TNF(alpha) modulation of visceral and spinal sensory processing.

The cytokine tumor necrosis factor(alpha) (TNF(alpha)) is associated with a constellation of physiological and behavioral characteristics that follow in response to infection such as fever, fatigue, listlessness, loss of appetite, malaise, and tactile hypersensitivity. These responses are examples of central nervous system (CNS) functions modified by the activated immune system. Our studies have focused on the involvement of TNF(alpha) in CNS control of gastrointestinal function and "visceral malaise". We have demonstrated that TNF(alpha) can elicit gastric stasis in a dose-dependent fashion via its interaction with vago-vagal neurocircuitry in the brainstem. Sensory elements of the vago-vagal reflex circuit (i.e., neurons of the solitary tract [NST] and area postrema [AP]) are activated by exposure to TNF(alpha), while the efferent elements (i.e., dorsal motor neurons of the vagus [DMN]) cause gastroinhibition. Transient exposure to low doses of TNF(alpha) cause potentiated (exaggerated) NST responses to stimulation. Subsequent studies suggest that TNF(alpha) presynaptically modulates the release of glutamate from primary afferents to the NST. Using immunohistochemical studies, we have observed the constitutive expression of the TNFR1 receptor on central vagal afferents and spinal trigeminal afferents in the medulla, as well as on cells and afferent fibers within the dorsal root ganglia and within laminae I and II of the dorsal horn throughout the spinal cord. The constitutive presence of these receptors on these afferents may explain why inflammatory or infectious processes that generate TNF(alpha) can disrupt gastrointestinal functions and cause tactile hypersensitivity. These receptors may also play a critical role in the chronic allodynia and hyper-reflexia observed after spinal cord injury or peripheral nerve damage.

Noradrenergic neurons in the rat solitary nucleus participate in the esophageal-gastric relaxation reflex.

Activation of esophageal mechanosensors excites neurons in and near the central nucleus of the solitary tract (NSTc). In turn, NSTc neurons coordinate the relaxation of the stomach [i.e., the receptive relaxation reflex (RRR)] by modulating the output of vagal efferent neurons of the dorsal motor nucleus of the vagus (DMN). The NSTc area contains neurons with diverse neurochemical phenotypes, including a large population of catecholaminergic and nitrergic neurons. The aim of the present study was to determine whether either one of these prominent neuronal phenotypes was involved in the RRR. Immunohistochemical techniques revealed that repetitive esophageal distension caused 53% of tyrosine hydroxylase-immunoreactive (TH-ir) neurons to colocalize c-Fos in the NSTc. No nitric oxide synthase (NOS)-ir neurons in the NSTc colocalized c-Fos in either distension or control conditions. Local brain stem application (2 ng) of alpha-adrenoreceptor antagonists (i.e., alpha1-prazosin or alpha2-yohimbine) significantly reduced the magnitude of the esophageal distension-induced gastric relaxation to approximately 55% of control conditions. The combination of yohimbine and prazosin reduced the magnitude of the reflex to approximately 27% of control. In contrast, pretreatment with either the NOS-inhibitor NG-nitro-l-arginine methyl ester or the beta-adrenoceptor antagonist propranolol did not interfere with esophageal distension-induced gastric relaxation. Unilateral microinjections of the agonist norepinephrine (0.3 ng) directed at the DMN were sufficient to mimic the transient esophageal-gastric reflex. Our data suggest that noradrenergic, but not nitrergic, neurons of the NSTc play a prominent role in the modulation of the RRR through action on alpha1- and alpha2-adrenoreceptors. The finding that esophageal afferent stimulation alone is not sufficient to activate NOS-positive neurons in the NSTc suggests that these neurons may be strongly gated by other central nervous system inputs, perhaps related to the coordination of swallowing or emesis with respiration.

Dissociation of the effects of nucleus raphe obscurus or rostral ventrolateral medulla lesions on eliminatory and sexual reflexes.

Rat preparations were used to investigate long-term changes in external anal sphincter (EAS) contractions and reflexive penile erection following electrolytic lesions of the nucleus raphe obscurus (nRO) or the rostral ventrolateral medulla. EAS contractions were measured electromyographically (EAS EMG) following distention of the EAS with a 5-mm probe. Penile erections were measured using a standard ex copula reflex testing paradigm. At 48 h postlesion, 100% of nRO-lesioned animals displayed reflexive erections and the magnitude of EAS EMG was significantly greater in lesioned animals than in sham controls. These results suggested EAS hyperreflexia following destruction of the nRO. By 14 days postlesion, EAS responsiveness in nRO-lesioned animals had returned to levels comparable to nonlesioned animals. No measures of penile erection were affected by nRO lesions. In animals with nucleus gigantocellularis (Gi) and lateral nucleus paragigantocellularis (Gi-lPGi) lesions, no significant changes to EAS reflexes were observed at any time point. At 48 h postoperative, Gi-lPGi lesions significantly reduced the latency to first erection and increased the number of erections elicited relative to controls. Similar facilitation of erection latency was observed at 14 days postlesion, while erection number and flip total were no longer significantly different from controls. These and previous studies suggest that the nRO regulates defecatory reflexes in the rat. These data further suggest that the comingled EAS and bulbospongiosus (BS) motoneurons are controlled by discrete and separate brainstem circuits and that increases in EAS and penile reflexes after spinal cord lesions are mediated by loss of different descending inputs.

TNF-alpha-induced c-Fos generation in the nucleus of the solitary tract is blocked by NBQX and MK-801.

Previous studies have shown that identified neurons of the nucleus of the solitary tract (NST) are excited by the cytokine tumor necrosis factor-alpha (TNF-alpha). Vagal afferent connections with the NST are predominantly glutaminergic. Therefore, we hypothesized that TNF-alpha effects on NST neurons may be via modulation of glutamate neurotransmission. The present study used activation of the immediate early gene product c-Fos as a marker for neuronal activation in the NST. c-Fos expression was evaluated after microinjections of TNF-alpha in the presence or absence of either the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX) or the N-methyl-D- aspartate (NMDA) antagonist MK-801. To assess the specificity of the interaction between TNF-alpha and glutamate, c-Fos expression was also evaluated after injection of oxytocin (OT) (which has a direct excitatory effect in this area of the brain stem) in the presence and absence of NBQX or MK-801. c-Fos labeling was significantly increased in the NST after TNF-alpha exposure. Coinjection of either NBQX or MK-801 with TNF-alpha prevented significant c-Fos induction in the NST. Microinjections of OT also induced significant NST c-Fos elevation, but this expression was unaffected by coinjection of either antagonist with OT. These data lead us to conclude that TNF-alpha activation of NST neurons depends on glutamate and such an interaction is not generalized to all agonists that act on the NST.

Tumor necrosis factor-alpha induces cFOS and strongly potentiates glutamate-mediated cell death in the rat spinal cord.

Excitotoxic cell death due to glutamate release is important in the secondary injury following CNS trauma or ischemia. Proinflammatory cytokines also play a role. Both glutamate and tumor necrosis factor-alpha (TNF(alpha)) are released immediately after spinal cord injury. Neurophysiological studies show that TNF(alpha) can potentiate the effects of glutamatergic afferent input to produce hyperactivation of brain-stem sensory neurons. Therefore, we hypothesized that TNF(alpha) might act cooperatively with glutamate to affect cell death in the spinal cord as well. Nanoinjections of either TNF(alpha) (60 pg) or kainate (KA; 32 ng) alone into the thoracic gray resulted in almost no tissue damage or cell death 90 min after injection. However, the combination of TNF(alpha) plus KA at these same doses produced a large area of tissue necrosis and neuronal cell death, an effect which was blocked by the AMPA receptor antagonist CNQX (17 ng). These results suggest that secondary injury may involve potentiation of AMPA receptor-mediated excitatory cell death by TNF(alpha).

c-Fos generation in the dorsal vagal complex after systemic endotoxin is not dependent on the vagus nerve.

The present study used activation of the c-Fos oncogene protein within neurons in the dorsal vagal complex (DVC) as a marker of neuronal excitation in response to systemic endotoxin challenge [i.e. , lipopolysaccharide (LPS)]. Specifically, we investigated whether vagal connections with the brain stem are necessary for LPS cytokine- induced activation of DVC neurons. Systemic exposure to LPS elicited a significant activation of c-Fos in neurons in the nucleus of the solitary tract (NST) and area postrema of all thiobutabarbital-anesthetized rats examined, regardless of the integrity of their vagal nerves. That is, rats with both vagi cervically transected were still able to respond with c-Fos activation of neurons in the DVC. Unilateral cervical vagotomy produced a consistent but small reduction in c-Fos activation in the ipsilateral NST of all animals within this experimental group. Given that afferent input to the NST is exclusively excitatory, it is not surprising that unilateral elimination of all vagal afferents would diminish NST responsiveness (on the vagotomized side). These data lead us to conclude that the NST itself is a primary central nervous system detector of cytokines.

TNF-alpha activates solitary nucleus neurons responsive to gastric distension.

Tumor necrosis factor-alpha (TNF-alpha) is liberated as part of the immune response to antigenic challenge, carcinogenesis, and radiation therapy. Previous studies have implicated elevated circulating levels of this cytokine in the gastric hypomotility associated with these disease states. Our earlier studies suggest that a site of action of TNF-alpha may be within the medullary dorsal vagal complex. In this study, we describe the role of TNF-alpha as a neuromodulator affecting neurons in the nucleus of the solitary tract that are involved in vago-vagal reflex control of gastric motility. The results presented herein suggest that TNF-alpha may induce a persistent gastric stasis by functioning as a hormone that modulates intrinsic vago-vagal reflex pathways during illness.

Induction of endogenous tumor necrosis factor-alpha: suppression of centrally stimulated gastric motility.

Gastric stasis is frequently seen in conjunction with critical infectious illness, chronic inflammatory disorders, radiation sickness, and carcinogenesis. These conditions are associated with elevated circulating levels of the cytokine tumor necrosis factor-alpha (TNF-alpha). The present studies examined the relationship between endogenously produced TNF-alpha and the central neural mechanisms that augment gastric motility. Systemic lipopolysaccharide (LPS) was employed to induce TNF-alpha production in thiobutabarbital-anesthetized rats. Sixty minutes after intravenous LPS injection, gastric motility could not be stimulated by a potent centrally acting gastrokinetic stimulant, thyrotropin-releasing hormone (TRH). This failure to elicit gastric motility via central mechanisms coincided with high circulating levels of TNF-alpha. However, intravenous injections of bethanecol, a peripherally acting cholinergic agonist with direct gastrokinetic effects, were still able to elicit normal increases in gastric motility in the presence of TNF-alpha and LPS. Therefore, the inability to stimulate gastric motility via central TRH could not be attributed to the direct inhibitory effects of either LPS or TNF-alpha on the stomach. If the production of endogenous TNF-alpha was suppressed via the use of urethan as the anesthetic agent, then intravenous injections of LPS were no longer effective in suppressing gastric motility. Thus these effects on gastric motility are not directly attributable to LPS nor are they due to direct effects on the gastric smooth muscle. Our previous study demonstrated that microinjection of femtomole quantities of TNF-alpha in the brain stem dorsal vagal complex (DVC) can modulate gastric motility. This central TNF-alpha effect on gastric motility was dose dependent and required an intact vagal efferent pathway. The results from these two studies suggest that systemically produced TNF-alpha may gain access to the DVC to modulate gastric function.

Brainstem pathways responsible for oesophageal control of gastric motility and tone in the rat.

1. Previous anatomical studies indicate that the nucleus of the solitary tract, pars centralis (NSTc) contains the neurones which receive vagal afferent input from the oesophagus. The purpose of the present study was to characterize the NSTc circuits in the medulla that may be responsible for oesophageal control of gastric motility. 2. Moderate balloon distension of the oesophagus of the rat (14-18 mmHg) provoked a significant reduction in gastric motility and tone recorded with strain gauges. This receptive relaxation effect was eliminated by bilateral lesions centred on the NSTc. 3. NSTc cells activated by oesophageal distension were labelled extracellularly and juxtacellularly with neurobiotin. NSTc neurones send axonal projections throughout the entire rostral-caudal extent of the dorsal motor nucleus of the vagus (DMN). These NSTc-DMN connections were confirmed by retrograde transport of neurobiotin from DMN to NSTc. NSTc neurones were observed with dendrites arborizing within the ependymal lining of the fourth ventricles. Thus, NSTc neurones may be in position to monitor blood-borne or ventricular agents and to alter the function of gastric-vago-vagal reflexes in response to these stimuli. 4. Neurophysiological recordings identified two subpopulations of DMN neurones which may be either activated or inhibited by oesophageal distension. Neurones excited by oesophageal distension were located mainly lateral and caudal in the DMN; neurones inhibited by oesophageal stimulation were located in medial and rostral DMN. 5. Our neurobiotin tracing results verified earlier studies showing that the NSTc projects to the intermediate reticular nucleus and the compact division of the nucleus ambiguus. Additionally, we found that the NSTc may be involved in reciprocal connections with the anterior, rostrolateral NST. 6. These results suggest that the gastric relaxation evoked by oesophageal distension is critically dependent on intact brainstem vago-vagal circuits. The NSTc, the recipient of oesophageal afferent projections from the vagus nerve, sends axons to the entire DMN, the source of parasympathetic control of the stomach. DMN neurones respond differentially to oesophageal distension, reinforcing the view that oesophageal afferents may provoke gastric relaxation by activating a vagal inhibitory pathway while simultaneously inhibiting a vagal excitatory pathway.

Descending projections from the nucleus raphe obscurus to pudendal motoneurons in the male rat.

Previous physiological and behavioral studies have shown that the nucleus raphe obscurus (nRO) modulates pelvic floor reflex function (Yamanouchi and Kakeyama [1992] Physiol. Behav. 51:575-579; Beattie et al. [1996] Soc. Neurosci. Abstr. 22:722.4; Holmes et al. [1997] Brain Res. 759:197-204). In the present study, small injections of fluorescent tracers were used to investigate direct descending projections from the rostral and caudal portions of the brainstem nRO to retrogradely labeled pudendal motoneurons (MN) in the male rat. The caudal nRO projects into the ventral and lateral funiculi of the spinal cord, with arborizations in the thoracic intermediolateral cell column; in laminae VII, IX, and X of the lumbosacral cord; and in the sacral parasympathetic nucleus (SPN). Many identified external anal sphincter and ischiocavernosus MNs appeared to be in direct apposition with fibers originating from the caudal nRO; and more than half of the bulbospongiosus MNs that were identified appeared to receive such descending input. In addition to the nRO spinal autonomic and pudendal motoneuronal targets, projections were observed to regions of the intermediate gray that contain interneurons organizing the pelvic floor reflexes and to MN pools that are involved in functionally related somatic activities. Finally, several neurons in the lumbar enlargement were labeled retrogradely with FluoroRuby after injections into the nRO and the immediately adjacent reticular formation. Thus, the nRO may be in a position to modulate the coordinated actions of autonomic preganglionic and functionally related skeletal MN activity involved in sexual and eliminative reflex functions.

Nucleus raphe obscurus (nRO) regulation of anorectal motility in rats.

Previous research has demonstrated that anorectal contractions in the rat are modulated by activation of spinal autonomic circuits. In the present study, anterograde tracing of descending pathways originating from the caudal nucleus raphe obscurus (nRO) revealed that this nucleus projects to cells within the intermediolateral (IML) cell column of the thoracic cord and the sacral parasympathetic nucleus (SPN). These anatomical studies suggested that the nRO may influence the regulation of spinal reflexes of the pelvic floor. In a second set of experiments, acute rat preparations were used to investigate changes in anorectal motility during electrical stimulation of the nRO. Anorectal contractions were measured by a fluid-filled manometer. Electrical stimulation of the nRO significantly reduced spontaneous anorectal activity when compared to baseline contractions recorded for 1 min prior to stimulation. Stimulation sites outside the nRO did not affect anorectal contractions when compared to either (a) the 1-min pre-stimulation baseline for that site or (b) the 1-min stimulation period for sites within the nRO. Stimulation of caudal portions of the nRO were more likely than the rostral nRO to reduce anorectal contractions. Given that the SPN contains preganglionic neurons which may be involved in control of anorectal contractions (mediated via the pelvic nerve), the studies presented here suggest a functional role for nRO regulation of preganglionic motoneurons innervating the distal gut of the rat.

Effect of pancreatic polypeptide on rat dorsal vagal complex neurons.

1. Pancreatic polypeptide (PP) microinjected into the dorsal vagal complex (DVC) elevates gastric activity through a vagal mechanism. Thus, it was hypothesized that PP alters the activity of nuclei comprising the DVC, i.e. the nucleus tractus solitarii (NTS) and the dorsal motor nucleus (DMN). 2. In vivo and in vitro approaches were used. For in vivo studies, micropipettes were used for recording and injecting vehicle or PP. Neurons were identified as NTS or DMN using orthodromic and antidromic activation, respectively, following vagal stimulation. Gastric-related DVC neurons were located using antral inflation. For in vitro studies, DMN neurons were recorded from medullary slices. 3. Of the twenty-eight NTS and DMN neurons identified, fifteen were activated, six inhibited and seven unaffected after PP microinjection. Forty-two gastric-related neurons were located in the DVC, of which twenty-five were stimulated by PP and seventeen exhibited no change. No gastric-related cells were inhibited. 4. For in vitro studies, 66% of DMN neurons were activated by PP (n = 27/47) while the remaining 33% were inhibited (n = 14/47). Similar results were obtained in normal or synaptic blockade media. 5. These results support the hypothesis that PP alters DVC neuronal activity, which may thereby lead to the previously observed alterations in gastric activity.

Vagal control of digestion: modulation by central neural and peripheral endocrine factors.

Vago-vagal reflex control circuits in the dorsal vagal complex of the brainstem provide overall coordination over digestive functions of the stomach, small intestine and pancreas. The neural components forming these reflex circuits are under significant descending neural control. By adjusting the excitability of the different components of the reflex, alterations in digestion control can be produced by the central nervous system. Additionally, the dorsal vagal complex is situated within a circumventricular region without an effective "blood-brain barrier". As a result, vago-vagal reflex circuitry is also exposed to humoral influences which profoundly alter digestive functions by acting directly on brainstem neurons. Behavioral and endocrine physiological observations suggest that this "humoral afferent pathway" may significantly alter the regulation of food intake.

Vagovagal reflex control of digestion: afferent modulation by neural and "endoneurocrine" factors.

Vagovagal reflex control circuits in the dorsal vagal complex of the brain stem provide overall coordination of gastric, small intestinal, and pancreatic digestive functions. The neural components forming these reflex circuits are under substantial descending neural control. By adjusting the excitability of the differing components of the reflex, significant alterations in digestion control can be produced by the central nervous system. Additionally, the dorsal vagal complex is situated within a circumventricular region without a "blood-brain barrier." As a result, vagovagal reflex circuitry is also exposed to humoral influences, which can profoundly alter digestive functions by acting directly on brain stem neurons.

Modulation of IgA synthesis by neuroendocrine peptides.

Prolactin, estrogen, and progesterone together induce proliferation of the mammary glands with a coincident increase in the IgA-secreting plasma cells in the tissue. Of these three hormones, Prolactin is the most effective single peptide in stimulating IgA production. Vasoactive intestinal peptide (VIP) and somatostatin have also been shown to modulate IgA production. Although more extensive investigation is required, delineation of an immune axis involving prolactin, VIP, and somatostatin in mucosal immune tissue such as mammary gland is a promising area of research with clinical relevance to neonatal resistance to infection.

Activation of the bed nucleus of the stria terminalis increases gastric motility in the rat.

The diencephalic bed nucleus of the stria terminalis is known to make direct, peptide-containing axonal connections with the brainstem dorsal vagal complex, i.e., the nucleus of the solitary tract and the dorsal motor nucleus of the vagus. Given these anatomical data, one would predict that the bed nucleus, like other forebrain nuclei with similar connections, should function to alter parasympathetic autonomic functions. To examine this possibility, we activated the bed nucleus via electrical microstimulation and glutamate microinjections while monitoring gastric motility with miniature extraluminal strain gauges. We found that activation of the bed nucleus produced an increase in gastric motility that was atropine sensitive. This finding raises the possibility that the bed nucleus may be a significant forebrain regulator of gastrointestinal function.

Nucleus raphe obscurus (nRO) influences vagal control of gastric motility in rats.

Because the nucleus raphe obscurus (nRO) maintains a direct connection with the dorsal vagal complex in the medulla, this nucleus has the potential to influence vagal control of gastric function. Both electrical- and glutamate-induced activation of the nRO were found to enhance gastric motility and tone in the rat. The gastric responses to nRO stimulation were abolished by peripheral muscarinic blockade.

Thyrotropin-releasing hormone: effects on identified neurons of the dorsal vagal complex.

Previous reports have demonstrated that intraventricular administration of thyrotropin-releasing hormone (TRH) markedly elevates parasympathetic efferent activity. The following study determined if this response could be attributed to an effect of TRH on the neurons in the dorsal motor nucleus of the vagus (DMN) and/or the nucleus tractus solitarius (NTS), the nuclei that comprise the dorsal vagal complex (DVC). Individual DMN or NTS units were identified electrophysiologically by using stimulating electrodes placed on the cervical vagus. Alterations in firing rate of identified cells in response to pressure injection of TRH (10-40 fmol in 10-40 pl) or equal volumes of artificial cerebrospinal fluid (ACSF) were monitored. Of the DMN cells that were responsive to TRH, all were excited, whereas all responsive NTS cells were inhibited by this peptide. TRH was characterized as potent and had long-lasting effects on cells in DMN and NTS. The action of TRH on both nuclei in the dorsal vagal complex may explain the powerful effects of this peptide on vagally mediated functions.

Dorsal medullary serotonin and gastric motility: enhancement of effects by thyrotropin-releasing hormone.

The effects of serotonin (5-HT) and thyrotropin-releasing hormone (TRH) on gastric motility patterns were investigated. Microinjection of serotonin (8 pmol in 4 nl) into the dorsal motor nucleus of the vagus produced a small increase in motility and tone, an effect which declined with repeated injections. As demonstrated previously, TRH (1 nmol in 1 microliter) applied to the surface of the dorsal medulla evoked a large increase in gastric motility and tone. After gastric motility returned to baseline following the TRH injection, we found that subsequent 5-HT injections, which previously evoked small changes in motility and tone, now evoked large increases in these indices. TRH augmentation of 5-HT-mediated effects on autonomic nuclei may be a significant feature in the alterations in gastric function that accompany the sleep-waking cycle and stress-related gastric pathology.

Oxytocin, oxytocin antagonist, TRH, and hypothalamic paraventricular nucleus stimulation effects on gastric motility.

The roles of thyrotropin releasing hormone (TRH) and oxytocin as central regulators of gastric motility were investigated. Picomolar (4 picomoles) quantities of TRH injected into the dorsal motor nucleus of the vagus (DMN) elicited a significant increase in gastric motility while the same quantity of oxytocin elicited a reduction in phasic contractile activity and tone. The action of these peptides mimics the excitatory and inhibitory effects of stimulating the paraventricular nucleus of the hypothalamus (PVN); it is likely that this hypothalamic structure regulates gastric function through its peptidergic connections with medullary vagal structures. This hypothesis is supported by our observations that injections of an oxytocin antagonist into the DMN produced a disinhibition of gastric motility and an increase in the motility evoked by subsequent PVN stimulation. Vagotomy eliminated all subsequent central effects on motility of these peptides.