DMV Neurons Research Articles

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

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. 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.

Maternal High Fat Diet Increases an Inhibitory Extrasynaptic Tonic GABAA Current in Dorsal Motor Nucleus of the Vagus Neurons Regulating Gastric Functions

The perinatal period is critically important in the development of autonomic neurocircuits innervating the gastrointestinal (GI) tract influencing gastric emptying and appetite regulation. Developmental alterations may affect these GI neurocircuits possibly contributing to the energy imbalance seen in obesity. Previous studies suggest there is an increased GABAergic inhibition of gastric projecting dorsal motor nucleus of the vagus nerve (DMV) neurons in the brainstem of rats fed a high fat diet (HFD) during the perinatal period, but the underlying neural mechanisms and the effects on gastric motility have yet to be clarified. The aims of this study was to assess the effects of perinatal HFD on (i) inhibitory synaptic inputs to gastric‐projecting DMV neurons and (ii) gastric tone and motility.Whole cell patch clamp recordings were made from gastric projecting DMV neurons in thin brainstem slices from Sprague‐Dawley rats (4–6 weeks of age) fed either a control or HFD (14 or 60%kcal from fat, respectively) from embryonic day 13.In the present study, perinatal HFD rats were not significantly heavier than age‐matched control rats (6 weeks; 232±17g vs 209±5g). Superfusion with the non‐selective GABAA receptor antagonist, bicuculline (50μM), induced a larger inward current in DMV neurons from perinatal HFD vs control animals (55.7±14.1pA vs 21.7±1.7pA, n=8, P<0.05), suggesting a greater overall tonic inhibition. While superfusion with the phasic (synaptic) GABAA receptor antagonist, gabazine (25μM), did not reveal any difference in the resulting inward current between perinatal HFD vs control DMV neurons (11.2±1.4pA vs 6.7±1.5pA, n=6, P>0.05), the addition of bicuculline into the bath uncovered a larger tonic (extrasynaptic) GABAA current in perinatal HFD vs control DMV neurons (36.4±3.47pA vs 20.8±2.24pA, n=6, P<0.05), suggesting that the greater overall inhibition of perinatal DMV neurons results from a larger tonic GABAA current.The studies imply that perinatal overnutrition increases the tonic GABAergic inhibition of gastric projecting DMV neurons via an increase in activation of extrasynaptic GABAA receptors. Such alterations in brainstem neurocircuit development as a consequence of maternal over‐nutrition may induce gastric dysmotility and dysregulated gastointestinal reflexes, hence influence offspring to develop obesity.Support or Funding InformationFunded by NIH 078364 and NSF IOS1148978

Maternal High Fat Diet Induces the Expression of an Inhibitory Glycinergic Current in Rat Dorsal Motor Nucleus of the Vagus Neurons Regulating Gastric Functions

The perinatal period is critically important in the development of autonomic neurocircuits and maternal overnutrition during this period has been shown to predispose offspring to obesity and associated comorbidities. Previous studies suggest there is an increased inhibition of gastric projecting dorsal motor nucleus of the vagus nerve (DMV) neurons in rats fed a high fat diet (HFD) during the perinatal period, but the underlying neural mechanisms and the effects on gastric motility have yet to be determined. This aim of this study was to assess the effects of perinatal HFD on (i) inhibitory synaptic inputs to gastric‐projecting DMV neurons and (ii) gastric tone and motility.Whole cell patch clamp recordings were made from gastric projecting DMV neurons in thin brainstem slices from Sprague‐Dawley rats (4–6 weeks of age) fed either a control or HFD (14 or 60%kcal from fat, respectively) from embryonic day 13. Gastric motility and tone was measured via miniature strain gauges attached to the antrum of anesthetized rats.In the present study, perinatal HFD rats were not significantly heavier than age‐matched control rats (6 weeks; 232±17g vs 209±5g). The glycine receptor antagonist, strychnine (1μM), induced a larger inward current in perinatal HFD vs control DMV neurons (23±2pA vs 3±1pA, n=8, P<0.05), suggesting that glycine is a tonically released neurotransmitter in perinatal HFD, but not control, rats. Strychnine also increased the rise time (5.0±0.3ms vs 3.5±0.2ms), decay time (9.5±0.8ms vs 5.3±0.6ms), and half width (8.8±0.9ms vs 5.0±0.5ms) of miniature inhibitory postsynaptic currents (mIPSCs) in perinatal HFD (n=5, P<0.05 vs baseline for each parameter) but had no effect in control neurons rise time (3.45±0.2ms vs 3.7±0.2ms), decay time (4.5±0.3ms vs 4.9±0.5ms), or half‐width (4.3±0.3ms vs 4.8±0.5ms) of miniature postsynaptic currents (n=5, P>0.05 for each). Microinjection of strychnine (300pmoles/60nl) into the DMV increased gastric tone and motility in the antrum in perinatal HFD rats (82–101mg, 20–25% of baseline, respectively, n=2) but not in control rats (11–21mg, 2–10% of baseline, n=2, P<0.05 vs perinatal HFD for each), suggesting the tonic glycinergic inputs onto DMV neurons decreased vagal efferent outflow and reduced basal gastric motility and tone.The studies suggest that perinatal overnutrition induces the expression of a novel tonic glycinergic input onto gastric projecting DMV neurons, increasing the inhibitory drive to the stomach. Such alterations in brainstem neurocircuit development as a consequence of maternal over‐nutrition may induce gastric dysmotility and dysregulated gastointestinal reflexes, hence influencing offspring to develop obesity.Support or Funding InformationFunded by NIH 078364 and NSF IOS1148978

Sex Differences in GABAergic Neurotransmission to Gastric‐Projecting DMV Neurons

Functional gastrointestinal disorders, including delayed gastric emptying and decreased gastric motility, are more prevalent in women than men. It is suggested that the abundance of circulating estrogen in women may account for this difference. Gastric motility is finely tuned by the inputs arising from the dorsal motor nucleus of the vagus (DMV), which is itself controlled by a tonic, inhibitory GABAergic input. Estrogen has been shown to increase GABA expression in various CNS area, however the effect of estrogen to modulate GABAergic inputs onto DMV neurons, has not been investigated.The aims of the present study were to test the hypotheses that: i) in female rats the GABAergic tone controlling gastric motility is higher than in male rats; ii) estrogen levels in female rats influence the gastric motility response; and, iii) the response to exogenous application of estrogen in the DMV varies between male and female rats.In vivo anesthetized preparations of Sprague‐Dawley rats were used to compare gastric tone and motility in age‐matched males and virgin females; female rats were further subdivided according to the high estrogen (pro‐estrus and estrus) or low‐estrogen (meta‐ and di‐estrus) period of their cycle. To record gastric tone and motility, miniature strain gauges were sutured onto both the anterior corpus and antrum portions of the stomach. Following exposure of the dorsal brainstem, microinjections of the GABA‐A selective antagonist bicuculline methiodide (BIC, 0.1–5 pmoles/60nL) increased gastric tone and motility in a dose‐dependent manner; the increase was significantly higher in high estrogen (BIC 0.1pmoles increased gastric tone by 192±51 mg and motility by 158±53%; N=5) than in low estrogen females (100±29 mg and 175±112%; N=4) or males (56±17 mg and 111±19%; N=6). Conversely, microinjections of 17b‐estradiol (10pmoles/60nl) decreased gastric motility and tone by −141±26 mg and 145±33% (N=9) in males and by −111±54 mg and 153±40% (N=4) and −205±64 mg and 84±15% (N=5) in low and high‐estrogen females, respectively. The tone and motility effects of 17b‐estradiol were antagonized by pretreatment with the GPER receptor antagonist ICI182,720 (3nmol).The data suggests that i) in female rats the GABAergic tone controlling gastric motility is higher than in male rats; ii) estrogen levels in female rats influence the gastric motility response; and, iii) the response to exogenous application of estrogen in the DMV varies between male and female rats. Taken together, the data supports the hypothesis that estrogen mediates a greater inhibitory input to the DMV through increased expression GABAergic inputs.Support or Funding InformationSupported by ANMS and by AHA grants

Developmental Regulation of Chloride Currents in the Dorsal Motor Nucleus of the Vagus of the Rat

Tonically firing parasympathetic neurons of the dorsal motor nucleus of the vagus (DMV) modulate the functions of the proximal gastrointestinal (GI) tract. The pacemaker activity of DMV neurons is modulated by synaptic inputs from adjacent autonomic areas, including the nuclei of the tractus solitarius. Functional maturation of these vagal sensorimotor neurocircuits results from intense synaptogenesis and neuronal reorganization in the first postnatal weeks and their maturation may be assessed by the analysis of their time constant of synaptic current decay.Prior studies have shown that, in adult rats, perfusion with the GABAA antagonist bicuculline (BIC) increases the firing rate of DMV neurons, and BIC microinjection in the DMV increase gastric motility and pancreatic exocrine secretions, indicating a prominent role of brainstem GABA synapses in the modulation of GI functions. Conversely, glycine synapses do not appear to be present in GI‐related brainstem neurocircuits of adult rats.The aim of the present study was to examine GABA and glycine‐ mediated chloride currents in the development of synaptic inputs onto DMV neurons. Using whole cell recordings, miniature inhibitory currents were recorded in rats from postnatal day 1 (P1) to postnatal day 30 (P30), and analyzed for decay constant and event frequency. Between P1–15, the baseline frequency was 1.9±0.35 events.s−1 and the current decay constant was 10.3±1.4 ms (N=30). These values were not altered by maturation (P>0.05 for both parameters).Perfusion of the slice with the GABAA antagonist bicuculline (1 or 10uM) either abolished completely or decreased the frequency and the decay constant of the inhibitory events to a similar extent in neurons from P1–15 rats or >P15 rats (N=37 and N=65 for 1 and 10uM, respectively). Conversely, while perfusion with the glycine antagonist strychnine (1μM) did not decrease event frequency significantly in any of the groups, it increased the current decay constant (from 8.2±0.65 to 11.9±0.75ms and from 7.6±0.57 to 10.5±0.79ms, in P1–15 and >P15,neurons, respectively). Strychnine altered inhibitory currents in a significantly larger percentage of P1–15 (N=67) neurons, and blocked all events in 3 cells from P1–15 rats, but not in >P15 rats (n=58; p<0.05 vs P1–15).These data suggest that the development of brainstem vagal neurocircuits involves an early phase, during which GABA and glycine neurotransmission appear co‐released by the same neurons. The role of glycine in synaptic transmission appears to start declining at approximately P15. Disruption of this developmental switch from GABA‐glycine to purely GABAergic transmission may occur in response to early life stress, infection or inflammation, which may lead to adverse consequences in vagal efferent control of visceral functions.Support or Funding InformationFunding: NIH DK 99350 and DK 78364 & NSF IOS‐1148978

Su1551 Sex Differences in Gabaergic Neurotransmission to Gastric-Projecting DMV Neurons

Su1551 Sex Differences in Gabaergic Neurotransmission to Gastric-Projecting DMV Neurons

Brain stem as a target site for the metabolic side effects of olanzapine.

Olanzapine, an atypical antipsychotic, is widely prescribed for the treatment of schizophrenia and bipolar disorder despite causing undesirable metabolic side effects. A variety of mechanisms and brain sites have been proposed as contributors to the side effects; however, the role of the dorsal motor nucleus of the vagus nerve (DMV), which plays a crucial role in the regulation of subdiaphragmatic organs and thus governs energy and glucose homeostasis, is largely unknown. Identifying the effect of olanzapine on the excitability of DMV neurons in both sexes is thus crucial to understanding possible underlying mechanisms. Whole cell patch-clamp electrophysiological recordings were conducted in stomach- and liver-related DMV neurons identified with retrograde viral tracers and in random DMV neurons. The effect of olanzapine on the neuronal excitability of DMV neurons both in male and female mice was established. Our data demonstrate that olanzapine hyperpolarizes the DMV neurons in both sexes and this effect is reversible. The hyperpolarization is associated with decreased firing rate and input resistance. Olanzapine also decreases the excitability of a subset of stomach- and liver-related DMV neurons. Our study demonstrates that olanzapine has a powerful effect on DMV neurons in both sexes, indicating its ability to reduce vagal output to the subdiaphragmatic organs, which likely contributes to the metabolic side effects observed in both humans and experimental models. These findings suggest that the metabolic side effects of olanzapine may partially originate in the DMV.

Presynaptic ionotropic glutamate receptors modulate GABA release in the mouse dorsal motor nucleus of the vagus

Regulation of GABA release in the dorsal motor nucleus of the vagus (DMV) potently influences vagal output to the viscera. The presence of functional ionotropic glutamate receptors (iGluRs) on GABAergic terminals that rapidly alter GABA release onto DMV motor neurons has been suggested previously, but the receptor subtypes contributing to the response are unknown. We examined the effect of selective activation and inhibition of iGluRs on tetrodotoxin-insensitive, miniature inhibitory postsynaptic currents (mIPSCs) in DMV neurons using patch-clamp recordings in brainstem slices from mice. Capsaicin, which activates transient receptor potential vanilloid type 1 (TRPV1) receptors and increases mIPSC frequency in the DMV via an iGluR-mediated, heterosynaptic mechanism, was also applied to assess GABA release subsequent to capsaicin-stimulated glutamate release. Application of glutamate, N-methyl-d-aspartate (NMDA), or kainic acid (KA), but not AMPA, resulted in increased mIPSC frequency in most neurons. Inhibition of AMPA/KA receptors reduced mIPSC frequency, but selective antagonism of AMPA receptors did not alter GABA release, implicating the presence of presynaptic KA receptors on GABAergic terminals. Whereas NMDA application increased mIPSC frequency, blocking NMDA receptors was without effect, indicating that presynaptic NMDA receptors were present, but not activated by ambient glutamate levels in the slice. The effect of NMDA was prevented by AMPA/KA receptor blockade, suggesting indirect involvement of NMDA receptors. The stimulatory effect of capsaicin on GABA release was prevented when AMPA/KA or NMDA, but not AMPA receptors were blocked. Results of these studies indicate that presynaptic NMDAR and KA receptors regulate GABA release in the DMV, representing a heterosynaptic arrangement for rapidly modulating parasympathetic output, especially when synaptic excitation is elevated.

Functional segregation of voltage-activated calcium channels in motoneurons of the dorsal motor nucleus of the vagus.

Calcium influx elevates mitochondrial oxidant stress (mOS) in dorsal motor nucleus of the vagus (DMV) neurons that are prone to Lewy body pathologies in presymptomatic Parkinson's disease (PD) patients. In experimental PD models, treatment with isradipine, the dihydropyridine with the highest affinity to Cav1.3 channels, prevents subthreshold calcium influx via Cav1.3 channels into midbrain dopamine neurons and protects them from mOS. In DMV neurons, isradipine is also effective in reducing mOS despite overwhelming evidence that subthreshold calcium influx is negligible compared with spike-triggered influx. To solve this conundrum we combined slice electrophysiology, two-photon laser scanning microscopy, mRNA profiling, and computational modeling. We find that the unusually depolarized subthreshold voltage trajectory of DMV neurons is positioned between the relatively hyperpolarized activation curve of Cav1.3 channels and that of other high-voltage activated (HVA) calcium channels, thus creating a functional segregation between Cav1.3 and HVA calcium channels. The HVA channels flux the bulk of calcium during spikes but can only influence pacemaking through their coupling to calcium-activated potassium currents. In contrast, Cav1.3 currents, which we show to be more than an order-of-magnitude smaller than the HVA calcium currents, are able to introduce sufficient inward current to speed up firing. However, Kv4 channels that are constitutively open in the subthreshold range guarantee slow pacemaking, despite the depolarizing action of Cav1.3 and other pacemaking currents. We propose that the efficacy of isradipine in preventing mOS in DMV neurons arises from its mixed effect on Cav1.3 channels and on HVA Cav1.2 channels.

Diabetes induces GABA receptor plasticity in murine vagal motor neurons.

Autonomic dysregulation accompanies type-1 diabetes, and synaptic regulation of parasympathetic preganglionic motor neurons in the dorsal motor nucleus of the vagus (DMV) is altered after chronic hyperglycemia/hypoinsulinemia. Tonic gamma-aminobutyric acid A (GABAA) inhibition prominently regulates DMV neuron activity, which contributes to autonomic control of energy homeostasis. This study investigated persistent effects of chronic hyperglycemia/hypoinsulinemia on GABAA receptor-mediated inhibition in the DMV after streptozotocin-induced type-1 diabetes using electrophysiological recordings in vitro, quantitative (q)RT-PCR, and immunohistochemistry. Application of the nonspecific GABAA receptor agonist muscimol evoked an outward current of significantly larger amplitude in DMV neurons from diabetic mice than controls. Results from application of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride (THIP), a δ-subunit agonist, suggested that GABAA receptors containing δ-subunits contributed to the enhanced inducible tonic GABA current in diabetic mice. Sensitivity to THIP of inhibitory postsynaptic currents in DMV neurons from diabetic mice was also increased. Results from qRT-PCR and immunohistochemical analyses indicated that the altered GABAergic inhibition may be related to increased trafficking of GABAA receptors that contain the δ-subunit, rather than an expression change. Overall these findings suggest increased sensitivity of δ-subunit containing GABAA receptors after several days of hyperglycemia/hypoinsulinemia, which dramatically alters GABAergic inhibition of DMV neurons and could contribute to diabetic autonomic dysregulation.

Olanzapine Reduces Neuronal Activity in the Dorsal Motor Nucleus of the Vagus

Olanzapine, a widely prescribed atypical antipsychotic, relieves the symptoms of schizophrenia and bipolar disorder. Despite its positive impact on mental health, patients treated with olanzapine experience side effects related to weight gain and impaired glucose homeostasis. A variety of mechanisms and brain areas underlying the metabolic side effects have been proposed; however, the involvement of the brainstem remained to be elucidated. Parasympathetic neurons in the dorsal motor nucleus of the vagus (DMV) set the vagal tone and thus control visceral functions including feeding, digestion, and glucose homeostasis. In this study, whole‐cell patch‐clamp recordings were performed to test the hypothesis that olanzapine modulates neuronal activity in the DMV of mice. Acute application of olanzapine (10 µM) hyperpolarized the majority (~70 %) of DMV neurons in male mice by an average of ‐7.77 ± 1.17 mV (n=20). This effect was concentration‐dependent (EC50 = 16.02 ± 1.78 µM), and was associated with a reduction of input resistance. Similarly, hyperpolarization was observed in female mice (n=9). The hyperpolarization also occurred in the presence of tetrodotoxin, suggesting a postsynaptic mechanism of action. Next, stomach‐ and liver‐projecting DMV neurons were identified with transsynaptic retrograde viral labeling. Acute olanzapine application resulted in hyperpolarization of the majority of both liver‐ and stomach‐related DMV neurons. In summary, our data demonstrate that in slices olanzapine administration hyperpolarizes the majority of DMV neurons via postsynaptic mechanisms. These data suggest that olanzapine has the potential to modulate vagal outflow to subdiaphragmatic organs.Supported by: Pilot Project Grant Tulane University School of Medicine

Sex Differences in GABAergic Neurotransmission to Gastric‐Projecting DMV Neurons

Women have a higher prevalence of functional gastrointestinal (GI) disorders compared to men. Gastric motility is modulated by neurons of the dorsal motor nucleus of the vagus (DMV) and the activity of these neurons is regulated by a tonic GABAergic input. In general, females have been shown to have a higher expression of GABA throughout the central nervous system. The aim of this study was to investigate sex differences in the basic properties and GABAergic inputs to gastric-projecting DMV neurons. In an in vivo anesthetized preparation, microinjections of bicuculline methiodide (BIC, 0.5-50pmoles/60 nl) into the DMV of Sprague-Dawley rats increased gastric tone in a dose-dependent manner. Microinjection of 0.5 pmoles of BIC elicited a 32.0±12 mg increase in gastric tone (N=2) in males and a significantly greater increase in gastric tone in females (200.5±37; N=3; p<0.05). Whole-cell patch-clamp recordings were made from gastric-projecting DMV neurons. Female rats displayed an increased afterhyperpolarization amplitude (19.8±1 mV, N=18) compared to males (15.3±1 mV N=18; p<0.05), and a lower action potential frequency response to injection of 280 pA of current (31.5±2 events/s in males; 25.5±2 events/s in females). Perfusion of slices with BIC (0.5-50 μM) increased the firing rate of gastric-projecting neurons. The increase in the firing rate during perfusion with 50 μM BIC was significantly greater in females (237±53% of baseline; n=5) than in males (136.2±24%, n=3; p<0.05). These data show that gastric-projecting DMV neurons in females are less excitable and receive a higher tonic GABAergic input compared to males and suggest that reduced gastric motility in females may be due to a an increased inhibitory input to gastric-projecting neurons. Supported by: NIH DK55330

Exposure to a high fat diet during the perinatal period alters vagal motoneurone excitability, even in the absence of obesity.

Obesity is recognized as being multifactorial in origin, involving both genetic and environmental factors. The perinatal period is known to be critically important in the development of neural circuits responsible for energy homeostasis and the integration of autonomic reflexes. Diet-induced obesity alters the biophysical, pharmacological and morphological properties of vagal neurocircuits regulating upper gastrointestinal tract functions, including satiety. Less information is available, however, regarding the effects of a high fat diet (HFD) itself on the properties of vagal neurocircuits. The present study was designed to test the hypothesis that exposure to a HFD during the perinatal period alters the electrophysiological, pharmacological and morphological properties of vagal efferent motoneurones innervating the stomach. Our data indicate that perinatal HFD decreases the excitability of gastric-projecting dorsal motor nucleus neurones and dysregulates neurotransmitter release from synaptic inputs and that these alterations occur prior to the development of obesity. These findings represent the first direct evidence that exposure to a HFD modulates the processing of central vagal neurocircuits even in the absence of obesity. The perinatal period is critically important to the development of autonomic neural circuits responsible for energy homeostasis. Vagal neurocircuits are vital to the regulation of upper gastrointestinal functions, including satiety. Diet-induced obesity modulates the excitability and responsiveness of both peripheral vagal afferents and central vagal efferents but less information is available regarding the effects of diet per se on vagal neurocircuit functions. The aims of this study were to investigate whether perinatal exposure to a high fat diet (HFD) dysregulated dorsal motor nucleus of the vagus (DMV) neurones, prior to the development of obesity. Whole cell patch clamp recordings were made from gastric-projecting DMV neurones in thin brainstem slices from rats that were exposed to either a control diet or HFD from pregnancy day 13. Our data demonstrate that following perinatal HFD: (i) DMV neurones had decreased excitability and input resistance with a reduced ability to fire action potentials; (ii) the proportion of DMV neurones excited by cholecystokinin (CCK) was unaltered but the proportion of neurones in which CCK increased excitatory glutamatergic synaptic inputs was reduced; (iii) the tonic activation of presynaptic group II metabotropic glutamate receptors on inhibitory nerve terminals was attenuated, allowing modulation of GABAergic synaptic transmission; and (iv) the size and dendritic arborization of gastric-projecting DMV neurones was increased. These results suggest that perinatal HFD exposure compromises the excitability and responsiveness of gastric-projecting DMV neurones, even in the absence of obesity, suggesting that attenuation of vago-vagal reflex signalling may precede the development of obesity.

Nicotine enhances inhibition of mouse vagal motor neurons by modulating excitability of premotor GABAergic neurons in the nucleus tractus solitarii.

The caudal nucleus of the solitary tract (NTS) serves as the site of the first synapse for visceral sensory inputs to the central nervous system. The NTS sends functional projections to multiple brain nuclei, with gastric-related projections primarily targeting the dorsal motor nucleus of the vagus (DMV). Previous studies have demonstrated that the majority of caudal NTS neurons that project to the DMV respond robustly to nicotine and express nicotinic acetylcholine receptors (nAChRs). However, the cytochemical identity and relationship with specific viscera of DMV-projecting, nicotine-responsive caudal NTS neurons have not been determined. The present study used transgenic mice that express enhanced green fluorescent protein (EGFP) under a GAD67 promoter in a subset of GABAergic neurons, in vivo retrograde pseudorabies viral labeling to identify gastric-related vagal complex neurons, and patch-clamp electrophysiology in acute brain stem slices to test the hypothesis that gastric-related and GABAergic inhibitory synaptic input to the DMV from the caudal NTS is under a robust modulatory control by nAChRs. Our results suggest that activation of nAChRs in the caudal NTS, but not DMV, potentiates GABAergic, but not glutamatergic, input to the DMV. Gastric-related caudal NTS and DMV neurons are directly involved in this nicotine-sensitive circuitry. Understanding the central patterns of nicotinic modulation of visceral sensory-motor circuitry may help develop therapeutic interventions to restore autonomic homeostasis in patients with autonomic impairments.

Gastric vagal motoneuron function is maintained following experimental spinal cord injury

Clinical reports indicate that spinal cord injury (SCI) initiates profound gastric dysfunction. Gastric reflexes involve stimulation of sensory vagal fibers, which engage brainstem circuits that modulate efferent output back to the stomach, thereby completing the vago-vagal reflex. Our recent studies in a rodent model of experimental high thoracic (T3-) SCI suggest that reduced vagal afferent sensitivity to gastrointestinal (GI) stimuli may be responsible for diminished gastric function. Nevertheless, derangements in efferent signals from the dorsal motor nucleus of the vagus (DMV) to the stomach may also account for reduced motility. We assessed the anatomical, neurophysiological, and functional integrity of gastric-projecting DMV neurons in T3-SCI rats using: (i) retrograde labeling of gastric-projecting DMV neurons; (ii) whole cell recordings from gastric-projecting neurons of the DMV; and, (iii) in vivo measurements of gastric contractions following unilateral microinjection of thyrotropin-releasing hormone (TRH) into the DMV. Immunohistochemical analysis of gastric-projecting DMV neurons demonstrated no difference between control and T3-SCI rats. Whole cell in vitro recordings showed no alteration in DMV membrane properties and the neuronal morphology of these same, neurobiotin-labeled, DMV neurons were unchanged after T3-SCI with regard to cell size and dendritic arborization. Central microinjection of TRH induced a significant facilitation of gastric contractions in both control and T3-SCI rats and there were no significant dose-dependent differences between groups. Our data suggest that the acute, 3 day to 1 week post-SCI, dysfunction of vagally mediated gastric reflexes do not include derangements in the efferent DMV motoneurons.

Regulation of leptin receptor-expressing neurons in the brainstem by TRPV1.

The central nervous system plays a critical role in the regulation of feeding behavior and whole‐body metabolism via controlling the autonomic output to the visceral organs. Activity of the parasympathetic neurons in the dorsal motor nucleus of the vagus (DMV) determines the vagal tone and thereby modulates the function of the subdiaphragmatic organs. Leptin is highly involved in the regulation of food intake and alters neuronal excitability of brainstem neurons. Transient receptor potential vanilloid type 1 (TRPV1) has also been shown to increase neurotransmission in the brainstem and we tested the hypothesis that TRPV1 regulates presynaptic neurotransmitter release to leptin receptor‐expressing (LepRbEGFP) DMV neurons. Whole‐cell patch‐clamp recordings were performed to determine the effect of TRPV1 activation on excitatory and inhibitory postsynaptic currents (EPSC, IPSC) of LepRbEGFP neurons in the DMV. Capsaicin, a TRPV1 agonist increased the frequency of miniature EPSCs in 50% of LepRbEGFP neurons without altering the frequency of miniature IPSCs in the DMV. Stomach‐projecting LepRbEGFP neurons were identified in the DMV using the transsynaptic retrograde viral tracer PRV‐614. Activation of TRPV1 increased the frequency of mEPSC in ~50% of stomach‐related LepRbEGFP DMV neurons. These data demonstrate that TRPV1 increases excitatory neurotransmission to a subpopulation of LepRbEGFP DMV neurons via presynaptic mechanisms and suggest a potential interaction between TRPV1 and leptin signaling in the DMV.

CAMP-dependent insulin modulation of synaptic inhibition in neurons of the dorsal motor nucleus of the vagus is altered in diabetic mice.

Pathologies in which insulin is dysregulated, including diabetes, can disrupt central vagal circuitry, leading to gastrointestinal and other autonomic dysfunction. Insulin affects whole body metabolism through central mechanisms and is transported into the brain stem dorsal motor nucleus of the vagus (DMV) and nucleus tractus solitarius (NTS), which mediate parasympathetic visceral regulation. The NTS receives viscerosensory vagal input and projects heavily to the DMV, which supplies parasympathetic vagal motor output. Normally, insulin inhibits synaptic excitation of DMV neurons, with no effect on synaptic inhibition. Modulation of synaptic inhibition in DMV, however, is often sensitive to cAMP-dependent mechanisms. We hypothesized that an effect of insulin on GABAergic synaptic transmission may be uncovered by elevating resting cAMP levels in GABAergic terminals. We used whole cell patch-clamp recordings in brain stem slices from control and diabetic mice to identify insulin effects on inhibitory neurotransmission in the DMV in the presence of forskolin to elevate cAMP levels. In the presence of forskolin, insulin decreased the frequency of inhibitory postsynaptic currents (IPSCs) and the paired-pulse ratio of evoked IPSCs in DMV neurons from control mice. This effect was blocked by brefeldin-A, a Golgi-disrupting agent, or indinavir, a GLUT4 blocker, indicating that protein trafficking and glucose transport were involved. In streptozotocin-treated, diabetic mice, insulin did not affect IPSCs in DMV neurons in the presence of forskolin. Results suggest an impairment of cAMP-induced insulin effects on GABA release in the DMV, which likely involves disrupted protein trafficking in diabetic mice. These findings provide insight into mechanisms underlying vagal dysregulation associated with diabetes.

TNFα Causes Thrombin-Dependent Vagal Neuron Apoptosis in Inflammatory Bowel Disease

BackgroundThe role of peripheral tumor necrosis factor alpha (TNFα) in inflammatory bowel disease (IBD) is well established, but its central nervous system (CNS) effects are not understood. Thrombin, another mediator of inflammation in IBD, has been implicated in CNS vagal neuron apoptosis in the dorsal motor nucleus of the vagus (DMV). This study evaluates DMV TNFα exposure, characterizes effects of TNFα on DMV neurons, and identifies a relationship between DMV TNFα and thrombin in IBD. Methods2,4,6-Trinitrobenzene sulfonic acid was administered via enema to induce colonic inflammation in rats. TNFα in serum, cerebrospinal fluid (CSF), and DMV tissues were determined by ELISA and DMV TNFα expression by quantitative reverse transcription PCR (RT-PCR). TNFα was administered into the fourth intracerebral ventricle (4 V) adjacent to the DMV, with and without blockade of TNF receptor 1 (TNFR1) and the thrombin receptor proteinase-activated receptor 1 (PAR1). Immunofluorescence was used to evaluate microglial activation (Cd11b) and prothrombin presence in DMV sections. Apoptosis was examined using terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) and activated caspase-3 immunofluorescence. ResultsIBD is associated with increased TNFα protein in serum, CSF, and DMV tissue; DMV TNFα transcription is also increased. TNFα (4 V) caused a 54 % increase in microglial activation, a 27 % increase in DMV prothrombin protein, and a 31 % increase in vagal neuron apoptosis by TUNEL. There was a 52 % increase in activated caspase-3 immunofluorescence in TNFα-treated animals (p < 0.05). All effects of 4 V TNFα were prevented by TNFR1 blockade. TNFα-induced apoptosis was prevented by PAR1 blockade. ConclusionsIBD is associated with DMV exposure to TNFα, causing excess DMV prothrombin and vagal apoptosis.

Thrombin Mediates Vagal Apoptosis and Dysfunction in Inflammatory Bowel Disease

BackgroundIn inflammatory bowel disease, autonomic dysfunction contributes to symptoms, morbidity, and health care resource utilization. Efferent vagal neurons, which provide the primary parasympathetic input to the gastrointestinal tract, are housed in the dorsal motor nucleus of the vagus (DMV) in the brainstem. This study seeks to characterize the effects of IBD on DMV neuronal survival and function. MethodsTNBS (picrylsulfonic acid) was administered by enema to induce colitis in rats. Brain sections through the DMV were examined for neuronal apoptosis using TUNEL labeling, and for glial cell activation by immunofluorescence. Prothrombin production was evaluated via quantitative RT-PCR from DMV tissue, as well as by double immunofluorescence in DMV sections. To investigate the effects of thrombin in the DMV, thrombin or thrombin and an antagonist to its receptor were administered into the fourth ventricle via a stereotactically placed cannula. DMV sections were then examined for apoptosis by TUNEL assay. To evaluate the effect of thrombin on DMV neuronal function, we examined calcium signaling in primary DMV neuron cultures following exposure to thrombin and other neurotransmitters. ResultsTNBS colitis is associated with significantly increased rates of DMV neuronal apoptosis, affecting 12.7 % of DMV neurons in animals with colitis, compared to 3.4 % in controls. There was a corresponding increase in DMV neuron activated caspase-3 immunoreactivity (14.8 vs. 2.6 % of DMV neurons). TNBS-treated animals also demonstrated significantly increased DMV astrocyte and microglial immunoreactivity, indicating glial cell activation. DMV prothrombin production was significantly increased in TNBS colitis, with a close anatomic relationship between prothrombin and microglia. Direct DMV exposure to thrombin replicated the apoptosis and activation of caspase-3 seen in TNBS colitis; these effects were prevented by coadministration of the PAR-1 inhibitor FR171113. Cultured DMV neurons exhibited impaired calcium signaling in response to neurotransmitters following exposure to thrombin. Glutamate-induced calcium transients decreased by 59 %, and those triggered by GABA were reduced by 61 %. PAR-1 antagonism prevented these thrombin-induced changes in calcium signaling. ConclusionsIBD is associated with DMV microglial activation and production of prothrombin. Thrombin in the DMV causes vagal neuron apoptosis and decreased sensitivity to neurotransmitters.

Tu1789 Brain Activation With Rectal Distention Distinguishes Irritable Bowel Syndrome From Healthy Controls and Is Independent of Psychiatric Comorbidity

gastric motility in females is due to a higher GABAergic input to gastricprojecting DMV neurons. In an in vivo anesthetized preparation, microinjections of bicuculline methiodide (BIC, 0.5-50pmoles in 60 nl) into the DMV of Sprague-Dawley rats increased gastric tone in a dose-dependent manner. In males, microinjection of 0.5 pmoles of BIC elicited a 32.0±12 mg increase in gastric tone (N=2), whereas microinjection of 50 pmoles of BIC elicited an increase of 186.0±23 mg (N=2). In females, DMV microinjections of 0.5 pmoles of BIC elicited a significantly greater increase in gastric tone compared to males (200.5±37; N=3; p<0.05). Whole-cell patch-clamp recordings were made from identified gastric-projecting DMV neurons. Female rats displayed an increased afterhyperpolarization amplitude and decay time (19.7±2mV;180.7±39ms; N=12) compared tomales (15.9±1mV afterhyperpolarization amplitude; 103.9±24 ms decay time; N=9; p<0.05), and a lower action potential frequency response to injection of 80 pA of current (13.6±1 events/s in males; 9.2±1 events/ s in females). These differences are indicative of a decreased excitability of neurons in females. Females also had a higher frequency of miniature inhibitory postsynaptic currents compared to males (1.2±0.3, N=7, and 0.7±0.04, N=4, events/s in females and males, respectively). These data indicate that gastric-projecting DMV neurons in females are less excitable and receive a higher tonic GABAergic input compared to males. These data suggest that reduced gastric motility in females may be due to an increased inhibitory input to gastric-projecting neurons.