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.

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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

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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

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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

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].

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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

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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:

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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:

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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

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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

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

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

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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

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

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

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.