Elsevier

Brain Research

Volume 957, Issue 2, 13 December 2002, Pages 298-310
Brain Research

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

https://doi.org/10.1016/S0006-8993(02)03640-5Get rights and content

Abstract

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.

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.

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