Nitrergic Neurons Research Articles

Quantitative Spatial Analysis of Neuroligin-3 mRNA Expression in the Enteric Nervous System Reveals a Potential Role in Neuronal-Glial Synapses and Reduced Expression in Nlgn3R451C Mice.

Mutations in the Neuroligin-3 (Nlgn3) gene are implicated in autism spectrum disorder (ASD) and gastrointestinal (GI) dysfunction, but cellular Nlgn3 expression in the enteric nervous system remains to be characterised. We combined RNAScope in situ hybridization and immunofluorescence to measure Nlgn3 mRNA expression in cholinergic and VIP-expressing submucosal neurons, nitrergic and calretinin-containing myenteric neurons and glial cells in both WT and Nlgn3R451C mutant mice. We measured Nlgn3 mRNA neuronal and glial expression via quantitative three-dimensional image analysis. To validate dual RNAScope/immunofluorescence data, we interrogated available single-cell RNA sequencing (scRNASeq) data to assess for Nlgn3, Nlgn1, Nlgn2 and their binding partners, Nrxn1-3, MGDA1 and MGDA2, in enteric neural subsets. Most submucosal and myenteric neurons expressed Nlgn3 mRNA. In contrast to other Nlgns and binding partners, Nlgn3 was strongly expressed in enteric glia, suggesting a role for neuroligin-3 in mediating enteric neuron-glia interactions. The autism-associated R451C mutation reduces Nlgn3 mRNA expression in cholinergic but not in VIPergic submucosal neurons. In the myenteric plexus, Nlgn3 mRNA levels are reduced in calretinin, nNOS-labelled neurons and S100 β -labelled glia. We provide a comprehensive cellular profile for neuroligin-3 expression in ileal neuronal subpopulations of mice expressing the R451C autism-associated mutation in Nlgn3, which may contribute to the understanding of the pathophysiology of GI dysfunction in ASD.

Synaptoporin and parathyroid hormone 2 as markers of multimodal inputs to the auditory brainstem

The distribution of the synaptic vesicle protein synaptoporin was investigated by immunofluorescence in the central auditory system of the mouse brainstem. Synaptoporin immunostaining displayed region-specific differences. High and moderate accumulations of were seen in the superficial layer of the dorsal cochlear nucleus, dorsal and external regions of the inferior colliculus, the medial and dorsal divisions of the medial geniculate body and in periolivary regions of the superior olivary complex (SOC). Low or absent labeling was observed in the more central parts of these structures such as the principal nuclei of the SOC. It was conspicuous that dense synaptoporin immunoreactivity was detected predominantly in areas, which are known to be synaptic fields of multimodal, extra-auditory inputs. Target neurons of synaptoporin-positive synapses in the SOC were then identified by double-labelling immunofluorescence microscopy. We thereby detected synaptoporin puncta perisomatically at nitrergic, glutamatergic and serotonergic neurons but none next to neurons immunoreactive for choline-acetyltransferase and calcitonin-gene related peptide. These results leave open whether functionally distinct neuronal groups are accessed in the SOC by synaptoporin-containing neurons. The last part of our study sought to find out whether synaptoporin-positive neurons originate in the medial paralemniscal nucleus (MPL), which is characterized by expression of the peptide parathyroid hormone 2 (PTH2). Anterograde neuronal tracing upon injection into the MPL in combination with synaptoporin- and PTH2-immunodetection showed that (1) the MPL projects to the periolivary SOC using PTH2 as transmitter, (2) synaptoporin-positive neurons do not originate in the MPL, and (3) the close juxtaposition of synaptoporin-staining with either the anterograde tracer or PTH2 reflect concerted action of the different inputs to the SOC.

Effects of prenatal hypoxia-ischemia on male rat periaqueductal gray matter: Hyperalgesia, astrogliosis and nitrergic system impairment.

Prenatal hypoxic-ischemic insult (HI) may lead to a variety of neurological consequences that may persist throughout adulthood. In the most severe cases, HI is known to increase pain sensitivity which profoundly impacts quality of life. Periaqueductal gray matter (PAG) is a relevant region of the descending pain pathway and its function may be modulated by a complex network that includes nitrergic neurons and glial response, among other factors. Astrocytes, central players in pain modulation, are known to respond to HI by inducing hyperplasia, hypertrophy and increasing the number of their processes and the staining of glial fibrillary acidic protein (GFAP). In this work we investigated the effects of prenatal HI on touch and pain sensitivity, besides the distribution of the neuronal isoform of Nitric Oxide Synthase (nNOS) and GFAP in the PAG of young and adult male rats. At 18 days of gestation, rats had their uterine arteries clamped for 45min (HI group). SHAM-operated animals were also generated (SHAM group). At post-natal day 30 (P30) or 90 (P90), the offspring was submitted to the behavioral tests of Von Frey and formalin or histological processing to perform immunohistochemistry for nNOS and GFAP. Although there was no significant difference between the groups concerning touch sensitivity, we observed an increase in pain sensitivity in HI P30 and HI P90. The number of nNOS+cells was reduced in HI adult animals in dlPAG and vlPAG. GFAP immunostaining was increased in HI P90 in dlPAG and dmPAG. Our results demonstrated for the first time an increase in pain sensitivity as a consequence of prenatal HI in an animal model. It reinforces the relevance of this model to mimic the effects of prenatal HI, as hyperalgesia.

Aryl hydrocarbon receptor activation affects nitrergic neuronal survival and delays intestinal motility in mice.

Despite progress describing the effects of persistent organic pollutants (POPs) on the central nervous system, the effect of POPs on enteric nervous system (ENS) function remains underexplored. We studied the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a POP, and a potent aryl hydrocarbon receptor (AHR) ligand, on the ENS and intestinal motility in mice. C57Bl/6J mice treated with TCDD (2.4 µg/kg body weight) for 8 weeks (once per week) exhibited significant delay in intestinal motility as shown by reduced stool frequency, prolonged intestinal transit time, and a persistence of dye in the jejunum compared to control mice with maximal dye retention in the ileum. TCDD significantly increased Cyp1a1 expression, an AHR target gene, and reduced the total number of neurons and affected nitrergic neurons in cells isolated from WT mice, but not Ahr-/- mice. In immortalized fetal enteric neuronal cells, TCDD-induced nuclear translocation of AHR as well as increased Cyp1a1 expression. AHR activation did not affect neuronal proliferation. However, AHR activation resulted in enteric neuronal toxicity, specifically, nitrergic neurons. Our results demonstrate that TCDD adversely affects nitrergic neurons and thereby contributes to delayed intestinal motility. These findings suggest that AHR signaling in the ENS may play a role in modulating TCDD-induced gastrointestinal pathophysiology.

Enteric neuroanatomy and smooth muscle activity in the western diamondback rattlesnake (Crotalus atrox)

BackgroundGastrointestinal (GI) functions are controlled by the enteric nervous system (ENS) in vertebrates, but data on snakes are scarce, as most studies were done in mammals. However, the feeding of many snakes, including Crotalus atrox, is in strong contrast with mammals, as it consumes an immense, intact prey that is forwarded, stored, and processed by the GI tract. We performed immunohistochemistry in different regions of the GI tract to assess the neuronal density and to quantify cholinergic, nitrergic, and VIPergic enteric neurons. We recorded motility patterns and determined the role of different neurotransmitters in the control of motility. Neuroimaging experiments complemented motility findings.ResultsA well-developed ganglionated myenteric plexus (MP) was found in the oesophagus, stomach, and small and large intestines. In the submucous plexus (SMP) most neurons were scattered individually without forming ganglia. The lowest number of neurons was present in the SMP of the proximal colon, while the highest was in the MP of the oesophagus. The total number of neurons in the ENS was estimated to be approx. 1.5 million. In all regions of the SMP except for the oesophagus more nitric oxide synthase+ than choline-acetyltransferase (ChAT)+ neurons were counted, while in the MP ChAT+ neurons dominated. In the SMP most nerve cells were VIP+, contrary to the MP, where numerous VIP+ nerve fibers but hardly any VIP+ neuronal cell bodies were seen. Regular contractions were observed in muscle strips from the distal stomach, but not from the proximal stomach or the colon. We identified acetylcholine as the main excitatory and nitric oxide as the main inhibitory neurotransmitter. Furthermore, 5-HT and dopamine stimulated, while VIP and the ß-receptor-agonist isoproterenol inhibited motility. ATP had only a minor inhibitory effect. Nerve-evoked contractile responses were sodium-dependent, insensitive to tetrodotoxin (TTX), but sensitive to lidocaine, supported by neuroimaging experiments.ConclusionsThe structure of the ENS, and patterns of gastric and colonic contractile activity of Crotalus atrox are strikingly different from mammalian models. However, the main excitatory and inhibitory pathways appear to be conserved. Future studies have to explore how the observed differences are an adaptation to the particular feeding strategy of the snake.

Effects of Ventromedial Hypothalamic Nucleus (VMN) Aromatase Gene Knockdown on VMN Glycogen Metabolism and Glucoregulatory Neurotransmission

The enzyme aromatase is expressed at high levels in the ventromedial hypothalamic nucleus (VMN), a principal component of the brain gluco-regulatory network. Current research utilized selective gene knockdown tools to investigate the premise that VMN neuroestradiol controls glucostasis. Intra-VMN aromatase siRNA administration decreased baseline aromatase protein expression and tissue estradiol concentrations and either reversed or attenuated the hypoglycemic regulation of these profiles in a VMN segment-specific manner. Aromatase gene repression down-regulated protein biomarkers for gluco-stimulatory (nitric oxide; NO) and -inhibitory (gamma-aminobutyric acid; GABA) neurochemical transmitters. Insulin-induced hypoglycemia (IIH) up- or down-regulated neuronal nitric oxide synthase (nNOS) and glutamate decarboxylase65/67 (GAD), respectively, throughout the VMN. Interestingly, IIH caused divergent changes in tissue aromatase and estradiol levels in rostral (diminished) versus middle and caudal (elevated) VMN. Aromatase knockdown prevented hypoglycemic nNOS augmentation in VMN middle and caudal segments, but abolished the GAD inhibitory response to IIH throughout this nucleus. VMN nitrergic and GABAergic neurons monitor stimulus-specific glycogen breakdown. Here, glycogen synthase (GS) and phosphorylase brain- (GPbb; AMP-sensitive) and muscle- (GPmm; noradrenergic -responsive) type isoform responses to aromatase siRNA were evaluated. Aromatase repression reduced GPbb and GPmm content in euglycemic controls and prevented hypoglycemic regulation of GPmm but not GPbb expression while reversing glycogen accumulation. Aromatase siRNA elevated baseline glucagon and corticosterone secretion and abolished hypoglycemic hyperglucagonemia and hypercorticosteronemia. Outcomes document the involvement of VMN neuroestradiol signaling in brain control of glucose homeostasis. Aromatase regulation of VMN gluco-regulatory signaling of hypoglycemia-associated energy imbalance may entail, in part, control of GP variant-mediated glycogen disassembly.

Sex-dimorphic hindbrain lactate regulation of ventromedial hypothalamic nucleus glucoregulatory neuron 5’-AMP-activated protein kinase activity and transmitter marker protein expression

BackgroundThe oxidizable glycolytic end-product L-lactate is a gauge of nerve cell metabolic fuel stability that metabolic-sensory hindbrain A2 noradrenergic neurons impart to the brain glucose-regulatory network. Current research investigated the premise that hindbrain lactate deficiency exerts sex-specific control of energy sensor and transmitter marker protein responses to hypoglycemia in ventromedial hypothalamic nucleus (VMN) glucose-regulatory nitrergic and γ-aminobutyric acid (GABA) neurons. MethodsNitric oxide synthase (nNOS)- or glutamate decarboxylase65/67 (GAD)-immunoreactive neurons were laser-catapult-microdissected from male and female rat VMN after subcutaneous insulin injection and caudal fourth ventricular L-lactate or vehicle infusion for Western blot protein analysis. ResultsHindbrain lactate repletion reversed hypoglycemia-associated augmentation (males) or inhibition (females) of nitrergic neuron nNOS expression, and prevented up-regulation of phosphorylated AMPK 5’-AMP-activated protein kinase (pAMPK) expression in those neurons. Hypoglycemic suppression of GABAergic neuron GAD protein was averted by exogenous lactate over the rostro-caudal length of the male VMN and in the middle region of the female VMN. Lactate normalized GABA neuron pAMPK profiles in hypoglycemic male (caudal VMN) and female (all VMN segments) rats. Hypoglycemic patterns of norepinephrine (NE) signaling were lactate-dependent throughout the male VMN, but confined to the rostral and middle female VMN. ConclusionsResults document, in each sex, regional VMN glucose-regulatory transmitter responses to hypoglycemia that are controlled by hindbrain lactate status. Hindbrain metabolic-sensory regulation of hypoglycemia-correlated nitric oxide or GABA release may entail AMPK-dependent mechanisms in specific VMN rostro-caudal segments in each sex. Additional effort is required to examine the role of hindbrain lactoprivic-sensitive VMN neurotransmitters in lactate-mediated attenuation of hypoglycemic hyperglucagonemia and hypercorticosteronemia in male and female rats.

In vitro high glucose increases apoptosis, decreases nerve outgrowth, and promotes survival of sympathetic pelvic neurons.

Diabetes mellitus (DM) is a common cause of erectile dysfunction (ED), yet the molecular basis of DM neurogenic ED remains unknown. In this study we examined the impact of high glucose on survival and growth of primary cultured pelvic neurons in a rat model and assessed whether coculturing with healthy Schwann cells (SCs) can rescue pelvic neuron growth in patients with DM. Major pelvic ganglia (MPGs) from adult male Sprague Dawley rats (n = 8) were dissociated and plated on coverslips. Neurons were exposed to high glucose (45mM) for 24 or 48hours and compared to time-matched controls (25mM). Neurons were stained for neuron-specific beta-tubulin, neuronal nitric oxide synthase, vesicular acetylcholine transferase, tyrosine hydroxylase, and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling) assay. Schwann cells were dissociated from MPGs of healthy male Sprague Dawley rats (n = 4) and grown to confluence. Additional Sprague Dawley rats were made diabetic with streptozotocin (50mg/kg, n = 4), and 5weeks later MPGs were collected from these rats, dissociated, and cocultured on healthy SCs. Neurons and SCs were stained with beta-tubulin and S100. Length, branching, and survival of nitrergic, parasympathetic, and sympathetic neurons was assessed in neurons exposed to normal or high glucose concentrations, and neuron length was measured in neuron-SC coculture. The total number of neurons and the length and number of branches were significantly decreased after 24 and 48hours of high glucose (P < .05). The percentage of nitrergic neurons decreased 10% after 24 hours and 50% after 48 hours of high glucose (P < .05). After 24 hours of high glucose, cholinergic-positive neurons were unchanged; however, these neurons decreased 30% after 48 hours (P < .05). The proportion of sympathetic neurons increased 25% after 48 hours of high glucose (P < .05). At both timepoints, there was a 2-fold increase in the total apoptotic neurons with high glucose (P < .05). Neurite outgrowth recovered to control lengths after coculture of diabetic neurons with healthy SCs (P < .05). Glucose can be used as a tool to investigate the direct effects of DM on neuritogenesis. Our data suggest that an effective treatment for DM ED protects and repairs the penile neuronal supply. Exposing MPG neurons to high glucose offers a quick and, inexpensive proxy for DM-related conditions. A limitation of our study is that our model reflects type 1 DM, whereas clinically, most diabetic ED patients have type 2 DM. Culturing pelvic neurons in high glucose can be used as a tool to elucidate how to protect proerectile neurons from cell death and may lead to new therapeutic strategies for diabetic men suffering from ED.

Vagal preganglionic axons arborize in the myenteric plexus into two types: nitrergic and non-nitrergic postganglionic motor pools?

Vagal preganglionic neurons innervate myenteric ganglia. These autonomic efferents are distributed so densely within the ganglia that it has been impractical to track individual vagal axons through the myenteric plexus with tracer labeling. To evaluate whether vagal efferent axons evidence selectivity, particularly for nitrergic or non-nitrergic myenteric neurons within the plexus, we limited the numbers and volumes of brainstem dextran biotin tracer injections per animal. Reduced labeling and the use of immunohistochemistry generated cases in which some individual axons could be distinguished and traced in three dimensions (Neurolucida) within and among successive (up to 46) myenteric ganglia. In the myenteric plexus of all stomach regions, the majority (∼86%) of vagal efferents were organized into two distinct subtypes. One subtype (∼24% of dextran-labeled efferents, designated "primarily nitrergic") selectively contacted and linked-both within and between ganglia-nitric oxide synthase positive (nNOS+) neurons into presumptive motor modules. A second subtype (∼62% of efferents, designated "primarily non-nitrergic") appeared to selectively contact and link-both within and between ganglia-non-nitrergic enteric neurons into a second type of effector ensemble. A third candidate type (∼14% of labeled preganglionics), appeared to lack "nitrergic selectivity" and to contact both nNOS+ and nNOS- enteric neurons. In addition to the quantitative assessment of the efferent axons in stomach, qualitative observations of the proximal duodenum indicated similar selective vagal efferent projections, in proportions comparable with those evaluated in the stomach. Limited injections of tracer, three-dimensional (3-D) tracing of individual axons, and histochemistry of myenteric neurons might distinguish additional efferent phenotypes.NEW & NOTEWORTHY The present study highlights the following: 1) one type of vagal efferent axon selectively innervates nitrergic upper gastrointestinal myenteric neurons; 2) a second type of vagal efferent selectively innervates non-nitrergic gastrointestinal myenteric neurons; and 3) the two types of vagal efferents might modulate peristalsis reciprocally and cooperatively.

Effects of the long-term influence of bisphenol A and bisphenol S on the population of nitrergic neurons in the enteric nervous system of the mouse stomach

Bisphenol A (BPA) is an endocrine disruptor commonly used in the production of plastics. Due to its relatively well-known harmful effects on living organisms, BPA is often replaced by its various analogues. One of them is bisphenol S (BPS), widely used in the plastics industry. Until recently, BPS was considered completely safe, but currently, it is known that it is not safe for various internal organs. However, knowledge about the influence of BPS on the nervous system is scarce. Therefore, the aim of this study was to investigate the influence of two doses of BPA and BPS on the enteric nitrergic neurons in the CD1 strain mouse stomach using the double-immunofluorescence technique. The study found that both substances studied increased the number of nitrergic neurons, although changes under the impact of BPS were less visible than those induced by BPA. Therefore, the obtained results, for the first time, clearly indicate that BPS is not safe for the innervation of the gastrointestinal tract.

Diazepam Binding Inhibitor Control of Eu- and Hypoglycemic Patterns of Ventromedial Hypothalamic Nucleus Glucose-Regulatory Signaling.

Pharmacological stimulation/antagonism of astrocyte glio-peptide octadecaneuropeptide signaling alters ventromedial hypothalamic nucleus (VMN) counterregulatory γ-aminobutyric acid (GABA) and nitric oxide transmission. The current research used newly developed capillary zone electrophoresis-mass spectrometry methods to investigate hypoglycemia effects on VMN octadecaneuropeptide content, along with gene knockdown tools to determine if octadecaneuropeptide signaling regulates these transmitters during eu- and/or hypoglycemia. Hypoglycemia caused dissimilar adjustments in the octadecaneuropeptide precursor, i.e., diazepam-binding-inhibitor and octadecaneuropeptide levels in dorsomedial versus ventrolateral VMN. Intra-VMN diazepam-binding-inhibitor siRNA administration decreased baseline 67 and 65 kDa glutamate decarboxylase mRNA levels in GABAergic neurons laser-microdissected from each location, but only affected hypoglycemic transcript expression in ventrolateral VMN. This knockdown therapy imposed dissimilar effects on eu- and hypoglycemic glucokinase and 5'-AMP-activated protein kinase-alpha1 (AMPKα1) and -alpha2 (AMPKα2) gene profiles in dorsomedial versus ventrolateral GABAergic neurons. Diazepam-binding-inhibitor gene silencing up-regulated baseline (dorsomedial) or hypoglycemic (ventrolateral) nitrergic neuron neuronal nitric oxide synthase mRNA profiles. Baseline nitrergic cell glucokinase mRNA was up- (ventrolateral) or down- (dorsomedial) regulated by diazepam-binding-inhibitor siRNA, but knockdown enhanced hypoglycemic profiles in both sites. Nitrergic nerve cell AMPKα1 and -α2 transcripts exhibited division-specific responses to this genetic manipulation during eu- and hypoglycemia. Results document the utility of capillary zone electrophoresis-mass spectrometric tools for quantification of ODN in small-volume brain tissue samples. Data show that hypoglycemia has dissimilar effects on ODN signaling in the two major neuroanatomical divisions of the VMN and that this glio-peptide imposes differential control of glucose-regulatory neurotransmission in the VMNdm versus VMNvl during eu- and hypoglycemia.

The Comparison of the Influence of Bisphenol A (BPA) and Its Analogue Bisphenol S (BPS) on the Enteric Nervous System of the Distal Colon in Mice.

Bisphenol A (BPA), commonly used as a plasticizer in various branches of industry has a strong negative effect on living organisms. Therefore, more and more often it is replaced in production of plastics by other substances. One of them is bisphenol S (BPS). This study for the first time compares the impact of BPA and BPS on the enteric neurons using double immunofluorescence technique. It has been shown that both BPA and BPS affect the number of enteric neurons containing substance P (SP), galanin (GAL), vasoactive intestinal polypeptide (VIP), neuronal isoform of nitric oxide synthase (nNOS-a marker of nitrergic neurons) and/or vesicular acetylcholine transporter (VAChT- a marker of cholinergic neurons). The changes noted under the impact of both bisphenols are similar and consisted of an increase in the number of enteric neurons immunoreactive to all neuronal factors studied. The impact of BPS on some populations of neurons was stronger than that noted under the influence of BPA. The obtained results clearly show that BPS (similarly to BPA) administered for long time is not neutral for the enteric neurons even in relatively low doses and may be more potent than BPA for certain neuronal populations.

In Vitro Monitoring of Nitric Oxide Release in the Mouse Colon Using a Boron-Doped Diamond Microelectrode Modified with Platinum Nanoparticles and Nafion.

This research reports on the preparation of a boron-doped diamond microelectrode modified with platinum nanoparticles and Nafion and its application for detecting nitric oxide (NO) in vitro in the mouse colon. Platinum nanoparticle deposition was performed potentiodynamically using a 2.0 mmol L-1 potassium hexachloroplatinate solution and cycling from -0.2 to 1.3 V vs Ag/AgCl at 0.01 V s-1 for 10 cycles. The Nafion overlayer was applied by immersion in a solution containing 2.5% (w/v) colloidal Nafion and drying overnight at 55 °C in a humid environment. The optimal microelectrode preparation conditions were chosen based on the electrode response for NO oxidation as well as rejection of nitrite (NO2-) oxidation, the main interferent in the electrochemical detection of NO in biological media. Detection figures of merit include a sensitivity of 16.7 ± 2.7 mA M-1 cm-2 (n = 3 electrodes), a detection limit of 0.5 μmol L-1 (S/N = 3), and an electrode response reproducibility of 2.5% (RSD). Electrical stimulation and continuous amperometry were used to measure NO release from myenteric ganglia in wild-type male and female mice in response to an increasing number of electrical stimuli to study nitrergic signaling in the colon. We also present preliminary data regarding the use of optogenetics to selectively stimulate nitrergic myenteric neurons using blue light stimulation with a goal of understanding how inhibitory neuromuscular signaling is involved in the myenteric plexus circuitry that controls intestinal motility.

G protein-coupled lactate receptor GPR81 control of ventrolateral ventromedial hypothalamic nucleus glucoregulatory neurotransmitter and 5'-AMP-activated protein kinase expression.

Astrocytes store glycogen as energy and promote neurometabolic stability through supply of oxidizable l-lactate. Whether lactate regulates ventromedial hypothalamic nucleus (VMN) glucostatic function as a metabolic volume transmitter is unknown. Current research investigated whether G protein-coupled lactate receptor GPR81 controls astrocyte glycogen metabolism and glucose-regulatory neurotransmission in the ventrolateral VMN (VMNvl), where glucose-regulatory neurons reside. Female rats were pretreated by intra-VMN GPR81 or scramble siRNA infusion before insulin or vehicle injection. VMNvl cell or tissue samples were acquired by laser-catapult- or micropunch microdissection for Western blot protein or uHPLC-electrospray ionization-mass spectrometric glycogen analyses. Data show that GPR81 regulates eu- and/or hypoglycemic patterns of VMNvl astrocyte glycogen metabolic enzyme and 5'-AMP-activated protein kinase (AMPK) protein expression according to VMNvl segment. GPR81 stimulates baseline rostral and caudal VMNvl glycogen accumulation but mediates glycogen breakdown in the former site during hypoglycemia. During euglycemia, GPR81 suppresses the transmitter marker neuronal nitric oxide synthase (nNOS) in rostral and caudal VMNvl nitrergic neurons, but stimulates (rostral VMNvl) or inhibits (caudal VMNvl) GABAergic neuron glutamate decarboxylase65/67 (GAD)protein. During hypoglycemia, GPR81 regulates AMPK activation in nitrergic and GABAergic neurons located in the rostral, but not caudal VMNvl. VMN GPR81 knockdown amplified hypoglycemic hypercorticosteronemia, but not hyperglucagonemia. Results provide novel evidence that VMNvl astrocyte and glucose-regulatory neurons express GPR81 protein. Data identify neuroanatomical subpopulations of VMNvl astrocytes and glucose-regulatory neurons that exhibit differential reactivity to GPR81 input. Heterogeneous GPR81 effects during eu- versus hypoglycemia infer that energy state may affect cellular sensitivity to or postreceptor processing of lactate transmitter signaling.

Effect of electroacupuncture at "Tianshu" (ST25) on the regulation of nitrergic neurons in different intestinal segments in streptozotocin induced type 2 diabetic rats

To observe the effect of electroacupuncture (EA) at "Tianshu" (ST25) on nitrergic neurons in jejunum and distal colon in type 2 diabetic rats, so as to explore its mechanism of regulating different intestinal segments. Twenty-four SD rats were randomly divided into control, model and EA groups (n=8 in each group). The diabetes model was established by intraperitoneal injection of streptozotocin (35 mg/kg) and high-sugar and high-fat diet for 2 weeks. EA (2 Hz/15 Hz, 2 mA) was applied to bilateral ST25 for 20 min, once a day, 6 days a week for 4 weeks. The intestinal motility was evaluated by observing the first red stool excretion time and the distal colon bead excretion time. HE staining was used to observe the histological changes of jejunum and distal colon. The positive expression and protein expression of intestinal total neuronal marker protein gene product 9.5（PGP9.5） and neuronal nitric oxide synthase (nNOS) in jejunum and distal colon were detected by immunofluorescence staining and Western blot, respectively. After modeling, the blood glucose was significantly increased (P<0.01), the first red stool excretion time and the distal colon bead excretion time were shortened (P<0.01), the expression levels of PGP9.5 and nNOS in jejunum and distal colon were decreased (P<0.01) in the model group relevant to the control group. After treatment, compared with the model group, the blood glucose was decreased (P<0.01), the first red stool excretion time and the distal colon bead excretion time were prolonged (P<0.01, P<0.05), and the expression levels of PGP9.5 and nNOS in jejunum and distal colon were increased (P<0.05, P<0.01) in the EA group. HE staining showed disordered structure in intestinal mucosa of the jejunum and distal colon, and reduction of the number of goblet cells in the model group, which was relatively milder in the EA group. EA can effectively improve the intestinal mucosal damage and restore intestinal motor function in type 2 diabetic rats, which may be related to its function in regulating the number of nitrergic neurons in the intestinal nervous system.

Involvement of nitrergic neurons in colonic motility in a rat model of ulcerative colitis.

BACKGROUNDThe mechanisms underlying gastrointestinal (GI) dysmotility with ulcerative colitis (UC) have not been fully elucidated. The enteric nervous system (ENS) plays an essential role in the GI motility. As a vital neurotransmitter in the ENS, the gas neurotransmitter nitric oxide (NO) may impact the colonic motility. In this study, dextran sulfate sodium (DSS)-induced UC rat model was used for investigating the effects of NO by examining the effects of rate-limiting enzyme nitric oxide synthase (NOS) changes on the colonic motility as well as the role of the ENS in the colonic motility during UC.AIMTo reveal the relationship between the effects of NOS expression changes in NOS-containing nitrergic neurons and the colonic motility in a rat UC model. METHODSMale rats (n = 8/each group) were randomly divided into a control (CG), a UC group (EG1), a UC + thrombin derived polypeptide 508 trifluoroacetic acid (TP508TFA; an NOS agonist) group (EG2), and a UC + NG-monomethyl-L-arginine monoacetate (L-NMMA; an NOS inhibitor) group (EG3). UC was induced by administering 5.5% DSS in drinking water without any other treatment (EG1), while the EG2 and EG3 were gavaged with TP508 TFA and L-NMMA, respectively. The disease activity index (DAI) and histological assessment were recorded for each group, whereas the changes in the proportion of colonic nitrergic neurons were counted using immunofluorescence histochemical staining, Western blot, and enzyme linked immunosorbent assay, respectively. In addition, the contractile tension changes in the circular and longitudinal muscles of the rat colon were investigated in vitro using an organ bath system. RESULTSThe proportion of NOS-positive neurons within the colonic myenteric plexus (MP), the relative expression of NOS, and the NOS concentration in serum and colonic tissues were significantly elevated in EG1, EG2, and EG3 compared with CG rats. In UC rats, stimulation with agonists and inhibitors led to variable degrees of increase or decrease for each indicator in the EG2 and EG3. When the rats in EGs developed UC, the mean contraction tension of the colonic smooth muscle detected in vitro was higher in the EG1, EG2, and EG3 than in the CG group. Compared with the EG1, the contraction amplitude and mean contraction tension of the circular and longitudinal muscles of the colon in the EG2 and EG3 were enhanced and attenuated, respectively. Thus, during UC, regulation of the expression of NOS within the MP improved the intestinal motility, thereby favoring the recovery of intestinal functions.CONCLUSIONIn UC rats, an increased number of nitrergic neurons in the colonic MP leads to the attenuation of colonic motor function. To intervene NOS activity might modulate the function of nitrergic neurons in the colonic MP and prevent colonic motor dysfunction. These results might provide clues for a novel approach to alleviate diarrhea symptoms of UC patients.

Neurochemical classification of serotonin-immunoreactive neurons co-innervating motor endplates in the mouse esophagus.

Serotonin immunoreactivity was previously found in myenteric neurons co-innervating motor endplates in the mouse esophagus striated muscle and an involvement in motility control was suggested. However, it is not known if other neuroactive substances are present in these neurons and to what extent they co-localize. First, vasoactive intestinal peptide (VIP) was established as a bona fide marker for putative inhibitory myenteric neurons by evaluating co-localization with neuronal nitric oxide synthase (nNOS) and neuropeptide Y (NPY). Then, co-localization of serotonin and VIP was tested in co-innervating axons on motor endplates, which were visualized with α-bungarotoxin (α-BT) by multilabel immunofluorescence. Myenteric ganglia were also surveyed for co-localization in neuronal perikarya and varicosities. nNOS, NPY, and VIP were completely co-localized in enteric co-innervating nerve terminals on motor endplates. After co-staining with VIP, we found (a) serotonin (5-HT)-positive nerve endings without VIP (44% of 5-HT-positively innervated endplates), (b) 5-HT- and VIP-positive endings without co-localization (35%), and (c) 5-HT- and VIP-positive endings with co-localization (21%). About one-fifth of nerve terminals on motor endplates containing 5-HT originate from putative inhibitory peptidegic nitrergic neurons. However, the majority represents a different population presumably subserving different functions.

Cocultured Schwann Cells Rescue Irradiated Pelvic Neuron Outgrowth and Increase Survival

BackgroundProstatic radiation therapy (RT) leads to erectile dysfunction by damaging peri-prostatic pro-erectile nerves of the pelvic ganglion. Schwann cells (SC) facilitate neuronal repair after mechanical injury, however, their role in repair of pelvic neurons post-radiation hasn't been explored. AimTo determine if SCs cocultured with primary pelvic neurons can rescue neuronal survival and growth after ex vivo RT. MethodsMajor pelvic ganglia (MPG) were collected from adult male Sprague-Dawley rats (n = 12) to isolate SCs. SCs received RT (0 or 8 Gy), were plated on coated coverslips and grown to confluence before the addition of neurons. Additional MPGs were irradiated (0 or 8 Gy) and digested to isolate pelvic neurons. Dissociated neurons were plated alone or atop SC-coated coverslips to create 6 experimental groups (n = 3/grp): (i) Control (CON) MPG, (ii) RT MPG, (iii) CON SC + CON MPG, (iv) CONSC + RT MPG, (v) RT SC + CON MPG, and (iv) RT SC + RT MPG. After 72 hours, coverslips were fixed and stained for beta-tubulin (neuron marker), S100 (SC marker), neuronal nitric oxide synthase (nitrergic marker), tyrosine hydroxylase (sympathetic marker), and terminal deoxynucleotidyl transferase dUTP nick-end labeling. OutcomesWe measured neurite length, branching, specific neuron populations and apoptosis. ResultsEx vivo RT decreased MPG neuron length, increased apoptosis and decreased nitrergic neurons in monoculture. Compared to all other groups, CON SC + RT MPG cocultures demonstrated increased neurite outgrowth (P < .001). Neurite branching was decreased in the RT MPG + RT SC coculture, but unchanged in other cocultures. Groups containing RT MPG neurons exhibited increased apoptosis, but coculture with CON SC reduced the degree of RT-induced apoptosis (P < .01). The number of tyrosine hydroxylase positive neurons was unchanged while nitrergic neurons were significantly lower in RT neurons and coculture with CON SCs was unable to prevent nitrergic loss. Clinical TranslationThese findings suggest that SCs may be an important target in prostate cancer patients with radiation-induced pelvic neuropathy to promote MPG neuron survival and neuronal repair after RT. Strengths and LimitationsThis is the first study to characterize the ex vivo ability of SCs to rescue pelvic nerve growth and survival. The study is limited by little supporting mechanistic molecular data and the need to confirm the ability of healthy SCs to promote pelvic neuron survival and repair following prostatic RT in vivo. ConclusionUnirradiated SCs partially mitigated RT-induced MPG apoptosis but did not affect the loss of nitrergic neuron populations suggesting that SCs promote irradiated MPG neuron survival and facilitate intrinsic repair functions.Randolph JT, Pak ES, McMains JC, et al. Cocultured Schwann Cells Rescue Irradiated Pelvic Neuron Outgrowth and Increase Survival. J Sex Med 2022;19:1333–1342.

Transduction of Systemically Administered Adeno-Associated Virus in the Colonic Enteric Nervous System and c-Kit Cells of Adult Mice.

Systemic delivery of adeno-associated virus (AAV) vectors transduces the enteric nervous system. However, less is known on the mapping and morphological and neurochemical characterization in the adult mouse colon. We used AAV9-CAG-GFP (AAV9) and AAV-PHP.S-hSyn1-tdTomato farnesylated (PHP.S-tdTf) to investigate the segmental distribution, morphologies and neurochemical coding of the transduction. The vectors were retro-orbitally injected in male and female adult mice, and 3 weeks later, the colon was prepared for microcopy with or without immunohistochemistry for neuronal and non-neuronal markers. In contrast to the distributions in neonatal and juvenile rodents, the AAV transduction in neurons and/or nerve fibers was the highest in the proximal colon, decreased gradually in the transverse, and was sparse in the distal colon without difference between sexes. In the proximal colon, the AAV9-transduced myenteric neurons were unevenly distributed. The majority of enteric neurons did not have AAV9 expression in their processes, except those with big soma with or without variously shaped dendrites, and a long axon. Immunolabeling demonstrated that about 31% neurons were transduced by AAV9, and the transduction was in 50, 28, and 31% of cholinergic, nitrergic, and calbindin-positive myenteric neurons, respectively. The nerve fiber markers, calcitonin gene-related peptide alpha, tyrosine hydroxylase or vasoactive intestinal polypeptide co-localized with AAV9 or PHP.S-tdTf in the mucosa, and rarely in the myenteric plexus. Unexpectedly, AAV9 expression appeared also in a few c-Kit immunoreactive cells among the heavily populated interstitial cells of Cajal (ICC). In the distal colon, the AAV transduction appeared in a few nerve fibers mostly the interganglionic strands. Other types of AAV9 and AAV-PHP vectors induced a similar colonic segmental difference which is not colon specific since neurons were transduced in the small intestine and gastric antrum, while little in the gastric corpus and none in the lower esophagus.ConclusionThese findings demonstrate that in adult mice colon that there is a rostro-caudal decrease in the transduction of systemic delivery of AAV9 and its variants independent of sex. The characterization of AAV transduction in the proximal colon in cholinergic and nitrergic myenteric neurons along with a few ICC suggests implications in circuitries regulating motility.

Castration mediated schwann cell dedifferentiation leads to slower nerve conduction, decreased neuritogenesis, and nitrergic neuron loss

ABSTRACT Introduction/Objectives Low levels of androgens due to androgen deprivation therapy for prostate cancer treatment or due to aging lead to erectile dysfunction (ED). Castration, a preclinical model of low androgens, induces ED due to damage to pelvic nerves and a loss of erectogenic nitrergic nerves. Schwann cells (SC) release neurotrophic factors to promote pelvic nerve growth and survival. It is unknown how testosterone deficiency impacts SC function. This study determines how testosterone deficiency influences SC phenotype and trophic support in neuronal regeneration. We hypothesize testosterone deficiency will decrease myelinated pelvic SC populations, delay penile nerve conduction and reduce pelvic nitrergic neurons. Methods Male Sprague Dawley rats (12 wks) were divided into control (CON) and surgical castration (CAST) groups. All experiments were performed six weeks following castration. Dorsal penile nerves (DPN; 15-16 mm) were excised, placed in a sylgard dish containing oxygenated buffer and conduction velocity of myelinated fast fibers was assessed (n=8 per group). The major pelvic ganglia (MPG) were collected and dissociated into SC or neurons. Dissociated neurons were grown on laminin coated coverslips. To determine whether SCs could provide trophic support, we co-cultured neurons on top of confluent SCs. We assessed the following co-culture groups: CON SC + CON MPG, CON SC + CAST MPG, CAST SC + CON MPG (n=4 per group, cultured in triplicate). After 72 hours, all cultures were fixed and stained with beta tubulin class III, neuronal nitric oxide synthase (nNOS), and tyrosine hydroxylase (TH) to assess neurite length and branching, nitrergic and sympathetic neuron populations, respectively. Dissociated SC were characterized via qPCR for markers of all SC (Sox10), myelinated SC (Krox20), and dedifferentiated SC (glial fibrillary acidic protein; GFAP). Results CAST slows DPN myelinated fast fiber conduction velocity. In dissociated cultured MPG neurons, castration reduces both neurite length and number of branches. Interestingly, CAST MPG neurons co-cultured with CON SC restore neurite length and branching to same level as CON MPG neurons co-cultured with CON SC; however, CAST SC do not impact CON MPG neurite length. CAST MPG neurons co-cultured with CON SC and CON MPG neurons co-cultured with CAST SC both have less nitrergic neurons than CON SC + CON MPG neurons. CAST does not impact the number of TH neurons in single or co-cultured conditions. CAST reduces gene expression of Sox10, myelinated SC, and dedifferentiated SC. Conclusions CAST markedly impairs penile nerve conduction, reduces neurite outgrowth and the proportion of pro-erectile nitrergic neurons. Healthy SC are able to rescue neuritogenesis but not the survival of nitrergic neurons. Additionally, healthy MPG neurons plated on CAST SC have a reduced number of nitrergic neurons. We believe CAST SC are undergoing a phenotypic switch from a myelinated neurotrophic form into dedifferentiated SC promoting neuritogenesis and loss of nitrergic neurons. Preserving the myelinated SC phenotype may prevent impaired nerve damage and subsequent ED in patients with testosterone deficiency. Disclosure Work supported by industry: no.