EFFECTS OF THE SELECTNE DOPAMINE D3 RECEPTOR ANTAGONIST, NAFADOTRIDE, ON THE DEVELOPMENT OF BEHAVIORAL SENSITIZATION TO COCAINE
A Thesis Presented to
the Faculty of the College of Science and Technology
Morehead State University
In Partial Fulfillment
of the Requirement for the Degree
Master of Arts
by
Yu Liu
March, 2003 rnsu ,HfS&S lo\'.). 20LJ.Q L.783e Accepted by the faculty of the College of Science and Technology, Morehead State University, in partial fulfillment of the requirements for the Master of Arts Degree.
Director of Thesis
Master's Committee
Date EFFECTS OF THE SELECTIVE DOPAMINE D3 RECEPTOR ANTAGONIST, NAFADOTRIDE, ON THE DEVELOPMENT OF BEHAVIORAL SENSITIZATION TO COCAINE
YuLiu,M.A. Morehead State University, 2003
ABSTRACT
It has recently been proposed that the development of behavioral sensitization to psychostimulant drugs is mediated by a repeated drug-induced alteration in dopamine D3 receptors. Consistent with this hypothesis, concurrent treatments with the preferential dopamine D3 receptors antagonist, nafadotride, has been reported to prevent the development of behavioral sensitization to amphetamine. This dopamine D3 hypothesis was further tested in the present study by determining whether nafadotride would also prevent the development of behavioral sensitization to cocaine.
At the beginning of this experiment, male Wistar rats (250-300g) were tested for activity after a saline injection on two days. Based on the distance traveled on the second day, the subjects were divided into four drug treatment groups: vehicle-vehicle, vehicle-cocaine, nafadotride-vehicle, and nafadotride-cocaine. From Day 3 to Day 6 (drug treatment phase), the subjects received daily I. P. injection with vehicle, cocaine, nafadotride, or cocaine+ nafadotride, respectively. Five minutes after each daily injection, the rats were placed into the activity boxes (Med-Associates model OFA-163) for 40 minutes. On the 7th and 8th days
(conditioning test phase), all rats received saline ittjections prior to activity testing. On the last day (Day 9), each subject was given a challenge injection of cocaine followed by activity testing.
The results indicated that repeated cocaine treatments produced a progressively greater increase in activity (i.e. behavioral sensitization). In contrast, repeated nafadotride treatments did not significantly affect locomotor activity. More important, concurrent treatment ofnafadotride with cocaine did not affect either the cocaine-induced acute or chronic locomotor effect. On the drug-withdrawal days, rats previously treated with cocaine displayed a conditioned hyperactivity response, which was not prevented by concurrent nafadotride treatment. Further, the cocaine challenge injection produced a significantly greater increase in activity in rats previously treated cocaine, compared to rats injected with cocaine for the first time. Finally, pretreatment with nafadotride and cocaine did not block the development of sensitization to cocaine. In fact, concurrent nafadotride-cocaine pretreatments tended to produce an even greater increase in cocaine-induced activity than the cocaine pretreatments alone. Thus, if anything, nafadotride treatments tended to enhance the development of the behavioral sensitization to cocaine.
These findings clearly do not support the hypothesis that the development of behavioral sensitization to cocaine is mediated through changes in dopamine D3 receptors.
Furthermore, these findings suggest that the development of behavioral sensitization to different psychostimulant"drugs may be mediated by different neurochemical mechanisms. - Accepted by: --Ld.~~f=.-,~~~~, Chair ttfio✓ ACKNOWLEDGMENTS
The first person I would like to thank is Dr. Bruce Mattingly, who patiently taught me throughout this whole project. Secondly, I also appreciate the help of my fellow graduate students (John Dunkrnan and Angela Duke) who assisted with the behavioral testing. Without their support, this project could not have been successfully completed. Finally, but most of all, I want to thank to my family, who support me with unconditioned and continuous love.
Morehead State University Y.L. TABLE OF CONTENTS
Page
Chapter I Introduction ...... I
Chapter 2 Method ...... 9
Chapter 3 Results ...... 13
Chapter 4 Discussion ...... 34
Chapter 5 Reference ...... 42
Appendix A: ANOVA Summary Tables ...... 48
Appendix B: Counterbalancing ...... 61
I LIST OF FIGURES
Figure Page
1. Med-Associates Activity Chamber ...... 10
2. Mean distance traveled (± SEM) across blocks of 10 min
on Day 1 and 2 for rats treated 5 min before each session
with saline (YEH) injection ...... 14
3. Mean number ofrears (± SEM) across blocks of 10 min
on Day 1 and 2 for rats treated 5 min before each session
with saline (YEH) injection ...... 15
4. Mean number of stereotypic counts (± SEM) across blocks
of 10 min on Day 1 and 2 for rats treated 5 min before each session
with saline (YEH) injection .. ·: ...... 17
5. Mean distance traveled(± SEM) across blocks of 10 min
on Day 3, 4, 5 and 6 for rats treated 5 min before each session
with saline (YEH), 15mgik:g Cocaine (COC),
0. lmgik:g Nafadotride (NAF) or 15mgik:g COC + 0. lmgik:g NAF ...... 18
6. Mean number ofrears.(± SEM) across blocks of 10 min
on Day 3, 4, 5 and 6 for rats treated 5 min before each session
with saline (YEH), 15mgik:g Cocaine (COC), 0.lmgik:gNafadotride
(NAF) or 15mgik:g COC + 0.lmgik:g NAF ...... 20
11 Figure
7. Mean number of stereotypic counts (± SEM) across blocks
of 10 min on Day 3, 4, 5 and 6 for rats treated 5 min
before each session with saline (YEH), 15mg/kg Cocaine (COC),
0.lmg/kgNafadotride (NAF) or 15mg/kg COC + 0.lmg/kgNAF ...... 22
8. Mean distance traveled(± SEM) across blocks of 10 min
on Day 7 and 8 for rats treated 5 min before each session
with saline (YEH) injection ...... 24
9. Mean number of rears(± SEM) across blocks of 10 min
on Day 7 and 8 for rats treated 5 min before each session
with saline (YEH) injection ...... 26
10. Mean stereotypic counts(± SEM) across blocks of 10 min
on Day 7 and 8 for rats treated 5 min before each session
with saline (YEH) injection ...... 28
11. Mean distance traveled(± SEM) after a cocaine challenge injection
(Day 9) for rats previously treated with either saline (YEH) or 15mg/kg
Cocaine (COC) in combination with either saline (YEH) orO.lmg/kg
Nafadotride (NAF). The left panel represents the total day activity and
the right panel presents the same data as a function of four IO min
blocks within the day ...... 29
lll Figure
12. Mean numbers of rears(± SEM) after a cocaine challenge injection
(Day 9) for the animals previously treated with either saline (VEH) or
15mgikg Cocaine (COC) in combination with either saline (YEH)
or 0. lmgikg Nafadotride (NAP). The left panel represents
the total day activity and the right pane) presents the same data as a
function of four 10 min blocks within the day ...... 31
13. Mean stereotypic counts (± SEM) after a cocaine challenge injection (Day 9)
for the animals previously treated either saline (VEH) or 15mgikg Cocaine
(COC) in combination with either saline (YEH) or 0.lmgikg Nafadotride
(NAP). The left panel represents the total day activity and the right panel
presents the same data as a function of four IO min blocks within the day...... 33
IV •
LIST OF TABLES
Table • 1. Experimental Design ...... 10
2. Summary of Analysis of Variance Performed on
Mean Distance Traveled: Habituation Phase, Days 1-2 ...... 49
3. Summary of Analysis of Variance Performed on
Mean Number of Rears: Habituation Phase, Days 1-2 ...... 50
4. Summary of Analysis of Variance Performed on
Mean Stereotypic Counts: Habituation Phase, Days 1-2 ...... 51
5. Summary of Analysis of Variance Performed on
Mean Distance Traveled: Drug Treatment Phase, Days 3-6 ...... 52
6. Summary of Analysis of Variance Perfo1med on
Mean Number of Rears: Drug Treatment Phase, Days, 3-6 ...... 53
7. Summary of Analysis of Variance Perfo1med on
Mean Stereotypic Counts: Drug Treatment Phase, Days, _3-6 ...... 54
8. Summary of Analysis of Variance Performed on
Mean Distance Traveled: Conditioning Test Phase, Days, 7-8 ...... 55
9. Summary of Analysis of Variance Perfo1med on
Mean Number of Rears: Conditioning Test Phase, Days, 7-8 ...... 56
10. Summary of Analysis of Variance Performed on
Mean Stereotypic Counts: Conditioning Test Phase, Days, 7-8 ...... 57
V Table Page
11. Summary of Analysis of Variance Performed on
Mean Distance Traveled: Cocaine Challenge Test, Day 9 ...... ;.... 58
12. Summary of Analysis of Variance Performed on
Mean Number of Rears: Cocaine Challenge Test Day 9 ...... 59
13. Summary of Analysis ofVariance·Perfo1med on
Mean Stereotypic Counts: Cocaine Challenge Test Day 9 ...... 60
VI Chapter 1
Introduction
Drug addiction
Drug abuse is a global phenomenon. It affects almost every country. Drug
abuse trends around the world, especially among youth, have become a very serious
issue in the last a few decades. For example, heroin abuse is reported in three-quarters
of all countries and abuse of cocaine is reported in two-thirds. Drug-related problems
include increased rates of crime and violence, susceptibility to HIV/AIDS and
hepatitis, demand for treatment and emergency-room visits and a breakdown in social
behavior (UNODCCP report, 2001 ).
Curativeness, religion, and recreation have been the main reasons for people
to use psychoactive drugs for many years. However, at the end of the last century, due
to the development of science, especially in the field of chemistry and pharmacology,
stronger, more pure and highly addictive substances such as cocaine and heroin were
synthesized. In addition, the invention of hypodermic syringes enabled people to
inject these drugs, making their effects more powerful and the risk of addiction more
serious (UNOCCP report, 2001).
Currently, the mechanism mediating drug addiction has become a major focus in the area of psychology and neuroscience. "The central feature of drug addiction is compulsive drug use-loss of control over apparently voluntary acts of drug seeking
1 and drug taking" (Goldstein, 1994). Drugs have both acute and chronic effects on
behavior. If they are used repeatedly, some acute drug effects may become less,
which is called tolerance; while others become higher which is considered
sensitization. Generally, drug addiction studies include drug-conditioning effect and
drug-brain adaptation. The former focuses on the role of classical and operant
conditioning mechanism in mediating drug addiction. The latter stresses on the role of
specific brain mechanisms to drug addiction. Two different types of brain responses
to drug addiction have been proposed for: 1) neural adaptations, which includes
homeostatic response to excessive stimulation; and 2) synaptic plasticity, which
associates drug related stimuli with specific learned behavior (Berke' & Hyman,
2000).
Behavioral Sensitization
Initially, repeated drug use produces withdrawal effects, such as craving in humans. In turn, addiction is observed, which is represented by the. compulsive drug seeking behaviors (Robinson & Berridge, 1993; Robinson & Becker, 1986).
However, it is difficult to experimentally study the neural and behavioral mechanism
of drug addiction in humans. Therefore, many researchers have developed animal models to study the different aspects of the drug addiction process. Behavioral sensitization is one of the most frequently observed behavioral effects with the repeated administration of psychostimulant drugs in animals (Kalivas & Stewart,
1991 ). Behavioral sensitization refers to "the finding that animals given several drug
2 injections spaced out at intervals of a day or more tend to show sensitized locomotor
activity and stereotypy, progressively increasing with each injection" (Goldstein,
1994). Due to the similarities between the drug addiction in humans and the
behavioral sensitization in animals, behavioral sensitization has become a widely
used model to study the mechanisms of drug addiction, particularly craving
(Robinson & Berridge, 1993).
Dopamine Receptor and Behavioral Sensitization
Dopamine is crucial to a wide variety of behaviors and functions, which
include the behavioral changes from sensitization to addiction, from movement to
emotion, and development to plasticity (Joyce, 2001). "In part, this diversity reflects
multiplicity of the organization and types of dopamine receptors, the neurons that
express those receptors, and their source of dopamine innervations" (Joyce, 2001).
Thus, dopamine receptors receive a lot of attention in drug abuse research.
There are at least five types of dopamine receptors in the vertebrate CNS, and
these fall into two classes: DI-type (DI and D5) and D2-type (D2, D3 and D4).
Dopamine DI-type receptors are localized on the membrane of postsynaptic neurons, while the D2-type receptors are found on the membrane of both pre and post synaptic neurons. Early research suggested that stimulation of dopamine receptors was crucial to the development of behavioral sensitization, particularly the repeated stimulation of DI-type receptors (Henry & White, 1991). For example, concurrent treatment with selective DI-type (SCH 23390), but not D2-type (Sulpride), dopamine antagonists prevents the development of behavioral sensitization to the direct dopamine agonist,
3 apomorphine (Mattingly, Rowlett, Graff & Hatton, 1991) and the indirect agonist,
amphetamine (Stewart & Vezina, 1989; Drew & Glick, 1990). In.addition, animals
repeatedly treated with the selective DI-type dopamine agonist, SKF 38393,
subsequently show a sensitized locomotor response to apomorphine (Mattingly,
Rowlett & Lovell, 1993). These and other similar findings led to the view that the
development of behavioral sensitization to different drugs was mediated by a
commpn neurochemical mechanism, especially stimulation of dopamine DI receptors
(White, Joshi, Koeltzow & Hu, 1998). However, recent studies with cocaine have
questioned this assumption. For example, concurrent treatment with the DI receptor
antagonist, SCH 23390, has consistently failed to prevent the development of the
cocaine-induced behavioral sensitization (Mattingly, Hart, Lim & Perkins, 1994;
Mattingly et al., 1996; White et al., 1998). Moreover, neither the D2 receptor
antagonist ( eticlopride) nor concurrent treatment with both DI and D2 receptor
antagonists (SCH 23390 + eticlopride) prevent the development of behavioral
sensitization to cocaine (White et al., 1998). Thus, treatments with DI-type, D2-type
or a combination ofDI- and D2-type dopamine receptor antagonists failed to prevent the development of behavioral sensitization to cocaine. These findings suggest that the development of behavioral sensitization to different drugs, such as cocaine, amphetamine, or apomorphine, may be mediated by different neurochemical mechanisms.
4 Dopamine D3 receptors and Behavioral Sensitization
. As mentioned previously, three individual dopamine receptors (D2, D3 and
D4) have been classified as D2-type receptors (Berke & Hyman, 2000). Recent
research suggests that these subtypes are differentially expressed in the regions of
human brain, providing a rationale for how they could mediate different actions of
dopamine (see Berke & Hyman, 2000, for review). For example, D3 receptors, in
contrast to D 1 and D2 dopamine receptors, have a restricted pattern of expression in
the nucleus accumbens, which appears to be a critical structure in the control of
motivation and the effects of drug-conditioned cues (Bouthenet, Souil, Martres,
Sokoloff, Giros & Schwartz, 1991 ). Because of this unique distribution, dopamine D3
receptors have been proposed to play a major role in various neuropsychiatric and
neurological disorders (Joyce, 2001).
Recently, it has been hypothesized that the development of behavioral
sensitization to psychostimulant drugs may be related to the differential down
regulation of dopamine D3 receptors compared to D 1/D2 receptors (Richtand, Logue,
Welge, Perdiue, Tubbs, Spitzer, Sethuraman & Geracioti, 2000). This hypothesis is
based upon several converging lines of evidence. First, considerable evidence exists
which suggests that dopamine D 1/D2 and dopamine D3 receptors play opposing roles
in the regulation oflocomotor behavior. Specifically, the selective stimulation of
dopamine D 1/D2 receptors results in the stimulation oflocomotor activity in rodents, whereas the selective stimulation of dopamine D3 receptors inhibit locomotor activity
(Ekman, Nissbrandt, Heilig, Dijkstra & Erikson, 1998; Sautel, Griffon, Sokoloff,
5 Schwartz, Launay, Simon, Costentin, Schoenfelder, Garrido & Mann, 1995). Thus,
the level of locomotor activity is dependent upon the relative levels of stimulation of
D l/D2 vs. D3 receptors. Second, a great deal of evidence suggests that repeated
stimulation ofreceptors results in a homeos!atic down-regulation in proportion to the
degree of overstimulation (Richtand, Woods, Berger & Strakowski, 200 I). Since
dopamine has been reported to have a 70-fold g_reater affinity for D3 receptors
compared to D 1/D2 receptors (Sololoff, Martres, Giros, Bouthenet & Schwartz,
1992), it has been hypothesized that D3 receptors should undergo greater down
regulation than D 1/D2 receptors with repeated psychostimulant treatments (Richtand
et al., 2000). Thus, the progressive increase in locomotor activity (i.e., sensitization)
observed with repeated psychostimulant administration might be due to a progressive
decrease in DJ-mediated behavioral inhibition (i.e., disinhibition) resulting from D3
receptor tolerance or down-regulation (Richtand et al., 2000). Consistent with this
hypothesis, Richtand et al. (2000) reported that concurrent treatment with the
selective dopamine D3 receptor antagonist, nafadotride, along with amphetamine
blocked the development of behavioral sensitization to amphetamine. Presumably,
nafadotride blocked the development of amphetamine-induced behavioral
sensitization by preventing the down-regulation of D3 receptors. In addition,
Mattingly, Fields, Langfels, Rowlett, Robinet and Bardo (1996) found that co
administration of cocaine and the dopamine D3 receptor agonist, 7-OH-DPAT
acutely attenuated cocaine-induced increases in motor behavior but enhanced the development of behavioral sensitization to cocaine. Based upon the above hypothesis,
6 this could be attributed to a 7-OH-DPAT-induced enhancement ofD3 receptor down
regulation or tolerance.
Although Richtand et al. (2000) present a persuasive argument for a role of
dopamine D3 receptors in the development of behavioral sensitization, a number of
findings.are inconsistent with their hypothesis. For example, the number of binding
sites for D3 receptors has been reported to· be significantly elevated, rather than
diminished, in cocaine-overdose victims (Staley & Mash, 1996). Furthermore, the
locomotor inhibitory effect ofa low dose of the selective D3 agonist PD128, 927 was
unaffected by prior sensitization to cocaine (Prinssen, Koek and Kleven 1998). In
addition, concurrent treatment with the selective dopamine D3 receptor antagonist
U99194A and cocaine does not affect the development of behavioral sensitization to
cocaine (Mattingly, Caudill, Abel, Sutherland, & Dong, 2000). Finally, repeated
treatment with the selective dopamine D3 receptor agonist 7-OH-DPAT does not
produce sensitization or cross-sensitization to cocaine or apmorphine (Mattingly et
al., 1996). Thus, the involvement of dopamine D3 receptors in the development of
behavioral sensitization to psychostimulant drugs remains unclear.
Purpose of Current Experiment
As noted previously, it has been reported that concurrent treatment with the
putatively selective dopamine D3 receptor antagonist nafadotride blocks the develqpment of behavioral sensitization to amphetamine (Richtand et al., 2000). The purpose of the present study, therefore, was to determine whether nafadotride
7 treatment would also block the development of behavioral sensitization to cocaine.
Consequently, groups ofrats were treated daily with cocaine or saline combined with either saline or nafadotride, and tested for activity. Following the chronic treatment and a withdrawal interval, all rats were tested for activity after a challenge injection of cocaine. If the dopamine D3 receptors stimulation is crucial the development of behavioral sensitization.to cocaine, then concurrent nafadotride treatment should block this effect.
8 Chapter 2
Method
Subjects
Thirty-one male Wistar albino rats (Harlan, Spraque Dawley, Indianapolis,
IN) weighing between 250-300g at the beginning ofthe experiment, served as
subjects. The rats were housed individually in standard wire-mesh cages in a colony
room, which was temperature-controlled, and maintained on a 12-hour light dark
cycle. All behavioral tests were conducted during the light cycle. Food and water
were available continuously.
Apparatus
Behavioral activity was measured in four open field chambers (see Figure 1, ' . Med Associates model OFA-163), and each chamber was located in an individual sound attenuated experimental cubicle. Each chamber was equipped with a 16 X 16 array of photocell beams and detectors that were 2.5cm above the floor respectively.
Another set of eight photocell beams and detectors were mounted 16 cm above the floor to measure rearing behavior. Signals from each individual photocell array were delivered to a Gateway-2000 computer with Med-Associate software placed in an adjacent room. Distance traveled (cm), number ofrears, and stereotypic counts
(small movements) were recorded at 10 min intervals.
9 Figure 1: Med-Associates locomotor activity testing chamber
Drug
Nafadotride was generously provided by Dr. Pierre Sokoloff (INSERM, Paris,
France) and was dissolved in 0.9% saline. Nafadotride concentration was described as free base. Cocaine hydrochloride was also dissolved in 0.9% saline. Cocaine concentration was described as chlorinate salt. All injections were I. P. in a final volume of 1 ml/kg. 0.9% saline was used as vehicle injection.
Design and Procedure
This experiment was a 2 (cocaine dose: 0 or 15 mg/kg) X 2 (nafadotride dose:
0 or 0.1 mg/kg) factorial design (see Table 1).
Table 1 Experimental Design Cocaine (I. P.) Vehicle Nafadotride Nafadotride (I.Pl Vehicle N = 7 N=B Cocaine N=B N=B
10 The experiment was conducted in four phases: the habituation phase, the drug
treatment phase, the conditioning test phase, and the cocaine challenge test.
The habituation phase lasted two days. Each animal received a saline
injection and 5 minutes later was placed in the activity chamber for 40 minutes. . . Animals were immediately returned to the home cages after the behavioral test. Based I on the distance traveled on day 2, the animals were assigned into the four drug I
treatment groups: vehicle (saline), cocaine (15mg/kg), nafadotride (0.lmg/kg), and
cocaine (15mg/kg) + nafadotride (0.lmg/kg).
The drug treatment phase was conducted from Day 3 to Day 6. The procedure
for this phase was the same as the habituation phase, except that the animals were
injected with the assigned drugs 5 minutes prior to behavioral testing.
th The conditioning tests were conducted on the ?1h and s day. The procedure
was the same as the habituation phase in that all the animals were injected with saline
before activity testing.
The cocaine challenge test was conducted on Day 9. All subjects were
injected with cocaine (15mg/kg). Five minutes after the injection, all the subjects
were placed into the activity chambers.
Data analysis
Significant differences among the groups in mean distance traveled, the i i number of rears, and stereotypic counts during the habituation, drug treatment, i conditioning phase, and the cocaine challenge day were analyzed using 2 X 2 multi I I factorial analyses of variance (ANOVA). Drug treatment conditions were used as the
11 between factors; days and blocks of 10 min within each day were used as the within factors. Significant interactions were analyzed with additional ANOVAs performed on individual session and/or block data, followed by Newman-Keuls tests.
12 Chapter3
RESULT
Habituation Phase: Day 1 and 2
Mean Distance Traveled
The mean distance traveled across blocks of ten minutes on the two habituation days for the four drug treatment groups is shown in Figure 2. A summary of the ANOVA performed on these data is displayed in Table 2 (Appendix A). As may be seen in Figure 2, the animals assigned to the four drug treatment conditions did not differ in distance traveled following the saline injection on Day 1 and 2. The
ANO VA performed on these data revealed no significant drug effects (nafadotride effect: F < 1.00, and cocaine effect: F < 1.00; Nafadotride X Cocaine interaction effect: F < 1.00). In addition, all the subjects tended to show declines in distance traveled across days (day effect: F (1, 27) = 3.88, p = 0.06). As expected, there was a significant decrease in mean distance traveled across blocks and days on some blocks
((block effect: F (3, 27) = 275.07, p < 0.001; Day X Block interaction effect: F (3, 27)
= 4.06, p < 0.05).
The mean number of rears for each of the four drug treatment groups across the four 10 min blocks of Day 1 and 2 is presented in Figure 3. A summary of the
ANOVA performed on both days is presented in Table 3 (Appendix A). As may be seen in Figure 3, the groups assigned to the four drug treatment conditions did not
13 HABITUATION SESSIONS
-O·VEH-VEH ---VEH-COC C w -O·NAF-VEH ..J w --e-- NAF-COC 1000
~1- w (J ~ I- 500 SQ C
w~ :5
01'------''------'------'--~---' 1 2 BLOCKS OF 10 MIN WITHIN EACH DAY
Figure 2. Mean distance traveled (+ SEM) across blocks of 10 min on Day 1 and 2 for rats treated 5 min before each session with saline (VEH) injection. HABITUATION SESSIONS 100 -O·VEH-VEH -a-VEH-COC -O·NAF-VEH 75 ....,.NAF-COC U) 0:: <( w 0:: 50 _. z CJ1 <( w \ \ \,_ \ ' ~ \ 25
1 2 BLOCKS OF 10 MIN WITHIN EACH DAY
Figure 3. Mean number of rears (+ SEM) across blocks of 1 O min on Day 1 and 2 for rats treated 5 min before each session with saline (VEH) injection. differ during these two sessions (nafadotride effect: F (1, 27) = 2.35, p > 0.05;
cocaine effect: F < 1.00; Nafadotride X Cocaine interaction effect: F < 1.00).
Overall, there was a significant decline in the mean number ofrears across blocks and
days for some blocks (day effect: F (I, 27) = 21.22, p < 0.0001; block effect: F (3, 27)
= 215.03, p < 0.0001; Day X Block interaction effect: F (3, 27) = 6.4, p < 0.01).
Stereotypic Counts
The mean stereotypic counts for each of the four drug treatment groups across
the four 10 min blocks of Day 1 and 2 are presented in Figure 4. A summary of the
ANOV A performed on these two days is presented in Table 4 (Appendix A). As may be seen in Figure 4, the four groups did not show significant difference in stereotypic
counts (nafadotride effect: F < 1.00; cocaine effect: F < 1.00; Nafadotride X Cocaine interaction effect: F < 1.00). In addition, there were declines in stereotypic counts across days and blocks within each day (day effect: F (1, 27) = 9.90, p < 0.05; block effect F (3, 27) = 189.35, p < 0.0001). As expected, there was no significant difference in the stereotypic counts among these four groups before the drug treatment phase.
Drug Treatment Phase: Day 3 to Day 6
Mean Distance Traveled
Figure 5 displays the mean distance traveled (in cm) for the four drug treatment groups on Day 3, 4, 5 and 6. A summary of the ANOVA performed on these four days is presented in Table 5 (Appendix A). As may be seen in Figure 5, cocaine injection (15mg/kg) acutely stimulated locomotor activity as represented by
16 HABITUATION SESSIONS 2500 -D·VEi-1-VEH (I) 1-z ---VEH~COC ::, 2000 -O·NAF-VEH 0 (.) -+-NAF-C0C ~ C.. 1500 1->- ..... 0 ---1 w 0:: 1000 w I- m
500 ::iw ~
1 2 BLOCKS OF'10 MIN WITHIN EACH DAY
Figure 4. Mean number of stereotypic counts (+ SEM) across blocks of 10 min on Day 1 and 2 for rats treated 5 min before each session with saline (VEH) injection. DRUG TREATMENT SESSIONS
-0-· NAF-VEH :E -DVEH-VEH (.) -a-VEH-COC ..,.NAF-COC c 2500 w ...J w ~ 2000 0:: I- ~ 1500
!en 1000 C w~ 500 :E
3 4 5 6
BLOCKS OF 10 MIN WITHIN EACH DAY Figure 5. Mean distance traveled (± SEM) across of 10 min on Day 3, 4, 5 and 6 for rats treated 5 min before test session with saline (VEH), 15 mg/kg cocaine (COC), 0.1 mg/kg nafadotride (NAF) or 15 mg/kg COC + 0.1 mg/kg NAF injections. distance traveled (cocaine effect: F (1, 27) = 126.76, p < 0.0001). More important,
the animals injected with cocaine tended to show increasing activities across days
(Cocaine X Day interaction effect: F (3, 81) = 2.57, p = 0.06). Based on Newman
Keuls"tests (p < 0.05), the subjects treated with cocaine produced a progressive
increase in distance traveled (i.e., sensitization), while the vehicle groups maintained
the same level of distance traveled. Although all drug treatments decreased distance
traveled across blocks within each day, and this decrease was greater for the animals
treated with cocaine (block effect: F (3, 27) = 201.91, p < 0.0001; Cocaine X Block
interaction effect: F (3, 81) = 23.22, p < 0.0001). In contrast, repeated treatment with
nafadotride did not significantly affect activity across either the days or blocks within
each day (nafadotride effect: F < 1.00; Nafadotride X Day interaction effect: F <
1.00; Nafadotride X Block interaction effect: F < 1.00). More important, nafadotride
did not significantly affect cocaine-induced changes in distance traveled (Cocaine X
Nafadotride interaction effect: F < 1.00).
Rears
Figure 6 shows the mean number ofrears for the four drug treatment groups on Day 3, 4, 5, and 6. A summary of the ANOVA performed on the four days is presented in Table 6 (Appendix A). As may be seen in Figure 6, there was an acute stimulation of the number of rears in the cocaine treatment groups, while injection with nafadotride did not significantly affect rearing behavior (cocaine effect: F (!, 27)
= 19.13, p < 0.01; nafadotride effect: F < 1.00). Further, the stimulating effects of cocaine on rearing tended to increase across days (Cocaine X Day interaction effect:
19 DRUG TREATMENT SESSIONS 120 -D-VEH-VEH -0-· NAF-VEH -a-VEH-COC -+-NAF-COC 100
. 0::en 80 <( .w ·. 0:: z 60 . <( w I\) 0 ::!: 40
20
0 3 4 5 6
BLOCKS OF 10 MIN WITHIN EACH DAY
Figure 6. Mean rears (± SEM) across of 10 min on Day 3, 4, 5 and 6 for rats treated 5 min before test session with saline (VEH), 15 mg/kg cocaine (COC), 0.1 mg/kg nafadotride (NAF) or 15 mg/kg COC + 0.1 mg/kg NAF injections. F (3, 81) = 2.60, p < 0.06). Indeed, analysis of this interaction with Newman-Keuls
tests (p<0.05) indicated that the cocaine treatment groups increased rearing from Day
3 to Day 5. Repeated treatment with nafadotride did not significantly affect the
number ofrears across either days or blocks within each day (Nafadotride X Day
interaction effect: F (3, 81) = 1.21, p > 0.05; Nafadotride X Block interaction effect: F
< 1.00). More important, nafadotride did not significantly affect cocaine-induced
changes in the number ofrears (Cocaine X Nafadotride interaction effect: F < 1.00).
Although the mean numbers ofrears overall did not significantly change across days,
there was a decline in the number of rears across blocks within each day ( day effect:
F (3, 81) = 1.83, p > 0.05; block effect: F (3, 81) = 66.69, p < 0.0001).
Stereotypic counts
Figure 7 presents the stereotypic counts for the four drug treatment groups
from Day 3 to 6. A summary of the ANOVA performed on these four days is
presented in Table 7 (Appendix A). As may be seen in Figure 7, the treatment with
cocaine acutely stimulated stereotypic counts, while the injection with nafadotride did not display this effect (cocaine effect: F (1, 28) = 295.10, p < 0.0001; nafadotride effect: F< 1.00). Further, ANOVA analysis revealed a significant interaction effect between the cocaine and day (Cocaine X Day interaction effect: F (3, 81) = 2.88, p <
0.05). This interaction, however, was due to the decline in stereotypes counts for the vehicle rats across days (Newman-Keuls, p < 0.05), rather than to an increase in stereotypic counts for the cocaine rats (Newman-Keuls, p > 0.05). In addition, there was a decline in the stereotypic counts for the subjects across the four blocks within
21 DRUG TREATMENT SESSIONS
·D.YEH-VEH ·O-·NAF-VEH en ---VEH-COC -+-NAF-COC !z 2500 :::, 0 (.J u 2000 a: >- b 1500 w 0:: w ti 1000 z <( w 500 :E
3 4 5 6
BLOCKS OF 10 MIN WITHIN EACH DAY
Figure 7. Mean stereotypic counts (± SEM) across blocks of 10 min on Day 3, 4, 5 and 6 for rats treated 5 min before test session with saline (VEH), 15 mg/kg cocaine (COG), 0.1 mg/kg nafadotride (NAF) or 15 mg/kg COG + 0.1 mg/kg NAF injections. each day, and this decrease was smaller for rats treated with cocaine (block effect: F
(3, 81}= 156.75, p < 0.0001; Cocaine X Block interaction effect: F (3, 81) = 22.93, P
< 0.0001 ). In contrast, repeated treatment with nafadotride did not significantly affect
stereotypic counts across either days or blocks within each day (Nafadotride X Day
interaction effect: F < 1.00, Nafadotride X Block interaction effect: F < 1.00). More
important, concurrent treatment of nafadotride and cocaine did not significantly affect
the cocaine-induced stereotypic counts of the animals (Cocaine X Nafadotride
interaction effect: F (1, 27) = 1.22, p > 0.05).
Conditioning Test Phase: Day 7 and 8
Mean Distance Traveled
The mean distance traveled for each of the four drug treatment groups across
the four 10 min blocks of Day 7 and 8 is presented in Figure 8. A summary of the
ANOVA performed on both two days is presented in Table 8 (Appendix A). As may
be seen in Figure 8, rats previously treated with cocaine were significantly more
active after the saline injection, compared to rats not previously exposed to cocaine
(cocaine effect: F (1, 27) = 33.15, p < 0.0001). As expected, this cocaine pre exposure effect was greatest during the first ten minutes of each session (Cocaine X
Block effect: F (3, 81) = 10.23, p < 0.0001), and greater overall on the first session than on the second conditioning test session (Cocaine X Day interaction effect: F (1,
27) = 36.35, p < 0.0001). Overall, nafadotride pretreatment did not significantly affect activity (nafadotride effect: F (1, 27) = 1.96, p > 0.05), but rats previously injected with nafadotride were more active than control rats on the first day (Nafadotride X
23 CONDITIONING TEST SESSIONS 1500 ~ (.) -D·VEH-VEH -C 1250 ---vEH-COC w ...I -O·NAF-VEH w ! 1000 ...... NAF-COC I- w (.) 750 z <( ~ I- 500 ~ ,, C \ ' ,, l\ ' ~- z \ ' \,\ <( \\~ \ w 250 :i!: \'b ,,,,. ,:a ----- ...... __ ',',, ~- 0 ------7 8
BLOCKS OF 10 MIN WITHIN EACH DAY
Figure 8. Mean distance traveled (± SEM) across blocks of 1O min on Day 7 and 8 for rats treated 5 min before each session with saline (VEH) injection. Day interaction effect: F (1, 27) = 5.45, p < 0.05). More important, concurrent
pretreatment with nafadotride did not block the development of cocaine-induced
conditioned hyperactivity (Cocaine X Nafadotride interaction effect: F (1, 27) = 1.12,
p > 0.05).
The number of rears for each of the four drug treatment groups across the four
10 min blocks on Day 7 and 8 is presented in Figure 9. A summary of the ANOVA
performed on these data is presented in Table 9 (Appendix A). As may be seen in
Figure 9, the groups previously treated with cocaine displayed significantly higher
number ofrears, suggesting a cocaine conditioning effect (cocaine effect: F (I, 27) =
20.02, p < 0.001). In contrast, the groups with previous nafadotride treatment did not
show significant change in the number ofrears (nafadotride effect: F (1, 27) = 1.43, p
> 0.05). The animals tended to show declines in the number of rears across the four
blocks within each day, and the animals previously treated with cocaine displayed a
greater a decrease (block effect: F (3, 27) = 114.61, p < 0.0001; Cocaine X Block
interaction effect: F (3, 81) = 6.39, p < 0.001). However, the subjects did not display
significant changes in the number of rears across days (day effect, F < 1.00).
Furthermore, previous repeated treatment with nafadotride did not significantly affect
the cocaine-induced changes in the number of rears (Cocaine X Nafadotride effect: F
< 1.00).
25 CONDITIONING TEST SESSIONS 100,-----r---,------.----,------, -□ ·VEH-VEH ... VEH-COC 80 -o•NAF-VEH ~ ...... NAF-COC <( w 60. 0:: z N O'l ~ 40 :l:: 20
7 8 BLOCKS OF 10 MIN WITHIN EACH DAY
Figure 9. Mean rears (± SEM) across blocks of 10 min on Day 7 and 8 for rats treated 5 min before each session with saline (VEH) injection. Stereotypic Counts
The mean stereotypic counts for each of the four drug treatment groups across these two days and four 10 min blocks within each day are presented in Figure 10. A summary of the ANOVA performed on both days is presented in Table 10 (Appendix
A). As may be seen in Figure 10, rats previously treated with cocaine displayed significantly greater stereotypic counts across both conditioning test days ( cocaine
effect: F (I, 27) = 55.42, p< 0.0001). As expected, this increased stereotypy decreased
across both days and blocks within sessions (Cocaine X Day X Block interaction effect, F (3, 81) = 3.40, p < 0.05). Interestingly, nafadotride pretreatment also
enhanced stereotypic counts (nafadotride effect: F (1, 27) = 3.98, p > 0.05), but only
on the first test session (Nafadotride X Day interaction effect: F (1, 27) =4.54, p <
0.05). Concurrent pretreatment with nafadotride and cocaine, however, did not affect the enhanced stereotypic response, compared to cocaine pretreatment alone
(Nafadotride X Cocaine interaction effect: F < 1.00).
Cocaine Challenge test: Day 9
Mean Distance Traveled
Figure 11 displays the mean distance traveled for the four pretreatment groups
after the cocaine challenge injection. A summary of the ANOVA performed on this
data is presented in Table 11 (Appendix A). Overall, as may be seen in the left panel, rats previously treated with cocaine were significantly more active following the
cocaine challenge injection than rats receiving cocaine for the first time, clearly
27 CONDITIONING TEST SESSIONS 2500.-----.--.-----.--.-----, u, -O·VEH-VEH l- -a-VEH-COC 2000 g -O·NAF-VEH (.) ~ -+-NAF-COC g:: 1500
!3w ~ I\) CXl w 1000 1- u,
w~ 500 ~
7 8 BLOCKS OF 10 MIN WITHIN EACH DAY
Figure 10. Mean stereotypic counts (± SEM) across blocks of 10 min on Day 7 and 8 for rats treated 5 min before each session with saline (VEH) injection. COCAINE CHALLENGE TEST 10000~------~ -O·VEH-VEH ---VEH-COC C -O·NAF-VEH ~ 7500 3000 w -+-NAF-COC
1-~ w 5000 2000 u ~ l'8 I SQ C 2500 1000
w~ ~
10 20 30 40 CHALLENGE DAY BLOCKS OF 10 MIN
Figure 11. Mean distance traveled (+ SEM) after a cocaine challenge injection (day 9) for the animals previously treated either saline (VEH) or 15mg/kg Cocaine (COG) in combination with either saline (VEH) or 0.1 mg/kg Nafadotride (NAF). The left panel represents the total day activity and the right panel presents the same data as a function of four 1O min blocks within the day. indicating the development of behavioral sensitization to cocaine ( cocaine effect: F
(1, 27) = 15.30, p < 0.00 I). Overall, nafadotride pretreatment did not significantly
affect responsiveness to the cocaine challenge injection (nafadotride effect: F (1, 27)
= 2.94, P > 0.05). However, as may be seen in the right panel of Figure 11,
nafadotride pretreated rats were more active than vehicle pretreated rats on the early
blocks of the test session (Nafadotride X Block interaction effect: F (3, 81) = 2.97, p
< 0.05). Indeed, Newman-Keuls analysis of this interaction indicated that nafadotride
pretreated rats were significantly more active than vehicle rats on the first three
blocks of this session (P < 0.05 in each case). Similarly, the effect of cocaine
pretreatment declined across blocks (Cocaine X Block interaction effect: F (3, 81) =
5.53, p < 0.01). Although rats pretreated with both cocaine and nafadotride displayed
the greatest responsiveness to the cocaine challenge injection, nafadotride did not
significantly enhance cocaine-induced sensitization (Nafadotride X Cocaine
interaction effect: F < 1.00; Nafadotride X Cocaine X Block interaction effect, F (3,
81) = 1.36, p > 0.05). It is clear from these results that concurrent pretreatment with
nafadotride did not block the development of behavioral sensitization to cocaine as
measured by horizontal locomotor activity.
Rears
Figure 12 displays the mean numbers ofrears for the four pretreatment groups
after the cocaine challenge injection. A summary of the ANOVA performed on this
data is presented in Table 12 (Appendix A). Overall, the animals treated previously with cocaine displayed an increase in subsequent behavioral sensitivity to cocaine on
30 COCAINE CHALLENGE TEST 400~------~ -□ ·VEH-VEH -e-VEH-COC 320 100 -O·NAF-VEH -+-NAF-C0C Cl) 0:: <( 240 75 w 0:: z <( t,)__,_ w 160 50 :e:
25
10 20 30 40 CHALLENGE DAY BLOCK OF 10 MIN
Figure 12. Mean rears(+ SEM) after a cocaine challenge injection (day 9) for the animals previously treated either saline (VEH) or 15mg/kg Cocaine (C0C) in combination with either saline (VEH) or 0.1 mg/kg Nafadotride (NAF). The left panel represents the total day activity and the right panel presents the same data as a function of four 1O min blocks within the day. the challenge day, while prior treatment with nafadotride did not significantly affect rearing behavior (cocaine effect: F (1, 27) = 4.16, p < 0.06; nafadotride effect: F <
1.00) .. As shown on the right panel, there tended to be declines in the number of rears among all the subjects across blocks (block effect, F (3, 27) = 27.47, p < 0.0001).
More important, the concurrent treatments of nafadotride and cocaine did not block the development of sensitization to cocaine (Nafadotride X Cocaine interaction effect:
F< 1.00).
Stereotypic Counts
Figure 13 displays the mean stereotypic for the four pretreatment groups after the cocaine challenge injection. A summary of the ANOVA performed on this data is presented in Table 13 (Appendix A). Overall, as may be seen in the left panel, prior treatment with cocaine also produced a significant increase in subsequent stereotypic counts, which was the proof of the behavioral sensitization to cocaine ( cocaine effect:
F (1, 27) = 4.31, p < 0.05). In addition, prior treatment with nafadotride also tended to increase cocaine-induced stereotypy (nafadotride effect: F (1, 27) = 4.13, P <
0.06). As shown on the right panel, each group tended to show a decrease in the stereotypic counts across blocks (block effect, F (3, 27) = 41.65, p < 0.0001 ). More important, concurrent treatments ofnafadotride and cocaine did not significantly block cocaine-induced sensitization of stereotypy (Nafadotride X Cocaine interaction effect: F < 1.00).
32 COCAINE CHALLENGE TEST 12000~------~ 4000,------,---,------,---,-----, en -O·VEH-VEH I- -a-VEH-COC ~ 9600 -O·NAF-VEH 0 3000 (.) -+-NAF-COC ~ a. 7200 1->- 0 2000 w w w 0:: 4800 w I t/) 1000 2400 ~w :!!:
10 20 30 40 CHALLENGE DAY BLOCK OF 10 MIN Figure 13. Mean stereotypic counts ( + SEM) after a cocaine challenge injection (day 9) for the animals previously treated either saline (VEH) or 15mg/kg Cocaine (COC) in combination with either saline (VEH) or 0.1 mg/kg Nafadotride (NAF). The left panel represents the total day activity and the right panel presents the same data as a function of four 1O min blocks within the day. Chapter4
Discussion
The current results clearly indicate that repeated exposure to cocaine results in
the development of behavioral sensitization. During the pretreatment sessions,
cocaine produced a progressive increase in distanced traveled and rearing, but not
stereotypic movements, compared to a steady decline in locomotor behaviors across
days for rats treated with only saline. Similarly, the subsequent challenge injection of
cocaine, after a two-day withdrawal interval, produced a significantly greater increase
in horizontal locomotor activity and rearing in rats previously treated with cocaine
compared to rats receiving cocaine for the first time. These results are consistent with
previous studies conducted in our laboratory (Mattingly et al., 1994; 1996) and others
(e.g., Kalivas, Duffy, DuMars & Skinner, 1988), suggesting that the development of
behavioral sensitization is a reliable and robust phenomenon.
In contrast to cocaine, the acute administration of the putative selective
dopamine D3 receptor antagonist, nafadotride, did not significantly affect any of the three measures of activity during the four pretreatment sessions. As noted previously, a great deal of evidence suggests that dopamine D3 receptors are inhibitory with respect to the generation oflocomotor behavior (Richtand, et al., 200 l ). Consistent with this view, a number of studies have reported that stimulation of dopamine D3 receptors with selective agonists such as 7-OH-DPAT (Mattingly et al., 1996) or PD
128, 907 (Prinssen et al., 1998), produced a significant suppression oflocomotor
34 activity. Based on these data, it would be predicted that selective antagonism of inhibitory dopamine D3 receptors with nafadotride should result in a significant increase in activity. However, this was not the case in the current experiment. In contrast to the lack oflocomotor effects with nafadotride, it has been reported that the putative selective dopamine D3 receptor antagonist U99194A does acutely increase locomotor activity (Kling-Petersen, Ljung & Svensson, 1995; Waters, Svensson,
Haadsma-Svensson, Smith & Carlsson, 1993; Mattingly et al., 2000). At present, the nature of this discrepancy is unclear. Although this conflict could be due to the different selective antagonists (i.e., nafadotride vs U99194A) used, it also could be due to the number of methodological differences among the studies reporting conflicting results. The current study used a dose of nafadotride slightly above that reported to block the development of behavioral sensitization to amphetamine
(Richtand et al., 2000). Unfortunately, in that study the effect of nafadotride alone on locomotor activity was not determined. It should be noted however, that although nafadotride did not enhance activity in the current study, unlike dopamine D2 receptor antagonist such as haloperidol and eticlopride (Mattingly, Rowlett, Ellison &
Rase, 1996; White et al., 1998), nafadotride did not decrease activity, either. The lack of a nafadotride-induced decrease in activity is consistent with the view that the functional properties of dopamine D3 receptors differ significantly from dopamine
D2 receptors (See Ekman et al., 1998; Sautel et al., 1995 for review).
Consistent with the lack of effect of nafadotride alone, the concurrent treatment of nafadotride with cocaine during the pretreatment sessions did not
35 significantly affect the level of cocaine-induced activity either acutely or across sessions. Again, based upon the hypothesis that dopamine D3 receptors are inhibitory with respect to locomotor behavior, it was expected that nafadotride administered with cocaine should have increased the stimulating effects of cocaine. However, this was not the case. Further, this finding is inconsistent with the previous finding in this lab that the selective dopamine D3 agonist, 7-OH-DPAT, attenuates the acute effects of cocaine (Mattingly et al., 1996). Likewise, it has been reported that co administration of the dopamine D3 antagonist U99194A with amphetamine enhances the locomotor-stimulating effects of amphetamine (Kling-Petersen et al. , 1995). It is, of course, possible that the dose of nafadotride used in the current study was too low to significantly affect activity alone or to affect cocaine-induced activity.
However, a lower dose than used in the present study was reported to block the development of sensitization to amphetamine (Richland et al., 200 t ), and the dose used in the current study was the highest possible to maintain selectivity for D3 receptors (Levant & Vansell, 1997). Doses greater that 0.10 mg/kg have been reported to have significant effects on dopamine D2-type receptors (Levant &
Vansell, 1997).
During the two drug withdrawal days, rats previously treated with cocaine were hyperactive, compared to saline control rats, when placed into the activity test chambers without cocaine. Indeed, cocaine pretreated rats displayed significantly greater levels of all three measures of locomotor behavior (distanced traveled, rearing, and stereotypy) after the saline injections compared to control rats. This
36 finding is consistent with previous research with cocaine and other psychostimulants
(e.g., Carey, Damianopoulos & DePalma, 1999), and has usµally been interpreted
within the context of classical conditioning (Le Foll, Frances, Diaz & Schwartz,
2002). In this model, it is assumed that_ cocaine serves as an unconditioned stimulus
(UCS) that elicits an unconditioned response (UCR) of hyperactivity. With repeated
cocaine treatments, the activity test environment becomes a conditioned stimulus
(CS), which can then elicit a conditioned response (CR) of hyperactivity in the
absence of cocaine. Although the current results are consistent with this
interpretation, alternative explanations cannot be eliminated with the current design
(see, Carey et al., 1999, for review). For example, in the current study, rats previously
treated with nafadotride displayed a significant increase in distanced traveled and
stereotypy counts, after the saline injections during the conditioning test. It is hard to
argue that this nafadotride-induced increased activity response is due to classical
conditioning since nafadotride did not affect activity during the pretreatment sessions.
As discussed previously, rats previously treated with cocaine displayed
significantly greater horizontal activity (distance traveled), vertical activity (rears),
and stereotypic small movements after the cocaine challenge injection than rats
receiving cocaine for the first time. In contrast, prior treatments with nafadotride
alone did not significantly affect subsequent sensitivity to cocaine as measured by either distance traveled or rearing. Nafadotride pretreatments did, however, result in a slightly enhanced increase in cocaine-induced stereotypy counts during the cocaine challenge test. As previously mentioned, rats previously treated with nafadotride
37 alone also displayed an increase in stereotypy counts after saline injections during the
withdrawal interval. Neither of these effects was expected, and at present, the
mechanism responsible for these effects is not clear.
During the pretreatment sessions, concurrent treatments with nafadotride and
cocaine did not attenuate or block the progressive increases observed in cocaine
induced locomotor activity over the four pretreatment days. This finding suggests that
nafadotride did not block the development of behavioral sensitization to cocaine. The
results of the cocaine challenge test confirm this interpretation. That is, rats treated
with nafadotride and cocaine during the pretreatment sessions did not significantly
differ from rats previously treated with only cocaine on any measure oflocomotor
behavior during the cocaine challenge test. In fact, rats pretreated with both
nafadotride and cocaine displayed greater (but not significantly) distance traveled
after the cocaine challenge injection than rats previously treated with cocaine alone.
These findings clearly indicate that nafadotride did not block the development of
behavioral sensitization to cocaine.
The present study was designed to test the hypothesis that repeated dopamine
D3 receptor stimulation is a critical factor mediating the development of behavioral
sensitization to psychostimulant drugs (Richtand et al., 2001 ). Assuming that nafadotride effectively blocked dopamine D3 receptors in the current study, the present results are clearly inconsistent with this hypothesis. These results contrast with a recent study that reported that the co-administration of nafadotride with amphetamine blocked the development of behavioral sensitization to amphetamine
38 (Richtand et al., 2001 ). Although the discrepant findings between the current
experiment using cocaine compared to those of the previous study using
amphetamine may be related to methodological factors, the design of the present
study was modeled after the amphetamine experiment (Richland ~t al., 2000). Indeed,
the number and duration of the habituation, pretreatment, withdrawal, and challenge
sessions was the same as in the previous study. Similarly, it is possible that
nafadotride did not sufficiently block dopamine D3 receptors in the current study to
prevent sensitization to cocaine. But as noted 'previously, we used the highest dose
reported to be selective for dopamini;; D3 receptors (Sautel, et al., 1995). Moreover, in
a previous study, we found nearly identical results using the preferential dopamine
D3 receptor antagonist U99 l 94A (Mattingly et al., 2000). Thus, it seems likely that
the differential effectiveness ofnafadotride in blocking the development of behavioral
sensitization to amphetamine and cocaine may be more related to differences in the
mechanism of action of the psychostimulants than to methodological or other factors.
Assuming that nafadotride in the current study and U99 l 94A in our previous study
(Mattingly et al., 2000), effectively blocked dopamine D3 receptors, the results of
these two studies suggest that a differential down-regulation of dopamine D3 vs D2
receptors is not responsible for the development of behavioral sensitization to
cocame.
The differential effects of the preferential dopamine D3 receptor antagonist, nafadotride, on amphetamine- and cocaine-induced behavioral sensitization is not the
only reported differences in dopamine receptor involvement in the induction of
39 behavioral sensitization to these two drugs and other dopamine agonists. For example, the dopamine DI receptor antagonist SCH 23390 has been reported to block the development of behavioral sensitization to the indirect agonist amphetamine
(Stewart & Vezina, 1989; Drew & Glick, 1990), as well as to the direct nonselective dopamine agonist apomorphine (Mattingly et al., 199 I). In contrast, several studies have demonstrated that although SCH 23390 blocks the acute effects of cocaine, concurrent treatments with SCH 23390 and cocaine does not prevent the development of behavioral sensitization to cocaine (Mattingly et al., 1994; 1996; White et al.,
1998). Thus, at present, both selective DI and D3, but not D2, dopamine receptor antagonists have been found to block the development of amphetamine-induced behavioral sensitization (Drew & Glick, 1990; Richland et al., 2000). In contrast, selective D 1, D2, and D3 dopamine antagonists are not effective in attenuating or blocking the development of behavioral sensitization to cocaine (Mattingly et al.,
I 994; 1996; 2000). Taken together, these findings suggest that the stimulation of dopamine receptors may not be necessary for the development of behavioral sensitization to cocaine. Since cocaine blocks the reuptake of other monoamines (e.g., serotonin, norepinephrine) besides dopamine, it is possible that these other neurotransmitters play a more important role in the development of behavioral sensitization to cocaine. Currently, several laboratories are evaluating this possibility
(e.g., Muller, Carey, De Souza Silva, Jocham, & Huston, 2002).
In conclusion, the current findings clearly indicate that concurrent nafadotride treatment does not block the development of behavioral sensitization to cocaine. This
40 finding is inconsistent with the hypothesis that the development of behavioral sensitization to cocaine is mediated by dopamine D3 receptor stimulation. Assuming that dopamine D3 receptor stimulation is critical to the development of behavioral sensitization to amphetamine and other psychostimulant drugs, the current results suggest that the development of behavioral sensitization to different psychostimulant drugs is not mediated by common neurochemical mechanisms.
41 Chapters
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47 APPENDIX A
ANOVASUMMARYTABLES
48 Table 2
Summary of Analysis of Variance Performed on Mean Distance Traveled during the
Habituation Phase (Day 1 and 2)
Source DF Mean Square FValue Pr>F
Between Groups Nafadotride (N) 1 55743 0.33 0.5710 Cocaine (C) 1 89156 0.53 0.4744 NXC 1 30042 0.18 0.6770 Error 27 169430
Within Groups Day(D) 1 160766 3.88 0.0593* NXD 1 2934 0.07 0.7923 CXD 1 440 0.01 0.9179 NXCXD 1 16742 0.40 0.5306 Error 27 41486
Block (B) 3 7771773 275.07 <.0001 * NXB 3 10336 0.37 0.7779 CXB 3 24757 0.88 0.4570 NXCXB 3 12957 0.46 0.7120 Error 81 28254
BXD 3 95905 4.06 0.0096* NXBXD 3 17798 0.75 0.5230 CXBXD 3 8586 0.36 0.7792 NXCXBXD 3 27141 1.15 0.3339 Error 81 23594
* Significant effect
49 Table 3
Summary of Analysis of Variance Performed on Mean Number of Rears during the
Habituation Phase (Day 1 and 2)
Source DF Mean Square FValue Pr>F
Between Groups Nafadotride (N) 1 1662 2.35 0.1368 Cocaine (C) 1 5 0.01 0.9348 NXC 1 112 0.16 0.6935 Error 27 707
Within Groups Day(D) 1 4722 21.22 <.0001 * NXD 1 2 0.01 0.9309 CXD 1 0 0.00 0.9699 NXCXD 1 15 0.07 0.7973 Error 27 223
Block (B) 3 48573 215.03 <.0001 * NXB 3 80 0.35 0.7874 CXB 3 165 0.73 0.5361 NXCXB 3 91 0.40 0.7520 Error 81 226
BXD 3 752 6.40 0.0006* NXBXD 3 204 1.74 0.1658 CXBXD 3 1 0.01 0.9982 NXCXBXD 3 275 2.34 0.0793 Error 81 118
* Significant effect
50 Table4
Summary of Analysis of Variance Performed on Mean Number of Stereotypic Counts during the Habituation Phase (Day 1 and 2)
Source DF Mean Square FValue Pr Between Groups Nafadotride (N) I 332047 0.64 0.4301 Cocaine (C) 1 195880 0.38 0.5435 NXC 1 312165 0.60 0.4441 Error 27 517399 Within Groups Day(D) 1 1574921 9.90 0.0040* NXD 1 37152 0.23 0.6328 CXD I 23168 0.15 0.7057 NXCXD 1 2162 0.01 0.9080 Error 27 159050 Block (B) 3 22541156 189.35 <.0001 * NXB 3 34663 0.29 0.8316 CXB 3 15487 0.13 0.9419 NXCXB 3 33215 0.28 0.8404 Error 81 119043 BXD 3 133016 1.49 0.2223 NXBXD 3 20001 0.22 0.8790 CXBXD 3 31269 0.35 0.7883 NXCXBXD 3 74782 0.84 0.4758 Error 81 89021 * Significant effect 51 Table 5 Summary of Analysis of Variance Performed on Mean Distance Traveled during the Drug Treatment Phase (Day 3 to 6) Source DF Mean Square FValue Pr>F Between Groups Nafadotride (N) I 91317 0.04 0.8406 Cocaine (C) I 280658911 126.76 <.0001 * NXC I 234854 0.11 0.7472 Error 27 2214097 Within Groups Day(D) 3 1014258 1.40 0.2478 NXD 3 512893 0.71 0.5490 CXD 3 1857080 2.57 0.0600* NXCXD 3 485236 0.67 0.5720 Error 81 722729 Block(B) 3 16377402 201.91 <.0001* NXB 3 37106 0.46 0.7128 CXB 3 1883520 23.22 <. 0001 * NXCXB 3 84108 1.04 0.3808 Error 81 81112 BXD 3 42882 1.02 0.4218 NXBXD 3 13244 0.32 0.9693 CXBXD 3 36752 0.88 0.5463 NXCXBXD 3 43076 1.03 0.4181 Error 81 41902 * Significant effect 52 Table 6 Summary of Analysis of Variance Performed on Mean Number of Rears during the Drug Treatment Phase (Day 3 to 6) Source DF Mean Square FValue Pr>F . Between Groups Nafadotride (N) 1 2 0.00 0.9887 Cocaine (C) 1 184291 19.13 0.0002* NXC 1 · 799 0.08 0.7756 Error 27 9636 Within Groups Day(D) 3 2224 1.83 0.1481 NXD 3 1472 1.21 0.3108 CXD 3 3160 2.60 0.0577* NXCXD 3 1423 1.17 0.3258 Error 81 1215 Block (B) 3 35172 66.69 <.0001 * NXB 3 83 0.16 0.9248 CXB 3 1251 2.37 0.0764 NXCXB 3 1285 2.44 0.0706 Error 81 527 BXD 3 535 2.83 0.0035* NXBXD 3 161 0.85 0.5898 CXBXD 3 49 0.26 0.9842 NXCXBXD 3 325 1.72 0.0853 Error· 81 189 * Significant effect 53 Table 7 Summary of Analysis of Variance Performed on Mean Number of Stereotypic Counts during the Drug Treatment Phase (Day 3 to 6) Source DF Mean Square FValue Pr>F Between Groups N afadotride (N) 1 34838 0.38 0,5415 Cocaine (C) I 268891784 295.10 <.0001 * NXC I 1109646 1.22 0.2795 Error 27 · 911189 Within Groups Day (D) 3 9181 0.02 0.9958 NXD 3 265896 0.61 0.6113 CXD 3 1259184 2.88 0.0408* NXCXD 3 537932 1.23 0.3037 Error 81 436841 Block (B) 3 18697052 156.75 <.0001 * NXB 3 87758 0.74 0.5337 CXB 3 2735617 22.93 <.0001 * NXCXB 3 307444 2.58 0.0594 Error 81 119281 BXD 3 79839 1.61 0.1138 NXBXD 3 52489 1.06 0.3960 CXBXD 3 16134 0.32 0.9664 NXCXBXD 3 106751 2.15 0.0263* Error 81 49687 * Significant effect 54 Table 8 Summary of Analysis of Variance Performed on Mean Distance Traveled during the Drug Conditioning Test Phase (Day 7 and 8) Source DF Mean Square FValue Pr>F Between Groups Nafadotride (N) 1 393209 1.96 0.1724 Cocaine (C) 1 6633233 33.15 <.0001 * NXC I 224251 1.12 0.2992 Error 27 200120 Within Groups Day (D) 1 87713 2.71 0.1110 NXD 1 176235 5.45 0.0272* CXD 1 20508 36.35 0.0180* NXCXD 1 26821 0.83 0.3703 Error 27 32315 Block (B) 3 5681737 149.91 <.0001 * NXB 3 12002 0.32 0.8133 CXB 3 387584 10.23 <.0001 * NXCXB 3 5060 0.13 0.9398 Error 81 37901 BXD 3 15286 0.93 0.4287 NXBXD 3 13715 0.84 0.4774 CXBXD 3 59258 3.62 0.0166* NXCXBXD 3 12608 0.77 0.5144 Error 81 16384 * Significant effect 55 Table 9 Summary of Analysis of Variance Performed on Mean Number of Rears during the Conditioning Test Phase (Day 7 and 8) Source DF Mean Square F Value Pr>F Between Groups Nafadotride (N) 1 1240 1.43 0.2422 Cocaine (C) 1 17363 20.02 0.0001 * NXC I 330 0.38 0.5425 Error 27 867 Within Groups Day(D) 1 6 0.03 0.8550 NXD 1 317 1.92 0.1777 CXD 1 49 0.30 0.5910 NXCXD 1 1 0.01 0.9316 Error 27 166 Block(B) 3 20739 114.61 <.0001* NXB 3 72 0.40 0.7541 CXB 3 1133 6.26 0.0007* NXCXB 3 48 0.27 0.8484 Error 81 181 BXD 3 11 0.12 0.9458 NXBXD 3 44 0.50 0.6809 CXBXD 3 102 1.15 0.3326 NXCXBXD 3 ·176 2.00 0.1202 Error 81 '88 * Significant effect 56 Table 10 Summary of Analysis of Variance Performed on Mean Number of Stereotypic Counts during the Conditioning Test Phase (Day 7 and 8) Source DF Mean Square FValue Pr>F Between Groups Nafadotride (N) I 1456187 3.98 0.0562* Cocaine (C) 1 20277548 55.42 <.0001 * NXC 1 242518 0.66 0.4227 Error 27 365857 Within Groups Day(D) 1 387366 3.90 0.0586* NXD 1 450729 4.54 0.0424* CXD 1 328433 3.31 0.0802 NXCXD 1 247486 2.49 0.1262 Error 27 99362 Block (B) 3 14032552 133.05 <.0001 * NXB 3 79067 0'.75 0.5257 CXB 3 121746 1.15 0.3324 NXCXB 3 50372 0.48 0.6987 Error 81 105468 BXD 3 5596 0.09 0.9664 NXBXD 3 47310 0.74 0.5288 CXBXD 3 215895 3.40 0.0217* NXCXBXD 3 38813 0.61 0.6101 Error 81 63574 * Significant effect 57 Table 11 Summary of Analysis of Variance Performed on Mean Distance Traveled during the Challenge Test (Day 9) Source DF Mean Square FValue Pr>F Between Groups Nafadotride (N) 1 6168669 2.94 0.0977 Cocaine (C) I 32072526 15.30 0.0006* NXC I 759789 0.36 0.5521 Error 27 2095596 Within Groups Block (B) 3 6494757 65.06 <.0001 * NXB 3 296209 2.97 0.0368* CXB 3 552306 5.53 0.0017* NXCXB 3 135897 1.36 0.2606 Error 81 99834 * Significant effect 58 Table 12 Summary of Analysis of Variance Performed on Mean Number of Rears during the Challenge Test (Day 9) Source DF Mean Square FValue Pr>F Between Groups Nafadotride (N) 1 1634 0.39 0.5383 Cocaine (C) 1 17508 4.16 0.0512* NXC 1 1 0.00 0.9887 Error 27 4204 Within Groups Block(B) 3 8717 27.47 <.0001 * NXB 3 292 0.92 0.4350 CXB 3 208 0.65 0.5826 NXCXB 3 82 0.26 0.8554 Error 81 317 * Significant effect 59 Table 13 Summary of Analysis of Variance Performed on Mean Number of Stereotypic Counts the Conditioning Effect (Day 9) Source DF Mean Square FValue Pr>F Between Groups Nafadotride (N) 1 4458084 4.13 0.0521 * Cocaine (C) 1 4653111 4.31 0.0475* NXC 1 184 0.00 0.9897 Error 27 1079372 Within Groups Block (B) 3 3073021 41.65 <.0001 * NXB 3 25362 0.34 0.7938 CXB 3 · 21566 0.29 0.8309 NXCXB 3 80799 1.09 0.3561 Error• 81 73791 * Significant effect 60 APPENDIXB COUNTERBANLANCING 61 Squad# Subject# Drug Treatment Group Chamber I I vehicle-vehicle 1 1 4 vehicle-cocaine 2 I 3 nafadotride-vehicle 3 1 2 nafadotride-cocaine 4 2 7 nafadotride-vehicle I 2 8 nafadotride-cocaine 2 2 6 vehicle-vehicle 3 2 5 vehicle-cocaine 4 3 11 vehicle-cocaine I 3 12 vehicle-vehicle 2 3 9· nafadotride-cocaine 3 3 10 nafadotride-vehicle 4 4 16 nafadotride-cocaine 1 4 15 nafadotride-vehicle 2 4 14 vehicle-cocaine 3 4 13 vehicle-vehicle 4 5 17 vehicle-cocaine 1 5 20 vehicle-vehicle 2 5 18 nafadotride-cocaine 3 5 19 nafadotride-vehicle 4 6 23 nafadotride-cocaine 1 6 21 nafadotride-vehicle 2 6 24 vehicle-cocaine 3 6 .22 vehicle-vehicle 4 7 26 vehicle-vehicle I 7· 27 vehicle-cocaine 2 7 28 nafadotride-vehicle 3 7 25 nafadotride-cocaine 4 8 29 nafadotride-vehicle 1 8 30 nafadotride-cocaine 2 8 31 vehicle-cocaine 4 62 ·