5-ht2 receptor antagonists given in the acute withdrawal from daily cocaine injections can reverse...

5-HT2 receptor antagonists given in the acute withdrawal from daily

cocaine injections can reverse established sensitization

Colin Davidsona, Cindy Lazarusa, Xueying Xionga, Tong H. Leea, Everett H. Ellinwooda,b,*

aDepartment of Psychiatry, Box 3870, Duke University Medical Center, Durham, NC 27710, USAbDepartment of Pharmacology, Box 3870, Duke University Medical Center, Durham, NC 27710, USA

Received 23 May 2002; received in revised form 2 September 2002; accepted 5 September 2002

Abstract

Male Sprague–Dawley rats were given two separate sensitizing regimens of cocaine (7 days on, 7 days off, 7 days on at 40 mg/kg/day,

s.c.) or saline injections. Half of the animals also received a drug with 5-hydroxytryptamine-2 (5-HT2) receptor antagonist properties

(clozapine, 3 mg/kg; mianserin 6 mg/kg; ketanserin 1 mg/kg, all s.c.) or saline during the second cocaine dosing regimen in the acute

withdrawal period, 3.5 h after each cocaine injection. On day 10 of withdrawal animals were challenged with cocaine (7.5 mg/kg, i.p.) and

assessed by a behavioral rating scale and locomotor activity monitoring. The 5-HT2 receptor antagonists, but not saline, reversed behavioral

sensitization and had little effect on behavior in the control animals. 5-HT2 receptor antagonists, therefore, may be a useful treatment for

cocaine addicts that have undergone previous sensitization periods. The pharmacological profile of these antagonists suggests that the 5-

HT2A receptor subtype may mediate this effect.

D 2002 Published by Elsevier Science B.V.

Keywords: Cocaine; Sensitization; 5-HT2; Withdrawal; Locomotion; Behavioral rating

1. Introduction

In the search for medications to treat cocaine and/or

amphetamine abuse, there have been many studies examin-

ing drugs that would either inhibit the induction or expres-

sion of stimulant sensitization (e.g., Pierce et al., 1998, Li et

al., 2000; King et al., 2000). The prevailing hypothesis is that

by blocking sensitization development and expression that

one can inhibit the augmented stimulant acquisition behav-

iors (Robinson and Berridge, 1993; Schenk and Partridge,

1997) noted in self-administration of stimulants. Sensitiza-

tion is a long-lasting (months), persistent phenomena. In

human high-dose cocaine abusers, many episodes of

repeated daily use or binges over a period of the days

followed by withdrawal is typical (Gawin and Ellinwood,

1988). Thus, when presenting for treatment, they would have

established a sensitized response weeks or months previ-

ously. There have only been a few studies demonstrating that

certain drugs when given to animals for several days in the

withdrawal period following a sensitization regimen can

reverse sensitization in contrast to blocking the induction

or expression of sensitization. The drugs that were found to

be effective by these investigators were a 5-HT3 receptor

antagonist (King et al., 1998, 2000), an N-methyl-D-aspartate

(NMDA) receptor antagonist + dopamine D2 receptor ago-

nist, or a dopamine D1 receptor agonist (Li et al., 2000).

More recently, Davidson et al. (2002b) reported that the 5-

HT3 receptor antagonist ondansetron reversed the estab-

lished sensitization from a previous sensitization regimen

when a second sensitization regimen was administered with

cocaine followed each day 3.5 h afterwards with an ondan-

setron dose. Similarly, when given 3.5 h after daily cocaine

self-administration, ondansetron inhibited the progressive

ratio self-administration the next day and for 3 days after

ondansetron was stopped (Davidson et al., 2002b). This type

of dosing regimen may prove useful to abusers who are

testing the effects of cocaine while in treatment. Previously

successful sensitization reversal antagonists of 5-HT3 and

NMDA receptors facilitate Ca2 +-protein kinase C (PKC)

pathways in extracellular signal-regulated kinase (ERK) map

kinase gene up-regulation; we therefore hypothesized that 5-

HT2A receptor antagonists which act through a different

phospholipase PKC, ERK map kinase activation would also

0014-2999/02/$ - see front matter D 2002 Published by Elsevier Science B.V.

PII: S0014 -2999 (02 )02390 -7

* Corresponding author. Tel.: +1-919-684-5225; fax: +1-919-681-

8369.

E-mail address: [email protected] (E.H. Ellinwood).

www.elsevier.com/locate/ejphar

European Journal of Pharmacology 453 (2002) 255–263

reverse sensitization (Greene et al., 2000). In addition,

several studies have indicated that 5-HT2A receptors take

part in: (1) the hyperlocomotion induced by cocaine (O’Neill

et al., 1999); (2) induce an augmented accumbens dopamine

release in cocaine sensitized rats (Yan et al., 2000); (3)

sensitization in that the 5-HT2A receptor antagonist ketan-

serin blocked the induction and/or expression of sensitization

in rats repeatedly dosed with cocaine (Filip et al. (2001).

Thus, drugs with 5-HT2A receptor antagonist properties

appeared to be excellent candidates for reversing previously

established sensitization when given in the acute withdrawal

phase of daily cocaine injections in a subsequent sensitiza-

tion regimen.

2. Methods and materials

2.1. Subjects

Male Sprague–Dawley rats (Charles River Laboratories)

weighing f 200 g at the start of cocaine treatment were

used throughout the study. Animals had unlimited access to

food (standard lab chow) and water and were on a 12/12 h

light/dark cycle with lights on at 7 a.m. Animals were

treated in accordance with the Guide for Care and Use of

Laboratory Animals (NIH). Animals were first acclimatized

to the vivarium for 1 week before dosing and kept 2 per

cage throughout the experiment with animals from the same

experimental group sharing a cage.

2.2. Drugs

Cocaine HCl (NIDA, Washington, DC) was dissolved

(20 mg/ml) in 0.9% sterile saline. Clozapine (Sigma/RBI,

Missouri) was initially dissolved in a few drops of 1 N HCl

then made up in 0.9% saline to 6 mg/ml prior to injections.

Mianserin (Sigma/RBI) was dissolved in saline to 3 mg/ml

and used at 6 mg/kg. Ketanserin (Sigma/RBI) was dissolved

in 0.9% saline to 1 mg/ml and used at 1 mg/kg. All doses

were calculated as the salt, and injection volume was based

on body weight. Clozapine was used at 3 mg/kg as it has an

ID50 of 2.89 mg/kg in inhibiting i.v. cocaine self-adminis-

tration in mice (Rasmussen et al., 2000) and at 3 mg/kg

inhibited cocaine self-administration in monkeys (Vanover

et al., 1993). Mianserin was used at 6 mg/kg as this dose

falls within the dose range used by Rocha et al. (1994) and

Lal et al. (1993) to show anxiolytic effects in rats. Ketan-

serin was used at 1 mg/kg, a dose which was found to block

the lysergic acid diethylamide (LSD; Cunningham and

Appel, 1987) and cocaine (McMahon and Cunningham,

2001) cue in a discriminative stimulus task and cocaine

induced hyperlocomotion (Herges and Taylor, 1998). We

have used these 5-HT2 receptor antagonists versus other

more selective antagonists as they are all clinically avail-

able, thus their potential use in human cocaine dependence

is expedited.

2.3. Pretreatment dosing regimen

There were four groups of animals—(1) saline controls

(saline/saline in the second regimen); (2) drug controls

(saline/DRUG); (3) cocaine-sensitized (cocaine/saline) and

(4) cocaine + drug (cocaine/DRUG)—in each of the three

experiments: (1) clozapine, (2) mianserin, and (3) ketan-

serin. Two groups received cocaine for 7 days were with-

drawn for 7 days, and were again given 7 days of cocaine

injections (first day: 7.5 mg/kg; other 6 days: 40 mg/kg/day,

s.c.); two groups received an identical regimen with saline

injections. These injections were given at 9 a.m. Further,

one cocaine group received a 5-HT2 receptor antagonist

during the second cocaine week 3.5 h after each cocaine

injection. The other cocaine group received parallel saline

injections. Similarly, the saline groups received 5-HT2

receptor antagonist or saline during the second dosing

regimen. None of the saline control animals received

cocaine until the test day when it received the 7.5 mg/kg

cocaine challenge. The dosing regimens are summarized in

Table 1. During the second week of cocaine injections, the

first injection was at 7.5 mg/kg; 60-min post-cocaine

challenge ambulations and behavior were monitored in

these cocaine-treated rats and they were ranked on total

ambulations in 60 min. The rats were then split into a

cocaine/saline or cocaine/DRUG group on the basis of these

ranks such that the two groups had an overlapping response

to the cocaine challenge. Any subsequent difference

between the two groups could therefore be attributed with

confidence to the test drug.

2.4. Behavioral testing

On day 10 of the second cocaine withdrawal, rats were

acclimated to the test room in their home cages (42�23� 22 cm) for 30 min under normal light conditions then

placed in their home cage within activity monitoring boxes

(Opto-Varimex ‘minor’ activity monitors; 15 photo beams,

2.5 cm apart, on each side, Columbus Instruments) for 30

Table 1

Pretreatment dosing regimen

Group Week 1 Week 2 Week 3 Week 4 Graph key

1 cocaine withdraw cocaine +

DRUG

withdraw

10 days

coc/DRUG

2 cocaine withdraw cocaine +

saline

withdraw

10 days

coc/sal

3 saline withdraw saline +

DRUG

withdraw

10 days

sal/DRUG

4 saline withdraw saline +

saline

withdraw

10 days

sal/sal

Summary of the different dosing regimens for the four experimental groups

in each experiment. Coc—cocaine injection (first day = 7.5, next 6

days = 40 mg/kg/day, s.c.). sal—daily saline injection. DRUG—clozapine,

mianserin or ketanserin injection (3, 6 or 1 mg/kg/day, s.c., respectively). In

week 3, the saline or DRUG injection was given 3.5 h after the cocaine or

saline injection. For more details, see Methods and materials.

C. Davidson et al. / European Journal of Pharmacology 453 (2002) 255–263256

min to acclimatize them to the test apparatus. For each

experiment behavioral rating and locomotion was moni-

tored for each animal for 15 min prior to cocaine challenge

to determine the baseline activity levels in the absence of

any drug. Animals were then given an acute cocaine

challenge (7.5 mg/kg, i.p.) and their behavior was moni-

tored over the next 60 min. Two indices of behavior were

taken: (1) a behavioral rating (see Table 2) based on the

Ellinwood and Balster (1974) rating scale and (2) ambula-

tory activity as assessed by two simultaneous beam breaks.

Ambulations were recorded in 5-min bins. A behavioral

rating was given to each of the animals at 5-min intervals,

the observation period was for 20 s for each rat. We note

that ambulatory activity in the home cage, versus larger

plexiglass boxes in previous experiments (e.g., King et al.,

1998), is less—probably due to the smaller area for

exploration and the reported effects of novelty on the

expression of cocaine sensitization (Badiani et al., 1995;

Browman et al., 1998).

2.5. Data analysis for cocaine challenge

The total 60 min ambulation data were first examined

for each group and any rat whose total ambulations fell out

with the 99% confidence intervals for the group were

dropped from further analysis. In most cases only 1 or 2

rats out of each group of 10 were dropped. The 60 min

ambulatory activity data were then analyzed using one-way

analysis of variance (ANOVA; for pretreatment groups).

The behavioral rating data were converted to incidence of

behavior (panel (B) in figures) by grouping the raw data

into three basic types of behavior: (1) inactive (asleep,

almost asleep, dystonic, inactive and inactive with oral

movements), (2) active (grooming and normal active

movement) and (3) hyperactive (slow and fast patterned

movements, hyperactivity and stereotypies). There were no

incidences of hyper-reactive behavior (see Table 2). These

data were analyzed by repeated measure two-way ANOVA

(pretreatment� (repeated measure) behavior). The bottom

panel (C) of each figure represents a plot of ambulations

per 5 min versus behavioral rating. This facilitates the

spread of the data, gives time-course information and we

feel this novel presentation shows that the cocaine sensi-

tized rats are clearly different from all other groups. The

significance level is set at P < 0.05 for all comparisons and

all post hoc P-values < 0.1 are reported. The data in the

figures are expressed as means and the standard error of

the mean (S.E.M.). Post hoc analysis was achieved using

Tukey’s test.

3. Results

Baseline activity levels prior to cocaine challenge were

not found to be different between any of the pretreatment

groups in either ambulatory activity or behavioral rating.

Throughout ‘cocaine + saline’ or ‘saline + ketanserin,’ etc.,

refers to the second dosing regimen when a 9 a.m. injection

of cocaine or saline is followed by an injection of saline or

treatment drug 3.5 h later. There are no statistics associated

with panel (C); however, we believe this novel presentation

(1) facilitates the visual representation of the data; (2)

provides time-course data lacking in the other panels and

(3) shows how locomotion and behavior are related, for

example, at certain times locomotor activity can be decreas-

ing while behavioral ratings increase (Fig. 1C), probably

due to in-place stereotypical activities.

3.1. Clozapine treatment

There was a significant difference between groups in

total ambulations in 60 min (F(3,26) = 8.578, P < 0.001).

Post hoc Tukey’s found the cocaine + saline-treated group to

exhibit a greater psychomotor effect versus all other groups

(Fig. 1A). There were no differences between the other three

treatment groups. Thus, the cocaine + saline group exhibited

locomotor sensitization while clozapine was able to reverse

sensitization, while having no effect on the saline control

group.

The behavioral rating data were analyzed by repeated

measure two-way ANOVA (pretreatment� behavior). There

was a main effect of ‘behavior’ (F(2,80) = 45.77, P < 0.001),

no effect of ‘pretreatment’ (because the sum of all behaviors

add up to 100% incidence) but there was a ‘pretreat-

ment’� ‘behavior’ interaction (F(6,80) = 4.79, P < 0.001).

Overall, there was a greater incidence of inactive versus

active (P < 0.001), inactive versus hyperactive (P < 0.001)

Table 2

Modified Ellinwood and Balster (1974) behavioral rating scale

Score Classification Definition

1 asleepa lying down, eyes closed

2 almost asleepa lying down, eyes partially shut

3 dystoniaa lying down, abnormal posture, tense muscles

4 inactivea lying down, eyes open, infrequent sniffing

5 inplace oral

behavioralying down, oral movements, e.g. yawning

6 groomingb grooming of face, body or groin

7 normal active

movementbinvestigation or sniffing of cage, rearing

8 hyperactivec running characterized by rapid

jerky positional changes

9 slow patterned

movementcrepetitive exploration of the cage at

normal activity level

10 fast patterned

movementcintense, rapid repetitive exploration of cage

11 stereotypyc the types of stereotypy are noted

12 hyper-reactivec jerky hyperactive, jumping, seizures,

obstinate regression

Each rat is examined for 20 s at 5-min intervals and its behavior noted. See

panel (B) in each figure.a These behaviors were grouped to give ‘inactive’ behaviors.b These behaviors were grouped to give ‘active’ behaviors.c These behaviors were grouped to give ‘hyperactive’ behaviors.

C. Davidson et al. / European Journal of Pharmacology 453 (2002) 255–263 257

and active versus hyperactive (P < 0.001) behaviors. Within

the inactive behaviors the cocaine + saline group showed

less of these versus all other groups (cocaine + saline versus

cocaine + clozapine, P < 0.001; cocaine + saline versus sa-

line + saline, P < 0.05; cocaine + saline versus saline + cloza-

pine, P= 0.002). There were no differences between groups

in ‘active’ behaviors. Within the ‘hyperactive’ behaviors,

the cocaine + saline group was again different from all other

groups displaying more hyperactive behaviors (cocaine + sa-

line versus cocaine + clozapine, P= 0.005; cocaine + saline

versus saline + saline, P= 0.004; cocaine + saline versus sal-

ine + clozapine, P= 0.004), there were no other group differ-

ences.

Within the saline + saline group, there was a greater

incidence of inactive versus hyperactive (P < 0.001) and

active versus hyperactive (P= 0.002) behaviors. Within the

saline + clozapine group there was a greater incidence of

inactive versus active (P= 0.001), inactive versus hyper-

active (P < 0.001) and active versus hyperactive (P= 0.023)

behaviors. Within the cocaine + saline group there were no

differences in the incidence of inactive, active or hyper-

active behaviors (P>0.85 for all comparisons). Within the

cocaine + clozapine group, there was a greater incidence of

inactive versus active (P < 0.001), inactive versus hy-

peractive (P < 0.001) but not active versus hyperactive

(P= 0.073) behaviors.

3.2. Mianserin treatment


total ambulations in 60 min (F(3,27) = 5.672, P= 0.004).

Post hoc Tukey’s found the cocaine + saline-treated group to

exhibit a greater psychomotor effect versus the saline + sa-

line (P < 0.05) and saline/mianserin groups (P < 0.005)

while the difference between the cocaine + saline and

cocaine + mianserin group was very nearly significant

(P= 0.056; Fig. 2A). There were no differences among

the other three treatment groups. Thus, the cocaine/saline

group exhibited locomotor sensitization while mianserin

tended to reverse sensitization, while having no effect on

the saline control group.

The behavioral rating data was analyzed by repeated



no effect of ‘pretreatment’ (because all incidences of behavior

add up to 100%) but there was a ‘pretreatment’� ‘behavior’

interaction (F(6,83) = 8.61, P < 0.001).


active (P= 0.009), inactive versus hyperactive (P < 0.001)

but not active versus hyperactive (P= 0.067) behaviors.

Within the inactive behaviors the cocaine + saline group

showed less of these versus all other groups (cocaine + saline

versus cocaine +mianserin, P= 0.003; cocaine + saline ver-

sus saline + saline, P < 0.001; cocaine + saline versus sali-

ne +mianserin, P < 0.001). The saline +mianserin group also

showed a greater incidence of inactive behaviors versus the

Fig. 1. Effect of clozapine pretreatment on cocaine challenge on day 10 of

withdrawal. Panel (A) shows the total 60-min ambulations after acute

cocaine challenge on day 10 of withdrawal. The P-values correspond to

differences versus the cocaine-sensitized rats (cocaine/saline). Panel (B)

describes the behavioral rating data. For each pretreatment group (saline/

saline; saline/clozapine; cocaine/saline; cocaine/clozapine), the incidence of

inactive, active and hyperactive behaviors is given. Within each pretreat-

ment group the single asterisk denotes statistical differences between the

incidence of inactive versus active or active versus hyperactive behaviors

while the double asterisk denotes statistical differences between the

incidence of inactive behaviors and both active and hyperactive behaviors.

The small ‘a’ denotes a decreased incidence in inactive behaviors in the

cocaine/saline group versus all other groups. The small ‘b’ denotes an

increased incidence in hyperactive behaviors in the cocaine/saline group

versus all other groups. The small ‘c’ denotes the near significant difference

( P= 0.073) between active versus hyperactive behaviors in the cocaine/

clozapine group. Panel (C) is a plot of ambulatory activity versus behavioral

rating over time. The hysteresis plot allows visualization of the variations in

the relationship of locomotion to behavior rating, e.g. locomotion may

decrease with stereotypy. The cocaine/saline group is clearly different from

all other groups. The numbers within the graph correspond to the time (min)

after acute cocaine challenge and the arrows show the direction of time.

coc: cocaine; sal: saline; CLOZ: clozapine.


cocaine +mianserin (P < 0.05) group. There were no differ-

ences between groups in ‘active’ behaviors. Within the

‘hyperactive’ behaviors the cocaine + saline group was again

different from all other groups displaying more hyperactive

behaviors (cocaine + saline versus cocaine + mianserin,

P= 0.002; cocaine + saline versus saline + saline, P < 0.001;

cocaine + saline versus saline +mianserin, P < 0.001), there

were no other group differences.

Within the sal/sal group, there was a greater incidence of

inactive versus hyperactive (P < 0.001) but not active versus

hyperactive (P= 0.067) behaviors. Within the saline +mian-

serin group there was a greater incidence of inactive versus

active (P < 0.001), inactive versus hyperactive (P < 0.001)

but no difference in active versus hyperactive (P= 0.371)

behaviors. Within the cocaine + saline group there was a

greater incidence of hyperactive versus inactive (P= 0.014)

but no other differences. Within the cocaine +mianserin

group there was a greater incidence of, inactive versus

hyperactive (P= 0.007) behaviors, and no other difference.

3.3. Ketanserin treatment


total ambulations in 60 min (F(3,28) = 4.374, P < 0.05). Post

hoc Tukey’s found the cocaine + saline-treated group to

exhibit a greater psychomotor effect versus the saline + sa-

line (P < 0.05) and saline + ketanserin (P < 0.05) but not the

cocaine + ketanserin (P= 0.373, see Fig. 3A) groups. There

were no differences among the other three treatment groups.

Thus, the cocaine + saline group exhibited locomotor sensi-

tization while ketanserin, although decreasing ambulations

substantially was unable to reverse sensitization in a statisti-

cally significant manner. Importantly ketanserin had no

effect on the saline control group.

The behavioral rating data was analyzed by repeated



no effect of ‘pretreatment’ (because all incidences of be-

havior add up to 100%) but there was a ‘pretreatment’�‘behavior’ interaction (F(6, 86) = 5.51, P < 0.001).


active (P < 0.001) and inactive versus hyperactive behaviors

(P < 0.001), while the incidence of active versus hyperactive

behaviors did not reach significance (P= 0.257). Within the

inactive behaviors the cocaine + saline group showed less of

these versus all other groups (cocaine + saline versus cocaine

+ ketanserin, P < 0.001; cocaine + saline versus saline + sa-

line, P= 0.037; cocaine + saline versus saline + ketanserin,

P < 0.001). There were no differences between groups in

‘active’ behaviors. Within the ‘hyperactive’ behaviors the

cocaine + saline group was again different from all other

groups, this time showing greater incidence of this behavior

(cocaine + saline versus cocaine + ketanserin, P= 0.005;

cocaine + saline versus saline + saline, P= 0.011; cocaine +

saline versus saline + ketanserin, P < 0.001), there were no

other group differences.

Within the saline + saline group there was a greater inci-

dence of inactive versus hyperactive behaviors (P= 0.005).

Within the saline + ketanserin group there was a greater

incidence of inactive versus active (P= 0.009), inactive

versus hyperactive (P < 0.001) but not active versus hyper-

active (P= 0.099) behaviors. Within the cocaine + saline

Fig. 2. Effect of mianserin pretreatment on cocaine challenge on day 10 of

withdrawal. For a description of panels (A), (B) and (C), see Fig. 1. Within

the saline/saline and cocaine/mianserin pretreatment groups, the single

asterisk above the ‘inactive’ bar denotes statistical differences between the

incidence of inactive versus active behaviors while the double asterisk

above the ‘inactive’ bar (saline/mianserin group) denotes statistical

differences between the incidence of inactive behaviors and both active

and hyperactive behaviors. The single asterisk above the ‘inactive’ bar

(cocaine/mianserin group) denotes statistical differences between the

incidence of inactive behaviors and hyperactive behaviors. The small ‘a’

above the saline/mianserin group denotes an increased incidence in inactive

behaviors in the saline/mianserin group versus all other groups. The small

‘b’ above the cocaine/saline group denotes a decreased incidence in

inactive behaviors versus all other groups. The small ‘c’ above the cocaine/

saline group denotes an increased incidence in hyperactive behaviors

versus all other groups. The small ‘d’ denotes near significant difference

(P= 0.055) between the incidence of active versus hyperactive behaviors in

the saline/saline group. coc: cocaine; sal: saline; MIAN: mianserin.


group there were no differences in the incidence of inactive,

active or hyperactive behaviors (P>0.28 for all compari-

sons). Within the cocaine + ketanserin group there was a

greater incidence of inactive versus active (P < 0.001), inac-

tive versus hyperactive (P < 0.001) but no difference in active

versus hyperactive (P= 0.725) behaviors.

4. Discussion

This repeated cocaine dosing regimen would have estab-

lished behavioral sensitization after the first dosing-with-

drawal regimen (Davidson et al., 2002a). Sensitization was

also evident when measured 10 days after the second with-

drawal period, as previously shown (Davidson et al.,

2002a). Importantly, each test drug, when given with the

second cocaine dosing regimen (3.5 h after cocaine),

reversed the expression of the previously established sensi-

tization, at least with respect to behavior. Ketanserin had

less effect than clozapine and mianserin in reducing loco-

motor sensitization, although it was used at the lowest dose.

It is important to consider that testing for sensitization took

place 10 days after the last treatment, when all drugs would

have been eliminated. Further, these antagonists, when

given to the control groups, did not reduce cocaine-induced

locomotor behavior. Their effect on behavioral rating in the

control animals was less clear cut in that mianserin treat-

ment caused an increase in inactive behavior versus the

saline control group. However, clozapine and ketanserin had

little effect on saline-treated animals in behavioral ratings.

Taken together then 5-HT2 receptor antagonists, when given

during acute cocaine withdrawal, act specifically to inhibit

the sensitized response and not locomotion or behavior per

se.

Others have given drugs with 5-HT2 receptor antagonis-

tic properties prior to or during cocaine in various drug

abuse models (see Table 3) primarily to reduce acute

cocaine effects. This is the first report of a 5-HT2 receptor

antagonist treatment given during repeated acute with-

drawals that can reverse the expression of cocaine sensiti-

zation previously established by a repeated dosing regimen

when tested 10 days later with cocaine alone (i.e., no

treatment drug on board). Given that cocaine abusers

usually have many sensitization and withdrawal episodes

in their progression from use to abuse, a multi-regimen

paradigm may provide the most appropriate animal model.

This would be especially true in testing treatment models in

which consolidation of sensitization has already taken place.

Thus, based on our data, we can state that drugs with 5-HT2

receptor antagonist properties reverse sensitization and not

just inhibit the development or expression of sensitization as

found by Filip et al., 2001. These clinically available drugs

include clozapine and mianserin, which are non-selective 5-

HT2 receptor antagonists, but ketanserin is selective for the

5-HT2A site, especially at the low dose used here.

The mechanism behind these findings may be related to

cocaine-induced sensitization of 5-HT2 receptors. 5-HT2

receptor–effector coupling was increased in the monkey

hippocampus after repeated cocaine (Farfel et al., 1991). An

increased head twitch response to 5-HT2A receptor agonists

was reported after a similar chronic cocaine regimen (Bau-

mann and Rothman, 1996). Binding studies also suggest an

increase in 5-HT2 sensitivity after chronic cocaine (Neise-

wander et al., 1994) although others disagree (Javaid et al.,

1993; Johnson et al., 1993) and differences may be due to

different treatment regimens and withdrawal times. In

microdialysis studies, Yan et al. (2000) found that the 5-

HT2A receptor agonist 2,5-dimethoxy-4-iodoamphetamine

Fig. 3. Effect of ketanserin pretreatment on cocaine challenge on day 10 of

withdrawal. For a description of panels (A), (B) and (C), see Fig. 1. Within

the saline/ketanserin and cocaine/ketanserin pretreatment groups, the

double asterisk above the ‘inactive’ bar denotes statistical differences

between the incidence of inactive behaviors and both active and hyperactive

behaviors. The small ‘a’ above the cocaine/saline group denotes a decreased

incidence in inactive behaviors versus all other groups. The small ‘b’ above

the cocaine/saline group denotes an increased incidence in hyperactive

behaviors versus all other groups. The small ‘c’ denotes the near significant

difference (P= 0.099) between the incidence of active versus hyperactive

behaviors in the saline/ketanserin group. coc: cocaine; sal: saline; KET:

ketanserin.


(DOI) augmented dopamine release in cocaine sensitized

rats, an effect blocked by ketanserin. Neuroendocrine stud-

ies (Levy et al., 1992; Baumann and Rothman, 1996) also

report increased 5-HT2A receptor sensitivity after intermit-

tent high dose (30 mg/kg) cocaine injections.

The data presented here are consistent with the hypoth-

esis that there are potent mechanisms which inhibit cocaine

sensitization in rats when 5-HT2 receptor antagonists are

given at some time point after (e.g., 3.5 h) cocaine admin-

istration. One possibility is that this antagonism dissociates

the previously established sensitization and hypothetical

opponent processes. Withdrawal opponent processes have

previously been postulated to be involved in psychostimu-

lant addiction (Koob and Bloom, 1988; Koob et al., 1997).

Acute adversive withdrawal effects have been shown for

cocaine with increased ultrasonic stress vocalizations

(Mutschler and Miczek, 1998) and decreased activity and

cortical h-wave function (Gauvin et al., 1997) while cocaine

‘crash’ is found in human abusers (Gawin and Ellinwood,

1988).

Both 5-HT2 and 5-HT3 receptor mechanisms may well be

involved in the sensitization process by: (1) mobilization of

Ca2 + (Zhang et al., 1995; Florian and Watts, 1998; Hagberg

et al., 1998; Coultrap et al., 1999) and (2) activation of PKC

(Greene et al., 2000; Ronde and Nichols, 1998) which, in

turn, leads to activation of mitogen activated protein extra-

cellular signal-regulated kinase (MAP-ERK) and effects on

gene expression and protein synthesis (Sweatt, 2001;

Adams and Sweatt, 2002). For reviews of the ERK/MAPK

calmodulin kinase activation of cyclic AMP response ele-

ment binding protein (CREB) and long-term sensitization in

addiction, see Hyman and Malenka (2001), Impey et al.

(1999) and Pittenger et al. (2002).

Genova and Hyman (1998) reported that the 5-HT3

receptor antagonist MDL-72222 blocked c-Fos and that

phosphorylation of cAMP response element-binding protein

(CREB) and activation of transcription factor-1 (ATF-1) by

serotonin requires 5-HT3 receptor activation and Ca2 +.

Another Ca2 + activating receptor implicated in sensitization

and learning, NMDA (Sweatt, 2001), can be manipulated in

the chronic withdrawal process to reverse sensitization (Li et

al., 2000). The NMDA receptor antagonists memantine or

MK-801 given along with a dopamine D2 receptor agonist

are also capable of reversing a previously established

sensitization, see Li et al. (2000).

Regardless of the mechanism, we suggest that the acute

withdrawal period represents an important window for the

administration of putative therapeutic agents as it allows for

the interaction of the agent with hypothesized ongoing

opponent processes caused by cocaine. This is consistent

with the potential for a means of treating cocaine abusers

who have already established a sensitized profile of self-

administration. It is well known that abusers in treatment

programs will frequently test for cocaine effects. Daily

Table 3

Spectrum of outcomes with drugs having 5-HT2 receptor antagonist properties when given prior to or with cocaine

Study Model Drug Species Dose (mg/kg) Route Time Result

Roberts and Vickers (1984) SA clozapine Wistar rat (M) 12.5 i.p. 1 h prior # FR SA

Loh et al. (1992) clozapine Wistar rat (M) 5–20 i.p. 1 h prior 10 mg/kg z coc PR SA

Lacosta and Roberts (1993) ketanserin Wistar rat (M) 0.4–6.4 i.p. 1 h prior no effect on PR SA

methysergide 2.5–20 i.p. 1 h prior no effect on PR SA

McMillen et al. (1993) amperozide S–D rat (M) 0.5–2.5 s.c. bid � 4 days #cocaine drinking

Vanover et al. (1993) clozapine monkey 0.3–1 i.m. 30 min prior z FR self-admin

Ufer et al. (1999) clozapine S–D rat (M) 2.6 p.o. drinking water #cocaine drinking in HCa

Rasmussen et al. (2000) clozapine mouse ED50 2.89 s.c. 30 min prior # FR SA

olanzapine ED50 0.15 s.c. 30 min prior # FR SA

risperidone ED50 0.08 s.c. 30 min prior # FR SA

Kosten and Nestler (1994) CPP clozapine S–D rat (M) 10 s.c. 1 h prior # cocaine CPP

Meil and Schechter (1997) olanzapine S–D rat (M) 3–4.5 i.p. 30 min prior # FR SAb # CPP

Meert et al. (1990) DD ritanserin Wistar rat (M) 0.63–40 s.c. 1 h prior no effect on cocaine DD

risperidone 2.5 s.c. 2 h prior # cocaine DD

Van Campenhout et al. (1999) risperidone Honover rat (M) 2.5 s.c. 2 but not 1 h prior #cocaine DDc

olanzapine 2.5 s.c. 1 but not 2 h prior #cocaine DDc

clozapine 0.16–20 s.c. 1 and 2 h prior no effect on cocaine DDc

Filip and Cunningham (2002) RS 102221 S–D rat (M) 1.5 Ag intra-NAc with cocaine injection #cocaine DD

Ellinwood and Kilbey (1979) SENS clozapine S–D rat (M) 20 i.p. 1 h prior #cocaine stereotypy

Filip et al. (2001) ketanserin Wistar rat (M) 3 i.p. 30 min prior #cocaine stereotypy

Summary of previous work testing drugs with 5-HT2 receptor antagonistic properties in various drug abuse models.

M: male; PR: progressive ratio; FR: fixed ratio; SA: self-administration; CPP: conditioned place-preference; DD: drug discrimination; SENS: sensitization;

S–D: Sprague–Dawley; HC: high consuming.a This study found an increased cocaine consumption in low consuming rats.b Olanzapine also produced a dose-dependent reduction in FR responding for food.c All drugs decrease response rate.


post-cocaine 5-HT2 or 5-HT3 receptor antagonist dosing

may represent a novel way of thinking about disassociation

of post-cocaine (opponent process) effects from cocaine’s

initial effects and its relationship in reversing sensitization

mechanisms.

In conclusion, our primary novel findings are that rats

behaviorally sensitized to a daily high-dose cocaine regimen

and withdrawn for 7 days to fully establish sensitization, then

given a second series of 7 days of high-dose cocaine, could

have sensitization reversed if they were given clozapine,

mianserin or ketanserin 3.5 h after the second cocaine

sensitization regimen. These drugs have all been used in

humans. These findings (1) provide evidence for a role for 5-

HT2A receptors in cocaine sensitization and (2) represent a

potential novel means of treating cocaine abusers. Further the

potency of clozapine in reversing sensitization may be

especially useful in schizophrenic substance abusers (Alba-

nese et al., 1994; Yovell and Opler, 1994; Farren et al., 2000).

Acknowledgements

This work was supported by NIDA grants to E.H.E.

(DA-12768, DA-10327) Reese Jones (EHE Co-PI, DA-

109396) and T.H.L. (DA-06519).

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