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Dopamine Antagonists Do Not Block

Conditioned Place Preference Induced by Paced
Mating Behavior in Female Rats

Article in Behavioral Neuroscience · May 2004

DOI: 10.1037/0735-7044.118.2.356 · Source: PubMed

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Behavioral Neuroscience Copyright 2004 by the American Psychological Association
2004, Vol. 118, No. 2, 356 –364 0735-7044/04/$12.00 DOI: 10.1037/0735-7044.118.2.356

Dopamine Antagonists Do Not Block Conditioned Place Preference

Induced by Paced Mating Behavior in Female Rats
Patricia Garcı́a Horsman and Raúl G. Paredes
Universidad Nacional Autónoma de México

The authors assessed the behavioral effects of dopamine (DA) receptor antagonists, Cis (Z) flupentixol
and S(⫹)-raclopride L-tartrate, on conditioned place preference (CPP) induced by paced mating behav-
ior. Ovariectomized female rats of the Wistar strain were used. The administration of amphetamine (1
mg/kg) induced a clear CPP that was completely blocked by the DA antagonists flupentixol (0.25 mg/kg)
or raclopride (0.125 mg/kg). These doses had no effect on motor coordination. Female rats that mated in
a pacing chamber developed a clear CPP. Neither flupentixol nor raclopride blocked the reward state
induced by paced mating behavior. These results indicate that DA is not involved in the reward state
induced by paced mating behavior in female rats.

One of the most important components of female sexual behav-
ior is the pattern of approaches and withdrawals from a sexually
active male to achieve the female’s preferred rate of copulation,
which in turn culminates in ejaculation (Bermant, 1961; Erskine,
Kornberg, & Cherry, 1989; Peirce, & Nuttall, 1961). This sponta-
neous behavior, known as pacing, occurs in seminatural conditions
(McClintock, & Adler, 1978) and laboratory conditions (Erskine,
1989; Paredes & Vazquez, 1999; Pfaus, Smith, & Byrne, 2000;
Pfaus, Smith, & Coopersmith, 1999). Under pacing conditions, the
female regulates, or paces, the rate of sexual stimulation received
from the male. The intervals between mounts, intromissions, and
ejaculation are proportional to the intensity of the stimulation,
such that return latencies after an ejaculation are longer than after
an intromission or a mount. Furthermore, paced coital stimula-
tion is more effective than nonpaced mating in abbreviating the
estrous cycle and inducing pregnancy (Blaustein & Erskine, 2002;
Erskine, 1989).
It has been proposed that sexual activity may function as a
reinforcer in a way similar to that of classical primary reinforcers
such as water or food (Bermant & Westbrook, 1966; Gilman &
Westbrook, 1978). The rewarding effects of sexual behavior, par-
ticularly paced copulation, have been clearly identified in the
laboratory through the use of conditioning procedures such as the
conditioning place preference paradigm (CPP; Martinez & Pare-
des, 2001; Meisel & Joppa, 1994; Paredes & Alonso, 1997; Pare-
des & Vázquez, 1999). Using this procedure, we have demon-
strated that female rats that regulate their coital interactions with a
stud male, in a two-compartment chamber in which only the
Patricia Garcı́a Horsman and Raúl G. Paredes, Instituto de Neurobiolo-
gı́a, Universidad Nacional Autónoma de México, Querétaro, Mexico.
This research was supported by Dirección General de Asuntos del
Personal Académico Grant IN227402 and Consejo Nacional de Ciencia y
Tecnologı́a Grant V40286M. We thank Francisco Camacho, Maria de
Lourdes Lara, Martı́n Garcı́a, and Pilar Galarza for their excellent technical
assistance.
Correspondence concerning this article should be addressed to Raúl G.
Paredes, Instituto de Neurobiologı́a, Apartado Postal 1-1141, Querétaro
76001, México. E-mail: rparedes@servidor.unam.mx
356
female can freely move between compartments, develop a clear
place preference (Martinez & Paredes, 2001; Paredes & Alonso,
1997; Paredes & Vázquez, 1999). Thus, the physiological state
induced by paced mating is associated with environmental cues,
and rats spend more time in the compartment paired with the
rewarding behavior.
Early pharmacological studies demonstrated that modifying do-
paminergic neurotransmission affected female sexual behavior.
Monoamine oxidase inhibitors decreased the lordosis response
(Meyerson, 1964), whereas amine synthesis inhibitors increased
the frequency of lordosis in ovariectomized (OVX), estrogen-
primed female rats (Ahlenius, Engel, Eriksson, Modigh, & Sod-
ersten, 1972; Meyerson & Lewander 1970). Latter studies using
drugs that specifically interacted with the dopaminergic system
demonstrated that pimozide, a D2 dopamine (DA) antagonist,
increased the lordosis quotient and duration (Everitt, Fuxe, Hok-
felt, & Jonsson, 1975). A dopamine receptor agonist had opposite
effects, that is, it reduced the lordosis quotient, intensity, and
duration (Everitt & Fuxe, 1977; Everitt, Fuxe, & Hokfelt, 1974).
These observations led the authors to suggest that DA had an
inhibitory function on feminine sexual behavior. More recent
studies have demonstrated that the role of DA in female sexual
behavior is not as clear as originally conceived, and contradictory
findings are common in the field. Even with a single compound,
the results are, at best, difficult to interpret. The selective D2
receptor agonist LY163502 increased the lordosis response in
OVX females primed with estrogen. These facilitatory effects were
blocked by prior treatment with the DA antagonists haloperidol
or flupentixol (Foreman & Hall, 1987). Contrary to the facili-
tatory effects on lordosis in estrogen-primed females, the same
compound produced a clear inhibition of lordosis in OVX
females primed with estradiol benzoate (EB) and progesterone
(P), supporting other observations (Eliasson & Meyerson, 1976;
Grierson, James, Pearson, & Wilson, 1988). Dual effects on
lordosis have also been described for the D2 antagonist
sulpiride. Small doses inhibited receptivity in OVX females
primed with EB and P but facilitated lordosis in OVX females
primed with estradiol (Grierson et al., 1988).
 PLACE PREFERENCE, DA, AND FEMALE SEXUAL BEHAVIOR
Microdialysis studies have demonstrated an increase in DA
release during copulation in the striatum and accumbens of female
rats (Pfaus, Damsma, Wetern, & Fibiger, 1995) and hamsters
(Kohlert & Meisel, 1999; Kohlert, Rowe, & Meisel, 1997; Meisel,
Camp, & Robinson, 1993). Moreover, female rats that paced their
sexual behavior showed significantly greater increases in DA
concentrations than females that could not pace their sexual con-
tacts (Mermelstein & Becker, 1995). In a follow-up study, the
preferred pacing interval was determined for a group of females.
This was defined as the average return latency after intromissions
in two pacing sessions. Significant increases in DA concentration
in both the striatum and accumbens was observed in the group that
mated at the preferred pacing interval, as well as in the group that
actually paced their sexual contacts (Jenkins & Becker, 2001).
Studies of CPP have found that female hamsters spend less time
in the compartment associated with sexual behavior when treated
with a D2 receptor antagonist, sulpiride or raclopride. Neither of
these D2 antagonists disrupted the ability of female hamsters to
display lordosis. Meisel and collaborators suggested that D2 re-
ceptors affected the expression of sexual behavior and prevented
the acquisition of CPP (Meisel, Joppa, & Rowe, 1996). Together,
these experiments suggest an important role of DA in motivated
behaviors. No studies have evaluated the effects of DA antagonists
on the reward process associated with mating in female rats. In the
present study, we examine the role of DA in the rewarding con-
sequences of paced mating behavior in female rats. We evaluated
two DA antagonists, cis(Z)flupentixol and S(⫹)-raclopride
L-tartrate, using the CPP paradigm. The hypothesis was that if the
increase in extracellular DA levels induced a reward state during
paced copulatory behavior, doses of DA antagonist that do not
interfere with motor execution would be able to block the CPP
induced by paced mating behavior. We performed four experi-
ments. In the first experiment, we determined the doses of the DA
antagonists that had no effect upon motor coordination. In the
second, we tested whether the DA antagonists, in doses that do not
have an effect on motor coordination, could block the CPP induced
by amphetamine (1 mg/kg). In the third experiment, we evaluated
whether the DA antagonists could block the CPP induced by paced
mating. In the last experiment, we determined whether the DA
antagonists themselves could induce CPP.
General Method
Subjects
Sexually naive female Wistar rats from a local colony weighting 250 –
350 g were used in all experiments. The rats were maintained under a
reversed 12-hr light– dark cycle at constant room temperature (approxi-
mately 22 °C), with free access to water and commercial rodent pellets
(LabDiet; Nutrition International, Brentwood, MO).
For all the experiments, the females were bilaterally ovariectomized
(OVX) 2 weeks before use. Anesthesia was induced by intraperitoneal
administration of 95 mg/kg ketamine and 12 mg/kg xylazine, in a volume
of 1 ml/kg. After surgery, they were housed in groups of 4. In the four
experiments, the rats received subcutaneous injections of 25 ␮g EB 48 –52
hr before testing, and 1mg P 4 hr before testing. Steroids were dissolved in
corn oil and injected in a volume of 0.2 ml per rat. Sexually experienced
males were used as stimuli.
357
Apparatus
Smart Rod. The Smart Rod (Accuscan Electronics, Columbus, OH)
was used to evaluate motor coordination. This apparatus consists of a
cylinder (rota-rod) with a diameter of 16 cm. The rats were placed on the
cylinder, which rotated at different speeds. Whenever the rat fell down, it
was replaced on the cylinder approximately 5 s later. Before drug treat-
ment, the rats were trained at a speed of 20 revolutions per minute (rpm).
The rats were tested on the rota-rod at a speed of 25 rpm. The number of
falls during a 3-min period was counted and provided the measure of motor
execution. This test was used to differentiate the effect of the drugs on
sexual activity from possible effects on motor coordination. In the case of
male rats, it has been shown that whenever motor execution is affected,
sexual behavior is inhibited (Ågmo, Paredes, & Fernández, 1987).
Place preference. The place preference apparatus consisted of a three-
compartment box made of wood. A central compartment (22 cm long ⫻ 24
cm high ⫻ 32 cm wide) connected the lateral compartments (23 cm long ⫻
37 cm high ⫻ 32 cm wide). One of the lateral compartments was painted
white, and the floor was covered with clean wood shavings. The opposite
lateral compartment was painted black and moistened with a 2% solution
of glacial acetic acid immediately before a rat was placed in it. The middle
compartment was painted gray and communicated with the lateral com-
partments through a 10 ⫻ 10-cm sliding door on each side. The front side
of the middle compartment was made of fine wire mesh, which allowed
observation of the rat inside the cage. The place preference cages were
located in a room illuminated with dim white light. The mating cages (40
cm long ⫻ 60 cm high ⫻ 40 cm wide) were located adjacent to the
preference cages. They had wood shavings on the floor, and the front wall
was made of glass for observation. When females were allowed to pace, a
removable wood partition was placed in the middle of the mating cage. The
partition had a small hole (4 cm diameter) through which only the female
could move freely from one side to the other; the hole was too small for the
male to go through. In each test, a male stimulus rat was placed in the
mating cage 2 min before the female was introduced. The test ended when
the female received 15 intromissions or one ejaculation.
Drugs
dl-amphetamine sulfate (salt; Sigma/RBI, St. Louis, MO) was dissolved
in physiological saline and injected intraperitoneally in a volume of 1
ml/kg 40 min before testing. Cis(Z) flupentixol (Lundbec, Copenhagen,
Denmark), a D1/D2 DA antagonist, was dissolved in physiological saline
and injected 30 min before testing in doses of 0.25 mg/kg, 0.50 mg/kg, and
1.00 mg/kg, in a volume of 1 ml/kg.
S(⫹)-raclopride L-tartrate (Sigma/RBI), a D2 DA antagonist, was dis-
solved in physiological saline and injected 15 min before testing in doses
of 0.125 mg/kg, 0.250 mg/kg, and 0.500 mg/kg, in a volume of 1 ml/kg.
Experiment 1
In the first experiment, we determined the dose of the DA
antagonists that had no effect upon motor coordination. Two
groups of 12 rats each were bilaterally ovariectomized and injected
with EB and P before testing.
Method
Design. All drugs were administered according to a Latin square
design. Each rat in a given group received all doses of a given drug plus
vehicle, and the effects of all doses plus vehicle were observed in a usually
equal number of rats at each session. In this way each subject served as its
own control. The interval between drug injection was 4 days, which was
sufficient to avoid any effects being carried over from the previous treat-
ment. No rat was treated with more than one drug; that is, one group was
358 HORSMAN AND PAREDES
treated with NaCl and all doses of flupenthixol, and the other group
received NaCl and all the doses of raclopride.
Procedure. Before drug treatments, the rats were trained on the cyl-
inder during four sessions of 5 min. During the first 20 s, the apparatus
rotated at 10 rpm; during the second 30 s, the speed was increased to 15
rpm; and in the last 250 s, the apparatus rotated at 20 rpm. The test was
repeated until they were able to walk with no falls during the 5-min test.
Motor coordination was quantified as the number of falls in 3 min with the
cylinder rotating at 25 rpm.
Statistical analysis. The data on the motor coordination test was eval-
uated with an analysis of variance (ANOVA) for repeated measures fol-
lowed by Fisher’s least significant difference procedure for each group.
Results
The ANOVA revealed significant differences between groups
for both drugs: flupentixol, F(3, 11) ⫽ 25.48, p ⬍ .0001, and
raclopride, F(3, 11) ⫽ 26.72, p ⬍ .0001. The post hoc test revealed
that the doses of 0.5 mg/kg and 1.0 mg/kg flupentixol, as well as
the highest doses of raclopride, 0.25 mg/kg and 0.50 mg/kg,
produced significant motor deficits. The lower doses of the antag-
onists had no effect on motor coordination. Rats injected with 0.25
mg/kg flupentixol and 0.125 mg/kg raclopride did not differ sig-
nificantly from their respective control group (see Figure 1).
Experiment 2
In the second experiment, we evaluated whether the doses of
DA antagonists that had no effect on motor coordination could
block the CPP induced by 1 mg/kg amphetamine.
Figure 1. Mean (⫾ SEM) number of falls in the 3-min test, in female rats treated with different doses of
flupentixol (top) and raclopride (bottom). n ⫽ 12 for each drug. Asterisks indicate significant difference from
saline (NaCl), *** p ⬍ .001.
Method
Procedure. Four groups of 12 OVX female rats were used. All groups
were primed with 25 ␮g EB 48 –52 hr, and 1 mg P 4 hr, before being placed
in the nonpreferred compartment (see below). The experimental treatments
were as follows: (a) NaCl ⫹ NaCl, (b) amphetamine (1 mg/kg) ⫹ NaCl, (c)
amphetamine (1 mg/kg) ⫹ flupentixol 0.25 mg/kg, (d) amphetamine (1
mg/kg) ⫹ raclopride 0.125 mg/kg (see Table 1). All drugs were given
intraperitoneally in a volume of 1 ml/kg, allowing enough time for the
drugs to have their maximum effect—amphetamine, 40 min; flupenthixol,
30 min; and raclopride, 15 min— before placing the subjects in the non-
preferred compartment of the CPP box.
Conditioning and testing procedures. Females were given a pretest,
which consisted of placing each subject in the central compartment of the
place preference cage and measuring the time spent in each lateral com-
partment during a 10-min session. The conditioning sessions were as
follows: On the first, third, and fifth sessions, all groups received two saline
injections and then were placed in their preferred (nonreinforced) compart-
ment for 30 min. On the second, fourth, and sixth sessions, rats of Group
1 were injected with NaCl and Groups 2, 3, and 4 were injected with their
corresponding drugs at their respective intervals and immediately trans-
ferred to their nonpreferred (reinforced) compartment (Table 1). When all
the rats had completed three nonreinforced and three reinforced sessions,
the subjects were tested as in the pretest. An interval of 24 hr separated
each session.
Statistical analysis. To evaluate place preference conditioning two
criteria were used: the time in the reinforced compartment between pretest
and test and the preference score [(time in reinforced compartment ⫼ (time
in reinforced compartment ⫹ time in nonreinforced compartment)] should
increase after conditioning. The use of both criteria reduces the possibility
of spurious effects. The data were analyzed by a 2 (test) ⫻ 4 (group)
 PLACE PREFERENCE, DA, AND FEMALE SEXUAL BEHAVIOR 359

Table 1 Experiment 3
Treatments for the Groups of Ovariectomized Females Before
Placement in the Preferred or Nonpreferred Compartment in Experiment 2 demonstrated that amphetamine clearly induced a
Experiments 2, 3, and 4 change of preference. Flupentixol and raclopride blocked the CPP
induced by amphetamine in doses that did not interfere with motor
Compartment execution. Experiment 3 was designed to evaluate whether the
same doses of the DA antagonists could block the CPP induced by
Preferred Nonpreferred paced mating behavior.
Group (nonreinforced) (reinforced)

Experiment 2 Method
NaCl NaCl ⫹ NaCl NaCl ⫹ NaCl Procedure. Four groups of 12 OVX, sexually naive female rats were
Amph NaCl ⫹ NaCl Amph ⫹ NaCl used: females not allowed to pace their sexual contacts and treated with
Amph ⫹ Flu NaCl ⫹ NaCl Amph ⫹ Flu NaCl before mating, females allowed to pace their sexual contacts and
Amph ⫹ Rac NaCl ⫹ NaCl Amph ⫹ Rac treated with NaCl before mating, females allowed to pace their sexual
contacts and treated with flupentixol 30 min before mating, and females
Experiment 3
allowed to pace their sexual contacts and treated with raclopride 15 min
NaCl ⫹ NP NaCl NaCl before nonpaced mating before mating (Table 1). As in Experiment 2, all females were primed with
NaCl ⫹ P NaCl NaCl before paced mating 25 ␮g EB 48 –52 hr before testing and 1 mg P 4 hr before testing.
Flu ⫹ P NaCl Flu before paced mating Sexual behavior testing procedures. The following parameters of sex-
Rac ⫹ P NaCl Rac before paced mating ual behavior were recorded:

Experiment 4 1. Latencies and number of mounts, intromissions, and

ejaculations;
NaCl NaCl NaCl
Flu NaCl Flu 2. Interintromission intervals (III; ejaculation latency divided by the
Rac NaCl Rac
number of intromissions);
Note. NaCl ⫽ saline; Amph ⫽ amphetamine; Flu ⫽ flupentixol; Rac ⫽
raclopride; NP ⫽ nonpaced; P ⫽ paced. 3. Mean lordosis intensity (MLI; sum of points of lordosis intensity
divided by the number of mounts plus intromissions received);

ANOVA with repeated measures on the test variable (pretest–test). All
probabilities were two-tailed.
Results
The results of the second experiment showed that amphetamine
(1 mg/kg) induced a clear CPP. The lower doses of the DA
antagonists that had no effect on motor coordination blocked the
CPP induced by amphetamine (see Figure 2). The ANOVA of the
preference score revealed a significant effect of session, F(1,
44) ⫽ 11.97, p ⫽ .001; group, F(3, 44) ⫽ 21.19, p ⬍ .001; and
Group ⫻ Session, F(3, 44) ⫽ 3.67, p ⬍ .05. Only the group
injected with amphetamine showed a significant increase in the
preference score, F(1, 44) ⫽ 17.30, p ⬍ .001. The analysis of time
in the reinforced compartment revealed a significant effect of
group, F(3, 44) ⫽ 15.33, p ⬍ .001; and Group ⫻ Session, F(3,
44) ⫽ 2.97, p ⬍ .05; but not of session, F(1, 44) ⫽ 1.56, p ⫽ .20.
The test for simple main effects showed that the time in the
reinforced compartment increased significantly in the group of
females injected with amphetamine, F(1, 44) ⫽ 7.87, p ⬍ .01. No
significant differences were observed in the other groups. Figure 2
illustrates the clear change of preference induced when females
were injected with amphetamine. Although the pretest value of the
amphetamine group appears to be low, it is important to consider
that the rats were randomly assigned to their corresponding group
before the pretest and hence the groups could not be matched for
their baseline levels. Because each subject was its own control, the
important comparison is pretest versus test within the same group.
The results in Figure 2 also show that the injection of flupentixol
and raclopride completely blocked the change of preference in-
duced by amphetamine.
4. Lordosis quotient (LQ, the total number of lordosis responses
divided by the number of mounts and intromissions multiplied by
100).
The CPP procedure was similar to the one used in Experiment 2. A
pretest was performed. In the first, third, and fifth sessions, the female rats
were injected with NaCl and placed in the preferred (nonreinforced)
compartment. On the second, fourth, and sixth sessions, females were
treated with their respective drug before mating. At the end of the mating
test, females were gently removed from the mating cage and placed in the
nonpreferred (reinforced) compartment of the place preference cage. The
first group did not control the rate of sexual stimulation (nonpaced copu-
lation). The other three groups mated with the wood partition in place,
allowing the females to pace their sexual contacts. The test ended after the
female received 15 intromissions or one ejaculation. Previous studies in our
laboratory have shown that at least 10 paced intromissions are necessary to
induce CPP (Paredes & Vázquez, 1999).
Statistical analysis. The CPP data were analyzed by a 2 (test) ⫻ 4
(group) ANOVA with repeated measures on the test variable (pretest–test).
The sexual behavior parameters were evaluated with a Kruskal–Wallis
one-way ANOVA followed by a Newman–Keuls multiple comparisons
post hoc test. We calculated the average of the three conditioning sessions.
Results
Sexual behavior. No differences were found in the number of
mounts and intromissions between groups. When mount intromis-
sion and ejaculation latencies were evaluated, significant differ-
ences were found for the groups that paced their sexual contacts
compared with the nonpacing group, H(3) ⫽ 26.66, p ⬍ .0001;
H(3) ⫽ 13.92, p ⫽ .003; H(3) ⫽ 12.40, p ⫽ .0061. The LQ was
similar for all groups of females. No significant differences were
observed between the groups that paced their behavior. The fe-
360 HORSMAN AND PAREDES

Figure 2. Mean (⫾ SEM) preference score and time in the reinforced compartment at pretest and test in the
different groups of female rats. n ⫽ 12 per group. NaCl⫹NaCl ⫽ injected with saline plus saline;
Amph⫹NaCl ⫽ injected with amphetamine (1 mg/kg) plus NaCl; Amph⫹Flu ⫽ injected with amphetamine plus
flupentixol (0.25 mg/kg); Amph⫹Rac ⫽ injected with amphetamine plus raclopride (0.125 mg/kg). Asterisks
indicate a significant difference from pretest, ** p ⬍ .01.

males that paced their sexual behavior and were injected with
raclopride had lower MLI than the other groups. No differences in
the III were found between the pacing females. However the III
was significantly longer for the groups that paced their sexual
contacts and were treated with flupentixol and raclopride. No
significant differences were found in mount and intromission
return latencies between the groups that paced their coital contacts.
The data are summarized in Table 2.
Place preference. The preference score in the groups that
paced their sexual interactions showed a significant increase. The
ANOVA revealed a significant effect of session, F(1, 44) ⫽ 92.23,
p ⬍ .001; and Group ⫻ Session, F(3, 44) ⫽ 18.07, p ⬍ .001; but
not group, F(3, 44) ⫽ 0.35, p ⫽ .79. The test of simple main
effects revealed a significant increase in the preference score in all
the groups that paced their sexual contacts, regardless of whether
they were treated with NaCl, F(1, 44) ⫽ 25.41, p ⬍ .001; flupen-
tixol, F(1, 44) ⫽ 21.46, p ⬍ .001; or raclopride, F(1, 44) ⫽ 99.39,
p ⬍ .001, before pacing (see Figure 3). The ANOVA of the time
in the reinforced compartment revealed a significant effect of
session, F(1, 44) ⫽ 20.77, p ⬍ .001; and Group ⫻ Session, F(3,
44) ⫽ 12.65, p ⬍ .001; but not group, F(3, 44) ⫽ 0.11, p ⫽ .95.
The test of simple main effects showed a significant increase in
time spent in the reinforced compartment for all groups that paced
their sexual contacts: NaCl, F(1, 44) ⫽ 6.67, p ⬍ .05; flupentixol,
F(1, 44) ⫽ 5.83, p ⬍ .05; and raclopride, F(1, 44) ⫽ 41.00, p ⬍
.001. Females that did not pace their sexual contacts and were
treated with NaCl showed a reduction in the time spent in the
reinforced compartment during the test, F(1, 44) ⫽ 5.23, p ⬍ .05
(see Figure 3).
Experiment 4
In order to evaluate the effect of the DA antagonists alone on
CPP, we performed a fourth experiment. We tested whether flu-
pentixol or raclopride could induce a change of preference.
Method
Procedure. Conditioning and testing procedures were the same as in
Experiment 2. All females were OVX and primed with EB and P before
being placed in the nonpreferred compartment. The rats were divided into
three groups: The first was injected with NaCl (1 ml/kg); the second, with
flupentixol (0.25 mg/kg); and the third, with raclopride (0.125 mg/kg).
After the pretest, in the first, third, and fifth sessions, the rats were injected
with NaCl and placed in the preferred (nonreinforced) compartment. On
 PLACE PREFERENCE, DA, AND FEMALE SEXUAL BEHAVIOR 361

Table 2
Mean (⫾ SEM) Parameters of Sexual Behavior Recorded in the Different Groups of Female
Rats in Experiment 3

Treatment

Nonpaced Paced

Behavior parameter NaCl NaCl Flupentixol Raclopride

NM 13 ⫾ 2 13 ⫾ 1 10 ⫾ 2 11 ⫾ 1
NI 10 ⫾ 1 13 ⫾ 1 12 ⫾ 1 12 ⫾ 1
ML 6⫾1 43 ⫾ 13** 56 ⫾ 16** 44 ⫾ 8**
IL 30 ⫾ 8 81 ⫾ 28* 84 ⫾ 16** 101 ⫾ 22**
EL 331 ⫾ 23 712 ⫾ 16* 566 ⫾ 63* 594 ⫾ 77*
LQ 96 ⫾ 1 97 ⫾ 1 95 ⫾ 1 97 ⫾ 1
MLI 1.50 ⫾ 0.02 1.70 ⫾ 0.05 1.40 ⫾ 0.04 1.20 ⫾ 0.04*
III 35 ⫾ 3 63 ⫾ 13 54 ⫾ 6* 56 ⫾ 9*
MRL — 18.2 ⫾ 3.2 18.8 ⫾ 6.8 62.0 ⫾ 32.2
IRL — 41.6 ⫾ 6.5 54.4 ⫾ 8.0 72.8 ⫾ 13.4

Note. The data represent the average of the three tests of coital behavior. All latencies are expressed in seconds.
Dashes indicate parameter not measured. NaCl ⫽ saline; NM ⫽ number of mounts; NI ⫽ number of
intromissions; ML ⫽ mount latency; IL ⫽ intromission latency; EL ⫽ ejaculation latency; LQ ⫽ lordosis
quotient; MLI ⫽ mean lordosis intensity; III ⫽ interintromission intervals; MRL ⫽ mount return latencies;
IRL ⫽ intromission return latencies.
* p ⬍ .05, ** p ⬍ .01, significantly different from nonpaced group (Kruskal–Wallis one-way analysis of variance
followed by a Newman–Keuls multiple comparisons post hoc test).

the second, fourth, and sixth sessions, females were treated with NaCl,
flupentixol or raclopride (depending on the group, see Table 1) before
being placed in the nonpreferred (reinforced) compartment. At the end of
conditioning, the rats were tested in the same way as in the pretest.
Statistical analysis. The data were analyzed by 2 (test) ⫻ 3 (group)
ANOVA with repeated measures on the test variable (pretest–test).
Results
The results of this experiment showed that the lower doses of
flupentixol and raclopride did not induce CPP. No significant
effect on time spent in the reinforced compartment was found:
NaCl, F(1, 32) ⫽ 2.76, p ⫽ .107; flupentixol, F(1, 32) ⫽ 0.23, p ⫽
.638; and raclopride, F(1, 32) ⫽ 3.23, p ⫽ .082 (data not shown).
Discussion
The results of the present study clearly indicate that doses of
0.25 mg/kg cis(Z) flupentixol and 0.125 mg/kg S(⫹)-raclopride
L-tartrate do not produce motor deficits that might interfere with
copulation. Amphetamine at a dose of 1 mg/kg induced a clear
CPP. The induction of CPP by amphetamine was blocked by the
DA antagonists flupentixol and raclopride. The same doses of the
antagonists were not able to block the CPP induced by paced
mating, suggesting that DA is not involved in the reward state
induced by pacing. The results of this study replicate previous
findings indicating that CPP is an effective procedure to study
appetitive or motivational components of sexual behavior (Ågmo
& Berenfeld, 1990; Meisel & Joppa 1994; Oldenburger, Everitt, &
De Jonge, 1992; Paredes & Martinez, 2001). Paced coital contacts
in the female rat produce a positive affect of sufficient intensity
and duration to induce conditioning (Martinez & Paredes, 2001;
Paredes & Alonso, 1997; Paredes & Vázquez, 1999). The results
of the present experiments further support this conclusion, since
only females that paced their coital contacts developed CPP. No
change of preference was observed in females that were not
allowed to control the rate of sexual stimulation.
The role of dopamine (DA) in female rat sexual behavior is
rather controversial. Dual effects upon lordosis behavior have been
reported after systemic injections of different dopaminergic com-
pounds. For example, when apomorphine (a DA agonist) is ad-
ministered to females that display high levels of lordosis behavior,
a clear inhibition of this behavior is observed (Everitt et al., 1975;
Meyerson, 1964). On the other hand, when apomorphine is in-
jected to females that display low levels of lordosis behavior,
facilitation is observed (Rodriguez-Sierra, Naggar, & Komisaruk,
1976). Similar contradictory results were obtained after adminis-
tration of the selective D2 receptor agonist LY1635602. In some
studies, an increase in lordosis response was described (Foreman
& Hall, 1987), whereas others reported a clear inhibition of lor-
dosis behavior (Eliasson & Meyerson, 1976; Grierson et al., 1988).
Dual effects on lordosis were also observed after injection of the
D2 antagonist sulpiride (Grierson et al., 1988). It is clear from
pharmacological studies that a compound in the same dose can
produce a different effect depending on the hormonal condition of
the animal, complicating the interpretation of the effects of DA in
female sexual behavior.
Results from studies of DA release are not all that conclusive.
For example, it is clear from early studies that the increase in DA
release is not necessarily associated with the display of feminine
sexual behavior. Significant increases in DA release were observed
in the nucleus accumbens of female rats when a male was pre-
sented behind a screen, preventing physical interaction (Pfaus et
al., 1995). In another study, DA increased in the nucleus accum-
bens in the second 15-min sampling period after the male had been
introduced. However, the female hamsters received the largest
number of intromissions during the first 15-min period with the
male (Meisel et al., 1993). As can be seen, there is no clear
362 HORSMAN AND PAREDES

Figure 3. Mean (⫾ SEM) preference score and time in the reinforced compartment at pretest and test in the
different groups of female rats. n ⫽ 12 per group. NaCl ⫽ saline; Flu ⫽ flupentixol; Rac ⫽ raclopride; NP ⫽
nonpaced. Asterisks indicate a significant difference from pretest, * p ⬍ .05, ** p ⬍ .01.

evidence that the increase in DA release is specifically associated
with a particular aspect of female sexual behavior. It has also been
proposed that increased extracellular DA in the accumbens and the
striatum is associated with the rewarding value of sex (Jenkins &
Becker, 2001). Two considerations can be made regarding this
hypothesis. First, as we have repeatedly demonstrated, sexual
behavior is rewarding for a female rat only if she is able to pace
(control) the rate of sexual stimulation (Martinez & Paredes, 2001;
Paredes & Alonso, 1997; Paredes & Vázquez, 1999). Although
pacing induces a higher release of DA in the striatum in compar-
ison with nonpacing, the changes in DA release in the accumbens
are very similar in pacing and nonpacing females (Mermelstein &
Becker, 1995). Out of four sample points in which the male was
present, the increase in DA release in the accumbens was very
similar between pacing and nonpacing females in three of them.
Only in one point was DA release significantly lower in nonpacing
females. Moreover, estrogen alone (without exposure to a male)
induced a significant increase in DA release in the accumbens and
striatum compared to oil treatment (Mermelstein & Becker, 1995).
As can be seen, not only pacing behavior, but other factors asso-
ciated with mating can also increase DA release in the accumbens
and the striatum. The second factor to consider is the alternate
mechanism by which DA might mediate the effects on female
sexual behavior. Mani and coworkers (Mani, 2003; Mani, Allen,
Clark, Blaustein, & O’Malley, 1994; Mani, Blaustein, Allen,
O’Malley, & Clark, 1994; Mani, Mitchell, & O’Malley, 2001)
have demonstrated that DA induces a ligand independent activa-
tion of P receptors. For example, the intraventricular administra-
tion of apomorphine in rats treated only with estrogen produced a
lordosis response equivalent to that seen after administration of E
and P. The selective D1 agonist, SKF 38393, also produced a
response similar to P in rats primed only with E (Mani, Allen, et
al., 1994). The facilitation of lordosis induced by the D1 agonist
was blocked by the D1 antagonist SCH 23390, whereas a D2
agonist, quinpirole, did not facilitate lordosis in E-primed females.
The authors interpreted these results as suggesting that P and DA
induce lordosis through effects on P receptors within the brain
(Mani, Allen, et al., 1994). In the same study, P receptor antago-
nists or an antisense oligonucleotide to P receptors blocked the
facilitation of lordosis induced by P or apomorphine, suggesting
that DA induces a ligand-independent activation of P receptors
(Mani, 2003). This cross-talk between P receptors and DA could
explain, at least in part, some of the early and contradictory
observations with respect to the pharmacological treatments. It
could also be argued that the increase in DA during sexual behav-
ior in female rats could be associated with this cross-talk mecha-
nism and not with a direct control of DA on female sexual
behavior or with the reward induced by paced mating. As well,
there is no clear evidence that EB or P alone induce CPP. We have
repeatedly shown that EB and P injections without mating do not
 PLACE PREFERENCE, DA, AND FEMALE SEXUAL BEHAVIOR
induce CPP (Martinez & Paredes 2001; Paredes & Alonso, 1997).
Moreover, it has been reported that high levels of EB disrupt CPP
(Galea et al., 2001).
Sexual behavior and aggression induced CPP in female Syrian
hamsters (Meisel & Joppa, 1994) that was blocked by the D2
receptor antagonist raclopride, suggesting that these DA receptor
subtypes are involved in the appetitive aspects of female sexual
behavior (Meisel et al., 1996). In the present study, raclopride
blocked the CPP induced by amphetamine but was not able to
block the CPP induced by paced mating. These apparently contra-
dictory findings could be related to the differences in reproductive
pattern between hamsters and rats. Whereas female rats exhibit
proceptive, receptive, and pacing behaviors during a sexual en-
counter, female hamsters maintain, if receptive, a lordosis posture
for most of the time during mating, with very low levels of motor
activity (Meisel et al., 1993; Meisel & Joppa, 1994). Another
important factor is that nonreceptive female hamsters are aggres-
sive toward males (Meisel et al., 1993; Meisel & Joppa, 1994).
Indeed, in the early study demonstrating a CPP in female hamsters
during sexual behavior, the authors acknowledged that they were
not able to dissociate whether aggression, sexual behavior, or a
combination of both induced CPP (Meisel & Joppa, 1994). More-
over, female hamsters do not pace their sexual interactions,
whereas in female rats this characteristic is crucial for sex to be
rewarding.
It has been recently argued that the preferred pacing interval
(PPI) induces a reward state when compared with nonpaced mat-
ing (Jenkins & Becker, 2003). However, the methodological lim-
itations for that experiment are evident: First, the sexual interaction
occurred in the conditioning chamber, making impossible to es-
tablish whether the change of preference was associated with
mating, the sociosexual interaction, or with other factors such as
the simple presence of a conspecific. Second, the authors exposed
the females, in one side of the preference cage, to a positive stimuli
(PPI), but, in the other side of the cage, the females were exposed
to a negative stimuli (nonpaced mating). It is therefore impossible
to dissociate whether the females did actually develop a CPP for
the preferred interval or whether they were simply avoiding the
negative stimuli induced by nonpaced mating. By mating the rats
in a mating cage and then placing them in the CPP apparatus, we
were able to evaluate the physiological state induced by mating,
free of distracting variables such as the presence of a conspecific.
Furthermore, giving the females only one stimulus during the CPP
provided clear and straightforward results that are easy to interpret.
In the present study, none of the DA antagonists could block the
CPP induced by paced mating in female rats. Neither flupentixol
(D1/D2 antagonist) or raclopride (D2 antagonist) were able to block
the CPP induced by paced mating behavior. Similar results have
been described in male rats in which a D2 DA antagonist, pimo-
zide, did not block the CPP induced by ejaculation (Ågmo &
Berenfeld, 1990). In the same study naloxone, an opiate antago-
nist, blocked the CPP induced by ejaculation, supporting early
observations that opioids are involved in sexual behavior (Ågmo &
Paredes, 1988; Hughes, Herbert, & Everitt, 1990; Mehrara &
Baum, 1990; Miller & Baum, 1987) and contribute to the reward-
ing consequences of mating (Ågmo, 1999). Results from our
laboratory indicate that CPP established by paced sexual contacts
is completely blocked by naloxone (Martinez & Paredes, 2001),
363
further supporting the involvement of opioids in the reward pro-
cesses associated with mating in male and female rats.
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Received August 28, 2003
Revision received October 13, 2003
Accepted October 14, 2003 䡲