Elsevier

Neuropharmacology

Volume 62, Issue 4, March 2012, Pages 1823-1833
Neuropharmacology

CART peptide in the nucleus accumbens shell acts downstream to dopamine and mediates the reward and reinforcement actions of morphine

https://doi.org/10.1016/j.neuropharm.2011.12.004Get rights and content

Abstract

The opioid-mesolimbic-dopamine circuitry operates between ventral tegmental area (VTA) and nucleus accumbens (Acb) and serves as a major reward pathway. We hypothesized that the neuropeptide cocaine- and amphetamine-regulated transcript (CART) is involved in the natural reward action mediated by the circuitry. Therefore, the modulation of opioid-mesolimbic-dopamine reward circuitry by CART was investigated using pellet self-administration paradigm in operant chamber. Morphine administered bilaterally in shell region of Acb (AcbSh) significantly increased active lever pressings and pellet self-administration. While CART given bilaterally in the AcbSh significantly increased pellet self-administration, CART antibody produced no effect. Morphine induced pellet self-administration was potentiated by CART, and antagonized by CART antibody administered in AcbSh. A close interaction between dopamine and CART systems was observed. Several tyrosine hydroxylase (marker for dopamine) immunoreactive fibers were seen contacting CART neurons in the AcbSh. Intraperitoneal administration of pramipexole, a dopamine agonist, increased pellet self-administration. The effect was blocked by prior treatment with CART antibody targeted at AcbSh. CART-immunoreactive cells and fibers in the AcbSh, and cells but not fibers in hypothalamic paraventricular nucleus (PVN), were significantly increased in the animals trained in operant chamber. However, CART-immunoreactive profile in the medial forebrain bundle, VTA and arcuate nucleus of hypothalamus did not respond. We suggest that CART, released from the axonal terminals in the framework of AcbSh, may serve as the final output of the endogenous opioid-mesolimbic-dopamine circuitry that processes natural reward.

Highlights

► CART treatment bilaterally in AcbSh increased pellet self-administration. ► Morphine administered in the AcbSh significantly increased active lever pressings. ► While CART potentiated, CART antibody antagonized morphine induced reward effect. ► CART neurons in AcbSh are contacted by tyrosine hydroxylase-containing axons. ► CART-immunoreactivity in the AcbSh was significantly increased in trained animals.

Introduction

Morphine triggered stimulation of mesolimbic-dopaminergic system produces sense of reward within the framework of nucleus accumbens (Acb). While opioid receptors are abundantly present in the shell region of Acb (AcbSh), opioid signaling in this region strongly modulated flavor-based food choice (Gracy et al., 1997, Woolley et al., 2007). The administration of μ-selective opioid agonists into AcbSh increased feeding (Znamensky et al., 2001). Activation of opioid receptors in ventral striatum resulted in the consumption of tasty and/or calorically dense foods (sugar and fat), and promoted behaviors associated with enhanced palatability (Kelley et al., 2002).

While opioids promoted firing rate of the dopaminergic neurons in ventral tegmental area (VTA), morphine administration in VTA triggered the release of dopamine in Acb (Matthews and German, 1984, Di Chiara and Imperato, 1988). Opioid μ-receptors specific peptide DAMGO ([D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin) injected in Acb did not influence the perception or recognition of taste per se, but the pleasure derived from tastes (Kelley et al., 2002). Lesions in the Acb interfered with intravenous morphine self-administration (Smith et al., 1985, Leone et al., 1991). Although Acb in rat is the site for generation of morphine evoked reward (Koob, 2000), the precise cascade of neural events is yet to be defined.

In recent years, much interest has been generated in the neuropeptide cocaine- and amphetamine-regulated transcript (CART). The discovery of the peptide was linked to the production of CART mRNA in the medial forebrain bundle (MFB), VTA and ventral palladium following cocaine or amphetamine treatment (Douglass et al., 1995, Hubert et al., 2008). Circumstantial evidences suggest a role for CART in reward. While intra-VTA CART treatment produced conditioned place preference and increased locomotor activity in rats (Kimmel et al., 2000), Jaworski et al. (2003) reported close interaction of CART with mesolimbic-dopamine and psychostimulants. However, the role of CART in processing of natural reward has not been investigated.

Interestingly, CART shows close interaction with dopamine, as well as morphine, in anatomical areas like VTA and Acb. Beaudry et al. (2004) reported that the CART mRNA expressing neurons in the AcbSh showed the presence of both dopamine D2 and D3 receptors transcripts, and that dopamine D3 receptors antisense oligonucleotide administration reduced CART expression in the AcbSh. Close interaction between CART and morphine, with reference to analgesia and addiction, is well established (Damaj et al., 2004, Dylag et al., 2006). CART in the Acb of rat is co-localized with substance P, gamma-aminobutyric acid (GABA) and dynorphin which are involved in reward and reinforcing actions of drugs of abuse (Dallvechia-Adams et al., 2002, Hubert and Kuhar, 2005, Roberts, 2005). Thus possibilities emerge that endogenous CART might serve as a mediator in the reward and reinforcement actions of morphine. The present study aims at defining the relationship of the endogenous CART circuitry, with the opioid-mesolimbic-dopamine system, in processing the hedonic property of food, within the framework of AcbSh.

Rats were trained in operant chamber to self-administer food pellets; a well recognized paradigm for reward and reinforcement (Ruffing and Domino, 1981, Hasenöhrl et al., 1994, Kinney et al., 2001). Each active lever press by the rat to obtain a pellet was considered as a unit of reward seeking behavior, while repetition of lever pressings suggested reinforcement. With a view to ensure that the parameter being evaluated is reward, and not some unspecific feeding-related effect, satiated rats were used in all experiments. Before the commencement of experiments, we attempted to train animals using non-sweetened chow pellets. Even after prolonged training sessions, these rats did not learn to press the lever, although they could be easily trained if the sweet food pellets were offered. Therefore, the count of lever pressings to obtain the sweet pellets seems to be a reliable measure of reward and reinforcement as reported in earlier studies (Ahn and Phillips, 1999, Bassareo and Di Chiara, 1999, Spangler et al., 2004). In addition, food self-administration is considered as a natural stimulus and has been widely used to assess reward behavior processed by the endogenous opioids and dopamine in the mesocorticolimbic system (Jaworski and Jones, 2006, Woolley et al., 2007, Lajtha and Sershen, 2010). Morphine injected in the AcbSh is known to increase the food intake; the effect was attributed to the hedonic reward properties of food (Peciña and Berridge, 2000). To find out if CART has a role in this action of morphine, the peptide was co-administered with morphine and the responses in operant chamber was evaluated. Since no antagonists for CART receptors are available, endogenous CART was immunoneutralized with CART antibody and the effects of morphine and dopamine were evaluated (Scruggs et al., 2003, Dandekar et al., 2008). In addition, activation of the dopamine system is known to produce reward behavior. Therefore, to find out if the output of dopamine system is modulated by CART, pramipexole (D2/D3 dopamine receptors agonist) was administered along with CART antibody and the response was evaluated in the operant chamber. Moreover, the association between the dopamine and CART-containing neurons in the AcbSh was observed using tyrosine hydroxylase (TH) antibody by double-labeling immunofluorescence technique.

Mattson and Morrell (2005) reported increased CART-immunoreactivity in the Acb following exposure of the rat to cues in the conditioned place preference paradigm. To find out if the CART elements in the brain are sensitive to the training in operant chamber, the sections of the rat brain were subjected to immunohistochemical labeling with antibodies against CART. In the present study, CART-immunoreactivity profile in the AcbSh, MFB, VTA, paraventricular nucleus of hypothalamus (PVN) and arcuate nucleus of hypothalamus (ARC) was investigated. These areas are known to contain CART, and participate in reward and reinforcement mechanisms (Hurd et al., 1999, Kuhar and Dall Vechia, 1999, Jaworski et al., 2002, Jaworski and Jones, 2006, Lajtha and Sershen, 2010).

Section snippets

Animals

Adult, male, Sprague–Dawley rats (240–260 g) were group housed and given free access to chow and drinking water. They were maintained on a 12 h light/dark cycle, in controlled temperature (25 ± 2 °C) and relative humidity (50–70%). All the experimental procedures were approved and carried out under strict compliance with the Institutional Animal Ethics Committee, Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, MS, India.

Operant test for food self-administration

The method described by

Effect of icv treatment with aCSF, CART or CART antibody on the pellet self-administration in the rats subjected to operant chamber

Treatment with aCSF or non-immune serum did not influence the lever press count. However, CART treatment significantly increased the active lever pressings in a dose-dependent manner as compared to that in the aCSF-treated group [F(3,31) = 82.6, P < 0.0001]. CART, at 50–100 ng/rat doses, showed significant increase (P < 0.001) in pellet self-administration, however, at lower dose (25 ng/rat), CART failed to produce any reward and reinforcement activity (P > 0.05) and therefore, considered as

Role of CART in natural reward

Attempts were made to clarify the role of CART in reward and reinforcement like effects. The peptide not only produced condition place preference comparable to that of psychostimulants (Kimmel et al., 2000), but also potentiated the effects of cocaine administered in VTA or Acb (Jaworski et al., 2003). This indicated that CART may mediate the reward-like effects of cocaine. In the present study, while pellet self-administration was significantly increased following CART administration, CART

Conclusion

Endogenous CART in the framework of AcbSh may be an essential component of the opioidergic-dopaminergic reward system that promotes hedonic component of food intake.

Acknowledgment

Supported by the grants from the Department of Science and Technology (SR/SO/AS/40/2007), Govt. of India, New Delhi, India. We are grateful to Dr. Lars Thim and Dr. Jes Thorn Clausen, Novo Nordisk, Denmark, for providing CART antibody. We also thank Institute of Life Science (ILS), Bhubaneswar, India for providing Confocal Microscopy imaging and other instrumentation facilities.

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