Prevertebral Sympathetic Ganglia Research Articles

Characterization of viscerofugal neurons in human colon by retrograde tracing and multi-layer immunohistochemistry.

Viscerofugal neurons (VFNs) have cell bodies in the myenteric plexus and axons that project to sympathetic prevertebral ganglia. In animals they activate sympathetic motility reflexes and may modulate glucose metabolism and feeding. We used rapid retrograde tracing from colonic nerves to identify VFNs in human colon for the first time, using ex vivo preparations with multi-layer immunohistochemistry. Colonic nerves were identified in isolated preparations of human colon and set up for axonal tracing with biotinamide. After fixation, labeled VFN cell bodies were subjected to multiplexed immunohistochemistry for 12 established nerve cell body markers. Biotinamide tracing filled 903 viscerofugal nerve cell bodies (n = 23), most of which (85%) had axons projecting orally before entering colonic nerves. Morphologically, 97% of VFNs were uni-axonal. Of 215 VFNs studied in detail, 89% expressed ChAT, 13% NOS, 13% calbindin, 9% enkephalin, 7% substance P and 0 of 123 VFNs expressed CART. Few VFNs contained calretinin, VIP, 5HT, CGRP, or NPY. VFNs were often surrounded by dense baskets of axonal varicosities, probably reflecting patterns of connectivity; VAChT+ (cholinergic), SP+ and ENK+ varicosities were most abundant around them. Human VFNs were diverse; showing 27 combinations of immunohistochemical markers, 4 morphological types and a wide range of cell body sizes. However, 69% showed chemical coding, axonal projections, soma-dendritic morphology and connectivity similar to enteric excitatory motor neurons. Viscerofugal neurons are present in human colon and show very diverse combinations of features. High proportions express ChAT, consistent with cholinergic synaptic outputs onto postganglionic sympathetic neurons in prevertebral ganglia.

Sympathetic Input to Multiple Cell Types in Mouse and Human Colon Produces Region-Specific Responses

The colon is innervated by intrinsic and extrinsic neurons that coordinate functions necessary for digestive health. Sympathetic input suppresses colon motility by acting on intrinsic myenteric neurons, but the extent of sympathetic-induced changes on large-scale network activity in myenteric circuits has not been determined. Compounding the complexity of sympathetic function, there is evidence that sympathetic transmitters can regulate activity in non-neuronal cells (such as enteric glia and innate immune cells). We performed anatomical tracing, immunohistochemistry, optogenetic (GCaMP calcium imaging, channelrhodopsin), and colon motility studies in mice and single-cell RNA sequencing in human colon to investigate how sympathetic postganglionic neurons modulate colon function. Individual neurons in each sympathetic prevertebral ganglion innervated the proximal or distal colon, with processes closely opposed to multiple cell types. Calcium imaging in semi-intact mouse colon preparations revealed changes in spontaneous and evoked neural activity, as well as activation of non-neuronal cells, induced by sympathetic nerve stimulation. The overall pattern of response to sympathetic stimulation was unique to the proximal or distal colon. Region-specific changes in cellular activity correlated with motility patterns produced by electrical and optogenetic stimulation of sympathetic pathways. Pharmacology experiments (mouse) and RNA sequencing (human) indicated that appropriate receptors were expressed on different cell types to account for the responses to sympathetic stimulation. Regional differences in expression of α-1 adrenoceptors in human colon emphasize the translational relevance of our mouse findings. Sympathetic neurons differentially regulate activity of neurons and non-neuronal cells in proximal and distal colon to promote distinct changes in motility patterns, likely reflecting the distinct roles played by these 2 regions.

Pursuit of Neurotransmitter Functions: Being Attracted with Fascination of the Synapse.

In the beginning of the 1970s, only two chemical substances, acetylcholine and γ-aminobutyric acid (GABA), had been definitely established as neurotransmitters. Under such circumstances, I started my scientific career in Professor Masanori Otsuka's lab searching for the transmitter of primary sensory neurons. Until 1976, lines of evidence had accumulated indicating that the undecapeptide substance P could be released as a transmitter from primary afferent fibers into spinal synapses, although the substance P-mediated synaptic response had yet to be identified. Peripheral synapses could serve as a good model and thus, it was demonstrated in the prevertebral sympathetic ganglia by1985 that substance P released from axon collaterals of primary sensory neurons acts as the transmitter mediating non-cholinergic slow excitatory postsynaptic potential (EPSP). At that time, we also found that autonomic synapses were useful to uncover the transmitter role of the opioid peptide enkephalins, whose functions had been unknown since their discovery in 1975. Accordingly, enkephalins were found to serve a transmitter role in mediating presynaptic inhibition of cholinergic fast and non-cholinergic slow transmission in the prevertebral sympathetic ganglia. In 1990s, we attempted to devise a combined technique of brain slices and patch-clamp recordings. We applied it to study the regulatory mechanisms that operate around cerebellar GABAergic inhibitory synapses, because most of the studies then had centered on excitatory synapses and because inhibitory synapses are crucially involved in brain functions and disorders. Consequently, we discovered novel forms of heterosynaptic interactions, dual actions of a single transmitter, and receptor crosstalk, the details of which are described in this review.

Targeted electrophysiological analysis of viscerofugal neurons in the myenteric plexus of guinea-pig colon

Enteric viscerofugal neurons are mechanosensory interneurons that form the afferent limb of intestino-intestinal reflexes involving prevertebral sympathetic neurons. Fast synaptic inputs to viscerofugal neurons arise from other enteric neurons, but their sources are unknown. We aimed to describe the origins of synaptic inputs to viscerofugal neurons by mapping the locations of their cell bodies within the myenteric plexus. Viscerofugal neuron somata were retrogradely traced with 1,1′-didodecyl-3,3,3′,3′-tetramethyl indocarbocyanine perchlorate (DiI) from colonic nerve trunks and impaled with microelectrodes, in longitudinal muscle/myenteric plexus preparations of the guinea-pig distal colon (39 impalements, n=14). Thirty-eight viscerofugal neurons were uni-axonal and had the electrophysiological characteristics of myenteric S-neurons; one neuron was multipolar with AH-neuron electrophysiological characteristics. Depolarizing current pulses evoked either single- or multiple action potentials in viscerofugal neurons (range 1–25 spikes, 500ms, 100–900pA, 21 cells). Electrical stimulation of internodal strands circumferential to viscerofugal neurons evoked fast excitatory postsynaptic potentials (EPSPs) in 19/24 cells. Focal pressure-ejection of the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP, 10μm) directly onto viscerofugal nerve cell bodies evoked large depolarizations and action potentials (23±10mV, latency 350±230ms, 21/22 cells). DMPP was then focally applied to multiple sites, up to 3mm from the recorded viscerofugal neuron, to activate other myenteric S-neurons. In a few sites in myenteric ganglia, DMPP evoked repeatable fast EPSPs in viscerofugal neurons (latency 300±316ms, 38/394 sites, 10 cells). The cellular sources of synaptic inputs to viscerofugal neurons were located both orally and aborally (19 oral, 19 aboral), but the amplitude of oral inputs was consistently greater than aboral inputs (13.1±4.3mV vs. 10.1±4.8mV, respectively, p<0.05, paired t-test, n=6). Most impaled viscerofugal neurons were nitric oxide synthase (NOS) immunoreactive (20/27 cells tested). Thus, the synaptic connections onto viscerofugal neurons within the myenteric plexus suggest that multiple enteric neural pathways feed into intestino-intestinal reflexes, involving sympathetic prevertebral ganglia.

P21 Endogenous H2S produced by CSE selectively facilitates acetylcholine release from synaptic terminals of central origin in mouse superior mesenteric ganglion

Abdominal sympathetic prevertebral ganglia in mammals play a major physiological role in regulating motility, absorption and secretion in the gastrointestinal tract and its accessory organs. Hydrogen sulfide (H2S) has emerged as an important messenger molecule in the central nervous system and enteric nervous system. We designed experiments to test the hypotheses that prevertebral ganglia have the enzymatic machinery to generate H2S, that H2S is continuously generated and released in the prevertebral ganglia and that H2S modulates fast cholinergic nicotinic excitatory synaptic input (F-EPSP). Methods The superior mesenteric ganglion (SMG) and attached splanchnic and colonic nerve trunks were dissected from adult SJL/J mice. The use of mouse tissue was approved by the Institutional Animal Care and Use Committee. Sharp glass microelectrodes were used for intracellular recordings and bi-polar stimulating electrodes were used to evoke F-EPSPs. Immunostaining was performed with antibodies for cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) and with vesicular acetylcholine transporter to identify preganglionic cholinergic terminals. Since the amount of tissue in mouse prevertebral ganglia was less than the amount required for the assay method (gas chromatography), we used guinea pig prevertebral ganglia to measure endogenous H2S generation. Results Immunoreactivity of CSE was found in neurons and glia cells. There was no colocalization of CSE with immunoreactivity of vesicular acetylcholine transporter. Immunoreactivity of CBS was only found in some glia cells. There was no colocalization between immunoreactivity of CBS and immunoreactivity of vesicular acetylcholine transporter. Endogenous H2S was generated and released from homogenized guinea pig sympathetic prevertebral ganglia at a rate of 2.52 ± 0.52 pmol/min/mg tissue weight, suggesting that H2S is generated in intact prevertebral ganglia. To determine whether the F-EPSPs are potentiated by endogenously released H2S in mouse SMG, we tested the effect of inhibiting H2S break-down using stigmatellin, a specific sulfide quinone reductase inhibitor. Stigmatellin (1 μM) significantly (p dl -propargylglycine (PAG) (1 μM) on F-EPSPs evoked by splanchnic and colonic nerve stimulation in the presence of PAG. The amplitude and area of F-EPSPs evoked by splanchnic nerve stimulation were significantly (p 0.05) effect on F-EPSPs evoked by colonic nerve stimulation (30.2 ± 6.1 mV and 808 ± 235 ms · mV with PAG vs. 26.9 ± 7.6 mV and 855 ± 241 ms · mV before PAG; n = 5). Conclusions H2S is enzymatically generated and released in the mouse prevertebral ganglia where it potentiates fast cholinergic synaptic input in a pathway specific manner.

489 Endogenous H2S Produced in Prevertebral Sympathetic Ganglia Predominantly by Cse in Neurons and Glia Cells Modulates Central Cholinergic Synaptic Input

Hydrogen sulfide(H2S), mainly produced endogenously by two enzymes, cystathionine γlyase(CSE) and cystathionine β-synthase(CBS), is a messenger molecule in the central nervous system, in the GI tract and in sympathetic prevertebral ganglia. Our previous study found, in mouse superior mesenteric ganglion(SMG), that exogenous H2S acts selectively on splanchnic nerve terminals to potentiate fast excitatory post synaptic potentials (F-EPSPs). In this study, we investigated the distribution of CSE and CBS and the role of endogenously produced H2S on central sympathetic input. Methods: The SMG, splanchnic nerve trunks and colonic nerve trunk were dissected from adult SJL/J mice. Immunostaining was performed with antibodies for CSE and CBS and for vesicular acetylcholine transporter(VAChT) to identify cholinergic nerve terminals. Microelectrodes were used for intracellular recordings. Results: CSE immunoreactivity(IR) was found in neurons and glia cells and did not co-localize with VAChT-IR. CBS-IR was found only in glia cells. To determine whether the F-EPSPs could be potentiated by endogenously released H2S, we tested the effect of inhibiting H2S breakdown with stigmatellin, a specific sulfide quinone reductase inhibitor, on F-EPSPs. Stigmatellin(1μM) significantly(P<0.05) increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(21.0±4.8mV and 441±107ms●mV with stigmatellin vs. 18.4±5.3mV and 338±108ms●mV before stigmatellin, n=4) but had no significant effect on F-EPSPs evoked by colonic nerve stimulation. In contrast, the CSE inhibitor, dl-propargylglycine(PAG, 1mM), significantly(P<0.05) decreased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.7±3.9mV and 824±288ms●mV with PAG vs. 27.0±4.1mV and 1230±414ms●mV before PAG, n=6), but had no effect on F-EPSPs evoked by colonic nerve stimulation. Surprisingly, the CBS inhibitor, aminooxyacetic acid(AOAA, 0.5mM), increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.6±12.1mV and 651±455ms●mV with AOAA vs. 20.2±10.9mV and 438±318ms●mV before AOAA, n=4, P<0.05) and increased the amplitude and the area of F-EPSPs evoked by colonic nerve stimulation(36.2±8.2mV and 1609±468ms●mV with AOAA vs. 28.9±8.1mV and 1084±402ms●mV before AOAA, n=6, P<0.05). Bicuculline(20μM), a specific GABA-A receptor inhibitor, blocked the potentiating effect of AOAA suggesting that the AOAA induced potentiation of F-EPSPs was due to the inhibition of GABA transaminase-induced accumulation of GABA. Conclusion: We conclude that endogenously produced H2S in the mouse SMG modulates fast cholinergic synaptic input in a pathwayspecific manner. H2S modulates fast cholinergic synaptic input from central splanchnic nerve terminals but has no affect on input from peripheral cholinergic input. (Funded by NIH DK17238)

490 Studies of the Psychophysiological Markers and the Brain Processing of Nausea in Healthy Humans Using a Novel Virtual Reality Video

Hydrogen sulfide(H2S), mainly produced endogenously by two enzymes, cystathionine γlyase(CSE) and cystathionine β-synthase(CBS), is a messenger molecule in the central nervous system, in the GI tract and in sympathetic prevertebral ganglia. Our previous study found, in mouse superior mesenteric ganglion(SMG), that exogenous H2S acts selectively on splanchnic nerve terminals to potentiate fast excitatory post synaptic potentials (F-EPSPs). In this study, we investigated the distribution of CSE and CBS and the role of endogenously produced H2S on central sympathetic input. Methods: The SMG, splanchnic nerve trunks and colonic nerve trunk were dissected from adult SJL/J mice. Immunostaining was performed with antibodies for CSE and CBS and for vesicular acetylcholine transporter(VAChT) to identify cholinergic nerve terminals. Microelectrodes were used for intracellular recordings. Results: CSE immunoreactivity(IR) was found in neurons and glia cells and did not co-localize with VAChT-IR. CBS-IR was found only in glia cells. To determine whether the F-EPSPs could be potentiated by endogenously released H2S, we tested the effect of inhibiting H2S breakdown with stigmatellin, a specific sulfide quinone reductase inhibitor, on F-EPSPs. Stigmatellin(1μM) significantly(P<0.05) increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(21.0±4.8mV and 441±107ms●mV with stigmatellin vs. 18.4±5.3mV and 338±108ms●mV before stigmatellin, n=4) but had no significant effect on F-EPSPs evoked by colonic nerve stimulation. In contrast, the CSE inhibitor, dl-propargylglycine(PAG, 1mM), significantly(P<0.05) decreased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.7±3.9mV and 824±288ms●mV with PAG vs. 27.0±4.1mV and 1230±414ms●mV before PAG, n=6), but had no effect on F-EPSPs evoked by colonic nerve stimulation. Surprisingly, the CBS inhibitor, aminooxyacetic acid(AOAA, 0.5mM), increased the amplitude and the area of F-EPSPs evoked by splanchnic nerve stimulation(23.6±12.1mV and 651±455ms●mV with AOAA vs. 20.2±10.9mV and 438±318ms●mV before AOAA, n=4, P<0.05) and increased the amplitude and the area of F-EPSPs evoked by colonic nerve stimulation(36.2±8.2mV and 1609±468ms●mV with AOAA vs. 28.9±8.1mV and 1084±402ms●mV before AOAA, n=6, P<0.05). Bicuculline(20μM), a specific GABA-A receptor inhibitor, blocked the potentiating effect of AOAA suggesting that the AOAA induced potentiation of F-EPSPs was due to the inhibition of GABA transaminase-induced accumulation of GABA. Conclusion: We conclude that endogenously produced H2S in the mouse SMG modulates fast cholinergic synaptic input in a pathwayspecific manner. H2S modulates fast cholinergic synaptic input from central splanchnic nerve terminals but has no affect on input from peripheral cholinergic input. (Funded by NIH DK17238)

Su2000 Reduced Small Bowel Transit is Associated With Enhanced Excitability of Sympathetic Prevertebral Ganglion Neurons but Not Ileum Myenteric Neurons During Colitis in Guinea Pigs

Background: Localized colitis causes a reduction in small bowel motor function in humans and non-human animals yet the mechanisms that contribute to this phenomenon is not completely understood. Aim: To determine the time course of changes in 1) small bowel motor function, 2) excitability of ileum myenteric plexus neurons and 3) excitability of inferior mesenteric ganglion (IMG) neurons following an inflammatory insult to the distal colon. Methods: Colitis was induced by a single enema of trinitrobenzene sulfonic acid (TNBS) dissolved in 30% EtOH. Small bowel transit was assessed by determining the location of the leading edge of charcoal 30min following gavage and reported as a percentage of the total length of the small intestine. Neuronal excitability was assessed by conventional intracellular electrophysiology. Results: Small bowel transit was reduced in guinea pigs 12h, and 24h post-TNBS, but returned to normal at 48h and 6d post-TNBS (control, 84±3;12h, 50±9; 24h, 44±3; 48h, 68±8; 6d, 75±2;P<0.05). AH neurons of the guinea pig ileum were hyperexcitable at 24h 48h and 6d post-TNBS, but were not different than controls at 12h post-TNBS. Evidences of excitability were a more depolarized membrane potential (mV: control, -76±3;12h, -73±2; 24h, -59±2; 48h, -63±2; 6d, -68±3;P<0.05), and increased number of action potentials (control, 1.2±0.2;12h, 1.1±0.7; 24h, 3.5±1.2; 48h, 4.5±1.3; 6d, 7.8±3.0;P<0.05). There was no change in the magnitude of the afterhyperpolarization or the threshold current. Tonic neurons of the IMG were hyperexcitable at 12h, 24h and 6d post-TNBS, the three time points tested. Evidences of excitability were a decreased threshold current (pA: control, 340±26;12h, 258±23; 24h, 192±17; 6d, 164±18;P<0.05), and increased number of action potentials (control, 3.1±0.1;12h, 5.7±0.7; 24h, 6.2±0.4; 6d, 5.1±0.3;P<0.05). There was no change in resting membrane potential. Conclusion: Reduced small bowel transit 12h following TNBS suggests rapid functional changes in response to the inflammatory insult. There is no evidence for excitability of myenteric AH neurons at this early time point so these cells may not directly contribute to the functional change. Conversely, PVG neurons are excitable at this early time point. This association supports the concept that PVG neurons contribute to small bowel motor dysfunction during colitis. The mechanisms contributing the later excitability of myenteric AH neurons and restoration of normal transit despite ongoing neuronal hyperexcitability remain unresolved. Supported by NIH Grant DK76665.

Different Roles of 12/15-Lipoxygenase in Diabetic Large and Small Fiber Peripheral and Autonomic Neuropathies

Up-regulation of 12/15-lipoxygenase, which converts arachidonic acid to 12(S)- and 15(S)-hydroxyeicosatetraenoic acids, causes impaired cell signaling, oxidative-nitrosative stress, and inflammation. This study evaluated the role for 12/15-lipoxygenase in diabetic large and small fiber peripheral and autonomic neuropathies. Control and streptozotocin-diabetic wild-type and 12/15-lipoxygenase-deficient mice were maintained for 14 to 16 weeks. 12/15-lipoxygenase gene deficiency did not affect weight gain or blood glucose concentrations. Diabetic wild-type mice displayed increased sciatic nerve 12/15-lipoxygenase and 12(S)-hydroxyeicosatetraenoic acid levels. 12/15-lipoxygenase deficiency prevented or alleviated diabetes-induced thermal hypoalgesia, tactile allodynia, motor and sensory nerve conduction velocity deficits, and reduction in tibial nerve myelinated fiber diameter, but not intraepidermal nerve fiber loss. The frequencies of superior mesenteric-celiac ganglion neuritic dystrophy, the hallmark of diabetic autonomic neuropathy in mouse prevertebral sympathetic ganglia, were increased 14.8-fold and 17.2-fold in diabetic wild-type and 12/15-lipoxygenase-deficient mice, respectively. In addition, both diabetic groups displayed small (<1%) numbers of degenerating sympathetic neurons. In conclusion, whereas 12/15-lipoxygenase up-regulation provides an important contribution to functional changes characteristic for both large and small fiber peripheral diabetic neuropathies and axonal atrophy of large myelinated fibers, its role in small sensory nerve fiber degeneration and neuritic dystrophy and neuronal degeneration characteristic for diabetic autonomic neuropathy is minor. This should be considered in the selection of endpoints for future clinical trials of 12/15-lipoxygenase inhibitors.

Multilevel interactions between the sympathetic and parasympathetic nervous systems: a minireview

In order to allow precise regulation of bodily functions, the activity of the autonomic nervous system must be precisely regulated. The traditional model concerning the regulation of norepinephrine and acetylcholine release in target tissues suggests that the activities of the efferent arms of the autonomic nervous system are more or less independent of each other. However, plenty of experimental and clinical studies have demonstrated the presence of multiple interactions between the sympathetic and parasympathetic nervous system that are mediated through several pathways and mechanisms at both central and peripheral levels of the neuraxis. Interactions within the central nervous system are mediated predominantly by neurons within the nucleus of the solitary tract and paraventricular hypothalamic nucleus. Peripheral interactions are based on the morphological-functional organization of the sympathetic and parasympathetic pathways at the levels of the sympathetic prevertebral ganglia or neuroeffector connections. Furthermore, evidence suggests that neuroeffector connections may be realized at the axo-axonal, presynaptic, postsynaptic, and post-receptor levels. Alterations in interactions between the sympathetic and parasympathetic nervous system can lead to unbalanced autonomic activities, which may influence the development of various disorders, including cardiovascular, inflammatory, metabolic, neurological, and psychiatric diseases. The aim of this article is to illustrate the complexity of interaction between the sympathetic and parasympathetic nervous systems and to describe the role of these interactions in the heart, adrenal medulla, and vagal trunk.

Accessory lumbar splanchnic ganglia in humans: a case report

The present study was designed and performed on 20 human adult cadavers at the level of the retroperitoneal space in order to provide evidence of the paravertebral and prevertebral sympathetic ganglia. Only one specimen, male, presented two ganglia on the left side located on the first lumbar splanchnic nerve, one proximal and the other distal. The macroscopically detected swellings were drawn and certified as autonomic ganglia after performing silver stains by the method of Bielschowsky (on blocks). No existing reference to date mentions such lumbar splanchnic ganglia, which could be the result of a fragmentation followed by displacement from the lumbar sympathetic trunk. Their demonstrated anatomical connections with the afferents of the spermatic ganglion and the intermesenteric plexus may indicate the involvement of these ganglia in vasomotor and gonadal functions.

Neuritic dystrophy and neuronopathy in Akita ( Ins2Akita) diabetic mouse sympathetic ganglia

Diabetic autonomic neuropathy is a debilitating, poorly studied complication of diabetes. Our previous studies of non-obese diabetic (NOD) and related mouse models identified rapidly developing, dramatic pathology in prevertebral sympathetic ganglia; however, once diabetic, the mice did not survive for extended periods needed to examine the ability of therapeutic agents to correct established neuropathy. In the current manuscript we show that the Akita ( Ins2 Akita ) mouse is a robust model of diabetic sympathetic autonomic neuropathy with unambiguous, spontaneous, rapidly-developing neuropathology which corresponds closely to the characteristic pathology of other rodent models and man. Akita mice diabetic for 2, 4 or 8 months of diabetes progressively developed markedly swollen axons and dendrites (“neuritic dystrophy”) in the prevertebral superior mesenteric (SMG) and celiac ganglia (CG). Comparable changes failed to develop in the superior cervical ganglia (SCG) of the Akita mouse or in any ganglia of non-diabetic mice. Morphometric studies demonstrate an overall increase in presynaptic axon terminal cross sectional area, including those without any ultrastructural features of dystrophy. Neurons in Akita mouse prevertebral sympathetic ganglia show an unusual perikaryal alteration characterized by the accumulation of membranous aggregates and minute mitochondria and loss of rough endoplasmic reticulum. These changes result in the loss of a third of neurons in the CG over the course of 8 months of diabetes. The extended survival of diabetic mice and robust pathologic findings provide a clinically relevant paradigm that will facilitate the analysis of novel therapeutic agents on the reversal of autonomic neuropathy.

Synaptic ultrastructural alterations anticipate the development of neuroaxonal dystrophy in sympathetic ganglia of aged and diabetic mice.

Neuroaxonal dystrophy, a distinctive axonopathy characterized by marked enlargement of distal axons, is the hallmark pathologic alteration in aged and diabetic human prevertebral sympathetic ganglia and in corresponding rodent models. Neuroaxonal dystrophy is thought to represent the abnormal outcome of cycles of synaptic degeneration and regeneration; a systematic study of identified axon terminals in aged and diabetic prevertebral ganglia, however, has not previously been performed. We examined the initial changes that develop in presynaptic and postsynaptic elements in sympathetic ganglia of aged and diabetic mice and found numerous synaptic changes involving both presynaptic and postsynaptic elements. Early alterations in presynaptic axon terminal size, vesicle content, and morphology culminate in the development of anastomosing membranous tubulovesicular aggregates, accumulation of autophagosomes, and amorphous debris that form a continuum with progressively larger classically dystrophic swellings. Dendritic changes consist of the development of swellings composed of delicate tubulovesicular elements and mitochondriopathy characterized by increased numbers of small mitochondria and, exclusively in aged ganglia, megamitochondria. These results support the hypothesis that neuroaxonal dystrophy results from progressive changes in presynaptic axon terminals that likely involve membrane dynamics and which are accompanied by distinctive changes in postsynaptic dendritic elements.

Increased superoxide levels in ganglia and sympathoexcitation are involved in sarafotoxin 6c-induced hypertension

Endothelin (ET) type B receptors (ET(B)R) are expressed in multiple tissues and perform different functions depending on their location. ET(B)R mediate endothelium-dependent vasodilation, clearance of circulating ET, and diuretic effects; all of these should produce a fall in arterial blood pressure. However, we recently showed that chronic activation of ET(B)R in rats with the selective agonist sarafotoxin 6c (S6c) causes sustained hypertension. We have proposed that one mechanism of this effect is constriction of capacitance vessels. The current study was performed to determine whether S6c hypertension is caused by increased generation of reactive oxygen species (ROS) and/or activation of the sympathetic nervous system. The model used was continuous 5-day infusion of S6c into male Sprague-Dawley rats. No changes in superoxide anion levels in arteries and veins were found in hypertensive S6c-treated rats. However, superoxide levels were increased in sympathetic ganglia from S6c-treated rats. In addition, superoxide levels in ganglia increased progressively the longer the animals received S6c. Treatment with the antioxidant tempol impaired S6c-induced hypertension and decreased superoxide levels in ganglia. Acute ganglion blockade lowered blood pressure more in S6c-treated rats than in vehicle-treated rats. Although plasma norepinephrine levels were not increased in S6c hypertension, surgical ablation of the celiac ganglion plexus, which provides most of the sympathetic innervation to the splanchnic organs, significantly attenuated hypertension development. The results suggest that S6c-induced hypertension is partially mediated by sympathoexcitation to the splanchnic organs driven by increased oxidative stress in prevertebral sympathetic ganglia.

Enteric nervous system

The enteric nervous system (ENS) derives from the neural crest and consists of neurons distributed in two ganglionated plexuses, myenteric and submucosal, located within the walls of the gut. The ENS contains as many neurons as the spinal cord (approximately 80-100 million neurons) and controls intestinal motility and secretion largely independently of influences from the CNS.1–5 The ENS is affected by Lewy body pathology at early stages of PD,6,7 and by many genetic8,9 or immune9,10 neurologic disorders associated with gastrointestinal dysmotility. The aim of this paper is to briefly review the functional and neurochemical organization of the ENS and its involvement in neurologic disorders. There are recent comprehensive reviews on these subjects.3,8,9,11,12 The control of motility and secretion in the gastrointestinal tract depends on both extrinsic parasympathetic and sympathetic innervation and intrinsic innervation, provided by the ENS. Extrinsic parasympathetic inputs originate in the dorsal motor nucleus of the vagus, which controls primarily the motility of the esophagus and stomach, and the sacral parasympathetic nucleus, which contributes to control of motility of the distal colon and rectum. The prevertebral sympathetic ganglia mediate peripheral reflexes that inhibit motility of the gut. The intrinsic innervation of the gut consists of the ganglia of the ENS, which are grouped into two plexuses: the myenteric plexus (of Auerbach), located between the outer longitudinal and the inner circular muscle layers, and submucosal plexus (of Meissner), located between the circular muscle and the muscularis mucosae (figure).1–4 The ENS controls intestinal motility and secretion largely independently of extrinsic parasympathetic and sympathetic innervation, although these extrinsic influences have a modulatory role on ENS activity. Neurons of the myenteric plexus control the activity of the smooth muscle of the gut whereas those in the submucosal plexus also regulate mucosal secretion and blood flow. …

The Continuing Saga of Neuronal Oxidative Stress in Hypertension

Numerous studies have demonstrated increased production of reactive oxygen species (ROS) in blood vessels and kidneys in hypertension and provided evidence that the oxidative stress in these organs causes or contributes to the elevated blood pressure.1,2 Recently, the nervous system has emerged as an additional site and target of oxidative stress in hypertension. The vast majority of these studies have focused on the central nervous system, where activation of reduced nicotinamide-adenine dinucleotide phosphate [NAD(P)H] oxidase has been shown to contribute to sympatho-excitation and increases in blood pressure.3 NAD(P)H oxidase is a major source of ROS in hypertension, in both peripheral tissues and brain1–3; and homologues of NAD(P)H oxidase (Nox) are differentially expressed in diverse cell types including neurons.3,4 NAD(P)H oxidase consists of 2 membrane-bound subunits (p22phox and Nox) and cytosolic components that are recruited to the membrane during activation (p47phox, p67phox, p40phox, and GTPase Rac).4 Nox homologues include Nox1, Nox2 (gp91phox), Nox3, Nox4, Nox5, Duox1, and Duox2.4 Neurohumoral regulation of arterial blood pressure is of course not only determined by the central nervous system but is also influenced by alterations in sensory nerve activity and the function of peripheral sympathetic and parasympathetic nerves. The article by Cao et al,5 published in this issue of Hypertension , is one of a series of articles from this group6–8 that describe NAD(P)H oxidase expression and regulation of ROS production in sympathetic neurons located in prevertebral sympathetic ganglia of normotensive and hypertensive (deoxycorticosterone acetate [DOCA]–salt) rats. The present results confirm earlier findings that Nox1, Nox2, Nox4, p22phox, and p47phox are expressed in sympathetic ganglia7–9 and that NAD(P)H oxidase activity in these ganglia is increased in DOCA-salt hypertension.7 Two important novel findings of …

Differential Regulation of NADPH Oxidase in Sympathetic and Sensory Ganglia in Deoxycorticosterone Acetate–Salt Hypertension

We demonstrated recently that superoxide anion levels are elevated in prevertebral sympathetic ganglia of deoxycorticosterone acetate-salt hypertensive rats and that this superoxide anion is generated by reduced nicotinamide-adenine dinucleotide phosphate oxidase. In this study we compared the reduced nicotinamide-adenine dinucleotide phosphate oxidase enzyme system of dorsal root ganglion (DRG) and sympathetic celiac ganglion (CG) and its regulation in hypertension. The reduced nicotinamide-adenine dinucleotide phosphate oxidase activity of ganglion extracts was measured using fluorescence spectrometry of dihydroethidine; the activity in hypertensive dorsal root ganglion was 34% lower than in normotensive DRG. In contrast, activity was 79% higher in hypertensive CG than normotensive CG. mRNA for the oxidase subunits NOX1, NOX2, NOX4, p47(phox), and p22(phox) were present in both CG and DRG; mRNA for NOX4 was significantly higher in CG than in DRG. The levels of mRNA and protein expression of the membrane-bound catalytic subunit p22(phox) and of the regulatory subunits p47(phox) and Rac-1 were measured in CG and DRG in normotensive and hypertensive rats. p22(phox) mRNA and protein expression was greater in CG of hypertensive rats but not in DRG. Compared with normotensive controls, p47(phox) mRNA and protein, as well as Rac-1 protein, were significantly decreased in hypertensive DRG but not in CG. Immunohistochemical staining of p47(phox) showed translocation from cytoplasm to membrane in hypertensive CG but not in hypertensive DRG. This suggests that reduced nicotinamide-adenine dinucleotide phosphate oxidase activation in sympathetic neurons and sensory neurons is regulated in opposite directions in hypertension. This differential regulation may contribute to unbalanced vasomotor control and enhanced vasoconstriction in the splanchnic circulation.

Presynaptic GABA‐A receptors prevent depression of nicotinic transmission in rabbit coeliac ganglion neurones

We investigated the involvement of GABA-A receptors in the modulation of the nicotinic transmission of central origin in isolated rabbit coeliac ganglia. Our study was performed in vitro and the electrical activity of the ganglionic neurones was recorded using intracellular recording techniques. During iterative stimulation of the splanchnic nerves, the synaptic action potential probability decreased gradually, indicating a depression of the nicotinic activation. Pharmacological agents acting at GABA-A receptors modulated the action potential probability during the train of pulses. Muscimol (a GABA-A receptor agonist), diazepam (a benzodiazepine site agonist) and 1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (a GABA uptake blocker) increased this probability. Conversely, gabazine or bicuculline (two GABA-A receptor antagonists), picrotoxin (a picrotoxin site agonist) and flumazenil (a benzodiazepine site antagonist) reduced it. These results demonstrate that endogenous GABA, released during the train of pulses, facilitates the central nicotinic activation of the ganglionic neurones by acting on GABA-A receptors. Muscimol also reduced the amplitude ratio of excitatory postsynaptic potentials triggered during the paired-pulse protocol without any change in postsynaptic properties. This result is consistent with a presynaptic action of GABA-A receptors. Our study shows that presynaptic GABA-A receptors facilitate the central nicotinic activation of prevertebral ganglionic neurones and thus play a novel role in the integrative properties of the sympathetic prevertebral ganglia.

Structure of peripheral synapses: autonomic ganglia

Final motor neurons in sympathetic and parasympathetic ganglia receive synaptic inputs from preganglionic neurons. Quantitative ultrastructural analyses have shown that the spatial distribution of these synapses is mostly sparse and random. Typically, only about 1%-2% of the neuronal surface is covered with synapses, with the rest of the neuronal surface being closely enclosed by Schwann cell processes. The number of synaptic inputs is correlated with the dendritic complexity of the target neuron, and the total number of synaptic contacts is related to the surface area of the post-synaptic neuron. Overall, most neurons receive fewer than 150 synaptic contacts, with individual preganglionic inputs providing between 10 and 50 synaptic contacts. This variation is probably one determinant of synaptic strength in autonomic ganglia. Many neurons in prevertebral sympathetic ganglia receive additional convergent synaptic inputs from intestinofugal neurons located in the enteric plexuses. The neurons support these additional inputs via larger dendritic arborisations together with a higher overall synaptic density. There is considerable neurochemical heterogeneity in presynaptic boutons. Some synapses apparently lack most of the proteins normally required for fast transmitter release and probably do not take part in conventional ganglionic transmission. Furthermore, most preganglionic boutons in the ganglionic neuropil do not form direct synaptic contacts with any neurons. Nevertheless, these boutons may well contribute to slow transmission processes that need not require conventional synaptic structures.

Fundamentals of Neurogastroenterology: Basic Science

The focus of neurogastroenterology in Rome II was the enteric nervous system (ENS). To avoid duplication with Rome II, only advances in ENS neurobiology after Rome II are reviewed together with stronger emphasis on interactions of the brain, spinal cord, and the gut in terms of relevance for abdominal pain and disordered gastrointestinal function. A committee with expertise in selective aspects of neurogastroenterology was invited to evaluate the literature and provide a consensus overview of the Fundamentals of Neurogastroenterology textbook as they relate to functional gastrointestinal disorders (FGIDs). This review is an abbreviated version of a fuller account that appears in the forthcoming book, Rome III. This report reviews current basic science understanding of visceral sensation and its modulation by inflammation and stress and advances in the neurophysiology of the ENS. Many of the concepts are derived from animal studies in which the physiologic mechanisms underlying visceral sensitivity and neural control of motility, secretion, and blood flow are examined. Impact of inflammation and stress in experimental models relative to FGIDs is reviewed as is human brain imaging, which provides a means for translating basic science to understanding FGID symptoms. Investigative evidence and emerging concepts implicate dysfunction in the nervous system as a significant factor underlying patient symptoms in FGIDs. Continued focus on neurogastroenterologic factors that underlie the development of symptoms will lead to mechanistic understanding that is expected to directly benefit the large contingent of patients and care-givers who deal with FGIDs.