Research reportCART in the dorsal vagal complex: sources of immunoreactivity and effects on Fos expression and food intake
Introduction
Cocaine and amphetamine-regulated transcript (CART) is widely expressed in the central and peripheral nervous system, suggesting multiple functional roles [25]. Intracerebroventricular injection of CART-peptide (55–102, CARTp) was found to suppress food intake [3], [24], [26], [36], [40] and stimulate expression of c-Fos in many brain areas, including the paraventricular and dorsomedial hypothalamic nuclei, central nucleus of the amygdala, and the parabrachial and solitary nuclei in the hindbrain [40]. Because CART is co-expressed with POMC in neurons of the lateral arcuate nucleus, with MCH in neurons of the perifornical hypothalamus, and with oxytocin and TRH in the paraventricular nucleus, it was implicitly assumed that ICV CARTp suppresses food intake by acting on downstream targets of these neuron populations in various parts of the hypothalamus. However, experimental testing of this assumption has been made difficult by lack of knowledge of CART-receptor(s).
An alternative site for CARTp to decrease food intake has been recently proposed [9]. Vagal afferent neurons in the rat nodose ganglia were found to express CART-immunoreactivity (CART-IR), and most CART-IR neurons also expressed CCKA-receptor mRNA. In addition there is a rich CART-IR fiber plexus in the dorsal medulla [9], [23]. Thus, CART released upon CCK-stimulation of gastrointestinal vagal afferents could function as a satiety transmitter at the level of the NTS [9]. In support of this hypothesis, we showed earlier that CARTp was more potent in suppressing food intake when injected into the 4th ventricle as compared to the lateral ventricle [43]. Subsequently it was shown that in rats with an aqueductal plug to interrupt flow of cerebrospinal fluid, CARTp injection into the 3rd ventricle was no longer able to suppress food intake, while CARTp injection into the 4th ventricle continued to suppress food intake [2]. The recent observation that CARTp injections into specific hypothalamic nuclei resulted in increased rather than decreased food intake [1], could be taken as further evidence that CARTp delivered into the cerebral ventricles acts at a hindbrain site to decrease food intake.
The general aim of the present study was to identify the site(s) in the hindbrain for CARTp to suppress food intake. Based on Broberger’s hypothesis that the site of action is in the NTS [9], we expected that injection of CARTp directly into the NTS would be more potent in suppressing food intake than 4th ventricular injection. Similarly, we expected that 4th ventricular CARTp injection would stimulate c-Fos expression in NTS neurons. Although Fos was stimulated in the NTS by CARTp injections into the 4th ventricle, direct injections into the NTS did not potently suppress food intake. These results prompted us to carry out a detailed analysis of the distribution of CART-IR neurons in the NTS and nodose ganglia and their axon terminals in the dorsal vagal complex. Specifically, our aim was to determine the sources of CART-IR fiber terminals in the dorsal vagal complex, combining retrograde tracing of potential inputs by means of Fluorogold injections into the NTS/area postrema, with CART immunohistochemistry, and using unilateral supranodose vagotomy. Furthermore, we determined the proportion of vagal afferent neurons in the nodose ganglia projecting to the stomach and duodenum that express CART-IR.
Section snippets
Animals
Adult male Sprague–Dawley rats (Harlan Industries, Indianapolis, IN) weighing 280–320 g were housed individually in hanging wire mesh cages in a climate controlled room (22±2 °C) on a 12:12 h light cycle with lights on at 07:00 h and lights off at 19:00 h. Food and water were available ad libitum except as specified below.
Intracranial injections
Animals were anesthetized with ketamine/xylazine/acepromazine (80/5/1.6 mg/kg, s.c.) and given atropine (1 mg/kg, i.p.). A 24-Ga stainless steel guide cannula (Plastics One,
Fourth ventricular but not direct NTS CARTp injections suppress food intake
Because of the high density of CART fibers contributed by vagal afferents we aimed the injector tip at a medial location in the commissural part of the NTS, just below the area postrema. Histological verification showed that injector tips were either in the commissural NTS or the ventral aspects of the area postrema near the midline (Fig. 1B). Injection of 80 pmol CARTp at this location was completely ineffective and 200 pmol only marginally suppressed sucrose intake [at 6 min: t28=1.82, P
Discussion
The fact that fourth ventricular CARTp injections suppress food intake [2], [43] and that vagal primary afferent neurons express CART [9] suggested that CART might play a physiological role in vagally-mediated gastrointestinal satiety [9]. Here we report that CARTp injected directly into an area of the NTS that receives gastrointestinal vagal afferent terminals in moderately hungry rats does not potently suppress short-term food intake. This finding prompted us to examine the extent of
Acknowledgements
We thank Tricia Antolik for help with measuring food intake and Richard C. Rogers for stimulating discussions. Research was supported by the National Institute for Diabetes and Digestive and Kidney Diseases, DK47348.
References (44)
- et al.
Intramedullary connections of the rostral nucleus of the solitary tract in the hamster
Brain Res.
(1991) - et al.
Food-related gastrointestinal signals activate caudal brainstem neurons expressing both NMDA and AMPA receptors
Brain Res.
(2001) - et al.
Effects of cholecystokinin (CCK-8) on two classes of gastroduodenal vagal afferent fibre
J. Auton. Nerv. Syst.
(1990) - et al.
MK-801 interferes with nutrient-related signals for satiation
Appetite
(1998) - et al.
Visceral afferent participation in delayed satiation following NMDA receptor blockade
Physiol. Behav.
(1998) - et al.
Mediation by nucleus tractus solitarii glutamatergic neurotransmission of the cardiovascular reflex evoked by distal esophageal distension
Auton. Neurosci.
(2002) - et al.
Cocaine- and amphetamine-regulated transcript peptide-immunoreactivity in dorsal motor nucleus of the vagus neurons of immature rats
Brain Res. Dev. Brain Res.
(2001) - et al.
Differential expression of cocaine- and amphetamine-regulated transcript-immunoreactivity in the rat spinal preganglionic nuclei
Neurosci. Lett.
(2000) - et al.
Cocaine- and amphetamine-regulated transcript peptide-immunoreactivity in adrenergic C1 neurons projecting to the intermediolateral cell column of the rat
J. Chem. Neuroanat.
(2002) - et al.
Leptin activates hypothalamic CART neurons projecting to the spinal cord
Neuron
(1998)
Glutaminergic vagal afferents may mediate both retching and gastric adaptive relaxation in dogs
Auton. Neurosci.
Long-term effects on feeding and body weight after stimulation of forebrain or hindbrain CRH receptors with urocortin
Brain Res.
Cooperation of NMDA and tachykinin NK(1) and NK(2) receptors in the medullary transmission of vagal afferent input from the acid-threatened rat stomach
Pain
CART peptides
Regul. Pept.
Stimulation of 5-HT2 receptors in the nucleus tractus solitarius enhances NMDA receptor-mediated reflex-evoked bradycardiac responses in the rat
Auton. Neurosci.
A fluorescent labeling strategy for staining the enteric nervous system
J. Neurosci. Methods
Cholecystokinin: proofs and prospects for involvement in control of food intake and body weight
Neuropeptides
Capsaicin application to central or peripheral vagal fibers attenuates CCK satiety
Peptides
Actions of cocaine- and amphetamine-regulated transcript (CART) peptide on regulation of appetite and hypothalamo–pituitary axes in vitro and in vivo in male rats
Brain Res.
Projections from the rostral parvocellular reticular formation to pontine and medullary nuclei in the rat: involvement in autonomic regulation and orofacial motor control
Neuroscience
Delay in meal termination follows blockade of N-methyl-d-aspartate receptors in the dorsal hindbrain
Brain Res.
Recombinant CART peptide induces c-Fos expression in central areas involved in control of feeding behaviour
Brain Res.
Cited by (59)
Functional anatomy of the vagus system: How does the polyvagal theory comply?
2022, Biological PsychologyDemystifying functional role of cocaine- and amphetamine-related transcript (CART) peptide in control of energy homeostasis: A twenty-five year expedition
2021, PeptidesCitation Excerpt :The NTS is the central site of sensory vagal afferent termination, and it has been shown that approximately 50 % vagal sensory neurons that reside in the NG express Cartpt [42,99]. In addition to the NG, CART + neuron cell bodies residing in the NTS itself and AP, the medullary reticular formation and the hypothalamus may all contribute the pool of CART + fibers in the NTS [68]. The fact that the knockdown of CARTp in the NG prevented antibody-induced hyperphagia [69] suggests that, the NG provide the main source of endogenous NTS CARTp that inhibit food intake [69].
CART in energy balance and drug addiction: Current insights and mechanisms
2020, Brain ResearchBlunted Vagal Cocaine- and Amphetamine-Regulated Transcript Promotes Hyperphagia and Weight Gain
2020, Cell ReportsCitation Excerpt :Clinical studies using vagal neuromodulation are showing early signs of success for treating obesity (Ikramuddin et al., 2014), highlighting the vagus nerve as a viable peripheral therapeutic target. The cocaine- and amphetamine-regulated transcript (CART), a neuropeptide transmitter that is expressed in a subpopulation of VANs (Broberger et al., 1999; de Lartigue et al., 2007; Kupari et al., 2019; Zheng et al., 2002) innervating the gut (Bai et al., 2019; Zheng et al., 2002), may be an important molecular signal for control of food intake. CART was originally discovered as a differentially expressed transcript in the striatum of rats in response to cocaine and amphetamine (Douglass et al., 1995) but was subsequently found to be distributed in regions of the brain associated with eating behavior (Koylu et al., 1997).
The Gastrointestinal Tract and Control of Food Intake
2018, Physiology of the Gastrointestinal Tract, Sixth EditionPlasticity of vagal afferent signaling in the gut
2017, Medicina (Lithuania)