1. 1
The Serotonin 1B receptor agonist CP 94253 reduces cocaine intake after a period of
protracted abstinence
ABSTRACT:
Cocaine is a highly addictive psychostimulant that is widely used around the world. It is far more
cost effective to curb this problem through treatment than by any other method as medicinal drug
treatment is 15 times more effective than law enforcement at reducing the societal costs of
cocaine use as determine by RAND corp. In a previous paper from our lab, it was found that via
virally mediated introduction of additional 5-HT1B receptors into the nucleus accumbens there
was a leftward shift in the cocaine intake dose-response curve in animals that were self-
administering cocaine by pressing a lever. These findings suggest that 5-HT1B receptor action
enhances the reinforcing effects of cocaine. However, when animals were given a 21-day period
of prolonged abstinence and then tested for cocaine intake, it was determined that 5-HT1B
receptor action had the opposite effect of decreasing cocaine intake presumably due to a decrease
in the reinforcing effects of cocaine: [16]. The experiment in the current paper was devised to
further test this finding via pharmacological means using the 5-HT1B agonist CP 94253 to
increase stimulation of 5-HT1B receptors. Animals were trained to self-administer by pressing a
lever on fixed ratio schedules of cocaine reinforcement given at 0.75 mg/kg and 0.075 mg/kg
doses of cocaine. These doses allowed us to examine changes in self-administration on both the
ascending and descending limbs of the inverted u-shaped cocaine dose-effect curve. Our results
indicated that in animals given CP 94253 exhibited a decrease in responding on both the
ascending and descending limbs of the dose response curve demonstrating a downward shift
after prolonged abstinence. These exciting results suggest that the agonist decreases cocaine
intake, and therefore, the agonist may be a useful treatment for cocaine dependence.
INTRODUCTION:
Cocaine is a psychostimulant that can be highly addictive with repeated use. According to
the department of Health and Human Services, though the treatment admission rate for cocaine
abuse was 23% lower in 2008 than in 1998, the drug is still a societal problem as it has become
the primary psychoactive drug in many Mid-Atlantic States. Approximately 35.3 million
Americans aged 12 and older were reported to have used cocaine, 8.5 million reported using the
inhalable and highly addictive form known as crack, and 2.4 million Americans were current
users of cocaine (National Survey on Drug Use and Health). According to the National Institute
on Drug Abuse, the annual financial costs of illicit drug use disorders and alcohol abuse in the
United States were estimated to be $181 and $185 billion. Such a problem needs to be addressed
as aggressively as possible.
Cocaine has a high affinity for the dopamine (DA) transporter (DAT; Ki = 277 nM),
serotonin (5-HT, 5-hydroxytryptamine) transporter (SERT; Ki= 217 nM) and norepinephrine
(NE) transporter (NET; Ki = 144 nM) [1]
. Psychostimulants, though they affect all monoamine
transporters, have different relative impacts with great efficacy at serotonin receptors as well as
in dopaminergic and adrenergic receptors for subjects given cocaine. This effect is achieved in
serotonin synapses by blocking serotonin reuptake transporters increasing the amount of
available serotonin in the synapse.

2. 2
With the exception of the 5-HT3 receptor, which is a ligand-gated ion channel, 5-HT
receptors belong to the class of G-protein-coupled receptors (GPCR) [2]
. The 5-HT family is
diverse, comprised of 7 families with a total of 15 subtypes of structurally and pharmacologically
different components in the mammalian pool of species [1] [2]
. 5-HT neurons originate in the raphe
nuclei with ascending and descending limbs innervating the majority of the brain and spinal
cord. Thus, 5-HT has been associated with regulations of vital autonomic functions and
psychological states [1]
.
The 5-HT system is strongly implicated in cocaine-associated motivational effects.
Following chronic cocaine self-administration, individuals in withdrawal exhibit a decrease in
extracellular levels of 5-HT in the nucleus accumbens [3]
. This decrease occurs at a time when
cravings for cocaine and the motivation to obtain it emerge during withdrawal, suggesting these
events are related. The progressive increase of cue induced drug craving after several weeks of
abstinence is a phenomenon referenced to in animal literature as “the incubation of drug
craving.” Investigators have identified analogous incubation phenomenon in rodents (time
dependent increases in cue-induced drug seeking) [4]
. Increases in craving has been previously
studied in the Neisewander lab in which Fos expression was measured, after a priming injection,
to be much greater in cocaine experienced rats relative to a control suggesting that this
enhancement reflects an experience-dependent motivational effect of the priming injections [5]
.
This drug seeking behavior increase over time is not specific to cocaine as similar findings have
been observed after self-administration of heroin, nicotine, methamphetamine and alcohol in rats,
further demonstrating the importance of craving during abstinence in drug experienced animals
(a predictable physiological response) [4]
.
Our animal model was used to measure cocaine-seeking and cocaine-taking behaviors.
The model allowed for self-administration, in which the animals were able to attain the drug
independently by pressing a lever. As such, animals learn to associate the pleasurable effects of
the drug with their actions and thus increase their lever responding. After a certain number of
infusions however, which varies slightly among animals, intake plateaus creating a predictable
level of lever pressing which can then be subject to external pressures. Cocaine-seeking behavior
involves the subject seeking to obtain the drug in the absence of reinforcement. It is interesting
as it can be used to signify motivation. Responding on the active lever in the absence of cocaine
reinforcement is the operational definition of cocaine-seeking behavior. As animals are more
motivated, it is asserted that they will be more active in their lever responding. As individuals
pursue the acquisition of more cocaine over time, they are seeking the drug indicating that the
lever press behavior for a cocaine reward was reinforced. In addition, testing for cue- and
cocaine primed reinstatement was done because such a model is the best method of simulating a
former drug addict sampling a small amount of drug, being around drug paraphernalia, or any
other exposure to cocaine associated cues that may induce motivation.
Testing happens on either a progressive ratio schedule (PR) or fixed ratio schedule (FR).
In the past, PR schedules have been used frequently to study the relative reinforcing strengths of
and motivation for intravenously delivered drugs because those animals given a drug with
heavier abuse potential would see a more constant progression across the schedule as drugs that
are more reinforcing produce higher break points (animals are willing to work more to obtain the
drug). Furthermore, as it becomes more difficult for rats to obtain the reward as their schedule
becomes more difficult; their progression is a measure of motivation for the cocaine while FR

3. 3
schedules can only demonstrate the former of these two occurrences. This is significant in
developing evaluation procedures for abuse liability of various psychoactive drugs [7]
.
An animal can be subject to extinction. This is a method by which subjects are re-
introduced to the drug environment with all cues but the reinforcing stimulus withheld, in an
effort to teach the subject to disassociate drug availability from the environment in which the
drug is acquired. Animals can also be subject to abstinence in which the subjects are not exposed
to the drug-taking environment for a predetermined period of time. After extinction or
abstinence, the animal can be triggered to relapse in which reintroduction of the reinforcing
stimulus produces a dramatic spike in subject lever pressing. Here, manipulations can be
conducted such as administering agents to observe their effects on the animals’ lever-pressing in
relapse.
During this time, 5-HT neurotransmitter levels drop and symptoms of withdrawal such as
“depression, anhedonia, insomnia, fatigue, hyperphagia and drug craving” [1] [8]
generally present
themselves. Though most symptoms fade within the first week of withdrawal, drug craving
persists. 5-HT1B has thus far proved to be a good target for the study of cocaine effects and as a
mode of regulation of cocaine induced hyperlocomotion and seeking.
The effect of 5-HT1B receptor [5-HT1B(R)] stimulation on dopamine-mediated
reinforcement in rats was investigated using intravenous self-administration of the selective
dopamine uptake inhibitor GBR-12909 on an FR5 schedule of reinforcement [9]
. The finding
showed that pretreatment with the selective 5-HT1A receptor agonist 8-OH-DPAT (0.03–1.0
mg/kg, subcutaneous [s.c.]) had no significant effect on GBR-12909 self-administration.
However, pretreatment with the 5-HT1A/1B receptor agonist CGS-12066B (1–10 mg/kg,
intraperitoneal [i.p.]) dose-dependently reduced the self-administration of GBR-12909 (83
mg/injection) by increasing the interval between drug injections, consistent with an enhancement
of the reinforcing effects of GBR-12909 [8]
. In a study examining the effect of prolonged
treatment with cocaine on 5-HT1B expression in the brain, a 5-HT1B/1D receptor agonist was used
in the presence of a 5-HT1D antagonist (to isolate 1B effects). The result was an up-regulation of
5-HT1BRs in a number of rat brain areas [15]
.
The dose-effect function was used in our experimentation as it provides a good model for
tracing changes in animal motivation over time For example, lower doses on a dose-effect curve
would not trigger cocaine intake as well as higher doses. However, at sufficiently high doses
animals need only a few doses, a value that varies between individuals, to obtain a “high” after
which cocaine intake is limited to the amount needed to maintain the high originally achieved.
Thus the dose-effect function, also known as that dose response curve, is shaped like an inverted
U. Shifts left or right indicated then increases or decreases in the efficacy of a dose of cocaine
when experimental drugs are onboard. A downward shift would indicate a decrease in intake at
all doses suggesting a decrease in overall motivation.
The human serotonin receptor 1B, encoded by the HTR1B (5-HT1B) gene, is a presynaptic
serotonin autoreceptor that plays an important role in regulating serotonin synthesis and release
[10]
. As a heteroreceptor, actions at 5-HT1B receptor reduce the release of dopamine, GABA,
glutamate, and acetylcholine [10]
. Interest in 5-HT1B receptors has grown in recent years as they

4. 4
have been attributed to decreases in motivation, both for cocaine (a re-uptake inhibitor) and
sucrose. The 5-HT1B receptor agonists 5-methoxy-3-1,2,3,6-tetrahydro-4-pyridinyl- 1H-indole
(RU 24969) (0.3–3 mg/kg), 3-(1,2,5,6-tetrahydro-4- pyridyl)-5-propoxypyrrolo[3,2-b]pyridine
(CP 94253) (0.3–3 mg/ kg), and 3-(1,2,5,6-tetrahydropyrid-4-yl) pyrrolo[3,2-b]pyridine (CP
93,129) (3 and 10 mg, intracerebroventricular [i.c.v.]) each dose-dependently reduced the self-
administration of a cocaine dose on the descending limb of the FR5 cocaine dose–effect function
[11]
, and all reduced the threshold dose of cocaine necessary to produce a reinforcing effect in rats
[11]
. These effects were also observed on a PR schedule of reinforcement in which as the unit
dose of CP 94253 pretreatment increased from .3 mg/kg to 3 mg/kg, the total number of cocaine
infusions obtained per 3 hour session increased (the lowest intake being on the saline dose)[11]
.
When 5-HT1B receptors are stimulated using the 5-HT1B/1A receptor agonist, RU24969, a
decrease in cue- and cocaine-primed cocaine-seeking behavior and, even sucrose seeking
behavior was observed in animals given RU24969 doses of .3, 1, and 3 [mg/kg] with no change
in locomotor activity in the lower two doses [12]
. These effects are completely reversed when the
5-HT1B antagonist GR127935 (3 mg/kg, s.c.) was administered suggesting that the observed
general decrease in motivation was 5-HT1B mediated [12]
. When CP 94253, a 5-HT1B receptor
agonist, is administered subcutaneously to rats before testing across a range of cocaine doses, it
dose dependently decreases reinstatement of cocaine-seeking behavior while increasing cocaine
reinforcement, indicating that stimulation of 5-HT1B receptors enhances sensitivity to the
reinforcing effects of cocaine but attenuates incentive motivation for cocaine [13]
. This pattern of
results is surprising given that most manipulations that enhance drug reinforcement also enhance
motivation for drug, but 5-HT1B receptor agonists produce opposing effects on these two
measures.
Cocaine can serve as a discriminative stimulus, meaning that animals can learn to
perform a particular response for reinforcement when they are in a cocaine state and a different
response for the reinforcement when they are in a saline state. In other words, the animal can use
the psychoactive effects of the drug as a cue for knowing which type of response is needed for
reinforcement. Through the study of this activity of cocaine, it was determined that this
discriminative stimulus effect was not caused by 5-HT1B receptors. Using the 5-HT1B agonist, it
was however determined that stimulation of this receptor enhances the discriminative stimulus
effects of cocaine producing a leftward shift in the dose response curve in rats given CP 94253
(2.5 and 5 mg/kg, s.c.) [14]
.
We have conducted research previously in attempting to further elucidate the role of
serotonin 1B receptors in cocaine abuse-related behaviors. This was done by using viral-
mediated gene transfer to increase 5-HT1BRs in the terminals of the medial NAsch neurons [16]
.
5-HT1BR gene transfer during maintenance shifted the dose-response curve to the left on a FR5
schedule and increased breakpoints and cocaine intake on a PR schedule. In contrast, the same
manipulation after prolonged abstinence attenuated breakpoints and cocaine intake on a PR
schedule of reinforcement, as well as cue- and cocaine-primed reinstatement of cocaine-seeking
behavior [16]
. This suggests that 5-HT1BRs differentially modulate cocaine abuse-related
behaviors depending on the stage of the addiction cycle, with enhanced reinforcing effects of
cocaine during maintenance and diminished reinforcing effects of cocaine following protracted
abstinence. The purpose of this honors thesis was to examine the reliability of the previous
findings using a pharmacological approach. For this reason, we decided to pretreat rats with a 5-

5. 5
HT1B agonist prior to tests for cocaine self-administration and cocaine-seeking behavior in the
hopes of mimicking the previous results. Additionally, we examined the effects of the agonist at
multiple cocaine doses on FR responding.
METHODS:
Subjects:
Adult male Sprague-Dawley rats (n = 124) weighing 263 g to 355 g at the time of surgery
were individually housed in a climate-controlled colony room with a reversed 10-hr light/ 14-hr
dark cycle (lights off at 6:00 AM). The Guide for the Care and Use of Laboratory Animals
(2011) was used to dictate all experimentation and animal husbandry practices. The Institutional
Animal Care and Use Committee at Arizona State University reviewed and approved all
experimentation.
Surgery:
Under 2-3% isoflurane anesthesia, catheters were implanted into the right interior jugular
vein of Spraug-Dawley rats [17]
. The catheter was then run underneath the skin of the rat to the
top of the skull where an oval incision was made half an inch in radius within which all
musculature and skin was removed so that the skull was exposed. To this skull were added four
equidistant screws. The end of the catheter was fitted with a cannula and fastened with super
glue on top of the skull using super glue. This apparatus was further secured with dental acrylic
to prevent dislodging. A cap was placed on top of each cannula and was only removed when the
animal’s catheter was being flushed with Timentin®
or when it was connected to the drug
infusion line in an operant box during self-administration. Rats were returned to home boxes for
6-7 days prior to self-administration during which time they were handled and flushed daily with
0.1 ml saline containing heparin sodium (70 U/ml; APP Pharmaceuticals, Schaumburg, IL),
Abbokinase (20 mg/ml; ImaRx Therapeutics, Tucson, AZ) and Timentin (66.7 mg/ml;
GlaxoSmithKline, Research Triangle Park, NC) for 5 days following surgery. After this period,
only heparinized saline with Timentin®
was given so as to maintain patency by preventing
clotting and biofilm growth inside of the catheter. Brevital®
, an ultra-short acting barbiturate,
was used as a measure of patency as it caused temporary muscle relaxation in rats with patent
catheters.
Drugs:
Cocaine hydrochloride (RTI International, Research Triangle Park, NC) was dissolved in
0.9% bacteriostatic saline (Hospira Inc., Lake Forest, IL) and filtered through 0.2 µm membrane
Acrodisc Syringe Filters (PALL Life Sciences, Ann Arbor, MI). CP 94253 was dissolved in
0.9% bacteriostatic saline. The dose of CP 94253 (5.6 mg/kg, s.c.) utilized in the present
experiments was based on previous research indicating that this is the minimally effective dose
capable of altering cocaine self-administration and cocaine-seeking behavior during
reinstatement testing[12], [18], [19]
. SB 224289, a selective 5-HT1Bantagonist [20],[21]
, was dissolved in
.9% bacteriostatic saline and was gently heated to achieve solubility using 5 mg/kg as this dosage
of drug has been shown to be effective as a pharmacological agent in previous studies[20]
.

6. 6
Self-Administration Training:
General Training:
Rats were trained to perform an operant response, lever press, which resulted in the
delivery of cocaine. During this phase of the experiment, rats gain extensive experience with
having daily access to cocaine. Self-administration training occurred in operant conditioning
chambers (30 × 25 × 25 cm; Med Associates, St. Albans, VT). Lines were attached to the
cannula portion of individual rat’s catheters. Two levers were presented to each rat, one referred
to as inactive and the other referred to as active. The active lever, when pressed a preset number
of times, would produce a 5 second infusion of the reinforcer. During and one second prior to
this infusion, a light directly above the active lever and a sound cue would be triggered ending
when the infusion did. After this period, a house light on the opposite wall of the chamber lit up
for 20 seconds during which time animals could not both receive a reinforcer and register active
lever presses to count toward the next infusion. This was implemented to prevent animals from
overdosing. A progression in necessary lever presses per infusion was used because it was
advantageous for early adoption in that the FR1 (an infusion per lever press barring the 20
second period of lever inactivity after an infusion) would increase the likelihood for a positive
association between lever pressing and reward as rats first begin self-administration. If an
animal, over a span of three days, pressed for a similar number of infusions with a 15% margin
for intake within this time period, their schedule would get more difficult (increased number of
active-lever presses for infusion) to a ceiling of FR5. The number of infusions varied among
individual subjects. Rats were progressed FR1-FR5 schedule in which they were placed in the
operant chambers for a two-hour period 6 days a week (on Sunday, rats were flushed with
Timentin®
and handled for two minutes each). This was done in order to make direct
comparisons with previous reports examining the effects of CP 94253on cocaine self-
administration and drug-seeking behavior.
To facilitate acquisition of cocaine self-administration, rats were initially restricted to 16
g of food/day beginning 2 days prior to training [22]
. Rats were maintained on food restriction
(16–22 g) until the final FR5 schedule was achieved for 2 consecutive days of adequate level
pressing, after which they were given ad libitum access to food throughout the rest of self-
administration training and testing.
Extinction Training:
Rats that were previously trained to perform an operant response, lever press, which
resulted in the delivery of cocaine, are trained here in their self-administration environment to
recognize that their drug environment is no longer providing them with any stimulus whether it
is cues or the reinforcer. After establishing a habit of cocaine self-administration, the rats then
underwent extinction training during which cocaine and the stimulus complex delivered with
cocaine were no longer available. Lever presses in the absence of cocaine reinforcement is
referred to as cocaine-seeking behavior and it is thought to be an index of how motivated the rat
is for cocaine (i.e., the more cocaine-seeking behavior, the more motivated). The reason rats are
given extinction training is that eventually their response rates drop during training but can be
reignited by exposing the rats to stimuli that increase their motivation for drug, such as a sample

7. 7
of the drug or seeing the light/tone cues that had been paired with the drug during self-
administration. Rats commenced extinction training 24 hr after their last operant
session. Extinction training consisted of daily 1 hour exposures to the unaltered self-
administration environment with the exception of the reward consequences from active lever
pressing: no cocaine infusion or light and tone cues. Extinction training for all animals was run
under the same conditions. In all rats, extinction training was successful with all subjects
showing minimal response rats by the end of training.
Rats tested for the effects of CP 94253 on cue- and cocaine-primed reinstatement of
extinguished drug-seeking behavior following acute (24 hr) or protracted (120 hr) abstinence
received a single 2-hr extinction session immediately prior to each of their 2 drug seeking test
sessions.
Testing:
After rats stabilized on the FR5 schedule with less than 15% variability per session across
3 consecutive days, rats tested for the effects of CP 94253 on progressive ratio (PR) responding
following 21 days of forced abstinence were given access to cocaine (0.375 mg/kg/0.1 ml,
intravenous [i.v.]; 3-9 PR sessions, 15-20 total sessions) on a schedule that progressed
exponentially from an FR1 according to the formula 5*e^(0.2n)-5 [20]
. In this model, n
represented the number of reinforcers the rat received. Following their last PR self-
administration training session rats began extinction training (17 sessions across 21 days of
abstinence) or were placed into forced abstinence (21 days of abstinence).
The training dose of cocaine (0.75 mg/kg/0.1 ml, i.v.; 15 sessions) used was intended to
capture the descending limb of the dose effect function. Prior to being placed into 21 days of
forced abstinence, rats were given a test dose of cocaine (0.075 mg/kg/0.1 ml, i.v.; 6 sessions).
Both concentrations were intended to measure the effects of CP-94,253 of the ascending and
descending limb of the dose response curve. The first trial of testing was done in a manner such
that all animals began abstinence on the same date. However, the second group tested was
allowed individual variability as different groups of rats began abstinence on different dates
based on when they were able to stabilize on the progressive ratio schedule.
Experiment 1:Effects of CP 94253 on PR and FR responding following forced abstinence or
extinction:
Following 21 days of extinction training (n = 22) or forced abstinence (n = 22), rats were
assigned to drug groups (either CP 94253 or saline) counterbalanced for previous total cocaine
intake. Parameters for PR testing were identical to those described above. Prior to self-
administration testing, rats were pre-treated with saline or CP 94253 15 min prior to being placed
in the operant chambers. To acclimate the rats to injections prior to PR testing, all rats received a
saline injection (1 ml/kg, s.c.) 15 min before their last two extinction sessions or in the home
cages on days 20 and 21 of forced abstinence. Test sessions were capped at 3 hr in order to
prevent CP-94,253 washout during PR-test sessions.
Experiment 2: Effects of CP 94253 on FR5 responding on the ascending and descending limbs of
the dose response curve:

8. 8
Following 21 days of forced abstinence, rats (n = 40) were assigned to drug groups (i.e.
CP 94253 or saline) counterbalanced for previous total cocaine intake. Parameters for FR5
testing were identical to those described above except that half of the rats (n = 20) were tested for
cocaine intake on the descending limb of the self-administration dose-effect function using the
training dose (0.75 mg/kg/ 0.1 ml infusion, i.v.), while the other half (n = 20) were tested for
cocaine intake using a dose on the ascending limb of the dose-effect function (0.075 mg/kg/ 0.1
ml infusion, i.v.). Rats were pre-treated with saline or CP 94253 15 min prior to the 1-hr self-
administration test session. Injection stress was controlled for by injecting all subjects with
saline (1 ml/kg, s.c.) during regular handling on the last two days of forced abstinence.
Experiment 3: Effects of SB 224289 and SB 224289 plus CP 94253 cocktail on FR5 responding:
This experiment was conducted to see if agonist effects could be reversed by the addition
of an antagonist to confirm that CP 94253 effects were mediated solely by the 5-HT1B receptor.
Following 21 days of forced abstinence, rats (n = 30) were assigned to drug groups (i.e. CP
94253, SB 224289, CP+SB, or saline) counterbalanced for previous total cocaine intake.
Parameters for FR5 testing were identical to those described above except that all rats were
testing using the training (0.75 mg/kg/ 0.1 ml infusion, i.v.) dose. Rats were pre-treated with
saline, CP 94253, SB 224289, or a cocktail of SB 224289 and CP 94253 prior to the 2-hr self-
administration test session. SB was given 1 hour before testing for those rats in the SB only and
cocktail groups. To maintain consistency, all rats were given two injections, one 1 hour before
testing and another 15 minutes before. Thus, CP experimental rats were injected with saline 1
hour before and CP 15 minutes before and saline rats were injected with saline at both time
periods. Injection stress was controlled for by injecting all subjects with saline (1 ml/kg, s.c.)
during regular handling on the last two days of forced abstinence.
Experiment 4: Drug-seeking behavior for cue-elicited and cocaine-primed drug seeking after 24
hours and 120 hours of abstinence:
General Protocol:
Following cocaine self-administration training, rats (n = 40) were assigned to drug groups (i.e.
CP 94253 or saline) counterbalanced for previous total cocaine intake. Tests for cue- (n = 20)
and cocaine-primed (n = 20) drug-seeking behavior were each examined twice using a within-
subjects design in which rats were pre-treated with saline or CP 94253 15 min prior to testing,
with order of pre-treatment counterbalanced. Within-subject testing occurred 24 hours and 120
hours following the last cocaine self-administration training session for both groups to quantify
changes in agonist effects at 5-HT1BRs during the course of withdrawal. Testing on both of these
periods of time began with a 2-hr extinction session. In order to control for possible injection
stress on responding during tests for cue- and cocaine-primed drug seeking, rats received a saline
injection (1 ml/kg, s.c.) 15 min before their last two self-administration training sessions and
before both extinction sessions.
Cue Elicited:

9. 9
On the days of cue-elicited test sessions, rats were connected to tethers in operant boxes and
were presented with the same stimulus complex that had been previously paired with cocaine
infusions on an FR1 reinforcement schedule. A non-contingent cue was presented if the rat did
not respond within the first 5 minutes in order to engage the rats’ interest.
Cocaine-Primed:
For the two cocaine-primed test sessions rats received a cocaine-priming injection (10 mg/kg,
i.p.) immediately before they were placed into the operant conditioning chambers for a 1-hr test
session, during which responses produced no consequences.
Statistical Analyses:
Infusion and response rates during self-administration and extinction training were analyzed
using independent-sample t tests. For PR testing following 21 days of forced abstinence, the
total number of infusions, active lever responses, and the highest ratios achieved were analyzed
using separate one-way ANOVAs with abstinence history (abstinence or extinction) and CP
94253 dose (saline or 5.6 mg/kg) as between subjects factors; significant effects were further
analyzed using independent sample t tests. For FR5 testing following 21 days of forced
abstinence, the total number of infusions were analyzed using a one-way ANOVA with cocaine
(0.075 mg/kg or 0.75 mg/kg) and CP 94253 (saline or 5.6 mg/kg) doses as between subjects
factors; significant effects were further analyzed using independent sample t tests. In order to
analyze the time-dependent effects of CP 94253 on drug-seeking behavior, response rates during
cue- and cocaine-primed drug seeking tests were conducted using independent-sample t tests.
Effects of SB 224289 and SB 224289 plus CP 94253 cocktail on FR5 responding following a 21
day period of forced abstinence was analyzed using an one way ANOVA followed by post hoc
Newman-Keuls tests. All statistics were conducted using SPSS, version 20 (IBM Corp.,
Armonk, NY); α was set at 0.05 for all comparisons.
RESULTS and DISCUSSION:
The results support the hypothesis that stimulation of 5-HT1BRs attenuates cocaine abuse
behaviors (seeking behavior and intake quantity) following a period of protracted abstinence.
Each cohort of animals was tested using a 95% confidence interval. Graphics A, B, and C of
Figure 1 represent rats with a history of extinction training while D, E, and F are representative
of rats that did not go through extinction training prior to PR testing. CP-94,253 was effective in
drastically reducing the number of cocaine reinforcers, the number of active lever presses, and
the highest break points in a 3 hour test session. There was no difference between animals with
or without a history of extinction (Figure 1A and 1D respectively). This suggests that on a PR
schedule regardless of whether subjects were abstinent or went through extinction, cocaine had a
diminished reinforcing and/or motivating effect. More importantly, rather than this result being a
product of the agonist’s effect on extinction learning, this appears to be derivative of being in
abstinence.
The results presented in Figure 2 were all measurements on a FR5 schedule of
reinforcement in rats with a history of forced abstinence (21 days). Following the period of

10. 10
forced abstinence, it was discovered that the agonist CP 94253 reduced cocaine intake on the
descending limb of the cocaine dose-response curve as was seen in our previous study[16]
. More
interesting is the effect after a period of protracted abstinence (21 days or longer) in which the
effect of CP 94253 on the descending limb of the dose response curve remains the same as when
measured under maintenance (i.e., decrease in cocaine intake) while the ascending limb seems to
switch CP 94253 effects with decreased responding during abstinence vs. increased responding
during maintenance [9][11]
. This finding suggests that during protracted abstinence CP 94253
administration most likely flattens the dose-response curve rather than creating a left-shift. This
similarity between PR and FR testing is significant because it indicates that reinforcement and
motivation were both attenuated by stimulating 5-HT1BRs following protracted abstinence rather
than producing differential effects on motivation versus reinforcement.
In experiment 3 as shown in Figure 3, SB 224289 group was added to measure the effect
of the antagonist alone, as well as a CP 94253 and SB cocktail to demonstrate that CP effects are
due to stimulation of 5-HT1B receptors. These four groups were designed so that there is one
control (saline) and three experimental drug groups. The saline control provided the same
function as in the first cohort. The antagonist was added in the second test group because it was
previously identified as a serotonin-1B antagonist. Our goal was to determine if CP 94253 was
having nonspecific effects at the doses we were using. When SB 224289 was administered,
blocking 5-HT1B receptors in fact did prevent the effects of CP 94253 (see figure 3), consistent
with both drugs acting at the 5-HT1B receptors. Furthermore, these results provided internal
replication for the effects of CP on the descending limb of the cocaine dose-response curve (i.e.,
two groups or rats run at different times in the chronological timescale provided similar results).
The fourth experiment is represented by figure 4; in this experiment, rats with the same
history of abstinence were tested with 24 hours and 120 hours of abstinence using a within
subjects design. This was intended to represent acute and prolonged abstinence. In both the cue
elicited reinstatement group and the cocaine primed groups, there was no measurable difference
in drug seeking between the CP 94253 and saline treated animals after 24 hours of acute
withdrawal. However, after 120 hours, both test modalities showed a marked decrease in drug
seeking in those animals given CP 94253 relative to the saline control. Again, the duel effect of
CP 94253 relative to time without the reward present is being demonstrated. This indicates that
such a change is initiated by changes in 5-HT1B alone (whether by increasing the functionality of
existing receptors via an agonist or by increasing the actual quantity of receptors via viral means
[16]
) and not the drug as saline had a negligible lever pressing response.
The Parsons group found that 5-HT1B(R) stimulation enhances cocaine reinforcement.
However, their only subjects of examination were rats in maintenance [11]
. A leftward shift of the
dose-response curve occurs when these rats are given CP 94253[11]
. This indicates that the peak
of infusions relative to concentration of cocaine (mg/kg) shifted to the left. There was an increase
on the left side (ascending limb) of the curve following this shift. This indicated that rats
responded as though they had received a higher dose of cocaine suggesting the reinforcer was
more attractive after CP 94253 injections. There was a difference in intake of cocaine between
control and CP 94253 rats on the right side (descending limb) of the curve; those rats given CP
were more satisfied early and thus need less cocaine. This is represented by the leftward shift in
which rats given higher doses of cocaine reduced their intake (they were satiated earlier). Other

11. 11
studies have shown similar results during stimulation of 5-HT1B receptors on enhancing the
discriminative stimulus effects of cocaine producing a leftward shift in the dose response curve
in rats given CP 94253 (2.5 and 5 mg/kg, s.c.) [14]
. Similar to the withdrawal study this paper
intended to pharmacologically clarify, the Neumaier group’s findings using intracranial virally
mediated gene transfer. Results after increasing 5-HT1B receptors were congruous with our
maintenance findings in which overexpression of 5-HT1B receptors also shifted the dose–
response curve for cocaine-conditioned place preference to the left, indicating alterations in the
rewarding effects of cocaine [23]
. Though in this case there is no agonist, the effect of an agonist
is congruent with increasing the quantity of 5-HT1B receptors.
At the Polish Academy of Sciences, research into the effect of CP 94,253 on
reinstatement of extinguished drug seeking behavior (after a minimum of 10 extinction days with
the responding on the active lever below 10% of the level observed during maintenance during at
least 3 consecutive days) found that the agonist given with a sub threshold dose of cocaine (2.5
mg/kg, IP) potentiated cocaine-seeking behavior while the opposite effect was observed when
injecting a higher (10mg/kg, IP) dose [24]
. Our previous data for cocaine-primed reinstatement
contradicts this finding as CP 94,253 attenuated reinstatement of extinguished cocaine-seeking
behavior using priming doses of 2.5 and 10 mg/kg, IP [13]
, effects that occur following 120 but
not 24 hrs of abstinence (see figure 4). In addition, the present results demonstrate that on a PR
schedule, rates of self-administration for a high dose of cocaine are attenuated in rats given CP
94253, and CP 94,253 reduced FR5 self-administration rates for both a high (0.75 mg/kg, IV)
and a low (0.075 mg/kg, IV) cocaine dose (i.e., reduced cocaine intake). Collectively, these data
strongly suggest that stimulation of 5-HT1B receptors following periods of protracted abstinence
attenuate both the reinforcing effects of cocaine and the incentive motivation to seek cocaine, an
effect that likely occurs on or around day five of withdrawal.
This fact that mesolimbic 5-HT1B(R)s differentially modulate cocaine abuse-related
behaviors was first observed in our lab as was discussed earlier (supported via VMGT);
facilitating it during periods of active drug use whilst inhibiting it during protracted withdrawal.
Those results have also been confirmed using this pharmacological approach. Importantly,
ratifying the protracted abstinence data also shows that there is no real disconnect between the
effects of 5-HT1BRs on reinforcement and motivation as rats showed no significant difference in
results between PR and FR testing following protracted. This suggests that both motivation and
reinforcement are attenuated by 5-HT1Bagonists following protracted abstinence.
The attenuation of cocaine abuse-related behavior following a period of forced abstinence
when given CP 94253 is mediated by the 5-HT1B(R). With Ki (nM) values of 2 at 5-HT1B(R)
compared to values of 89, 860, and 49 at 5-HT1A, 5-HT1C and 5-HT1D receptors, respectively,
and little to no affinity for 5-HT2, 5-HT3, D1, D2, 1, 2, -adrenergic, muscarinic,
benzodiazepine, and histamine or opiate receptors, it is clear that CP 94253 is a potent, selective
5-HT1B-receptor agonist [6]
. Increasing the expression of 5-HT1B receptors in terminals of nucleus
accumbens projection neurons produces effect similar to the effects of CP 94253 on cocaine
intake and cocaine-seeking behaviors following protracted abstinence as discussed earlier [16]
.
Furthermore, the present results support the premise that when the effects of CP 94253 are
blocked with SB 224289, no variation induced by 5-HT1B(R)s is recognized leading us to
conclude that CP 94253 is attenuating cocaine intake via the stimulation of this receptor type.

12. 12
These findings suggest that targeting 5-HT1B receptors may lead to a novel treatment for
cocaine dependence and that the therapeutic efficacy of these treatments may vary depending on
the stage of the addiction cycle (short term maintenance or protracted abstinence). What may be
important for future treatment is the plethora of 5-HT1B agonists (though not as selective for 5-
HT1B) that already exist on the market (specifically triptans). As they will work similarly to CP
94253, giving one of these triptan drugs to patients post detoxification may help in the recovery
process. Such a venture may be a place for future investigation as we seek to eliminate the drug
abuse ailment. As with many drugs however, triptans have some warnings in need of inspection;
especially FDA warnings that claim that its use has been associated with serotonin syndrome and
possibly cardiovascular (CV) problems. These claims however may be unfounded in that any CV
warning posted was done only because 5-HT1B/1D(R)s are located in blood vessels. Additionally,
serotonin syndrome instances have only occurred when this drug was paired with selective
serotonin reuptake inhibitors or selective noradrenergic reuptake inhibitors, or monoamine
oxidase inhibitors (as stated in the official FDA warning).
CONCLUSION:
The present findings, together with previous research[9],[12],[3],
, show that 5-HT1B agonist
effects are very much time dependent and have a suppressive effect after a period of
abstinence[12][16]
. Using the selective 5-HT1B antagonist SB 224289, it was concluded that CP-
94,253’s effects on behavior measured in this study are in fact mediated by the 5-HT1B(R) alone.
With the measurable aid that the receptor agonist provided during time of protracted abstinence
in the way of decreased cocaine-seeking behavior and intake during administration periods, it is
clear that targeting 5-HT1B(R) may be an effective method for treating cocaine dependence in the
future.

13. 13
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[10] Cao J; LaRocque E; Li D, Associations of the 5-Hydroxytryptamine (Serotonin) Receptor 1B
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[12]Acosta, J.I.; Boynton, F.A.; Kirschner, K.F.; Neisewander, J.L., Stimulation of 5-HT1B receptors
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behavior. Addict Biol 2009, 14 (4), 419-30.

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[14]Filip, M; Nowak, E; Papla, I; Przegalinski, E; Role of 5-Hydroxytryptamine1B Receptors and 5-
Hydroxytryptamine uptake inhibition in the cocaine-evoked discrimination stimulus effects in rats.
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Modulation of Cocaine Abuse-Related Behaviors. Biol Psychiatry 2012
[17] Pockros, L. A.; Pentkowski, N. S.; Swinford, S. E.; Neisewander, J. L., Blockade of 5-HT2A
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[18] Morikawa, H.; Manzoni, O. J.; Crabbe, J. C.; Williams, J. T., Regulation of central synaptic
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[19] Sari, Y., Serotonin1B receptors: from protein to physiological function and behavior.
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[21] J V Selkirk; C Scott; M Ho; M J Burton; J Watson; L M Gaster; L Collin; B J Jones; D N
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Expression of 5-HT1B Receptors in Nucleus AccumbensEfferents Sensitizes Animals to Cocaine.
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396, 2002.

15. 15
Figure 1. Cohort 1.Effects of the 5-HT1B receptor agonist CP 94253 on cocaine self-
administration (0.375 mg/kg/0.1 ml, i.v.) on an exponential progressive ratio (PR) schedule of
reinforcement in rats with (A, B and C) or without (D, E, and F) a history of extinction training
(17 sessions across 21 days). Prior to testing, there were no differences between saline and CP
94253 groups in the number of cocaine reinforcers obtained during self-administration training
(data not shown) or response rates during extinction training (data not shown). Rats (n = 9-
10/group) were pretreated with saline (1 ml/kg, s.c.; white bars) or CP 94253 (5.6 mg/kg, s.c.;
black bars) 15 min before PR testing. There were main effects of CP 94253 dose on cocaine
intake [F (1, 34) = 20.98, p < 0.001], active lever pressing [F (1, 34) = 10.75, p < 0.005] and the
highest ratio achieved [F (1, 34) = 10.31, p < 0.005]. Regardless of extinction history, CP 94253
attenuated cocaine intake [Extinction: t (18) = 2.74, p < 0.05; Abstinence: t (16) = 4.45, p <
0.001], active lever pressing [Extinction: t (18) = 2.49, p < 0.05; Abstinence: t (16) = 3.91, p <
0.005] and the highest ratio achieved [Extinction: t (18) = 2.40, p < 0.05; Abstinence: t (16) =
4.28, p < 0.005]. Asterisk (*) represents a difference from saline, independent sample t-tests.

16. 16
Figure 2. Effects of the 5-HT1B receptor agonist CP 94253 on cocaine self-administration
under a fixed ratio (FR) 5 schedule of reinforcement in rats with a history of forced abstinence
(21 days). Data are expressed as the number of cocaine reinforcers obtained at doses previously
found to be on the ascending (A; 0.075 mg/kg/0.1 ml, i.v.) and descending (B; 0.75 mg/kg/0.1
ml, i.v.) limbs of the cocaine dose-effect function. Prior to testing, there were no differences
between saline and CP 94253 groups in the number of cocaine reinforcers obtained during
cocaine self-administration training (data not shown). Rats (n = 7-9/group) were pretreated with
saline (1 ml/kg, s.c.; white bars) or CP 94253 (5.6 mg/kg, s.c.; black bars) 15 min before FR5
testing. There was a cocaine by CP 94253 dose interaction [F (1, 29) = 4.56, p < 0.05], with
subsequent analysis indicating that CP 94253 attenuated cocaine intake on both the ascending [t
(13) = 2.77, p < 0.05] and descending [t (16) = 2.62, p < 0.05] limbs of the cocaine dose-effect
function compared to saline treated rats. Asterisk (*) represents a difference from saline,
independent sample t-tests.

17. 17
Figure 3. Effects of the 5-HT1B receptor agonist CP-94,253 (5.6 mg/kg) on cocaine self-
administration following protracted abstinence. The ANOVA indicated a significant drug
interaction [F(3,38)=4.29, p< .05]. Post hoc analysis showed that CP 94253 attenuated the
number of cocaine infusions compared to the vehicle, antagonist (SB 224289), and cocktail
groups. SB was administered in combination with CP to demonstrate 5HT1B receptor
specificity. Asterisk (*) represents a difference from all groups, Newman-Keuls (p<.05).

18. 18
Figure 4. Effects of the 5-HT1B receptor agonist CP 94253 (5.6 mg/kg, s.c.) on cocaine-seeking
behavior following 1 or 5 days of abstinence, expressed as the number of active lever responses
emitted during tests for cue-elicited (A) and cocaine-primed (B) drug seeking. Rats (n =
10/group) were pretreated with saline (1 ml/kg, s.c.; white bars) or CP 94253 (5.6 mg/kg, s.c.;
black bars) 15 min before testing, with order of pretreatment (i.e. CP 94253 or saline)
counterbalanced for the number of cocaine reinforcers obtained during cocaine self-
administration training (data not shown). For cue-elicited reinstatement, cues were available
response-contingently during the 1-hr test session on a fixed ratio (FR) 1 schedule of
reinforcement. For cocaine-primed reinstatement, the cocaine prime (10 mg/kg, i.p.) was
administered immediately before testing and no cues were presented during the 1-hr test
sessions. Compared to saline treated rats, CP 94253 attenuated cue- [t (18) = 2.45, p < 0.05] and
cocaine-primed [t (15) = 2.49, p < 0.05] cocaine-seeking behavior following 5, but not 1, day of
abstinence. Asterisk (*) represents a difference from saline, paired sample t-tests.