Effects of vagal and splanchnic section on food intake, weight, serum leptin and hypothalamic neuropeptide Y in rat

Truncal vagotomy can cause reduced food intake and weight loss in humans and laboratory animals. In order to investigate some of the factors that might contribute to this effect, we studied changes in ingestive behaviour, whole body and organ weights, serum leptin and hypothalamic neuropeptide Y in rats with bilateral vagal section, bilateral splanchnic nerve section and combined vagotomy plus splanchnectomy. Pyloromyotomy was combined with vagotomy to lessen effects of vagotomy on gastric emptying. Animals with vagotomy or vagotomy plus splanchnectomy lost weight and decreased their daily food intake relative to animals with splanchnectomy alone, rats with bilateral sham exposure of one or both nerve, or rats with pyloromyotomy alone. Serum leptin and white fat mass, 4 weeks after vagotomy, were about 20% of the values in the sham-operated animals at this time. No effect for splanchnic nerve section alone was observed. Pyloromyotomy caused no reduction in weight or fat mass, but reduced serum leptin. Following vagotomy with or without splanchnic nerve section, neuropeptide Y was elevated in the arcuate nucleus relative to values for the other four groups. Changes in neuropeptide Y were inversely correlated with levels of serum leptin. It is concluded that the effect of vagotomy could be due to the loss of a feeding signal carried by vagal afferent neurons, or to changed humoral signals, for example, increased production of a satiety hormone. However, it cannot be attributed to signals that reduce feeding (for example, gastric distension) reaching the central nervous system via the splanchnic nerves. The changes were sufficient to cause weight loss even though serum leptin was decreased, a change that would be expected to increase food intake.     

Cardiovascular effects and c-Fos expression in the rat hindbrain in response to innocuous stomach distension

The present work was planned to study the effects of non-noxious gastric distension on hemodynamic variables and on cardiovascular hindbrain areas detected by means of c-Fos immunoreactivity, to determine the afferent and central mechanisms involved. In anesthetized rats, innocuous stomach distension increased arterial blood pressure and heart rate and induced c-Fos immunoreactivity within nucleus tractus solitarii, nucleus ambiguus, ventrolateral medulla and lateral reticular nucleus. Bilateral vagotomy abolished the pressor response and c-Fos immunoreactivity in nucleus ambiguus and ventrolateral medulla. Also, c-Fos immunoreactivity was significantly decreased in nucleus tractus solitarii and lateral reticular nucleus. After bilateral splanchnicotomy the pressor and tachycardic responses caused by gastric distension were reduced. c-Fos immunoreactivity in nucleus tractus solitarii, lateral reticular nucleus and nucleus ambiguus was reduced in comparison to the intact rats. In ventrolateral medulla a preferential localization of c-Fos immunoreactivity was found within its caudal portion. It was shown that such gastric distension, known to activate low threshold mechanoreceptors, induced cardiovascular effects via both vagal and splanchnic afferents and involving their central convergence and interaction in modulating the baroreceptor buffer system.     

Central and peripheral vagal transport of cholecystokinin binding sites occurs in afferent fibers

The effects of various vagal lesions on cholecystokinin (CCK) binding sites in the nucleus tractus solitarii (NTS) and area postrema (AP) and the peripheral transport of CCK binding sites in the cervical vagus were examined in rats by in vitro autoradiography with [125I]CCK-8. Unilateral supraganglionic, but not subdiaphragmatic vagotomy significantly reduced CCK binding in the ipsilateral NTS. Specific unilateral afferent, but not efferent, vagal rootlet transections also significantly reduced NTS CCK binding ipsilateral to the transections. None of the vagal lesions altered CCK binding in the AP. Infraganglionic but not supraganglionic vagotomy eliminated the peripheral transport of vagal CCK binding sites. Together these results demonstrate that CCK receptors in the NTS are located on vagal afferent terminals, that CCK receptors in the AP are likely postsynaptic to a vagal afferent input and that the peripheral and central transport of vagal CCK binding sites occurs in afferent fibers.     

Vagal afferents mediate early satiation and prevent flavour avoidance learning in response to intraperitoneally infused exendin-4

Glucagon‐like peptide‐1 receptor (GLP‐1R) agonists such as exendin‐4 (Ex‐4) affect eating and metabolism and are potential candidates for treating obesity and type II diabetes. In the present study, we tested whether vagal afferents mediate the eating‐inhibitory and avoidance‐inducing effects of Ex‐4. Subdiaphragmatic vagal deafferentation (SDA) blunted the short‐term (< 1 h) but not long‐term eating‐inhibitory effect of i.p.‐infused Ex‐4 (0.1 μg/kg) in rats. A dose of 1 μg/kg Ex‐4 reduced 0.5, 1, 2 and 4 h cumulative food intake in SDA and sham‐operated rats to a similar extent. Paradoxically, SDA but not sham rats developed a conditioned flavour avoidance (CFA) after i.p. Ex‐4 (0.1 μg/kg). SDA completely blunted the induction of c‐Fos expression by Ex‐4 in the hypothalamic paraventricular nucleus. Ex‐4, however, increased the number of c‐Fos expressing cells, independent of intact vagal afferents, in the nucleus accumbens and in the central nucleus of the amygdala, the lateral external parabrachial nucleus, the caudal ventrolateral medulla and the dorsal vagal complex. These data suggest that intact vagal afferents are only necessary for the full expression of the early satiating effect of Ex‐4 but not for later eating‐inhibitory actions, when circulating Ex‐4 might reach the brain via the circulation. Our data also dissociate the satiating and avoidance‐inducing effects of the low Ex‐4 dose tested under our conditions and suggest that vagal afferent signalling may protect against the development of CFA. Taken together, these findings reveal a complex role of vagal afferents in mediating the effects of GLP‐1R activation on ingestive behaviour.     

Effects of perivagal administration of capsaicin on post-surgical food intake

The vagus nerve has been related to the short-term control of food intake. This involvement has previously been explored by examining the food intake of animals after recovery from a vagotomy or immediately after the intervention, among other methods. In the present work, a study was conducted on the impact of the perivagal application of capsaicin (a specific neurotoxic treatment that destroys most of the vagal afferent pathways) on the intake of water and solid (experiment 1) or liquid (experiment 2) food presented after the surgery The results of experiment 1 showed that lesioned animals consume significantly larger amounts of food and water compared with controls at 6, 12, and 24 h (but not at 48 or 72 h) after the surgical intervention. Likewise, experiment 2 revealed a greater intake of liquid food by capsaicin-treated animals at the first post-surgical sessions. These data are discussed in terms of the role played by vagal afferent fibers in the control of short-term food intake.     

Medullary substrates mediating antinociception produced by electrical stimulation of the vagus

Electrical stimulation of afferents of the right cervical vagus inhibited the tail-flick reflex elicited by noxious heat in barbiturate-anesthetized rats. This inhibitory effect was eliminated in rats receiving local anesthetic blockade of either the nucleus tractus solitarii (NTS), the lateral reticular nuclei, the nucleus raphe magnus-medullary reticular formation, or nucleus raphe obscurus regions of the medulla. Similarly, the vasodepressor and bradycardic effects of vagal stimulation were either attenuated or eliminated by local anesthetic blockade of these regions. Microinjection of the non-specific glutamate antagonist gamma-D-glutamylglycine (DGG) into the NTS region also eliminated vagally evoked inhibition of the tail-flick reflex, hypotension, and bradycardia. Conversely, microinjection of glutamate into the NTS region resulted in inhibition of the tail-flick reflex, hypotension, and bradycardia. These findings with DGG and glutamate are consistent with the view that glutamate serves as a neurotransmitter of the primary vagal afferents mediating these antinociceptive and cardiovascular responses. These results are discussed in terms of vagal afferent influences on somatosensory, somatomotor, and cardiovascular function.     

CART in the dorsal vagal complex: sources of immunoreactivity and effects on Fos expression and food intake

CART-peptide (CARTp) has been shown to suppress food intake, particularly when injected into the 4th ventricle of rats, and the presence of CART in nodose ganglia suggested a role in satiation. Based on retrograde tracing from the DVC combined with CART immunohistochemistry and supranodose vagotomy, we found that CART immunoreactivity in varicose fibers of the dorsal vagal complex originates from vagal afferents, sparse projections from the medullary reticular formation and the arcuate/retrochiasmatic nucleus of the hypothalamus, and most likely also from local CART neurons in the area postrema and NTS. In the nodose ganglia, 17% of neurons with projections to the stomach and 41% to the duodenum express CART-IR. CART-IR vagal afferents significantly contribute to the rich fiber plexus in mainly the commissural NTS and the adjacent area postrema. Injections of CARTp into the 4th ventricle strongly suppressed sucrose drinking and stimulated expression of c-Fos in the NTS. Injections of CARTp directly into various subnuclei of the NTS were less effective in suppressing food intake. The findings suggest that the critical site for CART's suppression of food intake is not in the termination zone of CART-containing vagal afferents in the commissural NTS, and that CART release from vagal afferent terminals plays a minor role in satiation. The functional role of CART in vagal afferents and the site of food intake suppression by 4th ventricular CARTp remain to be determined.     

Jejunal or portal vein infusions of lipids increase hepatic vagal afferent activity

Jejunal infusions of linoleic acid, corn oil, or caprylic acid significantly increased hepatic vagal afferent activity, whereas saline infusions were ineffective. The magnitude of response was greatest with either linoleic acid or corn oil. Hepatic portal infusions of linoleic acid, Liposyn II, or caprylic acid significantly increased hepatic vagal afferent activity, whereas 5% albumin/phosphate buffer vehicle was ineffective. The magnitude of response was greatest with either linoleic acid or Liposyn II. These data show that either jejunal or portal infusions of lipids increase activity of hepatic vagal afferents and could potentially serve as a complementary and/or alternative substrate to celiac vagal afferents in mediating the effects of jejunal infusions of lipids in suppressing food intake.     

Contribution of the vagus nerve to angiotensin II binding sites in the canine medulla

Specific angiotensin II (Ang II) binding sites are present in the dorsal medulla of several species and dose-related cardiovascular effects are produced by microinjection of the peptide into this region. Because the anatomical location of Ang II binding sites in the area postrema (ap), nucleus tractus solitarii (nTS) and dorsal motor nucleus of the vagus (dmnX) coincides with the topography of vagal afferent fibers and efferent motor neurons, the effect of either nodose ganglionectomy or cervical vagotomy on Ang II binding sites in the dorsomedial medulla was investigated in dogs by in vitro receptor autoradiography. Two weeks after unilateral ganglionectomy, there was a marked reduction in the density of specific Ang II binding sites in the ipsilateral ap, nTS and dmnX and an absence of binding sites in the region where vagal afferent fibers course through the rostral medulla. Unilateral cervical vagotomy, which has been shown to spare central processes of afferent fibers, resulted in a loss of binding only in the ipsilateral dmnX. We also show that Ang II binding sites are present in the nodose ganglion and central and peripheral processes of the vagus nerve. The data indicate that medullary Ang II binding sites are associated with both vagal afferent fibers and efferent motor neurons.     

Activation of Nesfatin‐1‐Containing Neurones in the Hypothalamus and Brainstem by Peripheral Administration of Anorectic Hormones and Suppression of Feeding via Central Nesfatin‐1 in Rats

Peripheral anorectic hormones, such as glucagon‐like peptide (GLP)‐1, cholecystokinin (CCK)‐8 and leptin, suppress food intake. The newly‐identified anorectic neuropeptide, nesfatin‐1, is synthesised in both peripheral tissues and the central nervous system, particularly by various nuclei in the hypothalamus and brainstem. In the present study, we examined the effects of i.p. administration of GLP‐1 and CCK‐8 and co‐administrations of GLP‐1 and leptin at subthreshold doses as confirmed by measurement of food intake, on nesfatin‐1‐immunoreactive (‐IR) neurones in the hypothalamus and brainstem of rats by Fos immunohistochemistry. Intraperitoneal administration of GLP‐1 (100 μg/kg) caused significant increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the supraoptic nucleus (SON), the area postrema (AP) and the nucleus tractus solitarii (NTS) but not in the paraventricular nucleus (PVN), the arcuate nucleus (ARC) or the lateral hypothalamic area (LHA). On the other hand, i.p. administration of CCK‐8 (50 μg/kg) resulted in marked increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the SON, PVN, AP and NTS but not in the ARC or LHA. No differences in the percentage of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the nuclei of the hypothalamus and brainstem were observed between rats treated with saline, GLP‐1 (33 μg/kg) or leptin. However, co‐administration of GLP‐1 (33 μg/kg) and leptin resulted in significant increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the AP and the NTS. Furthermore, decreased food intake induced by GLP‐1, CCK‐8 and leptin was attenuated significantly by pretreatment with i.c.v. administration of antisense nesfatin‐1. These results indicate that nesfatin‐1‐expressing neurones in the brainstem may play an important role in sensing peripheral levels of GLP‐1 and leptin in addition to CCK‐8, and also suppress food intake in rats.     

Hepato-vagal pathway associated with nicotine's anorectic effect in the rat.

Nicotine reduces appetite and body weight. Because the hepato-portal area senses different types of nutrients that transmit signals via vagal afferent nerves to the hypothalamus to modify food intake and feeding pattern, we investigated the effect of nicotine on a hepato-vagal-hypothalamic pathway. Low doses of nicotine (< 10 ng) injected into portal vein (i.p.v.) decreased, while high doses of nicotine increased (> or = 10 ng) electrophysiological activity of hepatic vagal afferents. Stimulatory effect of high dose of nicotine on vagal hepatic afferents was blocked by a prior i.p.v. injection of curare (30 microg) or hexamethonium (1 mg). Furthermore, activities of gastric vagal and adrenal sympathetic efferents were suppressed by low-dose, but stimulated by high-dose i.p.v. nicotine. These reflex effects did not occur in hepatic vagotomized rats. Results of experiments demonstrate that in addition to nicotine's anorectic effect being mediated via a direct central action, nicotine also acts peripherally via hepatic vagal afferents from sensors of nicotine in the hepato-portal region.     

Effects of Cholecystokinin in the Supraoptic Nucleus and Paraventricular Nucleus are Negatively Modulated by Leptin in 24‐h Fasted Lean Male Rats

Cholecystokinin (CCK) and leptin are two important satiety factors that are considered to act in synergy to reduce meal size. Peripheral injection of CCK activates neurones in several hypothalamic nuclei, including the supraoptic (SON) and paraventricular (PVN) nuclei and neurones in the brainstem of fed rats. We investigated whether peripheral leptin would modulate the effects of CCK on neuronal activity in the hypothalamus and brainstem of fasted rats by investigating Fos expression in the PVN, SON, arcuate nucleus, ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), area postrema (AP) and the nucleus tractus solitarii (NTS). Male rats, fasted for 24 h, received either one i.p. injection of vehicle, leptin or CCK‐8 alone, or received one injection of vehicle or leptin before an i.p. injection of CCK‐8. We found that CCK increased Fos expression in the PVN and SON as well as in the NTS and AP, but had no effect on Fos expression in the arcuate nucleus, VMH or DMH compared to vehicle. Leptin injected alone significantly increased Fos expression in the arcuate nucleus but had no effect on Fos expression in the VMH, DMH, SON, PVN, AP or NTS compared to vehicle. Fos expression was significantly increased in the AP in rats injected with both leptin and CCK compared to rats injected with vehicle and CCK. Unexpectedly, there was significantly less Fos expression in the PVN and SON of fasted rats injected with leptin and CCK than in rats injected with vehicle and CCK, suggesting that leptin attenuated CCK‐induced Fos expression in the SON and PVN. However, Fos expression in the NTS was similar in fasted rats injected with vehicle and CCK or with leptin and CCK. Taken together, these results suggest that leptin dampens the effects of CCK on Fos expression in the SON and PVN, independently from NTS pathways, and this may reflect a direct action on magnocellular neurones.     

Induction of Fos-like protein in neurons of the medulla oblongata after electrical stimulation of the vagus nerve in anesthetized rabbit

Antibodies against the c-fos protein product Fos were used to map the first- and higher-order neurons in the rabbit medulla oblongata after electrical stimulation of the vagus nerve. Fos immunoreactivity appeared bilaterally except in the nucleus tractus solitarii. Seven areas were labeled: the nucleus tractus solitarii, the area postrema, the subnucleus lateralis caudalis magnocellularis medullar oblongata, the lateral reticular nucleus, the ambiguus nucleus, the dorsal part of the spinal trigeminal nucleus, the nucleus reticularis lateralis, the lateral border of the external cuneatus nucleus, the medial part of the inferior olivary nucleus (subnucleus β). The last two areas have never been visualized with conventional tracing techniques and may represent higher-order neurons connected to visceral vagal pathways. No labeling was observed in the nodose ganglion.     

Acute baroreceptor unloading evokes Fos expression in anesthetized rat brain

Functional organization of brain pathways subserving the baroreceptor reflex was investigated by mapping immunoreactivity of Fos protein, a neuronal activity marker, in response to acute baroreceptor unloading in anesthetized rats. Compared with normal control and sham operation, sinoaortic denervation (SAD) evoked a distinctive pattern of Fos expression in the nucleus tractus solitarii (NTS), the primary termination of baroreceptor afferents. The sinoaortic denervation also induced a prominent and reproducible Fos expression in specific regions of the brainstem and forebrain, which receive direct or indirect inputs from the nucleus tractus solitarii. These brain regions included the rostral ventrolateral medulla (RVLM), lateral parabrachial nucleus (LPBN), hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON), and central amygdaloid nucleus (CeA). These findings help us to identify brain regions that are specifically responsive to decreased arterial baroreceptor activity, without the accompanying confounding variables of behavioral arousal or stress.     

Withdrawal and restoration of central vagal afferents within the dorsal vagal complex following subdiaphragmatic vagotomy

Vagotomy, a severing of the peripheral axons of the vagus nerve, has been extensively utilized to determine the role of vagal afferents in viscerosensory signaling. Vagotomy is also an unavoidable component of some bariatric surgeries. Although it is known that peripheral axons of the vagus nerve degenerate and then regenerate to a limited extent following vagotomy, very little is known about the response of central vagal afferents in the dorsal vagal complex to this type of damage. We tested the hypothesis that vagotomy results in the transient withdrawal of central vagal afferent terminals from their primary central target, the nucleus of the solitary tract (NTS). Sprague–Dawley rats underwent bilateral subdiaphragmatic vagotomy and were sacrificed 10, 30, or 60 days later. Plastic changes in vagal afferent fibers and synapses were investigated at the morphological and functional levels by using a combination of an anterograde tracer, synapse‐specific markers, and patch‐clamp electrophysiology in horizontal brain sections. Morphological data revealed that numbers of vagal afferent fibers and synapses in the NTS were significantly reduced 10 days following vagotomy and were restored to control levels by 30 days and 60 days, respectively. Electrophysiology revealed transient decreases in spontaneous glutamate release, glutamate release probability, and the number of primary afferent inputs. Our results demonstrate that subdiaphragmatic vagotomy triggers transient withdrawal and remodeling of central vagal afferent terminals in the NTS. The observed vagotomy‐induced plasticity within this key feeding center of the brain may be partially responsible for the response of bariatric patients following gastric bypass surgery. J. Comp. Neurol. 521:3584‐3599, 2013. © 2013 Wiley Periodicals, Inc.     

Immunohistochemical detection of glutamate in rat vagal sensory neurons

Vagal primary afferent neurons have their cell bodies located in the nodose (inferior) and jugular (superior) vagal ganglia and send terminals into the nucleus tractus solitarii (NTS) which lies in the dorsomedial medulla. The presence of glutamate (Glu)-containing neurons in the rat nodose ganglion was investigated using immunohistochemistry. Glu-immunoreactivity on nodose sections was found in neuronal perikarya and nerve fibers, but not in non-neuronal elements such as Schwann cells and satellite cells. Both immunoreactive and non-immunoreactive ganglion cells were observed. The immunoreactive ganglion cells amounted to about 60% of the nodose population. No specific intraganglionic localization was observed for the non-immunoreactive cells. Immunoreactive perikarya were slightly smaller than the non-immunoreactive ones, but no relationship was found between size and staining intensities of immunoreactive neurons. The present data indicate that immunodetectable Glu is present in a large population of vagal afferent neurons. They therefore add to a growing body of evidence suggesting that Glu may be the main neurotransmitter released by vagal afferent terminals within the nucleus tractus solitarii.     

Differential content of vesicular glutamate transporters in subsets of vagal afferents projecting to the nucleus tractus solitarii in the rat

The vagus nerve contains primary visceral afferents that convey sensory information from cardiovascular, pulmonary, and gastrointestinal tissues to the nucleus tractus solitarii (NTS). The heterogeneity of vagal afferents and their central terminals within the NTS is a common obstacle for evaluating functional groups of afferents. To determine whether different anterograde tracers can be used to identify distinct subpopulations of vagal afferents within NTS, we injected cholera toxin B subunit (CTb) and isolectin B4 (IB4) into the vagus nerve. Confocal analyses of medial NTS following injections of both CTb and IB4 into the same vagus nerve resulted in labeling of two exclusive populations of fibers. The ultrastructural patterns were also distinct. CTb was found in both myelinated and unmyelinated vagal axons and terminals in medial NTS, whereas IB4 was found only in unmyelinated afferents. Both tracers were observed in terminals with asymmetric synapses, suggesting excitatory transmission. Because glutamate is thought to be the neurotransmitter at this first primary afferent synapse in NTS, we determined whether vesicular glutamate transporters (VGLUTs) were differentially distributed among the two distinct populations of vagal afferents. Anterograde tracing from the vagus with CTb or IB4 was combined with immunohistochemistry for VGLUT1 or VGLUT2 in medial NTS and evaluated with confocal microscopy. CTb‐labeled afferents contained primarily VGLUT2 (83%), whereas IB4‐labeled afferents had low levels of vesicular transporters, VGLUT1 (5%) or VGLUT2 (21%). These findings suggest the possibility that glutamate release from unmyelinated vagal afferents may be regulated by a distinct, non‐VGLUT, mechanism. J. Comp. Neurol. 522:642–653, 2014. © 2013 Wiley Periodicals, Inc.     

Evidence for vagal involvement in the eating elicited by adrenergic stimulation of the paraventricular nucleus

We examined the role of the vagus nerves in mediating the eating and preprandial drinking seen after injection of norepinephrine (NE) into the region of the paraventricular hypothalamic nucleus of satiated rats. Complete subdiaphragmatic vagotomy (confirmed by gastric secretion tests) abolished the NE-elicited eating response, whether the diet used was lab chow, milk, or a milk-chow mixture, and attenuated, by 38%, the NE-elicited drinking response. These effects occurred independently of changes in body weight or daily food intake imposed by vagal surgery. The vagotomized rats retained the capacity to rapidly increase eating in response to food deprivation or insulin injection challenges, indicating that the effect of vagotomy on NE-induced eating was not due to some non-specific impairment. Efferent vagal blockade of intact rats with systemic injections of atropine methyl nitrate (0.4 mg/kg) prior to central NE infusions yielded similar results. Finally-selective section of the coeliac branch of the vagus produced a 49% reduction of NE, elicited eating, as compared with a 29% reduction in water intake, while selective section of the gastric plus hepatic vagal branches, leaving only the coeliac branch intact, did not significantly affect either ingestive response. Both of these selectively vagotomized groups displayed an unimpaired capacity to increase food intake in response to systemic insulin injections. These results suggest participation of efferent vagal mechanisms in the adrenergic feeding, and, to a lesser extent, drinking phenomena and are consistent with a particular role for some function under coeliac vagal control (perhaps insulin secretion) in modulating the effects of NE on feeding behavior.     

Evidence that MK-801 stimulates intraoral intake by acting on hepatic afferents

Satiety signals from the gastrointestinal tract travel via vagal afferents to the nucleus of the solitary tract (NTS) in the brain stem, the first central relay in a neural network which controls food intake. The non-competitive NMDA antagonist MK-801 facilitates food intake in rats by acting on the NTS. Here we report that hepatic portal vein infusion of MK-801 (25 or 50 microg/kg) increases intake of an intraorally infused 1 M solution of sucrose (by 113 +/- 9 and 132 +/- 11%, respectively) and that this effect is prevented by hepatic vagotomy. By contrast, jugular vein infusion of MK-801 fails to increase sucrose intake but induces forward locomotion, indicating activation of a central mechanism. These data suggest that MK-801 can stimulate food intake by acting peripherally on hepatic vagal afferents.