Jason Q. Pilarski

Prenatal nicotine exposure and development of nicotinic and fast amino acid-mediated neurotransmission in the control of breathing

There is mounting evidence that neonatal animals exposed to nicotine in the prenatal period exhibit a variety of anatomic and functional abnormalities that adversely affect their respiratory and cardiovascular control systems, but how nicotine causes these developmental alterations is unknown. The principle that guides our work is that PNE impairs the ability of nicotinic acetylcholine receptors (nAChRs) to modulate the pre-synaptic release of both inhibitory (particularly GABA) and excitatory (glutamate) neurotransmitters, leading to marked alterations in the density and/or function of receptors on the (post-synaptic) membrane of respiratory neurons. Such changes could lead to impaired ventilatory responses to sensory afferent stimulation, and altered breathing patterns, including central apneic events. In this brief review we summarize the work that lead to the development of this hypothesis, and introduce some new data that support and extend it.

Developmental Nicotine Exposure Alters Neurotransmission and Excitability in Hypoglossal Motoneurons

Hypoglossal motoneurons (XII MNs) control muscles of the mammalian tongue and are rhythmically active during breathing. Acetylcholine (ACh) modulates XII MN activity by promoting the release of glutamate from neurons that express nicotinic ACh receptors (nAChRs). Chronic nicotine exposure alters nAChRs on neurons throughout the brain, including brain stem respiratory neurons. Here we test the hypothesis that developmental nicotine exposure (DNE) reduces excitatory synaptic input to XII MNs. Voltage-clamp experiments in rhythmically active medullary slices showed that the frequency of excitatory postsynaptic currents (EPSCs) onto XII MNs from DNE animals is reduced by 61% (DNE = 1.7 ± 0.4 events/s; control = 4.4 ± 0.6 events/s; P < 0.002). We also examine the intrinsic excitability of XII MNs to test whether cells from DNE animals have altered membrane properties. Current-clamp experiments showed XII MNs from DNE animals had higher intrinsic excitability, as evaluated by measuring their response to injected current. DNE cells had high-input resistances (DNE = 131.9 ± 13.7 MΩ, control = 78.6 ± 9.7 MΩ, P < 0.008), began firing at lower current levels (DNE = 144 ± 22 pA, control = 351 ± 45 pA, P < 0.003), and exhibited higher frequency-current gain values (DNE = 0.087 ± 0.012 Hz/pA, control = 0.050 ± 0.004 Hz/pA, P < 0.02). Taken together, our data show previously unreported effects of DNE on XII MN function and may also help to explain the association between DNE and the incidence of central and obstructive apneas.

Developmental Nicotine Exposure Alters AMPA Neurotransmission in the Hypoglossal Motor Nucleus and Pre-Bötzinger Complex of Neonatal Rats

Developmental nicotine exposure (DNE) impacts central respiratory control in neonates born to smoking mothers. We previously showed that DNE enhances the respiratory motor response to bath application of AMPA to the brainstem, although it was unclear which brainstem respiratory neurons mediated these effects (Pilarski and Fregosi, 2009). Here we examine how DNE influences AMPA-type glutamatergic neurotransmission in the pre-Bötzinger complex (pre-BötC) and the hypoglossal motor nucleus (XIIMN), which are neuronal populations located in the medulla that are necessary for normal breathing. Using rhythmic brainstem slices from neonatal rats, we microinjected AMPA into the pre-BötC or the XIIMN while recording from XII nerve rootlets (XIIn) as an index of respiratory motor output. DNE increased the duration of tonic activity and reduced rhythmic burst amplitude after AMPA microinjection into the XIIMN. Also, DNE led to an increase in respiratory burst frequency after AMPA injection into the pre-BötC. Whole-cell patch-clamp recordings of XII motoneurons showed that DNE increased motoneuron excitability but did not change inward currents. Immunohistochemical studies indicate that DNE reduced the expression of glutamate receptor subunits 2 and 3 (GluR2/3) in the XIIMN and the pre-BötC. Our data show that DNE alters AMPAergic synaptic transmission in both the XIIMN and pre-BötC, although the mechanism by which this occurs is unclear. We suggest that the DNE-induced reduction in GluR2/3 may represent an attempt to compensate for increased cell excitability, consistent with mechanisms underlying homeostatic plasticity.

Increased nicotinic receptor desensitization in hypoglossal motor neurons following chronic developmental nicotine exposure

Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed on hypoglossal motor neurons (XII MNs) that innervate muscles of the tongue. Activation of XII MN nAChRs evokes depolarizing currents, which are important for regulating the size and stiffness of the upper airway. Although data show that chronic developmental nicotine exposure (DNE) blunts cholinergic neurotransmission in the XII motor nucleus, it is unclear how nAChRs are involved. Therefore, XII MN nAChR desensitization and recovery were examined in tissues from DNE or control pups using a medullary slice preparation and tight-seal whole cell patch-clamp recordings. nAChR-mediated inward currents were evoked by brief pressure pulses of nicotine or the α4β2 nAChR agonist RJR-2403. We found that, regardless of treatment, activatable nAChRs underwent desensitization, but, following DNE, nAChRs exhibited increased desensitization and delayed recovery. Similar results were produced using RJR-2403, showing that DNE influences primarily the α4β2 nAChR subtype. These results show that while some nAChRs preserve their responsiveness to acute nicotine following DNE, they more readily desensitize and recover more slowly from the desensitized state. These data provide new evidence that chronic DNE modulates XII MN nAChR function, and suggests an explanation for the association between DNE and the incidence of central and obstructive apneas.

Developmental nicotine exposure enhances inhibitory synaptic transmission in motor neurons and interneurons critical for normal breathing

Nicotine exposure in utero negatively affects neuronal growth, differentiation, and synaptogenesis. We used rhythmic brainstems slices and immunohistochemistry to determine how developmental nicotine exposure (DNE) alters inhibitory neurotransmission in two regions essential to normal breathing, the hypoglossal motor nucleus (XIIn), and preBötzinger complex (preBötC). We microinjected glycine or muscimol (GABAA agonist) into the XIIn or preBötC of rhythmic brainstem slices from neonatal rats while recording from XII nerve roots to obtain XII motoneuron population activity. Injection of glycine or muscimol into the XIIn reduced XII nerve burst amplitude, while injection into the preBötC altered nerve burst frequency. These responses were exaggerated in preparations from DNE animals. Quantitative immunohistochemistry revealed a significantly higher GABAA receptor density on XII motoneurons from DNE pups. There were no differences in GABAA receptor density in the preBötC, and there were no differences in glycine receptor expression in either region. Nicotine, in the absence of other chemicals in tobacco smoke, alters normal development of brainstem circuits that are critical for normal breathing.

Prenatal development of respiratory chemoreceptors in endothermic vertebrates

Respiratory chemoreceptors are neurons that detect PCO(2), PO(2), and/or pH in body fluids and provide sensory feedback for the control of breathing. They play a critical role in coupling pulmonary ventilation to metabolic demand in endothermic vertebrates. During birth in mammals and hatching in birds, the state change from placental or chorioallantoic gas exchange to pulmonary respiration makes acute demands on the neonatal lungs and ventilatory control system, including the respiratory chemoreceptors. Here we review the literature on prenatal development of carotid body chemoreceptors, central chemoreceptors, and airway chemoreceptors, with emphasis on the histology, histochemistry, and neurophysiology of chemosensory cells or their afferents, and their physiological genomics if known. In general, respiratory chemoreceptors develop prenatally and are functional but immature at birth or hatching. Each type of respiratory chemoreceptor has a unique prenatal developmental time course, and all studied to date require a period of postnatal maturation to express the full adult response.

Effects of Aerobic and Anaerobic Metabolic Inhibitors on Avian Intrapulmonary Chemoreceptors

Birds have rapidly responding respiratory chemoreceptors [intrapulmonary chemoreceptors (IPC)] that provide vagal sensory feedback about breathing pattern. IPC are exquisitely sensitive to CO(2) but are unaffected by hypoxia. IPC continue to respond to CO(2) during hypoxic and even anoxic conditions, suggesting that they may generate ATP needed for signal transduction anaerobically. To assess IPC energy metabolism, single-cell action potential discharge and acid-base status were recorded from 26 pentobarbital-anesthetized Anas platyrhynchos before and after intravenous infusion of the glycolytic blocker iodoacetate (10-70 mg/kg), mitochondrial blocker rotenone (2 mg/kg), and/or mitochondrial uncoupler 2,4-dinitrophenol (5-15 mg/kg). After 5 min exposure at the highest dosages, iodoacetate inhibited IPC discharge 65% (15.9 +/- 0.3 s(-1) to 5.5 +/- 0.3 s(-1), P < 0.05), rotenone inhibited discharge 80% (12.9 +/- 0.5 s(-1) to 2.6 +/- 0.6 s(-1), P < 0.05), and 2,4-dinitrophenol inhibited discharge 19% (14.0 +/- 0.3 s(-1) to 11.3 +/- 0.3 s(-1), P < 0.05). These results suggest that IPC utilize glucose, require an intact glycolytic pathway, and metabolize the products of glycolysis to CO(2) and H(2)O by mitochondrial respiration. The small but significant effect of 2,4-dinitrophenol suggests that ATP production by glycolysis may be sufficient to meet IPC energy demands if NADH can be oxidized to NAD experimentally by uncoupling mitochondria, or physiologically by transient lactate production. A model for IPC spike frequency adaptation is proposed, whereby the rapid onset of phasic IPC discharge requires ATP from anaerobic glycolysis, using lactate as the electron acceptor, and the roll-off in IPC discharge reflects transient acidosis due to intracellular lactic acid accumulation.