Short photoperiods increase ultrasonic vocalization rates among male Syrian hamsters

Physiology & Behavior, Vol. 38, pp. 453-458. Copyright©Pergamon Press Ltd., 1986. Printed in the U.S.A.

0031-9384/86 $3.00 + .00

Short Photoperiods Increase Ultrasonic Vocalization Rates Among Male Syrian Hamsters I J O H N A . M A T O C H I K , M I C H A E L M I E R N I C K I , J. B R A D L E Y AND MAUREEN L. BERGONDY

POWERS 2

Department o f Psychology and The John F. Kennedy Center for Research on Education and Human Development Vanderbilt University, Nashville, T N 37240 R e c e i v e d 19 F e b r u a r y 1986 MATOCHIK, J. A., M. MIERNICKI, J. B. POWERS AND M. L. BERGONDY. Short photoperiods increase ultrasonic vocalization rates among male Syrian hamsters. PHYSIOL BEHAV 38(4) 453--458, 1986.--Two experiments investigated the effects of daylength on the emission of 35 kHz ultrasonic (US) calls among male hamsters. In Experiment 1, castrated males received Silastic implants subcutaneously that contained either low doses of testosterone in oil or oil alone; US calls were recorded when these males were paired with receptive females. Males exposed to eight hours of light per day (short photoperiod) called more often than males exposed to fourteen hours of light per day (long photoperiod). This was true whether or not they received testosterone. In Experiment 2, a similar testing and photoperiod exposure paradigm was used, but the subjects were gonadally intact. Among males exposed to short photoperiods, US call rates increased while endogenous testosterone levels decreased. In contrast, hamsters exposed to long photoperiods maintained stable calling rates and testosterone levels. These findings are related to recent studies concerning the neural mechanisms that regulate ultrasonic vocalizations and to the possible role of photoperiod in modulating conspecific aggression. Male hamsters

Photoperiod

Sexual behavior

Ultrasonic vocalizations

G O N A D A L hormones, odors, ultrasonic vocalizations, and daylength interact to affect reproduction in Syrian hamsters [6, 10, 23, 37, 39]. Hamsters, like a number of other mammals, use changes in daylength (photoperiod) to time their annual reproductive cycles [3, 18, 22, 32, 36] so that young are born when environmental conditions are most appropriate for their survival. Hamsters remain reproductively competent when exposed to photoperiods containing 12.5 or more hours of light per day. Shorter photoperiods cause infertility [5,31]. Among males, short days decrease testicular weight, lower testosterone (T) production and impair spermatogenesis [17]. The mechanisms by which these gonadal effects occur have not been fully established, although many of the associated neuroendocrine changes have been well documented [31,32]. One of these, a daily lengthening of the interval during which pineal melatonin secretion remains elevated, may mediate photoperiodic effects on reproduction among hamsters and several other mammalian species [3, 29, 33]. A variety of experiments have suggested that in hamsters, short day exposure increases the sensitivity of the brain to

Testosterone

Aggression

the feedback effects of gonadal steroids on gonadotropin secretion [36]. Low levels of T that suppress L H and F S H among males exposed to short days do not do so among males exposed to long days [7, 34, 35]. In contrast, short days may decrease the sensitivity of the brain to the effects of T on sexual behavior. Specifically, castrated male hamsters receiving exogenous T copulate less vigorously if exposed to short, rather than long days [4,25]. Photoperiodic effects on behavioral responsiveness to T also occur for other behaviors and species [8, 16, 21, 24]. In general, these reports suggest a decreased behavioral sensitivity to this hormone. However, Garrett and Campbell [16] reported that gonadally intact male hamsters became more aggressive during exposure to short photoperiods. Because conspecific aggression can be stimulated by T in hamsters [26--28, 38], these observations suggested that not all behaviors become less sensitive to T in short photoperiods. During recent experiments on the effects of daylength on sexual and other social behaviors of male hamsters, we observed that animals exposed to short photoperiods increased their ultrasonic (35 kHz) vocalization rates when they were

~Supported by HD 14535 to J.B.P., HD 15052 to the Kennedy Center, HD 05797 to the Hormone Assay Core Laboratory of the Center for Reproductive Biology Research of Vanderbilt University and NSF Predoctoral Fellowship to M.L.B. 2Requests for reprints should be addressed to Dr. J. Bradley Powers, Department of Psychology, Vanderbilt University, 134 Wesley Hall, Nashville, TN 37240.

453

454

MATOCH IK ETA L. TABLE 1 TESTING SCHEDULEAND IMPLANTCHANGES FOR EXPERIMENT I

ULTRASONIC CALL RATES WEEK 13

Hormone Irritant

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Male hamsters were castrated and assigned to LD 14:10 or LD 8:16 photoperiods (PP) on week 0. *Testosterone (T) or control (Empty) Silastic capsules implanted in relevant groups. flnitial T capsules replaced after week 11 behavior tests by ones containing higher T doses. ~T capsules replaced after week 15 behavior tests by ones containing crystalline T. §T males given either a copulatory behavior (CB) or an ultrasound (US) test; Empty males given a US test only. ¶Subjective day (SD) tests began 7 and 4 hours after lights on among LP and SP males, respectively. Subjective night (SN) tests began 4 and 7 hours after lights off among LP and SP males, respectively.

paired with receptive females. This behavior, like aggression, can be facilitated by T [15], and both appear to respond in the same way to short photoperiod exposure. This report summarizes our observations. GENERAL METHOD

Animals Male Syrian hamsters (Mesocricetus auratus) were obtained from Harlan Sprague Dawley (Indianapolis, IN) for Experiment 1 and from Charles River Laboratories (Wilmington, MA) for Experiment 2. Unless otherwise indicated, animals in both studies were housed 3 per cage and entrained to an LD 14:10 illumination cycle (lights on 0400) with food and water continuously available.

Behavior Tests Ultrasound (US) Tests. Ultrasounds were recorded while males were paired with sexually receptive females for 2 min. The males were placed individually into clear, Plexiglas boxes (30x36x30 cm) 2 min before the female was introduced. Stimulus females had been ovariectomized under Nembutal anesthesia (75 mg/kg) and implanted subcutaneously (SC) with 20-mm lengths of Silastic tubing (1.98 mm i.d.; 3.18 mm o.d) containing 25 mg estradiol/ml sesame oil. Approximately 3 hr before use, they were injected with 500 /xg progesterone to induce sexual receptivity. Ultrasounds were monitored by a QMC Mini Bat Detector set to an input frequency of 35 kHz and positioned approximately 35 cm above the animals. Since female hamsters do not emit US when in lordosis [12,14], calls were recorded only when the female was in this posture, assuring that only male calls were scored. We divided the number of calls by the amount of time the female was in lordosis to obtain calls/rain. During US tests, the male could copulate with the female, but these behaviors were not recorded.

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PHOTOPERIOD CONDITION

FIG. 1. Results from week 13 US tests (Experiment 1). On week 0, animals were castrated and placed into either long (LD 14:10) or short (LD 8:16) photoperiods. Nine weeks later, animals were administered Silastic capsules containing either low doses of testosterone in oil or oil alone (empty).

Copulatory Behavior (CB) Tests. Ultrasounds were also scored during CB tests. Males were placed individually into boxes identical to those used for US tests 10 min before a receptive female was introduced. CB tests lasted for 5 min (10 min for Experiment 2) or until the male achieved two ejaculations, whichever occurred first. During these tests, we scored the latencies and frequencies of various copulatory and chemoinvestigatory behaviors as well as the emission of ultrasonic calls, but these other measures are not reported here. The derivation of US call rates from CB tests was less accurate than those from US tests because we did not continually record when the female was or was not in lordosis. Statistical Analyses Group differences were evaluated using either twosample t-tests or t-tests for correlated means. The 2-tail probability values obtained are indicated in the text for significant group differences. SPECIFIC EXPERIMENTALPROCEDURES

Experiment I Male hamsters were castrated and separate groups were administered one of two doses of T to investigate the effects of short photoperiod exposure on the display of sociosexual behaviors. This paradigm allowed for the evaluation of behavioral effects independent of the decreased levels of systemic T which typically result among gonadally intact animals exposed to short photoperiods. The schedule for all behavioral testing and hormone implantation is presented in Table 1. However, due to the limited focus of this paper, only those aspects of the study pertinent to the analysis of ultrasonic calling will be described. Following acclimation to our colony for four weeks, 90 males were castrated under Nembutal anesthesia (75 mg/kg)

SHORT PHOTOPERIODS AND U L T R A S O N I C C A L L I N G ULTRASONIC CALL RATES 30

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times when T capsules were replaced.

and divided equally between long (LD 14:10, lights on at 0400) and short (LD 8:16, lights on at 0700) photoperiod conditions. An additional 10 animals in each photoperiod remained gonadally intact so that testicular regression could be monitored. For brevity, long and short photoperiods are referred to as LP and SP, respectively, and treatment or testing weeks refer to the number of weeks following entry into the assigned photoperiod condition. During week 9, 30 of the 45 castrated animals in each photoperiod were given 20-mm Silastic implants SC containing low doses of T (0.07 or 0.007 mg T/ml sesame oil); an additional 15 males received implants containing oil only. These initial concentrations of T were chosen to provide systemic levels considerably below those typically found in gonadally intact males, and were doses we had previously established to be above threshold for the androgendependent behaviors we measure. We wished to avoid the possibility that changes in responsiveness to T might be masked by excessive hormonal stimulation. However, the first series of behavior tests (week 11) indicated that these T doses were too low to reinstate copulatory behavior. Therefore, the implants were replaced with ones containing higher concentrations of T (0.3 and 0.03 mg T/ml oil). These implants were also ineffective for restoring mounts, intromissions and ejaculation. Thus, we abandoned the strategy of using subphysiological doses of T and replaced the solution-rifled capsules with 10-ram Silastic implants containing crystalline T following behavior tests on week 15; these implants produce systemic levels of T in the physiological range [4]. When Silastic capsules were either initially implanted, or were later exchanged for ones containing higher T concentrations, hamsters were anesthetized with Nembutal (75 mg/kg) and capsules were inserted SC into the dorsal neck region. During the weeks when implants were exchanged, the control males with empty implants were anesthetized, but their Silastic capsules were not otherwise manipulated.

455 Behavioral tests on weeks 11 and 13 were conducted during the animals' subjective day (SD); testing on weeks 15 and 18 was conducted during subjective night (SN) under dim red illumination (see Table 1 for testing times). During week 18, all of the males with empty implants and a subset of the males with T implants (LP: n= 14 of 28; SP: n= 12 of 27) were given US tests only. The remaining T males received CB tests only. Even though the copulatory and chemoinvestigatory behavior of T-treated males appeared unresponsive to this hormone until late in the experiment, they emitted US during tests on weeks 11, 13, 15 and 18, and differences between photoperiod conditions were clear. These are the behavioral data reported below. During weeks 10 and 21, the gonadally intact males that had not been behaviorally tested had the size of their left testes measured under ether anesthesia. A Testis Index (TI) was obtained, defined as testis length x width (mm)/body weight (g), and is highly correlated with the functional capacity of the gonads. Experiment 2

In this study, we used gonadally intact male hamsters (n=36; 7-9 weeks old) from Charles River Laboratories (Wilmington, MA), the supplier typically employed by researchers in the area of photoperiodic time measurement. The animals were initially housed under LP as described in Experiment 1. During the 16 weeks after their arrival they were given six, 50-min exposures to sexually receptive females. This provided them with sexual experience and allowed us to exclude non-copulators. Following the sixth experience session, males were given CB and US tests as described above. The behavioral scores obtained were used to assign animals to either LP (n=12) or SP (n=24) conditions on week 0 so that they were matched on appropriate behavioral measures. CB tests were given on weeks 3, 7 and 11; US tests were given on weeks 4, 8 and 12. All behavior tests were conducted during SD at approximately the same circadian time for both groups. During week 9, aggression among SP males resulted in the death of 4 animals which necessitated a switch to individual housing for both SP and LP animals during the remainder of the experiment. On week 13, all animals were sacrificed and physiological measures were obtained. From each animal we determined a TI as well as a Flank Gland index (FGI). The latter is an indicant of peripheral androgenic stimulation, and was defined as the area of the left flank gland (mm). Additionally, the seminal vesicles were weighed and blood samples taken by cardiac puncture for subsequent radioimmunoassay of T in serum. These assays were performed by the Hormone Assay Core Laboratory of the Vanderbilt University Center for Reproductive Biology. Briefly, T concentrations were determined by radioimmunoassay after ether extraction and purification of the steroid by celite column chromatography [1] with recovery of 80--85%; inter- and intra-assay coefficients of variation were 8.7 and 5.9%, respectively. RESULTS

Experiment 1

SP males emitted more calls than LP males during US tests on week 13 (Fig. 1). Because the low doses of T implanted on week 9 did not affect the call rates of hamsters in either photoperiod, groups were collapsed across hormone treatment; a significant difference was found between LP

456

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P H O T O P E R I O D CONDITION

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FIG. 3. US call rates of gonadally intact males during US tests (Experiment 2). For SP males, n=20 on week 12 tests. Week 0 values represent call rates obtained 1 week before entry into LP or SP conditions.

FIG 4. US call rates of gonadally intact males during CB tests (Experiment 2). For SP males, n=20 week 11 tests. Week 0 values represent call rates obtained 1 week before entry into LP or SP conditions.

and SP animals on week 13 tests (mean-+sem: 6.9-+1.0 vs. 12.0__ + 1.7 calls/rain, respectively; p <0.01). When the dose of T was raised following week 15 CB tests, all groups subsequently emitted more ultrasounds during week 18 US tests (Fig. 2). This in part reflected the shift to subjective night testing because the increase in US calling rates was shown by both hormone-treated and control animals. However, these increased calling rates were significant only for the SP males receiving T (0<0.01). During CB tests on weeks 11 and 15, SP males called more frequently than L P males, but only the week 11 comparison was statistically significant (0<0.05 and p =0.06 for weeks 11 and 15, respectively; data not shown). The withingroup increase in call rates between weeks 8 and 15, however, was significant for both photoperiod conditions (p<0.001), again in part reflecting the shift from SD to SN testing. F o r those animals given CB tests during week 18 after having their T implants replaced during week 15, US call rates increased significantly for SP males only (p<0.01), thus confirming in these CB tests what was observed in the 2-min US tests using a different subset of animals. Among the gonadally intact males that were not tested behaviorally, those exposed to SP had smaller testes than males exposed to L P on both weeks 10 and 21 [mean TI-+ sem: 0.59-+0.06 vs. 1.62-+0.07, p<0.001 (week 10); 1.31___0.08 vs. 1.75-+0.12, p<0.01 (week 21)]. The difference between weeks 10 and 21 mean TI values among the SP males indicates that their testes had partially recrudesced by week 21.

PERIPHERAL MEASURES OF ANDROGENIC STIMULATION FOR EXPERIMENT 2

TABLE 2

Experiment 2 The males in this study remained gonadally intact throughout; thus, animals exposed to SP conditions were influenced by changes in their endogenous T levels. US vocalizations rates displayed during US and CB tests are presented in Figs. 3 and 4. SP males called at increasingly higher rates during successive US tests whereas L P males did not; by week 12, the call rates of SP males were nearly 4

Peripheral Measures

Photoperiod Condition LP SP

TI

FGI

SVW

1.95 _+ 0.152 0.93 _+ 0.069

43.25 _+ 3.03 14.65 _+ 0.97

1.25 -+ 0.04 0.45 _+ 0.07

All values are means _+ sem and were obtained on week 13. TI--Testis Index (mmVg body wt.). FGI--Flank Gland Index (mm2). SVW--Seminal Vesicle Weight (g). LP--Long Photoperiod (LD 14:10). SPi-Short Photoperiod (LD 8:16).

times those of L P males (Fig. 3; p<0.01 for week 12). The same basic profile was also evident in the US call rates observed during CB tests. Again, SP males displayed increasingly higher rates during successive CB tests while the rates for L P males remained relatively unchanged; differences between photoperiod conditions were significant for weeks 7 and 11 (Fig. 4; p<0.01 and 0.001, respectively). Serum T levels, determined following the final behavioral tests (week 13), were lower among SP males (mean__-sem: 4.01___0.54 and 1.003-+0.34 ng/ml for L P and SP animals, respectively;p<0.001). SP males also had smaller testes, flank glands, and seminal vesicles (/9<0.001 in each case, see Table 2). GENERAL DISCUSSION Exposure of male hamsters to short photoperiods for 3-4 months significantly increased the number of US calls they made during tests with receptive females. This occurred both in the presence and absence of testosterone. Although T and

SHORT PHOTOPERIODS A N D U L T R A S O N I C C A L L I N G some of its metabolites stimulate ultrasonic calling in hamsters, it is clear from our findings that castrated males do emit calls and that exposure to SP enhances this response (Fig. 1). When a Silastic implant was used which finally achieved effective androgenic stimulation (based on other behavioral measures), week 18 US call rates increased significantly, but only among SP males (Fig. 2). The lack of T responsiveness among LP males in Experiment 1 could relate to their having been without effective androgenic stimulation for 15 weeks following castration, thus promoting a relative lack of sensitivity to testosterone's behavioral effects. Our results suggest that the added variable of short photoperiod exposure in some way overcame this insensitivity. The facilitation of ultrasonic calling by SP was confirmed in Experiment 2. US call rates progressively increased over the three month exposure period; concurrently, T levels were decreasing (Figs. 3 and 4). These results are consistent with the hypothesis that brain mechanisms regulating ultrasonic calling were becoming more responsive to T (or to other variables) as time in SP conditions progressed. The overall calling rate was lower in Experiment 2 than it was in Experiment 1. Although we have no explanation for this, the two sets of hamsters did come from different suppliers, and those used in Experiment 2 received extensive sexual experience prior to the beginning of the study, whereas the males in Experiment 1 did not. The behavioral differences observed in both Experiment 1 and 2 between LP and SP animals cannot be attributed to differences in the circadian times at which they were tested. Although circadian variations in hamster ultrasonic call rates have not been reported, such variations do occur in sexual behavior but they are small [9]. We tested each group at equivalent phases of their daily circadian cycles, based on the known relation between circadian time and photoperiod duration among hamsters [5]. Thus it is unlikely that the higher US call rates among SP males resulted because they were tested at a more responsive point on a daily cycle of responsiveness. Group differences in ultrasonic call rates could simply reflect group differences in sexual activity. It is likely, though not fully documented, that copulating males emit more calls than males that are not copulating. In Experiment 2, the copulatory behavior of SP males gradually worsened over successive tests, as would be expected on the basis of their declining T levels. By week 11, all LP males were ejaculating, whereas only 5 of 20 SP males still showed this behavior pattern. Indeed, only eight SP hamsters were intromitting. Thus, copulatory performance decreased while call rates increased. Yet the 5 males that continued to ejaculate also had elevated call rates that were not significantly different from the rates of the males whose copulatory performance deteriorated over time. Thus it is unlikely that differences in copulation per se can account for the SP effect on US vocalizations. Analysis of Experiment 1 also supports this conclusion. For example, US call rates were significantly higher among SP males during week 13 (Fig. 1), yet on mating tests both before and after week 13, copulation was displayed at such minimal levels by all of the animals, regardless of hormone or photoperiodic treatment (data not shown), that differences in this behavior could hardly have been responsible for the differences in US call rates among SP and LP males. What other factors might be relevant to the interpretation of our findings? One possibility stems from earlier observa-

457 tions concerning the effects of SP on aggressive interactions. Garrett and Campbell [16] reported that gonadally intact male hamsters exposed to short photoperiods increased the frequency of their aggressive responses towards conspecifics. Because this behavior can be facilitated by androgens [26-28, 38], the increase in aggression seemed paradoxical because, as expected, SP males had lower T levels than did LP males. In Experiment 2, which used gonadally intact males, we were made aware of an increase in conspecific aggression when, during week 9, four animals in the SP condition were killed by cagemates which necessitated a switch to individual housing for the remainder of the experiment. Because testosterone facilitates both aggressive interactions and the emission of US calls, these effects of SP may suggest that the two response systems are influenced by a common mechanism, and therefore might be functionally related. Could an increased rate of US calling have significance in situations where males might be competing for limited resources and the probability of intermale aggression is high? Unfortunately, there is little available evidence by which to evaluate this possibility, although attention has been drawn previously to an association between aggression and ultrasonic calling among rodents [30]. Some findings suggest that US calls can serve as attractants among hamsters and that these calls may facilitate the persistence of the lordosis response in receptive females [2, 12, 13]. Although these functions appear diametrically opposed in terms of response requirements, they could have interesting implications in the context of aggressive encounters. If among both males and females, US calls that were detected at some distance generated an attractant response, i.e., a seeking out of the source of stimulation, but at close range (perhaps at higher intensities) increased the probability of an immobilization response, the latter could be of significance if one animal was attempting to subdue or dominate another. Because conspecific aggression does occur when male hamsters are placed in short photoperiods [16], a higher US calling rate could be functional if it did serve, via an immobilization response, to facilitate the ease by which a dominant status could be achieved. Our findings may also relate to a recent report on the effects of hypothalamic lesions in hamsters. Floody [11] tested both copulatory and US vocalization behavior in males and females that had been lesioned in either the preoptic area (POA) or the ventromedial hypothalamus (VMH). Among males, damage to the POA impaired copulation without affecting the emission of US calls. In contrast, VMH lesions had no effect on CB but they increased vocalization behavior. Recently, this same laboratory has reported that lesions of the lateral septum/bed nucleus of the stria terminalis can also increase vocalization rates among females [20]. Thus, there is little doubt that mechanisms operate in the hamster brain to inhibit US vocalizations. It is a reasonable hypothesis that external (e.g., olfactory [19]), or internal (e.g., testosterone [15]) cues that stimulate ultrasonic calling may in part suppress this inhibitory mechanism. If this inhibition were also reduced by SP exposure, then ultrasonic calls should be more readily facilitated by the cues that normally stimulate them. This interpretation is then compatible with the general hypothesis that exposure of male hamsters to short photoperiods eventually leads to an alteration of brain mechanisms regulating the expression of important sociosexual behaviors. The precise nature of these neuroendocrine changes remains to be elucidated.

MATOCHIK ET AL.

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