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The smooth muscle cells surrounding small arteries and arterioles sense changes in local blood pressure and reflexively adjust their contractility to regulate vessel caliber. This autoregulatory property, known as myogenic tone, helps maintain a constant capillary pressure, which is important for tissue perfusion, fluid balance and vessel integrity. Here, we show that in addition to pressure, temperature is a critical determinant of myogenic tone. Heating steeply activates tone in mouse skeletal muscle, brain, gut, and skin arteries with a temperature co-efficient ( Q (10) ) of ~11-20, reflecting a highly thermosensitive process. Further, the temperatures for half-maximal tone correspond closely to resting tissue temperatures, indicating that arterial tone is sensitive to small thermal fluctuations. In skeletal muscle arteries, we show that temperature and intraluminal pressure are sensed largely independently yet integrated to trigger contractility. Further, we reveal key roles for heat-sensitive channels TRPV1 and TRPM4 in heat-induced myogenic tone in the skeletal muscle, both in vitro and in vivo . Elevations in temperature are known to increase tissue perfusion by altering blood viscosity; remarkably, we demonstrate that thermosensitive tone counterbalances this effect. Thus, the thermosensitivity of myogenic tone is tuned to tissue temperature and serves as a fundamental mechanism to regulate local blood flow.

This review is on how current knowledge of brainstem control of gastric mechanical function unfolded over nearly four decades from the perspective of our research group. It describes data from a multitude of different types of studies involving retrograde neuronal tracing, microinjection of drugs, whole-cell recordings from rodent brain slices, receptive relaxation reflex, accommodation reflex, c-Fos experiments, immunohistochemical methods, electron microscopy, transgenic mice, optogenetics, and GABAergic signaling. Data obtained indicate the following: (1) nucleus tractus solitarius (NTS)-dorsal motor nucleus of the vagus (DMV) noradrenergic connection is required for reflex control of the fundus; (2) second-order nitrergic neurons in the NTS are also required for reflex control of the fundus; (3) a NTS GABAergic connection is required for reflex control of the antrum; (4) a single DMV efferent pathway is involved in brainstem control of gastric mechanical function under most experimental conditions excluding the accommodation reflex. Dual-vagal effectors controlling cholinergic and non-adrenergic and non-cholinergic (NANC) input to the stomach may be part of the circuitry of this reflex. (5) GABAergic signaling within the NTS via Sst-GABA interneurons determine the basal (resting) state of gastric tone and phasic contractions. (6) For the vagal-vagal reflex to become operational, an endogenous opioid in the NTS is released and the activity of Sst-GABA interneurons is suppressed. From the data, we suggest that the CNS has the capacity to provide region-specific control over the proximal (fundus) and distal (antrum) stomach through engaging phenotypically different efferent inputs to the DMV.

Key points The functional roles of the capsaicin receptor, TRPV1, outside of sensory nerves are unclear. We mapped TRPV1 in the mouse circulation, revealing extensive expression in the smooth muscle of resistance arterioles supplying skeletal muscle, heart and adipose. Activation of TRPV1 in vascular myocytes constricted arteries, reduced coronary flow in isolated hearts, and increased systemic blood pressure. These functional effects were retained after sensory nerve ablation, indicating specific signalling by arterial TRPV1. TRPV1 mediated the vasoconstrictive and blood pressure responses to the endogenous inflammatory lipid, lysophosphatidic acid. These results show that TRPV1 in arteriolar myocytes modulates regional blood flow and systemic blood pressure, and suggest that TRPV1 may be a target of vasoactive inflammatory mediators. Abstract The capsaicin receptor, TRPV1, is a key ion channel involved in inflammatory pain signalling. Although mainly studied in sensory nerves, there are reports of TRPV1 expression in isolated segments of the vasculature, but whether the channel localizes to vascular endothelium or smooth muscle is controversial and the distribution and functional roles of TRPV1 in arteries remain unknown. We mapped functional TRPV1 expression throughout the mouse arterial circulation. Analysis of reporter mouse lines TRPV1PLAP-nlacZ and TRPV1-Cre:tdTomato combined with Ca2+ imaging revealed specific localization of TRPV1 to smooth muscle of terminal arterioles in the heart, fat and skeletal muscle. Capsaicin evoked inward currents (current density ∼10% of sensory neurons) and raised intracellular Ca2+ levels in arterial smooth muscle cells, constricted arterioles ex vivo and in vivo and increased systemic blood pressure in mice and rats. Further, capsaicin markedly and dose-dependently reduced coronary flow. Pharmacologic and/or genetic disruption of TRPV1 abolished all these effects of capsaicin as well as vasoconstriction triggered by lysophosphatidic acid, a bioactive lipid generated by platelets and atherogenic plaques. Notably, ablation of sensory nerves did not affect the responses to capsaicin revealing a vascular smooth muscle-restricted signalling mechanism. Moreover, unlike in sensory nerves, TRPV1 function in arteries was resistant to activity-induced desensitization. Thus, TRPV1 activation in vascular myocytes enables a persistent depolarizing current, leading to constriction of coronary, skeletal muscle, and adipose arterioles and a sustained increase in systemic blood pressure. This article is protected by copyright. All rights reserved.

Activity in the dorsal vagal complex (DVC) is essential to gastric motility regulation. We and others have previously shown that this activity is greatly influenced by local GABAergic signaling, primarily because of somatostatin (SST)-expressing GABAergic neurons. To further understand the network dynamics associated with gastric motility control in the DVC, we focused on another neuron prominently distributed in this complex, neuropeptide-Y (NPY) neurons. However, the effect of these neurons on gastric motility remains unknown. Here, we investigate the anatomic and functional characteristics of the NPY neurons in the nucleus tractus solitarius (NTS) and their interactions with SST neurons using transgenic mice of both sexes. We sought to determine whether NPY neurons influence the activity of gastric-projecting neurons, synaptically interact with SST neurons, and affect end-organ function. Our results using combined neuroanatomy and optogenetic in vitro and in vivo show that NPY neurons are part of the gastric vagal circuit as they are trans-synaptically labeled by a viral tracer from the gastric antrum, are primarily excitatory as optogenetic activation of these neurons evoke EPSCs in gastric-antrum-projecting neurons, are functionally coupled to each other and reciprocally connected to SST neurons, whose stimulation has a potent inhibitory effect on the action potential firing of the NPY neurons, and affect gastric tone and motility as reflected by their robust optogenetic response in vivo. These findings indicate that interacting NPY and SST neurons are integral to the network that controls vagal transmission to the stomach. SIGNIFICANCE STATEMENT The brainstem neurons in the dorsal nuclear complex are essential for regulating vagus nerve activity that affects the stomach via tone and motility. Two distinct nonoverlapping populations of predominantly excitatory NPY neurons and predominantly inhibitory SST neurons form reciprocal connections with each other in the NTS and with premotor neurons in the dorsal motor nucleus of the vagus to control gastric mechanics. Light activation and inhibition of NTS NPY neurons increased and decreased gastric motility, respectively, whereas both activation and inhibition of NTS SST neurons enhanced gastric motility

Arterioles maintain blow flow by adjusting their diameter in response to changes in local blood pressure. In this process called the myogenic response, a vascular smooth muscle mechanosensor controls tone predominantly through altering the membrane potential. In general, myogenic responses occur slowly (minutes). In the heart and skeletal muscle, however, tone is activated rapidly (tens of seconds) and terminated by brief (100 ms) arterial constrictions. Previously, we identified extensive expression of TRPV1 in the smooth muscle of arterioles supplying skeletal muscle, heart and fat. Here we reveal a critical role for TRPV1 in the rapid myogenic tone of these tissues. TRPV1 antagonists dilated skeletal muscle arterioles in vitro and in vivo, increased coronary flow in isolated hearts, and transiently decreased blood pressure. All of these pharmacologic effects were abolished by genetic disruption of TRPV1. Stretch of isolated vascular smooth muscle cells or raised intravascular pressure in arteries triggered Ca

The capsaicin receptor, TRPV1, is a key ion channel involved in inflammatory pain signaling. Although mainly studied in sensory nerves, there are reports of TRPV1 expression in isolated segments of the vasculature, but whether the channel localizes to vascular endothelium or smooth muscle is controversial and the distribution and functional roles of TRPV1 in arteries remain unknown. We mapped functional TRPV1 expression throughout the mouse arterial circulation. Analysis of reporter mouse lines TRPV1PLAP-nlacZ and TRPV1-Cre:tdTomato combined with Ca2+ imaging revealed specific localization of TRPV1 to smooth muscle of terminal arterioles in the heart, fat and skeletal muscle. Capsaicin evoked inward currents and raised intracellular Ca2+ levels in arterial smooth muscle cells, constricted arterioles ex vivo and in vivo and increased systemic blood pressure in mice and rats. Further, capsaicin markedly and dose-dependently reduced coronary flow. Pharmacologic and/or genetic disruption of TRPV1 abolished all these effects of capsaicin as well as vasoconstriction triggered by lysophosphatidic acid, a bioactive lipid generated by platelets and atherogenic plaques. Notably, ablation of sensory nerves did not affect the responses to capsaicin revealing a vascular smooth muscle-restricted signaling mechanism. Moreover, unlike in sensory nerves, TRPV1 function in arteries was resistant to activity-induced desensitization. Thus, TRPV1 activation in vascular myocytes of resistance arterioles enables a persistent depolarizing current, leading to constriction of coronary, skeletal muscle, and adipose arterioles and a sustained increase in systemic blood pressure.

The α4β2 nicotinic acetylcholine receptor (nAChR) is important in central nervous system physiology and in mediating several of the pharmacological effects of nicotine on cognition, attention, and affective states. It is also the likely receptor that mediates nicotine addiction. This receptor assembles in two distinct stoichiometries: (α4)2(β2)3 and (α4)3(β2)2, which are referred to as high-sensitivity (HS) and low-sensitivity (LS) nAChRs, respectively, based on a difference in the potency of acetylcholine to activate them. The physiologic and pharmacological differences between these two receptor subtypes have been described in heterologous expression systems. However, the presence of each stoichiometry in native tissue currently remains unknown. In this study, different ratios of rat α4 and β2 subunit cDNA were transfected into human embryonic kidney 293 cells to create a novel model system of HS and LS α4β2 nAChRs expressed in a mammalian cell line. The HS and LS nAChRs were characterized through pharmacological and biochemical methods. Isolation of surface proteins revealed higher amounts of α4 or β2 subunits in the LS or HS nAChR populations, respectively. In addition, sazetidine-A displayed different efficacies in activating these two receptor stoichiometries. Using this model system, a neurophysiological "two-concentration" acetylcholine or carbachol paradigm was developed and validated to determine α4/β2 subunit stoichiometry. This paradigm was then used in layers I-IV of slices of the rat motor cortex to determine the percent contribution of HS and LS α4β2 receptors in this brain region. We report that the majority of α4β2 nAChRs in this brain region possess a stoichiometry of the (α4)3(β2)2 LS subtype.

Desensitization of the nicotinic acetylcholine receptor (nAChR) containing the β2 subunit is a potentially critical mechanism underlying the body weight (BW) reducing effects of nicotine. The purpose of this study was a) to determine the α subunit(s) that partners with the β2 subunit to form the nAChR subtype that endogenously regulates energy balance and b) to probe the extent to which nAChR desensitization could be involved in the regulation of BW. We demonstrate that deletion of either the α4 or the β2, but not the α5, subunit of the nAChR suppresses weight gain in a sex-dependent manner. Furthermore, chronic treatment with the β2-selective nAChR competitive antagonist dihydro-β-erythroidine (DHβE) in mice fed a high-fat diet suppresses weight gain. These results indicate that heteromeric α4β2 nAChRs play a role as intrinsic regulators of energy balance and that desensitizing or inhibiting this nAChR is likely a relevant mechanism and thus could be a strategy for weight loss.

Central nervous system regulation of the gastric tone and motility is primarily mediated via preganglionic neurons of the dorsal motor nucleus of the vagus (DMV). This is thought to occur by simultaneous engagement of both independent excitatory and inhibitory pathways from the DMV and has been proposed to underlie the opposing effects seen on gastric tone and motility in a number of in vivo models. Contrary to this view, we have been unable to find any evidence for this “dual effector” pathway. Since this possibility is so fundamental to how the brain-gut axis may interact in light of both peripheral and central demands, we decided to explore it further in two separate animal models previously used in conjunction with GABAB signaling to report the existence of a “dual effector” pathway. Using anesthetized rats or ferrets, we microinjected baclofen (7.5 pmol; n = 6), a GABAB agonist into the DMV of rats or intravenously administered it (0.5 mg/kg; n = 4) in ferrets. In rats, unilateral microinjection of baclofen into the DMV caused a robust dose-dependent increase in gastric tone and motility that was abolished by ipsilateral vagotomy and counteracted by pretreatment with atropine (0.1 mg/kg; IV). Similarly, as microinjection in the rats, IV administration of baclofen (0.5 mg/kg) in the ferrets induced its characteristic excitatory effects on gastric tone and motility, which were blocked by either pre- or post-treatment with atropine (0.1 mg/kg; IV). Altogether, our data provide evidence that the gastric musculature (other than the gastric sphincters) is regulated by a “single effector” DMV pathway using acetylcholine.

Nicotine's effect on food intake and body weight has been well documented; however, the relevant receptors underlying these effects have not been firmly established. The purpose of the present study was to: (1) identify the nicotinic acetylcholine receptor (nAChR) subtype involved in food intake and body weight; (2) establish whether food intake and body weight reduction produced by nicotinic drugs are due to activation or desensitization of nAChRs; and, (3) assess the role of the melanocortin system in nicotinic drug effects on food intake and body weight. To identify the nAChR, we tested the effect of sazetidine-A (SAZ-A), a relatively selective ligand of β2-containing nAChRs, on food intake and body weight in obese mice. SAZ-A (3 mg/kg; SC) administered twice-daily significantly decreased food intake and body weight. To assess whether these effects involved desensitization, SAZ-A was administered to non-obese mice via osmotic pump, which, due to its slow sustained drug delivery method, causes prolonged desensitization. SAZ-A via osmotic pump delivery significantly decreased the gain in body weight and reduced food intake. In contrast, body weight was unaffected by SAZ-A in β2−/− mice or in mice lacking the melanocortin 4 receptor (MC4R). These results indicate that β2 containing nAChRs are essential to SAZ-A's inhibitory effect on body weight and food intake and engage the melanocortin system.

Key points The dorsal motor nucleus of the vagus (DMV) in the brainstem consists primarily of vagal preganglionic neurons that innervate postganglionic neurons of the upper gastrointestinal tract. The activity of the vagal preganglionic neurons is predominantly regulated by GABAergic transmission in the DMV. The present findings indicate that the overwhelming GABAergic drive present at the DMV is primarily from somatostatin positive GABA (Sst-GABA) DMV neurons. Activation of both melanocortin and μ-opioid receptors at the DMV inhibits Sst-GABA DMV neurons. Sst-GABA DMV neurons may serve as integrative targets for modulating vagal output activity to the stomach. Abstract We have previously shown that local GABA signalling in the brainstem is an important determinant of vagally-mediated gastric activity. However, the neural identity of this GABA source is currently unknown. To determine this, we focused on the somatostatin positive GABA (Sst-GABA) interneuron in the dorsal motor nucleus of the vagus (DMV), a nucleus that is intimately involved in regulating gastric activity. Also of particular interest was the effect of melanocortin and μ-opioid agonists on neural activity of Sst-GABA DMV neurons because their in vivo administration in the DMV mimics GABA blockade in the nucleus. Experiments were conducted in brain slice preparation of transgenic adult Sst-IRES-Cre mice expressing tdTomato fluorescence, channelrhodopsin-2, archaerhodopsin or GCaMP3. Electrophysiological recordings were obtained from Sst-GABA DMV neurons or DiI labelled gastric-antrum projecting DMV neurons. Our results show that optogenetic stimulation of Sst-GABA neurons results in a robust inhibition of action potentials of labelled premotor DMV neurons to the gastric-antrum through an increase in inhibitory post-synaptic currents. The activity of the Sst-GABA neurons in the DMV is inhibited by both melanocortin and μ-opioid agonists. These agonists counteract the pronounced inhibitory effect of Sst-GABA neurons on vagal pre-motor neurons in the DMV that control gastric motility. These observations demonstrate that Sst-GABA neurons in the brainstem are crucial for regulating the activity of gastric output neurons in the DMV. Additionally, they suggest that these neurons serve as targets for converging CNS signals to regulate parasympathetic gastric function.

Background/Objectives: We tested the hypothesis that abolishing vagal nerve activity will reverse the obesity phenotype of melanocortin 4 receptor knockout mice (Mc4r-/-). Subjects/Methods: In two separate studies, we examined the efficacy of bilateral subdiaphragmatic vagotomy (SDV) with pyloroplasty in the prevention and treatment of obesity in Mc4r-/- mice. Results: In the first study, SDV prevented >20% increase in body weight (BW) associated with this genotype. This was correlated with a transient reduction in overall food intake (FI) in the preventative arm of the study. Initially, SDV mice had reduced weekly FI; however, FI normalized to that of controls and baseline FI within the 8-week study period. In the second study, the severe obesity that is characteristic of the adult Mc4r-/- genotype was significantly improved by SDV with a magnitude of 30% loss in excess BW over a 4-week period. Consistent with the first preventative study, within the treatment arm, SDV mice also demonstrated a transient reduction in FI relative to control and baseline levels that normalized over subsequent weeks. In addition to the accompanying loss in weight, mice subjected to SDV showed a decrease in respiratory exchange ratio (RER), and an increase in locomotor activity (LA). Analysis of the white fat-pad deposits of these mice showed that they were significantly less than the control groups. Conclusions: Altogether, our data demonstrates that SDV both prevents gain in BW and causes weight loss in severely obese Mc4r-/- mice. Moreover, it suggests that an important aspect of weight reduction for this type of monogenic obesity involves loss of signaling in vagal motor neurons.

The α4β2 nicotinic acetylcholine receptor (nAChR) is important in central nervous system physiology and in mediating several of the pharmacological effects of nicotine on cognition, attention, and affective states. It is also the likely receptor that mediates nicotine addiction. This receptor assembles in two distinct stoichiometries: (α4)2(β2)3 and (α4)3(β2)2, which are referred to as high-sensitivity (HS) and low-sensitivity (LS) nAChRs, respectively, based on a difference in the potency of acetylcholine to activate them. The physiologic and pharmacological differences between these two receptor subtypes have been described in heterologous expression systems. However, the presence of each stoichiometry in native tissue currently remains unknown. In this study, different ratios of rat α4 and β2 subunit cDNA were transfected into human embryonic kidney 293 cells to create a novel model system of HS and LS α4β2 nAChRs expressed in a mammalian cell line. The HS and LS nAChRs were characterized through pharmacological and biochemical methods. Isolation of surface proteins revealed higher amounts of α4 or β2 subunits in the LS or HS nAChR populations, respectively. In addition, sazetidine-A displayed different efficacies in activating these two receptor stoichiometries. Using this model system, a neurophysiological “two-concentration” acetylcholine or carbachol paradigm was developed and validated to determine α4/β2 subunit stoichiometry. This paradigm was then used in layers I–IV of slices of the rat motor cortex to determine the percent contribution of HS and LS α4β2 receptors in this brain region. We report that the majority of α4β2 nAChRs in this brain region possess a stoichiometry of the (α4)3(β2)2 LS subtype.

AT-1001 [N-(2-bromophenyl)-9-methyl-9-azabicyclo[3.3.1] nonan-3-amine] is a high-affinity and highly selective ligand at α3β4 nicotinic cholinergic receptors (nAChRs) that was reported to decrease nicotine self-administration in rats. It was initially reported to be an antagonist at rat α3β4 nAChRs heterologously expressed in HEK293 cells. Here we compared AT-1001 actions at rat and human α3β4 and α4β2 nAChRs similarly expressed in HEK 293 cells. We found that, as originally reported, AT-1001 is highly selective for α3β4 receptors over α4β2 receptors, but its binding selectivity is much greater at human than at rat receptors, because of a higher affinity at human than at rat α3β4 nAChRs. Binding studies in human and rat brain and pineal gland confirmed the selectivity of AT-1001 for α3β4 nAChRs and its higher affinity for human compared with rat receptors. In patch-clamp electrophysiology studies, AT-1001 was a potent partial agonist with 65-70% efficacy at both human and rat α3β4 nAChRs. It was also a less potent and weaker (18%) partial agonist at α4β2 nAChRs. Both α3β4 and α4β2 nAChRs are upregulated by exposure of cells to AT-1001 for 3 days. Similarly, AT-1001 desensitized both receptor subtypes in a concentration-dependent manner, but it was 10 and 30 times more potent to desensitize human α3β4 receptors than rat α3β4 and human α4β2 receptors, respectively. After exposure to AT-1001, the time to recovery from desensitization was longest for the human α3β4 nAChR and shortest for the human α4β2 receptor, suggesting that recovery from desensitization is primarily related to the dissociation of the ligand from the receptor.

For over a century, the behavior of the aorta and other large arteries has been described as passive elastic tubes in which no active contraction occurs in the smooth muscle wall. In response to pulsatile pressure changes, the vessels undergo a 'passive' elastic dilatation-contraction cycle, described as a "Windkessel" effect. However, Mangel and colleagues have presented evidence that is contrary to this view. They reported that in the rabbit, the aorta undergoes rhythmic 'active' (contraction) during the cardiac cycle; but these findings have been largely ignored. In the present study, we observed spontaneous contractions in synchrony with the heartbeat in another species (rat). In addition we demonstrate that aorta contractions are of neurogenic origin. Electrical stimulation of the aorta evoked contractions that occur at a rate that is in the range of the animal's heartbeat and are suppressed by tetrodotoxin and the alpha-adrenergic receptor blocker, phentolamine. Altogether, these findings indicate that aortic contractions are under neural control from the heart.