Dorsal Motor Nucleus Research Articles

Perinatal High Fat Diet Decreases Oxytocinergic Innervation to the Brainstem

IntroductionPerinatal high fat diet (pHFD) exposure is known to increase anxiety in offspring, and reduce stress resilience, however the mechanism that causes this functional change is unknown. Oxytocin is the prototypical anti‐stress peptide, and the paraventricular nucleus of the hypothalamus (PVN) provides descending inputs to the dorsal vagal complex (DVC) within the brainstem. The DVC provides parasympathetic extrinsic control over the gastrointestinal tract. The present study was designed to investigate the hypothesis that changes in oxytocin hypothalamic‐brainstem innervation may be partially responsible for the lack of stress resilience observed following a pHFD.MethodsRats were fed control or high fat diet (14 or 60% kcal from fat, respectively) from embryonic day 13, and weaned onto the same diet at postnatal day 21. At 6‐8 weeks of age, 7 rats were anesthetized, perfused transcardially and brains removed for immunohistochemical processing. In another set of experiments 8 rats (N=4 control, 4 pHFD) the retrograde tracer cholera toxin B (CTB; 0.5%) was injected into the DVC (5 injections of 120nl throughout the rostro‐caudal extent of the DVC). After recovery for 10 days, rats were perfused transcardially and brains removed as above. Coronal slices (50um) were processed for immunohistochemical detection of oxytocin in the brainstem, as well as and oxytocin, CTB, and oxytocin/CTB co‐localization in the hypothalamus. Results were compared using an unpaired t‐test.ResultsIn naïve rats, total oxytocin fluorescence in the DMV was 5.754 arbitrary units/area (AU) in control rats vs. 1.547 AU/area in pHFD rats (p=0.0272). Within the hypothalamus, it was found that there were significantly fewer (p=0.0006) oxytocinergic cells innervating the DVC in pHFD rats (2.250 cells/mid‐PVN coronal slice) compared to control (11.17 cells/mid‐PVN coronal slice). There was also a significant reduction in CTB‐positive neurons in pHFD (71.60 cells/mid‐PVN coronal slice) rats compared to control (185.5 cells/mid‐PVN coronal slice) rats (p < 0.001). In contrast, no significant differences were found in the number of PVN oxytocin‐positive cells when comparing control versus pHFD rats (126.5 cells vs. 88.87 cells, respectively; p=0.0904).ConclusionsThese data suggest that pHFD decreases oxytocin innervation to the DVC. Previous studies have shown that oxytocin is important in regulating the gastrointestinal response to stress; given the decrease in oxytocin immunoreactivity observed following pHFD this may provide a potential mechanism to explain the increased anxiety and decreased stress resilience under these conditions. Following this study, it will be important to correlate the results with their functional significance through electrophysiology and behavioral assays.AcknowledgementsThe project described was supported by NIH 1R25DK113652 and DK111667. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Diabetes and Digestive and Kidney Diseases or the National Institutes of Health.

Electrical Stimulation of the Dorsal Motor Nucleus of the Vagus Attenuates Serum Pro‐Inflammatory Cytokine Levels in Murine Models of Endotoxemia and Sepsis

The inflammatory reflex is a vagus nerve‐mediated and brainstem‐integrated physiological mechanism that regulates immunity and inflammation (Annu Rev Immunol, 2018, 36:783). The efferent vagus nerve originates in brainstem nuclei, including the dorsal motor nucleus of the vagus (DMN). Recently, it was shown that optogenetic stimulation of cholinergic DMN neurons inhibits serum TNF levels during endotoxemia (Proc Natl Acad Sci USA, 2020, 117(47):29803). However, the effects of electrical DMN stimulation on cytokine levels in inflammatory conditions have not been previously evaluated. To provide insight, we examined the effect of electrical DMN stimulation in mice with endotoxemia and cecal ligation and puncture (CLP)‐induced polymicrobial sepsis. In the endotoxemia model, 10–12‐week‐old male C57BL/6 mice (n=14‐20 per group) were anesthetized using isoflurane and the head was positioned on a stereotaxic frame. A surgical intervention and a stereotaxic‐guided approach were used to insert a concentric bipolar electrode into the left DMN. Electrical stimulation (50 μA, 30 Hz, 260 μs) or sham stimulation was performed for five mins, followed by lipopolysaccharide (LPS, 0.5 mg/kg) i.p. administration. The mice were euthanized 90 mins after LPS and blood (via cardiac puncture) was collected and processed for cytokine analysis. In the CLP model, DMN stimulation or sham stimulation was performed as described above immediately after subjecting mice (n=14‐20 per group) to CLP, i.e., following a midline laparotomy in anesthetised mice, the cecum was isolated and the distal end was ligated and punctured. 24h later, the mice were euthanised and blood was collected and processed for analysis. In mice with endotoxemia, electrical DMN stimulation significantly decreased serum TNF (p = 0.0069) and IL‐6 (p = 0.0427) levels compared with sham stimulation. In addition, electrical DMN stimulation increased the serum levels of the anti‐inflammatory cytokine IL‐10 (p = 0.0284). In mice with CLP, electrical DMN stimulation resulted in significantly lower serum TNF levels compared with sham stimulation (p = 0.0291). These results indicate for the first time that electrical DMN stimulation alters circulating pro‐inflammatory and anti‐inflammatory levels in murine endotoxemia and sepsis. These findings are of interest for further development of bioelectronic approaches targeting DMN for therapeutic benefit in conditions, characterised by immune dysregulation and inflammation.

Anatomical and Molecular Phenotypes of Fast and Slow Vagal Targets in the Intrinsic Cardiac Nervous System

The vagus nerve coordinates the communication between the brain and most of the visceral organs. Vagus nerve activity is critical to promote cardiac health and cardioprotective response in heart failure. The vagal motor outflow involves two nuclei, the nucleus ambiguous (NA) and the dorsal motor nucleus of the vagus (DMV). NA acts via nicotinic cholinergic processes to elicit fast kinetics of the intrinsic cardiac nervous system (ICN) response at the heart. By contrast, the DMV influences the ICN neurons via muscarinic cholinergic processes yielding slower kinetics of response. Interestingly, NA and DMV project to distinct neuronal populations in the ICN, providing an anatomical basis for the fast versus slow vagal action on the heart. We aim to delineate the ICN neuronal network mediating the two lanes of vagal inputs for regulating distinct cardiac functions with lane‐specific transcriptomic identities of ICN neuron targets. We performed immunohistochemistry (IHC) on the rat heart tissue sections embedded in the optimal cutting temperature (OCT) followed by confocal microscopy imaging. We found that while the ICN neurons express muscarinic and nicotinic acetylcholine receptors (AChRs) broadly across the ICN, with several neurons predominantly expressing either muscarinic or nicotinic AChRs. We used Laser Capture Microscopy (LCM) to acquire neurons that predominantly express muscarinic or nicotinic AChRs. We are profiling the transcriptomic identities of these LCM‐captured neuron classes. Our results suggest that mAChR‐only and nAChR‐only neurons differentially express neuromodulatory processes, including receptors, neurotransmitter synthesis enzymes, and neuropeptides. Further, we incorporate a systems approach combining spatial single‐cell transcriptomics, neuronal network modeling, and physiological control systems modeling to identify selective targets for novel neuromodulatory interventions that improve heart health.

Ciprofloxacin Permanently Accelerates Gastrointestinal Transit in a Rodent Model of Fluoroquinolones Associated Disability

Fluoroquinolones (FQs) are a class of antibiotics intended to be prescribed for life‐threatening infections. However, they are often administered for less severe infections, which they are not meant to treat. In addition, studies suggest that FQs can lead to severe long‐lasting complications described as Fluoroquinolone Associated Disability (FQAD). Some of these permanent issues affect the central nervous system, cardiac, and musculoskeletal systems among others. Gastrointestinal issues have also been considered a possible side effect due to how FQs may negatively modulate the vagus nerve, which plays a major role in digestion and regulation of satiety. Indeed, the inhibitory neurotransmitter GABA regulates the motor portion of the vagus nerve, and FQs act as a selective inhibitor of GABAA receptors. This blockade of GABAA receptors is believed to contribute to the GI issues that seem to arise after taking FQs.To measure possible changes in digestion, Sprague Dawley rats were treated daily for 14 consecutive days with either 0.3ml of 0.9% saline (CTL n=6) or 20mg/kg of ciprofloxacin (CPX) via oral gavage. On experiment day (day 14 or 28; n=6 for both), rats were administered phenol red dye, sacrificed, and the stomach, proximal, middle, and distal small intestine were harvested to measure dye recovery via spectrophotometry as a measure of gastrointestinal transit. Results showed that both male and female rats in the 28 day group had significantly less dye recovered from their stomach compared to the 14 day and CTL group, with female rats being disproportionately affected compared to males (P<0.05). This data provides strong evidence that FQs lead to permanent acceleration in digestive patterns. Further experiments will analyze how the phenotype of enteric motor neurons, as well as the phenotype of neurons within the Dorsal Motor Nucleus of the vagus in the medulla oblongata, is affected by this drug. This will contribute to determining the mechanism underlying the observed digestion change in rats treated with ciprofloxacin.

High Fat Diet‐Induced Neuroplasticity in Cardiac‐Projecting Dorsal Motor Nucleus of the Vagus (DMV) Motoreurons

The DMV is critical in the maintenance of gastrointestinal (GI) function and metabolism through its axonal projections to subdiaphragmatic organs. However, there is a subpopulation of choline acetyltransferase (ChAT) positive DMV neurons that send projections to cardiac tissue. Given their anatomical apposition to nuclei regulating GI and metabolic function, it is likely that cardiac vagal motorneurons (CVNs) in the DMV are sensitive to nutrient signals such as high fat diet (HFD). We hypothesized that HFD exposure for 15 days modulates the electrophysiological properties of DMV CVNs, thus restraining their ability to discharge.Mice underwent retrograde tracing to identify DMV CVNs, were challenged with HFD (60%) or control chow, and were used for whole‐cell patch‐clamp electrophysiology. When comparing DMV CVNs from control and HFD‐fed animals under current clamp conditions, no significant differences in firing frequency, resting membrane potential, or input resistance were found under normal conditions. However, DMV CVNs from HFD‐fed mice exhibited a significant increase in evoked action potentials (APs) following blockade of inhibitory, gamma‐aminobutyric acid type‐A receptor (GABAAR) neurotransmission with bicuculline methiodide (BIC; 30 µM; n=10; 5.02±1.23 APs) compared to the previous artificial cerebrospinal fluid conditions (aCSF; 4.2±0.92 APs; p<0.0001). CVNs from controls demonstrated no significant differences between BIC and aCSF conditions (n=10; p=0.1194). Taken together, HFD increased CVN sensitivity to antagonism of GABAARs. Additional studies using voltage clamp configuration assessed differences in the two signaling modalities of GABA: phasic/synaptic and tonic/extrasynaptic. Spontaneous phasic inhibitory post‐synaptic currents were not different in frequency, amplitude, or decay time. However, DMV CVNs from HFD animals had a significant increase in tonic GABAAR current after the application of BIC (n=9; 1.35±0.19 pA/pF) compared to controls (n=7; 0.8±0.11 pA/pF; p=0.0361). Given that δ subunit‐containing GABAARs (GABAA(δ)Rs) are theorized to mediate tonic GABAergic current, follow‐up experiments assessed the application of the specific GABAA(δ)R agonist, THIP (3 µM) followed by BIC. Although BIC‐sensitive increases in tonic current remained in CVNs from HFD mice, no significant currents were induced in either group after THIP application, suggesting that all GABAA(δ)Rs are actively contributing to tonic currents under these conditions. Since no antagonists exist for GABAA(δ)Rs, we developed a transgenic mouse line where the δ subunit is knocked out of ChAT motorneurons (i.e. ChAT/GABAA(δ)R‐null mice) to determine whether the electrophysiological changes induced by HFD could be prevented. Preliminary findings indicate that ablation of GABAA(δ)Rs in ChAT positive DMV CVNs was enough to prevent HFD‐induced increases in evoked APs following BIC application (n=6; 4.58±1.05 APs) compared to aCSF (4.5±0.98 APs; p=0.9559). These data suggest that DMV CVNs express more GABAA(δ)Rs responsible for the generation of long‐term inhibition after HFD exposure, thus restraining their discharge and potentially reducing their capacity for cardiovascular regulation. These early changes in CVN electrophysiology could impact not only cardiovascular function, but lead to other comorbidities, including obesity, since exercise capacity is linked to vagal regulation from the DMV. Further studies are warranted to determine the mechanistic underpinnings of HFD‐induced CVN neuroplasticity.

Crosstalk in the Central Autonomic Neurocircuitry Underlying Cardiovascular and Metabolic Control

Obesity associated cardiovascular risks such as hypertension are serious medical conditions that increase mortality. The brain autonomic networks play an important role in the regulation of cardiovascular function and metabolism. Therefore, obesity may represent a cardiovascular‐metabolic coupling and possible crosstalk in the central autonomic networks. We and others have previously shown the existence of shared nuclei in the autonomic networks innervating cardiovascular organs such as the kidney and metabolic organs such as the liver and brown adipose tissue (BAT). However, whether there are shared neurons within these nuclei associated with these three organs is unknown. To address this, two groups of mice received injections of pseudorabies virus (PRV) expressing a green fluorescent protein (GFP) into both kidneys and PRV expressing a red fluorescent protein (RFP) in either the interscapular BAT (iBAT) or the left lobe of the liver. The animals were sacrificed 5‐7 days post‐injection and perfused. The brains were extracted, sectioned at 50 µm thickness, stained, and imaged with confocal microscopy. Sections were matched to the mouse atlas (Franklin & Paxinos) and neurons co‐expressing GFP and RFP throughout the brain were identified. We found several nuclei in which neurons were co‐labeled with GFP (kidney) and RFP (BAT or liver). Interestingly, co‐expression does not appear limited to nuclei of specific regions in the brain. Instead, co‐expression was observed in many nuclei from cortical and subcortical regions to hypothalamic areas, midbrain, and brainstem nuclei. Indeed, co‐expressing neurons were observed in areas such as the cortical regions (motor cortex and amygdala), hypothalamic nuclei (paraventricular nucleus (PVN), lateral hypothalamus (LH), and dorsomedial hypothalamus (DMH)), midbrain regions such as the periaqueductal gray, and brainstem nuclei such as the locus coeruleus (LC). In general, there appeared to be two scenarios for shared nuclei: 1) in regions such as LH, nucleus of the solitary tract, and dorsal motor nucleus of vagus there was very little co‐expression, and 2) in regions such as PVN, DMH, LC, and motor cortex there was moderate to high levels of co‐expression. This is the first study to provide anatomical evidence for crosstalk between the autonomic networks regulating cardiovascular and metabolic functions. Moreover, our data demonstrate that crosstalk between the autonomic networks controlling cardiovascular and metabolic functions is distributed throughout the central nervous system.

Hypoglycemia: Control of counter‐regulatory hormone secretion by the brain

Hypoglycaemia is a common side‐effect of the treatment of type 1 diabetes with insulin. The Diabetes Control and Complications Trial indicated that, while intensive insulin therapy reduced the incidence and severity of diabetic complications such as retinopathy, neuropathy and nephropathy, the incidence of severe hypoglycaemia was increased 2‐3 fold. In non‐diabetic individuals, hypoglycaemia triggers the glucose counter‐regulatory response. This response consists of inhibition of insulin secretion and activation of glucagon secretion. Prolonged hypoglycaemia also evokes adrenaline secretion and less importantly, growth hormone and cortisol secretion. However, in individuals that have type 1 diabetes, the glucagon response to hypoglycaemia is lost. In these patients, the pancreatic α‐cells continue to synthesise glucagon since glucagon secretagogues such as arginine produce a robust glucagon secretory response. Our research has examined the hypothalamic and medullary neurocircuits that mediate adrenaline and glucagon secretion under conditions of neuroglucoprivation or hypoglycaemia. These studies have shown that the response to neuroglucoprivation involves specialised sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM) that mediate adrenaline secretion. These neurons are readily distinguishable from sympathetic vasomotorneurons also located in the RVLM. In addition, we have described the properties of perifornical hypothalamic (PeH) orexinergic (Ox) neurons that are activated by insulin‐induced hypoglycaemia (IHH) or neuroglucoprivation. PeH neurons project to many different targets including the RVLM and the dorsal motor nucleus of the vagus (DMV). Ox2‐R in the RVLM mediate the adrenaline secretory response. PeH neurons also mediate the glucagon response to IHH since the release of glucagon in response to IHH is diminished in Ataxin3 rats which have an age‐related loss of PeH orexin neurons.

Topographical Organization and Morphology of Substance‐P Axons in the Flat‐Mounts of the Mouse Whole Stomach

More than 50 million Americans suffer from chronic pain. However, the anatomical and physiological mechanisms of peripheral nociceptive processes have not been well elucidated which has seriously impeded the progress of designing novel bioengineering manipulations/treatments for chronic pain. In this study, we performed a comprehensive topographical mapping of pain‐related neural circuitry in the flat‐mount of whole mouse stomach (male, n=8, 3‐5 months). We used Substance P (SP) as a marker for nociceptive axons and applied a combination of state‐of‐the‐art techniques, including flat‐mount tissue processing and immunohistochemistry of the whole stomach, confocal microscopy, Zeiss Imager microscopy to determine the distribution and morphology of SP‐IR axons and terminals in the whole stomach. We found that 1) SP‐IR axons formed extensive terminal networks in both the ventral and dorsal stomachs. 2) SP‐IR axons were much denser in the antrum and corpus regions than in the fundus and cardia. 3) SP‐IR varicose axons ran in parallel with the circular and longitudinal muscle layers. 4) SP‐IR axons innervated blood vessels. 5) SP‐IR varicose axons formed terminals wrapping around individual myenteric neurons. 6) There were no confirmed SP‐IR myenteric neurons. 7) SP‐IR afferent innervation of the stomach showed a similar pattern of SP‐IR axons and terminals between the ventral and dorsal stomachs. In control mice (n=8), we injected tracer DiI into the ventral and dorsal stomach muscular layers or Fluorogold injection (i.p) and found that the main extrinsic source of SP‐IR afferent axons in the stomach was from the T7‐T11 DRG and to a lesser extent the VNG, but not from the celiac ganglia and dorsal motor nucleus of vagus. Our data provide for the first time a comprehensive topographical map for SP‐IR axons and terminals in the whole stomach with single cell/axon/synapse resolution. This work will contribute to a 3D digital representation of a brain‐stomach nociceptive atlas in a stomach scaffold, thus providing a novel anatomical foundation for functional mapping of nociceptive afferent axons and their pathological remodeling in the stomach. The first two authors contributed equally to this work.

Central Activation of the Cholinergic Anti‐Inflammatory Pathway Improves Outcome in a Murine Model of Systemic Lupus Erythematosus

The cholinergic anti‐inflammatory pathway is an endogenous vagus‐to‐spleen mechanism that regulates the immune system and when activated, results in decreases in systemic inflammation. Previous studies from our group demonstrated that the pharmacological potentiation of the efferent vagus nerve via systemic administration of galantamine reduces renal inflammation and blood pressure in systemic lupus erythematosus (SLE), suggesting an impairment in the cholinergic anti‐inflammatory pathway in this model. In this study, we aimed to delineate specific pathways and we hypothesized that central stimulation of vagal efferents by selective activation of neurons within the dorsal motor nucleus of the vagus (DMVN) would similarly protect SLE mice and confirm the role of the cholinergic anti‐inflammatory in controlling inflammation. For that, we selectively activated DMVN neurons using designer receptors exclusively activated by designer drugs (DREADDs). Female NZBWF1 (SLE) and parental control – NZW (Control) mice (n=3‐8) received bilateral microinjections of pAAV‐hSyn‐hM3D(Gq)‐mCherry or pAAV‐hSyn‐mCherry (control virus) into the DMVN at 31 weeks of age. Two weeks post‐microinjection, DREADD agonist CNO (3mg/kg) was administered subcutaneously for 2 weeks starting at 33 weeks. At 35 weeks, mice were housed in metabolic cages for urine collection and catheters were implanted in the carotid to access mean arterial pressure (MAP) in conscious mice. Mice were subsequently euthanized, spleen was collected for Western blotting, blood for double‐stranded (ds) DNA autoantibody quantification and brain to confirm the site of virus microinjection. Selective activation of DMVN neurons did not alter MAP (mmHg) in SLE mice (SLE: 146 ± 6 vs SLE/Gq DREADD: 142 ± 3) or controls (Control: 126 ±4 vs Control/Gq DREADD: 132 ± 2), and also did not change dsDNA autoantibodies (activity units) in SLE mice (SLE: 4.7e5± 1.1e4 vs SLE/Gq DREADD: 3.7e8 ± 1.3e6) or controls (Control: 1.8e5± 5.3e4 vs Control/Gq DREADD: 2.8e5± 1.3e5). Splenic TNFα protein expression was increased in SLE mice compared to controls (SLE: 1.9e6 ± 3.6e5 vs Control: 6.5e5 ± 8.6e4, p=0.001) and DMVN neuron activation decreased splenic TNFα expression in SLE mice (SLE: 1.9e6 ± 3.6e5 vs SLE/Gq DREADD: 1.1e6 ± 1.1e5, p=0.05) but not controls (Control: 6.1e6 ± 3.1e6 vs Control /Gq DREADD: 1.7e6±4.4e5). Albumin excretion rate (µg/day) was higher in SLE mice compared to controls (SLE: 10210±4015 vs Control 97.1±54.3, p=0.07) and DMVN neuron stimulation decreased albumin excretion rate in SLE mice although not significantly (SLE/Gq DREADD: 207.4±142.1, p=0.07). In addition, selective activation of DMV neurons decreased urinary leukocytes (62.5% vs 50%) and blood in the urine (50% vs 16%) in SLE mice. The present study showed that boosting the cholinergic anti‐inflammatory pathway via selective activation of vagal efferents lowered splenic inflammation and protected the kidney without altering blood pressure suggesting an important role of the cholinergic anti‐inflammatory pathway in regulating inflammation and renal injury in SLE.

Perinatal High Fat Diet and Acute Stress alters the Chloride Gradient in Dorsal Motor Nucleus of the Vagus Neurons Controlling Gastric Function

The US is currently facing an obesity epidemic, though the concurrent rise in maternal obesity rates receives less attention. Maternal obesity is an important factor that influences intrauterine and early postnatal environment and is associated with developmental changes in the offspring, including alterations in metabolic function and susceptibility to anxiety and depression, all of which are associated with abnormal gastrointestinal (GI) tract function. Previous work from this lab has shown that perinatal high fat diet (pHFD) leads to developmental changes to vagal neurocircuits, including changes to GABAA receptor subunits on dorsal motor nucleus of the vagus (DMV) neurons. However, the manner of neuronal recordings (whole‐cell patch clamp) disrupted internal chloride (Cl‐) concentration, preventing accurate assessment of the extent of GABA‐mediated inhibition. KCC2, a Cl‐ transporter, is important in determining GABAergic inhibition and stress outcomes in hypothalamic neurons, however its role in regulating these factors in DMV neurons is unknown. The current study was designed to test the hypothesis that pHFD reduces KCC2 levels in DMV neurons, subsequently collapsing the Cl‐ gradient and leading to altered gastric output.Sprague‐Dawley rats were fed either a control (14% kcal from fat) or high fat (60% kcal from fat) diet starting on embryonic day 13. After weaning on postnatal day 28, brainstem slices were made to conduct patch clamp recordings on DMV neurons. This was done in both naïve (unstressed) or stressed conditions, in which rats underwent a 10 minute forced swim (FS). To determine the Cl‐ reversal, perforated patch clamp recordings were performed in order to preserve the internal Cl‐ concentration. Then, DMV neurons were voltage clamped from ‐90 to ‐40 mV and the amplitude of the current response to picospritz of the GABAAagonist muscimol (100 μM) was measured. These events were plotted on an I‐V curve, with the x‐intercept representing the reversal of Cl‐. To determine a potential mediator in the shift in the Cl‐ reversal, changes in KCC2 levels following pHFD exposure were evaluated immunohistochemically. Lastly, gastric emptying (GE) assays were performed using the 13C octanoic acid breath assessment.In both pHFD and control diet + FS DMV neurons, there was a depolarizing shift in the Cl‐ reversal compared to naïve rats (‐43.05 mV and ‐44.58 mV respectively, compared to ‐64.56 mV in naïve conditions; p < 0.0001). It was also found that there is a significant reduction in KCC2 expression in pHFD DMV region compared to controls (2.237% area fluorescence in pHFD compared to 5.335% in control diet; p=0.0147). Lastly, pHFD rats had significantly delayed GE rates compared to those fed a control diet (83.7 minutes for half‐decay in pHFD compared to 66.7 minutes in controls; p < 0.05), resembling previously published data which showed a delay in GE following an acute stress. These results demonstrate that both a pHFD and acute stress lead to depolarizing shifts in the Cl‐ reversal and that this may be through a mechanism involving a reduction in KCC2 expression. As the gastric emptying rates demonstrate, these changes could lead to a maladaptive GI stress response in pHFD rats.Determining the mechanism in which pHFD and acute stress modulate DMV function, as well as the impact of their intersection on the gastric stress response, will better inform our understanding of the reduced stress resiliency seen in pHFD offspring.

A 3D Anatomical and Molecular Map of Cardiac Vagal Motoneurons

Recent efforts through the NIH SPARC program have opened new opportunities for harnessing vagal activity to promote cardioprotection. However, cardiac‐projecting vagal motoneurons of the Dorsal Motor Nucleus of the Vagus (DMV) are relatively understudied, even as recent studies show that DMV activity is required for cardioprotection induced by physiological interventions. By combining principles of neuroscience and single cell transcriptomics, we begin to explore the transcriptomic phenotypes of cardiac‐projecting DMV neurons. Neuromodulatory co‐transmission is an emerging discipline of neuroscience, and the work of the Allen Institute suggests transcriptomics of neuropeptides and corresponding receptors may characterize cell types. We find that DMV neurons are not solely cholinergic, but simultaneously catecholaminergic and GABAergic, including those that project to the heart. These findings challenge the conventional neuronal nomenclature. We propose that inputs determine phenotype, and work done on the Mouse Brain Atlas supports this premise, suggesting a transcriptomic diversification, less region‐specific, results from interaction with the environment. We present here a characterization of DMV cell types by input‐output transcriptomics. We combine high throughput transcriptomics, 3D anatomical mapping, and neural tracing in 12 week old Sprague Dawley male and female rats to interrogate the phenotypes of DMV neurons. To distinguish potential mechanisms of cardioprotection, we highlight the functional significance of several transcripts for secreted proteins in cardiac‐projecting DMV neurons. We have spatially mapped the location and molecular phenotypes of these single cells in the DMV and Nucleus Ambiguus (NA). The cardiac‐projecting neurons of the DMV extend approximately 2.5 mm rostrocaudally. The cardiac‐projecting neurons of the left DMV are particularly prominent caudally, while the cardiac‐projecting neurons of the right DMV are particularly prominent rostrally. A unique phenotype, with transcriptomic markers of strong cardioprotective potential, is present at the rostrocaudal position of left DMV previously shown to affect ventricular contractility. There is consistency between our findings from three spatial transcriptomics techniques. Based on these results, we propose a 3D anatomical and molecular map of cardiac vagal motoneurons.

OC-0926 Dose to the dorsal vagal complex is predictive of radiation induced nausea

OC-0926 Dose to the dorsal vagal complex is predictive of radiation induced nausea

Etonogestrel Administration Reduces the Expression of PHOX2B and Its Target Genes in the Solitary Tract Nucleus.

Heterozygous mutations of the transcription factor PHOX2B are responsible for Congenital Central Hypoventilation Syndrome, a neurological disorder characterized by inadequate respiratory response to hypercapnia and life-threatening hypoventilation during sleep. Although no cure is currently available, it was suggested that a potent progestin drug provides partial recovery of chemoreflex response. Previous in vitro data show a direct molecular link between progestins and PHOX2B expression. However, the mechanism through which these drugs ameliorate breathing in vivo remains unknown. Here, we investigated the effects of chronic administration of the potent progestin drug Etonogestrel (ETO) on respiratory function and transcriptional activity in adult female rats. We assessed respiratory function with whole-body plethysmography and measured genomic changes in brain regions important for respiratory control. Our results show that ETO reduced metabolic activity, leading to an enhanced chemoreflex response and concurrent increased breathing cycle variability at rest. Furthermore, ETO-treated brains showed reduced mRNA and protein expression of PHOX2B and its target genes selectively in the dorsal vagal complex, while other areas were unaffected. Histological analysis suggests that changes occurred in the solitary tract nucleus (NTS). Thus, we propose that the NTS, rich in both progesterone receptors and PHOX2B, is a good candidate for ETO-induced respiratory modulation.

Substance P Increases the Excitability of Dorsal Motor Nucleus of the Vagus Nerve via Inhibition of Potassium Channels

Increases in the substance P (SP) concentration in the medial portion of the dorsal motor nucleus of the vagus nerve (mDMV) in the brainstem are closely associated with chemotherapy induced nausea and vomiting (CINV). However, the underlying cellular and molecular mechanisms of action are not well understood. In this study, we investigated the effects of SP on mDMV neurons using whole-cell patch-clamp recordings from rat brainstem slices. Application of different concentrations of SP induced tonic and phasic responses. Submicromolar concentrations of induced an inward shift of the holding current by increasing membrane input resistance. The response was mimicked by acidification of the extracellular solution and inhibited by a neurokinin type 1 receptor antagonist. These responses have equilibrium potentials close to the K+ equilibrium potential. In addition, a TWIK-related acid-sensitive K+ channel 3 (TASK-3) inhibitor, PK-THPP, induced responses similar to those produced by submicromolar SP concentrations. Micromolar concentrations of SP facilitated γ-aminobutyric acid (GABA) release but diminished glutamate release; these changes were blocked by a GABAB receptor antagonist and a neurokinin type 3 receptor antagonist, respectively. In current-clamp recordings, submicromolar SP concentrations increased neuronal excitability by depolarizing membrane potentials. However, neither the increase in SP concentration to the micromolar range nor the addition of GABAA and ionotropic glutamate receptor antagonists affected neuronal excitability. Thus, SP increases the excitability of mDMV neurons by inhibiting K+ conductance.

Plasma autoantibodies to glial fibrillary acidic protein (GFAP) react with brain areas according to Braak staging of Parkinson’s disease

Idiopathic Parkinson’s disease (PD) is characterized by a progredient degeneration of the brain, starting at deep subcortical areas such as the dorsal motor nucleus of the glossopharyngeal and vagal nerves (DM) (stage 1), followed by the coeruleus–subcoeruleus complex; (stage 2), the substantia nigra (SN) (stage 3), the anteromedial temporal mesocortex (MC) (stage 4), high-order sensory association areas and prefrontal fields (HC) (stage 5) and finally first-order sensory association areas, premotor areas, as well as primary sensory and motor field (FC) (stage 6). Autoimmunity might play a role in PD pathogenesis. Here we analyzed whether anti-brain autoantibodies differentially recognize different human brain areas and identified autoantigens that correlate with the above-described dissemination of PD pathology in the brain. Brain tissue was obtained from deceased individuals with no history of neurological or psychiatric disease and no neuropathological abnormalities. Tissue homogenates from different brain regions (DM, SN, MC, HC, FC) were subjected to SDS-PAGE and Western blot. Blots were incubated with plasma samples from 30 PD patients and 30 control subjects and stained with anti-IgG antibodies to detect anti-brain autoantibodies. Signals were quantified. Prominent autoantigens were identified by 2D-gel-coupled mass spectrometry sequencing. Anti-brain autoantibodies are frequent and occur both in healthy controls and individuals with PD. Glial fibrillary acidic protein (GFAP) was identified as a prominent autoantigen recognized in all plasma samples. GFAP immunoreactivity was highest in DM areas and lowest in FC areas with no significant differences in anti-GFAP autoantibody titers between healthy controls and individuals with PD. The anti-GFAP autoimmunoreactivity of different brain areas correlates with the dissemination of histopathological neurodegeneration in PD. We hypothesize that GFAP autoantibodies are physiological but might be involved as a cofactor in PD pathogenesis secondary to a leakage of the blood–brain barrier.

Distribution of leptin receptors in the brain stem: possible route in the pathophysiology of neuromuscular control of airway resistance during sleep

IntroductionLeptin, a hormone related to satiety, has been studied because of its association with obesity and sleep apnea. The distribution of leptin receptors in the brain stem, and in the hypoglossal nucleus, has not yet been described. The stimulation of these muscles has been studied in the treatment of sleep apnea. Objectiveto detail the presence of leptin receptors in the nuclei of these nerves to enable studies of stimulation of this region through leptin. Methods: the brains of five cadavers, removed during necropsy, collected at the Death Verification Service were included. An informed consent was signed by a family member (wife, mother or son/daughter) who answered specific questionnaire concerning comorbities. Anthropometric measurements were recorded. The medulla oblongata and pons fragments were identified. Immunohistochemical staining analysis was performed to identify the location of the leptin receptors. ResultsIn the immunohistochemical analysis an intense staining signal of the brownish coloration of neurons was evidenced in the hypoglossal nerve nucleus, moderate in the olivary nucleus and mild in the dorsal nucleus of the vagus and trigeminal nucleus. In motor neurons, more intense brown pigmentation can be observed in the nucleus and cytoplasm when compared to sensory neurons. ConclusionThe immunoexpression of leptin receptor was demonstrated in the motor neurons of the human hypoglossal nucleus. These results may contribute to unravel details of the pathophysiology of neuromuscular control of airway collapse during sleep and to the development of new drugs capable of improving the neuromuscular tone of upper airway in apneic individuals.

Involvement of the Dorsal Vagal Complex in Alcohol-Related Behaviors

The neurobiological mechanisms that regulate the development and maintenance of alcohol use disorder (AUD) are complex and involve a wide variety of within and between systems neuroadaptations. While classic reward, preoccupation, and withdrawal neurocircuits have been heavily studied in terms of AUD, viable treatment targets from this established literature have not proven clinically effective as of yet. Therefore, examination of additional neurocircuitries not classically studied in the context of AUD may provide novel therapeutic targets. Recent studies demonstrate that various neuropeptides systems are important modulators of alcohol reward, seeking, and intake behaviors. This includes neurocircuitry within the dorsal vagal complex (DVC), which is involved in the control of the autonomic nervous system, control of intake of natural rewards like food, and acts as a relay of interoceptive sensory information via interactions of numerous gut-brain peptides and neurotransmitter systems with DVC projections to central and peripheral targets. DVC neuron subtypes produce a variety of neuropeptides and transmitters and project to target brain regions critical for reward such as the mesolimbic dopamine system as well as other limbic areas important for the negative reinforcing and aversive properties of alcohol withdrawal such as the extended amygdala. This suggests the DVC may play a role in the modulation of various aspects of AUD. This review summarizes the current literature on neurotransmitters and neuropeptides systems in the DVC (e.g., norepinephrine, glucagon-like peptide 1, neurotensin, cholecystokinin, thyrotropin-releasing hormone), and their potential relevance to alcohol-related behaviors in humans and rodent models for AUD research. A better understanding of the role of the DVC in modulating alcohol related behaviors may lead to the elucidation of novel therapeutic targets for drug development in AUD.

Mapping Motor Neuron Vulnerability in the Neuraxis of Male SOD1G93A Mice Reveals Widespread Loss of Androgen Receptor Occurring Early in Spinal Motor Neurons.

Sex steroid hormones have been implicated as disease modifiers in the neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Androgens, signalling via the androgen receptor (AR), predominate in males, and have widespread actions in the periphery and the central nervous system (CNS). AR translocates to the cell nucleus when activated upon binding androgens, whereby it regulates transcription of target genes via the classical genomic signalling pathway. We previously reported that AR protein is decreased in the lumbar spinal cord tissue of symptomatic male SOD1G93A mice. Here, we further explored the changes in AR within motor neurons (MN) of the CNS, assessing their nuclear AR content and propensity to degenerate by endstage disease in male SOD1G93A mice. We observed that almost all motor neuron populations had undergone significant loss in nuclear AR in SOD1G93A mice. Interestingly, loss of nuclear AR was evident in lumbar spinal MNs as early as the pre-symptomatic age of 60 days. Several MN populations with high AR content were identified which did not degenerate in SOD1G93A mice. These included the brainstem ambiguus and vagus nuclei, and the sexually dimorphic spinal MNs: cremaster, dorsolateral nucleus (DLN) and spinal nucleus of bulbocavernosus (SNB). In conclusion, we demonstrate that AR loss directly associates with MN vulnerability and disease progression in the SOD1G93A mouse model of ALS.

Rifaximin modulates TRH and TRH-like peptide expression throughout the brain and peripheral tissues of male rats

BackgroundThe TRH/TRH-R1 receptor signaling pathway within the neurons of the dorsal vagal complex is an important mediator of the brain-gut axis. Mental health and protection from a variety of neuropathologies, such as autism, Attention Deficit Hyperactivity Disorder, Alzheimer’s and Parkinson’s disease, major depression, migraine and epilepsy are influenced by the gut microbiome and is mediated by the vagus nerve. The antibiotic rifaximin (RF) does not cross the gut-blood barrier. It changes the composition of the gut microbiome resulting in therapeutic benefits for traveler’s diarrhea, hepatic encephalopathy, and prostatitis. TRH and TRH-like peptides, with the structure pGlu-X-Pro-NH2, where “X” can be any amino acid residue, have reproduction-enhancing, caloric-restriction-like, anti-aging, pancreatic-β cell-, cardiovascular-, and neuroprotective effects. TRH and TRH-like peptides occur not only throughout the CNS but also in peripheral tissues. To elucidate the involvement of TRH-like peptides in brain-gut-reproductive system interactions 16 male Sprague–Dawley rats, 203 ± 6 g, were divided into 4 groups (n = 4/group): the control (CON) group remained on ad libitum Purina rodent chow and water for 10 days until decapitation, acute (AC) group receiving 150 mg RF/kg powdered rodent chow for 24 h providing 150 mg RF/kg body weight for 200 g rats, chronic (CHR) animals receiving RF for 10 days; withdrawal (WD) rats receiving RF for 8 days and then normal chow for 2 days.ResultsSignificant changes in the levels of TRH and TRH-like peptides occurred throughout the brain and peripheral tissues in response to RF. The number of significant changes in TRH and TRH-like peptide levels in brain resulting from RF treatment, in descending order were: medulla (16), piriform cortex (8), nucleus accumbens (7), frontal cortex (5), striatum (3), amygdala (3), entorhinal cortex (3), anterior (2), and posterior cingulate (2), hippocampus (1), hypothalamus (0) and cerebellum (0). The corresponding ranking for peripheral tissues were: prostate (6), adrenals (4), pancreas (3), liver (2), testis (1), heart (0).ConclusionsThe sensitivity of TRH and TRH-like peptide expression to RF treatment, particularly in the medulla oblongata and prostate, is consistent with the participation of these peptides in the therapeutic effects of RF.

Neural control of pancreatic peptide hormone secretion

Pancreatic peptide hormone secretion is inextricably linked to maintenance of normal levels of blood glucose. In animals and man, pancreatic peptide hormone secretion is controlled, at least in part, by input from parasympathetic (vagal) premotor neurons that are found principally in the dorsal motor nucleus of the vagus (DMV). Iatrogenic (insulin-induced) hypoglycaemia evokes a homeostatic response commonly referred to as the glucose counter-regulatory response. This homeostatic response is of particular importance in Type 1 diabetes in which episodes of hypoglycaemia are common, debilitating and lead to suboptimal control of blood glucose. Glucagon is the principal counterregulatory hormone but for reasons unknown, its secretion during insulin-induced hypoglycaemia is impaired. Pancreatic parasympathetic neurons are distinguishable electrophysiologically from those that control other (e.g. gastric) functions and are controlled by supramedullary inputs from hypothalamic structures such as the perifornical region. During hypoglycaemia, glucose-sensitive, GABAergic neurons in the ventromedial hypothalamus are inhibited leading to disinhibition of perifornical orexin neurons with projections to the DMV which, in turn, leads to increased secretion of glucagon.