Interactions between Brainstem Neurons That Regulate the Motility to the Stomach.

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.

TRPV1 in arteries enables a rapid myogenic tone.

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 Ca2+ signalling and vasoconstriction. The majority of these stretch-responses were TRPV1-mediated, with the remaining tone being inhibited by the TRPM4 antagonist, 9-phenantrol. Notably, tone developed more quickly in arteries from wild-type compared with TRPV1-null mice. Furthermore, the immediate vasodilation following brief constriction of arterioles depended on TRPV1, consistent with a rapid deactivation of TRPV1. Pharmacologic experiments revealed that membrane stretch activates phospholipase C/protein kinase C signalling combined with heat to activate TRPV1, and in turn, L-type Ca2+ channels. These results suggest a critical role, for TRPV1 in the dynamic regulation of myogenic tone and blood flow in the heart and skeletal muscle. KEY POINTS: We explored the physiological role of TRPV1 in vascular smooth muscle. TRPV1 antagonists dilated skeletal muscle arterioles both ex vivo and in vivo, increased coronary perfusion and decreased systemic blood pressure. Stretch of arteriolar myocytes and increases in intraluminal pressure in arteries triggered rapid Ca2+ signalling and vasoconstriction respectively. Pharmacologic and/or genetic disruption of TRPV1 significantly inhibited the magnitude and rate of these responses. Furthermore, disrupting TRPV1 blunted the rapid vasodilation evoked by arterial constriction. Pharmacological experiments identified key roles for phospholipase C and protein kinase C, combined with temperature, in TRPV1-dependent arterial tone. These results show that TRPV1 in arteriolar myocytes dynamically regulates myogenic tone and blood flow in the heart and skeletal muscle.

Brainstem Neuronal Circuitries Controlling Gastric Tonic and Phasic Contractions: A Review.

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.

TRPV1 expressed throughout the arterial circulation regulates vasoconstriction and blood pressure.

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 tissue.  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, adipose tissue 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. Pharmacological 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.

The (α4)3(ß2)2 Stoichiometry of the Nicotinic Acetylcholine Receptor Predominates in the Rat Motor Cortex.

The α4ß2 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(ß2)3 and (α4)3(ß2)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 ß2 subunit cDNA were transfected into human embryonic kidney 293 cells to create a novel model system of HS and LS α4ß2 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 ß2 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/ß2 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 α4ß2 receptors in this brain region. We report that the majority of α4ß2 nAChRs in this brain region possess a stoichiometry of the (α4)3(ß2)2 LS subtype.

Optogenetic and pharmacological evidence that somatostatin-GABA neurons are important regulators of parasympathetic outflow to the stomach.

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.

AT-1001 Is a Partial Agonist with High Affinity and Selectivity at Human and Rat α3ß4 Nicotinic Cholinergic Receptors.

AT-1001 [N-(2-bromophenyl)-9-methyl-9-azabicyclo[3.3.1] nonan-3-amine] is a high-affinity and highly selective ligand at α3ß4 nicotinic cholinergic receptors (nAChRs) that was reported to decrease nicotine self-administration in rats. It was initially reported to be an antagonist at rat α3ß4 nAChRs heterologously expressed in HEK293 cells. Here we compared AT-1001 actions at rat and human α3ß4 and α4ß2 nAChRs similarly expressed in HEK 293 cells. We found that, as originally reported, AT-1001 is highly selective for α3ß4 receptors over α4ß2 receptors, but its binding selectivity is much greater at human than at rat receptors, because of a higher affinity at human than at rat α3ß4 nAChRs. Binding studies in human and rat brain and pineal gland confirmed the selectivity of AT-1001 for α3ß4 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 α3ß4 nAChRs. It was also a less potent and weaker (18%) partial agonist at α4ß2 nAChRs. Both α3ß4 and α4ß2 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 α3ß4 receptors than rat α3ß4 and human α4ß2 receptors, respectively. After exposure to AT-1001, the time to recovery from desensitization was longest for the human α3ß4 nAChR and shortest for the human α4ß2 receptor, suggesting that recovery from desensitization is primarily related to the dissociation of the ligand from the receptor.

Melanocortin signaling in the brainstem influences vagal outflow to the stomach.

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 an in vivo approach, 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 and MC4-R(-/-) mice. Bath application of MT-II or α-MSH significantly reduced spontaneous action potentials (but not in MC4-R(-/-) mice). However, in low-calcium ACSF, MT-II decreased neuronal firing, whereas α-MSH increased it. These effects mirror those of our in vivo DMV 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.

Chronic sazetidine-A maintains anxiolytic effects and slower weight gain following chronic nicotine without maintaining increased density of nicotinic receptors in rodent brain.

Chronic nicotine administration increases the density of brain α4ß2* 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 α4ß2* 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 α4ß2 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.

Arteries are finely tuned thermosensors regulating myogenic tone and blood flow.

In response to changing blood pressure, arteries adjust their caliber to control perfusion. This vital autoregulatory property, termed vascular myogenic tone, stabilizes downstream capillary pressure. We discovered that tissue temperature critically determines myogenic tone. Heating steeply activates tone in skeletal muscle, gut, brain and skin arteries with temperature coefficients ( Q 10 ) of ∼11-20. Further, arterial thermosensitivity is tuned to resting tissue temperatures, making myogenic tone sensitive to small thermal fluctuations. Interestingly, temperature and intraluminal pressure are sensed largely independently and integrated to trigger myogenic tone. We show that TRPV1 and TRPM4 mediate heat-induced tone in skeletal muscle arteries. Variations in tissue temperature are known to alter vascular conductance; remarkably, thermosensitive tone counterbalances this effect, thus protecting capillary integrity and fluid balance. In conclusion, thermosensitive myogenic tone is a fundamental homeostatic mechanism regulating tissue perfusion. One-Sentence Summary: Arterial blood pressure and temperature are integrated via thermosensitve ion channels to produce myogenic tone.

CNS sites controlling the gastric pyloric sphincter: Neuroanatomical and functional study in the rat.

The pyloric sphincter receives parasympathetic vagal innervation from the dorsal motor nucleus of the vagus (DMV). However, little is known about its higher-order neurons and the nuclei that engage the DMV neurons controlling the pylorus. The purpose of the present study was twofold. First, to identify neuroanatomical connections between higher-order neurons and the DMV. This was carried out by using the transneuronal pseudorabies virus PRV-152 injected into rat pylorus torus and examining the brains of these animals for PRV labeling. Second, to identify the specific sites within the DMV that functionally control the motility and tone of the pyloric sphincter. For these studies, experiments were performed to assess the effect of DMV stimulation on pylorus activity in urethane-anesthetized male rats. A strain gauge force transducer was sutured onto the pyloric tonus to monitor tone and motility. L-glutamate (500 pmol/30 nL) was microinjected unilaterally into the rostral and caudal areas of the DMV. Data from the first study indicated that neurons labeled with PRV occurred in the DMV, hindbrain raphe nuclei, midbrain Edinger-Westphal nucleus, ventral tegmental area, lateral habenula, and arcuate nucleus. Data from the second study indicated that microinjected L-glutamate into the rostral DMV results in contraction of the pylorus blocked by intravenously administered atropine and ipsilateral vagotomy. L-glutamate injected into the caudal DMV relaxed the pylorus. This response was abolished by ipsilateral vagotomy but not by intravenously administered atropine or L-NG-nitroarginine methyl ester (L-NAME). These findings identify the anatomical and functional brain neurocircuitry involved in controlling the pyloric sphincter. Our results also show that site-specific stimulation of the DMV can differentially influence the activity of the pyloric sphincter by separate vagal nerve pathways.

Tonic GABAA receptor conductance in medial subnucleus of the tractus solitarius neurons is inhibited by activation of µ-opioid receptors.

Our laboratory previously reported that gastric activity is controlled by a robust GABA(A) 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 GABA(A) 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 GABA(A) 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 GABA(A) 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 GABA(A) receptors containing the δ-subunit, that determines membrane excitability, and that is partially regulated by µ-opioid receptors.

Human induced pluripotent stem-derived retinal pigment epithelium (RPE) cells exhibit ion transport, membrane potential, polarized vascular endothelial growth factor secretion, and gene expression pattern similar to native RPE.

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.

Brainstem activation of GABAB receptors in the nucleus tractus solitarius increases gastric motility.

Background and aim: Local GABAergic signaling in the dorsal vagal complex (DVC) is essential to control gastric function. While the inhibitory GABAA receptor action on motility in the DVC is well-documented, the role of the GABAB receptor on gastric function is less well-established. Microinjection of baclofen, a selective GABAB receptor agonist, in the dorsal motor nucleus of the vagus (DMV) increases gastric tone and motility, while the effect on motility in the nucleus tractus solitarius (NTS) needs to be investigated. Previous in vitro studies showed that GABAB receptors exert a local inhibitory effect in unidentified NTS neurons. Since the NTS and DMV nuclei have differential control of gastric motility, we compared GABAB receptor activation in the NTS to that reported in the DMV. We microinjected baclofen unilaterally in the NTS while monitoring intragastric pressure and compared its action to optogenetic activation of somatostatin (SST) neurons in transgenic sst-Cre::channelrhodopsin-2 (ChR2) mice. We also performed patch-clamp recordings from SST and DMV neurons in brainstem slices from these mice. Methods: In vivo drug injections and optogenetic stimulation were performed in fasted urethane/α-chloralose anesthetized male mice. Gastric tone and motility were monitored by an intragastric balloon inserted in the antrum and inflated with warm water to provide a baseline intragastric pressure (IGP). Coronal brainstem slices were obtained from the sst-Cre::ChR2 mice for interrogation with optogenetics and pharmacology using electrophysiology. Results: The unilateral microinjection of baclofen into the NTS caused a robust increase in gastric tone and motility that was not affected by ipsilateral vagotomy. Optogenetic activation of SST neurons that followed baclofen effectively suppresses the gastric motility in vivo. In brain slices, baclofen suppressed spontaneous and light-activated inhibitory postsynaptic currents in SST and gastrointestinal-projection DMV neurons and produced outward currents. Conclusion: Our results show that GABAB receptors in the NTS strongly increase gastric tone and motility. Optogenetic stimulation in vivo and in vitro suggests that these receptors activated by baclofen suppress the glutamatergic sensory vagal afferents in the NTS and also inhibit the interneurons and the inhibitory neurons that project to the DMV, which, in turn, increase motility via a cholinergic excitatory pathway to the stomach.

General anesthetics activate a nociceptive ion channel to enhance pain and inflammation.

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.

Somatostatin Neurons in the Mouse Pontine Nucleus Activate GABAA Receptor Mediated Synaptic Currents in Locus Coeruleus Neurons.

The pontine nuclei comprising the locus coeruleus (LC) and Barrington's nucleus (BRN) amongst others form the neural circuitry(s) that coordinates arousal and voiding behaviors. However, little is known about the synaptic connectivity of neurons within or across these nuclei. These include corticotropin-releasing factor (CRF+) expressing neurons in the BRN that control bladder contraction and somatostatin expressing (SST+) neurons whose role in this region has not been discerned. To determine the synaptic connectivity of these neurons, we employed optogenetic stimulation with recordings from BRN and LC neurons in brain stem slices of channelrhodopsin-2 expressing SST or CRF neurons. Optogenetic stimulation of CRF+ BRN neurons of Crf Cre ;chr2-yfp mice had little effect on either CRF+ BRN neurons, CRF- BRN neurons, or LC neurons. In contrast, in Sst Cre ;chr2-yfp mice light-activated inhibitory postsynaptic currents (IPSCs) were reliably observed in a majority of LC but not BRN neurons. The GABAA receptor antagonist, bicuculline, completely abolished the light-induced IPSCs. To ascertain if these neurons were part of the neural circuitry that controls the bladder, the trans-synaptic tracer, pseudorabies virus (PRV) was injected into the bladder wall of Crf Cre ;tdTomato or Sst Cre ;tdTomato mice. At 68-72 h post-viral infection, PRV labeled neurons were present only in the BRN, being preponderant in CRF+ neurons with few SST+ BRN neurons labeled from the bladder. At 76 and 96 h post-virus injection, increased labeling was observed in both BRN and LC neurons. Our results suggest SST+ neurons rather than CRF+ neurons in BRN can regulate the activity of LC neurons.

Countermeasures for Preventing and Treating Opioid Overdose.

The only medication available currently to prevent and treat opioid overdose (naloxone) was approved by the US Food and Drug Administration (FDA) nearly 50 years ago. Because of its pharmacokinetic and pharmacodynamic properties, naloxone has limited utility under some conditions and would not be effective to counteract mass casualties involving large-scale deployment of weaponized synthetic opioids. To address shortcomings of current medical countermeasures for opioid toxicity, a trans-agency scientific meeting was convened by the US National Institute of Allergy and Infectious Diseases/National Institutes of Health (NIAID/NIH) on August 6 and 7, 2019, to explore emerging alternative approaches for treating opioid overdose in the event of weaponization of synthetic opioids. The meeting was initiated by the Chemical Countermeasures Research Program (CCRP), was organized by NIAID, and was a collaboration with the National Institute on Drug Abuse/NIH (NIDA/NIH), the FDA, the Defense Threat Reduction Agency (DTRA), and the Biomedical Advanced Research and Development Authority (BARDA). This paper provides an overview of several presentations at that meeting that discussed emerging new approaches for treating opioid overdose, including the following: (1) intranasal nalmefene, a competitive, reversible opioid receptor antagonist with a longer duration of action than naloxone; (2) methocinnamox, a novel opioid receptor antagonist; (3) covalent naloxone nanoparticles; (4) serotonin (5-HT)1A receptor agonists; (5) fentanyl-binding cyclodextrin scaffolds; (6) detoxifying biomimetic "nanosponge" decoy receptors; and (7) antibody-based strategies. These approaches could also be applied to treat opioid use disorder.

micro-Opioid receptor stimulation in the medial subnucleus of the tractus solitarius inhibits gastric tone and motility by reducing local GABA activity.

We examined the effects of altering mu-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-Ala(2),MePhe(4),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, GABA(A) receptors, 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.

α4ß2 nicotinic acetylcholine receptors intrinsically influence body weight in mice.

Desensitization of the nicotinic acetylcholine receptor (nAChR) containing the ß2 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 ß2 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 ß2, but not the α5, subunit of the nAChR suppresses weight gain in a sex-dependent manner. Furthermore, chronic treatment with the ß2-selective nAChR competitive antagonist dihydro-ß-erythroidine (DHßE) in mice fed a high-fat diet suppresses weight gain. These results indicate that heteromeric α4ß2 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.

GABAB Receptor Signaling in the Dorsal Motor Nucleus of the Vagus Stimulates Gastric Motility via a Cholinergic Pathway.

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.