Exploring the potential neurotoxicity of vaping vitamin E or vitamin E acetate.

Serious adverse health effects have been reported with the use of vaping products, including neurologic disorders and e-cigarette or vaping product use-associated lung injury (EVALI). Vitamin E acetate, likely added as a diluent to cannabis-containing products, was linked to EVALI. Literature searches were performed on vitamin E and vitamin E acetate-associated neurotoxicity. Blood brain barrier (BBB) penetration potential of vitamin E and vitamin E acetate were evaluated using cheminformatic techniques. Review of the literature showed that the neurotoxic potential of inhalation exposures to these compounds in humans is unknown. Physico-chemical properties demonstrate these compounds are lipophilic, and molecular weights indicate vitamin E and vitamin E acetate have the potential for BBB permeability. Computational models also predict both compounds may cross the BBB via passive diffusion. Based on literature search, no experimental nonclinical studies and clinical information on the neurotoxic potential of vitamin E via inhalation. Neurotoxic effects from pyrolysis by-product, phenyl acetate, structurally analogous to vitamin E acetate, suggests vitamin E acetate has potential for central nervous system (CNS) impairment. Cheminformatic model predictions provide a theoretical basis for potential CNS permeability of these inhaled dietary ingredients suggesting prioritization to evaluate for potential hazard to the CNS.

Anti-inflammatory effect of caffeine is associated with improved lung function after lipopolysaccharide-induced amnionitis.

BACKGROUND: Although caffeine enhances respiratory control and decreases the need for mechanical ventilation and resultant bronchopulmonary dysplasia, it may also have anti-inflammatory properties in protecting lung function. OBJECTIVE: We hypothesized that caffeine improves respiratory function via an anti-inflammatory effect in lungs of a lipopolysaccharide (LPS)-induced pro-inflammatory amnionitis rat pup model. METHODS: Caffeine was given orally (10 mg/kg/day) from postnatal day (p)1 to p14 to pups exposed to intra-amniotic LPS or normal saline. Expression of IL-1ß was assessed in lung homogenates at p8 and p14, and respiratory system resistance (Rrs) and compliance (Crs) as well as CD68 cell counts and radial alveolar counts were assessed at p8. RESULTS: In LPS-exposed rats, IL-1ß and CD68 cell counts both increased at p8 compared to normal saline controls. These increases in pro-inflammatory markers were no longer present in caffeine-treated LPS-exposed pups. Rrs was higher in LPS-exposed pups (4.7 ± 0.9 cm H2O/ml·s) at p8 versus controls (1.6 ± 0.3 cm H2O/ml·s, p < 0.01). LPS-exposed pups no longer exhibited a significant increase in Rrs (2.8 ± 0.5 cm H2O/ml·s) after caffeine. Crs did not differ significantly between groups, although radial alveolar counts were lower in both groups of LPS-exposed pups. CONCLUSIONS: Caffeine promotes anti-inflammatory effects in the immature lung of prenatal LPS-exposed rat pups associated with improvement of Rrs, suggesting a protective effect of caffeine on respiratory function via an anti-inflammatory mechanism.

Repeated ß2-adrenergic receptor agonist therapy attenuates the response to rescue bronchodilation in a hyperoxic newborn mouse model.

BACKGROUND: Preterm infants with neonatal lung injury are prone to wheezing and are often treated with ß2-adrenergic receptor (ß-AR) agonists although the benefits of ß-AR agonists may be lost with chronic use. OBJECTIVE: To investigate if repeated ß-AR agonist exposure downregulates ß-ARs in the immature lung resulting in a decreased response to bronchodilator rescue and whether hyperoxic exposure aggravates this response. METHODS: Newborn mice were raised for 21 days in 60 or 21% oxygen and received daily aerosols of formoterol or saline. Respiratory system resistance (Rrs) and compliance (Crs) were measured in response to methacholine challenge and rescue bronchodilation with levalbuterol. Western blot analysis quantified the relative amount of lung ß-ARs. RESULTS: Hyperoxia increased the airway reactivity to methacholine. Animals raised in hyperoxia that received daily formoterol were most sensitive to methacholine and exhibited a blunted response to levalbuterol bronchodilation. Hyperoxia-exposed animals receiving daily formoterol versus saline showed a significant decrease in the relative amount of lung ß-ARs. CONCLUSIONS: In this hyperoxia-exposed neonatal mouse model, repeated ß-AR agonist treatments increased the airway reactivity and attenuated the response to a rescue bronchodilator. The blunted bronchodilator response could be explained by a reduced quantity of lung ß-ARs. Our findings may account for the time-dependent decrease in the therapeutic benefit of ß-AR agonists in preterm infants with neonatal lung injury, which may have clinical consequences for patients already prone to airway hyperreactivity.

Intrapulmonary lipopolysaccharide exposure upregulates cytokine expression in the neonatal brainstem.

UNLABELLED: Perinatal inflammation and neonatal sepsis trigger lung and brain injury. We hypothesized that endotoxin exposure in the immature lung upregulates proinflammatory cytokine expression in the brainstem and impairs respiratory control. Lipopolysaccharide (LPS) or saline was administered intratracheally to vagal intact or denervated rat pups. LPS increased brainstem IL-1ß and vagotomy blunted this response. There was an attenuated ventilatory response to hypoxia and increased brainstem IL-1ß expression after LPS. CONCLUSION: Intratracheal endotoxin exposure in rat pups is associated with upregulation of IL-1ß in the brainstem that is vagally mediated and associated with an impaired hypoxic ventilatory response.

Intermittent hypoxic episodes in preterm infants: do they matter?

Intermittent hypoxic episodes are typically a consequence of immature respiratory control and remain a troublesome challenge for the neonatologist. Furthermore, their frequency and magnitude are underestimated by clinically employed pulse oximeter settings. In extremely low birth weight infants the incidence of intermittent hypoxia progressively increases over the first 4 weeks of postnatal life, with a subsequent plateau followed by a slow decline beginning at weeks 6-8. Such episodic hypoxia/reoxygenation has the potential to sustain a proinflammatory cascade with resultant multisystem morbidity. This morbidity includes retinopathy of prematurity and impaired growth, as well as possible longer-term cardiorespiratory instability and poor neurodevelopmental outcome. Therapeutic approaches for intermittent hypoxic episodes comprise determination of optimal baseline saturation and careful titration of supplemental inspired oxygen, as well as xanthine therapy to prevent apnea of prematurity. In conclusion, characterization of the pathophysiologic basis for such intermittent hypoxic episodes and their consequences during early life is necessary to provide an evidence-based approach to their management.

Vagal afferents modulate cytokine-mediated respiratory control at the neonatal medulla oblongata.

Perinatal sepsis and inflammation trigger lung and brain injury in preterm infants, and associated apnea of prematurity. We hypothesized that endotoxin exposure in the immature lung would upregulate proinflammatory cytokine mRNA expression in the medulla oblongata and be associated with impaired respiratory control. Lipopolysaccharide (LPS, 0.1mg/kg) or saline was administered intratracheally to rat pups and medulla oblongatas were harvested for quantifying expression of mRNA for proinflammatory cytokines. LPS-exposure significantly increased medullary mRNA for IL-1ß and IL-6, and vagotomy blunted this increase in IL-1ß, but not IL-6. Whole-body flow plethysmography revealed that LPS-exposed pups had an attenuated ventilatory response to hypoxia both before and after carotid sinus nerve transection. Immunochemical expression of IL-1ß within the nucleus of the solitary tract and area postrema was increased after LPS-exposure. In summary, intratracheal endotoxin-exposure in rat pups is associated with upregulation of proinflammatory cytokines in the medulla oblongata that is vagally mediated for IL-1ß and associated with an impaired hypoxic ventilatory response.

Modulation of cardiorespiratory function mediated by the paraventricular nucleus.

The hypothalamic paraventricular nucleus (PVN) coordinates autonomic and neuroendocrine systems to maintain homeostasis and to respond to stress. Neuroanatomic and neurophysiologic experiments have provided insight into the mechanisms by which the PVN acts. The PVN projects directly to the spinal cord and brainstem and, specifically, to sites that control cardio-respiratory function: the intermediolateral cell columns and phrenic motor nuclei in the spinal cord and rostral ventrolateral medulla (RVLM) and the rostral nuclei in the ventral respiratory column (rVRC) in the brainstem. Activation of the PVN increases ventilation (both tidal volume and frequency) and blood pressure (both heart rate and sympathetic nerve activity). Excitatory and inhibitory neurotransmitters including glutamate and GABA converge in the PVN to influence its neuronal activity. However, a tonic GABAergic input to the PVN directly modulates excitatory outflow from the PVN. Further, even within the PVN, microinjection of GABA(A) receptor blockers increases glutamate release suggesting an indirect mechanism by which GABA control contributes to PVN functions. PVN activity alters blood pressure and ventilation during various stresses, such as maternal separation, chronic intermittent hypoxia (CIH), dehydration and hemorrhage. Among the several PVN neurotransmitters and neurohormones, vasopressin and oxytocin modulate ventilation and blood pressure. Here, we review our data indicating that increases in vasopressin and vasopressin type 1A (V(1A)) receptor signalling in the RVLM and rVRC are mechanisms increasing blood pressure and ventilation after exposure to CIH. That blockade of V(1A) receptors in the medulla normalizes baseline blood pressure as well as blunts PVN-evoked blood pressure and ventilatory responses in CIH-conditioned animals indicate the role of vasopressin in cardiorespiratory control. In summary, morphological and functional studies have found that the PVN integrates sensory input and projects to the sympathetic and respiratory control systems with descending projections to the medulla and spinal cord.

Role of central neurotransmission and chemoreception on airway control.

This review summarizes work on central neurotransmission, chemoreception and CNS control of cholinergic outflow to the airways. First, we describe the neural transmission of bronchoconstrictive signals from airway afferents to the airway-related vagal preganglionic neurons (AVPNs) via the nucleus of the solitary tract (nTS) and, second, we characterize evidence for a modulatory effect of excitatory glutamatergic, and inhibitory GABAergic, noradrenergic and serotonergic pathways on AVPN output. Excitatory signals arising from bronchopulmonary afferents and/or the peripheral chemosensory system activate second order neurons within the nTS, via a glutamate-AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor signaling pathway. These nTS neurons, using the same neurotransmitter-receptor unit, transmit information to the AVPNs, which in turn convey the central command through descending fibers and airway intramural ganglia to airway smooth muscle, submucosal secretory glands, and the vasculature. The strength and duration of this reflex-induced bronchoconstriction is modulated by GABAergic-inhibitory inputs. In addition, central noradrenergic and serotonergic inhibitory pathways appear to participate in the regulation of cholinergic drive to the tracheobronchial system. Down-regulation of these inhibitory influences results in a shift from inhibitory to excitatory drive, which may lead to increased excitability of AVPNs, heightened airway responsiveness, greater cholinergic outflow to the airways and consequently bronchoconstriction. In summary, centrally coordinated control of airway tone and respiratory drive serve to optimize gas exchange and work of breathing under normal homeostatic conditions. Greater understanding of this process should enhance our understanding of its disruption under pathophysiologic states.

Increased vasopressin transmission from the paraventricular nucleus to the rostral medulla augments cardiorespiratory outflow in chronic intermittent hypoxia-conditioned rats.

A co-morbidity of sleep apnoea is hypertension associated with elevated sympathetic nerve activity (SNA) which may result from conditioning to chronic intermittent hypoxia (CIH). Our hypothesis is that SNA depends on input to the rostral ventrolateral medulla (RVLM) from neurons in the paraventricular nucleus (PVN) that release arginine vasopressin (AVP) and specifically, that increased SNA evoked by CIH depends on this excitatory input. In two sets of neuroanatomical experiments, we determined if AVP neurons project from the PVN to the RVLM and if arginine vasopressin (V(1A)) receptor expression increases in the RVLM after CIH conditioning (8 h per day for 10 days). In the first set, cholera toxin beta subunit (CT-beta) was microinjected into the RVLM to retrogradely label the PVN neurons. Immunohistochemical staining demonstrated that 14.6% of CT-beta-labelled PVN neurons were double-labelled with AVP. In the second set, sections of the medulla were immunolabelled for V(1A) receptors, and the V(1A) receptor-expressing cell count was significantly greater in the RVLM (P < 0.01) and in the neighbouring rostral ventral respiratory column (rVRC) from CIH- than from room air (RA)-conditioned rats. In a series of physiological experiments, we determined if blocking V(1A) receptors in the medulla would normalize blood pressure in CIH-conditioned animals and attenuate its response to disinhibition of PVN. Blood pressure (BP), heart rate (HR), diaphragm (D(EMG)) and genioglossus muscle (GG(EMG)) activity were recorded in anaesthetized, ventilated and vagotomized rats. The PVN was disinhibited by microinjecting a GABA(A) receptor antagonist, bicuculline (BIC, 0.1 nmol), before and after blocking V(1A) receptors within the RVLM and rVRC with SR49059 (0.2 nmol). In RA-conditioned rats, disinhibition of the PVN increased BP, HR, minute D(EMG) and GG(EMG) activity and these increases were attenuated after blocking V(1A) receptors. In CIH-conditioned rats, a significantly greater dose of blocker (0.4 nmol) was required to blunt these physiological responses (P < 0.05). Further, this dose normalized the baseline BP. In summary, AVP released by a subset of PVN neurons modulates cardiorespiratory output via V(1A) receptors in the RVLM and rVRC, and increased SNA in CIH-conditioned animals depends on up-regulation of V(1A) receptors in the RVLM.

Developmental changes in brainstem neurons regulating lower airway caliber.

Premature infants are at risk for lower airway obstruction; however, maturation of reflex pathways regulating lower airway patency is inadequately studied. We hypothesized that postnatal maturation causes developmental change in brainstem efferent airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) that project to the airways and in pulmonary afferent fibers that terminate in the nucleus tractus solitarius (NTS). Ferrets aged 7, 14, 21, and 42 d received intrapulmonary injection of cholera toxin (CT)-beta subunit, a transganglionic retrograde tracer. Five days later, their brainstem was processed for dual immunolabeling of CT-beta and the cholinergic marker, choline acetyl transferase. CT-beta-labeled AVPNs and CT-beta-labeled afferent fiber optical density (OD) were analyzed. There was a significantly higher CT-beta-labeled cell number within the rNA at the youngest compared with older ages. All efferent CT-beta-labeled cells expressed choline acetyl transferase. OD of CT-beta-labeled afferent fibers was also higher at 7 d compared with 14 d. We conclude that the number of efferent AVPNs and afferent fiber OD both diminish over the second postnatal week. We speculate that exposure to injurious agents in early postnatal life may inhibit natural remodeling and thereby enhance later vulnerability to airway hyperreactivity.

Neuroanatomical relationships of substance P-immunoreactive intrapulmonary C-fibers and nicotinic cholinergic receptors.

Previous studies have suggested that sensory mechanisms may be important components of addiction to, and withdrawal from, cigarette smoking. The sensory and respiratory responses to nicotine are mediated, in part, by bronchopulmonary C-fiber afferents. Nicotine has a direct stimulatory effect on pulmonary sensory neurons, and nicotinic cholinergic receptors (nAChRs) composed of various combinations of alpha and beta subunits are known to be present in pulmonary ganglia. At the subcellular level, however, little is known about expression of nAChRs on sensory fibers in the intrapulmonary airways. The present study was therefore designed to evaluate the expression of nAChRs on a subset of intrapulmonary sensory nerve endings known to exhibit immunoreactivity for substance P (SP). The presence of nAChR subunits was first confirmed at the mRNA and protein levels in rat lung tissues by using RT-PCR and Western blot techniques. Then, double labeling of SP-immunoreactive (-IR) C-fibers and different nAChR subunits was performed. Alpha2, alpha3, alpha4, alpha5, alpha7, and beta2 subunits were detected at all levels of the intrapulmonary airways; including bronchi, terminal and respiratory bronchioles, alveolar walls, and alveolar macrophages. None of the nAChR subunits studied was expressed by the SP-IR C-fibers. However, SP-expressing C-fibers were observed in close proximity to and intermingling with nAChR-expressing airway epithelial cells. The close proximity of C-fibers to nAChR-expressing airway epithelial cells suggests that a component of nicotinic stimulation of SP-IR C-fiber afferents may be mediated by endogenous chemical substances released by nAChR-expressing epithelial cells.

Modeling of sleep-induced changes in airway function: implication for nocturnal worsening of bronchial asthma.

Here we describe the model of sleep-induced worsening of airway function in patients with airway disorders. Our model is based on the noradrenergic pathways that link central neuronal structures responsible for alternating wakefulness and sleep with the neuronal networks regulating the activity of airway-related vagal preganglionic neurons (AVPNs). Our previous studies showed that cholinergic outflow to the airways depend on the activity of inhibitory inputs to AVPNs. Major inhibitory cell groups, regulating AVPNs discharge, include brainstem noradrenaline (NA)-containing cells receiving projections from the hypothalamic sleep-promoting neurons of the ventrolateral preoptic region (VLPO). When activated, VLPO cells, using GABA and/or galanin as mediators, downregulate the activity of inhibitory NA neurons projecting to AVPNs. Therefore, changes that occur during sleep lead to a shift from inhibitory to excitatory transmission of the AVPNs, thereby increasing cholinergic outflow to the airways. Our model, based on neuroanatomical and molecular studies, and physiology experiments, can be used to explain sleep-related worsening of bronchial asthma and might contribute to development of clinically meaningful treatment for patients with sleep-induced worsening of airway function and respiratory symptoms.

Allergic lung inflammation affects central noradrenergic control of cholinergic outflow to the airways in ferrets.

Brain stem noradrenergic cell groups mediating autonomic responses to stress project to airway-related vagal preganglionic neurons (AVPNs). In ferrets, their activation produces withdrawal of cholinergic outflow to the airways via release of norepinephrine and activation of alpha(2A)-adrenergic receptors (alpha(2A)-AR) expressed by AVPNs. In these studies, we examined the effects of allergen exposure of the airway (AE) with ovalbumin on noradrenergic transmission regulating the activity of AVPNs and, consequently, airway smooth muscle tone. Experiments were performed in vehicle control (Con) and AE ferrets. Microperfusion of an alpha(2A)-AR agonist (guanabenz) in close proximity to AVPNs elicited more pronounced effects in Con than AE ferrets, including a decrease in unit activity and reflexly evoked responses of putative AVPN neurons with a corresponding decrease in cholinergic outflow to the airways. Although no differences were found in the extent of noradrenergic innervation of the AVPNs, RT-PCR and Western blot studies demonstrated that AE and repeated exposure to antigen significantly reduced expression of alpha(2A)-ARs at message and protein levels. These findings indicate that, in an animal model of allergic asthma, sensitization and repeated challenges with a specific allergen diminish central inhibitory noradrenergic modulation of AVPNs, possibly via downregulation of alpha(2A)-AR expression by these neurons.

Airway-related vagal preganglionic neurons express multiple nicotinic acetylcholine receptor subunits.

Nicotine acting centrally increases bronchomotor tone and airway secretion, suggesting that airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) express nicotinic acetylcholine receptors (nAChRs). In the present study, we examined the three main functionally characterized subtypes of nAChRs in the CNS, the alpha7 homomeric and alpha4beta2 heteromeric receptors. First, we characterized the expression of these subunits at the message (mRNA) and protein levels in brain tissues taken from the rNA region, the site where AVPNs are located. In addition, double labeling fluorescent immunohistochemistry and confocal laser microscopy were used to define the presence of alpha7, alpha4, and beta2 nAChRs on AVPNs that were retrogradely labeled with cholera toxin beta subunit (CTb), injected into the upper lung lobe (n=4) or extrathoracic trachea (n=4). Our results revealed expression of all three studied subunits at mRNA and protein levels within the rNA region. Furthermore, virtually all identified AVPNs innervating intrapulmonary airways express alpha7 and alpha4 nAChR subunits. Similarly, a majority of labeled AVPNs projecting to extrathoracic trachea contain alpha7 and beta2 subunits, but less than half of them show detectable alpha4 nAChR traits. These results suggest that AVPNs express three major nAChR subunits (alpha7, alpha4, and beta2) that could assemble into functional homologous or heterologous pentameric receptors, mediating fast and sustained nicotinic effects on cholinergic outflow to the airways.

Phenotypic traits of the hypothalamic PVN cells innervating airway-related vagal preganglionic neurons.

The paraventricular nucleus of the hypothalamus (PVN) integrates multiple inputs via projections from arginine vasopressin (AVP)- and oxytocin (OXT)-containing neurons to the brain stem and spinal cord as well as regulates respiratory and cardiovascular stress-related responses, which also affect airway function. In the present study, we used immunocytochemistry and the retrograde transneuronal tracer, Bartha strain of pseudorabies virus expressing green fluorescent protein (PRV-GFP), to localize AVP- and OXT-producing neurons that project to airway-related vagal preganglionic neurons (AVPNs) innervating intrapulmonary airways. PRV-GFP was microinjected into the upper right lung lobe, and after 4 days survival, hypothalamic tissue sections were processed for co-expression of PRV-GFP and AVP or PRV-GFP and OXT. In addition, in a separate group of five rats, Phaseolus vulgaris leucoagglutinin (PHAL), an anterograde tracer, was injected unilaterally into the PVN and cholera toxin beta subunit was microinjected into the tracheal wall. Analysis of five successfully infected animals showed that 14% of PRV-GFP labeled neurons express AVP traits and 18% of transneuronally-labeled neurons contain OXT. Furthermore, the identified AVPNs innervating extrathoracic trachea receive axon terminals of the PVN neurons. The results indicate that AVP- and OXT-producing PVN cells, via direct projections to the AVPNs, could modulate cholinergic outflow to the airways, as a part of overall changes in response to stress.

CNS determinants of sleep-related worsening of airway functions: implications for nocturnal asthma.

This review summarizes the recent neuroanatomical and physiological studies that form the neural basis for the state-dependent changes in airway resistance. Here, we review only the interactions between the brain regions generating quiet (non-rapid eye movement, NREM) and active (rapid eye movement, REM) sleep stages and CNS pathways controlling cholinergic outflow to the airways. During NREM and REM sleep, bronchoconstrictive responses are heightened and conductivity of the airways is lower as compared to the waking state. The decrease in conductivity of the lower airways parallels the sleep-induced decline in the discharge of brainstem monoaminergic cell groups and GABAergic neurons of the ventrolateral periaqueductal midbrain region, all of which provide inhibitory inputs to airway-related vagal preganglionic neurons (AVPNs). Withdrawal of central inhibitory influences to AVPNs results in a shift from inhibitory to excitatory transmission that leads to an increase in airway responsiveness, cholinergic outflow to the lower airways and consequently, bronchoconstriction. In healthy subjects, these changes are clinically unnoticed. However, in patients with bronchial asthma, sleep-related alterations in lung functions are troublesome, causing intensified bronchopulmonary symptoms (nocturnal asthma), frequent arousals, decreased quality of life, and increased mortality. Unquestionably, the studies revealing neural mechanisms that underlie sleep-related alterations of airway function will provide new directions in the treatment and prevention of sleep-induced worsening of airway diseases.

Ablation of vagal preganglionic neurons innervating the extra-thoracic trachea affects ventilatory responses to hypercapnia and hypoxia.

This study tested the hypothesis that during hypercapnia or hypoxia, airway-related vagal preganglionic neurons (AVPNs) of the nucleus ambiguus (NA) release acetylcholine (ACh), which in a paracrine fashion, activates ACh receptors expressed by inspiratory rhythm generating cells. AVPNs in the NA were ablated by injecting a saporin- (SA) cholera toxin b subunit (CTb-SA) conjugate into the extra-thoracic trachea (n=6). Control animals were injected with free CTb (n=6). In CTb treated rats, baseline ventilation and ventilatory responses to hypercapnia (5 and 12% CO(2) in O(2)) or hypoxia (8% O(2) in N(2)) were similar (p>0.05) prior to and 5 days after injection. CTb-SA injected rats maintained rhythmic breathing patterns 5 days post injection, however, tachypneic responses to hypercapnia or hypoxia were significantly reduced. The number of choline acetyltransferase (ChAT) immunoreactive cells in the NA was much lower (p<0.05) in CTb-SA rats as compared to animals receiving CTb only. These results suggest that AVPNs participate in the respiratory frequency response to hypercapnia or hypoxia.

Brain stem excitatory and inhibitory signaling pathways regulating bronchoconstrictive responses.

This review summarizes recent work on two basic processes of central nervous system (CNS) control of cholinergic outflow to the airways: 1) transmission of bronchoconstrictive signals from the airways to the airway-related vagal preganglionic neurons (AVPNs) and 2) regulation of AVPN responses to excitatory inputs by central GABAergic inhibitory pathways. In addition, the autocrine-paracrine modulation of AVPNs is briefly discussed. CNS influences on the tracheobronchopulmonary system are transmitted via AVPNs, whose discharge depends on the balance between excitatory and inhibitory impulses that they receive. Alterations in this equilibrium may lead to dramatic functional changes. Recent findings indicate that excitatory signals arising from bronchopulmonary afferents and/or the peripheral chemosensory system activate second-order neurons within the nucleus of the solitary tract (NTS), via a glutamate-AMPA signaling pathway. These neurons, using the same neurotransmitter-receptor unit, transmit information to the AVPNs, which in turn convey the central command to airway effector organs: smooth muscle, submucosal secretory glands, and the vasculature, through intramural ganglionic neurons. The strength and duration of reflex-induced bronchoconstriction is modulated by GABAergic-inhibitory inputs and autocrine-paracrine controlling mechanisms. Downregulation of GABAergic inhibitory influences may result in a shift from inhibitory to excitatory drive that may lead to increased excitability of AVPNs, heightened airway responsiveness, and sustained narrowing of the airways. Hence a better understanding of these normal and altered central neural circuits and mechanisms could potentially improve the design of therapeutic interventions and the treatment of airway obstructive diseases.

Orexin stimulates breathing via medullary and spinal pathways.

A central neuronal network that regulates respiration may include hypothalamic neurons that produce orexin, a peptide that influences sleep and arousal. In these experiments, we investigated 1) projections of orexin-containing neurons to the pre-Botzinger region of the rostral ventrolateral medulla that regulates rhythmic breathing and to phrenic motoneurons that innervate the diaphragm; 2) the presence of orexin A receptors in the pre-Botzinger region and in phrenic motoneurons; and 3) physiological effects of orexin administered into the pre-Botzinger region and phrenic nuclei at the C3-C4 levels. We found orexin-containing fibers within the pre-Botzinger complex. However, only 0.5% of orexin-containing neurons projected to the pre-Botzinger region, whereas 2.9% of orexin-containing neurons innervated the phrenic nucleus. Neurons of the pre-Botzinger region and phrenic nucleus stained for orexin receptors, and activation of orexin receptors by microperfusion of orexin in either site produced a dose-dependent, significant (P <0.05) increase in diaphragm electromyographic activity. These data indicate that orexin regulates respiratory activity and may have a role in the pathophysiology of sleep-related respiratory disorders.

Chemical profile of vagal preganglionic motor cells innervating the airways in ferrets: the absence of noncholinergic neurons.

In ferrets, we investigated the presence of choline acetyltransferase (ChAT), vasoactive intestinal peptide (VIP), and markers for nitric oxide synthase (NOS) in preganglionic parasympathetic neurons innervating extrathoracic trachea and intrapulmonary airways. Cholera toxin beta-subunit, a retrograde axonal transganglionic tracer, was used to identify airway-related vagal preganglionic neurons. Double-labeling immunohistochemistry and confocal microscopy were employed to characterize the chemical nature of identified airway-related vagal preganglionic neurons at a single cell level. Physiological experiments were performed to determine whether activation of the VIP and ChAT coexpressing vagal preganglionic neurons plays a role in relaxation of precontracted airway smooth muscle tone after muscarinic receptor blockade. The results showed that 1) all identified vagal preganglionic neurons innervating extrathoracic and intrapulmonary airways are acetylcholine-producing cells, 2) cholinergic neurons innervating the airways coexpress ChAT and VIP but do not contain NOS, and 3) chemical stimulation of the rostral nucleus ambiguus had no significant effect on precontracted airway smooth muscle tone after muscarinic receptor blockade. These studies indicate that vagal preganglionic neurons are cholinergic in nature and coexpress VIP but do not contain NOS; their stimulation increases cholinergic outflow, without activation of inhibitory nonadrenergic, noncholinergic ganglionic neurons, stimulation of which induces airway smooth muscle relaxation. Furthermore, these studies do not support the possibility of direct inhibitory innervation of airway smooth muscle by vagal preganglionic fibers that contain VIP.