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Female rats exhibit a conditioned place preference for nonpaced mating
Hormones and Behavior 51 (2007) 89 – 94 www.elsevier.com/locate/yhbeh
Female rats exhibit a conditioned place preference for nonpaced mating Sarah H. Meerts, Ann S. Clark ⁎ Department of Psychological and Brain Sciences, Dartmouth College, Hanover, NH 03755, USA Received 26 July 2006; revised 29 August 2006; accepted 29 August 2006 Available online 3 October 2006
Abstract Paced, but not nonpaced, mating behavior is reported to induce a conditioned place preference (CPP) in female rats. Contrary to these previous findings, Experiment 1 showed that female rats that received 15 intromissions from a single male rat during each of five conditioning sessions exhibited a CPP for the compartment associated with mating when the intromissions were delivered via a paced or nonpaced paradigm. Experiment 2 demonstrated that nonpaced mating induced a CPP when a single male delivered the 15 intromissions but not when the male was replaced following ejaculation and a new male allowed to complete the requisite number of intromissions. These findings invite reevaluation of the reinforcing aspects of mating behavior in female rats. © 2006 Elsevier Inc. All rights reserved. Keywords: Paced mating behavior; Intromission; Post-ejaculatory interval; Sexual behavior
Paced mating behavior is the pattern of approach and withdrawal exhibited by a female rat during a sexual encounter with a male rat observed in a semi-natural environment (McClintock and Adler, 1978) or in a testing arena that allows the female to escape (for review, Erskine, 1989). Nonpaced mating has been contrasted with paced mating; in nonpaced mating tests the male controls the tempo of sexual interactions because the female rat cannot escape (Erskine, 1989). Stimuli received during paced and nonpaced mating may elicit different neuroendocrine and physiological responses in female rats. Circulating levels of luteinizing hormone are elevated in female rats mated under paced compared to nonpaced mating conditions (Erskine and Kornberg, 1992). Paced mating behavior also enhances reproductive success; a greater percentage of rats become pregnant or pseudopregnant and have larger litters when they receive paced rather than nonpaced mating stimulation (Coopersmith and Erskine, 1994; Erskine, 1989). Conditioned place preference (CPP) is a form of classical conditioning used to assess the reinforcing properties of a given stimulus (Carlezon, 2003). Typically, animals express a preference for a distinct environment associated with adminis⁎ Corresponding author. Fax: +1 603 646 1419. E-mail address: [email protected] (A.S. Clark). 0018-506X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2006.08.007
tration of a drug or behavior that is reinforcing (Bardo and Bevins, 2000; Carlezon, 2003). Several researchers have demonstrated that female rats exhibit a CPP for a context associated with paced, but not nonpaced, mating (Gans and Erskine, 2003; Jenkins and Becker, 2003; Paredes and Alonso, 1997; cf., Oldenburger et al., 1992). The reinforcing effects of paced mating have been linked with the female's role in controlling the timing of mating stimulations, the slower rate of intromissions and the increased intensity of cervical stimulation (Jenkins and Becker, 2003; Martinez and Paredes, 2001; Paredes and Alonso, 1997; Paredes and Vazquez, 1999). We recently conducted a study examining the reinforcing aspects of mating and designed to follow the procedures outlined originally by Paredes and Alonso (1997) and updated by Paredes and Vazquez (1999). These authors reported that female rats that received 10–15 paced intromissions expressed a CPP for the environment paired with paced mating behavior. In our laboratory, sexually experienced Long-Evans rats typically deliver ∼ 6 intromissions in the first ejaculatory series (Clark, unpublished) compared with the 8–15 intromissions characteristic of an ejaculatory series in males used in Paredes and Vazquez (1999). Based on these observations we considered two experimental approaches to ensure that female rats received 15 intromissions during the conditioning sessions. First, an individual male could deliver all 15 intromissions sequentially;
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in this approach the male and female would remain in the mating arena following an ejaculation until the male eventually resumed mating and completed the 15 intromissions. Under paced mating conditions, the female rat would have the opportunity to withdraw to a separate compartment at any time during the test, whereas under nonpaced mating conditions the male and female rat would remain together in a single arena throughout the test. The second approach would stipulate that following ejaculation the male would be removed and a new male introduced (Bermant, 1961); this could be repeated until the female received 15 intromissions. In Experiment 1, using the first approach, we demonstrated that a CPP was induced in female rats when the reinforcing stimulus was composed of 15 intromissions received from a single male rat under mating conditions in which either the female can escape, paced mating, or cannot escape, nonpaced mating. Because the induction of a CPP to nonpaced mating was unexpected, Experiment 2 was conducted to test directly the role of using the same male or different males to deliver the 15 nonpaced intromissions. The results of Experiment 2 showed that nonpaced mating induced a CPP when the 15 intromissions (including ejaculations) were received from the same male, but not when the male was replaced following an ejaculation, thereby abbreviating the length of the post-ejaculatory interval for the female. Materials and methods Subjects Female Long-Evans rats (n = 58) weighing approximately 200 g were obtained from Harlan (Indianapolis, IN). Rats were housed individually in a hanging metal cage in a light (12:12, lights off at 1000 h)- and temperaturecontrolled vivarium. Commercial rat pellets and water were available ad libitum. Rats were ovariectomized under sodium pentobarbital anesthesia (50 mg/kg, Nembutal; Henry Schein, Indianapolis, IN) 7–10 days before the first mating test. At the start of the experiment none of the experimental rats had been tested previously for sexual behavior. Experimental rats received 10 μg estradiol benzoate (Sigma, St. Louis, MO) 48 h and 1 mg progesterone (Sigma) 4 h, prior to each mating test. Hormones were administered s.c. in a sesame oil vehicle. Sexually experienced male Long-Evans rats, aged 3–4 months, were used as stimulus rats. The Institutional Animal Care and Use Committee at Dartmouth College approved the use of rats in these studies and all procedures were conducted in accordance with NIH guidelines.
Experiment 1: paced or nonpaced mating with the same male Place preference Place preference conditioning and tests took place in an apparatus with three distinct compartments (Med Associates, St. Albans, VT). The middle compartment (12 × 21 × 21 cm high) was gray with a smooth Plexiglas floor and was connected to the two outer compartments (28 × 21 × 21 cm high) by manually controlled sliding guillotine doors. One side compartment was illuminated, white and had a metal bar floor. Fresh aspen bedding was placed in the waste pan beneath the bar floor before each conditioning session. The other side compartment was black, with metal grid flooring and had a 2% glacial acetic acid solution swabbed along the top of the box. The waste pan under the black compartment was empty. In each compartment, photobeams spaced at 5cm intervals along the bottom recorded the time spent in each compartment using MED-PC software. Place preference conditioning and tests were conducted in a sound-attenuated room with dim red lighting overhead. Following the CPP procedures outlined by Paredes and Alonso (1997) each rat was tested for an initial place preference during a 10-min pretest. Rats were placed in the middle compartment of the place preference apparatus and allowed
to freely explore the entire apparatus; the compartment in which the rat spent the majority of time was designated the preferred compartment. Assignments to the Paced Mating, Nonpaced Mating and No Mating groups were made by matching on the initial place preference, defined as time spent in each compartment. Each experimental rat underwent a total of 10 conditioning sessions: five reinforced and five nonreinforced sessions. Following the methods of Paredes and Alonso (1997), all rats were hormone primed on reinforced conditioning sessions; the rats did not receive hormone priming before the pretest or test. For each rat, the reinforcing stimulus (Paced Mating, Nonpaced Mating or No Mating; n = 10 per group) was paired with the nonpreferred compartment. The nonreinforcing stimulus was placement into the preferred compartment directly from the homecage (Paced and Nonpaced Mating) or following saline injection (No Mating). A conditioning session began when the experimental rat was placed in the appropriate compartment of the place preference apparatus following a mating test (reinforced sessions) or following transport from the homecage (nonreinforced sessions). The No Mating group received saline injections 1 min before being placed in a compartment for both reinforced and nonreinforced sessions following the procedures described for the control group in Paredes and Alonso (1997). Conditioning sessions lasted 30 min and were regularly spaced (Table 1). The walls and waste pans were wiped with distilled water after each conditioning session. Twenty-four hours after the final conditioning session the experimental rats were given a second place preference test following the same procedures outlined above for the pretest.
Mating tests Mating tests (paced and nonpaced) were conducted in the same room as the place preference apparatus under dim red illumination. Paced mating was observed in clear Plexiglas arenas (75 × 37.5 × 32 cm high) with aspen bedding covering the floor. The arena was divided into two equally sized compartments using a clear partition (36.5 × 31.7 cm) with a 5.0-cm hole in each bottom corner. The partition allowed the experimental female rats to approach and withdraw from the male rat that could not cross the divider. Nonpaced mating tests were conducted in clear Plexiglas arenas (39.4 × 22.9 × 31.1 cm high) with aspen bedding covering the floor; the absence of a divider meant that experimental females could not elude the male during the test. Rats were acclimated to the paced mating arena on two separate occasions (15 min each) prior to conditioning. During acclimation, the experimental rats were permitted to explore the entire extent of the arena whereas the stimulus rats were placed in one compartment and tapped gently on the nose if they tried to exit the compartment (Clark et al., 2004). The parameters of the mating tests were modeled after those described by Paredes and Alonso (1997). Briefly, the experimental rat was placed into one compartment of the testing arena, the stimulus male was placed in the other, and both rats were allowed to habituate for 5 min. Paced mating tests began with the removal of an opaque Plexiglas partition (36.5 × 31.7 cm) and continued until the experimental female received 15 intromissions from the same male, including ejaculations (Paredes and Vazquez, 1999). Rats tested for nonpaced mating were placed in their respective mating arena alone for 5 min. During this period the stimulus male rat was permitted one intromission with a hormone-primed stimulus female in a separate arena. The nonpaced mating test began when the male rat was placed into the arena with the
Table 1 Experimental timeline Experimental day
Hormone treatment Session
1
2
None Pretest
EB
3
4a
...17
Nonreinforced conditioning session 1
P Reinforced conditioning session 1
None Test
a Hormone treatment, mating tests and conditioning were repeated five times, for a total of 10 conditioning sessions. Twenty-four hours after the 5th reinforced conditioning session, rats were again given a 10-min place preference test. EB = 10 μg/rat estradiol benzoate; P = 1 mg/rat progesterone.
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experimental rat and concluded when the female rat received 15 intromissions from the same male, including ejaculations (Paredes and Vazquez, 1999). In either mating group, if an ejaculation occurred before the 15th intromission, then the male rat remained in the arena with the female until he resumed mating. At the conclusion of a mating test, the female rat was gently transferred to the appropriate compartment of the nearby place preference apparatus for the 30min conditioning session. Number and timing of mounts, intromissions, and ejaculations as well as lordosis responses were recorded (Hardy and DeBold, 1971). The following measures were calculated: a lordosis quotient, the contact-return latencies and percentage of exits in response to each type of mating stimulation, the interintromission interval, defined as the mean length of time between intromissions, not including ejaculations, the post-ejaculatory interval, defined as the mean time elapsed after an ejaculation and before the next intromission, and the time with the male, operationally defined as the time spent in the male compartment during paced mating or the test duration during nonpaced mating (because the female could not withdraw from the male) (Coopersmith et al., 1996; Guarraci et al., 2004; Meisel and Sachs, 1994). The frequencies of proceptive (hops, darts and ear wiggling) and rejection (kicks and defensive postures) behaviors (Madlafousek and Hlinak, 1977) were also noted. Because the rats were fully receptive on all tests and the expected intensity-related response to mating stimulations was observed for both contact-return latencies and percentage of exits, no further mention is made of these data.
Experiment 2: nonpaced mating with the same or changed male Experiment 2 was identical to Experiment 1 except that the female rats were assigned to receive, as the reinforcing stimulus, 15 nonpaced intromissions with the same male (Same Male, n = 10), 15 nonpaced intromissions with a new male introduced as soon as possible after an ejaculation (approximately 15 s) (Changed Male, n = 9), or No Mating (n = 9). The groups were matched on initial place preference as in Experiment 1. Rats in the No Mating group received saline injections before reinforced and nonreinforced conditioning sessions.
Data analysis To assess whether the induction of a CPP varied as a function of mating group we calculated a preference score, defined as time in reinforced compartment / (time in reinforced compartment + time in nonreinforced compartment), and a difference score, defined as the time in the nonreinforced compartment − time in reinforced compartment (Paredes and Alonso, 1997). Paired t-tests were used to evaluate the change in preference score and difference score from the pretest to the test (Dominguez-Salazar et al., 2005; Kohlert and Olexa, 2005; Meisel and Joppa, 1994). A multivariate analysis of variance (MANOVA) with repeated measures was used to assess the effects of group (Paced Mating versus Nonpaced Mating; Same Male versus Changed Male) across conditioning session on three dependent measures: the mean interintromission interval, the post-ejaculatory interval, and time with the male; these measures were examined because of their potential role in the induction of CPP (Jenkins and Becker, 2003; Kohlert and Olexa, 2005; Meisel and Joppa, 1994). A significant overall MANOVA was followed up with univariate ANOVAs. The alpha level was set at P < 0.05.
Results Experiment 1: paced or nonpaced mating with the same male Female rats that received paced or nonpaced mating exhibited a CPP (Fig. 1). From pretest to test, both the Paced and Nonpaced Mating groups showed a significant increase in the preference score (t(10) = 3.5, and t(10) = 2.7, both P < 0.05) in tandem with a significant decrease in the difference score (t(10) = 3.9, and t(10) = 2.7, both P < 0.05). There were no changes in the preference and difference scores of the No Mating group from pretest to test.
Fig. 1. Mean ± SEM preference score (top) and difference score (bottom) on pretest (white bars) and test (black bars) are shown for female rats that received paced, nonpaced or no mating behavior as the reinforcing stimulus. N = 10 rats per group. Asterisks indicate that groups receiving either paced or nonpaced mating behavior displayed a CPP for the compartment associated with mating: a significant increase in preference score coincident with a significant decrease in difference score, P < 0.05.
Mating tests ended following the receipt of the 15th intromission. Table 2 illustrates that rats in the Paced Mating group received intromissions at a slower rate, had longer post-ejaculatory intervals and spent less time with the male during the mating test relative to the Nonpaced Mating group. The overall MANOVA revealed a significant effect of group (F(3,15) = 17.6) and a significant effect of conditioning session (F(12,6) = 7.3) in the absence of a conditioning session × group interaction (Table 2). A univariate ANOVA revealed that the effect of conditioning session was not significant for any individual dependent measure. Although females in both mating groups received equivalent numbers of intromissions, significantly more ejaculations were received by the Paced than Nonpaced Mating group (F(1,18) = 12.9, data not shown), consistent with other published studies (Becker et al., 2001; Erskine, 1989). Experiment 2: nonpaced mating with the same or changed male Under nonpaced mating conditions, only those females in the Same Male group demonstrated a CPP for the context associated with mating (Fig. 2). In the Same Male group the preference score increased significantly from pretest to test (t (10) = 3.4), partnered with a significant reduction in difference
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Table 2 Summary of mating data from conditioning sessions
Experiment 1 Paced mating behavior
Nonpaced mating behavior
Experiment 2 Same male
Changed male
Conditioning session
Interintromission interval (seconds)
Post-ejaculatory interval (seconds)
Time with male (seconds)
1 2 3 4 5 Mean 1 2 3 4 5 Mean
132.3 ± 40.5 59.6 ± 14.0 55.5 ± 10.4 64.9 ± 14.2 53.7 ± 12.4 73.2 ± 11.6 ⁎ 38.6 ± 6.8 41.7 ± 9.4 41.0 ± 9.2 37.1 ± 7.9 36.8 ± 14.2 39.1 ± 5.1
543.3 ± 49.2 478.1 ± 65.8 576.0 ± 38.6 624.0 ± 69.5 475.5 ± 25.8 539.4 ± 26.9 ⁎ 477.9 ± 20.5 452.3 ± 34.0 522.1 ± 54.6 447.9 ± 25.1 421.4 ± 37.8 464.3 ± 16.1
895.6 ± 134.4 695.9 ± 107.1 925.0 ± 168.7 884.1 ± 140.1 760.4 ± 136.9 832.2 ± 89.3 ⁎ 1360.9 ± 178.5 1421.3 ± 273.9 1530.4 ± 276.4 1066.7 ± 155.3 1387.3 ± 333.5 1353.3 ± 120.9
1 2 3 4 5 Mean 1 2 3 4 5 Mean
34.3 ± 4.6 41.2 ± 7.4 36.1 ± 8.5 24.3 ± 3.8 28.9 ± 3.8 32.9 ± 2.4 45.9 ± 5.3 35.3 ± 8.6 28.7 ± 4.5 33.1 ± 11.0 16.8 ± 1.4 32.0 ± 4.3
480.9 ± 37.6 459.8 ± 44.9 444.1 ± 41.3 401.4 ± 45.1 455.3 ± 43.5 448.3 ± 20.0 ⁎⁎ 105.1 ± 22.4 85.1 ± 25.8 34.8 ± 4.6 57.6 ± 21.8 22.8 ± 1.8 61.1 ± 7.0
1359.6 ± 106.5 1243.2 ± 152.7 1241.9 ± 211.4 931.3 ± 180.1 1221.7 ± 199.0 1199.5 ± ± 63.6 ⁎⁎ 770.8 ± 98.5 617.1 ± 127.1 407.8 ± 64.9 519.2 ± 159.2 250.1 ± 18.2 513.0 ± 62.5
Means ± SEM are shown. ⁎ P < 0.05 vs. nonpaced mating. ⁎⁎ P < 0.05 vs. changed male.
score (t(10) = 3.5, both P < 0.05). Preference and difference scores did not differ from pretest to test for the Changed Male or the No Mating groups. In Experiment 2, mating tests also concluded when the female had received 15 intromissions. The overall MANOVA revealed a significant effect of group (F(3,14) = 113.3), with no effect of conditioning session or a conditioning session × group interaction (Table 2). Although there were no group differences in the interintromission interval, the introduction of a new (Changed) male significantly reduced the interval between the female's receipt of an ejaculation from the original male and the first post-ejaculatory intromission delivered by the changed male. In addition, and related to the abbreviated postejaculatory interval, the time spent with the male was significantly shorter in the Changed Male than in the Same Male group. Discussion The results of the present study demonstrate that both paced and nonpaced mating behaviors induced a CPP in female rats. It is noteworthy that nonpaced mating behavior did not induce a CPP when the same male did not deliver the 15 intromissions. Our results suggest that introducing a new male and abbreviating the post-ejaculatory period may interfere with the reinforcing effects of mating in female rats.
The observation that female rats display a CPP for a context associated with nonpaced mating behavior prompts reconsideration of the reinforcing aspects of sexual behavior for female rats. The specific testing procedures employed may allow us to better examine the reinforcing elements of mating for female rats. Most recent studies have not observed a CPP to nonpaced mating (Dominguez-Salazar et al., 2005; Gans and Erskine, 2003; Jenkins and Becker, 2003; Martinez and Paredes, 2001; Paredes and Alonso, 1997; Paredes and Vazquez, 1999; cf., Oldenburger et al., 1992), leading researchers to speculate about what makes paced mating, and not nonpaced mating, reinforcing. It has been proposed that female rats display a CPP for mating only when they can control the timing of sexual contacts during a mating bout (Martinez and Paredes, 2001; Paredes and Vazquez, 1999). This view is challenged by the observation that female rats exhibit a CPP for a context paired with mating at their preferred interval over a context paired with nonpaced mating behavior even when females do not actively pace sexual contact (Jenkins and Becker, 2003). Data from the present study showing that female rats developed a CPP for mating that occurs at the male rate (nonpaced mating), further support the notion that control of the sexual encounter per se is not necessary to induce a CPP for mating. The outcomes of the present study may also allow us to rule out two other previously hypothesized bases for the differential reinforcing effects of paced and nonpaced mating. First, the rate
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Fig. 2. Mean ± SEM preference score (top) and difference score (bottom) on pretest (white bars) and test (black bars) are shown for female rats that received nonpaced mating with the same male, nonpaced mating with the male changed following an ejaculation, or no mating behavior as the reinforcing stimulus. N = 9–10 rats per group. The asterisk indicates that only female rats in the Same Male group developed a CPP for the compartment paired with mating: a significant increase in preference score coincident with a significant decrease in difference score, P < 0.05.
of sexual contacts has been posited as an essential component of the reinforcing effect of mating in females (Jenkins and Becker, 2003). Specifically, the longer interintromission interval associated with paced compared to nonpaced mating may be important (Jenkins and Becker, 2003). Because in the present study female rats expressed a CPP for a context associated with longer (∼ 70 s) and shorter (∼ 40 s) interintromission intervals, it appears that the interval between intromissions, within the timeframe in the present study, is not critical for induction of a CPP. Second, intromissions delivered in a paced mating paradigm may be of a longer duration than intromissions delivered in nonpaced mating (Bermant, 1964; Erskine et al., 1989). Others have suggested that the increased intromission duration, and resultant heightened cervical stimulation, may contribute to the enhanced reinforcing effects of paced mating (Erskine et al., 1989; Martinez and Paredes, 2001; Paredes and Alonso, 1997). It is unlikely that the potential disparity in cervical stimulation in the two mating formats contributed to the reinforcing effects of mating given that both paced and nonpaced mating induced a CPP in the present study. In the present study the variable that was manipulated to influence the reinforcing effects of mating was the continued presence of the same male throughout the post-ejaculatory period until he resumed mating, or changing the male following ejaculation and introducing a new male until the female
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received 15 intromissions. The Same versus the Changed Male groups differed on at least three dimensions: the number of partners per mating test, time with the male and the length of the post-ejaculatory interval. The number of partners per test was significantly greater for the Changed Male than the Same Male group (2.5 versus 1). Although it is possible that female rats find mating reinforcing only when paired with a single mating partner (Same Male) as opposed to multiple partners (Changed Male), it seems unlikely that access to multiple partners during a mating encounter interferes with the reinforcing effects of mating, because female rats readily mate with multiple partners when given the opportunity (McClintock and Adler, 1978; McClintock et al., 1982). Kippin and Pfaus (2001) raised the issue that discrepancies in social contact resulting from the copulatory criteria may contribute to differences in observed conditioned partner preference in male rats. Whereas in Experiment 1 time spent with the male was significantly less in the Paced Mating group than in the Nonpaced Mating group and both groups developed a CPP, in Experiment 2 time spent with the male was significantly less in the Changed Male group than in the Same Male group and only the Same Male group showed a CPP. Social stimuli can induce a CPP in female rodents; female hamsters prefer a context associated with a male hamster even when a vaginal mask prevents receipt of vaginal stimulation (Kohlert and Olexa, 2005) or when the interaction is aggressive in nature (Meisel and Joppa, 1994). The additional social contact afforded the Same Male group may have facilitated the development of a CPP. In addition, it remains to be tested whether a minimum amount (threshold) of contact with stimulus animals is required for a mating-induced CPP. We hypothesize that a key factor in determining whether a mating encounter induces a CPP in female rats may be the conditions accompanying the post-ejaculatory period. Specifically, we propose that mating is reinforcing in female rats when a sufficient period of time elapses between the receipt of an ejaculation and the first post-ejaculatory intromission. If an uninterrupted post-ejaculatory interval is reinforcing to female rats, then preserving this interval may enhance reproductive success. Sperm transport occurs during the 6–10 min following ejaculation and can be reduced by the receipt of an intromission too soon after an ejaculation (Matthews and Adler, 1977). Thus, pregnancy/pseudopregnancy is more likely when the sperm plug is undisturbed following ejaculation. Rats in the present study received sufficient stimulation (15 intromission) to initiate the neuroendocrine response required for pregnancy/pseudopregnancy (Erskine et al., 2004; Erskine, 1989), therefore the reinforcing effects of preserving the post-ejaculatory interval may confer a reproductive advantage. Potential procedural differences between the present study and those that do not find a CPP to nonpaced mating behavior may account for disparate outcomes (Dominguez-Salazar et al., 2005; Gans and Erskine, 2003; Jenkins and Becker, 2003; Martinez and Paredes, 2001; Paredes and Alonso, 1997; Paredes and Vazquez, 1999). As stated earlier, in our laboratory male rats typically deliver ∼ 6 intromissions in the first ejaculatory series. Supposing that in other laboratories male
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rats exhibit similar mating patterns, then either a single male could remain in the test chamber following ejaculation and ultimately deliver all 15 stimulations or the male could be replaced following ejaculation. Data from the present study would lead us to predict that if the male is changed following ejaculations, then nonpaced mating would not induce a CPP. Unfortunately because the use of a single or multiple males to deliver the requisite number of intromissions is not specified, it is not possible to ascertain the role of differences in methodology to the study outcomes. In some nonpaced mating experiments, the female rat is transferred to the CPP apparatus immediately following ejaculation and thus the issue of the Same/Changed male following ejaculation is moot (Martinez and Paredes, 2001; Paredes and Alonso, 1997; Paredes and Vazquez, 1999). It is not clear why nonpaced mating failed to induce a CPP under these conditions. As mentioned above, the role of social contact in CPP is not clear, and it is possible that the additional time spent with a sexually refractory male (in the Same Male group) may enhance the reinforcing aspects of mating. Along these same lines, Kippin and Pfaus (2001) and Pfaus et al. (2001) report that male rats develop a conditioned partner preference for a female that is present during the period of time immediately following an ejaculation; a mating-induced partner preference does not develop when the female is removed during the post-ejaculatory interval. Moreover, males develop a preference for a female present only during the post-ejaculatory interval. An experiment in which the male is removed following ejaculation and then reintroduced after an average post-ejaculatory interval would address this issue in female rats. The results of the present study demonstrated that female rats developed a CPP for nonpaced mating behavior except when a new male was introduced after the female received an ejaculation. By delineating conditions that interfere with a mating-induced CPP, our findings extend our understanding of what makes mating reinforcing to female rats. Acknowledgments We thank Dr. William A. Carlezon for his advice on these studies and Drs. Joel Brown and David Bucci for their comments on the manuscript. We also thank Adam Christensen, Katherine Lang, Catherine Marantz and Kevin Wolfson for the technical assistance. This work was supported by DA08574 to A.C. References Bardo, M.T, Bevins, R.A., 2000. Conditioned place preference: what does it add to our preclinical understanding of drug reward? Psychopharmacology 153, 31–43. Becker, J.B., Rudick, C.N., Jenkins, W.J., 2001. The role of dopamine in the nucleus accumbens and striatum during sexual behavior in the female rat. J. Neurosci. 21, 3236–3241. Bermant, G., 1961. Response latencies of female rats during sexual intercourse. Science 133, 1771–1773. Bermant, G., 1964. Effects of single and multiple enforced intercopulatory intervals on the sexual behavior of male rats. J. Comp. Physiol. Psychol. 57, 398–403.
Carlezon, W.A., 2003. Place conditioning to study drug reward and aversion. Methods Mol. Med. 84, 243–249. Clark, A.S., Kelton, M.C., Guarraci, F.A., Clyons, E.Q., 2004. Hormonal status and test condition, but not sexual experience, modulate partner preference in female rats. Horm. Behav. 45, 314–323. Coopersmith, C., Erskine, M.S., 1994. Influence of paced mating and number of intromissions on fertility in the laboratory rat. J. Reprod. Fertil. 102, 451–458. Coopersmith, C., Candurra, C., Erskine, M.S., 1996. Effects of paced mating and intromissive stimulation on feminine sexual behavior and estrus termination in the cycling rat. J. Comp. Psychol. 110, 176–186. Dominguez-Salazar, E., Camacho, F.J., Paredes, R.G., 2005. Prenatal blockade of androgen receptors reduces the number of intromissions needed to induce conditioned place preference after paced mating in female rats. Pharmacol. Biochem. Behav. 81, 871–878. Erskine, M.S., 1989. Solicitation behavior in the estrous female rat: a review. Horm. Behav. 23, 473–502. Erskine, M.S., Kornberg, E., 1992. Acute luteinizing hormone and prolactin responses to paced mating stimulation in the estrous female rat. J. Neuroendocrinol. 4, 173–179. Erskine, M.S., Kornberg, E., Cherry, J.A., 1989. Paced copulation in rats: effects of intromission frequency and duration on luteal activation and estrus length. Physiol. Behav. 45, 33–39. Erskine, M.S., Lehmann, M.L., Cameron, N.M., Polston, E.K., 2004. Coregulation of female sexual behavior and pregnancy induction: an exploratory synthesis. Behav. Brain Res. 153, 295–315. Gans, S., Erskine, M.S., 2003. Effects of neonatal testosterone treatment on pacing behaviors and development of a conditioned place preference. Horm. Behav. 44, 354–364. Guarraci, F.A., Megroz, A.B., Clark, A.S., 2004. Paced mating behavior in the female rat following lesions of three regions responsive to vaginocervical stimulation. Brain Res. 999, 40–52. Hardy, D.F., DeBold, J.F., 1971. Effects of mounts without intromissions upon the behavior of female rats during the onset of estrogen-induced heat. Physiol. Behav. 7, 643–645. Jenkins, W.J., Becker, J.B., 2003. Female rats develop conditioned place preferences for sex at their preferred interval. Horm. Behav. 43, 503–507. Kippin, T.E., Pfaus, J.G., 2001. The development of olfactory conditioned ejaculatory preferences in the male rat: I. Nature of the unconditioned stimulus. Physiol. Behav. 73, 457–469. Kohlert, J.G., Olexa, N., 2005. The role of vaginal stimulation for the acquisition of conditioned place preference in female Syrian hamsters. Physiol. Behav. 84, 135–139. Madlafousek, J., Hlinak, Z., 1977. Sexual behavior of the female laboratory rat: inventory, patterning, and measurement. Behavior 63, 129–174. Martinez, I., Paredes, R.G., 2001. Only self-paced mating is rewarding in rats of both sexes. Horm. Behav. 40, 510–517. Matthews, M., Adler, N.T., 1977. Facilitative and inhibitory influences of reproductive behavior on sperm transport in rats. J. Comp. Physiol. Psychol. 91, 727–741. McClintock, M.K., Adler, N.T., 1978. The role of the female during copulation in wild and domestic Norway rats (Rattus norvegicus). Behaviour 67, 67–96. McClintock, M.K., Anisko, J.J., Adler, N.T., 1982. Group mating among Norway rats: II. The social dynamics of copulation: competition, cooperation and mate choice. Anim. Behav. 30, 410–425. Meisel, R.L., Joppa, M.A., 1994. Conditioned place preference in female hamsters following aggressive or sexual encounters. Physiol. Behav. 56, 1115–1118. Meisel, R.L., Sachs, B.D., 1994. The physiology of male sexual behavior, In: Knobil, E., Neill, J.D. (Eds.), The Physiology of Reproduction, 2nd ed. Raven Press Ltd, New York, NY, pp. 3–105. Oldenburger, W.P., Everitt, B.J., de Jonge, F.H., 1992. Conditioned place preference induced by sexual interaction in female rats. Horm. Behav. 26, 214–228. Paredes, R.G., Alonso, A., 1997. Sexual behavior regulated (paced) by the female induces conditioned place preference. Behav. Neurosci. 111, 123–128. Paredes, R.G., Vazquez, B., 1999. What do female rats like about sex? Paced mating. Behav. Brain. Res. 105, 117–127. Pfaus, J.G., Kippin, T.E., Centeno, S., 2001. Conditioning and sexual behavior: a review. Horm. Behav. 40, 291–321.
Female rats exhibit a conditioned place preference for nonpaced mating Sarah H. Meerts, Ann S. Clark ⁎ Department of Psychological and Brain Sciences, Dartmouth College, Hanover, NH 03755, USA Received 26 July 2006; revised 29 August 2006; accepted 29 August 2006 Available online 3 October 2006
Abstract Paced, but not nonpaced, mating behavior is reported to induce a conditioned place preference (CPP) in female rats. Contrary to these previous findings, Experiment 1 showed that female rats that received 15 intromissions from a single male rat during each of five conditioning sessions exhibited a CPP for the compartment associated with mating when the intromissions were delivered via a paced or nonpaced paradigm. Experiment 2 demonstrated that nonpaced mating induced a CPP when a single male delivered the 15 intromissions but not when the male was replaced following ejaculation and a new male allowed to complete the requisite number of intromissions. These findings invite reevaluation of the reinforcing aspects of mating behavior in female rats. © 2006 Elsevier Inc. All rights reserved. Keywords: Paced mating behavior; Intromission; Post-ejaculatory interval; Sexual behavior
Paced mating behavior is the pattern of approach and withdrawal exhibited by a female rat during a sexual encounter with a male rat observed in a semi-natural environment (McClintock and Adler, 1978) or in a testing arena that allows the female to escape (for review, Erskine, 1989). Nonpaced mating has been contrasted with paced mating; in nonpaced mating tests the male controls the tempo of sexual interactions because the female rat cannot escape (Erskine, 1989). Stimuli received during paced and nonpaced mating may elicit different neuroendocrine and physiological responses in female rats. Circulating levels of luteinizing hormone are elevated in female rats mated under paced compared to nonpaced mating conditions (Erskine and Kornberg, 1992). Paced mating behavior also enhances reproductive success; a greater percentage of rats become pregnant or pseudopregnant and have larger litters when they receive paced rather than nonpaced mating stimulation (Coopersmith and Erskine, 1994; Erskine, 1989). Conditioned place preference (CPP) is a form of classical conditioning used to assess the reinforcing properties of a given stimulus (Carlezon, 2003). Typically, animals express a preference for a distinct environment associated with adminis⁎ Corresponding author. Fax: +1 603 646 1419. E-mail address: [email protected] (A.S. Clark). 0018-506X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2006.08.007
tration of a drug or behavior that is reinforcing (Bardo and Bevins, 2000; Carlezon, 2003). Several researchers have demonstrated that female rats exhibit a CPP for a context associated with paced, but not nonpaced, mating (Gans and Erskine, 2003; Jenkins and Becker, 2003; Paredes and Alonso, 1997; cf., Oldenburger et al., 1992). The reinforcing effects of paced mating have been linked with the female's role in controlling the timing of mating stimulations, the slower rate of intromissions and the increased intensity of cervical stimulation (Jenkins and Becker, 2003; Martinez and Paredes, 2001; Paredes and Alonso, 1997; Paredes and Vazquez, 1999). We recently conducted a study examining the reinforcing aspects of mating and designed to follow the procedures outlined originally by Paredes and Alonso (1997) and updated by Paredes and Vazquez (1999). These authors reported that female rats that received 10–15 paced intromissions expressed a CPP for the environment paired with paced mating behavior. In our laboratory, sexually experienced Long-Evans rats typically deliver ∼ 6 intromissions in the first ejaculatory series (Clark, unpublished) compared with the 8–15 intromissions characteristic of an ejaculatory series in males used in Paredes and Vazquez (1999). Based on these observations we considered two experimental approaches to ensure that female rats received 15 intromissions during the conditioning sessions. First, an individual male could deliver all 15 intromissions sequentially;
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in this approach the male and female would remain in the mating arena following an ejaculation until the male eventually resumed mating and completed the 15 intromissions. Under paced mating conditions, the female rat would have the opportunity to withdraw to a separate compartment at any time during the test, whereas under nonpaced mating conditions the male and female rat would remain together in a single arena throughout the test. The second approach would stipulate that following ejaculation the male would be removed and a new male introduced (Bermant, 1961); this could be repeated until the female received 15 intromissions. In Experiment 1, using the first approach, we demonstrated that a CPP was induced in female rats when the reinforcing stimulus was composed of 15 intromissions received from a single male rat under mating conditions in which either the female can escape, paced mating, or cannot escape, nonpaced mating. Because the induction of a CPP to nonpaced mating was unexpected, Experiment 2 was conducted to test directly the role of using the same male or different males to deliver the 15 nonpaced intromissions. The results of Experiment 2 showed that nonpaced mating induced a CPP when the 15 intromissions (including ejaculations) were received from the same male, but not when the male was replaced following an ejaculation, thereby abbreviating the length of the post-ejaculatory interval for the female. Materials and methods Subjects Female Long-Evans rats (n = 58) weighing approximately 200 g were obtained from Harlan (Indianapolis, IN). Rats were housed individually in a hanging metal cage in a light (12:12, lights off at 1000 h)- and temperaturecontrolled vivarium. Commercial rat pellets and water were available ad libitum. Rats were ovariectomized under sodium pentobarbital anesthesia (50 mg/kg, Nembutal; Henry Schein, Indianapolis, IN) 7–10 days before the first mating test. At the start of the experiment none of the experimental rats had been tested previously for sexual behavior. Experimental rats received 10 μg estradiol benzoate (Sigma, St. Louis, MO) 48 h and 1 mg progesterone (Sigma) 4 h, prior to each mating test. Hormones were administered s.c. in a sesame oil vehicle. Sexually experienced male Long-Evans rats, aged 3–4 months, were used as stimulus rats. The Institutional Animal Care and Use Committee at Dartmouth College approved the use of rats in these studies and all procedures were conducted in accordance with NIH guidelines.
Experiment 1: paced or nonpaced mating with the same male Place preference Place preference conditioning and tests took place in an apparatus with three distinct compartments (Med Associates, St. Albans, VT). The middle compartment (12 × 21 × 21 cm high) was gray with a smooth Plexiglas floor and was connected to the two outer compartments (28 × 21 × 21 cm high) by manually controlled sliding guillotine doors. One side compartment was illuminated, white and had a metal bar floor. Fresh aspen bedding was placed in the waste pan beneath the bar floor before each conditioning session. The other side compartment was black, with metal grid flooring and had a 2% glacial acetic acid solution swabbed along the top of the box. The waste pan under the black compartment was empty. In each compartment, photobeams spaced at 5cm intervals along the bottom recorded the time spent in each compartment using MED-PC software. Place preference conditioning and tests were conducted in a sound-attenuated room with dim red lighting overhead. Following the CPP procedures outlined by Paredes and Alonso (1997) each rat was tested for an initial place preference during a 10-min pretest. Rats were placed in the middle compartment of the place preference apparatus and allowed
to freely explore the entire apparatus; the compartment in which the rat spent the majority of time was designated the preferred compartment. Assignments to the Paced Mating, Nonpaced Mating and No Mating groups were made by matching on the initial place preference, defined as time spent in each compartment. Each experimental rat underwent a total of 10 conditioning sessions: five reinforced and five nonreinforced sessions. Following the methods of Paredes and Alonso (1997), all rats were hormone primed on reinforced conditioning sessions; the rats did not receive hormone priming before the pretest or test. For each rat, the reinforcing stimulus (Paced Mating, Nonpaced Mating or No Mating; n = 10 per group) was paired with the nonpreferred compartment. The nonreinforcing stimulus was placement into the preferred compartment directly from the homecage (Paced and Nonpaced Mating) or following saline injection (No Mating). A conditioning session began when the experimental rat was placed in the appropriate compartment of the place preference apparatus following a mating test (reinforced sessions) or following transport from the homecage (nonreinforced sessions). The No Mating group received saline injections 1 min before being placed in a compartment for both reinforced and nonreinforced sessions following the procedures described for the control group in Paredes and Alonso (1997). Conditioning sessions lasted 30 min and were regularly spaced (Table 1). The walls and waste pans were wiped with distilled water after each conditioning session. Twenty-four hours after the final conditioning session the experimental rats were given a second place preference test following the same procedures outlined above for the pretest.
Mating tests Mating tests (paced and nonpaced) were conducted in the same room as the place preference apparatus under dim red illumination. Paced mating was observed in clear Plexiglas arenas (75 × 37.5 × 32 cm high) with aspen bedding covering the floor. The arena was divided into two equally sized compartments using a clear partition (36.5 × 31.7 cm) with a 5.0-cm hole in each bottom corner. The partition allowed the experimental female rats to approach and withdraw from the male rat that could not cross the divider. Nonpaced mating tests were conducted in clear Plexiglas arenas (39.4 × 22.9 × 31.1 cm high) with aspen bedding covering the floor; the absence of a divider meant that experimental females could not elude the male during the test. Rats were acclimated to the paced mating arena on two separate occasions (15 min each) prior to conditioning. During acclimation, the experimental rats were permitted to explore the entire extent of the arena whereas the stimulus rats were placed in one compartment and tapped gently on the nose if they tried to exit the compartment (Clark et al., 2004). The parameters of the mating tests were modeled after those described by Paredes and Alonso (1997). Briefly, the experimental rat was placed into one compartment of the testing arena, the stimulus male was placed in the other, and both rats were allowed to habituate for 5 min. Paced mating tests began with the removal of an opaque Plexiglas partition (36.5 × 31.7 cm) and continued until the experimental female received 15 intromissions from the same male, including ejaculations (Paredes and Vazquez, 1999). Rats tested for nonpaced mating were placed in their respective mating arena alone for 5 min. During this period the stimulus male rat was permitted one intromission with a hormone-primed stimulus female in a separate arena. The nonpaced mating test began when the male rat was placed into the arena with the
Table 1 Experimental timeline Experimental day
Hormone treatment Session
1
2
None Pretest
EB
3
4a
...17
Nonreinforced conditioning session 1
P Reinforced conditioning session 1
None Test
a Hormone treatment, mating tests and conditioning were repeated five times, for a total of 10 conditioning sessions. Twenty-four hours after the 5th reinforced conditioning session, rats were again given a 10-min place preference test. EB = 10 μg/rat estradiol benzoate; P = 1 mg/rat progesterone.
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experimental rat and concluded when the female rat received 15 intromissions from the same male, including ejaculations (Paredes and Vazquez, 1999). In either mating group, if an ejaculation occurred before the 15th intromission, then the male rat remained in the arena with the female until he resumed mating. At the conclusion of a mating test, the female rat was gently transferred to the appropriate compartment of the nearby place preference apparatus for the 30min conditioning session. Number and timing of mounts, intromissions, and ejaculations as well as lordosis responses were recorded (Hardy and DeBold, 1971). The following measures were calculated: a lordosis quotient, the contact-return latencies and percentage of exits in response to each type of mating stimulation, the interintromission interval, defined as the mean length of time between intromissions, not including ejaculations, the post-ejaculatory interval, defined as the mean time elapsed after an ejaculation and before the next intromission, and the time with the male, operationally defined as the time spent in the male compartment during paced mating or the test duration during nonpaced mating (because the female could not withdraw from the male) (Coopersmith et al., 1996; Guarraci et al., 2004; Meisel and Sachs, 1994). The frequencies of proceptive (hops, darts and ear wiggling) and rejection (kicks and defensive postures) behaviors (Madlafousek and Hlinak, 1977) were also noted. Because the rats were fully receptive on all tests and the expected intensity-related response to mating stimulations was observed for both contact-return latencies and percentage of exits, no further mention is made of these data.
Experiment 2: nonpaced mating with the same or changed male Experiment 2 was identical to Experiment 1 except that the female rats were assigned to receive, as the reinforcing stimulus, 15 nonpaced intromissions with the same male (Same Male, n = 10), 15 nonpaced intromissions with a new male introduced as soon as possible after an ejaculation (approximately 15 s) (Changed Male, n = 9), or No Mating (n = 9). The groups were matched on initial place preference as in Experiment 1. Rats in the No Mating group received saline injections before reinforced and nonreinforced conditioning sessions.
Data analysis To assess whether the induction of a CPP varied as a function of mating group we calculated a preference score, defined as time in reinforced compartment / (time in reinforced compartment + time in nonreinforced compartment), and a difference score, defined as the time in the nonreinforced compartment − time in reinforced compartment (Paredes and Alonso, 1997). Paired t-tests were used to evaluate the change in preference score and difference score from the pretest to the test (Dominguez-Salazar et al., 2005; Kohlert and Olexa, 2005; Meisel and Joppa, 1994). A multivariate analysis of variance (MANOVA) with repeated measures was used to assess the effects of group (Paced Mating versus Nonpaced Mating; Same Male versus Changed Male) across conditioning session on three dependent measures: the mean interintromission interval, the post-ejaculatory interval, and time with the male; these measures were examined because of their potential role in the induction of CPP (Jenkins and Becker, 2003; Kohlert and Olexa, 2005; Meisel and Joppa, 1994). A significant overall MANOVA was followed up with univariate ANOVAs. The alpha level was set at P < 0.05.
Results Experiment 1: paced or nonpaced mating with the same male Female rats that received paced or nonpaced mating exhibited a CPP (Fig. 1). From pretest to test, both the Paced and Nonpaced Mating groups showed a significant increase in the preference score (t(10) = 3.5, and t(10) = 2.7, both P < 0.05) in tandem with a significant decrease in the difference score (t(10) = 3.9, and t(10) = 2.7, both P < 0.05). There were no changes in the preference and difference scores of the No Mating group from pretest to test.
Fig. 1. Mean ± SEM preference score (top) and difference score (bottom) on pretest (white bars) and test (black bars) are shown for female rats that received paced, nonpaced or no mating behavior as the reinforcing stimulus. N = 10 rats per group. Asterisks indicate that groups receiving either paced or nonpaced mating behavior displayed a CPP for the compartment associated with mating: a significant increase in preference score coincident with a significant decrease in difference score, P < 0.05.
Mating tests ended following the receipt of the 15th intromission. Table 2 illustrates that rats in the Paced Mating group received intromissions at a slower rate, had longer post-ejaculatory intervals and spent less time with the male during the mating test relative to the Nonpaced Mating group. The overall MANOVA revealed a significant effect of group (F(3,15) = 17.6) and a significant effect of conditioning session (F(12,6) = 7.3) in the absence of a conditioning session × group interaction (Table 2). A univariate ANOVA revealed that the effect of conditioning session was not significant for any individual dependent measure. Although females in both mating groups received equivalent numbers of intromissions, significantly more ejaculations were received by the Paced than Nonpaced Mating group (F(1,18) = 12.9, data not shown), consistent with other published studies (Becker et al., 2001; Erskine, 1989). Experiment 2: nonpaced mating with the same or changed male Under nonpaced mating conditions, only those females in the Same Male group demonstrated a CPP for the context associated with mating (Fig. 2). In the Same Male group the preference score increased significantly from pretest to test (t (10) = 3.4), partnered with a significant reduction in difference
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Table 2 Summary of mating data from conditioning sessions
Experiment 1 Paced mating behavior
Nonpaced mating behavior
Experiment 2 Same male
Changed male
Conditioning session
Interintromission interval (seconds)
Post-ejaculatory interval (seconds)
Time with male (seconds)
1 2 3 4 5 Mean 1 2 3 4 5 Mean
132.3 ± 40.5 59.6 ± 14.0 55.5 ± 10.4 64.9 ± 14.2 53.7 ± 12.4 73.2 ± 11.6 ⁎ 38.6 ± 6.8 41.7 ± 9.4 41.0 ± 9.2 37.1 ± 7.9 36.8 ± 14.2 39.1 ± 5.1
543.3 ± 49.2 478.1 ± 65.8 576.0 ± 38.6 624.0 ± 69.5 475.5 ± 25.8 539.4 ± 26.9 ⁎ 477.9 ± 20.5 452.3 ± 34.0 522.1 ± 54.6 447.9 ± 25.1 421.4 ± 37.8 464.3 ± 16.1
895.6 ± 134.4 695.9 ± 107.1 925.0 ± 168.7 884.1 ± 140.1 760.4 ± 136.9 832.2 ± 89.3 ⁎ 1360.9 ± 178.5 1421.3 ± 273.9 1530.4 ± 276.4 1066.7 ± 155.3 1387.3 ± 333.5 1353.3 ± 120.9
1 2 3 4 5 Mean 1 2 3 4 5 Mean
34.3 ± 4.6 41.2 ± 7.4 36.1 ± 8.5 24.3 ± 3.8 28.9 ± 3.8 32.9 ± 2.4 45.9 ± 5.3 35.3 ± 8.6 28.7 ± 4.5 33.1 ± 11.0 16.8 ± 1.4 32.0 ± 4.3
480.9 ± 37.6 459.8 ± 44.9 444.1 ± 41.3 401.4 ± 45.1 455.3 ± 43.5 448.3 ± 20.0 ⁎⁎ 105.1 ± 22.4 85.1 ± 25.8 34.8 ± 4.6 57.6 ± 21.8 22.8 ± 1.8 61.1 ± 7.0
1359.6 ± 106.5 1243.2 ± 152.7 1241.9 ± 211.4 931.3 ± 180.1 1221.7 ± 199.0 1199.5 ± ± 63.6 ⁎⁎ 770.8 ± 98.5 617.1 ± 127.1 407.8 ± 64.9 519.2 ± 159.2 250.1 ± 18.2 513.0 ± 62.5
Means ± SEM are shown. ⁎ P < 0.05 vs. nonpaced mating. ⁎⁎ P < 0.05 vs. changed male.
score (t(10) = 3.5, both P < 0.05). Preference and difference scores did not differ from pretest to test for the Changed Male or the No Mating groups. In Experiment 2, mating tests also concluded when the female had received 15 intromissions. The overall MANOVA revealed a significant effect of group (F(3,14) = 113.3), with no effect of conditioning session or a conditioning session × group interaction (Table 2). Although there were no group differences in the interintromission interval, the introduction of a new (Changed) male significantly reduced the interval between the female's receipt of an ejaculation from the original male and the first post-ejaculatory intromission delivered by the changed male. In addition, and related to the abbreviated postejaculatory interval, the time spent with the male was significantly shorter in the Changed Male than in the Same Male group. Discussion The results of the present study demonstrate that both paced and nonpaced mating behaviors induced a CPP in female rats. It is noteworthy that nonpaced mating behavior did not induce a CPP when the same male did not deliver the 15 intromissions. Our results suggest that introducing a new male and abbreviating the post-ejaculatory period may interfere with the reinforcing effects of mating in female rats.
The observation that female rats display a CPP for a context associated with nonpaced mating behavior prompts reconsideration of the reinforcing aspects of sexual behavior for female rats. The specific testing procedures employed may allow us to better examine the reinforcing elements of mating for female rats. Most recent studies have not observed a CPP to nonpaced mating (Dominguez-Salazar et al., 2005; Gans and Erskine, 2003; Jenkins and Becker, 2003; Martinez and Paredes, 2001; Paredes and Alonso, 1997; Paredes and Vazquez, 1999; cf., Oldenburger et al., 1992), leading researchers to speculate about what makes paced mating, and not nonpaced mating, reinforcing. It has been proposed that female rats display a CPP for mating only when they can control the timing of sexual contacts during a mating bout (Martinez and Paredes, 2001; Paredes and Vazquez, 1999). This view is challenged by the observation that female rats exhibit a CPP for a context paired with mating at their preferred interval over a context paired with nonpaced mating behavior even when females do not actively pace sexual contact (Jenkins and Becker, 2003). Data from the present study showing that female rats developed a CPP for mating that occurs at the male rate (nonpaced mating), further support the notion that control of the sexual encounter per se is not necessary to induce a CPP for mating. The outcomes of the present study may also allow us to rule out two other previously hypothesized bases for the differential reinforcing effects of paced and nonpaced mating. First, the rate
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Fig. 2. Mean ± SEM preference score (top) and difference score (bottom) on pretest (white bars) and test (black bars) are shown for female rats that received nonpaced mating with the same male, nonpaced mating with the male changed following an ejaculation, or no mating behavior as the reinforcing stimulus. N = 9–10 rats per group. The asterisk indicates that only female rats in the Same Male group developed a CPP for the compartment paired with mating: a significant increase in preference score coincident with a significant decrease in difference score, P < 0.05.
of sexual contacts has been posited as an essential component of the reinforcing effect of mating in females (Jenkins and Becker, 2003). Specifically, the longer interintromission interval associated with paced compared to nonpaced mating may be important (Jenkins and Becker, 2003). Because in the present study female rats expressed a CPP for a context associated with longer (∼ 70 s) and shorter (∼ 40 s) interintromission intervals, it appears that the interval between intromissions, within the timeframe in the present study, is not critical for induction of a CPP. Second, intromissions delivered in a paced mating paradigm may be of a longer duration than intromissions delivered in nonpaced mating (Bermant, 1964; Erskine et al., 1989). Others have suggested that the increased intromission duration, and resultant heightened cervical stimulation, may contribute to the enhanced reinforcing effects of paced mating (Erskine et al., 1989; Martinez and Paredes, 2001; Paredes and Alonso, 1997). It is unlikely that the potential disparity in cervical stimulation in the two mating formats contributed to the reinforcing effects of mating given that both paced and nonpaced mating induced a CPP in the present study. In the present study the variable that was manipulated to influence the reinforcing effects of mating was the continued presence of the same male throughout the post-ejaculatory period until he resumed mating, or changing the male following ejaculation and introducing a new male until the female
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received 15 intromissions. The Same versus the Changed Male groups differed on at least three dimensions: the number of partners per mating test, time with the male and the length of the post-ejaculatory interval. The number of partners per test was significantly greater for the Changed Male than the Same Male group (2.5 versus 1). Although it is possible that female rats find mating reinforcing only when paired with a single mating partner (Same Male) as opposed to multiple partners (Changed Male), it seems unlikely that access to multiple partners during a mating encounter interferes with the reinforcing effects of mating, because female rats readily mate with multiple partners when given the opportunity (McClintock and Adler, 1978; McClintock et al., 1982). Kippin and Pfaus (2001) raised the issue that discrepancies in social contact resulting from the copulatory criteria may contribute to differences in observed conditioned partner preference in male rats. Whereas in Experiment 1 time spent with the male was significantly less in the Paced Mating group than in the Nonpaced Mating group and both groups developed a CPP, in Experiment 2 time spent with the male was significantly less in the Changed Male group than in the Same Male group and only the Same Male group showed a CPP. Social stimuli can induce a CPP in female rodents; female hamsters prefer a context associated with a male hamster even when a vaginal mask prevents receipt of vaginal stimulation (Kohlert and Olexa, 2005) or when the interaction is aggressive in nature (Meisel and Joppa, 1994). The additional social contact afforded the Same Male group may have facilitated the development of a CPP. In addition, it remains to be tested whether a minimum amount (threshold) of contact with stimulus animals is required for a mating-induced CPP. We hypothesize that a key factor in determining whether a mating encounter induces a CPP in female rats may be the conditions accompanying the post-ejaculatory period. Specifically, we propose that mating is reinforcing in female rats when a sufficient period of time elapses between the receipt of an ejaculation and the first post-ejaculatory intromission. If an uninterrupted post-ejaculatory interval is reinforcing to female rats, then preserving this interval may enhance reproductive success. Sperm transport occurs during the 6–10 min following ejaculation and can be reduced by the receipt of an intromission too soon after an ejaculation (Matthews and Adler, 1977). Thus, pregnancy/pseudopregnancy is more likely when the sperm plug is undisturbed following ejaculation. Rats in the present study received sufficient stimulation (15 intromission) to initiate the neuroendocrine response required for pregnancy/pseudopregnancy (Erskine et al., 2004; Erskine, 1989), therefore the reinforcing effects of preserving the post-ejaculatory interval may confer a reproductive advantage. Potential procedural differences between the present study and those that do not find a CPP to nonpaced mating behavior may account for disparate outcomes (Dominguez-Salazar et al., 2005; Gans and Erskine, 2003; Jenkins and Becker, 2003; Martinez and Paredes, 2001; Paredes and Alonso, 1997; Paredes and Vazquez, 1999). As stated earlier, in our laboratory male rats typically deliver ∼ 6 intromissions in the first ejaculatory series. Supposing that in other laboratories male
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rats exhibit similar mating patterns, then either a single male could remain in the test chamber following ejaculation and ultimately deliver all 15 stimulations or the male could be replaced following ejaculation. Data from the present study would lead us to predict that if the male is changed following ejaculations, then nonpaced mating would not induce a CPP. Unfortunately because the use of a single or multiple males to deliver the requisite number of intromissions is not specified, it is not possible to ascertain the role of differences in methodology to the study outcomes. In some nonpaced mating experiments, the female rat is transferred to the CPP apparatus immediately following ejaculation and thus the issue of the Same/Changed male following ejaculation is moot (Martinez and Paredes, 2001; Paredes and Alonso, 1997; Paredes and Vazquez, 1999). It is not clear why nonpaced mating failed to induce a CPP under these conditions. As mentioned above, the role of social contact in CPP is not clear, and it is possible that the additional time spent with a sexually refractory male (in the Same Male group) may enhance the reinforcing aspects of mating. Along these same lines, Kippin and Pfaus (2001) and Pfaus et al. (2001) report that male rats develop a conditioned partner preference for a female that is present during the period of time immediately following an ejaculation; a mating-induced partner preference does not develop when the female is removed during the post-ejaculatory interval. Moreover, males develop a preference for a female present only during the post-ejaculatory interval. An experiment in which the male is removed following ejaculation and then reintroduced after an average post-ejaculatory interval would address this issue in female rats. The results of the present study demonstrated that female rats developed a CPP for nonpaced mating behavior except when a new male was introduced after the female received an ejaculation. By delineating conditions that interfere with a mating-induced CPP, our findings extend our understanding of what makes mating reinforcing to female rats. Acknowledgments We thank Dr. William A. Carlezon for his advice on these studies and Drs. Joel Brown and David Bucci for their comments on the manuscript. We also thank Adam Christensen, Katherine Lang, Catherine Marantz and Kevin Wolfson for the technical assistance. This work was supported by DA08574 to A.C. References Bardo, M.T, Bevins, R.A., 2000. Conditioned place preference: what does it add to our preclinical understanding of drug reward? Psychopharmacology 153, 31–43. Becker, J.B., Rudick, C.N., Jenkins, W.J., 2001. The role of dopamine in the nucleus accumbens and striatum during sexual behavior in the female rat. J. Neurosci. 21, 3236–3241. Bermant, G., 1961. Response latencies of female rats during sexual intercourse. Science 133, 1771–1773. Bermant, G., 1964. Effects of single and multiple enforced intercopulatory intervals on the sexual behavior of male rats. J. Comp. Physiol. Psychol. 57, 398–403.
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