Hormonal and testing conditions for the induction of conditioned place preference by paced mating

The ability to control or pace the sexual interaction has important physiological and behavioral consequences for the female rat. Paced mating favors reproduction and induces a positive affective state as revealed by conditioned place preference (CPP). In the present experiment we evaluated: 1) If paced mating induces CPP in naturally cycling females; 2) If females developed a positive affective state if they paced the sexual interaction through a 1- or a 3-hole pacing chamber; 3) If females that mate with the same male without pacing the sexual interaction develop CPP. In the first experiment intact females were divided in 4 different groups; 2 paced the sexual interaction until receiving 1 or 3 ejaculations; the other 2 groups mated, without pacing the sexual interaction, until receiving 1 or 3 ejaculations. Only the group that paced the sexual interaction until receiving 3 ejaculations developed a positive affective state. In experiments 2 and 3 hormonally treated ovariectomized females were used. In experiment 2 females were allowed to pace the sexual interaction through a 1- or a 3-hole pacing chamber: A clear positive affective state was induced in both testing conditions. Finally, in experiment 3 females did not develop CPP for non-paced sex despite the fact that they mated with the same male in the conditioning sessions. These results demonstrate that the pattern of vaginocervical stimulation that the females received by engaging in approach and avoidance behaviors to pace the sexual interaction can induce a positive affective state in naturally cycling females. They also confirm the existence of a threshold of vaginocervical stimulation for paced mating to induce CPP in female rats.     

Female rats exhibit a conditioned place preference for nonpaced mating

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.     

Expression of FOS, EGR-1, and ARC in the amygdala and hippocampus of female rats during formation of the intromission mnemonic of pseudopregnancy

Pseudopregnancy (PSP) in the female rat is a neuroendocrine condition that is induced by repeated and intermittent vaginocervical stimulation received during mating and involves the expression of bicircadian prolactin surges and cessation of normal estrous cyclicity for 10-12 days postmating. The temporal patterning and number of intromissions received by the female are critical for PSP initiation, and thus, short-term encoding of VCS occurs during transduction of intromissions into PSP. In this experiment, we characterized and compared the mating-induced neural activation patterns within amygdalar and hippocampal regions using expression of the immediate early genes FOS, EGR-1, and ARC. Cycling female rats mated on proestrus received 15 or 5 intromissions under paced or nonpaced mating conditions. High numbers of intromissions during nonpaced mating or low numbers received during paced mating are sufficient to induce PSP, while five nonpaced intromissions and mounts without intromission are insufficient. Here we demonstrate that the CA1 region of the hippocampus was selectively sensitive to PSP-sufficient but not PSP-insufficient mating stimulation by showing significant effects of paced mating for all three IEGs. Paced mating also stimulated the expression of ARC within the basolateral, cortical, and central nuclei of the amygdala. The posterodorsal medial amygdala also showed selective EGR-1 responses to PSP-sufficient mating stimulation. There was no effect of hemisphere on IEG expression. The postmating expression profiles of these IEGs provide evidence that limbic areas involved in encoding and consolidation of memory are involved in initiating the neuroendocrine memory of PSP.     

Only Self-Paced Mating Is Rewarding in Rats of Both Sexes

When rats are mated in a traditional mating chamber (with one male and one female) in which the male dictates the pace of the copulatory sequence, males develop a reward state as evaluated by conditioned place preference (CPP). In this mating situation no reward state is induced in females. However, when female rats are able to control (pace) the rate of sexual stimulation, thereby reducing the aversive consequences associated with mating, a clear CPP is observed. In the present study the CPP paradigm was used to determine whether if the reinforced state induced by coital interactions in male rats can be maintained when females pace the sexual interaction. Adult male and female rats were mated in one of two different conditions: (1) where subjects were able to pace their coital interactions or (2) where subjects were not able to pace their sexual contacts. The results showed that when males had control over the sexual interaction they developed a clear place preference while males that mated with females that paced their coital contacts did not develop CPP. Similarly, only females that were able to pace their sexual contacts developed place preference. These results suggest that coital interactions in males, as well as in females, can induce a reward state only when they are able to control the sexual interaction. Under seminatural conditions sexual behavior in rats is highly promiscuous, they mate in groups and repeatedly change partners in the middle of copulation. This behavioral sequence allows both, male and female to control the rate of sexual interaction, assuring the induction of a reward state outlasting the actual performance of coital responses.     

The effects of mating stimulation on c-Fos immunoreactivity in the female hamster medial amygdala are region and context dependent

During mating in hamsters, both tactile and nontactile sensory stimulation experienced by the female affect sexual behavior and progestational neuroendocrine reflexes. To test the interactions of these types of mating stimulation, c-Fos immunohistochemistry measured brain cellular activity during sexual behavior under conditions that included combinations of tactile and nontactile mating stimulation. Test groups received: (1) mating stimulation from a male, females being either fully mated or mated while wearing a vaginal mask, or (2) experimenter applied manual vaginocervical stimulation (VCS)-with or without males present, or (3) handling similar to VCS but without insertions-with or without males present. Numbers of c-Fos immunoreactive cells were counted in specific subdivisions of the posterior medial amygdala (MeP) and ventromedial hypothalamus (VMH). The medial amygdala dorsal and ventral subdivisions responded differentially to components of mating stimulation. The posterodorsal Me (MePD) cellular activation was greatest during mating conditions that included VCS and/or males present. However, the posteroventral Me (MePV) was sensitive to male exposure and not to VCS. Also, MePV and VMH shell responses mirrored each other, both being primarily sensitive to male exposure. In separate tests, manual VCS induced pseudopregnancy, though the procedure was most effective with additional nontactile stimulation from males present. In summary, contextual cues provided by nontactile male stimulation enhance the effect of vaginocervical and other tactile stimulation on reproductive processes. Furthermore, c-Fos expression in the female hamster medial amygdala is region and context dependent.     

Different doses of estradiol benzoate induce conditioned place preference after paced mating

The ovarian hormones estrogen and progesterone are required for the complete display of sexual behavior in female rats. Paced mating produces a reward state in intact cycling and ovariectomized (OVX), hormonally primed females as evaluated by the conditioned place preference (CPP) paradigm. Most of the studies that have evaluated CPP induced by paced mating in OVX females have used relatively high doses of estradiol benzoate (EB). In the present study we determined if different doses of EB, combined with progesterone (P), could induce CPP after paced mating. For this purpose OVX female rats were divided in five groups that received one of different doses of estradiol benzoate (5, 2.5, 1.25 or 0.625 μg estradiol + 0.5 mg of progesterone) before being allowed to pace the sexual interaction and conditioned in a CPP paradigm. We found that the lowest dose of EB used (0.625 μg) significantly reduced the lordosis quotient and the lordosis coefficient. Even though these females paced the sexual interaction, they didn't change its original preference, suggesting that sexual interaction did not induce a positive affective, reward state. Females allowed to pace the sexual interaction with higher doses of EB developed CPP after paced mating. These results indicate that a threshold of estradiol is required for paced mating to induce CPP.     

Female rats develop conditioned place preferences for sex at their preferred interval

Female rats engage in approach and avoidance behaviors directed toward the male to "pace" the rate of copulation. These pacing behaviors result in a pattern of vaginocervical stimulation that triggers a neuroendocrine reflex that is important for pregnancy to result from insemination. Each female rat has a preferred pacing interval, and females develop conditioned place preferences for paced sex versus nonpaced sex. Research from this laboratory has reported that extracellular dopamine concentrations in striatum and nucleus accumbens are greater in female rats that are engaging in paced sex compared with those engaging in nonpaced sex. Furthermore, females who have males removed at their preferred intervals during a copulatory bout show extracellular dopamine concentrations comparable to females engaging in paced sex. It is unclear, however, whether they would also develop a conditioned place preference for sex under such conditions. This experiment was designed to address this question. Female rats had six exposures each to a chamber in which they engaged in nonpaced sex and a chamber in which they engaged in paced or preferred pacing interval sex. Following conditioning trials, females were tested for a conditioned place preference. The findings indicate that female rats develop conditioned place preferences for paced sex and for sex in which the male is removed at her preferred interval. This suggests that sexual behavior is reinforcing to female rats when their preferred interval is achieved, whether or not they are actively controlling the rate of copulation.     

Stimulus requirements for pregnancy initiation in the golden hamster (Mesocricetus auratus) change with time of mating during the receptive period

When maintained under a 14L:10D photoperiod, the duration of behavioural receptivity in female golden hamsters was about 18-21 h depending on age and/or parity. The effectiveness of mating stimuli in initiating pregnancy was shown to be a function of when in the receptive period (early, middle, late) that mating occurred. During the 9-h period before ovulation, 5 ejaculatory series were sufficient to produce a nearly 100% pregnancy rate and maximum litter size. During the ovulation period, however, high pregnancy rates were achieved only when mating continued to satiety (12-15 ejaculatory series plus 10-24 long intromissions). Late in the receptive period even mating to satiety failed to result in a pregnancy. In general, pregnancy rates were significantly higher for young virgin than for older multiparous females when mating occurred during or after the ovulation period. The reduced fecundity of females mating during or after ovulation was due to insufficient vaginocervical stimulation to induce functional luteal activity and not to lack of spermatozoa. Females mating late in the receptive period did not show a cessation of oestrous cycles which characteristically follows the induction of a luteal phase. Greater amounts of vaginocervical stimulation during this time increased the number of females which delivered litters but had no significant effect on litter size. These results suggest that levels of male copulatory behaviour considered 'excessive' when mating occurs early in the receptive period are essential for pregnancy initiation when mating occurs later.     

Influence of mating and vaginocervical stimulation on rat uterine activity

Twenty female rats were implanted with myometrial electrodes and intrauterine balloons to study the effects of mating on uterine contractile activity. Mating produced both immediate and delayed effects. Uterine activity typically doubled during the period of active mating (the immediate effect), then briefly returned to the premating activity level after ejaculation. Beginning 5 min after the ejaculation, the delayed effect of mating was observed: contractile activity increased when the baseline activity had been low, but decreased when baseline was fast. This delayed effect lasted for 30 min to several hours. Vaginocervical stimulation seemed to play a critical role in these responses: artificial vaginocervical stimulation mimicked the response while vigorous mounting by males that were prevented from intromitting did not evoke these responses. Vaginocervical stimulation, whether mating-derived or experimenter-delivered, produced immediate and delayed effects on uterine contraction rate.     

Progestins and place preference conditioning after paced mating

When female rats pace their coital interaction, a reward state evaluated by conditioned place preference is induced. Progesterone (P) is essential for the expression of proceptive behavior and for the induction of CPP. However, the functional significance of ring A reduction of P for the induction of this state during estrous is unsettled. In the present study, we evaluated whether ring A-reduced metabolites of P are involved in the reward state induced when the females are allowed to pace their sexual contacts. Ovariectomized (ovx) female rats treated with estradiol benzoate (EB, 5 microg) and P (13 microg), Megestrol acetate (MA; 13 microg ), 5 alpha-pregnan-20 dione (5 alphaDHP; 3 microg), or 5 beta-pregnan-3 alpha-ol-20-one (5 beta,3 alpha-Pgl; 3 microg) were used. Progestins were dissolved in propylene glycol and intravenously (iv) injected through an indwelling jugular catheter before females were tested for pacing behavior. After 15 intromissions or one ejaculation, females were gently placed in the nonpreferred compartment of a CPP box. Paced mating in all groups treated with progestins induced a clear change of preference. The administration of progestins alone did not induce CPP. These results suggest that P and ring A-reduced metabolites facilitate the reward state following pacing.     

不同背景的同伴强化及催产素处理对雌性棕色田鼠可卡因条件位置偏爱的影响

同伴间的社会互作是一种天然奖赏,能够影响成瘾药物使用的敏感性。催产素（Oxytocin,OT)能够调节社会行为,并提高社会互作的奖赏价值。然而不同背景的同伴互作以及与OT合并使用是否对可卡因的奖赏效应有不同的影响尚不清楚。棕色田鼠(Microtus mandarinus)是一种单配制田鼠,个体间具有较复杂的社会行为。利用雌性棕色田鼠,我们首先检测了可卡因(20 mg/kg)单独强化以及与不同背景关系的同伴（熟性雌性、陌生雌性和陌生雄性）同时强化诱导的条件位置偏爱（conditioned place preference, CPP）及持续时间;其次检测了实验鼠外周注射OT (1 mg /kg)并给予不同背景同伴强化对可卡因CPP的影响。结果表明,可卡因单独强化时,实验鼠能够形成可卡因CPP并能持续至3周;用熟悉的雌性同伴强化时,实验鼠对可卡因CPP的维持时间缩短;用陌生的雌性或雄性同伴强化时可抑制可卡因CPP的形成。实验鼠注射OT后,用熟悉雌性或陌生雄性同伴分别强化时会抑制或反转可卡因CPP。这些结果表明不同背景关系的同伴强化对可卡因奖赏效应的影响不同。OT可促进同伴强化的奖赏价值,降低动物对可卡因的偏爱,且该效应因强化同伴的背景而不同。     

Influence of time of mating and paced copulation on induction of pseudopregnancy in cyclic female rats

The present experiment was designed to examine whether changes occur during the course of behavioural oestrus in the sensitivity of the female to copulatory stimulation and in patterns of sexual behaviour which might influence the likelihood of luteal maintenance. Cyclic female rats were mated on the evening of pro-oestrus (21:00 h) or early on the morning of oestrus (05:00 h) and received either 5 or 10 intromissions from males under conditions which allowed or did not allow pacing of contacts with males to occur. During mating, the levels of sexual receptivity, the timing of sexual mounts from males, and pacing behaviours were measured. The occurrence of pseudopregnancy or pregnancy in each animal was determined by examining vaginal smears for 14 days after mating and by examining the uterus for the presence of fetuses at the end of the experiment. At both mating times, pacing of copulation with males increased the likelihood of prolonged luteal activity. However, females were more likely to become pseudopregnant following non-paced mating at 05:00 h than at 21:00 h the previous evening. Of those females receiving an ejaculation during mating, no difference were seen between groups in the incidence of pregnancy. This change in sensitivity to cervical-vaginal stimulation during oestrus was not associated with changes in sexual receptivity or pacing behaviour. The ability of cervical-vaginal stimulation to induce pseudopregnancy therefore increases toward the end of the period of oestrus, but the behavioural mechanisms which regulate receipt of such stimulation remain constant during that time.     

Previous sexual experience alters the display of paced mating behavior in female rats

The present study tested whether the display of paced mating behavior in female rats over four weekly tests is affected by sexual experience and whether test parameters, i.e., ending the test based on time or number of stimulations received, influence behavioral changes. In Experiment 1A rats with nonpaced sexual experience returned to the male more quickly overall compared to sexually naïve rats in a 30-min test of paced mating behavior. In Experiment 1B, rats received four weekly 30-min tests with one, different, male rat partner each week. Over the four tests, rats returned to the male significantly more quickly after intromissions, but significantly more slowly after ejaculations. Experiment 2A tested whether sexual experience would influence paced mating behavior in tests with a 15-intromission end criterion and the male replaced after ejaculation. Rats tested weekly under 15-intromission test conditions returned to the male significantly more quickly after intromissions, but no behavioral change was observed after ejaculations. When those same rats were given a 30-min test of paced mating behavior (Experiment 2B), they returned to the male significantly more slowly after ejaculations. Collectively, these data show that sexual experience influences the display of paced mating behavior in female rats and that the test parameters interact with sexual experience to influence the nature of the changes. Sexual experience may facilitate behaviors that promote reproductive success in female rats.     

Extended paced mating tests induces conditioned place preference without affecting sexual arousal

One way to evaluate sexual arousal is by measuring approach behavior to sexual incentive stimuli. In our case we measure approach behavior to an originally non-preferred compartment which is associated with the physiological state induced by mating. This change of preference indicative of a positive affective (reward) state can be evaluated by conditioned place preference (CPP). We have shown that the CPP induced by paced mating is mediated by opioids. The administration of opioids also induces a reward state. The present study was designed to compare the rewarding properties of paced mating and a morphine injection. One group of females was allowed to pace the sexual interaction before being placed in the non-preferred compartment. In alternate sessions they received a morphine injection before being placed in the preferred compartment. In another group of females, the treatments were reversed. Only the females placed in the originally non-preferred compartment after paced mating changed their original preference, suggesting that paced mating induces a positive affective, reward, state of higher intensity than a morphine injection of 1 mg/kg. In a second experiment we determined if females allowed to pace the sexual interaction for 1 h would still developed CPP. No change in preference was observed in the females that mated for 1 h without pacing the sexual interaction. On the other hand, females that received between 10 and 15 paced intromissions as well as females that paced the sexual interaction for 1 h developed a clear CPP. The second experiment demonstrated that pacing is rewarding even in an extended mating session in which the females received around 25 intromissions and several ejaculations. These results further demonstrate the biological relevance associated with the ability of the female to space coital stimulation received during mating. This positive affective state will contribute to increase sexual arousal the next time a rat finds an appropriate mate.     

Cuticular lipids and odors induce sex-specific behaviors in the male cricket Gryllus bimaculatus

Male crickets display sex-specific (e.g., mating and agonistic) behaviors towards conspecific individuals. One of the key signals for these behaviors is the chemical substance on the cricket body surface. In the present study, we analyzed female and male cuticular substances in behavioral assays. Antennal contact stimulation using female forewings elicited a mating behavior in males, while that using male forewings elicited an agonistic behavior in males. Thin-layer-chromatographic and other techniques analysis showed that saturated cuticular lipids were present in both female and male cuticles and that unsaturated lipids were present only in the male cuticle. Filter papers soaked with saturated or unsaturated cuticular lipids were applied to antennae of male crickets. Males showed mating behavior in response to stimulation with saturated lipids from both females and males but showed avoidance behavior in response to stimulation with male unsaturated lipids. Because cuticular lipids did not induce agonistic behavior in males, we collected odors from male crickets and found that these odors induced agonistic behavior in males. Therefore, we concluded that the key signals for mating, avoidance and agonistic behaviors of male crickets are comprised of at least three different components, saturated and unsaturated cuticular lipids and male odors, respectively.     

Infusions of naloxone into the medial preoptic area, ventromedial nucleus of the hypothalamus, and amygdala block conditioned place preference induced by paced mating behavior

Paced mating induces positive affect as revealed by conditioned place preference (CPP) in female rats. It has been suggested that endogenous opioids are involved in the generation of this positive affect since systemic administration of the opioid antagonist naloxone blocks mating-induced CPP. Several brain structures, including the medial preoptic area (mPOA), the ventromedial nucleus of the hypothalamus (VMH), the amygdala (Me), and the nucleus accumbens (Acb) have been implicated in the control of female sexual behavior. However, it is not known if these structures also participate in the positive affect produced by paced mating. To this end we determined the effects of intracranial administration of naloxone methiodide into the mPOA, VMH, Me and Acb on conditioned place preference induced by paced mating in female rats. Regardless of the site of infusion 5 μg of naloxone did not affect any of the sexual behavior parameters measured during copulation. When CPP was evaluated, the groups infused with naloxone into the mPOA, the VMH, and the Me before each conditioning session did not develop place preference. Only the group infused with naloxone in the Acb and the control groups did so. These results demonstrate that opioid receptors within the mPOA, VMH and Me are necessary for the rewarding aspects of paced mating. We suggest that the Me and VMH are important for the transmission of sensory information produced by copulation while the mPOA is the site where the positive affect is originated.     

Infusions of naloxone into the medial preoptic area,ventromedial nucleus of the hypothalamus,and amygdala block conditioned place preference induced by paced mating behavior

Paced mating induces positive affect as revealed by conditioned place preference (CPP) in female rats. It has been suggested that endogenous opioids are involved in the generation of this positive affect since systemic administration of the opioid antagonist naloxone blocks mating-induced CPP. Several brain structures, including the medial preoptic area (mPOA), the ventromedial nucleus of the hypothalamus (VMH), the amygdala (Me), and the nucleus accumbens (Acb) have been implicated in the control of female sexual behavior. However, it is not known if these structures also participate in the positive affect produced by paced mating. To this end we determined the effects of intracranial administration of naloxone methiodide into the mPOA, VMH, Me and Acb on conditioned place preference induced by paced mating in female rats. Regardless of the site of infusion 5 μg of naloxone did not affect any of the sexual behavior parameters measured during copulation. When CPP was evaluated, the groups infused with naloxone into the mPOA, the VMH, and the Me before each conditioning session did not develop place preference. Only the group infused with naloxone in the Acb and the control groups did so. These results demonstrate that opioid receptors within the mPOA, VMH and Me are necessary for the rewarding aspects of paced mating. We suggest that the Me and VMH are important for the transmission of sensory information produced by copulation while the mPOA is the site where the positive affect is originated.     

Induction of pseudopregnancy using artificial VCS: importance of lordosis intensity and prestimulus estrous cycle length

In cycling female rats, vaginocervical stimulation (VCS) received naturally during mating or by artificial mechanical stimulation induces neuroendocrine and behavioral responses that are critical for reproduction, including bi-circadian prolactin surges which result in pregnancy or an 8-14-day diestrous period called pseudopregnancy (PSP). Following mating, the incidence of PSP is higher when females receive high (10) as opposed to low (3-5) numbers of intromissions. Therefore, a threshold level of VCS must be exceeded before hypothalamic changes required for PSP can occur. This study characterized the threshold curve for PSP induction for artificial VCS (VCS-a). Proestrous females were given 1, 2, 3, 4, or 8 VCS-a applied with a glass rod using 200 g of force for 2 s, with an 8-min interval between stimulations. The lordosis response (LR) to the stimulus was measured on a scale of increasing intensity from 0 to 3, and the occurrence of PSP was measured by daily vaginal lavage. In contrast to previous findings, VCS-a induced robust lordosis responses without concurrent flank and perineal stimulation. The frequency of PSP induction did not increase in females as a function of amounts of VCS-a. However, the occurrence of PSP was strongly tied to the maximum lordosis response (LR(max)) observed. PSP was observed only among multiply stimulated females that showed the highest LR(max) (3.0) to at least one of the stimulations. Multiply stimulated females that showed a LR(max) < 3.0 or females that received only one VCS-a never became PSP. PSP and a stronger LR(max) were more likely to occur in females that had 5-day compared to 4-day prestimulus estrous cycle lengths. We conclude that central mechanisms important for VCS-induced PSP and lordosis may be potentiated by estradiol's actions in estrogen-concentrating forebrain areas.     

Glutamate release in the ventromedial hypothalamus of the female rat during copulation: Modulation by estradiol

Binding of glutamate or its ionotropic receptor agonists in the ventromedial hypothalamus (VMH) of female rats inhibits both appetitive and consummatory aspects of sexual behavior. Because vaginocervical stimulation activates glutamate neurons in the VMH, and administration of estradiol benzoate (EB) and progesterone (P) delays this effect, the present study examined the effects of hormonal priming on glutamate release within the VMH of female rats paired with sexually vigorous males. Ovariectomized, sexually experienced rats were implanted with guide cannula aimed at the ventrolateral VMH, through which microdialysis probes were inserted prior to testing. Females were assigned randomly to one of three hormone treatment conditions: EB + P, EB alone, or the oil vehicle. Testing was conducted over 5 h, including a 120-min period of habituation to the testing chamber, a 60-min period of baseline sample collection, and a 120-min period during which a sexually vigorous male was introduced into the testing chamber. Dialysates were collected every 20 min during the test and were analyzed for glutamate using HPLC. Females primed with oil had large and significant increases in glutamate release from baseline once the male was introduced to the chamber. Treatment with EB alone decreased glutamate release in response to male cues. Although treatment with EB + P did not differ significantly from EB alone, the degree of reduced glutamate release was less than with EB alone. These results indicate that priming with EB reduces glutamate transmission in the VMH in response to male cues. Taken together with our previous findings, estradiol blunts the activation of glutamate neurons in the VMH thus allowing female rats to copulate.     

Male–Female Communication in the Crayfish Orconectes rusticus: The Use of Urinary Signals in Reproductive and Non-Reproductive Pairings

Animals use sensory communication to locate conspecifics, food, shelter, and avoid predators. Using urine visualization techniques as well as Digital Particle Illumination Velocimetry, we examined the role of urinary signals and current generations during social interactions of male and female crayfish. Both reproductive and non‐reproductive crayfish were paired to gain a better understanding of how reproductive state influences communication. Analyses of agonistic and mating events were paired in time with recorded urine release and current generation, illustrating a correlation of chemical communication with ritualized social behavior. Four treatment groups were run with specific combinations of different reproductive status: (1) both opponents reproductively active, (2) only the male in reproductive, (3) only the female reproductive, or (4) both opponents non‐reproductive. Results showed differences between treatment groups in urine release, current generation, and social behavior. Within reproductive pairings, both the male and female crayfish generated currents and released urine at higher rates than those in other treatment groups. Urine was released most often when opponents were in chelae contact with each other and these releases were often accompanied by anterior current generation. In addition, communication was different in reproductive trials where mating occurred. Overall, the results indicate that the use of hydrodynamic and chemical signals changes as a function of reproductive state and that this change in communication probably indicates readiness to mate.