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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 294:784–792, 2000 /2578/840556
Vol. 294, No. 2
Printed in U.S.A.
Intra-Ventral Tegmental Area Injection of Rat Cocaine and
Amphetamine-Regulated Transcript Peptide 55-102 Induces
Locomotor Activity and Promotes Conditioned Place
Preference1
HEATHER L. KIMMEL, WENHE GONG, STEPHANIE DALL VECHIA, RICHARD G. HUNTER, and MICHAEL J. KUHAR
Yerkes Regional Primate Research Center, Emory University, Atlanta, Georgia
Accepted for publication May 4, 2000
This paper is available online at http://www.jpet.org
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
Received for publication February 9, 2000.
1
This study was supported by National Institutes of Health Grants
RR00165, DA00418, DA10732, and DA005935.
suggesting that CART 55-102 is reinforcing. Increases in locomotor activity after each of these CART 55-102 injections were
similar and did not show tolerance or sensitization. This treatment regimen of CART 55-102 also did not produce sensitization to locomotor activity after a subsequent challenge with
cocaine or amphetamine. When CART 55-102 (0.2–1.0 ␮g/side)
was injected into the substantia nigra, no significant change in
motor activity was observed. However, a higher dose of CART
55-102 (5.0 ␮g/side) induced a delayed increase in motor activity, suggesting a possible diffusion from the substantia nigra
into the ventral tegmental area. Our findings suggest that CART
55-102 is behaviorally active and may be involved in the actions
of psychostimulants. This is the first demonstration of the psychostimulant-like effects of CART peptides.
(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).
ABBREVIATIONS: CART, cocaine- and amphetamine-regulated transcript; VTA, ventral tegmental area; GABA, ␥-aminobutyric acid; CPP,
conditioned place preference; SN, substantia nigra.
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ABSTRACT
Cocaine- and amphetamine-regulated transcript (CART) is a
novel mRNA that has been reported to be increased by acute
psychostimulant administration, and that may be involved in
the effects of psychostimulants. In this study, we examined the
effect of centrally administered CART peptides on locomotor
activity and conditioned place preference in the rat. CART
peptide fragments were bilaterally injected into the ventral tegmental area. CART 55-102 (0.2–5.0 ␮g/side), an endogenously
occurring peptide, dose dependently increased locomotor activity, whereas CART 1-26 (0.1–2.5 ␮g/side; not found endogenously) did not. The locomotor effects of CART 55-102 were
dose dependently blocked by the dopamine D2 receptor antagonist haloperidol (0.03–1.0 mg/kg i.p.). Four injections of 1.0
␮g/side CART 55-102 induced a significant place preference,
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Intra-VTA CART Produces Psychostimulant Effects in Rat
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
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-␮l 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-␮l 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 ␮g CART 55-102 counterbalanced for order in a withinsubject design. Once this dose-response curve was established, all
animals received an intra-VTA injection of 5.0 ␮g of CART 55-102. A
dose higher than 5.0 ␮g 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 ␮g/0.5 ␮l/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 ␮g 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 ␮g
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 ␮g 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 ␮l/side)
once every other day for 8 days. A second group (n ⫽ 6) received
CART 55-102 (1.0 ␮g/0.5 ␮l/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
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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.
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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
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
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
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 ␮g/5.0 ␮l 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-␮m-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.
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 ␮g 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.
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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 ␮g of
CART through the cannula (1.0 ␮g/0.5 ␮l/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.
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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 ␮g/side) CART 55-102 were
comparatively less effective (data not shown), whereas the
highest dose (5.0 ␮g/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.
Total Motor Activity. Intra-VTA injections of CART 55102 (0.2–5.0 ␮g) 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 ␮g of intra-VTA CART 55-102
increased all three behavioral measures greater than saline,
whereas 1.0 ␮g increased the distance traveled and stereotypy greater than saline, and 0.2 ␮g increased stereotypy
greater than saline. Doses higher than 5.0 ␮g of CART 55102 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.
The time course of distance traveled after intra-VTA injection of CART 55-102 was analyzed by a two-way ANOVA
with repeated measures on drug doses and 5-min intervals
(Fig. 2D). There was a significant effect of dose [F(4,65) ⫽
46.48, P ⬍ .0001] and time [F(11,715) ⫽ 45.90, P ⬍ .0001] as
well as a significant drug ⫻ time interaction [F(44,715) ⫽
360, P ⬍ .0001]. As shown in Fig. 2, intra-VTA injection of 1.0
or 5.0 ␮g of CART 55-102 increased locomotor activity within
minutes after administration.
Because several peptides that cause increased locomotor
activity do so through dopaminergic neurons (Longoni et al.,
1991; Florin et al., 1996), haloperidol (0.03–1.0 mg/kg i.p.)
was administered 30 min before intra-VTA injection of 1.0
␮g/0.5 ␮l/side CART 55-102. The total distance traveled was
measured for 60 min after the administration of CART (Fig.
3). A two-way ANOVA with repeated measures on drug doses
revealed a significant effect of intra-VTA injection of CART
[F(1,22) ⫽ 55.05, P ⬍ .0001], a significant effect of haloperidol dose [F(3,66) ⫽ 56.4, P ⬍ .0001, and a significant interaction between intra-VTA injection of CART ⫻ haloperidol
[F(3,66) ⫽ 19.21, P ⬍ .0001]. Post hoc tests showed that the
1.0 ␮g of intra-VTA CART 55-102 ⫹ saline (i.p.) or 0.03 mg/kg
haloperidol (i.p.) combination produced significantly more
activity than intra-VTA saline ⫹ saline (i.p.) or 0.03 mg/kg
haloperidol (i.p.) combination, respectively. Although 0.03
mg/kg haloperidol (i.p.) did not alter CART 55-102-induced
locomotor activity, 0.1 and 0.3 mg/kg haloperidol significantly reduced activity produced by CART 55-102 (Fig. 3).
Intra-SN injections of CART 55-102 (0.2–5.0 ␮g) also produced significant increases in the distance traveled
[F(4,16) ⫽ 11.34, P ⫽ .0001; Fig. 4], number of rearings
[F(4,16) ⫽ 5.80, P ⫽ .004; Fig. 4], and number of stereotyped
movements [F(4,16) ⫽ 43.89, P ⬍ .0001; Fig. 4]. However,
post hoc tests revealed that only 5.0 ␮g of CART 55-102
produced significant increases in all three behaviors. Lower
doses of CART 55-102 (0.2 and 1.0 ␮g) tended to decrease
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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.). 䡺, saline; f, 0.04 ␮g; Œ, 0.2 ␮g;
, 1.0 ␮g; F, 5.0 ␮g. *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.
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Fig. 3. Blockade of CART-induced (1.0 ␮g/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 (⽧), 0.1
(f), and 0.3 (F) mg/kg i.p.]. 䡺, 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.
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.). 䡺, saline; f, 0.04 ␮g; Œ,
0.2 ␮g; , 1.0 ␮g; F, 5.0 ␮g. *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.
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locomotor activity slightly, but this was not statistically significant.
The time course of distance traveled after intra-SN administration of CART 55-102 was analyzed by a two-way ANOVA
with repeated measures on drug doses and 5-min intervals
(Fig. 4). A significant effect of dose [F(4,65) ⫽ 35.63, P ⬍
.0001] and time [F(11,495) ⫽ 87.65, P ⬍ .0001] were noted as
well as a significant drug ⫻ time interaction [F(44,495) ⫽
9.39, P ⬍ .0001]. When injected into the SN, 1.0 ␮g of CART
55-102 did not increase locomotor activity at any time point
and 5.0 ␮g of CART 55-102 did not increase activity until 25
min postinjection. This delayed activation found after injection of the highest dose of CART 55-102 into the SN suggests
that this peptide may have diffused to an active site distant
from the site of injection.
Intra-VTA administration of CART 1-26 (up to 2.5 ␮g) did
not significantly alter the distance traveled [F(3,15) ⫽ 1.70,
NS; Fig. 5], number of rearings [F(3,15) ⫽ 1.38, NS; Fig. 5],
or number of stereotyped movements [F(3,15) ⫽ 1.88, NS;
Fig. 5]. Thus, not all CART peptide fragments increase these
behavioral measures.
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.
A separate group of animals was then prepared for testing
for CPP as described under Materials and Methods. Cart
55-102 (1.0 ␮g) or vehicle was paired with the white compartment four times over a period of 8 days. During the postconditioning trial, animals previously given intra-VTA CART
55-102 spent more time in the white compartment compared
with animals previously given saline (Fig. 6). A significant
treatment ⫻ trial interaction was found [F(1,10) ⫽ 9.389, P ⬍
.05]. In addition, animals previously given CART 55-102
significantly increased the time spent in the white compartment compared with the time observed in the preconditioning trial.
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 ␮g 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 55102 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
Intra-VTA CART Produces Psychostimulant Effects in Rat
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.
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 ␮g 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.
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Kimmel et al.
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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
␮g/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.
Discussion
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.,
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 ␮g/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.
2000
791
present at the time of testing and does not interfere with
behavioral measurements (Stolerman, 1992). Repeated injections of 1.0 ␮g 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 ␮g 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 ␮g 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
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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 ␮g 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 subtypeselective 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 ␮g) dose
dependently increased locomotor and stereotypic activities.
CART 55-102, at a high dose (5.0 ␮g), also increased rearing
activity. However, when CART 1-26 (up to 2.5 ␮g) 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 ␮g. When 5.0 ␮g 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 ␮g) 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
Intra-VTA CART Produces Psychostimulant Effects in Rat
792
Kimmel et al.
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.
Acknowledgments
We thank Elizabeth Nadler and Melody Elsley for administrative
assistance in the preparation of this manuscript and Dr. Nick Ling
for CART 1-26.
References
Koylu EO, Couceyro PR, Lambert PD and Kuhar MJ (1998) Cocaine- and amphetamine-regulated transcript peptide immunohistochemical localization in the rat
brain. J Comp Neurol 391:115–132.
Kristensen P, Judge M, Thim L, Ribel U, Christjansen K, Wulff B, Clausen J, Jensen
P, Madsen O, Vrang N, Larsen P and Hastrup S (1998) Hypothalamic CART is a
new anorectic peptide regulated by leptin. Nature (Lond) 393:72–76.
Kuhar M and Dall Vechia S (1999) CART peptides: Novel addiction- and feedingrelated neuropeptides. Trends Neurosci 22:316 –320.
Kuhar M and Yoho L (1999) CART peptide analysis by Western blotting. Synapse
33:163–171.
Lambert P, Couceyro P, Koylu E, Ling N, DeSouza E and Kuhar M (1997) A role for
novel CART peptide fragments in the central control of food intake. Neuropeptides
31:620 – 621.
Lambert P, Couceyro P, McGirr K, Dall Vechia S, Smith Y and Kuhar M (1998)
CART peptides in the central control of feeding and interactions with neuropeptide
Y. Synapse 29:293–298.
Longoni R, Spina L, Mulas A, Carboni E, Garau L, Melchiorri P and Di Chiara G
(1991) (D-Ala2)deltorphin II: D1-dependent stereotypies and stimulation of dopamine release in the nucleus accumbens. J Neurosci 11:1565–1576.
Louis J, inventor (1996) Methods of preventing neuron degeneration and promoting
neuron regeneration, in Amgen, Inc. International Patent Application. Patent
PCT/US96/05894.
O’Neill M and Shaw G (1999) Comparison of dopamine receptor antagonists on
hyperlocomotion induced by cocaine, amphetamine, MK-801 and the dopamine D1
agonist C-APB in mice. Psychopharmacology 145:237–250.
Paxinos G and Watson C (1986) The Rat Brain in Stereotaxic Coordinates. Academic
Press, Sydney.
Pellegrino LJ, Pelligrino AS and Cushman AJ (1979) Stereotaxic Atlas of the Rat
Brain. Plenum Press, New York.
Phillips A and LePiane F (1980) Reinforcing effects of morphine microinjection into
the ventral tegmental area. Pharmacol Biochem Behav 12:965–968.
Sanberg P, Moran T, Kubos K and Coyle J (1984) Automated measurement of
rearing behavior in adult and neonatal rats. Behav Neurosci 98:743–746.
Shippenberg TS, Bals-Kubik R and Herz A (1993) Examination of the neurochemical
substrates mediating the motivational effects of opioids: Role of the mesolimbic
dopamine system and D-1 vs. D-2 dopamine receptors. J Pharmacol Exp Ther
265:53–59.
Smith Y, Kieval J, Couceyro P and Kuhar M (1999) CART peptide-immunoreactive
neurones in the nucleus accumbens in monkeys: Ultrastructural analysis, colocalization studies, and synaptic interactions with dopaminergic afferents. J Comp
Neurol 407:491–511.
Spanagel R, Herz A and Shippenberg T (1992) Opposing tonically active endogenous
opioid systems modulate the mesolimbic dopaminergic pathway. Proc Natl Acad
Sci USA 89:2046 –2050.
Spiess J, Villarreal J and Vale W (1981) Isolation and sequence analysis of a
somatostatin-like polypeptide from ovine hypothalamus. Biochemistry 20:1982–
1988.
Stinus L, Koob G, Ling N, Bloom F and LeMoal M (1980) Locomotor activation
induced by infusion of endorphins into the ventral tegmental area: Evidence for
opiate-dopamine interactions. Proc Natl Acad Sci USA 77:2323–2327.
Stolerman I (1992) Drugs of abuse: behavioral principles, methods and terms. Trends
Pharmacol Sci 13:170 –176.
Thim L, Kristensen P, Nielsen P, Wulff B and Clausen J (1999) Tissue-specific
processing of cocaine- and amphetamine-regulated transcript peptides in the rat.
Proc Natl Acad Sci USA 96:2722–2727.
Thim L, Nielsen P, Judge M, Andersen A, Diers I, Egel-Mitani M and Hastrup S
(1998) Purification and characterisation of a new hypothalamic satiety peptide,
cocaine and amphetamine regulated transcript (CART), produced in yeast. FEBS
Lett 428:263–268.
Van Ree J, Gerrits M and Vanderschuren L (1999) Opioids, reward and addiction: An
encounter of biology, psychology, and medicine. Pharmacol Rev 51:341–396.
Vrang N, Tang-Christensen M, Larsen P and Kristensen P (1999) Recombinant
CART peptide induces c-Fos expression in central areas involved in control of
feeding behaviour. Brain Res 818:499 –509.
Send reprint requests to: Heather L. Kimmel, Ph.D., Yerkes Regional
Primate Research Center, Emory University, 954 Gatewood Rd. NE, Atlanta,
GA 30329. E-mail: [email protected]
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 8, 2017
Adams L, Gong W, Dall Vechia S, Hunter R and Kuhar M (1999) CART: From gene
to function. Brain Res 848:137–140.
Bozarth M (1987) Neuroanatomical boundaries of the reward-relevant opiatereceptor field in the ventral tegmental area as mapped by the conditioned place
preference method in rats. Brain Res 414:77– 84.
Cabib S, Castellano C, Cestari V, Filibeck U and Puglisi-Allegra S (1991) D1 and D2
receptor antagonists differently affect cocaine-induced locomotor hyperactivity in
the mouse. Psychopharmacology 105:335–339.
Couceyro P, Koylu E and Kuhar M (1997) Further studies on the anatomical distribution of CART by in situ hybridization. J Chem Neuroanat 12:229 –241.
Douglass J, McKinzie A and Couceyro P (1995) PCR differential display identifies a
rat brain mRNA that is transcriptionally regulated by cocaine and amphetamine.
J Neurosci 15:2471–2481.
DuMars L, Rodger L and Kalivas P (1988) Behavioral cross-sensitization between
cocaine and enkephalin in the A10 dopamine region. Behav Brain Res 27:87–91.
Elliott P and Nemeroff C (1986) Repeated neurotensin administration in the ventral
tegmental area: Effects on baseline and D-amphetamine-induced locomotor activity. Neurosci Lett 68:239 –244.
Florin S, Suaudeau C, Meunier J and Costentin J (1996) Nociceptin stimulates
locomotion and exploratory behavior in mice. Eur J Pharmacol 317:9 –13.
Gautvik K, de Lecea L, Gautvik V, Danielson P, Tranque P, Dopazo A, Bloom F and
Sutcliffe J (1996) Overview of the most prevalent hypothalamus-specific mRNAs,
as identified by directional tag PCR subtraction. Proc Natl Acad Sci USA 93:8733–
8738.
Hooks M, Jones G, Neill D and Justice J Jr (1991) Individual differences in amphetamine sensitization: Dose-dependent effects. Pharmacol Biochem Behav 41:203–
210.
Kalivas P (1985) Interactions between neuropeptides and dopamine neurons in the
ventromedial mesencephalon. Neurosci Biobehav Rev 9:573–587.
Kalivas P (1993) Neurotransmitter regulation of dopamine neurons in the ventral
tegmental area. Brain Res Rev 18:75–113.
Kalivas P and Steketee J (1992) Possible transduction mechanisms mediating the
acute and sensitized response to neurotensin in the ventral tegmental area. Ann
NY Acad Sci 668:157–164.
Kalivas P, Taylor S and Miller J (1985) Sensitization to repeated enkephalin administration into the ventral tegmental area of the rat. I. Behavioral characterization.
J Pharmacol Exp Ther 235:537–543.
Kelley A and Cador M (1988) Behavioral evidence for differential neuropeptide
modulation of the mesolimbic dopamine system. Ann NY Acad Sci 537:415– 434.
Kelley A and Delfs J (1991) Dopamine and conditioned reinforcement. II. Contrasting effects of amphetamine microinjection into the nucleus accumbens with peptide microinjection into the ventral tegmental area. Psychopharmacology 103:197–
203.
Kelley A, Stinus L and Iverson S (1980) Interactions between D-ala-met-enkephalin,
A10 dopaminergic neurones and spontaneous behaviour in the rat. Behav Brain
Res 1:3–24.
Kelsey J and Grabarek J (1999) Medial septal lesions in rats enhance locomotor
sensitization to amphetamine. Psychopharmacology 146:233–240.
Klitenick MA, De Witte P and Kalivas PW (1992) Regulation of somatodendritic
dopamine release in the ventral tegmental area by opioids and GABA: An in vivo
microdialysis study. J Neurosci 12:2623–2632.
Koylu E, Couceyro P, Lambert P, Ling N, DeSouza E and Kuhar M (1997) Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland. J Neuroendocrinol 9:823– 833.
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