Kannan V. Balan

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 (V1A) receptor expression increases in the RVLM after CIH conditioning (8 h per day for 10 days). In the first set, cholera toxin β subunit (CT‐β) was microinjected into the RVLM to retrogradely label the PVN neurons. Immunohistochemical staining demonstrated that 14.6% of CT‐β‐labelled PVN neurons were double‐labelled with AVP. In the second set, sections of the medulla were immunolabelled for V1A receptors, and the V1A 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 V1A 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 (DEMG) and genioglossus muscle (GGEMG) activity were recorded in anaesthetized, ventilated and vagotomized rats. The PVN was disinhibited by microinjecting a GABAA receptor antagonist, bicuculline (BIC, 0.1 nmol), before and after blocking V1A receptors within the RVLM and rVRC with SR49059 (0.2 nmol). In RA‐conditioned rats, disinhibition of the PVN increased BP, HR, minute DEMG and GGEMG activity and these increases were attenuated after blocking V1A 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 V1A receptors in the RVLM and rVRC, and increased SNA in CIH‐conditioned animals depends on up‐regulation of V1A receptors in the RVLM.

Neuronal expression of bitter taste receptors and downstream signaling molecules in the rat brainstem

Previous studies have shown that molecules of the taste transduction pathway may serve as biochemical markers for chemoreceptive cells in respiratory and gastrointestinal tracts. In this study, we tested the hypothesis that brainstem neurons contain signaling molecules similar to those in taste buds which may sense the chemical composition of brain extracellular fluids. We used the reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunohistochemical techniques to evaluate presence of different bitter-responsive type 2 taste receptors (T2Rs), their associated G-protein α-gustducin, the downstream signaling molecules phospholipase C isoform β2 (PLC-β2) and transient receptor potential melastatin 5 (TRPM5) in the brainstem of rats. RT-PCR confirmed the mRNA coding for α-gustducin, PLC-β2, TRPM5 and rT2R1 but not that of rT2R16, rT2R26 and rT2R38 in the medulla oblongata. Western blotting confirmed the presence of α-gustducin at the protein level in rat brainstem. Immunohistochemistry identified cells expressing α-gustducin and PLC-β2 at multiple cardiorespiratory and CO(2)/H(+) chemosensory sites, including rostral ventral medulla, facial, parapyramidal, solitary tract, hypoglossal and raphe nuclei. In the medullary raphe, α-gustducin and PLC-β2 were colocalized with a subpopulation of tryptophan hydroxylase (TPH)-immunoreactive serotonergic neurons, a subset of which has respiratory CO(2)/H(+) chemosensitivity. Presence of the T2R1 gene and other genes and proteins of the bitter taste transduction pathway in the brainstem implies additional functions for taste receptors and their effector molecules apart from their gustatory function.

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-1β and IL-6, and vagotomy blunted this increase in IL-1β, 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-1β 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-1β and associated with an impaired hypoxic ventilatory response.

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-1β 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-1β 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.

Co-expression of nAChRs and molecules of the bitter taste transduction pathway by epithelial cells of intrapulmonary airways.

AIMS The ability to sense the bitter taste of nicotine is an important component of addiction to, and withdrawal from, cigarette smoking. alpha-Gustducin and phospholipase C-beta2 (PLC-beta2), molecules involved in the taste transduction pathway, have been identified in airway epithelial solitary chemosensory cells (SCCs). Airway epithelial cells also express multiple nicotinic acetylcholine receptors (nAChRs). However, the relationship between nAChRs and molecules of taste transduction in response to nicotine is not known. This study was designed to determine whether nAChRs and the taste transduction molecules alpha-gustducin, PLC-beta2 and bitter taste receptors (T2R38) reside at sites of the intrapulmonary airways where interaction with the nicotine components of cigarette smoke is likely. MAIN METHODS We used the reverse transcription-polymerase chain reaction (RT-PCR) to detect alpha-gustducin, PLC-beta2 and T2R38 mRNA and immunohistochemistry to localize expression of these proteins by nAChR expressing cells of the airway. KEY FINDINGS RT-PCR demonstrated the presence of mRNA for alpha-gustducin, PLC-beta2 and T2R38. Immunohistochemistry showed the expression of alpha-gustducin, PLC-beta2 and T2R38 by subsets of epithelial cells at all levels of the intrapulmonary airways including bronchi, terminal and respiratory bronchioles. Double labeling demonstrated the co-expression of alpha-gustducin with alpha3, alpha4, alpha5, alpha7 and beta2, as well as, PLC-beta2 and T2R38 with alpha4, alpha5 and beta2 nAChR subunits. SIGNIFICANCE These findings provide morphological evidence for the presence of molecules of the bitter taste transduction pathway in nAChR expressing SCCs of the intrapulmonary airways. These SCCs may, thus, constitute a peripheral component of the bitter taste signal transduction pathway for nicotine.

Intrapulmonary lipopolysaccharide exposure upregulates cytokine expression in the neonatal brainstem

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‐1β and vagotomy blunted this response. There was an attenuated ventilatory response to hypoxia and increased brainstem IL‐1β expression after LPS.

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 α and β 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. α2, α3, α4, α5, α7, and β2 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.

Chronic Intermittent Hypoxia-Induced Augmented Cardiorespiratory Outflow Mediated by Vasopressin-V1A Receptor Signaling in the Medulla

A co-morbidity of sleep-disordered breathing is hypertension associated with elevated sympathetic nerve activity, which may result from chronic intermittent hypoxia (CIH). CIH evokes plasticity in cardiorespiratory regulating sites, including the paraventricular nucleus (PVN), which acts to sustain increased sympathetic nerve activity. Our working hypothesis is that vasopressin neurons mediate the sustained increase in blood pressure and altered breathing associated with CIH. In a series of neuroanatomical experiments, we determined if vasopressin-containing PVN neurons innervate rostral ventrolateral medulla (RVLM), and altered cardiorespiratory responses induced by CIH conditioning (8h/day for 10 days) is mediated by vasopressin-V(1A ) receptor signaling in the medulla. In the first set of experiments, cholera toxin β subunit was microinjected into the RVLM to delineate innervation of the PVN. Immunohistochemistry data showed vasopressin-containing PVN neurons were double-labeled with cholera toxin β subunit, indicating vasopressin projection to the RVLM. In the second set, sections of the medulla were immunolabeled for vasopressin V(1A ) receptor, and its expression was significantly higher in the RVLM and in the neighboring rostral ventral respiratory column in CIH- than from RA-conditioned rats. In a series of physiological experiments,we determined if blocking the vasopressin V(1A )receptor in the medulla would normalize blood pressure in CIH-conditioned rats and also attenuate the evoked responses to PVN disinhibition.Blood pressure, heart rate, diaphragmatic and genioglossus muscle activity were recorded in anesthetized, ventilated and vagotomized rats. The PVN was disinhibited by microinjecting bicuculline before and after blocking vasopressin V(1A ) receptors in the RVLM/rostral ventral respiratory column. In RA-conditioned rats, PVN disinhibition increased blood pressure, heart rate, minute diaphragmatic and genioglossus muscle activity, and these increases were attenuated after blocking the vasopressin V(1A ) receptor. In CIH-conditioned rats, a significantly greater dose of blocker was required to blunt these physiological responses and it also normalized the baseline blood pressure. Our findings indicate that vasopressin is the neuropeptide released from PVN neurons that modulates cardiorespiratory output via the RVLM and rostral ventral respiratory column.

Mutation in the myelin proteolipid protein gene alters BK and SK channel function in the caudal medulla

Proteolipid protein (Plp) gene mutation in rodents causes severe CNS dysmyelination, early death, and lethal hypoxic ventilatory depression (Miller et al., 2004). To determine if Plp mutation alters neuronal function critical for control of breathing, the nucleus tractus solitarii (nTS) of four rodent strains were studied: myelin deficient rats (MD), myelin synthesis deficient (Plp(msd)), and Plp(null) mice, as well as shiverer (Mbp(shi)) mice, a myelin basic protein mutant. Current-voltage relationships were analyzed using whole-cell patch-clamp in 300 microm brainstem slices. Voltage steps were applied, and inward and outward currents quantified. MD, Plp(msd), and Plp(null), but not Mbp(shi) neurons exhibited reduced outward current in nTS at P21. Apamin blockade of SK calcium-dependent currents and iberiotoxin blockade of BK calcium-dependent currents in the P21 MD rat demonstrated reduced outward current due to dysfunction of these channels. These results provide evidence that Plp mutation specifically alters neuronal excitability through calcium-dependent potassium channels in nTS.