Laurel M. Patterson

Orexin Signaling in the Ventral Tegmental Area Is Required for High-Fat Appetite Induced by Opioid Stimulation of the Nucleus Accumbens

The overriding of satiety and homeostatic control mechanisms by cognitive, rewarding, and emotional aspects of palatable foods may contribute to the evolving obesity crisis, but little is known about neural pathways and mechanisms responsible for crosstalk between the “cognitive” and “metabolic” brain in the control of appetite. Here we show that neural connections between the nucleus accumbens and hypothalamus might be part of this link. Using the well known model of selective stimulation of high-fat intake induced by intra-accumbens injection of the μ-opioid receptor agonist d-Ala2-N-Me-Phe4-gly5-ol-enkephalin (DAMGO), we demonstrate that orexin signaling in the ventral tegmental area is important for this reward-driven appetite to override metabolic repletion signals in presatiated rats. We further show that accumbens DAMGO in the absence of food selectively increases the proportion of orexin neurons expressing c-Fos in parts of the perifornical hypothalamus and that neural projections originating in DAMGO-responsive sites of the nucleus accumbens make close anatomical contacts with hypothalamic orexin neurons. These findings suggest that direct accumbens–hypothalamic projections can stimulate hypothalamic orexin neurons, which in turn through orexin-1 receptor signaling in the ventral tegmental area and possibly other sites interfaces with the motivational and motor systems to increase intake of palatable food.

Distribution and structure of vagal afferent intraganglionic laminar endings (IGLEs) in the rat gastrointestinal tract.

Abstract Intraganglionic laminar endings (IGLEs) are special terminal structures of vagal afferent fibers and have been demonstrated in the myenteric plexus of esophagus and stomach. In order to quantitatively map their presence and distribution over the entire gastrointestinal tract, including the small and large intestines, vagal afferents were anterogradely labeled in vivo by microinjections of the fluorescent carbocyanine dye DiI into the left or right nodose ganglion of adult male rats. In the most successfully labeled cases the highest density of IGLEs was found in the stomach, with about half to one-third of the myenteric ganglia receiving at least one IGLE. The proportion of myenteric ganglia innervated by IGLEs decreased in the small intestine; however, because of its large surface area this gut segment was estimated to contain the highest total number of IGLEs. Both the cecum and colon also contained significant numbers of IGLEs. In the stomach, this vagal afferent innervation by IGLEs was more or less lateralized, with less than 20% of labeled IGLEs found on the contralateral side with respect to the injection. The left/ventral vagus contributed a larger proportion of IGLEs to the proximal duodenum, while the right/dorsal vagus contributed a larger proportion of IGLEs to the distal duodenum and jejunum. Laser scanning confocal microscopy on select specimens revealed further structural details. The parent axon typically formed two or more branches that flanked the ganglia laterally, and in turn produced numerous highly arborizing laminar terminal branches that covered one or both flat sides of the ganglion in a dome-like fashion. The similar distribution patterns and structural details suggest a uniform function for the IGLEs throughout the gastrointestinal tract, but there is as yet no clear proof for any of the hypothesized roles as specialized mechanosensors or local effector terminals.

Vanilloid receptor (VR1) expression in vagal afferent neurons innervating the gastrointestinal tract.

The vanilloid receptor VR1 is a nonselective cation channel activated by capsaicin as well as increases in temperature and acidity, and can be viewed as molecular integrator of chemical and physical stimuli that elicit pain. The distribution of VR1 receptors in peripheral and central processes of rat primary vagal afferent neurons innervating the gastrointestinal tract was investigated by immunohistochemistry. Forty-two percent of neurons in the nodose ganglia retrogradely labeled from the stomach wall expressed low to moderate VR1 immunoreactivity (VR1-IR). VR1-IR was considerably lower in the nodose ganglia as compared to the jugular and dorsal root ganglia. In the vagus nerve, strongly VR1-IR fibers ran in separate fascicles that supplied mainly cervical and thoracic targets, leaving only weakly VR1-IR fibers in the subdiaphragmatic portion. Vagal afferent intraganglionic laminar endings (IGLEs) in the gastric and duodenal myenteric plexus did not express VR1-IR. Similarly, VR1-IR was contained in fibers running in perfect register with vagal afferents, but was not colocalized with horseradish peroxidase in the same varicosities of intramuscular arrays (IMAs) and vagal afferent fibers in the duodenal submucosa anterogradely labeled from the nodose ganglia. Only in the gastric mucosa did we find evidence for colocalization of VR1-IR in vagal afferent terminals. In contrast, many nerve fibers coursing through the myenteric and submucosal plexuses contained detectable VR1-IR, the majority of which colocalized calcitonin gene-related peptide immunoreactivity. In the dorsal medulla there was a dense plexus of VR1-IR varicose fibers in the commissural, dorsomedial and gelatinosus subnuclei of the medial NTS and the lateral aspects of the area postrema, which was substantially reduced, but not eliminated on the ipsilateral side after supranodose vagotomy. It is concluded that about half of the vagal afferents innervating the gastrointestinal tract express low levels of VR1-IR, but that presence in most of the peripheral terminal structures is below the immunohistochemical detection threshold.

Orexin-A projections to the caudal medulla and orexin-induced c-Fos expression, food intake, and autonomic function.

Orexin‐expressing neurons in the hypothalamus project throughout the neuraxis and are involved in regulation of the sleep/wake cycle, food intake, and autonomic functions. Here we specifically analyze the anatomical organization of orexin projections to the dorsal vagal complex (DVC) and raphé pallidus and effects on ingestive behavior and autonomic functions of local orexin‐A administration in nonanesthetized rats. Retrograde tracing experiments revealed that as many as 20% of hypothalamic orexin neurons project to the DVC, where they form straight varicose axon profiles, some of which are in close anatomical apposition with tyrosine hydroxylase (TH)‐, glucagon‐like peptide‐1‐, γ‐aminobutyric acid‐, and nitric oxide synthase‐immunoreactive neurons in a nonselective manner. Similar contacts were frequently observed with neurons of the nucleus of the solitary tract whose activation by gastrointestinal food stimuli was demonstrated by the expression of nuclear c‐Fos immunoreactivity. Orexin‐A administration to the fourth ventricle induced significant Fos‐expression throughout the DVC compared with saline control injections, with about 20–25% of TH‐ir neurons among the stimulated ones. Fourth ventricular orexin injections also significantly stimulated chow and water intake in nonfood‐deprived rats. Direct bilateral injections of orexin into the DVC increased intake of palatable high‐fat pellets. Orexin‐ir fibers also innervated raphé pallidus. Fourth ventricular orexin‐A (1 nmol) activated Fos expression in the raphé pallidus and C1/A1 catecholaminergic neurons in the ventral medulla and increased body temperature, heart rate, and locomotor activity. The results confirm that hypothalamomedullary orexin projections are involved in a variety of physiological functions, including ingestive behavior and sympathetic outflow. J. Comp. Neurol. 485:127–142, 2005.

Brainstem mechanisms integrating gut-derived satiety signals and descending forebrain information in the control of meal size

Ingestive behavior is controlled by a complex interplay between signals conveying availability of (1) potentially ingestible food in the environment, (2) digestible food in the alimentary canal, (3) circulating fuels and (4) stored fuels. Each of these four classes of signals interact with specific sensors and neural circuits whose integrated output determines when food intake is initiated and when it is stopped. Because the final common path responsible for oromotor control is contained within complex neural pattern generators within the brainstem and is intimately linked to sensory information from the alimentary canal, at least part of the integration between the four classes of signals is thought to take place at the level of the caudal brainstem. Here we show that CCK, representing a class 2, or direct signal, and MC4-melanocortin receptor activity, representing a second order class 3/4, or indirect signal, converge in the nucleus of the solitary tract where they modulate activity of the mitogen-activated, extracellular-signal regulated kinases 1 and 2 (ERK) pathway to determine the level of satiation. Blockade of this signaling pathway attenuates suppression of deprivation-induced food intake by intraperitoneal CCK and fourth ventricular MTII injection. Additional findings suggest that specific ERK-phosphorylation sites on ion channels and enzymes involved in catecholamine synthesis of NTS neurons may be involved in ERK-mediated satiation and meal termination. Longer-term downstream effects of ERK activation might involve CREB-mediated gene transcription known to produce plasticity changes in neurocircuitry that could determine inter-meal intervals and the size of future meals.

Capsaicin-resistant vagal afferent fibers in the rat gastrointestinal tract: anatomical identification and functional integrity.

The presence and distribution of vagal fibers and terminals throughout esophagus and gastrointestinal tract that could be anterogradely labeled by nodose ganglion tracer injections was quantitatively assessed in capsaicin- and vehicle-pretreated adult rats, in order to identify the capsaicin-resistant population. Up to 90% of the intraganglionic laminar endings (IGLEs), in the myenteric plexus of the esophagus, and 70-90% in the stomach, as well as 57% of the intramuscular endings or arrays (IMAs) in the fundic stomach survived the capsaicin treatment, while in the upper small intestine only few and in the lower small intestine, the cecum and colon, virtually no IGLEs survived capsaicin treatment. Intramucosal terminals were not assessed. Furthermore, gastric balloon distension-induced c-Fos expression in the dorsal vagal complex was not significantly decreased in capsaicin-treated rats. It is concluded that among primary vagal afferents there is a capsaicin-resistant population that primarily innervates the esophagus and upper gastrointestinal tract, and a capsaicin-sensitive population that innervates mainly the lower tract. At least vagal gastric tension-sensitive afferents also seems to be functionally intact in that they may be capable of synaptically activating second-order neurons in the brainstem.

Orexin inputs to caudal raphé neurons involved in thermal, cardiovascular, and gastrointestinal regulation

Orexin-expressing neurons in the lateral hypothalamus with their wide projections throughout the brain are important for the regulation of sleep and wakefulness, ingestive behavior, and the coordination of these behaviors in the environmental context. To further identify downstream effector targets of the orexin system, we examined in detail orexin-A innervation of the caudal raphé nuclei in the medulla, known to harbor sympathetic preganglionic motor neurons involved in thermal, cardiovascular, and gastrointestinal regulation. All three components of the caudal raphé nuclei, raphé pallidus, raphé obscurus, and parapyramidal nucleus, are innervated by orexin-A-immunoreactive fibers. Using confocal microscopy, we demonstrate close anatomical appositions between varicose orexin-A immunoreactive axon profiles and sympathetic premotor neurons identified with either a transneuronal retrograde pseudorabies virus tracer injected into the interscapular brown fat pads, or with in situ hybridization of pro-TRH mRNA. Furthermore, orexin-A injected into the fourth ventricle induced c-Fos expression in the raphé pallidus and parapyramidal nucleus. These findings suggest that orexin neurons in the hypothalamus can modulate brown fat thermogenesis, cardiovascular, and gastrointestinal functions by acting directly on neurons in the caudal raphé nuclei, and support the idea that orexin’s simultaneous stimulation of food intake and sympathetic activity might have evolved as a mechanism to stay alert while foraging.

GLP-1 receptor signaling is not required for reduced body weight after RYGB in rodents

Exaggerated GLP-1 and PYY secretion is thought to be a major mechanism in the reduced food intake and body weight after Roux-en-Y gastric bypass surgery. Here, we use complementary pharmacological and genetic loss-of-function approaches to test the role of increased signaling by these gut hormones in high-fat diet-induced obese rodents. Chronic brain infusion of a supramaximal dose of the selective GLP-1 receptor antagonist exendin-9-39 into the lateral cerebral ventricle significantly increased food intake and body weight in both RYGB and sham-operated rats, suggesting that, while contributing to the physiological control of food intake and body weight, central GLP-1 receptor signaling tone is not the critical mechanism uniquely responsible for the body weight-lowering effects of RYGB. Central infusion of the selective Y2R-antagonist BIIE0246 had no effect in either group, suggesting that it is not critical for the effects of RYGB on body weight under the conditions tested. In a recently established mouse model of RYGB that closely mimics surgery and weight loss dynamics in humans, obese GLP-1R-deficient mice lost the same amount of body weight and fat mass and maintained similarly lower body weight compared with wild-type mice. Together, the results surprisingly provide no support for important individual roles of either gut hormone in the specific mechanisms by which RYGB rats settle at a lower body weight. It is likely that the beneficial effects of bariatric surgeries are expressed through complex mechanisms that require combination approaches for their identification.

Roux-en-Y gastric bypass surgery increases number but not density of CCK-, GLP-1-, 5-HT-, and neurotensin-expressing enteroendocrine cells in rats.

Background  Roux‐en‐Y gastric bypass (RYGB) surgery is very effective in reducing excess body weight and improving glucose homeostasis in obese subjects. Changes in the pattern of gut hormone secretion are thought to play a major role, but the mechanisms leading to both changed hormone secretion and beneficial effects remain unclear. Specifically, it is not clear whether changes in the number of hormone‐secreting enteroendocrine cells, or changes in the releasing stimuli, or both, are important.

Extracellular Signal-Regulated Kinase 1/2 Signaling Pathway in Solitary Nucleus Mediates Cholecystokinin-Induced Suppression of Food Intake in Rats

Increased food intake is a major factor in the development of obesity, and the control of meal size is a valid approach to reduce food intake in humans. Meal termination, or satiety, is thought to be organized within the caudal brainstem where direct signals from the food handling alimentary canal and long-term signals from the forebrain converge in the solitary nucleus. Cholecystokinin (CCK) released from the gut after ingestion of food has been strongly implicated in nucleus tractus solitarius (NTS)-mediated satiation, but the exact cellular and intracellular signaling events are not understood. Using Western blotting and immunohistochemistry with phosphospecific antibodies, we demonstrate here that peripheral administration of CCK in rats leads to rapid activation of the extracellular signal-regulated kinase (ERK) signaling cascade in NTS neurons and that blockade of ERK signaling with microinfusion of a selective mitogen-activated ERK kinase inhibitor into the fourth ventricle attenuates the capacity of CCK to suppress food intake. In addition, we show that CCK-induced activation of ERK results in phosphorylation of the voltage-dependent potassium channel Kv4.2 and the nuclear transcription factor CREB (cAMP response element-binding protein). The results demonstrate that ERK signaling is necessary for exogenous CCK to suppress food intake in deprived rats and suggest that this pathway may also be involved in natural satiation and the period of satiety between meals through coupling of ERK activation to both cytosolic and nuclear effector mechanisms that have the potential to confer acute and long-term changes in neuronal functioning.