Brainstem Dbh+ neurons control allergen-induced airway hyperreactivity.

Exaggerated airway constriction triggered by repeated exposure to allergen, also called hyperreactivity, is a hallmark of asthma. Whereas vagal sensory neurons are known to function in allergen-induced hyperreactivity1-3, the identity of downstream nodes remains poorly understood. Here we mapped a full allergen circuit from the lung to the brainstem and back to the lung. Repeated exposure of mice to inhaled allergen activated the nuclei of solitary tract (nTS) neurons in a mast cell-, interleukin-4 (IL-4)- and vagal nerve-dependent manner. Single-nucleus RNA sequencing, followed by RNAscope assay at baseline and allergen challenges, showed that a Dbh+ nTS population is preferentially activated. Ablation or chemogenetic inactivation of Dbh+ nTS neurons blunted hyperreactivity whereas chemogenetic activation promoted it. Viral tracing indicated that Dbh+ nTS neurons project to the nucleus ambiguus (NA) and that NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that directly drive airway constriction. Delivery of noradrenaline antagonists to the NA blunted hyperreactivity, suggesting noradrenaline as the transmitter between Dbh+ nTS and NA. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. This knowledge informs how neural modulation could be used to control allergen-induced airway hyperreactivity.

Short-lived sensitization of cardiovascular outcomes of postpartum endotoxemia in preeclamptic rats: Role of medullary solitary tract neuroinflammation.

Preeclampsia (PE) is a pregnancy-related disorder with serious maternal complications. Considering the increased importance of postpartum infection in maternal morbidity and mortality, we investigated whether preeclamptic maternal programming alters cardiovascular consequences of endotoxemia in rats and the role of cardiac and brainstem neuroinflammation in this interaction. Preeclampsia was induced by oral administration of L-NAME (50 mg/kg/day) for 7 days starting from day 14 of conception. Changes in blood pressure, heart rate, and cardiac autonomic function caused by lipopolysaccharide (LPS, 5 mg/kg i.v.) were assessed in mothers at 3 weeks (weaning time) and 9 weeks postnatally. Compared with respective non-PE counterparts, LPS treatment of weaning PE mothers caused significantly greater (i) falls in blood pressure, (ii) rises in heart rate and left ventricular contractility (dP/dtmax), (iii) reductions in time and frequency domain indices of heart rate variability and shifts in cardiac sympathovagal balance (low-frequency/high-frequency ratio, LF/HF) towards parasympathetic dominance, and (iv) attenuation of reflex bradycardic responses measured by the vasoactive method. The intensified LPS effects in weaning PE rats subsided after 9 weeks of delivery. Immunohistochemical studies showed increased protein expression of nuclear factor kappa B (NF-κB) in brainstem neuronal pools of the nucleus of the solitary tract (NTS), but not rostral ventrolateral medulla (RVLM), in endotoxic PE weaning rats compared with non-PE rats. Cardiac NF-κB expression was increased by LPS but this was similarly noted in PE and non-PE rats. Together, preeclamptic maternal programming elicits short-term exacerbation of endotoxic cardiovascular and autonomic derangements due possibly to exaggerated NTS neuroinflammatory insult.

Cytokine Storm in COVID19: A Neural Hypothesis.

Cytokine storm in COVID-19 is characterized by an excessive inflammatory response to SARS-CoV-2 that is caused by a dysregulated immune system of the host. We are proposing a new hypothesis that SARS-CoV-2 mediated inflammation of nucleus tractus solitarius (NTS) may be responsible for the cytokine storm in COVID 19. The inflamed NTS may result in a dysregulated cholinergic anti-inflammatory pathway and hypothalamic-pituitary-adrenal axis.

Pulmonary Edema in COVID19-A Neural Hypothesis.

In COVID-19, lung manifestations present as a slowly evolving pneumonia with insidious early onset interstitial pulmonary edema that undergoes acute exacerbation in the late stages and microvascular thrombosis. Currently, these manifestations are considered to be only consequences of pulmonary SARS-CoV-2 virus infection. We are proposing a new hypothesis that neurogenic insult may also play a major role in the pathogenesis of these manifestations. SARS-CoV-2 mediated inflammation of the nucleus tractus solitarius (NTS) may play a role in the acute exacerbation of pulmonary edema and microvascular clotting in COVID-19 patients.

Proinflammatory Cytokines in the Nucleus of the Solitary Tract of Hypertensive Rats Exposed to Chronic Intermittent Hypoxia.

Obstructive sleep apnea (OSA) is characterized by chronic intermittent hypoxia (CIH), which is considered the main factor for developing hypertension. Sympathetic overflow, oxidative stress and inflammation have been associated with the CIH-induced hypertension. In rats exposed to CIH mimicking OSA, intermittent hypoxia enhanced carotid body (CB) chemosensory discharge, leading to an increase in arterial blood pressure in 3-5 days. In addition, CIH increases the CB levels of proinflammatory cytokines IL-1ß, IL-6 and TNF-α in the CB. Proinflammatory molecules have been also involved in neurogenic hypertension acting on brain cardiovascular centers, like the nucleus of the solitary tract (NTS), which is the primary site for afferent CB inputs. Accordingly, we aim to study if proinflammatory cytokines in the NTS may play a role in the hypertension induced by CIH. Male Sprague-Dawley rats 250 g were exposed to CIH (5% O2, 12 times/h, 8 h/day) for 7-28 days. Brains were removed and processed to measure IL-1ß, IL-6 and TNF-α in the NTS using qPCR and immunofluorescence. The mRNA levels were significantly augmented in the NTS of rats exposed during 21 days to CIH compared with control animals. In addition, a significant increase of IL-1ß, IL-6 and TNF-α immunofluorescence was found in the NTS at day 28 of CIH exposure compared with control rats. Present results suggest that proinflammatory cytokines in the NTS may contribute to the maintenance of hypertension in CIH-exposed animals.

House-dust mite allergen and ozone exposure decreases histamine H3 receptors in the brainstem respiratory nuclei.

Allergic airway diseases in children are a common and a growing health problem. Changes in the central nervous system (CNS) have been implicated in contributing to some of the symptoms. We hypothesized that airway allergic diseases are associated with altered histamine H3 receptor expression in the nucleus tractus solitarius (NTS) and caudal spinal trigeminal nucleus, where lung/airway and nasal sensory afferents terminate, respectively. Immunohistochemistry for histamine H3 receptors was performed on brainstem sections containing the NTS and the caudal spinal trigeminal nucleus from 6- and 12-month-old rhesus monkeys who had been exposed for 5 months to house dust mite allergen (HDMA)+O3 or to filtered air (FA). While histamine H3 receptors were found exclusively in astrocytes in the caudal spinal trigeminal nucleus, they were localized to both neuronal terminals and processes in the NTS. HDMA+O3 exposure significantly decreased histamine H3 receptor immunoreactivity in the NTS at 6 months and in the caudal spinal trigeminal nucleus at 12 months of age. In conclusion, exposing young primates to HDMA+O3 changed histamine H3 receptor expression in CNS pathways involving lung and nasal afferent nerves in an age-related manner. Histamine H3 receptors may be a therapeutic target for allergic asthma and rhinitis in children.

Immune challenge and satiety-related activation of both distinct and overlapping neuronal populations in the brainstem indicate parallel pathways for viscerosensory signaling.

Caudal brainstem viscerosensory nuclei convey information about the body's internal state to forebrain regions implicated in feeding behavior and responses to immune challenge, and may modulate ingestive behavior following immune activation. Illness-induced appetite loss might be attributed to accentuated "satiety" pathways, activation of a distinct "danger channel" separate from satiety pathways, or both. To evaluate neural substrates that could mediate the effects of illness on ingestive behavior, we analyzed the pattern and phenotypes of medullary neurons responsive to consumption of a preferred food, sweetened milk, and to intraperitoneal lipopolysaccharide challenge that reduced sweetened milk intake. Brainstem sections were stained for c-Fos, dopamine beta-hydroxylase, phenylethanolamine-N-methyltransferase, and glucagon-like peptide-1 (GLP-1) immunoreactivity. Sweetened milk intake activated many neurons throughout the nucleus of the solitary tract (NTS), including A2 noradrenergic neurons in the caudal half of the NTS. LPS challenge activated a similar population of neurons in the NTS, in addition to rostral C2 adrenergic and mid-level A2 noradrenergic neurons in the NTS, many C1 and A1 neurons in the ventrolateral medulla, and in GLP-1 neurons in the dorsal medullary reticular nucleus. Increased numbers of activated GLP-1 neurons in the NTS were only associated with sweetened milk ingestion. Evidence for parallel processing was reflected in the parabrachial nucleus, where sweetened milk intake resulted in activation of the inner external lateral, ventrolateral and central medial portions, whereas LPS challenge induced c-Fos expression in the outer external lateral portions. Thus, signals generated in response to potentially dangerous physiological conditions seem to be propagated via specific populations of catecholaminergic neurons in the NTS and VLM, and likely include a pathway through the external lateral PBN. The data indicate that immune challenge engages multiple ascending neural pathways including both a distinct catecholaminergic "danger" pathway, and a possibly multimodal pathway derived from the NTS.

Specific inflammatory condition in nucleus tractus solitarii of the SHR: novel insight for neurogenic hypertension?

Human essential hypertension is a complex polygenic trait with underlying genetic components that remain unknown. Since the brainstem structure--the nucleus of the solitary tract (NTS)--is a pivotal region for regulating the set-point of arterial pressure, we proposed a role for it in the development of primary hypertension. Using microarray and real-time RT-PCR, we have recently identified that some pro-inflammatory molecules, such as junctional adhesion molecule-1 (JAM-1; a leukocyte/platelet adhesion molecule), were over expressed in endothelial cells in the NTS of an animal model of human essential hypertension--the spontaneously hypertensive rat (SHR) compared to normotensive Wistar Kyoto rats (WKY). Adenoviral mediated over expression of JAM-1 in NTS of WKY rats produced both hypertension and localized leukocyte adherence to the microvasculature. With a known effect of leukocyte adhesion causing cytokine release, we predicted differences in the level of gene expression of cytokines in the NTS of SHR relative to WKY. Gene expression of monocyte chemoattractant protein-1 (MCP-1) was higher in the NTS of SHR while inter-leukin-6 (IL-6) was lower in the NTS of SHR compared to the WKY. Because both inflammatory molecules are known to affect neural functions, our data suggest that the microvasculature of NTS of the SHR exhibits a specific inflammatory state. We propose a new hypothesis that as a consequence of enhanced expression of leukocyte adhesion molecules within the microvasculature of NTS there is a specific inflammatory response that leads to cardiovascular autonomic dysfunction contributing to neurogenic hypertension.

Gene expression profiles of major cytokines in the nucleus tractus solitarii of the spontaneously hypertensive rat.

Since the nucleus of the solitary tract (NTS) is a pivotal region for regulating the set-point of arterial pressure, we proposed a role for it in the development of neurogenic hypertension. Recent studies have suggested that pro-inflammatory molecules are highly expressed in the NTS of an animal model of human essential hypertension--the spontaneously hypertensive rat (SHR), compared to normotensive Wistar-Kyoto rat (WKY). Based on this evidence, we hypothesized that inflammatory mediators such as cytokines are up-regulated in the hypertensive NTS. In the present study, we have assessed the level of gene expression of some cytokines in the NTS of SHR compared to WKY. In addition, for further confirmation of abnormal inflammatory condition within the NTS of SHR, we identified gene expression levels of an inflammatory marker, glycoprotein-39 (gp39) precursor, which is homologous to chitinase 3-like protein 1, human cartilage-gp39 or YKL40. The NTS was micro-dissected from 15-week-old male SHR and WKY rats. Total RNA was extracted and quantitative RT-PCR was performed. Gene expression of gp39 precursor and monocyte chemoattractant protein-1 were higher in the NTS of SHR while inter-leukin-6 was lower in the NTS of SHR compared to the WKY. In contrast, there were no significant differences in the expression of other cytokines including: inter-leukin-1 beta, tumor necrosis factor-alpha and transforming growth factor beta 1. These data together with our previous published finding of an over expression of junctional adhesion molecule-1 suggest that the NTS of the SHR exhibits a specific inflammatory state.

Dual modulation of synaptic transmission in the nucleus tractus solitarius by prostaglandin E2 synthesized downstream of IL-1beta.

The activation of the innate immune system induces the production of blood-borne proinflammatory cytokines like interleukin-1beta (IL-1beta), which in turn triggers brain-mediated adaptative responses referred to as sickness behaviour. These responses involve the modulation of neural networks in key regions of the brain. The nucleus tractus solitarius (NTS) of the brainstem is a key nucleus for immune-to-brain signalling. It is the main site of termination of vagal afferents and is adjacent to the area postrema, a circumventricular organ allowing blood-borne action of circulating IL-1beta. Although it is well described that IL-1beta activates cerebral endothelial and glial cells, it is still unknown if and how IL-1beta or downstream-synthesized molecules impact NTS synaptic function. In this study we report that IL-1beta did not modulate NTS synaptic transmission per se, whereas prostaglandin E(2) (PGE(2)), which is produced downstream of IL-1beta, produced opposite effects on spontaneous and evoked release. On the one hand, PGE(2) facilitated glutamatergic transmission between local NTS neurons by enhancing the frequency of spontaneous excitatory postsynaptic currents through a presynaptic receptor different from the classical EP1-4 subtypes. On the other hand, PGE(2) also depressed evoked excitatory input from vagal afferent terminals through presynaptic EP3 receptors coupled to G-proteins linked to adenylyl cyclase and protein kinase A activity. Our data show that IL-1beta-induced PGE(2) can modulate evoked and spontaneous release in the NTS differentially through different mechanisms. These data unravel some molecular mechanisms by which innate immune stimuli could signal to, and be integrated within, the brainstem to produce central adaptative responses.

Synergistic and additive actions of a psychosocial stressor and endotoxin challenge: Circulating and brain cytokines, plasma corticosterone and behavioral changes in mice.

Activation of the inflammatory immune response may provoke neuroendocrine and central neurochemical effects that are reminiscent of those elicited by traditional stressors, and when administered concurrently may have synergistic effects. The present investigation assessed whether a psychosocial stressor, comprising social disruption, would augment the effects of lipopolysaccharide in mice. It was indeed observed that the social disruption engendered by a period of 2-4 weeks of social isolation (but not 1-7 days of this treatment) followed by regrouping, enhanced the effects of lipopolysaccharide (LPS: 10mug) in the provocation of sickness behavior, as well as plasma corticosterone, IL-6, TNF-alpha and IL-10 levels. Similar effects were not apparent with respect to IL-1beta, IL-4, or IFN-gamma. Synergy between LPS and other stressors (restraint, tail pinch, and loud noise) was not apparent with respect to sickness or plasma corticosterone, provisionally suggesting that social stressors, such as regrouping, may be more powerful or may engage unique neural or neuroendocrine circuits that favour synergistic outcomes. Within the CNS, the LPS and the regrouping stressor synergistically enhanced NE utilization within the prefrontal cortex, and additively influenced hippocampal NE utilization. In contrast to the effects on circulating cytokines, the LPS-induced elevation of IL-1beta, IL-6 and TNF-alpha mRNA expression in the hippocampus, PFC and nucleus tractus solitarius was diminished in animals that had experienced the regrouping stressor. In view of the combined actions of LPS challenge and a social stressor, these data are interpreted as suggesting that models of depression based on immune activation ought to consider the stressor backdrop upon which immune challenges are imposed.

Brain response to cecal infection with Campylobacter jejuni: analysis with Fos immunohistochemistry.

Infections with bacterial pathogens can induce increased anxiety-like behaviors in rodents without otherwise noticeable behavioral or physiological symptoms of sickness, as shown with the food-borne pathogen Campylobacter jejuni. This observation implicates the ability of the brain to sense, and respond to, such an infection. We tested our hypothesis that intestinal infection with the gram-negative bacterium C. jejuni leads to activation of certain brain regions that process gastro-intestinal sensory information. The induction of c-Fos protein as a marker for neuronal activation was assessed in the brains of mice inoculated orally with live C. jejuni, as compared to saline-treated controls. Upon colonization of the intestines, C. jejuni activated visceral sensory nuclei in the brainstem (the nucleus of the solitary tract and the lateral parabrachial nucleus) both one and two days after the oral challenge. In addition, increased c-Fos expression occurred in the hypothalamic paraventricular nucleus on the second day. This neural response occurred in the absence of measurable systemic immune activation, as serum levels of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 were undetectable and/or unchanged. These findings support the notion that information about infection with C. jejuni in the gut is indeed relayed to the visceral sensory structures in the brain. The brain responses observed could contribute to changes in behavior observed after infection.

Reversible inactivation of the dorsal vagal complex blocks lipopolysaccharide-induced social withdrawal and c-Fos expression in central autonomic nuclei.

Peripheral administration of lipopolysaccharide (LPS), a potent activator of the immune system, induces symptoms of behavioral depression, such as social withdrawal, concommitant with increases in c-Fos expression in central autonomic network nuclei. Previous studies implicated vagal visceral sensory nerves in transduction of immune-related signals relevant to for the induction of social withdrawal, a symptom of behavioral depression. Vagal sensory nerves terminate in the dorsal vagal complex (DVC) of the brainstem, a region that functions to integrate visceral signals and may also play a role in modulating arousal and affect. The objective of the current study was to determine whether the DVC contributes to immunosensory pathways driving symptoms of social withdrawal associated with LPS-induced behavioral depression, using a reversible lesion technique to temporarily inactivate the DVC. To assess the effects of DVC inactivation on LPS-induced social withdrawal and the subsequent changes in brain activation, we used behavioral assessment of social withdrawal, and analyzed c-Fos expression, a marker of neuronal activation, in the central nucleus of the amygdala (CEA), bed nucleus of the stria terminalis (BST), hypothalamic paraventricular nucleus (PVN), and ventromendial preoptic area (VMPO). Two hours following intraperitoneal LPS injection, there was a significant increase in c-Fos immunoreactivity in forebrain regions in animals treated with LPS. DVC inactivation completely blocked LPS-induced social withdrawal and dramatically reduced LPS-induced Fos expression in all four forebrain regions assessed. Collectively, these findings support the idea that the DVC acts as an immune-behavior interface between the peripheral stimuli and brain areas involved in modulating social behavior.

Effect of nucleus tractus solitarius lesions on fever produced by interleukin-1beta.

Partial elimination of vagal sensory afferents by subdiaphragmatic vagal section has variously been reported to eliminate, to reduce, or to have no effect on fever produced by peripheral lipopolysaccharide and interleukin-1beta (IL-1beta). However, to adequately test the idea that vagal afferents convey immune information to the brain, all vagal input to the central nervous system must be eliminated. This was accomplished by bilateral electrolytic lesions of the nucleus tractus solitarius (NTS). Reflex bradycardia evoked by intravenous phenylbiguanide was eliminated in NTS-lesioned rats, verifying the lesion's effectiveness. IL-1beta (2 microg/kg) was given to conscious, unrestrained rats via an indwelling intraperitoneal catheter and produced rapid fever (approximately 1 degree C) with an onset latency of 15 min and peak response at 30 min, with a second, smaller peak at 130 min. NTS lesions attenuated the first fever peak, with a lesser, non-significant effect on the second peak. The thermogenic capacity of NTS-lesioned rats was evaluated using 3 different strategies: (1) thermogenesis evoked by CNS injections of prostaglandin E2, (2) 3 h exposure to a 4 degrees C environment, and (3) heat production of intrascapular brown fat produced by intravenous infusion of the beta3-adrenergic agonist BRL 37344. NTS-lesioned rats were equivalent, or even superior to control animals in their thermogenic response to these non-immune-related stimuli. Therefore, the impaired febrile response of NTS-lesioned rats to IL-1beta cannot be attributed to reduced thermogenic capacity. Finally, these results suggest that fever elicited by intraperitoneal IL-1beta is, at least in part, dependent on the integrity of NTS neurons, but also that mechanisms independent of vagal afferent projections to the NTS must also play a role in immune-to-brain signaling.

Induction of pro-inflammatory cytokine mRNAs in the brain after peripheral injection of subseptic doses of lipopolysaccharide in the rat.

Although it is generally accepted that pro-inflammatory cytokines produced by cells of the central nervous system play important roles in the communication between the central nervous system and the immune system during sepsis, it is not clear whether these cytokines are produced in the brain under subseptic conditions. In this study, we used in situ hybridization to examine the mRNA expression of the pro-inflammatory cytokines IL-1beta and TNFalpha in the brains of rats 2 and 12 h after they were challenged by peripheral injections of lipopolysaccharide (LPS) ranging from 0.01 to 1000 microg/kg. Unlike septic doses of LPS (> 500 microg/kg), which induce global expression of pro-inflammatory cytokines in the brain, subseptic doses of LPS (0.01-10 microg/kg) induced IL-1beta and TNFalpha mRNA expression only in the choroid plexus, the circumventricular organs, and meninges. The expression of the cytokine-responsive immediate early gene I kappaB alpha was induced in the brain after doses of LPS as low as 0.1 microg/kg. I kappaB alpha mRNA expression was confined to sites where IL-1beta and TNFalpha were expressed. These results indicate that the induction and action of pro-inflammatory cytokines during subseptic infection occur at the blood-brain barrier and at circumventricular organs, which may be sites for elaboration of signal molecules that communicate peripheral immune status to the brain.

Substance P receptor-expressing neurons in the medullary and spinal dorsal horns projecting to the nucleus of the solitary tract in the rat.

By using substance P receptor (SPR) immunofluorescence histochemistry combined with fluorescent retrograde labeling, we examined the distribution of the trigeminal and spinal neurons with SPR-like immunoreactivity (-LI) projecting to the nucleus of the solitary tract in the rat. After injection of Fluoro-Gold (FG) into the nucleus of the solitary tract, FG-labeled neurons showing SPR-LI were mainly seen in lamina I of the medullary and spinal dorsal horns, lamina V and the lateral spinal nucleus of the spinal cord. The present results suggest that the trigeminal and spinal neurons with SPR-LI, especially those in lamina I may be involved in the transmission of somatic and/or visceral nociceptive information from the medullary and spinal dorsal horns to the nucleus of the solitary tract.