Activation of Intra-nodose Ganglion P2X7 Receptors Elicit Increases in Neuronal Activity.

Vagus nerve innervates several organs including the heart, stomach, and pancreas among others. Somas of sensory neurons that project through the vagal nerve are located in the nodose ganglion. The presence of purinergic receptors has been reported in neurons and satellite glial cells in several sensory ganglia. In the nodose ganglion, calcium depletion-induced increases in neuron activity can be partly reversed by P2X7 blockers applied directly into the ganglion. The later suggest a possible role of P2X7 receptors in the modulation of neuronal activity within this sensory ganglion. We aimed to characterize the response to P2X7 activation in nodose ganglion neurons under physiological conditions. Using an ex vivo preparation for electrophysiological recordings of the neural discharges of nodose ganglion neurons, we found that treatments with ATP induce transient neuronal activity increases. Also, we found a concentration-dependent increase in neural activity in response to Bz-ATP (ED50 = 0.62 mM, a selective P2X7 receptor agonist), with a clear desensitization pattern when applied every ~ 30 s. Electrophysiological recordings from isolated nodose ganglion neurons reveal no differences in the responses to Bz-ATP and ATP. Finally, we showed that the P2X7 receptor was expressed in the rat nodose ganglion, both in neurons and satellite glial cells. Additionally, a P2X7 receptor negative allosteric modulator decreased the duration of Bz-ATP-induced maximal responses without affecting their amplitude. Our results show the presence of functional P2X7 receptors under physiological conditions within the nodose ganglion of the rat, and suggest that ATP modulation of nodose ganglion activity may be in part mediated by the activation of P2X7 receptors.

Lipopolysaccharides and trophic factors regulate the LPS receptor complex in nodose and trigeminal neurons.

Binding of bacterial lipopolysaccharides (LPS) to toll-like receptor 4 (TLR4) triggers an innate immunoresponse associated with pain and inflammation. The expression, and to a greater extent the regulation of TLR4 and its auxiliary proteins (myeloid differentiation protein 1 (MD1), myeloid differentiation protein 2 (MD2) and cluster of differentiation 14 (CD14)), are both poorly understood in trigeminal and nodose neurons. We used a combination of Western blotting, semi-quantitative polymerase chain reaction (PCR), pharmacological manipulation and immunohistochemistry. The expression pattern and regulation by LPS and trophic factors of TLR4/MD2/CD14 and radioprotective protein of 105kDa (RP105)/MD1 were determined in neonatal trigeminal and nodose mice neurons. We found that all these proteins were expressed in both trigeminal and nodose neurons. The trophic factors Artemin and nerve growth factor (NGF) up-regulated MD2 and RP105 mRNA levels in trigeminal neurons. In nodose neurons the trophic factors brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) up-regulated MD1 and RP105 mRNA levels. Also we observed that in both neuronal types LPS acutely (within 20 min) down-regulated CD14 and MD2 mRNAs. In addition, LPS increased significantly the proportion of trigeminal and nodose neurons expressing nociceptin/orphanin FQ in culture probably acting via TLR4/MD2. Although the exact mechanisms underlying the regulation by trophic factors and LPS require further elucidation, the findings of this study indicate that LPS acts through its archetypical receptor in trigeminal and nodose neurons.

Lipopolysaccharide signaling in the carotid chemoreceptor pathway of rats with sepsis syndrome.

In addition to their role in cardiorespiratory regulation, carotid body (CB) chemoreceptors serve as sensors for inflammatory status and as a protective factor during sepsis. However, lipopolysaccharide-induced sepsis (LPS) reduces CB responsiveness to excitatory or depressant stimuli. We tested whether LPS exerts a direct effect on the carotid chemoreceptor pathway, the CB and its sensory ganglion. We determined that the rat CB and nodose-petrosal-jugular ganglion complex (NPJgc) express TLR4, TNF-α and its receptors (TNF-R1 and TNF-R2). LPS administration (15mg/kg intraperitoneally) evoked MyD88-mechanism pathway activation in CB and NPJgc, with NF-κB p65, p38 MAPK, and ERK activation. Consistently, LPS increased TNF-α and TNF-R2. Double-labeling studies showed that the aforementioned pathway occurs in TH-containing glomus cells and NPJgc neurons, components of the chemosensitive neural pathway. Thus, our results suggest that LPS acting directly through TLR4/MyD88-mechanism pathways increases TNF-α and TNF-R2 expression in the carotid chemoreceptor pathway. These results show a novel afferent pathway to the central nervous system during endotoxemia, and could be relevant in understanding sepsis pathophysiology and therapy.

Alterations in the brain-gut axis underlying visceral chemosensitivity in Nippostrongylus brasiliensis-infected mice.

BACKGROUND & AIMS: Visceral hypersensitivity, a hallmark of irritable bowel syndrome, is generally considered to be mechanosensitive in nature and mediated via spinal afferents. Both stress and inflammation are implicated in visceral hypersensitivity, but the underlying molecular mechanisms of visceral hypersensitivity are unknown. METHODS: Mice were infected with Nippostrongylus brasiliensis (Nb) larvae, exposed to environmental stress and the following separate studies performed 3-4 weeks later. Mesenteric afferent nerve activity was recorded in response to either ramp balloon distention (60 mm Hg), or to an intraluminal perfusion of hydrochloric acid (50 mmol/L), or to octreotide administration (2 micromol/L). Intraperitoneal injection of cholera toxin B-488 identified neurons projecting to the abdominal viscera. Fluorescent neurons in dorsal root and nodose ganglia were isolated using laser-capture microdissection. RNA was hybridized to Affymetrix Mouse whole genome arrays for analysis to evaluate the effects of stress and infection. RESULTS: In mice previously infected with Nb, there was no change in intestinal afferent mechanosensitivity, but there was an increase in chemosensitive responses to intraluminal hydrochloric acid when compared with control animals. Gene expression profiles in vagal but not spinal visceral sensory neurons were significantly altered in stressed Nb-infected mice. Decreased afferent responses to somatostatin receptor 2 stimulation correlated with lower expression of vagal somatostatin receptor 2 in stressed Nb-infected mice, confirming a link between molecular data and functional sequelae. CONCLUSIONS: Alterations in the intestinal brain-gut axis, in chemosensitivity but not mechanosensitivity, and through vagal rather than spinal pathways, are implicated in stress-induced postinflammatory visceral hypersensitivity.

Angiotensin II inhibition of Ca2+ currents is independent of ATR1 angiotensin II receptor activation in rat adult vagal afferent neurons.

Angiotensin II (ANG II) has the ability to modulate the activity of neurons involved in the cardiovascular regulation. One effective way of doing that is by changing calcium currents. In the present study, we investigated the effects of ANG II on high-voltage-activated (HVA) Ca2+ currents measured in adult vagal afferent neurons using the whole-cell patch-clamp technique. In addition, we demonstrated the presence of ATR1 and ATR2 receptors mRNA at nodose neurons using conventional reverse transcriptase-polymerase chain reaction (RT-PCR). ANG II (100 nM) decreased the HVA Ca2+ current (peak current recorded at 0 mV: -60.9+/-8.7 pA/pF in control conditions versus -31.9+/-5.7 pA/pF in the presence of ANG II) and shifted the Ca2+ current activation to a more negative membrane potential (control V0.5=-12.5+/-1.5 mV versus -18.4+/-2.8 mV during perfusion with ANG II). Losartan (500 nM) was not able to prevent the ANG II effect on the HVA Ca2+ current making unlikely the involvement of the ATR1 receptor. When ANG II was perfused in the continuous presence of saralasin, a non-selective ANG II receptor antagonist, we observed a faster but transient inhibition of HVA Ca2+ current. The inhibition was not sustained as observed when we applied ANG II alone and the HVA Ca2+ current recovered with time reaching levels close to the control. Unexpectedly, treatment with the ATR2 blocker PD 123,319 (500 nM) caused a significant inhibition on the HVA Ca2+ current making rather difficult any further conclusions. The above results allow us to conclude that ANG II induced inhibition on the HVA Ca2+ current is probably not via ATR1 receptor activation.

Electrophysiological properties of rat nodose ganglion neurons co-transplanted with carotid bodies into the chick chorioallantoic membrane.

The electrophysiological properties of nodose ganglion neurons were evaluated immediately after removing nodose ganglia from young adult rats and 3 to 10 days after nodose ganglia implantation -either alone or co-implanted with carotid bodies- onto the chick chorioallantoic membrane. Implanted and co-implanted nodose neurons were less excitable than acutely recorded nodose neurons. Co-implanted neurons also showed reduced amplitudes for both action potentials and spike after-hyperpolarizations relative to those found in. acutely recorded nodose ganglion neurons and a smaller time constant (T) than that found in implanted neurons. In addition, no spontaneous activity was recorded from nodose ganglion neurons co-implanted with carotid bodies during 3-9 days, which suggests that functional synapses between carotid glomus cells and nodose neurons were not yet established. Results indicate the feasibility of obtaining viable nodose neurons for up to 10 days grafted onto the chick chorioallantoic membrane, where they can conserve most of their passive and active membrane properties and also are susceptible to carotid bodies trophic influences. They also suggest that nodose neurons would need more time for the development of functional synapses when grafted with carotid body glomus cells.

Decreases in the expression of CGRP and galanin mRNA in central and peripheral neurons related to the control of blood pressure following experimental hypertension in rats.

Calcitonin gene-related peptide (alpha CGRP) and galanin (GAL) are peptides known to participate in central mechanisms of blood pressure control. Nonetheless, variations in the synthesis of the peptides in response to a hypertensive challenge are not well described, specially using a model, which allows acute and chronic analyses. In this study, we have employed in situ hybridization to analyse changes in mRNA expression of alpha CGRP and GAL in the nucleus tractus solitarii (NTS), hypothalamic paraventricular nucleus (PVN) as well as petrosal and nodose ganglia after aortic coarctation-induced hypertension in rats. Acute (2h) and chronic (3 and 7 days) analyses were performed in order to evaluate the involvement of both peptides in different periods of hypertension. The analysis of relative mRNA levels showed significant differences between sham-operated and aortic coarcted hypertensive rats. alpha CGRP mRNA expression was decreased 2h (40%) and 3 days (42%) in nodose and petrosal ganglia, respectively, after coarctation. No changes in CGRP mRNA signal were seen in the NTS and PVN in the analysed periods. GAL mRNA expression was decreased in the NTS (19%) and PVN (55%), 3 and 7 days, respectively, after coarctation-induced hypertension. No changes in GAL mRNA expression were observed in petrosal and nodose ganglia following aortic coarctation. Data suggest that alpha CGRP and GAL may participate in the mechanisms involved in the establishment/maintenance of hypertension induced by aortic coarctation. Acute changes might be involved with the adaptation to the hypertensive state, while changes at the chronic phase might be related to counteraction of hypertension.

Change in the expression of NPY receptor subtypes Y1 and Y2 in central and peripheral neurons related to the control of blood pressure in rats following experimental hypertension.

Neuropeptide Y (NPY) is known to participate in central mechanisms of blood pressure control. However, variations on the expression of its receptors in response to a hypertensive challenge are not well defined, specially when considering that Y1 and Y2 often mediate opposite responses. In this study we have employed in situ hybridization to analyze changes in mRNA expression of NPY receptor subtypes Y1 and Y2 in the nucleus tractus solitarii (NTS), paraventricular nucleus of the hypothalamus (PVN) and petrosal and nodose ganglions 2 h, 3 and 7 days after aortic coarctation induced hypertension. Quantification by image analysis showed significant differences between sham-operated and aortic-coarcted hypertensive rats. Y1 receptor mRNA expression was increased (39%) in petrosal ganglion, 3 days after surgery. Y2 receptor mRNA expression was increased (143%) in the NTS of hypertensive compared with sham rats 2 h after surgery. Y2 receptor mRNA was decreased (62%) in the nodose ganglion of hypertensive compared with sham rats 2 h after surgery. No change was seen in Y1 and Y2 mRNA expression in the PVN in any analyzed period. The data suggest that NPY Y1 and Y2 receptors might participate in the mechanisms involved in the establishment/maintenance of hypertension induced by aortic coarctation. Acute changes seem to be involved with the adaptation to the new hypertensive state.

Macrostructure of the cranial cervical ganglionar complex and distal vagal ganglion during post natal development in dogs

Doze cães domésticos (Canis familiaris) foram dissecados para o estudo da situação, arranjo e ramificação nervosa do gânglio distal do nervo vago e gânglio cervical cranial. Os gânglios apresentaram-se fusiformes e recobertos pelo músuculo digástrico. Os principais ramos do gânglio cervical cranial observados foram os ramos para a artéria carótida externa e artéria carótida interna. Destacou-se o nervo laringeal cranial como ramo do gânglio distal do nervo vago. O estudo revelou gânglio cervical cranial e o gânglio distal do vago eram estruturas bem desenvolvidas e não encontrou-se diferenças anatômicas entre os gânglios observados em ambos antímeros.

Macrostructure of the cranial cervical ganglionar complex and distal vagal ganglion during post natal development in dogs

Doze cães domésticos (Canis familiaris) foram dissecados para o estudo da situação, arranjo e ramificação nervosa do gânglio distal do nervo vago e gânglio cervical cranial. Os gânglios apresentaram-se fusiformes e recobertos pelo músuculo digástrico. Os principais ramos do gânglio cervical cranial observados foram os ramos para a artéria carótida externa e artéria carótida interna. Destacou-se o nervo laringeal cranial como ramo do gânglio distal do nervo vago. O estudo revelou gânglio cervical cranial e o gânglio distal do vago eram estruturas bem desenvolvidas e não encontrou-se diferenças anatômicas entre os gânglios observados em ambos antímeros.(AU)

Slow inactivation of sodium currents in the rat nodose neurons.

Nodose neurons express sodium currents that can be differentiated based on their sensitivity to tetrodotoxin. Several studies have demonstrated significant differences in voltage-dependence and kinetics of activation and inactivation between tetrodotoxin-sensitive and tetrodotoxin-resistant currents. However, little is known about the slow inactivation. Using whole cell patch-clamp technique fast and slow inactivation of sodium currents were studied in cultured rat nodose neurons. Tetrodotoxin-resistant currents recovered much more rapidly after a 15-ms depolarization than tetrodotoxin-sensitive currents. However, repeated 5-ms depolarizations at 10 Hz induced a cumulative inhibition that was more prolonged in tetrodotoxin-resistant compared to tetrodotoxin-sensitive currents. Consistent with these findings, slow inactivation proceeded more rapidly and was more complete for the tetrodotoxin-resistant than for tetrodotoxin-sensitive currents. While the voltage-dependence of fast inactivation differed significantly between the pharmacologically distinct currents, the voltage-dependence of slow inactivation was similar for both sodium currents. We conclude that slow inactivation of sodium currents can be triggered by trains of brief depolarizations. The resulting prolonged decrease in membrane excitability may contribute to the different patterns of action potential generation observed in primary afferent neurons.

Núcleos do tracto solitário: além da primeira sinapse: funçöes neurovegetativas e hemeostáticas / Nuceus of solitary tract: beyond the first sinapse: autonomic and homeostasis functions

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On the asymmetry of the primary branching of vagal sensory axons: possible role of the supporting tissue.

Central and peripheral nonmedullated processes of vagal nodosal neurons of the cat were studied in normal nerves and after regeneration along their anatomical course and along the hypoglossal nerve. Nonmedullated fibers above the ganglion and in the root had comparable sizes (approximately 0.37 micron2) and caliber distribution. Below the ganglion, the cross-sectional area increased to 1.0 micron2. In axons of equal caliber, supranodosal and radicular fibers had similar microtubular densities while infranodosal fibers had two- to threefold that of the former. Regenerated fibers were studied after a recovery period of 6-9 months. Regrown axons were smaller than their parent axons; in turn, these were smaller than normal axons. This holds for central and peripheral nodosal branches, for homologous and heterologous regeneration. Regrown peripheral branches, either along their anatomical pathway or along the hypoglossal nerve, showed no change in microtubular density. Central branches exhibited their characteristic microtubular content when they regenerated along their anatomical course, but when regrowth took place along the hypoglossal nerve, the original low microtubular content of these branches increased to match the high content of peripheral fibers; parent central axons also shifted their microtubular content toward the pattern of peripheral fibers. We propose that the supporting tissue participates in specifying the organization of axonal microtubules.