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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.

Objective: Brain-derived neurotrophic factor (BDNF) is a potent regulator of neuronal development, and the Bdnf gene produces two populations of transcripts with either a short or long 3′ untranslated region (3′ UTR). Deficiencies in BDNF signaling have been shown to cause severe obesity in humans; however, it remains unknown how BDNF signaling impacts the organization of neuronal circuits that control energy balance. Methods: We examined the role of BDNF on survival, axonal projections, and synaptic inputs of neurons in the arcuate nucleus (ARH), a structure critical for the control of energy balance, using Bdnfklox/klox mice, which lack long 3′ UTR Bdnf mRNA and develop severe hyperphagic obesity. Results: We found that a small fraction of neurons that express the receptor for BDNF, TrkB, also expressed proopiomelanocortin (POMC) or neuropeptide Y (NPY)/agouti-related protein (AgRP) in the ARH. Bdnfklox/klox mice had normal numbers of POMC, NPY, and TrkB neurons in the ARH; however, retrograde labeling revealed a drastic reduction in the number of ARH axons that project to the paraventricular hypothalamus (PVH) in these mice. In addition, fewer POMC and AgRP axons were found in the dorsomedial hypothalamic nucleus (DMH) and the lateral part of PVH, respectively, in Bdnfklox/klox mice. Using immunohistochemistry, we examined the impact of BDNF deficiency on inputs to ARH neurons. We found that excitatory inputs onto POMC and NPY neurons were increased and decreased, respectively, in Bdnfklox/klox mice, likely due to a compensatory response to marked hyperphagia displayed by the mutant mice. Conclusion: This study shows that the majority of TrkB neurons in the ARH are distinct from known neuronal populations and that BDNF plays a critical role in directing projections from these neurons to the DMH and PVH. We propose that hyperphagic obesity due to BDNF deficiency is in part attributable to impaired axonal growth of TrkB-expressing ARH neurons.

The α7-nicotinic cholinergic receptor (α7-nAChR) is a key mediator of brain communication and has been implicated in a wide variety of central nervous system disorders. None of the currently available PET radioligands for α7-nAChR are suitable for quantitative PET imaging, mostly because of insufficient specific binding. The goal of this study was to evaluate the potential of 18F-ASEM (18F-JHU82132) as an α7-nAChR radioligand for PET. Methods: The inhibition binding assay and receptor functional properties of ASEM were assessed in vitro. The brain regional distribution of 18F-ASEM in baseline and blockade were evaluated in DISC1 mice (dissection) and baboons (PET). Results: ASEM is an antagonist for the α7-nAChR with high binding affinity (Ki 5 0.3 nM). 18F-ASEM readily entered the baboon brain and specifically labeled α7-nAChR. The in vivo specific binding of 18F-ASEM in the brain regions enriched with α7-nAChRs was 80%–90%. SSR180711, an α7-nAChR–selective partial agonist, blocked 18F-ASEM binding in the baboon brain in a dose-dependent manner, suggesting that the binding of 18F-ASEM was mediated by α7-nAChRs and the radioligand was suitable for drug evaluation studies. In the baboon baseline studies, the brain regional volume of distribution (VT )v alues for 18 F-ASEM were 23 (thalamus), 22 (insula), 18 (hippocampus), and 14 (cerebellum), whereas in the binding selectivity (blockade) scan, all regional VT values were reduced to less than 4. The range of regional binding potential values in the baboon brain was from 3.9 to 6.6. In vivo cerebral binding of 18 FASEM and α7-nAChR expression in mutant DISC1 mice, a rodent model of schizophrenia, was significantly lower than in control animals, which is in agreement with previous postmortem human data. Conclusion: 18 F-ASEM holds promise as a radiotracer with suitable imaging properties for quantification of α7-nAChR in the human brain.

Chronic nicotine administration increases the density of brain α4β2* nicotinic acetylcholine receptors (nAChRs), which may contribute to nicotine addiction by exacerbating withdrawal symptoms associated with smoking cessation. Varenicline, a smoking cessation drug, also increases these receptors in rodent brain. The maintenance of this increase by varenicline as well as nicotine replacement may contribute to the high rate of relapse during the first year after smoking cessation. Recently, we found that sazetidine-A (saz-A), a potent partial agonist that desensitizes α4β2* nAChRs, does not increase the density of these receptors in brain at doses that decrease nicotine self-administration, increase attention in rats, and produce anxiolytic effects in mice. Here, we investigated whether chronic saz-A and varenicline maintain the density of nAChRs after their up-regulation by nicotine. In addition, we examined the effects of these drugs on a measure of anxiety in mice and weight gain in rats. After increasing nAChRs in the rodent brain with chronic nicotine, replacing nicotine with chronic varenicline maintained the increased nAChR binding, as well as the α4β2 subunit proteins measured by western blots. In contrast, replacing nicotine treatments with chronic saz-A resulted in the return of the density of nAChRs to the levels seen in saline controls. Nicotine, saz-A and varenicline each demonstrated anxiolytic effects in mice, but only saz-A and nicotine attenuated the gain of weight over a 6-week period in rats. These findings suggest that apart from its modest anxiolytic and weight control effects, saz-A, or drugs like it, may be useful in achieving long-term abstinence from smoking.

Developing novel and selective compounds that desensitize α4β2 nicotinic acetylcholine receptors (nAChRs) could provide new effective treatments for nicotine addiction, as well as other disorders. Here we report a new class of nAChR ligands that display high selectivity and picomolar binding affinity for α4β2 nicotinic receptors. The novel compounds have Ki values in the range of 0.031-0.26 nM and properties that should make them good candidates as drugs acting in the CNS. The selected lead compound 1 (VMY-2-95) binds with high affinity and potently desensitizes α4β2 nAChRs. At a dose of 3 mg/kg, compound 1 significantly reduced rat nicotine self-administration. The overall results support further characterizations of compound 1 and its analogues in preclinical models of nicotine addiction and perhaps other disorders involving nAChRs.

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.

Our laboratory previously reported that gastric activity is controlled by a robust GABAA receptor-mediated inhibition in the medial nucleus of the tractus solitarius (mNTS) ( Herman et al. 2009 ), and that μ-opioid receptor activation inhibits gastric tone by suppression of this GABA signaling ( Herman et al. 2010 ). These data raised two questions: 1) whether any of this inhibition was due to tonic GABAA receptor-mediated conductance in the mNTS; and 2) whether μ-opioid receptor activation suppressed both tonic and phasic GABA signaling. In whole cell recordings from rat mNTS neurons, application of three GABAA receptor antagonists (gabazine, bicuculline, and picrotoxin) produced a persistent reduction in holding current and decrease in population variance or root mean square (RMS) noise, suggesting a blockade of tonic GABA signaling. Application of gabazine at a lower concentration abolished phasic currents, but had no effect on tonic currents or RMS noise. Application of the δ-subunit preferring agonist gaboxadol (THIP) produced a dose-dependent persistent increase in holding current and RMS noise. Pretreatment with tetrodotoxin prevented the action of gabazine, but had no effect on the THIP-induced current. Membrane excitability was unaffected by the selective blockade of phasic inhibition, but was increased by blockade of both phasic and tonic currents. In contrast, activation of tonic currents decreased membrane excitability. Application of the μ-opioid receptor agonist DAMGO produced a persistent reduction in holding current that was not observed following pretreatment with a GABAA receptor antagonist and was not evident in mice lacking the δ-subunit. These data suggest that mNTS neurons possess a robust tonic inhibition that is mediated by GABAA receptors containing the δ-subunit, that determines membrane excitability, and that is partially regulated by μ-opioid receptors.

We examined the effects of altering μ-opioid receptor (MOR) activity in the medial subnucleus of the tractus solitarius (mNTS) on several gastric end points including intragastric pressure (IGP), fundus tone, and the receptive relaxation reflex (RRR). Microinjection of the MOR agonist [d-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO; 1–10 fmol) into the mNTS produced dose-dependent decreases in IGP. Microinjection of the endogenous MOR agonists endomorphin-1 and endomorphin-2 (20 fmol) into the mNTS mimicked the effects of 10 fmol DAMGO. Microinjection of 1 and 100 pmol DAMGO into the mNTS produced a triphasic response consisting of an initial decrease, a transient increase, and a persistent decrease in IGP. The increase in IGP appeared to be due to diffusion to the dorsal motor nucleus of the vagus. The effects of 10 fmol DAMGO in the mNTS were blocked by vagotomy and by blockade of MORs, GABAAreceptors, and ionotropic glutamate receptors in the mNTS. The RRR response was abolished by bilateral microinjection of the opioid receptor antagonist naltrexone into the mNTS and reduced by intravenous administration of naltrexone. Our data demonstrate that 1) activation of MORs in the mNTS with femtomole doses of agonist inhibits gastric motility, 2) the mechanism of MOR effects in the mNTS is through suppression of local GABA activity, and 3) blockade of MORs in the mNTS prevents the RRR response. These data suggest that opioids play an important role in mediating a vagovagal reflex through release of an endogenous opioid in the mNTS, which, in turn, inhibits ongoing local GABA activity and allows vagal sensory input to excite second-order mNTS neurons.

We reported pharmacological data suggesting that stimulation of the vago-vagal reflex activates noradrenergic neurons in the hindbrain that inhibit dorsal motor nucleus of the vagus (DMV) neurons projecting to the fundus, but not to the antrum [Ferreira Jr., M., Sahibzada, N., Shi, M., Panico, W., Neidringhaus, M., Wasserman, A., Kellar, K.J., Verbalis, J., Gillis, R.A., 2002. CNS site of action and brainstem circuitry responsible for the intravenous effects of nicotine on gastric tone. J. Neurosci. 22, 2764–2779.]. The purpose of this study was to use an ultrastructural approach to test the hypothesis that noradrenergic terminals form synapses with DMV fundus-projecting neurons, but not with DMV antrum-projecting neurons. A retrograde tracer, CTβ-HRP, was injected into the gastric smooth muscle of either the fundus or the antrum of rats. Animals were re-anesthetized 48 h later and perfusion-fixed with acrolein and paraformaldehyde. Brainstems were processed histochemically for CTβ-HRP, and immunocytochemically for either DβH or PNMT by dual-labeling electron microscopic methods. Most cell bodies and dendrites of neurons that were retrogradely labeled from the stomach occurred at the level of the area postrema. Examination of 482 synapses on 238 neurons that projected to the fundus revealed that 17.4 +/− 2.7% (n = 4) of synaptic contacts were with DβH-IR terminals. Of 165 fundus-projecting neurons, 4.4 +/− 1.5% (n = 4) formed synaptic contacts with PNMT-IR terminals. In contrast, the examination of 384 synapses on 223 antrum-projecting neurons revealed no synaptic contact with DβH-IR terminals. These data provide proof that norepinephrine containing nerve terminals synapse with DMV fundus-projecting neurons but not with DMV antrum-projecting neurons. These data also suggest that brainstem circuitry controlling the fundus differs from circuitry controlling the antrum.

Our primary purpose was to characterize vagal pathways controlling gastric motility by microinjecting l-glutamate into the dorsal motor nucleus of the vagus (DMV) in the rat. An intragastric balloon was used to monitor motility. In 39 out of 43 experiments, microinjection of l-glutamate into different areas of the DMV rostral to calamus scriptorius (CS) resulted in vagally mediated excitatory effects on motility. We observed little evidence for inhibitory effects, even with intravenous atropine or with activation of gastric muscle muscarinic receptors by intravenous bethanechol. Inhibition of nitric oxide synthase with Nω-nitro-l-arginine methyl ester (l-NAME) HCl did not augment DMV-evoked excitatory effects on gastric motility. Microinjection of l-glutamate into the DMV caudal to CS produced vagally mediated gastric inhibition that was resistant to l-NAME. l-Glutamate microinjected into the medial subnucleus of the tractus solitarius (mNTS) also produced vagally mediated inhibition of gastric motility. Motility responses evoked from the DMV were always blocked by ipsilateral vagotomy, while responses evoked from the mNTS required bilateral vagotomy to be blocked. Microinjection of oxytocin into the DMV inhibited gastric motility, but the effect was never blocked by ipsilateral vagotomy, suggesting that the effect may have been due to diffusion of oxytocin to the mNTS. Microinjection of substance P and N-methyl-d-aspartate into the DMV also produced inhibitory effects attributable to excitation of nearby mNTS neurons. Our results do not support previous studies indicating parallel vagal excitatory and inhibitory pathways originating in the DMV rostral to CS. Our results do support previous findings of vagal inhibitory pathways originating in the DMV caudal to CS.

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.

We reported previously that intravenously administered d-glucose acts in the central nervous system to inhibit gastric motility induced by hypoglycemia in anesthetized rats. The purpose of this study was to determine whether this effect is due to inhibition of dorsal motor nucleus of the vagus (DMV) cholinergic motoneurons, which synapse with postganglionic cholinergic neurons, or to excitation of DMV cholinergic neurons, which synapse with postganglionic nonadrenergic, noncholinergic (NANC) neurons, particularly nitrergic neurons. Three approaches were employed: 1) assessment of the efficacy of d-glucose-induced inhibition of gastric motility in hypoglycemic rats with and without inhibition of nitric oxide synthase [10 mg/kg iv nitro-l-arginine methyl ester (l-NAME)], 2) assessment of the efficacy of intravenous bethanechol (30 μg·kg−1·min−1) to stimulate gastric motility in hypoglycemic rats during the time of d-glucose-induced inhibition of gastric motility, and 3) determination of c-Fos expression in DMV neurons after intravenous d-glucose was administered to normoglycemic rats. Results obtained demonstrated that l-NAME treatment had no effect on d-glucose-induced inhibition of gastric motility; there was no reduction in the efficacy of intravenous bethanechol to increase gastric motility, and c-Fos expression was not induced by d-glucose in DMV neurons that project to the stomach. These findings indicate that excitation of DMV cholinergic motoneurons that synapse with postganglionic NANC neurons is not a significant contributing component of d-glucose-induced inhibition of gastric motility.

Our purposes were to 1) develop an animal model where intravenously (iv) administered d-glucose consistently inhibited antral motility, and 2) use this model to assess whether iv glucose acts to inhibit motility from a peripheral or a central nervous system site and to elucidate the factor(s) that determine(s) whether stomach motor function is sensitive to changes in blood glucose. Rats were anesthetized with α-chloralose-urethane, and antral motility was measured by a strain-gauge force transducer sutured to the antrum. In some cases, antral motility and gastric tone were measured by monitoring intragastric balloon pressure. Increases in blood glucose were produced by continuous iv infusion of 25% d-glucose at 2 ml/h. Inhibition of antral motility and gastric tone was observed when gastric contractions were induced by hypoglycemia (subcutaneously administered insulin, 2.5 IU/animal). In contrast, no inhibition of gastric motor function was observed when glucose infusion was tested on gastric contractions that were 1) spontaneously occurring, 2) evoked by iv administered bethanechol in vagotomized animals, and 3) evoked by the TRH analog RX77368, microinjected into the dorsal motor nucleus of the vagus. Using the model of insulin-induced hypoglycemia to increase gastric motor activity, we found that neither sectioning the hepatic branch of the vagus ( n = 5), nor treating animals with capsaicin to destroy sensory vagal afferent nerves ( n = 5) affected the ability of iv d-glucose to inhibit gastric motor function. Our results indicate that an important factor determining whether stomach motor function will be sensitive to changes in blood glucose is the method used to stimulate gastric contractions, and that the primary site of the inhibitory action of iv glucose on gastric motility is the central nervous system rather than the periphery.

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.

To determine the predominant nicotinic ACh receptor (nAChR) located on neurones in the dorsal motor nucleus of the vagus (DMV) that project to the gastrointestinal tract, we used the rat brainstem slice preparation and whole-cell recordings of DMV neurones identified by retrograde DiI tracing to pharmacologically characterize nAChRs. Pressure ejection of acetylcholine (ACh, 250 microM for 200 ms) from a patch pipette placed approximately 10-20 microm from the surface of the recorded cell produced an inward current in most DMV neurones sampled. The average currents for neurones projecting to the fundus, antrum and caecum were 149 +/- 38 (n = 25), 115 +/- 18 (n = 29) and 117 +/- 23 pA (n = 6), respectively. Blockade of the alpha7 subtype of nAChR with either alpha-bungarotoxin (alpha-BGT) or methyllycaconitine (MLA) counteracted 60-75 % of the ACh-evoked current in DMV neurones projecting to the fundus, antrum and caecum. In neurones projecting to the fundus and the antrum, currents resistant to alpha-BGT were significantly blocked by dihydro-beta-erythroidine (10-20 nM), an antagonist of the alpha4beta2 subtype of nAChR. In neurones projecting to the caecum, currents resistant to alpha-BGT were significantly depressed by a low concentration of mecamylamine (1 microM). Cytisine (100 microM), an agonist of nAChRs that contain the alpha7 or the beta4 subunit, evoked significant currents in caecum-projecting neurones that were previously exposed to alpha-BGT. In contrast, cytisine had no effect on DMV neurones previously exposed to alpha-BGT that project to the fundus or antrum. Our data indicate that the prevailing nAChR subtype in DMV neurones projecting to the GI tract is the alpha7 subtype. In addition, we obtained evidence for the co-expression of the alpha4beta2 nAChR subtype on DMV neurones projecting to the fundus and antrum, and the alpha3beta4 nAChR subtype on DMV neurones projecting to the caecum.

SummaryBrain-derived neurotrophic factor (BDNF) is a key regulator of energy balance; however, its underlying mechanism remains unknown. By analyzing BDNF-expressing neurons in paraventricular hypothalamus (PVH), we have uncovered neural circuits that control energy balance. The Bdnf gene in the PVH was mostly expressed in previously undefined neurons, and its deletion caused hyperphagia, reduced locomotor activity, impaired thermogenesis, and severe obesity. Hyperphagia and reduced locomotor activity were associated with Bdnf deletion in anterior PVH, whereas BDNF neurons in medial and posterior PVH drive thermogenesis by projecting to spinal cord and forming polysynaptic connections to brown adipose tissues. Furthermore, BDNF expression in the PVH was increased in response to cold exposure, and its ablation caused atrophy of sympathetic preganglionic neurons. Thus, BDNF neurons in anterior PVH control energy intake and locomotor activity, whereas those in medial and posterior PVH promote thermogenesis by releasing BDNF into spinal cord to boost sympathetic outflow.

1. To examine the effects of glucose on the central components of the vago-vagal reflex control of gastric function, we performed both in vivo and in vitro experiments on neurones in the medial nucleus of the tractus solitarius (mNTS) and in the dorsal motor nucleus of the vagus (DMV). 2. In the in vivo anaesthetized rat preparation, unilateral microinjection of D-glucose (10 or 50 mM (60 nl)(-1)) in mNTS produced inhibition of gastric motility and an increase in intragastric pressure. D-glucose had no effect in the DMV. 3. In the in vitro rat brainstem slice preparation, whole-cell recordings of DMV neurones showed that increasing the glucose concentration of the perfusion solution from 5 mM to 15 or 30 mM produced outward currents of 35 +/- 5 pA (n = 7) and 51 +/- 10 pA (n = 11), respectively. These were blocked by tetrodotoxin and picrotoxin, indicating that glucose was acting indirectly to cause the release of GABA. Decreasing the glucose concentration of the perfusing solution by one-half produced an inward current of 36 +/- 5 pA (n = 7). 4. Stimulation of the NTS evoked inhibitory postsynaptic currents (IPSCs) in DMV neurones. The amplitude of the evoked IPSCs was positively correlated with glucose concentration. Perfusion with the ATP-sensitive K(+) (K(ATP)) channel opener diazoxide mimicked the effect of reduced glucose, while perfusion with the K(ATP) channel blocker glibenclamide mimicked the effects of increased glucose. 5. Our data indicate that glucose had no direct excitatory effect on DMV neurones, but DMV neurones appear to be affected by an action of glucose on cell bodies of mNTS neurones via effects on an ATP-sensitive potassium channel.

Activation of melanocortin 4 receptors (MC4-Rs) in brain nuclei associated with food intake profoundly influences consummatory behavior. Of these nuclei, the dorsal motor vagal nucleus (DMV), which has a dense concentration of MC4-Rs, is an important regulator of gastric tone and motility. Hence, the present study sought to examine the role of MC4-Rs in this nucleus on these activities. Using anin vivoapproach, MC4-R agonists, melanotan-II (MT-II) or α-melanocyte stimulating hormone (α-MSH), were unilaterally microinjected into the DMV of rats, and their effects were noted on gastric activity. MT-II decreased phasic contractions, whereas α-MSH increased their amplitude. Both effects were blocked by the MC4-R antagonist SHU9119 or by ipsilateral vagotomy. Microinjection of the agonists (MT-II and α-MSH) into the overlying nucleus of the solitary tract (NTS), an important component of “vago-vagal” gastric circuitry, decreased phasic contractions. In addition, α-MSH reduced gastric tone and mean arterial blood pressure. To study the underlying mechanisms of the effect of MC4-R stimulation on gastric activity, electrophysiological recordings were made from labeled DMV antrum neurons in rat pups andMC4-R−/−mice. Bath application of MT-II or α-MSH significantly reduced spontaneous action potentials (but not inMC4-R−/−mice). However, in low-calcium ACSF, MT-II decreased neuronal firing, whereas α-MSH increased it. These effects mirror those of ourin vivoDMV studies. Altogether, our novel findings show that activation of MC4-Rs in the brainstem, particularly in the medial NTS by the endogenous peptide α-MSH, modulates gastric activity, which may have physiological relevance for food intake and gastric function.

Neuronal acetylcholine receptors mediate the addictive effects of nicotine and may also be involved in alcohol addiction. Varenicline, an approved smoking cessation medication, showed clear efficacy in reducing alcohol consumption in heavy-drinking smokers. More recently, sazetidine-A, which selectively desensitizes α4β2 nicotinic receptors, was shown to significantly reduce alcohol intake in a rat model. To develop novel therapeutics for treating alcohol use disorder, we designed and synthesized novel sazetidine-A analogs containing a methyl group at the 2-position of the pyridine ring. In vitro pharmacological studies revealed that some of the novel compounds showed similar overall pharmacological property profiles with that of sazetidine-A, but exhibited reduced agonist activity across all nicotinic receptor subtypes tested. In animal studies, compound (S)-9 significantly reduced alcohol uptake in rats. More importantly, preliminary results from studies in a ferret model indicate that these novel nAChR ligands have an improved adverse side-effect profile in comparison with that of varenicline.

Age-related macular degeneration (AMD) is one of the major causes of blindness in aging population that progresses with death of retinal pigment epithelium (RPE) and photoreceptor degeneration inducing impairment of central vision. Discovery of human induced pluripotent stem (hiPS) cells has opened new avenues for the treatment of degenerative diseases using patient-specific stem cells to generate tissues and cells for autologous cell-based therapy. Recently, RPE cells were generated from hiPS cells. However, there is no evidence that those hiPS-derived RPE possess specific RPE functions that fully distinguish them from other types of cells. Here, we show for the first time that RPE generated from hiPS cells under defined conditions exhibit ion transport, membrane potential, polarized vascular endothelial growth factor secretion, and gene expression profile similar to those of native RPE. The hiPS-RPE could therefore be a very good candidate for RPE replacement therapy in AMD. However, these cells show rapid telomere shortening, DNA chromosomal damage, and increased p21 expression that cause cell growth arrest. This rapid senescence might affect the survival of the transplanted cells in vivo and therefore, only the very early passages should be used for regeneration therapies. Future research needs to focus on the generation of “safe” as well as viable hiPS-derived somatic cells.

We reported pharmacological data suggesting that stimulation of a vago-vagal reflex activates GABAergic neurons in the hindbrain that inhibit dorsal motor nucleus of the vagus (DMV) neurons projecting to the antrum, but not to the fundus (Ferreira et al., 2002). The purpose of this study was to use an ultrastructural approach to test the hypothesis that GABAergic terminals form synapses with DMV antrum-projecting neurons, but not with DMV fundus-projecting neurons. A retrograde tracer, CTB-HRP, was injected into the gastric smooth muscle of either the fundus or the antrum of anesthetized rats. Animals were re-anethetized 48 hours later and perfusion-fixed with acrolein and paraformaldehyde. Brainstems were processed histochemically for CTB-HRP, and immunocytochemically for glutamic acid decarboxylase isoenzyme 67 immunoreactivity (GAD67-IR) by dual-labeling electron microscopic methods. Most cell bodies and dendrites of neurons that were retrogradely labeled from the stomach occurred at the level of the area postrema. Examination of 214 synapses on 195 neurons that projected to the antrum revealed that 23.0 +/− 3.6% (n=4) of synaptic contacts were with GAD67-IR terminals. The examination of 220 synapses on 203 fundus-projecting neurons revealed that only 7.9 +/− 3.1% (n=4) of synaptic contacts were with GAD67-IR terminals. The difference between GAD67-IR synaptic contacts with antrum- and fundus-projecting neurons was statistically significant (p < 0.05). These data suggest that brainstem circuitry controlling the antrum involves GABAergic transmission.

It has been proposed that there is an “apparent monosynaptic” connection between gastric vagal afferent nerve terminals and inhibitory projection neurons in the nucleus tractus solitarius (NTS) and that two efferent parallel pathways from the dorsal motor nucleus of the vagus (DMV) influence peripheral organs associated with these reflexes ( 6 ). The purpose of our study was to verify the validity of these views as they relate to basal control of gastric motility. To test the validity of a direct connection of vagal afferent terminals (known to release l-glutamate) directly impacting second-order projection neurons, we evaluated the effect of GABAA receptor blockade in the area of the medial subnucleus of the tractus solitarius (mNTS) on gastric motility. Microinjection of bicuculline methiodide into the mNTS produced robust decreases in gastric motility (−1.6 ± 0.2 mmHg, P < 0.05, n = 23), which were prevented by cervical vagotomy and by pretreatment with kynurenic acid microinjected into the mNTS. Kynurenic acid per se had no effect on gastric motility. However, after GABAA receptor blockade in the mNTS, kynurenic acid produced a robust increase in gastric motility. To test for the contribution of two parallel efferent DMV pathways, we assessed the effect of either intravenous atropine methylbromide or NG-nitro-l-arginine methyl ester on baseline motility and on decreases in gastric motility induced by GABAA receptor blockade in the mNTS. Only atropine methylbromide altered baseline motility and prevented the effects of GABAA receptor blockade on gastric motility. Our data demonstrate the presence of intra-NTS GABAergic signaling between the vagal afferent nerve terminals and inhibitory projection neurons in the NTS and that the cholinergic-cholinergic excitatory pathway comprises the functionally relevant efferent arm of the vagovagal circuit.

General anesthetics (GAs) have transformed surgery through their actions to depress the central nervous system and blunt the perception of surgical insults. Counterintuitively, many of these agents activate peripheral nociceptive neurons. However, the underlying mechanisms and significance of these effects have not been explored. Here, we show that clinical concentrations of noxious i.v. and inhalation GAs excite sensory neurons by selectively activating TRPA1, a key ion channel in the pain pathway. Further, these GAs induce pain-related responses in mice that are abolished in TRPA1-null animals. Significantly, TRPA1-dependent neurogenic inflammation is greater in mice anesthetized with pungent compared with nonpungent anesthetics. Thus, our results show that TRPA1 is essential for sensing noxious GAs. The pronociceptive effects of GAs combined with surgical tissue damage could lead to a paradoxical increase in postoperative pain and inflammation.

The lower esophageal sphincter (LES) and the crural diaphragm (CD) surrounding the esophagogastric junction are key components of the gastroesophageal reflex mechanism, which engages the vago-vagal brainstem circuitry. Although both components work in conjunction to prevent gastroesophageal reflux, little is known about the brain area(s) where this integration takes place. The aims of this study were to: (1) trace the brainstem circuitry associated with the CD and the LES, and (2) determine possible sites of convergence. Experiments were done in adult male ferrets. Under isoflurane anesthesia, recombinant strains of the transneuronal pseudorabies virus (PRV-151 or PRV-Bablu) or the monosynaptic retrograde tracer cholera toxin beta-subunit (CTb) were injected into either the CD or the LES. Following a survival period of 5-7 days, animals were euthanized, perfused and their brains removed for dual-labeling immunofluorescence processing. In animals injected with recombinants of PRV into the CD and the LES, distinct labeling was found in various brainstem nuclei including: area postrema, DMV, nucleus tractus solitarius (NTS), medial reticular formation (MRF) and nucleus ambiguous (NA). Double-labeled cells were only evident in the DMV, NTS and MRF. Injections of CTb into the CD or the LES resulted in retrograde labeling only in the DMV. These findings demonstrate the presence of a direct projection from the DMV to the CD. They further suggest that the neuronal connections responsible for CD or LES function are contained in circuitries that, though largely independent, may converge at the level of DMV, NTS and MRF.

Quantitative analysis of innervation to dorsal motor nucleus of the vagus (DMV) fundus-projecting neurons indicates that ∼17% of input neurons are noradrenergic. To determine whether this small percentage of neurons innervating DMV output to the stomach is physiologically relevant, we evaluated the role of norepinephrine at the DMV in mediating a vagovagal reflex controlling the fundus. A strain gauge was sutured onto the fundus of isoflurane-anesthetized rats to monitor changes in tone evoked by esophageal distension (ED). ED produced a decrease in fundus tone of 0.31 ± 0.02 g ( P < 0.05), which could be reproduced after a 30-min interval between distensions. Bilateral cervical vagotomy and/or pretreatment with intravenous atropine methylbromide prevented the reflex-induced fundus relaxation. In contrast, intravenous NG-nitro-l-arginine methyl ester had no effect. Bilateral microinjection of α2-adrenoreceptor antagonists (yohimbine and RS-79948) into the DMV also prevented the response. Before microinjection of α2-adrenoreceptor antagonists, ED decreased fundus tone by 0.33 ± 0.05 g ( P < 0.05). After antagonist microinjection, ED decreased fundus tone by only 0.05 ± 0.06 g ( P > 0.05). Bilateral microinjection of prazosin into the DMV had no effect on the response. Microinjection of norepinephrine into the DMV mimicked the effect of ED and was also prevented by prior microinjection of an α2-adrenoreceptor antagonist. Our results indicate that noradrenergic innervation of DMV fundus-projecting neurons is physiologically important and suggest that norepinephrine released at the DMV acts on α2-adrenoreceptors to inhibit activity in a cholinergic-cholinergic excitatory pathway to the fundus.

Immunocytochemistry using antisera directed against dopamine-beta-hydroxylase (DBH) was used to determine the organization of the noradrenergic (NE) input to the hamster's superior colliculus (SC). Immunocytochemistry for DBH was combined with retrograde transport of fluorogold (FG) to determine the sources of NE input to SC. Microiontophoretic techniques were used together with extracellular single unit recording and receptive field mapping techniques to determine the manner in which NE influenced the responses of individual SC neurons. The hamster's SC contained numerous DBH-positive fibers but no immunopositive cells. These fibers formed a plexus that was most dense in the lower stratum griseum superficiale (SGS). The density of DBH-positive fibers was very low in the stratum opticum (SO) and increased in density in the stratum griseum intermediale (SGI) and the other deep layers. When FG injections into the SC were combined with immunocytochemical detection of DBH, double-labeled cells were observed in the contralateral locus ceruleus. DBH-positive neurons were observed in several other portions of the mesencephalon and pons, but none of these were labelled with FG. The effects of NE iontophoresis were assessed for a total of 135 SC neurons. In 74% (N = 100), NE reduced spontaneous and/or stimulus evoked activity. In 3% (N = 4 cells), NE increased activity, and in 23% (N = 31 cells) it had no effect. These percentages were essentially the same for superficial layer visual cells and somatosensory neurons in the deep laminae. The effect of NE iontophoresis upon signal to noise ratios was assessed for 46 visual and 56 somatosensory neurons. For 54% (N = 25) of the visual cells and 16% (N = 9) of the somatosensory cells, NE iontophoresis decreased signal to noise ratios. For 13% (N = 6) of the visual cells and 21% (N = 12) of the somatosensory cells, NE iontophoresis increased signal to noise ratios. The effects of NE on the responsivity of SC neurons were antagonized by propranolol (86% of the 21 cells tested), sotalol (67% of the six cells tested), and atenolol (effective in the single cell tested). All these agents are beta-adrenergic antagonists. The single alpha-adrenergic antagonist that we evaluated, corynanthine, potentiated the effects of NE on the responsivity of the two SC neurons that we tested.

The sphincter mechanism at the esophagogastric junction includes smooth muscle of the lower esophagus and skeletal muscle of the crural diaphragm (CD). Smooth muscle is known to be under the control of the dorsal motor nucleus of the vagus (DMV), while central nervous system (CNS) control of the CD is unknown. The main purposes of our study were to determine the CNS site that controls the CD and whether simultaneous changes in lower esophageal sphincter (LES) pressure and CD activity occur when this site is activated. Experiments were performed on anesthetized male ferrets whose LES pressure, CD activity, and fundus tone were monitored. To activate DMV neurons, l-glutamate was microinjected unilaterally into the DMV at three areas: intermediate, rostral, and caudal. Stimulation of the intermediate DMV decreased CD activity (−4.8 ± 0.1 bursts/min and −0.3 ± 0.01 mV) and LES pressure (−13.2 ± 2.0 mmHg; n = 9). Stimulation of this brain site also produced an increase in fundus tone. Stimulation of the rostral DMV elicited increases in the activity of all three target organs ( n = 5). Stimulation of the caudal DMV had no effect on the CD but did decrease both LES pressure and fundus tone ( n = 5). All changes in LES pressure, fundus tone, and some DMV-induced changes in CD activity (i.e., bursts/min) were prevented by ipsilateral vagotomy. Our data indicate that simultaneous changes in activity of esophagogastric sphincters and fundus tone occur from rostral and intermediate areas of the DMV and that these changes are largely mediated by efferent vagus nerves.

The purpose of this study was to activate a vagovagal reflex by using esophageal distension and nicotine and test whether hindbrain nitric oxide and norepinephrine are involved in this reflex function. We used double-labeling immunocytochemical methods to determine whether esophageal distension (and nicotine) activates c-Fos expression in nitrergic and noradrenergic neurons in the nucleus tractus solitarii (NTS). We also studied c-Fos expression in the dorsal motor nucleus of the vagus (DMV) neurons projecting to the periphery. Esophageal distension caused 19.7 ± 2.3% of the noradrenergic NTS neurons located 0.60 mm rostral to the calamus scriptorius (CS) to be activated but had little effect on c-Fos in DMV neurons. Intravenous administration of nicotine caused 19.7 ± 4.2% of the noradrenergic NTS neurons 0.90 mm rostral to CS to be activated and, as reported previously, had no effect on c-Fos expression in DMV neurons. To determine whether norepinephrine and nitric oxide were central mediators of esophageal distension-induced decrease in intragastric pressure (balloon recording), NG-nitro-l-arginine methyl ester microinjected into the NTS ( n = 5), but not into the DMV, blocked the vagovagal reflex. Conversely, α2-adrenergic blockers microinjected into the DMV ( n = 7), but not into the NTS, blocked the vagovagal reflex. These data, in combination with our earlier pharmacological microinjection data with nicotine, indicate that both esophageal distension and nicotine produce nitric oxide in the NTS, which then activates noradrenergic neurons that terminate on and inhibit DMV neurons.

Our purpose was to determine whether endogenously released GABA in the ventrolateral nucleus of the solitary tract (vlNTS) of the rat influences respiration. Experiments were carried out in anesthetized, vagotomized and spontaneously breathing rats, and diaphragm electromyogram activity was measured while drugs affecting GABAergic neurotransmission were microinjected into the vlNTS and medial NTS (mNTS). Bilateral microinjection of nipecotic acid, 5 or 25 nmol, into the vlNTS (but not in the mNTS) produced dose-dependent increases in inspiratory duration (Ti) frequently culminating in apneustic breathing. Neither unilateral microinjection of bicuculline nor CGP-35348 (GABAB receptor antagonist) reversed this response; however, a combination of both GABA receptor antagonists effectively reversed apneustic breathing. Bilateral microinjection of either muscimol or baclofen into the vlNTS mimicked the effect of nipecotic acid. Microinjection of the bicuculline produced apnea, whereas microinjection of CGP-35348 produced a decrease in Ti and an increase in expiratory duration. Immunohistochemical analysis of the vlNTS region revealed GABAA receptors densely localized to processes, whereas GABAB immunoreactivity was localized to cell bodies. Our data indicate that GABA activity in the vlNTS is important for respiratory function.

The purposes of our study were to determine (1) the effects of intravenous (i.v.) nicotine on gastric mechanical function of anesthetized rats, (2) the CNS site of action of nicotine to produce these effects, (3) the CNS nicotinic acetylcholine receptor (nAChR) subtype(s) responsible for mediating the i.v. effects of nicotine, and (4) the brainstem neurocircuitry engaged by i.v. nicotine for eliciting its gastric effects. This was accomplished by monitoring intragastric pressure (gastric tone) and contractility of the fundus and antrum while administering five doses of i.v. nicotine and microinjecting nicotine into specific brainstem nuclei. Additionally, c-Fos expression in the brainstem after i.v. nicotine and pharmacological agents were used as tools to identify the CNS site and circuitry and reveal the nAChR subtype(s) mediating the gastric effects of nicotine. Using these experimental approaches, we found the following. (1) When given intravenously in doses of 56.5, 113, 226, 452, and 904 nmol/kg, nicotine elicited only inhibitory effects on gastric mechanical function. The most sensitive area of the stomach to nicotine was the fundus, and this effect was mediated by the vagus nerve at doses of 56.5, 113, and 226 nmol/kg. (2) The CNS site of action and nAChR subtype responsible were glutamatergic vagal afferent nerve terminals in the medial subnucleus of the tractus solitarious (mNTS) and α4β2, respectively. (3) The brainstem neurocircuitry that was involved appeared to consist of a mNTS noradrenergic pathway projecting to the dorsal motor nucleus of the vagus (DMV). This pathway seems to be activated via nitriergic interneurons engaged by vagally released glutamate in the mNTS and results in α2 adrenergic receptor-mediated inhibition of DMV neurons projecting to the fundus and controlling gastric tone.

Our previous data obtained in the cat suggest that the neurons of the ventrolateral subnucleus of the tractus solitarius (vlNTS) act as an inspiratory off-switch and terminate the inspiratory phase of the respiratory cycle (Berger et al., Eur. J. Pharmacol. 277 (1995) 195-208; Gillis et al., Neurosci. Abstr. 23 (1997) 725). The purpose of the present study was to determine whether inhibition of the region of the vlNTS of the rat using drugs that hyperpolarize, disfacilitate or block both axonal conduction and action potential generation would alter the inspiratory phase of the respiratory cycle. Experiments were conducted in anesthetized, vagotomized and spontaneously breathing rats while monitoring diaphragmatic electromyogram activity. Vagus nerves were sectioned in order to rule out prolongation of inspiration evoked by microinjection of agents into the vlNTS which block excitatory drive from lung afferent inputs. Bilateral microinjection of the inhibitory amino acid gamma-aminobutyric acid (GABA) 25 nmol/45 nl produced an immediate prolongation of inspiratory duration (484+/-18 to 1291+/-84 ms) and an apneustic pattern of breathing. Other effects observed were a significant shortening of expiratory duration (778+/-36 to 432+/-38 ms), rise in blood pressure (83+/-4 to 108+/-6 mmHg) and a small but significant increase in heart rate (439+/-17 to 452+/-18 beats/min). Bilateral microinjection of the ionotropic glutamate receptor antagonist kynurenic acid (1 nmol) and the Na(+) channel blocker tetrodotoxin (10 pmol) into the region of the vlNTS consistently produced a similar prolongation of inspiratory duration and an apneustic pattern of breathing. These results support the hypothesis that neurons in the region of the vlNTS promote the transition from inspiration to expiration and function as part of the 'Inspiratory Off Switch'.

The purpose of our study was to test the hypothesis that 5-hydroxytryptamine (5-HT)(1A) receptor agonists counteract morphine-induced respiratory depression. Studies were conducted in anesthetized rats, and respiratory activity was monitored with diaphragm electromyography. Morphine was administered i.v. in doses that produce apnea. Once apnea was established, i.v. administration of the 5-HT(1A) receptor agonist drug 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) at 10 or 100 microgram/kg restored normal breathing in each animal (n = 24). This antagonistic effect of 8-OH-DPAT on morphine-induced respiratory depression was observed in both spontaneously breathing and artificially ventilated animals. Results obtained with 8-OH-DPAT were mimicked by buspirone (50 microgram/kg i.v.), another 5-HT(1A) receptor agonist drug. Pretreatment with 4-(2'-methoxyphenyl)-1-[2'[N-(2'-pyridinyl]-p-iodo-benzamido]ethyl]pi perazine, an antagonist of 5-HT(1A) receptors, prevented 8-OH-DPAT from counteracting morphine-induced apnea. These results indicate that activation of central nervous system 5-HT(1A) receptors is an effective way of reversing morphine-induced respiratory depression. Most important, this is the third model of disturbed respiratory function in which drugs that stimulate 5-HT(1A) receptors have been shown to restore breathing to near-normal levels.