Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Cocaine- and amphetamine-regulated transcript (CART) peptides are putative brain/gut neurotransmitters with purported neurotrophic and satiety effects (Louis, 1996; Lambert et al., 1997, 1998; Kristensen et al., 1998; Kuhar and Dall Vechia, 1999). In addition, their regional localization in brain is compatible with a role in sensory processing and in hypothalamic-pituitary-adrenal function (Couceyro et al., 1997; Koylu et al., 1997, 1998). Evidence also suggests a role for CART peptide in psychostimulant-related reward and reinforcement. This evidence includes 1) a report of CART mRNA elevation after acute cocaine or amphetamine administration (Douglass et al., 1995); 2) the presence of CART mRNA and peptides in neurons and processes of the shell of the nucleus accumbens, a region associated with reward (Douglass et al., 1995; Koylu et al., 1998; Smith et al., 1999); 3) the presence of CART peptide-positive axons and terminals in the ventral tegmental area (VTA), which also is associated with reward (Douglass et al., 1995; Koylu et al., 1998; Smith et al., 1999); and 4) the identification of CART peptides in a subpopulation of -aminobutyric acid (GABA) projection neurons in the nucleus accumbens, and the presence of dopaminergic inputs on these neurons (Smith et al., 1999). These findings suggest that CART peptides may somehow mediate or modulate the action of psychostimulant drugs after their inhibition of uptake or release of dopamine.

Several peptides in the VTA have been found to be behaviorally active after injection into this brain region (Kalivas, 1985, 1993; Kelley and Cador, 1988; Kelley and Delfs, 1991). For example, intra-VTA injection of opioid peptides increases locomotor activity (Stinus et al., 1980; DuMars et al., 1988), induces conditioned place preference (CPP) (Phillips and LePiane, 1980; Bozarth, 1987; Shippenberg et al., 1993), and induces locomotor sensitization of subsequent challenge of cocaine or amphetamine (Kalivas et al., 1985; DuMars et al., 1988). CART 55-102 is a peptide fragment (Kuhar and Dall Vechia, 1999) that is endogenously occurring in the rat brain (Spiess et al., 1981; Kuhar and Yoho, 1999; Thim et al., 1999). Although i.c.v. injection of CART 55-102 has been reported to inhibit feeding and cause tremor (Kristensen et al., 1998; Thim et al., 1998; Adams et al., 1999), the effects of injection into specific brain regions have not been reported. In this study, we examined the behavioral effects of injection of rat CART 55-102 into the rat VTA. We also examined the behavioral effects of intra-VTA injection of CART 1-26, a peptide fragment that has not been found in the brain or to be physiologically active (Thim et al., 1999). If we can determine the sites of activity and the forms of the peptide that are behaviorally active, we can begin to elucidate the actions and significance of CART peptides.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN) weighing 275 to 325 g at the beginning of testing were used. Animals were housed singly and maintained on a 12-h normal light/dark cycle (lights on at 7:00 AM) with food and water available ad libitum. All experimental evaluations were conducted during the light phase of the cycle. All experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Surgical and Infusion Procedures

Subjects were anesthetized with ketamine (75 mg/kg i.p.) and medetomidine (0.5 mg/kg i.p.). All rats were placed in a Kopf stereotaxic frame and implanted with a 22-gauge bilateral guide cannula assembly (Plastics One, Inc., Roanoke, VA) with a center-to-center distance of 1.5 mm (VTA) or 3.8 mm (substantia nigra; SN). Tips of cannulas were aimed at the dorsal surface of the VTA or SN to allow injector tips to extend 2 mm beyond the guides, thus reaching the target regions. Target stereotaxic coordinates relative to bregma for the VTA were A/P, 3.2 mm; M/L, 0.75 mm; and D/V, 8.5 mm; and for the SN were A/P, 3.2 mm; M/L, 1.9 mm; and D/V, 8.0 mm (Pellegrino et al., 1979). The incisor bar was placed at +5.0 mm. Guide cannulas were secured with skull screws and dental cement. Dummy cannulas (28 gauge), extending 2 mm beyond the guide cannula tips, were inserted to prevent blockage and a dust cap was attached to the top of the cannulas' assembly. Animals were allowed to recover for a minimum of 10 days before testing.

Stainless steel injector cannulas (28 gauge) were cut to protrude 2 mm beyond the tips of the guide cannulas. Polyethylene-10 tubing was used to connect injectors to 25-µl syringes (Hamilton Co., Reno, NV) mounted on infusion pumps (Harvard Apparatus, Cambridge, MA). During the infusion procedure, rats were confined in a small polyethylene box. Left and right VTA or SN was simultaneously infused with a 0.5-µl solution over a 60-s time period. Infusion cannulas were left in place for an additional 30 s to allow diffusion of the solution and to prevent backflow through the cannulas. Then dummy cannulas were reinserted into the guide cannulas and the dust cap secured.

Measurement of Locomotor Activity

Spontaneous and pharmacologically induced motor activity was measured with a photocell cage (Omnitech Electronics, Columbus, OH), operated by an IBM computer. Photocell cages measured 40 × 40 × 30 cm. Each cage had 32 horizontal photocells (16 front to back and 16 side to side, located every 2.4 cm) positioned 5 cm off the cage floor. Each cage was isolated in a separate steel box equipped with a 10-W light bulb, an air supply, and a door with a keyhole that allowed the experimenter to observe the rats. The distance traveled in centimeters was quantified by measuring the consecutive breaks of adjacent photocell beams. Rearing was quantified by counting the number of times that the animal interrupted the vertical photocell beams located 18 cm above the floor. A stereotypic episode was defined as a repetitive interruption of the same beam (Sanberg et al., 1984).

Before experiments, all rats were habituated to the activity chambers and the injection procedure by daily sham injections for 3 days. The rats were placed in the photocell cages for 1 h, given a sham microinjection, and returned to the cages for another hour. On days when experimental data were collected, the rats were habituated to the photocell cage for 1 h, given a single microinjection of the test compound, and then returned to the photocell cage. Distance traveled, number of rearings, and stereotypic counts were measured for 1 h immediately after the microinjection. After these behavioral parameters were recorded, rats were returned to their home cages for a minimum of 48 h before the next testing period. One group of rats (n = 7) received intra-VTA injections of 0.9% saline and 0.04, 0.2, and 1.0 µg CART 55-102 counterbalanced for order in a within-subject design. Once this dose-response curve was established, all animals received an intra-VTA injection of 5.0 µg of CART 55-102. A dose higher than 5.0 µg of CART 55-102 was not administered because several rats developed seizure activity after receiving this dose.

To determine whether intra-VTA CART 55-102 was producing its locomotor effects through a dopaminergic mechanism, the dopamine receptor antagonist haloperidol (0.03-0.3 mg/kg i.p.) was administered 30 min before intra-VTA administration of 1.0 µg/0.5 µl/side CART 55-102 (n = 5). Immediately after CART administration, activity was measured in 5-min increments for 1 h.

A second group of rats (n = 6) received intra-VTA injections of saline and 0.1, 0.5, and 1.0 µg of CART 1-26 counterbalanced for order in a within-subject design. A third group of animals (n = 5) received intra-SN injections of 0.9% saline and 0.04, 0.2, and 1.0 µg of CART 55-102 counterbalanced for order in a within-subject design. Once this dose-response curve was established, all animals received an intra-SN injection of 5.0 µg of CART 55-102.

CPP Test

CPP tests were performed in opaque Plexiglas chambers divided into two separate (75 × 37.5 × 75 cm) compartments. One of the compartments had black walls and a floor of 6.4-mm-diameter metal rods spaced 25 mm apart; the other had white walls and a metal mesh floor consisting of 1.0-mm-diameter wires spaced 6.4 mm apart. The removable partition dividing the two compartments was painted black on one side and white on the other. During pre- and postconditioning tests, this partition was removed and replaced with a similar partition that had a 15- × 15-cm hole, allowing the animals to move between the two compartments.

The CPP chamber was placed in a room lit with 20-W red lights. Behavioral activities of the animals in the CPP chambers were recorded by a video camera mounted on the ceiling. This camera was connected to a computer, which determined the location and movements of the animals with the Etho Vision 1.95 tracking program (Noldus Information Technology, Wageningen, the Netherlands).

The CPP test consisted of three phases: preconditioning, conditioning, and postconditioning. For the preconditioning phase (1 day), subjects were placed in the white compartment and the dividing partition was replaced with the partial partition to allow access to the entire apparatus for 15 min. The amount of time spent in each compartment was monitored and used to assess unconditioned preferences. During the conditioning phase (8 days), one group of rats (n = 6) was given an intra-VTA injection of 0.9% saline (0.5 µl/side) once every other day for 8 days. A second group (n = 6) received CART 55-102 (1.0 µg/0.5 µl/side) once every other day for 8 days. Immediately after the drug (or saline) injections, subjects were confined to the white compartment for 30 min. On alternative days between drug (or saline) injections, animals received sham injections. Immediately after the sham injections, subjects were confined to the black compartment for 30 min. Each subject received four drug (or saline) and four sham pairings. Half of each treatment group received drug (or saline) injections on the first, third, fifth, and seventh days, whereas the remaining subjects received drug (or saline) injections on the second, fourth, sixth, and eighth days. For the testing phase (1 day), the day after the last conditioning trial, subjects were tested for their preference in a drug-free state. Each rat was placed in the white compartment and the dividing partition was replaced with the partial partition to allow access to both sides of the apparatus for 15 min. The amount of time spent in each compartment was used to assess postconditioning (conditioned) preferences.

Locomotor Sensitization

Fourteen rats were implanted with a guide cannula aimed at the dorsal surface of the VTA as described earlier, and allowed to recover for 2 weeks. On each testing day, animals were habituated to the photocell chambers for 1 h before any experimental manipulations. After drug administration, animals were returned to the photocell chambers and locomotor activity was recorded for 1 h. On sham days 1 and 2, all of the animals were handled as though they were receiving a drug injection through the cannula, but no drug was administered. Two days later, seven animals received 2.0 µg of CART through the cannula (1.0 µg/0.5 µl/side), whereas the other seven animals received saline through the cannula. This treatment was repeated every 48 h for a total of four injections. Animals were then left untreated for 10 days after the final CART or saline injection. On days 11, 12, and 13 after the last drug injection, all animals were challenged with saline (i.p.), 10 mg/kg cocaine (i.p.), and 1.0 mg/kg amphetamine (i.p.), respectively.

CART Peptides and Nomenclature

The drugs used in this study were rat CART 55-102 (American Peptide, Sunnyvale, CA) and rat CART 1-26 (Neurocrine, San Diego, CA). All doses are expressed as the salt. Drugs were dissolved in sterile 0.9% saline.

CART peptide fragments were tested for biological activity by examining their effects on feeding. As previously reported, CART 55-102 inhibited food intake by 78% at a dose of 2.0 µg/5.0 µl i.c.v. (Kristensen et al., 1998), and our results (Adams et al., 1999) were comparable. Aliquots of the same batch of CART 55-102 that caused an inhibition of feeding were used in the experiments reported herein.

The assignation of numbers to CART amino acid sequences has varied in the literature. The numbering used herein corresponds to that for the long form of pro-CART protein with 102 amino acids as found in the rat (Kuhar and Dall Vechia, 1999). Rat CART 55-102 peptide begins with the amino acids IPIYE and continues to the terminal leucine. In general, the use of the acronym CART in this article refers to the peptide.

Histology

At the conclusion of behavioral studies, all rats were deeply anesthetized with sodium pentobarbital and intracardially perfused with PBS followed by 10% buffered formalin. The fixed brains were blocked in the plane of the atlas (Paxinos and Watson, 1986) and 40-µm-thick frozen sections were taken through the area of the guide cannulas. These sections were subsequently mounted on slides, stained with thionin, and examined under a microscope.

Statistics

Motor Activity. Each 1-h total activity measure (distance traveled, number of rearings, stereotypic counts) after drug or vehicle injection was subjected to a one-way ANOVA with repeated measures on drug doses, followed by a Tukey's post hoc test. The temporal data of distance traveled was analyzed by a two-way ANOVA with repeated measures on drug doses and 5-min intervals. A significant drug × time interaction was followed by a Tukey's post hoc test. Statistics were significant at P < .05.

CPP. Time spent in the white compartment was subjected to a two-way ANOVA with treatment (vehicle versus CART 55-102) as between-subject variable, and trial (preconditioning versus postconditioning) as within-subject variable. A significant trial × compartment interaction was followed by Student's t test to compare time spent in the white compartment by the CART-treated group with that of the vehicle-treated group; paired t-tests were used to compare time spent in the white compartment during the preconditioning test versus during the postconditioning test.

Sensitization. Each 1-h total activity measure (distance traveled, number of rearings, number of stereotypic movements) after drug or vehicle injection were subjected to a Student's t test comparing the CART-treated group with that of the vehicle-treated group.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Histology. Animals were prepared with bilateral cannulas as described under Materials and Methods. The placement of the cannulas' tips are shown in Fig. 1 for animals used to generate data for Figs. 2, 4, 5, and 6. Rats with cannula tips located outside of VTA or SN were excluded. However, Fig. 1 includes three rats whose VTA cannula tips were found deep and shifted; one in the mamillary body, and one in between the VTA and the SN. For these three rats, injections of low dose (0.2-1.0 µg/side) CART 55-102 were comparatively less effective (data not shown), whereas the highest dose (5.0 µg/side) gradually induced prolonged seizure activity (see below). The behavioral data of these three rats was not included in subsequent data analysis. Injections were bilateral in all cases, and doses given refer to the dose/side. Thus, the total dose given per animal was twice the stated dose in all cases.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Location of all injection cannula tips in the VTA (circle) or SN (star). All rats used for data analysis had injector tips localized to the VTA or SN (Paxinos and Watson, 1986). However, three rats whose VTA cannula tips were shifted, one in the mamillary body, and one in between the VTA and SN (triangle), are shown. For these three rats, injections of 5.0 µg of CART 55-102 per side induced a prolonged seizure activity. The behavioral data of these three rats was not included in data analysis in subsequent figures. Anatomical levels are according to Paxinos and Watson (1986). See text for details.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effects of CART 55-102 (dose/side) on locomotor effects [distance traveled (A), rearing counts (B), and stereotypic counts (C)] after injection into the VTA (mean ± S.E.). Locomotor effects were measured for 1 h as described under Materials and Methods. *P < .05, comparing all drug injections to saline injection with a one-way ANOVA (see text for F value). D, time course of motor activity after injection of CART 55-102 (dose/side) into the VTA (mean ± S.E.). , saline; , 0.04 µg; , 0.2 µg; , 1.0 µg; , 5.0 µg. *P < .05, comparing all doses to saline at each 5-min interval with a one-way ANOVA (see text for F value), followed by a Tukey's post hoc test. See text for details.

Total Motor Activity. Intra-VTA injections of CART 55-102 (0.2-5.0 µg) produced dose-dependent and significant increases in all three behavioral measures recorded: distance traveled [F(4,24) = 18.16, P < .0001; Fig. 2A], number of rearings [F(4,24) = 2.87, P = .044; Fig. 2B], and number of stereotyped movements [F(4,24) = 14.64, P < .0001; Fig. 2C]. Post hoc tests revealed that 5.0 µg of intra-VTA CART 55-102 increased all three behavioral measures greater than saline, whereas 1.0 µg increased the distance traveled and stereotypy greater than saline, and 0.2 µg increased stereotypy greater than saline. Doses higher than 5.0 µg of CART 55-102 were not used because this dose caused prolonged seizures in some animals. A postural change and tremor was observed immediately before the onset of seizures. Several rats that did not develop seizures also showed tremor-like head shaking in response to this dose of CART 55-102.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Blockade of CART-induced (1.0 µg/side intra-VTA) motor activity by increasing doses of haloperidol (i.p.). Animals were given haloperidol or saline i.p. and then CART peptide or saline intra-VTA. A, total distance traveled was measured for 1 h and analyzed with a two-way ANOVA with repeated measures on intra-VTA injection (CART, dark column versus saline, lighter column) and haloperidol doses [0.03 (), 0.1 (), and 0.3 () mg/kg i.p.]. , saline. *P < .05, points that are different from that intra-VTA saline + i.p. haloperidol at that haloperidol dose. +P < .05, points that are different from intra-VTA CART alone. B, time course of the locomotor effects induced by various doses of haloperidol (i.p.) administration followed by saline (intra-VTA) administration. C, time course of the locomotor effects induced by haloperidol (i.p.) administration followed by CART (intra-VTA) administration.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effects of CART 55-102 (dose/side) on locomotor effects [distance traveled (A), rearing counts (B), and stereotypic counts (C)] after injection into the SN (mean ± S.E.). Locomotor effects were measured for 1 h as described under Materials and Methods. *P < .05, comparing all drug injections to saline injection with a one-way ANOVA (see text for F value). D, time course of motor activity after injection of saline or various doses of CART 55-102 into the SN (mean ± S.E.). , saline; , 0.04 µg; , 0.2 µg; , 1.0 µg; , 5.0 µg. *P < .05, comparing all doses to saline at each 5-min interval with a one-way ANOVA (see text for F value), followed by a Tukey's post hoc test. See text for details.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effect of CART 1-26 on motor activity after injection into the VTA (mean ± S.E.). CART 1-26 did not produce any locomotor effects over a 60-min period at any dose tested. See text for details.

CPP Test. In a pilot study, untrained rats showed equal unconditioned preference for the two compartments (black, 451± 16 s versus white, 449 ± 16 s; n = 12). This unconditioned equal preference was remarkably stable when tested again a day later (black, 465 ± 34 s versus white, 434 ± 34 s) and a week later (black, 463 ± 34 s versus white, 437 ± 34 s). Subsequently, four intermittent pairings of 1.0 mg/kg i.p. amphetamine with the white compartment significantly increased the time animals spent in the white compartment on the postconditioning test (694 ± 37 s; n = 6), whereas four pairings of saline with the same compartment failed to produce a similar increase (457 ± 79 s; n = 6; data not shown). These results indicate that the psychomotor stimulant amphetamine produced a positive association with the white compartment, whereas the inert substance, saline, did not.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Intra-VTA administration of CART 55-102 induces CPP. Data are time (mean ± S.E.) spent in the white compartment. CART 55-102 (1.0 µg/side; dark column) or vehicle (light column) was injected into the VTA four times over 8 days (preconditioning). After the intra-VTA injection of CART, the animals were paired with the white compartment. On the test day (postconditioning), animals were allowed free access to both compartments and the amount of time spent in the white compartment was measured. *P < .01, compared with preconditioning and +P < .05, compared with vehicle-treated rats. See text for details.

Sensitization Test. Two groups of seven animals each were prepared for intra-VTA injections as described under Materials and Methods. Animals that received repeated injections of saline exhibited a similar level of activity produced by sham injections in the CART treatment group (Fig. 7). Intra-VTA injection of 1.0 µg of CART 55-102 on four separate days significantly enhanced all measures of activity compared with that produced by intra-VTA saline. Activities increased to about the same extent after each dose of CART 55-102, suggesting that repeated treatment with CART 55-102 was not producing tolerance or sensitization to these effects. When both treatment groups were challenged with saline, cocaine (10 mg/kg i.p.) or amphetamine (1.0 mg/kg i.p.), there were no significant differences between the saline or CART treatment groups; cocaine and amphetamine challenge produced a similar increase in motor activity in both groups of animals (Fig. 7). Thus, 1.0 µg of CART 55-102 intra-VTA, 10 mg/kg cocaine (i.p.), and 1.0 mg/kg (i.p.) amphetamine produced about the same increase in distance traveled. In an earlier study with different animals, the amphetamine challenge was given 1 day before the cocaine challenge, but again, CART pretreatment did not produce a sensitized response to amphetamine (data not shown). Thus, pretreatment with CART, under these conditions, did not sensitize the animals to the locomotor effects produced by cocaine or amphetamine.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Motor response (mean ± S.E.) to cocaine and amphetamine challenge after CART or vehicle treatment. On "sham" days animals were handled but no drug was given intra-VTA. CART 55-102 (1.0 µg/side) or vehicle was then injected four times over 8 days (treatments; tx.). *P < .05, compared with saline-treated animals on that treatment day. Eleven days later a subsequent challenge by cocaine (10 mg/kg i.p.) or amphetamine (12 days, 1.0 mg/kg i.p.) both groups did not reveal a sensitized response compared with vehicle-treated rats. See text for details.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

CART was identified by polymerase chain reaction differential display as an mRNA that was elevated in the rat striatum after acute systemic injection of cocaine or amphetamine (Douglass et al., 1995). This mRNA has been found in many brain areas, including those associated with reward and reinforcement (Douglass et al., 1995), and has been shown to be highly abundant relative to other mRNAs (Gautvik et al., 1996). The deduced amino acid sequence suggested that the protein product was a prepropeptide. It contained an N-terminal leader sequence indicating involvement in the secretion pathway along with several pairs of basic amino acids suggesting subsequent processing and cleavage. Indeed, a fragment beginning at amino acid 55 (Kuhar and Dall Vechia, 1999) was found in ovine hypothalamus (Spiess et al., 1981). More recently, several CART peptide fragments have been found in rat brains by Western blotting (Kuhar and Yoho, 1999) and some of these have been purified and sequenced (Thim et al., 1999). CART 55-102 has been shown to be present in rat brain (Kuhar and Yoho, 1999; Thim et al., 1999), including the VTA (Koylu et al., 1998), and to inhibit feeding in the rat after i.c.v. injection (Kristensen et al., 1998; Thim et al., 1998). Significant levels of CART peptide in the rat VTA are contained within neuronal axons and terminals (Koylu et al., 1998). Evidence that CART peptides may be neurotransmitters/cotransmitters has recently been summarized (Kuhar and Dall Vechia, 1999).

To determine whether CART peptides are behaviorally active with a possible role as mediators or modulators of the effects of psychostimulant drugs, we tested whether rat CART 55-102 injections into the VTA induced psychostimulant-like activity in the rat. Various neuropeptides in the VTA area have been found to have psychostimulant-like effects in the rat (Kalivas, 1985, 1993; Kelley and Cador, 1988; Kelley and Delfs, 1991; Kalivas and Steketee, 1992). For example, intra-VTA injections of opioid peptides, which colocalize with GABA, increased locomotor activity (Stinus et al., 1980; DuMars et al., 1988), induced CPP (Phillips and LePiane, 1980; Bozarth, 1987; Shippenberg et al., 1993), and induced locomotor sensitization to a subsequent challenge of cocaine or amphetamine (Kalivas et al., 1985; DuMars et al., 1988). The mechanism of these actions of opiates is thought to be due to the attenuation of the GABAergic inhibition of VTA dopaminergic neurons by GABA-containing nerve terminals (Klitenick et al., 1992; Spanagel et al., 1992).

The dopamine D₂ receptor antagonist haloperidol dose dependently attenuated the increase in locomotor activity produced by 1.0 µg of CART 55-102. This attenuation suggests that CART 55-102 produced the observed effects through a mechanism that involves dopamine receptors. The doses of haloperidol selected for testing in this study have been shown previously to block cocaine-induced locomotor activity in rats and mice (Cabib et al., 1991; O'Neill and Shaw, 1999). High doses of haloperidol (e.g., 1.0 mg/kg) have been shown to attenuate baseline locomotor activity in rodents (Cabib et al., 1991; O'Neill and Shaw, 1999). In this study, 0.1 and 0.3 mg/kg haloperidol attenuated the locomotor activity observed after intra-VTA administration of saline, but this attenuation was not statistically significant. Haloperidol, at those doses, significantly attenuated CART-induced locomotor activity. Therefore, the attenuation of CART-induced increases in locomotor activity is not a reflection of a general depression of motor activity but, rather, a pharmacological antagonism of the effects of CART.

The attenuation of CART-induced increases in locomotor activity by haloperidol suggests that CART is producing its effects through dopamine receptors, either directly or indirectly. As described, CART mRNA has been localized to the nucleus accumbens (Douglass et al., 1995; Koylu et al., 1998; Smith et al., 1999), a region that is rich in dopaminergic nerve terminals. In addition, CART peptides have been localized to GABAergic projection neurons in the nucleus accumbens (Smith et al., 1999). The anatomical localization of CART suggests that CART may enhance dopaminergic transmission by inhibiting the inhibitory GABAergic neurons in these brain regions. Further studies with additional subtype-selective dopamine antagonists and GABA antagonists will aid in elucidating the mechanism of action of CART peptides.

Intra-VTA injections of CART 55-102 (0.2-5.0 µg) dose dependently increased locomotor and stereotypic activities. CART 55-102, at a high dose (5.0 µg), also increased rearing activity. However, when CART 1-26 (up to 2.5 µg) was injected into the VTA, there was no significant change in the distance traveled, number of rearings, or number of stereotypic counts. These data suggest that the locomotor activating effect is at least somewhat specific for CART 55-102.

When CART 55-102 was injected bilaterally into the SN, there was no significant increase in motor activity after administration of doses up to 1.0 µg. When 5.0 µg was administered, there was a significant increase in all behavioral measures, but these increases in activity occurred in a delayed fashion, possibly reflecting diffusion of the peptide from the SN to VTA sites. It has been previously reported that injection of CART 55-102 (2.0 µg) into the lateral ventricle, a more distant site, did not significantly alter locomotor activity (Kristensen et al., 1998). These data indicate that sensitivity to CART 55-102 seems to be a result of action in the VTA instead of in the SN. The sensitivity of other brain regions such as the nucleus accumbens to CART 55-102 will be tested in future studies.

CPP is a measure of the reinforcing properties of drugs with classical conditioning techniques. An advantage of this paradigm over other behavioral tests is that the drug is not present at the time of testing and does not interfere with behavioral measurements (Stolerman, 1992). Repeated injections of 1.0 µg of CART 55-102 into the VTA induced a CPP for the environment associated with CART injections. In these studies, rats were placed into the white compartment after CART injections, to control for a natural preference for one compartment over the other. Previous studies have shown that rats usually have a natural preference for the darker compartment (Van Ree et al., 1999). Other groups of animals were given repeated intermittent intra-VTA injections of saline or CART 55-102, then challenged with cocaine (10 mg/kg i.p.) or amphetamine (1.0 mg/kg i.p.). No sensitization or tolerance to cocaine or amphetamine was found in CART-treated animals compared with those treated with saline, at least under these conditions. Moreover, four repeated injections of 1.0 µg of CART 55-102 each increased activities to a similar degree and failed to produce tolerance or sensitization to the locomotor-stimulating effects of CART itself. Although our paradigm of every-other-day injection will produce sensitization to psychostimulants (Hooks et al., 1991; Kelsey and Grabarek, 1999) it may not be adequate to produce sensitization by CART peptide. For example, Elliott and Nemeroff (1986) found that daily injection of neurotensin produced behavioral sensitization but injections into VTA every other day did not. Thus, we cannot rule out that another schedule of CART peptide injection would produce behavioral tolerance or sensitization.

Doses of 5.0 µg of CART 55-102 produced apparent seizures in some rats when the injection cannulas were misplaced and shifted from the VTA to the mamillary body or to a site in between the VTA and SN. Thus, CART 55-102 may be seizuregenic, although the specific site mediating the effect is not clear and will be resolved in future experiments. Possible mechanisms could include a disinhibition (of GABA) or promotion of excitatory transmission. Before the onset of seizures, and sometimes at higher doses without seizures, a postural change and tremor was observed. Tremor after administration of CART 55-102 has been observed previously in the rat (Kristensen et al., 1998).

The complete mechanism by which CART 55-102 elicits these behavioral effects is unknown, although activation of D₂ dopamine receptors appears to be involved. Other peptides with psychostimulant-like effects require intact A10 neurons (Kelley et al., 1980; Shippenberg et al., 1993) and may involve presynaptic inhibition of the GABAergic input to the VTA (Stinus et al., 1980; Klitenick et al., 1992; Spanagel et al., 1992). Regarding the latter, it is notable that CART peptides may be found in GABAergic neuronal afferents to the VTA (Smith et al., 1999) from the accumbens and thus could be strategically placed to inhibit GABA release via autoreceptors. CART receptors have not yet been identified by binding, but i.c.v. injection of CART 55-102 into the rat brain induces c-fos in a variety of neurons (Vrang et al., 1999), which is indicative of the existence of receptors and signal transduction cascades.

Our findings that intra-VTA injections of CART 55-102 peptide induced locomotor activation and CPP in the rat support the hypothesis that this peptide mediates or modulates the locomotor-activating and -rewarding mechanisms of psychostimulants. Although the involvement of CART peptides in the actions of psychostimulants has been suspected since the identification of CART (Douglass et al., 1995), this is the first demonstration that CART 55-102 is behaviorally active in this regard. Antagonists of CART peptides, although they have not yet been identified, will be needed to determine whether endogenous CART peptides are involved in the actions of cocaine and amphetamine.