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CART peptides are modulators of mesolimbic dopamine and psychostimulants
Life Sciences 73 (2003) 741 – 747 www.elsevier.com/locate/lifescie
CART peptides are modulators of mesolimbic dopamine and psychostimulants J.N. Jaworski *, A. Vicentic, R.G. Hunter, H.L. Kimmel, M.J. Kuhar Yerkes National Primate Research Center of Emory University, 954 Gatewood Road NE, Atlanta, GA 30329, USA
Abstract CART peptide produces behavioral effects when injected into the VTA or nucleus accumbens. In the VTA, the peptide behaves like an endogenous psychostimulant and produces increased locomotor activity and conditioned place preference. Since this is blocked by dopamine receptor blockers, it presumably involves release of dopamine. But in the nucleus accumbens, CART peptide reduces the locomotor-increasing effects of cocaine. This suggests that the peptide is an interesting target for medications development. D 2003 Elsevier Science Inc. All rights reserved. Keywords: CART peptides; Dopamine; Psychostimulants
Introduction CART peptides are peptide neurotransmitters involved in reward and reinforcement, feeding and satiety, stress, endocrine regulation, sensory processing and other physiological processes (for reviews see (Kuhar et al., 2000; Kuhar and Dall Vechia, 1999; Adams et al., 1999; Hurd et al., 1999; Couceyro and Lambert, 1999; Thim et al., 1998a)). They are among the newest of the neuroactive peptides and have rapidly become a focus largely due to their effects on feeding and satiety. In addition, emerging data indicates that these peptides are powerful modulators of mesolimbic dopamine. As such, they are involved in mechanisms of reward and reinforcement and may be useful targets for medications development. The existence of a CART peptide fragment was first reported by Spiess et al. (Spiess et al., 1981), who found, in extracts of hypothalamus, a fragment of a CART peptide corresponding to the first 30 * Corresponding author. Tel.: +1-404-727-8276; fax: +1-404-727-3278. E-mail address: [email protected] (J.N. Jaworski). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00394-1
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amino acids of rlCART 55-102 (for nomenclature, see (Kuhar et al., 2000)). It took another 15 years or so before this peptide was again noticed when Douglass et al. (Douglass et al., 1995), found that an mRNA increased after acute cocaine and amphetamine administration (Note that this is disputed by Vrang et al. (Vrang et al., 2002)). This mRNA was termed CART, (for cocaine-and amphetamineregulated transcript) and coded for a novel neuropeptide that included the fragment identified by Spiess et al. (Spiess et al., 1981). Both in situ hybridization and immunohistochemical studies showed identical results in that CART was expressed in specific nuclei throughout the brain, gut and spinal cord (Douglass et al., 1995; Koylu et al., 1998, 1997; Couceyro et al., 1998). These studies examining the distribution of the peptide led to the first proposal that CART peptides were involved in feeding (Koylu et al., 1997) and also revealed that CART peptides were found in brain regions associated with reward and reinforcement, including the ventral tegmental area (VTA), the nucleus accumbens and the amygdala. The role of CART in feeding has been firmly established (Kuhar et al., 2000; Kuhar and Dall Vechia, 1999; Adams et al., 1999; Hurd et al., 1999; Couceyro and Lambert, 1999; Thim et al., 1998a,b) and has led to the semi-serious suggestion that ‘‘CART’’ should stand for crepes are really tasty! Because of the association established between CART and psychostimulant drugs by Douglass et al. (Douglass et al., 1995), and the anatomical localization of CART peptides, many studies have been directed at the issue of CART’s involvement in reward and reinforcement.
Localization of CART peptides in the mesolimbic system One of the first observations deemed highly significant for reward and reinforcement was the high concentration of CART mRNA in the shell of the nucleus accumbens and other areas associated with reinforcement (Douglass et al., 1995). Shortly thereafter, similar high levels of CART peptide immunoreactivity (ir) were found in both the shell and the core of the nucleus accumbens in the rat (Koylu et al., 1998) and monkey (Smith et al., 1997, 1999). In addition to CART-containing cell bodies, numerous neuronal processes were found in these regions as well. In contrast, in the VTA, no CART mRNA-containing cell bodies or CART peptide ir cell bodies were found. Rather, CART-containing axons and nerve terminals were found throughout various subregions of the VTA, such as the paranigral nucleus and the interfascicular nucleus. The amygdala, a region associated with various psychostimulant-related processes, had high levels of staining in the medial nucleus, the basal lateral nucleus and the central nucleus. The occurrence of CART in mesolimbic regions was explored further in electronic microscopic and track-tracing studies. DallVechia-Adams (Dallvechia-Adams et al., 2001, 2002) found that cell bodies in the nucleus accumbens core region projected to the substantia nigra zona compacta and reticulata rather than the VTA. In the more medial regions of the nucleus accumbens, that is the shell, CART was also found to be highly concentrated in medium spiny projection neurons that contain GABA. Lesions of the neurones in the shell did not result in a striking depletion of CART-containing processes in the VTA. Thus, the CART-containing neurons in the shell do not seem to overwhelmingly project to the VTA, and their destination remains to be elucidated. In retrograde tracing studies, many CART-containing neurons in the perifornical region of the lateral hypothalamus were found to project to the VTA (DallVechia-Adams in preparation). The finding that CART-containing neurons in lateral hypothalamus projects to the VTA suggests a CART-mediated connection between feeding and drug-related reward. In the VTA, only a small number of the CART-
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peptide ir nerve terminal profiles apposed dopaminergic cell bodies, suggesting that a large fraction of the CART input to the VTA terminated on GABA-ergic interneurons. This raises the possibility that the mechanism of action of CART in the VTA could be primarily through the disinhibition of GABA-ergic interneurons (see below).
Injection of exogenous CART peptide into the VTA Because endogenous CART peptide is highly concentrated in the VTA, it is of interest to test whether or not injection of exogenous CART induces behavioral effects. Accordingly, Kimmel et al.
Fig. 1. Dose-dependent increase in locomotion by CART 55-102 (intra-VTA) and blockade of CART-induced locomotion by haloperidol (mean F SEM). Locomotor effects were measured for 1 h. A, *P < 0.05, comparing all drug injections to saline injection with a one-way ANOVA. B, animals were given haloperidol or saline i.p. and then CART peptide or saline intra-VTA. Data were analyzed with a two-way ANOVA with repeated measures on intra-VTA injection (CART, dark column versus saline, lighter column) and haloperidol doses. *P < 0.05, points that are different from intra-VTA saline + i.p. haloperidol at that haloperidol dose. + P < 0.05, points that are different from intra-VTA CART alone. Data reproduced from (Kimmel et al., 2000).
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(Kimmel et al., 2000), examined the effects of intra-VTA injection of CART 55-102. They found that these injections increased locomotor activity in a dose-dependent and time-limited fashion that was blocked by dopamine receptor antagonists (see Fig. 1). Moreover, another CART peptide fragment not known to have any activity in other experiments was without activity in the VTA. Also, injection of the CART peptide into the substantia nigra rather than the VTA produced no increase in locomotor activity. These experiments suggest that CART peptide could be an endogenous psychostimulant and be involved in mediating or modulating the actions of psychostimulant drugs. Additionally, injection of CART into the VTA was found to be reinforcing as it produced a conditioned place preference (Kimmel et al., 2000). These findings are strong evidence that CART peptides in the VTA could be involved in the action of psychostimulants. The converse experiment, injection of CART antibodies into the VTA, did not appear to have any effect on locomotor activity induced by injections of cocaine. This could mean that endogenously-occurring CART, as opposed to exogenously-administered CART, is not involved in the actions of cocaine. Alternatively, the large antibody molecules may have had difficulty diffusing to and blocking an adequate amount of cocaine-mediated released CART in order to have an effect on behavior. In any case, these experiments clearly show that CART can have an effect after injection into the VTA, and that these effects are psychostimulant-like. A study of CART peptide knockout mice (Asnicar et al., 2001) or of CART receptor antagonists (which are not currently known to exist) would be important in studying the role of endogenous CART in the action of psychostimulant drugs.
Injection of CART peptide into the nucleus accumbens Because of the importance of the nucleus accumbens in the action of psychostimulants, and because CART peptides are highly concentrated in this region, experiments were carried out where effects of bilateral injections of CART peptides into the nucleus accumbens were examined. While these injections had little effect on basal locomotor activity, it was found that they decreased cocaine-induced locomotor activity (Jaworski et al., 2002). This was quite different than effects found after intra-VTA injection. This ‘‘limiting’’ effect of CART peptide in the accumbens was dose-dependent and time-limited, as expected. Moreover, this property of limiting the effect of psychostimulant-induced locomotor activity suggests that CART agonists may have a useful role as medications that would blunt at least some of the effects of cocaine. In any case, additional studies on this topic will be useful, and it has recently been reported in an abstract that CART has the same effect on amphetamine-induced locomotor activity (Vezina et al., 2002). While many peptides will increase locomotor activity after injection into the VTA, only few peptides such as CART and neurotensin will blunt the effects of cocaine after accumbal injection (Binder et al., 2001).
CART receptors and mechanisms in VTA and accumbens Central to advancing our knowledge of the role of CART peptides would be the identification of and study of CART peptide receptors. So far, this has not been achieved, and binding studies using radio-labeled CART have not been successful in obtaining specific binding. The reasons for this are
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unknown, but could include high levels of nonspecific binding or disruption of the active binding site by radio-labeling. One paper in the literature suggests that CART peptide application to hippocampal cells results in increased mobilization of calcium (Yermolaieva et al., 2001). These studies need to be repeated and extended. One question has been whether or not CART peptide might interact at the receptor for another known neurotransmitter and alter its binding. Accordingly, we have examined the effect of CART peptides at 1 nM on the binding of a large number of ligands to their cognate receptors in Novascreen. These receptors include: opiate, sigma, estrogen, glucocorticoid, progesterone, testosterone (cytosolic and nuclear), calcium channels (types L, N), GABA (TBOB site), leukotriene B4, leukotriene D4, thromboxane A2, ANP, CRF, EGF, oxytocin, PAF, TRH, angiotensin II (AT1, AT2), bradykinin (BK2), CCKA, CCKB, endothelin (A, B), galanin, neurokinin (NK1, 2, 3), NPY, neurotensin, somatostatin, VIP and vasopressin 1. CART peptide 55-102 did not have any effect on any of the binding of ligands to these receptors. Our lab has also found that CART peptide (1 and 10 nM) had no effect on ligand binding to the D2 receptor (RG Hunter, unpublished data). In summary, these data, along with the behavioral effects induced by injection, specific receptors for CART must exist in the brain and have yet to be discovered. There is evidence suggesting multiple receptors for CART peptide. Different CART peptides have different relative potencies in different tests (Kimmel et al., 2002; Thim et al., 1998b; Bannon et al., 2001). This would increase the attraction to CART for developing new medications. It was mentioned above that many CART-containing nerve terminals in the VTA not only synapse on dopamine-containing neurons, but also impinge on GABA-ergic neurons and may disinhibit these neurons. In the accumbens, CART colocalizes with GABA and could possibly inhibit GABA output via receptors on GABA-ergic neurons. Thus, GABA could be central to CART’s effects in reward pathways. CART has also been reported to inhibit dopamine release from hypothalamic synaptosones (Brunetti et al., 2000), but this has not been demonstrated in mesolimbic areas.
Conclusion In conclusion, CART mRNA increases have been observed after administration of psychostimulants ((Douglass et al., 1995; Brenz Verca et al., 2001; Fagergren and Hurd, 1999); but challenged by Vrang et al. (Vrang et al., 2002)). In addition, CART peptide ir has been found in brain regions known to be associated with the actions of pychostimulant and other drugs that produce abuse and dependence. Exogenously applied into the VTA, CART peptides produce psychostimulant-like effects that include increased locomotor activity (blocked by dopamine receptor antagonists) and the development of conditioned place preference. Injection of CART peptide into the nucleus accumbens, on the other hand, inhibits the full effects of cocaine by blunting increases in locomotor activity. In the VTA and accumbens, CART is likely to act at least partly on GABA-containing neurons. Also in the VTA, the effect on GABA neurons is likely to disinhibit the dopamine neurons, and the DA neurons also receive a small input of CART-containing nerve terminals. Taken together, these studies suggest that CART peptides play a role in the action of psychostimulants and may somehow modulate the effects or the actions of these drugs through direct or indirect interactions with mesolimbic dopamine.
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Acknowledgements The authors acknowledge the assistance of Susan Marshall and Dr. Steve Holtzman, and the support of NIH grants DA10732, RR00165, DA00418, DA15593, DA15279 and DA15277. References Adams, L.D., Gong, W., Vechia, S.D., Hunter, R.G., Kuhar, M.J., 1999. CART: from gene to function. Brain Res. 848 (1 – 2), 137 – 140. Asnicar, M.A., Smith, D.P., Yang, D.D., Heiman, M.L., Fox, N., Chen, Y.F., Hsiung, H.M., Koster, A., 2001. Absence of cocaine- and amphetamine-regulated transcript results in obesity in mice fed a high caloric diet. Endocrinology 142 (10), 4394 – 4400. Bannon, A.W., Seda, J., Carmouche, M., Francis, J.M., Jarosinski, M.A., Douglass, J., 2001. Multiple behavioral effects of cocaine- and amphetamine-regulated transcript (CART) peptides in mice: CART 42-89 and CART 49-89 differ in potency and activity. J. Pharmacol. Exp. Ther. 299 (3), 1021 – 1026. Binder, E., Kinkead, B., Nemeroff, C., 2001. Neuropeptides. In: Breier, A., Tran, P., Herrea, J., Tollefson, G., Bymaster, F. (Eds.), Current Issues in the Psychopharmacology of Schizophrenia, Lippincott. Williams and Wilkins, Philadelphia, PA, pp. 349 – 371. Brenz Verca, M.S., Widmer, D.A., Wagner, G.C., Dreyer, J., 2001. Cocaine-induced expression of the tetraspanin CD81 and its relation to hypothalamic function. Mol. Cell Neurosci. 17 (2), 303 – 316. Brunetti, L., Orlando, G., Michelotto, B., Recinella, L., Vacca, M., 2000. Cocaine- and amphetamine-regulated transcript peptide-(55 – 102) and thyrotropin releasing hormone inhibit hypothalamic dopamine release. Eur. J. Pharmacol. 409 (2), 103 – 107. Couceyro, P.R., Lambert, P.D., 1999. CART peptides: therapeutic potential in obesity and feeding disorders. Drug News Perspect. 12, 133 – 136. Couceyro, P., Paquet, M., Koylu, E., Kuhar, M.J., Smith, Y., 1998. Cocaine- and amphetamine-regulated transcript (CART) peptide immunoreactivity in myenteric plexus neurons of the rat ileum and co-localization with choline acetyltransferase. Synapse 30 (1), 1 – 8. Dallvechia-Adams, S., Smith, Y., Kuhar, M.J., 2001. CART peptide-immunoreactive projection from the nucleus accumbens targets substantia nigra pars reticulata neurons in the rat. J. Comp. Neurol. 434 (1), 29 – 39. Dallvechia-Adams, S., Kuhar, M.J., Smith, Y., 2002. Cocaine- and amphetamine-regulated transcript peptide projections in the ventral midbrain: colocalization with gamma-aminobutyric acid, melanin-concentrating hormone, dynorphin, and synaptic interactions with dopamine neurons. J. Comp. Neurol. 448 (4), 360 – 372. Douglass, J., McKinzie, A.A., Couceyro, P., 1995. PCR differential display identifies a rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine. J. Neurosci. 15 (3 Pt 2), 2471 – 2481. Fagergren, P., Hurd, Y.L., 1999. Mesolimbic gender differences in peptide CART mRNA expression: effects of cocaine. Neuroreport 10 (16), 3449 – 3452. Hurd, Y.L., Svensson, P., Ponten, M., 1999. The role of dopamine, dynorphin, and CART systems in the ventral striatum and amygdala in cocaine abuse. Ann. N.Y. Acad. Sci. 877, 499 – 506. Jaworski, J.N., Kozel, M.A., Kuhar, M.J., 2002. CART injection into nucleus accumbens reduces cocaine-induced locomotion in rats. Society for Neuroscience, Orlando, Florida, USA. Neuroscience Society Abstract. Program No. 499.12. Kimmel, H.L., Gong, W., Vechia, S.D., Hunter, R.G., Kuhar, M.J., 2000. Intra-ventral tegmental area injection of rat cocaine and amphetamine-regulated transcript peptide 55 – 102 induces locomotor activity and promotes conditioned place preference. J. Pharmacol. Exp. Ther. 294 (2), 784 – 792. Kimmel, H.L., Thim, L., Kuhar, M.J., 2002. Activity of various CART peptides in changing locomotor activity in the rat. Neuropeptides 36 (1), 9 – 12. Koylu, E.O., Couceyro, P.R., Lambert, P.D., Ling, N.C., DeSouza, E.B., Kuhar, M.J., 1997. Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland. J. Neuroendocrinol. 9 (11), 823 – 833. Koylu, E.O., Couceyro, P.R., Lambert, P.D., Kuhar, M.J., 1998. Cocaine- and amphetamine-regulated transcript peptide immunohistochemical localization in the rat brain. J. Comp. Neurol. 391 (1), 115 – 132.
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Kuhar, M.J., Dall Vechia, S.E., 1999. CART peptides: novel addiction- and feeding-related neuropeptides. Trends Neurosci. 22 (7), 316 – 320. Kuhar, M.J., Adams, L.D., Hunter, R.G., Vechia, S.D., Smith, Y., 2000. CART peptides. Regul. Pept. 89 (1 – 3), 1 – 6. Smith, Y., Koylu, E.O., Couceyro, P., Kuhar, M.J., 1997. Ultrastructural localization of CART (cocaine- and amphetamineregulated transcript) peptides in the nucleus accumbens of monkeys. Synapse 27 (1), 90 – 94. Smith, Y., Kieval, J., Couceyro, P.R., Kuhar, M.J., 1999. CART peptide-immunoreactive neurones in the nucleus accumbens in monkeys: ultrastructural analysis, colocalization studies, and synaptic interactions with dopaminergic afferents. J. Comp. Neurol. 407 (4), 491 – 511. Spiess, J., Villarreal, J., Vale, W., 1981. Isolation and sequence analysis of a somatostatin-like polypeptide from ovine hypothalamus. Biochemistry 20 (7), 1982 – 1988. Thim, L., Kristensen, P., Larsen, P.J., Wulff, B.S., 1998a. CART, a new anorectic peptide. Int. J. Biochem. Cell Biol. 30 (12), 1281 – 1284. Thim, L., Nielsen, P.F., Judge, M.E., Andersen, A.S., Diers, I., Egel-Mitani, M., Hastrup, S., 1998b. Purification and characterisation of a new hypothalamic satiety peptide, cocaine and amphetamine regulated transcript (CART), produced in yeast. FEBS Lett. 428 (3), 263 – 268. Vezina, P., Creekmore, E., Kim, J.H., 2002. Microinjection of CART peptide 55 – 102 into the nucleus accumbens blocks amphetamine-induced locomotion. Society for Neuroscience, Orlando, Florida, USA. Neuroscience Society Abstract. Program No. 901.11. Vrang, N., Larsen, P.J., Kristensen, P., 2002. Cocaine-amphetamine regulated transcript (CART) expression is not regulated by amphetamine. Neuroreport 13 (9), 1215 – 1218. Yermolaieva, O., Chen, J., Couceyro, P.R., Hoshi, T., 2001. Cocaine- and amphetamine-regulated transcript peptide modulation of voltage-gated Ca2 + signaling in hippocampal neurons. J. Neurosci. 21 (19), 7474 – 7480.
CART peptides are modulators of mesolimbic dopamine and psychostimulants J.N. Jaworski *, A. Vicentic, R.G. Hunter, H.L. Kimmel, M.J. Kuhar Yerkes National Primate Research Center of Emory University, 954 Gatewood Road NE, Atlanta, GA 30329, USA
Abstract CART peptide produces behavioral effects when injected into the VTA or nucleus accumbens. In the VTA, the peptide behaves like an endogenous psychostimulant and produces increased locomotor activity and conditioned place preference. Since this is blocked by dopamine receptor blockers, it presumably involves release of dopamine. But in the nucleus accumbens, CART peptide reduces the locomotor-increasing effects of cocaine. This suggests that the peptide is an interesting target for medications development. D 2003 Elsevier Science Inc. All rights reserved. Keywords: CART peptides; Dopamine; Psychostimulants
Introduction CART peptides are peptide neurotransmitters involved in reward and reinforcement, feeding and satiety, stress, endocrine regulation, sensory processing and other physiological processes (for reviews see (Kuhar et al., 2000; Kuhar and Dall Vechia, 1999; Adams et al., 1999; Hurd et al., 1999; Couceyro and Lambert, 1999; Thim et al., 1998a)). They are among the newest of the neuroactive peptides and have rapidly become a focus largely due to their effects on feeding and satiety. In addition, emerging data indicates that these peptides are powerful modulators of mesolimbic dopamine. As such, they are involved in mechanisms of reward and reinforcement and may be useful targets for medications development. The existence of a CART peptide fragment was first reported by Spiess et al. (Spiess et al., 1981), who found, in extracts of hypothalamus, a fragment of a CART peptide corresponding to the first 30 * Corresponding author. Tel.: +1-404-727-8276; fax: +1-404-727-3278. E-mail address: [email protected] (J.N. Jaworski). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00394-1
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amino acids of rlCART 55-102 (for nomenclature, see (Kuhar et al., 2000)). It took another 15 years or so before this peptide was again noticed when Douglass et al. (Douglass et al., 1995), found that an mRNA increased after acute cocaine and amphetamine administration (Note that this is disputed by Vrang et al. (Vrang et al., 2002)). This mRNA was termed CART, (for cocaine-and amphetamineregulated transcript) and coded for a novel neuropeptide that included the fragment identified by Spiess et al. (Spiess et al., 1981). Both in situ hybridization and immunohistochemical studies showed identical results in that CART was expressed in specific nuclei throughout the brain, gut and spinal cord (Douglass et al., 1995; Koylu et al., 1998, 1997; Couceyro et al., 1998). These studies examining the distribution of the peptide led to the first proposal that CART peptides were involved in feeding (Koylu et al., 1997) and also revealed that CART peptides were found in brain regions associated with reward and reinforcement, including the ventral tegmental area (VTA), the nucleus accumbens and the amygdala. The role of CART in feeding has been firmly established (Kuhar et al., 2000; Kuhar and Dall Vechia, 1999; Adams et al., 1999; Hurd et al., 1999; Couceyro and Lambert, 1999; Thim et al., 1998a,b) and has led to the semi-serious suggestion that ‘‘CART’’ should stand for crepes are really tasty! Because of the association established between CART and psychostimulant drugs by Douglass et al. (Douglass et al., 1995), and the anatomical localization of CART peptides, many studies have been directed at the issue of CART’s involvement in reward and reinforcement.
Localization of CART peptides in the mesolimbic system One of the first observations deemed highly significant for reward and reinforcement was the high concentration of CART mRNA in the shell of the nucleus accumbens and other areas associated with reinforcement (Douglass et al., 1995). Shortly thereafter, similar high levels of CART peptide immunoreactivity (ir) were found in both the shell and the core of the nucleus accumbens in the rat (Koylu et al., 1998) and monkey (Smith et al., 1997, 1999). In addition to CART-containing cell bodies, numerous neuronal processes were found in these regions as well. In contrast, in the VTA, no CART mRNA-containing cell bodies or CART peptide ir cell bodies were found. Rather, CART-containing axons and nerve terminals were found throughout various subregions of the VTA, such as the paranigral nucleus and the interfascicular nucleus. The amygdala, a region associated with various psychostimulant-related processes, had high levels of staining in the medial nucleus, the basal lateral nucleus and the central nucleus. The occurrence of CART in mesolimbic regions was explored further in electronic microscopic and track-tracing studies. DallVechia-Adams (Dallvechia-Adams et al., 2001, 2002) found that cell bodies in the nucleus accumbens core region projected to the substantia nigra zona compacta and reticulata rather than the VTA. In the more medial regions of the nucleus accumbens, that is the shell, CART was also found to be highly concentrated in medium spiny projection neurons that contain GABA. Lesions of the neurones in the shell did not result in a striking depletion of CART-containing processes in the VTA. Thus, the CART-containing neurons in the shell do not seem to overwhelmingly project to the VTA, and their destination remains to be elucidated. In retrograde tracing studies, many CART-containing neurons in the perifornical region of the lateral hypothalamus were found to project to the VTA (DallVechia-Adams in preparation). The finding that CART-containing neurons in lateral hypothalamus projects to the VTA suggests a CART-mediated connection between feeding and drug-related reward. In the VTA, only a small number of the CART-
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peptide ir nerve terminal profiles apposed dopaminergic cell bodies, suggesting that a large fraction of the CART input to the VTA terminated on GABA-ergic interneurons. This raises the possibility that the mechanism of action of CART in the VTA could be primarily through the disinhibition of GABA-ergic interneurons (see below).
Injection of exogenous CART peptide into the VTA Because endogenous CART peptide is highly concentrated in the VTA, it is of interest to test whether or not injection of exogenous CART induces behavioral effects. Accordingly, Kimmel et al.
Fig. 1. Dose-dependent increase in locomotion by CART 55-102 (intra-VTA) and blockade of CART-induced locomotion by haloperidol (mean F SEM). Locomotor effects were measured for 1 h. A, *P < 0.05, comparing all drug injections to saline injection with a one-way ANOVA. B, animals were given haloperidol or saline i.p. and then CART peptide or saline intra-VTA. Data were analyzed with a two-way ANOVA with repeated measures on intra-VTA injection (CART, dark column versus saline, lighter column) and haloperidol doses. *P < 0.05, points that are different from intra-VTA saline + i.p. haloperidol at that haloperidol dose. + P < 0.05, points that are different from intra-VTA CART alone. Data reproduced from (Kimmel et al., 2000).
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(Kimmel et al., 2000), examined the effects of intra-VTA injection of CART 55-102. They found that these injections increased locomotor activity in a dose-dependent and time-limited fashion that was blocked by dopamine receptor antagonists (see Fig. 1). Moreover, another CART peptide fragment not known to have any activity in other experiments was without activity in the VTA. Also, injection of the CART peptide into the substantia nigra rather than the VTA produced no increase in locomotor activity. These experiments suggest that CART peptide could be an endogenous psychostimulant and be involved in mediating or modulating the actions of psychostimulant drugs. Additionally, injection of CART into the VTA was found to be reinforcing as it produced a conditioned place preference (Kimmel et al., 2000). These findings are strong evidence that CART peptides in the VTA could be involved in the action of psychostimulants. The converse experiment, injection of CART antibodies into the VTA, did not appear to have any effect on locomotor activity induced by injections of cocaine. This could mean that endogenously-occurring CART, as opposed to exogenously-administered CART, is not involved in the actions of cocaine. Alternatively, the large antibody molecules may have had difficulty diffusing to and blocking an adequate amount of cocaine-mediated released CART in order to have an effect on behavior. In any case, these experiments clearly show that CART can have an effect after injection into the VTA, and that these effects are psychostimulant-like. A study of CART peptide knockout mice (Asnicar et al., 2001) or of CART receptor antagonists (which are not currently known to exist) would be important in studying the role of endogenous CART in the action of psychostimulant drugs.
Injection of CART peptide into the nucleus accumbens Because of the importance of the nucleus accumbens in the action of psychostimulants, and because CART peptides are highly concentrated in this region, experiments were carried out where effects of bilateral injections of CART peptides into the nucleus accumbens were examined. While these injections had little effect on basal locomotor activity, it was found that they decreased cocaine-induced locomotor activity (Jaworski et al., 2002). This was quite different than effects found after intra-VTA injection. This ‘‘limiting’’ effect of CART peptide in the accumbens was dose-dependent and time-limited, as expected. Moreover, this property of limiting the effect of psychostimulant-induced locomotor activity suggests that CART agonists may have a useful role as medications that would blunt at least some of the effects of cocaine. In any case, additional studies on this topic will be useful, and it has recently been reported in an abstract that CART has the same effect on amphetamine-induced locomotor activity (Vezina et al., 2002). While many peptides will increase locomotor activity after injection into the VTA, only few peptides such as CART and neurotensin will blunt the effects of cocaine after accumbal injection (Binder et al., 2001).
CART receptors and mechanisms in VTA and accumbens Central to advancing our knowledge of the role of CART peptides would be the identification of and study of CART peptide receptors. So far, this has not been achieved, and binding studies using radio-labeled CART have not been successful in obtaining specific binding. The reasons for this are
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unknown, but could include high levels of nonspecific binding or disruption of the active binding site by radio-labeling. One paper in the literature suggests that CART peptide application to hippocampal cells results in increased mobilization of calcium (Yermolaieva et al., 2001). These studies need to be repeated and extended. One question has been whether or not CART peptide might interact at the receptor for another known neurotransmitter and alter its binding. Accordingly, we have examined the effect of CART peptides at 1 nM on the binding of a large number of ligands to their cognate receptors in Novascreen. These receptors include: opiate, sigma, estrogen, glucocorticoid, progesterone, testosterone (cytosolic and nuclear), calcium channels (types L, N), GABA (TBOB site), leukotriene B4, leukotriene D4, thromboxane A2, ANP, CRF, EGF, oxytocin, PAF, TRH, angiotensin II (AT1, AT2), bradykinin (BK2), CCKA, CCKB, endothelin (A, B), galanin, neurokinin (NK1, 2, 3), NPY, neurotensin, somatostatin, VIP and vasopressin 1. CART peptide 55-102 did not have any effect on any of the binding of ligands to these receptors. Our lab has also found that CART peptide (1 and 10 nM) had no effect on ligand binding to the D2 receptor (RG Hunter, unpublished data). In summary, these data, along with the behavioral effects induced by injection, specific receptors for CART must exist in the brain and have yet to be discovered. There is evidence suggesting multiple receptors for CART peptide. Different CART peptides have different relative potencies in different tests (Kimmel et al., 2002; Thim et al., 1998b; Bannon et al., 2001). This would increase the attraction to CART for developing new medications. It was mentioned above that many CART-containing nerve terminals in the VTA not only synapse on dopamine-containing neurons, but also impinge on GABA-ergic neurons and may disinhibit these neurons. In the accumbens, CART colocalizes with GABA and could possibly inhibit GABA output via receptors on GABA-ergic neurons. Thus, GABA could be central to CART’s effects in reward pathways. CART has also been reported to inhibit dopamine release from hypothalamic synaptosones (Brunetti et al., 2000), but this has not been demonstrated in mesolimbic areas.
Conclusion In conclusion, CART mRNA increases have been observed after administration of psychostimulants ((Douglass et al., 1995; Brenz Verca et al., 2001; Fagergren and Hurd, 1999); but challenged by Vrang et al. (Vrang et al., 2002)). In addition, CART peptide ir has been found in brain regions known to be associated with the actions of pychostimulant and other drugs that produce abuse and dependence. Exogenously applied into the VTA, CART peptides produce psychostimulant-like effects that include increased locomotor activity (blocked by dopamine receptor antagonists) and the development of conditioned place preference. Injection of CART peptide into the nucleus accumbens, on the other hand, inhibits the full effects of cocaine by blunting increases in locomotor activity. In the VTA and accumbens, CART is likely to act at least partly on GABA-containing neurons. Also in the VTA, the effect on GABA neurons is likely to disinhibit the dopamine neurons, and the DA neurons also receive a small input of CART-containing nerve terminals. Taken together, these studies suggest that CART peptides play a role in the action of psychostimulants and may somehow modulate the effects or the actions of these drugs through direct or indirect interactions with mesolimbic dopamine.
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