Nitrergic Neurons Research Articles

Circadian rhythm disruption modulates enteric neural precursor cells differentiation leading to gastrointestinal motility dysfunction via the NR1D1/NF-κB axis

ObjectivesCircadian rhythm disruption (CRD) is implicated with numerous gastrointestinal motility diseases, with the enteric nervous system (ENS) taking main responsibility for the coordination of gastrointestinal motility. The purpose of this study is to explore the role of circadian rhythms in ENS remodeling and to further elucidate the underlying mechanisms.MethodsFirst, we established a jet-lagged mice model by advancing the light/dark phase shift by six hours every three days for eight weeks. Subsequent changes in gastrointestinal motility and the ENS were then assessed. Additionally, a triple-transgenic mouse strain (Nestin-creERT2 × Ngfr-DreERT2: DTRGFP) was utilized to track the effects of CRD on the differentiation of enteric neural precursor cells (ENPCs). RNA sequencing was also performed to elucidate the underlying mechanism.ResultsCompared to the control group, CRD significantly accelerated gastrointestinal motility, evidenced by faster intestinal peristalsis (P < 0.01), increased fecal output (P < 0.01), and elevated fecal water content (P < 0.05), as well as enhanced electrical field stimulation induced contractions (P < 0.05). These effects were associated with an increase in the number of glial cells and nitrergic neurons in the colonic myenteric plexus. Additionally, ENPCs in the colon showed a heightened differentiation into glial cells and nitrergic neurons. Notably, the NR1D1/nuclear factor-kappaB (NF-κB) axis played a crucial role in the CRD-mediated changes in ENPCs differentiation. Supplementation with NR1D1 agonist or NF-κB antagonist was able to restore gastrointestinal motility and normalize the ENS in jet-lagged mice.ConclusionsCRD regulates the differentiation of ENPCs through the NR1D1/NF-κB axis, resulting in dysfunction of the ENS and impaired gastrointestinal motility in mice.

Pharmacological characterization of alpha adrenoceptor-mediated motor responses in the rat colon.

Inhibitory neuromuscular transmission in the gastrointestinal tract is mediated by intrinsic nitrergic and purinergic neurons. Purines activate G protein-coupled receptor P2Y1 receptors, increasing intracellular Ca2+ that activates small conductance calcium-activated potassium (SKCa) channels. Little is known about the effect of adrenergic receptor activation on intestinal smooth muscle. In vascular tissue, stimulation of α-adrenoceptors causes smooth muscle contraction, while their effect on intestinal tissue is poorly understood. This study aimed to pharmacologically characterize the effect of α-adrenoceptor activation in the rat colon, which shares similar inhibitory pathways to the human colon. Muscle bath experiments were performed with the rat proximal, mid, and distal colon oriented both circularly and longitudinally. The α1-adrenoceptor agonist phenylephrine (PE) (10-8-10-5 M) evoked concentration-dependent relaxations of the intestinal smooth muscle from all regions and orientations. However, in the mid-circular colon at low PE concentrations, a contraction sensitive to 10-5 M phentolamine (non-selective α-adrenoceptor blocker), the neural blocker tetrodotoxin (TTX; 10-6 M), and atropine (10-6 M) was recorded. PE-induced relaxations were insensitive to TTX (10-6 M) and the nonselective β-adrenoceptor blocker propranolol (10-6 M). In contrast, PE-induced relaxations were blocked by phentolamine (10-5 M), prazosin (10-6 M) (α1-adrenoceptor blocker), and RS17053 (10-6 M) (α1A-blocker), but not by yohimbine (10-6 M) (α2-adrenoceptor blocker). Apamin (10-6 M), a SKCa channel blocker, abolished PE-induced relaxations. Contractile responses in the circular muscle of the mid colon could be attributed to α-adrenoceptors located on enteric cholinergic neurons. Stimulation of α1A-adrenoreceptors activates SKCa channels to cause smooth muscle relaxation, which constitutes a signaling pathway that shares similarities with P2Y1 receptors.

Intestinal Motility Dysfunction in Goto-Kakizaki Rats: Role of the Myenteric Plexus.

The morpho-quantitative analysis of myenteric plexus neurons in the small and large intestines of 120-day-old male GK rats was investigated. The diabetes was confirmed by high fasting blood glucose levels. The myenteric plexus was evaluated through wholemount immunofluorescence. The morpho-quantitative analyses included evaluating neuronal density (neurons per ganglion) of the total neuronal population, the cholinergic and nitrergic subpopulations, and enteric glial cells per ganglion. The cell body area of 100 neurons per segment per animal was measured. The total neurons and nitrergic subpopulation were unaltered in the GK rats' small and large intestines. The cholinergic subpopulation exhibited decreased density in the three segments of the small intestine and an increased number in the proximal colon of the GK rats. The number of enteric glial cells increased in the ileum of the GK rats, which could indicate enteric gliosis caused by the intestinal inflammatory state. The area of the cell body was increased in the total neuronal population of the jejunum and ileum of the GK rats. Frequency histograms of the cell body area distribution revealed the contribution of cholinergic neurons to larger areas in the jejunum and nitrergic neurons in the ileum. The constipation previously reported in GK rats might be explained by the decrease in the density of cholinergic neurons in the small intestine of this animal model.

Interindividual Variation in Gut Nitrergic Neuron Density Is Regulated By GDNF Levels and ETV1

The size and function of the enteric nervous system (ENS) can vary substantially between individuals. Because ENS function is involved in the etiology of a growing number of common human diseases, understanding mechanisms that regulate ENS variation is important. We analyzed RNAseq data from 41 normal adult human colon biopsies and single-cell RNA-seq data from human and mouse developing gut. To establish cause-consequence relationship we used alleles in mice that allow levels change of the candidate effector molecule in the comparable range to human samples. We used siRNA and primary neuronal cultures to define downstream molecular events and characterized gut functional changes in mice where molecular phenotypes paralleled findings in humans. We found that glial cell line-derived neurotrophic factor (GDNF) levels in the human colon vary about 5-fold and correlate strongly with nitrergic marker expression. In mice, we defined that GDNF levels are regulated via its 3' untranslated region (3' UTR) in the gastrointestinal tract and observed similar correlation between GDNF levels and nitrergic lineage development. We identified miR-9 and miR-133 as evolutionarily conserved candidates for negative regulation of GDNF expression in the gastrointestinal tract. Functionally, an increase in inhibitory nitrergic innervation results in an increase in gastrointestinal tract transit time, stool size, and water content accompanied with modestly reduced epithelial barrier function. Mechanistically, we found that GDNF levels regulate nitrergic lineage development via induction of transcription factor ETV1, corroborated by single-cell gene expression data in human and mouse developing enteric neurons. Our results reveal how normal variation in GDNF levels influence ENS size, composition, and gut function, suggesting a mechanism for well-known interindividual variation among those parameters.

The impact of long-course chemoradiotherapy on the myenteric plexus, neuromuscular functions and responses to prokinetic drugs in the human rectum.

The long-term effects of chemoradiotherapy on human rectum are poorly understood. The aims were to investigate changes in inflammatory status, myenteric neuron numbers/phenotype, neuromuscular functions and prokinetic drug efficacy. Macroscopically normal proximal-to-mid rectum was obtained from 21 patients undergoing surgery for bowel cancer, 98days (range: 63-350) after concurrent capecitabine and pelvic radiotherapy, and 19 patients without chemoradiotherapy. Inflammatory status was measured by H&E, CD45 staining and qPCR. Myenteric neurons were examined by immunohistochemistry. Neuromuscular functions and drug efficacy were studied using exogenous agents and electrical field stimulation (EFS) to activate intrinsic nerves. Inflammation was not detected. Numbers of myenteric ganglia/neurons were unchanged (11.7±2.4 vs. 10.3±2.2 neurons/mm myenteric plexus with/without chemoradiotherapy) as were the numbers of cholinergic/nitrergic neurons. EFS stimulated cholinergic and nitrergic neurons so the contractile response of the muscle was the sum of both but dominated by cholinergic (causing contraction) or less often, nitrergic activity (relaxation), followed, after termination of EFS, by neuronally mediated contraction. Inhibition of nitric oxide synthase (by L-NAME 300μM) more clearly defined EFS-evoked contractions. The 5-HT4 agonist prucalopride 10μM and the cholinesterase inhibitor donepezil 1µM, respectively increased and greatly increased the composite contractile response to EFS (measured as 'area-under-the curve') and the contractions isolated by L-NAME (respectively, by 22±14% and 334±87%; n=11/8). After chemoradiotherapy, nitrergic-mediated muscle relaxations occurred more often during EFS (in 29.8±6.1% preparations vs. 12.6±5.1% without chemoradiotherapy, n=21/18). With L-NAME, the ability of prucalopride to facilitate EFS-evoked contraction was lost and that of donepezil approximately halved (contractions increased by 132±36%; n=8). Several months after chemoradiotherapy, the rectum was not inflamed and myenteric neuron numbers/phenotype unchanged. However, nitrergic activity was increased relative to cholinergic activity, and prokinetic-like drug activity was lost or greatly reduced. Thus, chemoradiotherapy causes long-term changes in neuromuscular functions and markedly reduces the efficacy of drugs for treating constipation.

Effect of microwave radiation on adult neurogenesis and behavior of prenatally exposed rats

Postnatal neurogenesis appears to be highly sensitive to environmental factors, including microwave electromagnetic radiation (MWR). Here, we investigated the impact of MWR during intrauterine development on juvenile and adult neurogenesis in the rostral migratory stream (RMS) and the dentate gyrus of the hippocampus in the rat brain, as well as its effect on animal behavior. Female rats were exposed to MWR at a frequency of 2.45 GHz for 2 hours daily throughout pregnancy. The offspring of irradiated mothers survived to either juvenile age or adulthood. The brains of the rats were subjected to morphological analysis, assessing cell proliferation and death in both neurogenic regions. In the RMS, the differentiation of nitrergic neurons was also investigated. The effect of MWR on behavior was evaluated in rats surviving to adulthood. Prenatal MWR exposure caused significant changes in the number of proliferating and dying cells, depending on the age of the animals and the observed neurogenic region. In addition, MWR attenuated the maturation of nitrergic neurons in the RMS in both juvenile and adult rats. Morphological alterations in neurogenesis were accompanied by changes in animals’ behavior. Affected neurogenesis and changes in animal behavior suggest a high sensitivity of the developing brain to MWR.

Oxidative Stress and Erectile Dysfunction: Pathophysiology, Impacts, and Potential Treatments.

Erectile dysfunction (ED) is a prevalent condition affecting men's sexual health, with oxidative stress (OS) having recently been identified as a significant contributing causative factor. This narrative review aims to elucidate the role of OS in the pathophysiology of ED, focusing on impact, mechanisms, and potential therapeutic interventions. Key findings indicate that OS disrupts endothelial function and nitric oxide (NO) signaling, crucial for erectile function. Various sources of reactive oxygen species (ROS) and their detrimental effects on penile tissue are discussed, including aging, diabetes mellitus, hypertension, hyperlipidemia, smoking, obesity, alcohol consumption, psychological stress, hyperhomocysteinemia, chronic kidney disease, and sickle cell disease. Major sources of ROS, such as NADPH oxidase, xanthine oxidase, uncoupled endothelial NO synthase (eNOS), and mitochondrial electron transport, are identified. NO is scavenged by these ROS, leading to endothelial dysfunction characterized by reduced NO availability, impaired vasodilation, increased vascular tone, and inflammation. This ultimately results in ED due to decreased blood flow to penile tissue and the inability to achieve or maintain an erection. Furthermore, ROS impact the transmission of nitrergic neurotransmitters by causing the death of nitrergic neurons and reducing the signaling of neuronal NO synthase (nNOS), exacerbating ED. Therapeutic approaches targeting OS, including antioxidants and lifestyle modifications, show promise in ameliorating ED symptoms. The review underscores the need for further research to develop effective treatments, emphasizing the interplay between OS and vascular health in ED. Integrating pharmacological and non-pharmacological strategies could enhance clinical outcomes for ED patients, advocating for OS management in ED treatment protocols to improve patient quality of life.

A chemogenetic screen reveals that Trpv1-expressing neurons control regulatory T cells in the gut.

Neuroimmune cross-talk participates in intestinal tissue homeostasis and host defense. However, the matrix of interactions between arrays of molecularly defined neuron subsets and of immunocyte lineages remains unclear. We used a chemogenetic approach to activate eight distinct neuronal subsets, assessing effects by deep immunophenotyping, microbiome profiling, and immunocyte transcriptomics in intestinal organs. Distinct immune perturbations followed neuronal activation: Nitrergic neurons regulated T helper 17 (TH17)-like cells, and cholinergic neurons regulated neutrophils. Nociceptor neurons, expressing Trpv1, elicited the broadest immunomodulation, inducing changes in innate lymphocytes, macrophages, and RORγ+ regulatory T (Treg) cells. Neuroanatomical, genetic, and pharmacological follow-up showed that Trpv1+ neurons in dorsal root ganglia decreased Treg cell numbers via the neuropeptide calcitonin gene-related peptide (CGRP). Given the role of these neurons in nociception, these data potentially link pain signaling with gut Treg cell function.

Nitric oxide mediated kisspeptin regulation of steroidogenesis and gametogenesis in the catfish, Clarias batrachus.

Nitric oxide (NO) is a gaseous molecule that regulates various reproductive functions. It is a well-recognized regulator of GnRH-FSH/LH-sex steroid secretion in vertebrates including fish. Kisspeptin is a recently discovered neuropeptide which also regulates GnRH secretion. Nitrergic and kisspeptin neurons are reported in close physical contact in the mammalian brain suggesting their interactive role in the release of GnRH. The existence of kisspeptin and NOS is also demonstrated in vertebrate gonads, but information on their reciprocal relation in gonads, if any, is obscure. Therefore, attempts were made to evaluate the functional reciprocal relation between nitric oxide and kisspeptin in the catfish gonads, if any, by administering the nitric oxide synthase (NOS) inhibitor, L-NAME {N(G)-nitro-L-arginine methyl ester}, which reduces NO production, and kisspeptin agonist (KP-10) and assessing their impacts on the expressions of kisspeptin1, different NOS isoforms, NO and steroid production in the gonadal tissue. The results revealed that L-NAME suppressed the expression of kiss1 in gonads of the catfish establishing the role of NO in kisspeptin expression. However, KP-10 increased the expression of all the isoforms of NOSs (iNOS, eNOS,nNOS) and concurrently NO and steroids in the ovary and testis. In vitro studies also indicate that kisspeptin stimulates the production of NO and estradiol and testosterone levels in the gonadal explants and medium. Thus, in vivo results clearly suggest a reciprocal interaction between kisspeptin and NO to regulate the gonadal activity of the catfish. The in vitro findings further substantiate our contention regarding the interactive role of kisspeptin and NO in gonadal steroidogenesis.

Inhibition of calcium-sensing receptor by its antagonist promotes gastrointestinal motility in a Parkinson’s disease mouse model

BackgroundThe Calcium-sensing receptor (CaSR) participates in the regulation of gastrointestinal (GI) motility under normal conditions and might be involved in the regulation of GI dysmotility in patients with Parkinson's disease (PD). MethodsCaSR antagonist-NPS-2143 was applied in in vivo and ex vivo experiments to study the effect and underlying mechanisms of CaSR inhibition on GI dysmotility in the MPTP-induced PD mouse model. FindingsOral intake of NPS-2143 promoted GI motility in PD mice as shown by the increased gastric emptying rate and shortened whole gut transit time together with improved weight and water content in the feces of PD mice, and the lack of influence on normal mice. Meanwhile, the number of cholinergic neurons, the proportion of serotonergic neurons, as well as the levels of acetylcholine and serotonin increased, but the numbers of nitrergic and tyrosine hydroxylase immunoreactive neurons, and the levels of nitric oxide synthase and dopamine decreased in the myenteric plexus in the gastric antrum and colon of PD mice in response to NPS-2143 treatment. Furthermore, the numbers of c-fos positive neurons in the nucleus tractus solitarius (NTS) and cholinergic neurons in the dorsal motor nucleus of the vagus (DMV) increased in NPS-2143 treated PD mice, suggesting the involvement of both the enteric (ENS) and central (CNS) nervous systems. However, ex vivo results showed that NPS-2143 directly inhibited the contractility of antral and colonic strips in PD mice via a non-ENS mediated mechanism. Further studies revealed that NPS-2143 directly inhibited the voltage gated Ca2+ channels, which might, at least in part, explain its direct inhibitory effects on the GI muscle strips. InterpretationCaSR inhibition by its antagonist ameliorated GI dysmotility in PD mice via coordinated neuronal regulation by both ENS and CNS in vivo, although the direct effects of CaSR inhibition on GI muscle strips were suppressive.

Effect of leptin on nitrergic neurons in the lateral hypothalamic area and the supraoptic nucleus of rats

ABSTRACT The adipocyte-derived hormone, leptin, plays a key role in the maintenance of energy homeostasis. Leptin binds to the long form of its receptor, which is predominantly expressed in various hypothalamic regions, including the lateral hypothalamic area (LH) and supraoptic nucleus (SO). Several studies have suggested that leptin directly activates neuronal nitric oxide synthase, leading to increased nitric oxide production. We used histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) as a marker for nitric oxide synthase activity and assessed the effect of leptin on nitrergic neurons in the LH and SO of rats. We found that intraperitoneal administration of leptin led to a significant increase in the number of NADPH-d-positive neurons in the LH and SO. In addition, the intensity (optical density) of NADPH-d staining in LH and SO neurons was significantly elevated in rats that received leptin compared with saline-treated rats. These findings suggest that nitrergic neurons in the LH and SO may be implicated in mediating the central effects of leptin.

Characterization of Sonic Hedgehog transcripts in the adult mouse brain: co-expression with neuronal and oligodendroglial markers

In the adult mammalian brain, astrocytes are proposed to be the major Sonic Hedgehog (Shh)-responsive cells. However, the sources of the Shh molecule mediating activation of the pathway are still poorly characterized. The present work investigates the distribution and phenotype of cells expressing Shh mRNA in the adult mouse brain. Using single-molecule fluorescent in situ hybridization (smfISH), we report much broader expression of Shh transcripts in almost all brain regions than originally reported. We identify Shh mRNA in HuC/D+ neuronal populations, including GABAergic (glutamic acid decarboxylase 67, Gad67), cholinergic (choline acetyltransferase, ChAT), dopaminergic (tyrosine hydroxylase, TH), nitrergic (neuronal nitric oxide synthase, nNOS), and in a small population of oligodendroglial cells expressing Sox10 and Olig2 mRNA transcription factors. Further analysis of Shh mRNA in cerebral cortical and hypothalamic neurons suggests that Shh is also expressed by glutamatergic neurons. Interestingly, we did not observe substantial Desert Hedgehog and Indian Hedgehog mRNA signals, nor Shh signals in S100β+ astrocytes and Iba1+ microglial cells. Collectively, the present work provides the most robust central map of Shh-expressing cells to date and underscores the importance of nitrergic neurons in regulating Shh availability to brain cells. Thus, our study provides a framework for future experiments aimed at better understanding of the functions of Shh signaling in the brain in normal and pathological states, and the characterization of novel regulatory mechanisms of the signaling pathway.

The anterior cingulate cortex and its interface with the dorsal periaqueductal grey regulating nitric oxide-mediated panic-like behaviour and defensive antinociception

The anterior cingulate cortex (ACC) Cg1 (24b) area modulates glutamate-mediated unconditioned fear and antinociception organised by hypothalamus. However, it remains unknown whether 24b area also modulates these latter defensive responses through connections with the dorsal periaqueductal grey matter (dPAG), a midbrain structure implicated in the genesis of innate fear-induced defence. The aim of this work is to examine the correlation between the behavioural effects of intra-ACC microinjections of vehicle, NMDA (1 nmol) or lidocaine (2%) with Fos protein expression and nitrergic activity in the dPAG of male C57BL/6 mice that were threatened by snakes. In addition, the 24b area-dPAG pathways were also characterised by neural tract tracing procedures. Finally, the effect of dPAG pretreatment with the neuronal nitric oxide synthase inhibitor N(omega)-propyl-l-arginine (NPLA; 0.2, 0.4 or 0.8 nmol) 10 min before 24b area treatment with NMDA on behavioural and nociceptive responses of threatened mice was studied. The activation of 24b area N-methyl-d-aspartic acid receptors facilitated escape and freezing rather than risk assessment, and enhanced Fos expression and nitrite levels in dPAG, while lidocaine decreased escape and risk assessment as well as Fos and nitrergic activity in dPAG. In addition, dPAG pretreatment with NPLA suppressed intra-24b NMDA-facilitated panicogenic effects while increased nociception. Infusions of an antegrade neurotracer into 24b area showed axonal fibres surrounding both dorsomedial and dorsolateral PAG perikarya. Neurons were identified in 24b area after deposits of a retrograde neurotracer into dPAG. Our findings suggest that the ACC/24b area modulates innate defensive responses through the recruitment of dPAG nitrergic neurons.

Bisphenol A Effects on Neurons’ Neurochemical Character in the Urinary Bladder Intramural Ganglia of Domestic Pigs

Bisphenol A (BPA), a substance globally used to produce plastics, is part of many everyday items, including bottles, food containers, electronic elements, and others. It may penetrate the environment and living organisms, negatively affecting, among others, the nervous, immune, endocrine, and cardiovascular systems. Knowledge of the impact of BPA on the urinary bladder is extremely scarce. This study investigated the influence of two doses of BPA (0.05 mg/kg body weight (b.w.)/day and 0.5 mg/kg b.w./day) given orally for 28 days on the neurons situated in the ganglia located in the urinary bladder trigone using the typical double immunofluorescence method. In the study, an increase in the percentage of neurons containing substance P (SP), galanin (GAL), a neuronal isoform of nitric oxide synthase (nNOS—used as the marker of nitrergic neurons), and/or cocaine- and amphetamine-regulated transcript (CART) peptide was noted after BPA administration. The severity of these changes depended on the dose of BPA and the type of neuronal factors studied. The most visible changes were noted in the cases of SP- and/or GAL-positive neurons after administering a higher dose of BPA. The results have shown that oral exposure to BPA, lasting even for a short time, affects the intramural neurons in the urinary bladder wall, and changes in the neurochemical characterisation of these neurons may be the first signs of BPA-induced pathological processes in this organ.

L-glutathione 1% promotes neuroprotection of nitrergic neurons and reduces the oxidative stress in the jejunum of rats with Walker-256-bearing tumor.

Our main goals were to investigate the effects of L-glutathione (1%) treatment in Walker-256 tumor-bearing rats by analyzing immunoreactive neurons (IR), responsive to the nNOS enzyme and 3-Nitrotyrosine, in their jejunum myenteric plexus. Moreover, the oxidative state and inflammatory process in these animals were investigated. Four experimental groups were utilized: control (C), control treated with L-glutathione (CGT), Walker-256 tumor-bearing rats (TW), and Walker-256 tumor-bearing rats treated with L-glutathione (TWGT). After 14 days of tumor inoculation, the jejunum was collected for immunohistochemical techniques and assessment of oxidative status. Plasma was collected to evaluate oxidative status and measure cytokines. The TW group exhibited a decrease of reduced glutathione in their jejunum, which was prevented in the L-glutathione treated TWGT group. TW animals presented pronounced oxidative stress by increasing levels of lipoperoxidation in their jejunum and malondialdehyde in their plasma; however, the L-glutathione treatment in TWGT group was not able to avoid it. The total antioxidant capacity was altered in groups TW and TWGT, yet the last one had a better index in their plasma. The IL-10, and TNF-α levels increased in TWGT animals. The nNOS-IR neuron density decreased in the jejunum myenteric plexus of the TW group, which was avoided in the TWGT group. The nNOS +3-Nitrotyrosine neurons quantification did not show significative alterations. The treatment with L-glutathione (1%) imposed an important defense to some parameters of oxidative stress induced by TW-256, leading to neuroprotection to the loss in the nNOS-IR neuron density.

Differential G Protein-Coupled Estrogen Receptor-1 Regulation of Counter-Regulatory Transmitter Marker and 5′-AMP-Activated Protein Kinase Expression in Ventrolateral versus Dorsomedial Ventromedial Hypothalamic Nucleus

Introduction: The ventromedial hypothalamic nucleus (VMN) is an estrogen receptor (ER)-rich structure that regulates glucostasis. The role of nuclear but not membrane G protein-coupled ER-1 (GPER) in that function has been studied. Methods: Gene silencing and laser-catapult microdissection/immunoblot tools were used to examine whether GPER regulates transmitter and energy sensor function in dorsomedial (VMNdm) and/or ventrolateral (VMNvl) VMN counter-regulatory nitrergic and γ-Aminobutyric acid (GABA) neurons. Results: Intra-VMN GPER siRNA administration to euglycemic animals did not affect VMNdm or -vl nitrergic neuron nitric oxide synthase (nNOS), but upregulated (VMNdm) or lacked influence on (VMNvl) GABA nerve cell glutamate decarboxylase_65/67 (GAD) protein. Insulin-induced hypoglycemia (IIH) caused GPER knockdown-reversible augmentation of nNOS, 5′-AMP-activated protein kinase (AMPK), and phospho-AMPK proteins in nitrergic neurons in both divisions. IIH had dissimilar effects on VMNvl (unchanged) versus VMNdm (increased) GABAergic neuron GAD levels, yet GPER knockdown affected these profiles. GPER siRNA prevented hypoglycemic upregulation of VMNvl and -dm GABA neuron AMPK without altering pAMPK expression. Conclusions: Outcomes infer that GPER exerts differential control of VMNdm versus -vl GABA transmission during glucostasis and is required for hypoglycemic upregulated nitrergic (VMNdm and -vl) and GABA (VMNdm) signaling. Glycogen metabolism is reported to regulate VMN nNOS and GAD proteins. Data show that GPER limits VMNvl glycogen phosphorylase (GP) protein expression and glycogen buildup during euglycemia but mediates hypoglycemic augmentation of VMNvl GP protein and glycogen content; VMNdm glycogen mass is refractory to GPER control. GPER regulation of VMNvl glycogen metabolism infers that this receptor may govern local counter-regulatory transmission in part by astrocyte metabolic coupling.

Transplanted ENSCs form functional connections with intestinal smooth muscle and restore colonic motility in nNOS-deficient mice

BackgroundEnteric neuropathies, which result from abnormalities of the enteric nervous system, are associated with significant morbidity and high health-care costs, but current treatments are unsatisfactory. Cell-based therapy offers an innovative approach to replace the absent or abnormal enteric neurons and thereby restore gut function.MethodsEnteric neuronal stem cells (ENSCs) were isolated from the gastrointestinal tract of Wnt1-Cre;R26tdTomato mice and generated neurospheres (NS). NS transplants were performed via injection into the mid-colon mesenchyme of nNOS−/− mouse, a model of colonic dysmotility, using either 1 (n = 12) or 3 (n = 12) injections (30 NS per injection) targeted longitudinally 1–2 mm apart. Functional outcomes were assessed up to 6 weeks later using electromyography (EMG), electrical field stimulation (EFS), optogenetics, and by measuring colorectal motility.ResultsTransplanted ENSCs formed nitrergic neurons in the nNOS−/− recipient colon. Multiple injections of ENSCs resulted in a significantly larger area of coverage compared to single injection alone and were associated with a marked improvement in colonic function, demonstrated by (1) increased colonic muscle activity by EMG recording, (2) faster rectal bead expulsion, and (3) increased fecal pellet output in vivo. Organ bath studies revealed direct neuromuscular communication by optogenetic stimulation of channelrhodopsin-expressing ENSCs and restoration of smooth muscle relaxation in response to EFS.ConclusionsThese results demonstrate that transplanted ENSCs can form effective neuromuscular connections and improve colonic motor function in a model of colonic dysmotility, and additionally reveal that multiple sites of cell delivery led to an improved response, paving the way for optimized clinical trial design.

Hypoxia-Inducible Factor 1α Stabilization Restores Epigenetic Control of Nitric Oxide Synthase 1 Expression and Reverses Gastroparesis in Female Diabetic Mice

Although depletion of neuronal nitric oxide synthase (NOS1)-expressing neurons contributes to gastroparesis, stimulating nitrergic signaling is not an effective therapy. We investigated whether hypoxia-inducible factor 1α (HIF1A), which is activated by high O2 consumption in central neurons, is a Nos1 transcription factor in enteric neurons and whether stabilizing HIF1A reverses gastroparesis. Mice with streptozotocin-induced diabetes, human and mouse tissues, NOS1+ mouse neuroblastoma cells, and isolated nitrergic neurons were studied. Gastric emptying of solids and volumes were determined by breath test and single-photon emission computed tomography, respectively. Gene expression was analyzed by RNA-sequencing, microarrays, immunoblotting, and immunofluorescence. Epigenetic assays included chromatin immunoprecipitation sequencing (13 targets), chromosome conformation capture sequencing, and reporter assays. Mechanistic studies used Cre-mediated recombination, RNA interference, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated epigenome editing. HIF1A signaling from physiological intracellular hypoxia was active in mouse and human NOS1+ myenteric neurons but reduced in diabetes. Deleting Hif1a in Nos1-expressing neurons reduced NOS1 protein by 50% to 92% and delayed gastric emptying of solids in female but not male mice. Stabilizing HIF1A with roxadustat (FG-4592), which is approved for human use, restored NOS1 and reversed gastroparesis in female diabetic mice. In nitrergic neurons, HIF1A up-regulated Nos1 transcription by binding and activating proximal and distal cis-regulatory elements, including newly discovered super-enhancers, facilitating RNA polymerase loading and pause-release, and by recruiting cohesin to loop anchors to alter chromosome topology. Pharmacologic HIF1A stabilization is a novel, translatable approach to restoring nitrergic signaling and treating diabetic gastroparesis. The newly recognized effects of HIF1A on chromosome topology may provide insights into physioxia- and ischemia-related organ function.

Neural targets of the enteric dopaminergic system in regulating motility of rat proximal colon.

In isolated segments of the rat proximal colon, the dopamine reuptake inhibitor GBR 12909 (GBR) causes a dilatation, while the D1-like receptor antagonist SCH 23390 (SCH) induces a tonic constriction, suggesting that neurally released dopamine tonically stimulates enteric inhibitory efferent neurons. Here, the targets of the enteric dopaminergic neurons were investigated. Cannulated segments of rat proximal colon were bathed in physiological salt solution and luminally perfused with 0.9% saline, while all drugs were applied to the bath. Spatio-temporal maps of colonic motility were constructed from video recordings of peristaltic contractions, and the maximum diameter was measured as an index of colonic contractility. GBR (1μM)-induced dilatations of colonic segments were prevented by SCH (5μM), L-nitro arginine (L-NA; 100μM), a nitric oxide synthase inhibitor, or tetrodotoxin (0.6μM). In contrast, constrictions induced by a higher concentration of SCH (20μM) were unaffected by either L-NA or tetrodotoxin. The vasoactive intestinal peptide (VIP) receptor antagonist VIP10-28 (3μM) or P2Y1 receptor antagonist MRS 2500 (1μM) had no effect on either the GBR-induced dilatation or the SCH-induced constriction. In colonic segments that had been pretreated with 6-hydroxydopamine (100μM, 3h) to deplete enteric dopamine, GBR failed to increase the colonic diameter, while SCH was still capable of constricting colonic segments. Enteric dopaminergic neurons appear to project to nitrergic neurons to dilate the proximal colon by activating neuronal D1-like receptors. In addition, constitutively activated D1-like receptors expressed in cells yet to be determined may provide a tonic inhibition on colonic constrictions.

Sex-dependent endozepinergic regulation of ventromedial hypothalamic nucleus glucose counter-regulatory neuron aromatase protein expression in the adult rat

The hypothalamic brain cell types that produce estradiol from testosterone remain unclear. Aromatase inhibition affects ventromedial hypothalamic nucleus (VMN) glucose-stimulatory nitric oxide (NO) and glucose-inhibitory γ-aminobutyric acid (GABA) transmission during insulin (INS)-induced hypoglycemia (IIH). Pure GABA and NO nerve cell samples acquired by laser-catapult-microdissection from consecutive rostro-caudal segments of the VMN were analyzed by Western blot to investigate whether regional subpopulations of each cell type contain machinery for neuro-estradiol synthesis. Astrocyte endozepinergic signaling governs brain steroidogenesis. Pharmacological tools were used here to determine if the glio-peptide octadecaneuropeptide (ODN) controls aromatase expression in GABA and NO neurons during eu- and/or hypoglycemia. Intracerebroventricular administration of the ODN G-protein coupled-receptor antagonist cyclo(1−8)[DLeu5]OP (LV-1075) decreased (male) or enhanced (female) VMN GABAergic neuron aromatase expression, but increased or reduced this profile in nitrergic neurons in a region-specific manner in each sex. IIH suppressed aromatase levels in GABA neurons located in the middle segment of the male VMN or distributed throughout this nucleus in the female. This inhibitory response was altered by the ODN isoactive surrogate octapeptide (OP) in female, but was refractory to OP in male. NO neuron aromatase protein in hypoglycemic male (middle and caudal VMN) and female (rostral and caudal VMN) rats, but was normalized in OP- plus INS-treated rats of both sexes. Results provide novel evidence that VMN glucose-regulatory neurons may produce neuro-estradiol, and that the astrocyte endozepine transmitter ODN may impose sex-specific control of baseline and/or hypoglycemic patterns of aromatase expression in distinct subsets of nitrergic and GABAergic neurons in this neural structure.