline (control group) in utero. These studies are clinically important because DNE in human infants is associated with an abnormally high incidence of breathing, feeding, swallowing and cardiovascular abnormalities that affects the health and well-being of millions of human infants in infancy and childhood. It is therefore crucial to begin establishing the mechanisms that lead to abnormal development of the brainstem neurons that regulate these critical homeostatic functions.

Public Health Relevance

Our goal is to better understand how exposure to nicotine in utero and in the early neonatal period (Developmental Nicotine Exposure, DNE) alters development of nervous system structure and function in infants and young children, with specific focus on the hypoglossal motoneurons, which are critical for normal breathing, swallowing, sucking and chewing. Clinical studies have shown that DNE can lead to learning and memory problems, a greater rate of nicotine addiction, growth abnormalities, feeding and swallowing disorders, sleep abnormalities, and an increased risk of asthma, sudden infant death syndrome (SIDS) and both central and obstructive sleep apnea. Over the last several years, our laboratory has produced considerable evidence showing that DNE neonates exhibit a variety of abnormal anatomic and functional changes that adversely affect normal breathing control. We seek to understand the cellular mechanisms that cause these developmental changes. Here we propose a series of detailed experiments that will examine how DNE alters the structure and function of hypoglossal motor neurons, and correlate them with behavioral measures in neonatal rats studied in vivo. Hypoglossal motor neurons, located in the brainstem, control muscles of the tongue. Altered control of the tongue muscles is the major cause of breathing problems, including obstructive sleep apnea, as well as swallowing, sucking and chewing abnormalities. Moreover, because these neurons are large and readily accessible in slices of the neonatal rat brainstem, that produces a ventilatory rhythm;detailed in vitro studies of their structure and function are possible. We will determine how DNE influences the normal release of neurotransmitters onto the motoneurons;the function of inhibitory and excitatory neurotransmitter receptors on the neurons;the location and number of synapses;and the growth and development of dendrites. The in vivo experiments will allow us to determine how DNE influences the frequency and duration of central and obstructive apnea, and the reflex control of the tongue muscles. Techniques used include patch clamp electrophysiology of single hypoglossal motor neurons, immunohistochemistry, measures of neuron size and shape, and measures of tongue muscle activity and breathing in anesthetized neonatal rats. For all studies, neurons from DNE neonates will be compared to age-matched, saline-exposed control animals.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Pregnancy and Neonatology Study Section (PN)
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Urv, Tiina K
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University of Arizona
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Cholanian, Marina; Powell, Gregory L; Levine, Richard B et al. (2017) Influence of developmental nicotine exposure on glutamatergic neurotransmission in rhythmically active hypoglossal motoneurons. Exp Neurol 287:254-260
Powell, Gregory L; Gaddy, Joshua; Xu, Fei et al. (2016) Developmental nicotine exposure disrupts dendritic arborization patterns of hypoglossal motoneurons in the neonatal rat. Dev Neurobiol 76:1125-37
Jaiswal, Stuti J; Wollman, Lila Buls; Harrison, Caitlyn M et al. (2016) Developmental nicotine exposure enhances inhibitory synaptic transmission in motor neurons and interneurons critical for normal breathing. Dev Neurobiol 76:337-54
Wakefield, Hilary E; Fregosi, Ralph F; Fuglevand, Andrew J (2016) Current injection and receptor-mediated excitation produce similar maximal firing rates in hypoglossal motoneurons. J Neurophysiol 115:1307-13
Wollman, Lila B; Haggerty, Jarl; Pilarski, Jason Q et al. (2016) Developmental nicotine exposure alters cholinergic control of respiratory frequency in neonatal rats. Dev Neurobiol 76:1138-49
Powell, Gregory L; Levine, Richard B; Frazier, Amanda M et al. (2015) Influence of developmental nicotine exposure on spike-timing precision and reliability in hypoglossal motoneurons. J Neurophysiol 113:1862-72
Ferng, Jonathan; Fregosi, Ralph F (2015) Influence of developmental nicotine exposure on the ventilatory and metabolic response to hyperthermia. J Physiol 593:5201-13
Fregosi, Ralph F; Ludlow, Christy L (2014) Activation of upper airway muscles during breathing and swallowing. J Appl Physiol (1985) 116:291-301
Jaiswal, Stuti J; Pilarski, Jason Q; Harrison, Caitlyn M et al. (2013) Developmental nicotine exposure alters AMPA neurotransmission in the hypoglossal motor nucleus and pre-Botzinger complex of neonatal rats. J Neurosci 33:2616-25
Pilarski, Jason Q; Wakefield, Hilary E; Fuglevand, Andrew J et al. (2012) Increased nicotinic receptor desensitization in hypoglossal motor neurons following chronic developmental nicotine exposure. J Neurophysiol 107:257-64

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