Hormones and Behavior 65 (2014) 42–46
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Hormones and Behavior journal homepage: www.elsevier.com/locate/yhbeh
Relationship between 22-kHz calls and testosterone in male rats Hideaki Inagaki ⁎, Yuji Mori Laboratory of Veterinary Ethology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
a r t i c l e
i n f o
Article history: Received 22 August 2013 Revised 8 November 2013 Accepted 15 November 2013 Available online 23 November 2013 Keywords: Hormone receptor Sex hormone Stress response Ultrasonic vocalization Vocal communication
a b s t r a c t Ultrasonic calls in rats induced by the presence of a predator, referred to as “22-kHz calls,” are mainly emitted by socially dominant male rats. Testosterone levels are closely related to social dominance in male rats. In the present study, we investigated the relationship between the emission of stress-induced 22-kHz calls and circulating testosterone levels in male rats, using a combination of surgery (castration or sham operation) and chronic steroid administration (testosterone or cholesterol) to modify circulating testosterone levels. We also assessed the effects of androgen and/or estrogen receptor antagonists on the emission of 22-kHz calls in male rats. An air puff stimulus, known to reliably induce 22-kHz calls in rats, was used as a stressor. Castrated rats with cholesterol implants exhibited significantly fewer 22-kHz calls than rats that had received a sham operation and cholesterol implants, and there was no significant difference between castrated rats with testosterone implants and rats that had received a sham operation and cholesterol implants. Only male rats pretreated with a binary mixture of androgen and estrogen antagonists exhibited significantly fewer 22-kHz calls than controls. These results show that testosterone in male rats has a positive effect on the emission of stress-induced 22-kHz calls, and the calls may be regulated by the activation of both androgen and estrogen receptors. © 2013 Elsevier Inc. All rights reserved.
Introduction Rats emit social vocalizations in the ultrasonic range (above 20 kHz) in a variety of aversive or appetitive situations (Brudzynski, 2009). Adult rats emit long (usually between 0.5 and 3.0 s for a single call) ultrasonic bouts of 20–30 kHz with a narrow bandwidth of 1–4 kHz, referred to as “22-kHz calls”. These ultrasonic calls are typically responses to potentially harmful or life-threatening situations or to the expectation of a known unpleasant stimulus even if the rat lacks exact information about when it will happen. For example, rats call in the presence of a predator (Blanchard et al., 1991), when they confront a dominant and aggressive rat (Panksepp et al., 2004), and even when they receive a light but sudden and unpredictable air puff (Knapp and Pohorecky, 1995) or tactile stimulus (Inagaki et al., 2005). Moreover, it is widely accepted that 22-kHz calls serve as alarm calls to warn conspecifics of external danger; namely, 22-kHz calls are considered to be one of social communicative behaviors (Litvin et al., 2007). A male rat's emission of 22-kHz calls can be influenced by the rat's social hierarchical position; when a predator (such as a cat) is presented to groups that include several male and female rats in seminatural visible burrow systems, it is usually the socially dominant males that emit 22-kHz calls (Blanchard et al., 1991). It is also well known that social status is closely related to testosterone levels in male rats (Bernhardt, 1997); for example, testosterone removal (castration) causes socially dominant rats to become submissive (Albert et al., 1986), and chronic ⁎ Corresponding author. Fax: +81 3 5841 8190. E-mail address: [email protected] (H. Inagaki). 0018-506X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yhbeh.2013.11.003
administration of testosterone to subordinate rats significantly increases their dominant behaviors (Bonson and Winter, 1992). On the basis of this information, we hypothesized that the testosterone can influence the emission of 22-kHz calls in male rats, although no systematic analysis regarding such relationship has been reported. To test our hypothesis, we investigated the relationship between the total number and duration of stress-induced 22-kHz calls and circulating testosterone levels in male rats using a combination of surgery (castration or sham operation) and chronic steroid administration (testosterone or cholesterol) (Experiment 1). We also assessed the effects of androgen and/or estrogen receptor antagonists on the emission of 22-kHz calls in male rats (Experiment 2). Materials and methods Animals A total of 98 male Wistar rats (Clea Japan, Tokyo, Japan) were used in this study. All animals were housed in pairs in wire-topped transparent cages (400 × 250 × 180 mm) with wood shavings for bedding. They were provided with water and food ad libitum and maintained on a 12-h light–dark cycle (lights turned off at 20:00). The cages were maintained at a constant temperature (24 ± 1 °C) and humidity (40–45%). Pretreatment before experiments In Experiment 1, 40 rats aged 8 weeks received one of three treatments. A total of 12 rats were castrated and implanted with silicon
H. Inagaki, Y. Mori / Hormones and Behavior 65 (2014) 42–46
tubes (1.57-mm inner diameter, 3.18-mm outer diameter; Kaneka Medix Corporation, Osaka, Japan) containing cholesterol (Wako Pure Chemical Industries, Osaka, Japan). A total of 14 rats were castrated and implanted with silicon tubes containing testosterone (Wako Pure Chemical Industries), and 14 rats received sham operations and were implanted with silicon tubes containing cholesterol. We packed a 10-mm length of each tube with cholesterol or testosterone and closed each end of the tube with a glass bead (3 mm in diameter). Each subject received a single implantation of either tube. All operations were performed under inhalation anesthesia with ethyl ether. In Experiment 2, 58 rats aged 10 weeks received one of four types of treatment featuring subcutaneous injections once a day for 7 consecutive days. A total of 14 rats received an androgen receptor antagonist, flutamide (Wako Pure Chemical Industries; 50 mg/kg, 50 mg flutamide dissolved in 1 ml of vehicle); 12 rats received an estrogen receptor antagonist, tamoxifen (MP Biomedicals, Solon, OH, USA; 1 mg/kg, 1 mg tamoxifen dissolved in 1 ml of vehicle); 16 rats were treated with a binary mixture of the two drugs (flutamide 50 mg/kg and tamoxifen 1 mg/kg, 50 mg flutamide and 1 mg tamoxifen dissolved in 1 ml of vehicle), and 16 rats received corn oil as a control (Wako Pure Chemical Industries; 1 ml/kg). Experimental apparatus and procedures All experiments were performed when subjects were 11 weeks of age. At the beginning of the experiment they were moved to a soundproofed experimental room and remained in their own cages for at least 60 min. Each animal was then transferred to a wire-topped transparent experimental cage (400 × 250 × 200 mm) and habituated to the cage for 5 min. After 5 min, the wire lid was removed and the animal received an air puff stimulus, which is known to reliably induce 22-kHz calls in rats (Brudzynski and Holland, 2005; Inagaki et al., 2012; Knapp and Pohorecky, 1995). A total of 30 air puffs with an interstimulus interval of 2 s were directed to the nape of each subject's neck, delivered from a nozzle (10 mm outer diameter and 2 mm caliber) at a distance of approximately 50 mm from the subject. The pressure of the air puff was maintained at 0.3 MPa by a pressure valve, following procedures used in earlier studies (Brudzynski and Holland, 2005; Knapp and Pohorecky, 1995). Immediately after the air puff stimuli, we placed the wire lid on the experimental cage and recorded 22-kHz calls for 5 min using an ultrasound microphone (Condenser Microphone CM16/CMPA; Avisoft Bioacoustics, Berlin, Germany) set at a distance of 50 mm from the top of the wire lid. Data acquisition hardware (UltraSoundGate 116Hbm; Avisoft Bioacoustics) and recording software (Avisoft-RECORDER Version 4.0; Avisoft Bioacoustics) on a personal computer were used. Settings included a sampling rate of 100 kHz and a 16-bit format. The abovementioned sequence of air puff stimuli and the recording of 22kHz calls was repeated three times for each subject. During the recording of 22-kHz calls, the behaviors of subjects were simultaneously videorecorded (HDR-SR12; Sony, Tokyo, Japan). After the experiment, the number of fecal boli excreted by each subject was counted. All experimental procedures were conducted between 10:00 and 15:00. For Experiment 1 only, 7 days after the experiment, we anesthetized each subject with ethyl ether and collected blood (6 − 8 ml, for euthanasia) from the abdominal aorta into a heparinized conical tube. Each blood sample was maintained at 4 °C and centrifuged to obtain plasma, which was then frozen at − 80 °C. We collected all blood samples between 13:00 and 15:00. This study was approved by the Animal Care and Use Committee of the Faculty of Agriculture, The University of Tokyo. Data analyses For spectrogram generation, recordings were transferred to AvisoftSASLab Pro (Version 5.1; Avisoft Bioacoustics) and a fast Fourier transformation (FFT) was conducted. Spectrograms were generated with
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an FFT-length of 512 points and a time window overlap of 50% (100% Frame, FlatTop window). We defined 22-kHz calls as long calls (0.5 − 3.0 s) in the ultrasonic range within a narrow band of peak frequencies (20 − 23 kHz) with a narrow bandwidth (1 − 4 kHz) according to an earlier report (Brudzynski, 2009).We recorded the total number of 22-kHz calls and also added together all calls from each subject to generate the total duration of calls. The calls were measured automatically using Avisoft-SASLab Pro. In addition, we calculated the duration of video-recorded freezing responses during the time that 22-kHz calls were recorded (15 min). Freezing in this study was defined as an immobile posture with cessation of skeletal and vibrissae movement except for respiration. Plasma testosterone levels in Experiment 1 were measured by enzyme immunoassay (Testosterone EIA kit; Cayman Chemical Company, Ann Arbor, MI, USA). Statistical analyses All data were displayed as the mean ± standard error. Statistical comparisons were performed using a one-way analysis of variance (ANOVA) followed by the post hoc Dunnett test. The criterion for statistical significance was p b 0.05 for all comparisons. In experiment 2, effect sizes (η2 for the one-way ANOVA and Cohen's d for the post hoc test) were calculated to evaluate magnitudes of effects of drug treatments on emission of 22-kHz calls (η2: large = 0.14, medium = 0.06, small = 0.01; Cohen's d: large = 0.80, medium = 0.50, small = 0.20). Results Experiment 1: Relationship between the emission of 22-kHz calls and circulating testosterone levels The total duration of air puff-induced 22-kHz calls was significantly affected by treatment (F2,37 = 6.65, p b 0.01). The post hoc test indicated that castrated rats with cholesterol implants exhibited significantly shorter 22-kHz calls than rats that had received a sham operation and cholesterol implants (p b 0.01). There was no significant difference between castrated rats with testosterone implants and rats that had received a sham operation and cholesterol implants (Fig. 1a). The total number of air puff-induced 22-kHz calls was also significantly affected by treatment (F2,37 = 4.48, p b 0.05). The post hoc test indicated that castrated rats with cholesterol implants exhibited significantly fewer 22-kHz calls than rats that had received sham operations and cholesterol implants (p b 0.05). There was no significant difference between castrated rats with testosterone implants and rats that had received sham operations and cholesterol implants (Fig. 1b). In contrast, the total duration of freezing and the total number of fecal boli produced were not significantly affected by treatment (freezing: F2,37 = 1.94, p = 0.14, fecal boli: F2,37 = 0.33, p = 0.72) (Fig. 1c, d). Plasma testosterone levels were significantly affected by treatment (F2,37 = 30.5, p b 0.01). The post hoc test indicated that castrated rats with cholesterol implants had significantly lower plasma testosterone levels (1.60 ± 0.11 × 10−2 ng/ml) than rats that had received a sham operation and cholesterol implants (2.42 ± 0.34 ng/ml) (p b 0.01). There was no difference between castrated rats with testosterone implants (2.11 ± 0.17 ng/ml) and rats that had received a sham operation and cholesterol implants. Experiment 2: Effects of sex hormone receptor antagonists on the emission of 22-kHz calls The total duration of air puff-induced 22-kHz calls was significantly affected by treatment (F3,54 = 3.08, p b 0.05). The effect size was large (η2 = 0.15). The post hoc test indicated that only rats pretreated with a binary mixture of flutamide and tamoxifen exhibited significantly shorter 22-kHz calls than control rats (p b 0.05) (Fig. 2a), and the effect size for paired comparisons was larger (Cohen's d = 1.10) than those of
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Fig. 1. The total duration of 22-kHz calls (a), the total number of 22-kHz calls (b), the total duration of freezing (c), and the total number of fecal boli (d) observed in male rats after they received an air puff stimulus in Experiment 1. Each subject received one of three types of pretreatment 3 weeks before the experiment: a sham operation with a cholesterol implant (Sham + C, n = 14), castration with a cholesterol implant (Cast + C, n = 12), castration with a testosterone implant (Cast + T, n = 14). Each bar represents the mean ± standard error; **p b 0.01, *p b 0.05.
other two paired comparisons (pretreatment with flutamide and control: Cohen's d = 0.27, pretreatment with tamoxifen and control: Cohen's d = 0.02). Although the total number of air puff-induced 22kHz calls was not significantly affected by treatment (F3,54 = 2.07, p = 0.12) and the effect size was medium (η2 = 0.10), the post hoc test indicated that rats pretreated with a binary mixture of flutamide and tamoxifen exhibited significantly fewer 22-kHz calls than control rats (p b 0.05) (Fig. 2b). The effect size for paired comparisons between the two was larger (Cohen's d = 0.86) than those of other two paired comparisons (pretreatment with flutamide and control: Cohen's d = 0.24, pretreatment with tamoxifen and control: Cohen's d = 0.15). The total duration of freezing and the total number of fecal boli produced were not significantly affected by treatment (freezing: F3,54 = 0.27, p = 0.85, fecal boli: F3,54 = 0.60, p = 0.61) (Fig. 2c, d). Discussion This study measured the role of testosterone in the emission of stress-induced 22-kHz calls in male rats using an air puff stimulus as a stressor. In Experiment 1, removal of testosterone by castration significantly reduced the rats' emission of air puff-induced 22-kHz calls, but the calls returned to pre-castration levels when castrated rats received testosterone implants. In Experiment 2, rats displayed significantly fewer air puff-induced 22-kHz calls when they were pretreated with both an androgen receptor antagonist and an estrogen receptor antagonist simultaneously, although pretreatment with only one of the
two drugs did not have a significant and effective effect. The results of this study show that testosterone in male rats has a positive effect on emission of stress-induced 22-kHz calls, and calling behavior may be regulated by the activation of both androgen and estrogen receptors. Although behavioral, physiological, neuroanatomical, and pharmacological studies indicate that 22-kHz calls are a valid measure of stress-induced negative affective states in rats (Sanchez, 2003), it is probable that the reduction in 22-kHz calls induced by low testosterone levels was not due to a reduction in stress responses in the castrated animals. Earlier studies have demonstrated that testosterone levels influence stress responses in rats. For example, rats show an increased acoustic startle reflex in the presence of bright ambient light, a behavior called light-enhanced startle (LES); LES is seen reliably in castrated male rats but not as reliably in gonadally intact male rats, and testosterone replacement attenuates the LES in the castrated rats (Toufexis et al., 2005). A footshock or exposure to a novel open field increases the incidence of a well-known hormonal stress response, the hypothalamic pituitary adrenal (HPA) response. The HPA response is greater in castrated rats than in intact rats, and testosterone treatment of castrated animals returns their post-stress display of the HPA response to intact levels (Handa et al., 1994). Testosterone had the opposite effect in these studies to its effect on stress-induced 22-kHz calls in our present study. Furthermore, low testosterone levels in our present study did not affect rats' freezing responses or defecation, both of which are well known to be typical behavioral stress responses in rats (Million et al., 2003; Rodgers, 1997). These findings suggest that the reduction
H. Inagaki, Y. Mori / Hormones and Behavior 65 (2014) 42–46
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Fig. 2. The total duration of 22-kHz calls (a), the total number of 22-kHz calls (b), the total duration of freezing (c), and the total number of fecal boli (d) observed in male rats after they received an air puff stimulus in Experiment 2. Each subject received one of four types of subcutaneous injection once a day for 7 consecutive days before the experiment: corn oil (Corn oil; n = 16), flutamide (FL, 50 mg/kg; n = 14), tamoxifen (TM, 1 mg/kg; n = 12), a binary mixture of flutamide and tamoxifen (FL + TM, 50 mg/kg and 1 mg/kg respectively; n = 16). Each bar represents the mean ± standard error; *p b 0.05.
in 22-kHz calls induced by low testosterone levels in this study was regulated by other mechanisms with little relation to the difference in testosterone-dependent stress responses in male rats. In the present study, we observed testosterone-dependent changes in the emission of 22-kHz calls that were not obviously correlated with typical stress responses (freezing and defecation) in male rats. One possible explanation of this is that stress-induced 22-kHz calls in male rats can be characterized not only as a simple stress response but also as a typical masculine behavior in rats. One observation that supports this suggestion is that dominant male rats in seminatural visible burrow systems are more likely than subordinate males to engage in the risk assessment component of sentinel behaviors (Blanchard et al., 1991). Their behavior may be related to the link between dominance and the emission of 22-kHz alarm calls, because dominant animals are more likely to be the first ones to detect danger (i.e., the existence of a predator) (Blanchard et al., 1991). Moreover, it is known that dominant–subordinate relationships can influence the emission of stress-induced 22-kHz calls in pair-housed male rats; the duration of 22-kHz calls may be associated with submissive behavior (Inagaki et al., 2005). These facts indicate that 22-kHz calls would possess an aspect of social biological function in contrast to stress-induced individual reactions such as the freezing behavior. Accordingly, it may be important to consider social hierarchical positions when using 22-kHz calls to evaluate negative affective states in male rats; however, future studies are of course necessary to confirm this because there could be complex interactions between steroid treatments and dominance status on 22-kHz calls.
This study shows that testosterone-dependent emission of stressinduced 22-kHz calls in male rats may be mediated via both testosterone and its metabolites, because testosterone can be aromatized to 17β-estradiol that binds to estrogen receptors, although testosterone does not bind to estrogen receptors in physiological concentrations (MacLusky et al., 1987). Similar hormonal mechanisms regulated by estrogen receptors have been reported in earlier studies that investigated the inhibitory effects of testosterone on HPA responses (Lund et al., 2006) and anxiety-related behaviors (Frye et al., 2008) in male rats. Other studies have demonstrated that the emission of 22-kHz calls is mediated by cholinergic projections to the hypothalamic preoptic area (Brudzynski, 1994; Brudzynski and Barnabi, 1996), which contains not only androgen and estrogen receptors but also the cytochrome P450 enzyme aromatase (Shughrue et al., 1997; Simerly et al., 1990; Zhao et al., 2007). Therefore, it is conceivable that complex mechanisms involving the interaction between two types of sex hormone ligands and receptors in the hypothalamus may be involved in testosteronedependent emission of stress-induced 22-kHz calls in male rats. On the other hand, there is also a possibility that the effects of two drugs may be attributable to an additive effect of one of two sex hormone receptors. Further studies are necessary to clarify this issue. In conclusion, the present study shows that testosterone may play an important role in the regulation of stress-induced 22-kHz calls in male rats. Recent studies have shown that 22-kHz calls are inextricably linked to negative affective states in rats (Brudzynski, 2009; Sanchez, 2003). Our data may provide new insight into the neurohormonal background of the negative affective vocalizations in rats. Further research in
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this area may shed light on the underpinning of human psychiatric conditions associated with fear and anxiety. Acknowledgment This study was supported by Grant-in-Aid for Scientific Research (C) (22500382) from the Japan Society for the Promotion of Science. References Albert, D.J., Walsh, M.L., Gorzalka, B.B., Siemens, Y., Louie, H., 1986. Testosterone removal in rats results in a decrease in social aggression and a loss of social dominance. Physiol. Behav. 36, 401–407. Bernhardt, P.C., 1997. Influences of serotonin and testosterone in aggression and dominance: convergence with social psychology. Curr. Dir. Psychol. Sci. 6, 44–48. Blanchard, R.J., Blanchard, D.C., Agullana, R., Weiss, S.M., 1991. Twenty-two kHz alarm cries to presentation of a predator, by laboratory rats living in visible burrow systems. Physiol. Behav. 50, 967–972. Bonson, K.R., Winter, J.C., 1992. Reversal of testosterone-induced dominance by the serotonergic agonist quipazine. Pharmacol. Biochem. Behav. 42, 809–813. Brudzynski, S.M., 1994. Ultrasonic vocalization induced by intracerebral carbachol in rats: localization and a dose-response study. Behav. Brain Res. 63, 133–143. Brudzynski, S.M., 2009. Communication of adult rats by ultrasonic vocalization: biological, sociobiological, and neuroscience approaches. ILAR J. 50, 43–50. Brudzynski, S.M., Barnabi, F., 1996. Contribution of the ascending cholinergic pathways in the production of ultrasonic vocalization in the rat. Behav. Brain Res. 80, 145–152. Brudzynski, S.M., Holland, G., 2005. Acoustic characteristics of air puff-induced 22-kHz alarm calls in direct recordings. Neurosci. Biobehav. Rev. 29, 1169–1180. Frye, C.A., Koonce, C.J., Edinger, K.L., Osborne, D.M., Walf, A.A., 2008. Androgens with activity at estrogen receptor beta have anxiolytic and cognitive-enhancing effects in male rats and mice. Horm. Behav. 54, 726–734. Handa, R.J., Nunley, K.M., Lorens, S.A., Louie, J.P., Mcgivern, R.F., Bollnow, M.R., 1994. Androgen regulation of adrenocorticotropin and corticosterone secretion in the male rat following novelty and foot shock stressors. Physiol. Behav. 55, 117–124.
Inagaki, H., Kuwahara, M., Kikusui, T., Tsubone, H., 2005. The influence of social environmental condition on the production of stress-induced 22 kHz calls in adult male Wistar rats. Physiol. Behav. 84, 17–22. Inagaki, H., Takeuchi, Y., Mori, Y., 2012. Close relationship between the frequency of 22-kHz calls and vocal tract length in male rats. Physiol. Behav. 106, 224–228. Knapp, D.J., Pohorecky, L.A., 1995. An air-puff stimulus method for elicitation of ultrasonic vocalizations in rats. J. Neurosci. Methods 62, 1–5. Litvin, Y., Blanchard, D.C., Blanchard, R.J., 2007. Rat 22 kHz ultrasonic vocalizations as alarm cries. Behav. Brain Res. 182, 166–172. Lund, T.D., Hinds, L.R., Handa, R.J., 2006. The androgen 5α-dihydrotestosterone and its metabolite 5α-androstan-3β,17β-diol inhibit the hypothalamo pituitary-adrenal response to stress by acting through estrogen receptor β-expressing neurons in the hypothalamus. J. Neurosci. 26, 1448–1456. MacLusky, N.J., Clark, A.S., Naftolin, F., Goldman-Rakic, P.S., 1987. Estrogen formation in the mammalian brain: possible role of aromatase in sexual differentiation of the hippocampus and neocortex. Steroids 50, 459–474. Million, M., Grigoriadis, D.E., Sullivan, S., Crowe, P.D., McRoberts, J.A., Zhou, H.P., Saunders, P.R., Maillot, C., Mayer, E.A., Tache, Y., 2003. A novel water-soluble selective CRF1 receptor antagonist, NBI 35965, blunts stress-induced visceral hyperalgesia and colonic motor function in rats. Brain Res. 985, 32–42. Panksepp, J., Burgdorf, J., Beinfeld, M.C., Kroes, R.A., Moskal, J.R., 2004. Regional brain cholecystokinin changes as a function of friendly and aggressive social interactions in rats. Brain Res. 1025, 75–84. Rodgers, R.J., 1997. Animal models of 'anxiety': where next? Behav. Pharmacol. 8, 477–496. Sanchez, C., 2003. Stress-induced vocalisation in adult animals. A valid model of anxiety? Eur. J. Pharmacol. 463, 133–143. Shughrue, P.J., Lane, M.V., Merchenthaler, I., 1997. Comparative distribution of estrogen receptor-α and -β mRNA in the rat central nervous system. J. Comp. Neurol. 388, 507–525. Simerly, R.B., Chang, C., Muramatsu, M., Swanson, L.W., 1990. Distribution of androgen and estrogen receptor messenger RNA-containing cells in the rat-brain: an in situ hybridization study. J. Comp. Neurol. 294, 76–95. Toufexis, D., Davis, C., Hammond, A., Davis, M., 2005. Sex differences in hormonal modulation of anxiety measured with light-enhanced startle: possible role for arginine vasopressin in the male. J. Neurosci. 25, 9010–9016. Zhao, C.J., Fujinaga, R., Tanaka, M., Yanai, A., Nakahama, K.I., Shinoda, K., 2007. Regionspecific expression and sex-steroidal regulation on aromatase and its mRNA in the male rat brain: immunohistochemical and in situ hybridization analyses. J. Comp. Neurol. 500, 557–573.
Contents lists available at ScienceDirect
Hormones and Behavior journal homepage: www.elsevier.com/locate/yhbeh
Relationship between 22-kHz calls and testosterone in male rats Hideaki Inagaki ⁎, Yuji Mori Laboratory of Veterinary Ethology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
a r t i c l e
i n f o
Article history: Received 22 August 2013 Revised 8 November 2013 Accepted 15 November 2013 Available online 23 November 2013 Keywords: Hormone receptor Sex hormone Stress response Ultrasonic vocalization Vocal communication
a b s t r a c t Ultrasonic calls in rats induced by the presence of a predator, referred to as “22-kHz calls,” are mainly emitted by socially dominant male rats. Testosterone levels are closely related to social dominance in male rats. In the present study, we investigated the relationship between the emission of stress-induced 22-kHz calls and circulating testosterone levels in male rats, using a combination of surgery (castration or sham operation) and chronic steroid administration (testosterone or cholesterol) to modify circulating testosterone levels. We also assessed the effects of androgen and/or estrogen receptor antagonists on the emission of 22-kHz calls in male rats. An air puff stimulus, known to reliably induce 22-kHz calls in rats, was used as a stressor. Castrated rats with cholesterol implants exhibited significantly fewer 22-kHz calls than rats that had received a sham operation and cholesterol implants, and there was no significant difference between castrated rats with testosterone implants and rats that had received a sham operation and cholesterol implants. Only male rats pretreated with a binary mixture of androgen and estrogen antagonists exhibited significantly fewer 22-kHz calls than controls. These results show that testosterone in male rats has a positive effect on the emission of stress-induced 22-kHz calls, and the calls may be regulated by the activation of both androgen and estrogen receptors. © 2013 Elsevier Inc. All rights reserved.
Introduction Rats emit social vocalizations in the ultrasonic range (above 20 kHz) in a variety of aversive or appetitive situations (Brudzynski, 2009). Adult rats emit long (usually between 0.5 and 3.0 s for a single call) ultrasonic bouts of 20–30 kHz with a narrow bandwidth of 1–4 kHz, referred to as “22-kHz calls”. These ultrasonic calls are typically responses to potentially harmful or life-threatening situations or to the expectation of a known unpleasant stimulus even if the rat lacks exact information about when it will happen. For example, rats call in the presence of a predator (Blanchard et al., 1991), when they confront a dominant and aggressive rat (Panksepp et al., 2004), and even when they receive a light but sudden and unpredictable air puff (Knapp and Pohorecky, 1995) or tactile stimulus (Inagaki et al., 2005). Moreover, it is widely accepted that 22-kHz calls serve as alarm calls to warn conspecifics of external danger; namely, 22-kHz calls are considered to be one of social communicative behaviors (Litvin et al., 2007). A male rat's emission of 22-kHz calls can be influenced by the rat's social hierarchical position; when a predator (such as a cat) is presented to groups that include several male and female rats in seminatural visible burrow systems, it is usually the socially dominant males that emit 22-kHz calls (Blanchard et al., 1991). It is also well known that social status is closely related to testosterone levels in male rats (Bernhardt, 1997); for example, testosterone removal (castration) causes socially dominant rats to become submissive (Albert et al., 1986), and chronic ⁎ Corresponding author. Fax: +81 3 5841 8190. E-mail address: [email protected] (H. Inagaki). 0018-506X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yhbeh.2013.11.003
administration of testosterone to subordinate rats significantly increases their dominant behaviors (Bonson and Winter, 1992). On the basis of this information, we hypothesized that the testosterone can influence the emission of 22-kHz calls in male rats, although no systematic analysis regarding such relationship has been reported. To test our hypothesis, we investigated the relationship between the total number and duration of stress-induced 22-kHz calls and circulating testosterone levels in male rats using a combination of surgery (castration or sham operation) and chronic steroid administration (testosterone or cholesterol) (Experiment 1). We also assessed the effects of androgen and/or estrogen receptor antagonists on the emission of 22-kHz calls in male rats (Experiment 2). Materials and methods Animals A total of 98 male Wistar rats (Clea Japan, Tokyo, Japan) were used in this study. All animals were housed in pairs in wire-topped transparent cages (400 × 250 × 180 mm) with wood shavings for bedding. They were provided with water and food ad libitum and maintained on a 12-h light–dark cycle (lights turned off at 20:00). The cages were maintained at a constant temperature (24 ± 1 °C) and humidity (40–45%). Pretreatment before experiments In Experiment 1, 40 rats aged 8 weeks received one of three treatments. A total of 12 rats were castrated and implanted with silicon
H. Inagaki, Y. Mori / Hormones and Behavior 65 (2014) 42–46
tubes (1.57-mm inner diameter, 3.18-mm outer diameter; Kaneka Medix Corporation, Osaka, Japan) containing cholesterol (Wako Pure Chemical Industries, Osaka, Japan). A total of 14 rats were castrated and implanted with silicon tubes containing testosterone (Wako Pure Chemical Industries), and 14 rats received sham operations and were implanted with silicon tubes containing cholesterol. We packed a 10-mm length of each tube with cholesterol or testosterone and closed each end of the tube with a glass bead (3 mm in diameter). Each subject received a single implantation of either tube. All operations were performed under inhalation anesthesia with ethyl ether. In Experiment 2, 58 rats aged 10 weeks received one of four types of treatment featuring subcutaneous injections once a day for 7 consecutive days. A total of 14 rats received an androgen receptor antagonist, flutamide (Wako Pure Chemical Industries; 50 mg/kg, 50 mg flutamide dissolved in 1 ml of vehicle); 12 rats received an estrogen receptor antagonist, tamoxifen (MP Biomedicals, Solon, OH, USA; 1 mg/kg, 1 mg tamoxifen dissolved in 1 ml of vehicle); 16 rats were treated with a binary mixture of the two drugs (flutamide 50 mg/kg and tamoxifen 1 mg/kg, 50 mg flutamide and 1 mg tamoxifen dissolved in 1 ml of vehicle), and 16 rats received corn oil as a control (Wako Pure Chemical Industries; 1 ml/kg). Experimental apparatus and procedures All experiments were performed when subjects were 11 weeks of age. At the beginning of the experiment they were moved to a soundproofed experimental room and remained in their own cages for at least 60 min. Each animal was then transferred to a wire-topped transparent experimental cage (400 × 250 × 200 mm) and habituated to the cage for 5 min. After 5 min, the wire lid was removed and the animal received an air puff stimulus, which is known to reliably induce 22-kHz calls in rats (Brudzynski and Holland, 2005; Inagaki et al., 2012; Knapp and Pohorecky, 1995). A total of 30 air puffs with an interstimulus interval of 2 s were directed to the nape of each subject's neck, delivered from a nozzle (10 mm outer diameter and 2 mm caliber) at a distance of approximately 50 mm from the subject. The pressure of the air puff was maintained at 0.3 MPa by a pressure valve, following procedures used in earlier studies (Brudzynski and Holland, 2005; Knapp and Pohorecky, 1995). Immediately after the air puff stimuli, we placed the wire lid on the experimental cage and recorded 22-kHz calls for 5 min using an ultrasound microphone (Condenser Microphone CM16/CMPA; Avisoft Bioacoustics, Berlin, Germany) set at a distance of 50 mm from the top of the wire lid. Data acquisition hardware (UltraSoundGate 116Hbm; Avisoft Bioacoustics) and recording software (Avisoft-RECORDER Version 4.0; Avisoft Bioacoustics) on a personal computer were used. Settings included a sampling rate of 100 kHz and a 16-bit format. The abovementioned sequence of air puff stimuli and the recording of 22kHz calls was repeated three times for each subject. During the recording of 22-kHz calls, the behaviors of subjects were simultaneously videorecorded (HDR-SR12; Sony, Tokyo, Japan). After the experiment, the number of fecal boli excreted by each subject was counted. All experimental procedures were conducted between 10:00 and 15:00. For Experiment 1 only, 7 days after the experiment, we anesthetized each subject with ethyl ether and collected blood (6 − 8 ml, for euthanasia) from the abdominal aorta into a heparinized conical tube. Each blood sample was maintained at 4 °C and centrifuged to obtain plasma, which was then frozen at − 80 °C. We collected all blood samples between 13:00 and 15:00. This study was approved by the Animal Care and Use Committee of the Faculty of Agriculture, The University of Tokyo. Data analyses For spectrogram generation, recordings were transferred to AvisoftSASLab Pro (Version 5.1; Avisoft Bioacoustics) and a fast Fourier transformation (FFT) was conducted. Spectrograms were generated with
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an FFT-length of 512 points and a time window overlap of 50% (100% Frame, FlatTop window). We defined 22-kHz calls as long calls (0.5 − 3.0 s) in the ultrasonic range within a narrow band of peak frequencies (20 − 23 kHz) with a narrow bandwidth (1 − 4 kHz) according to an earlier report (Brudzynski, 2009).We recorded the total number of 22-kHz calls and also added together all calls from each subject to generate the total duration of calls. The calls were measured automatically using Avisoft-SASLab Pro. In addition, we calculated the duration of video-recorded freezing responses during the time that 22-kHz calls were recorded (15 min). Freezing in this study was defined as an immobile posture with cessation of skeletal and vibrissae movement except for respiration. Plasma testosterone levels in Experiment 1 were measured by enzyme immunoassay (Testosterone EIA kit; Cayman Chemical Company, Ann Arbor, MI, USA). Statistical analyses All data were displayed as the mean ± standard error. Statistical comparisons were performed using a one-way analysis of variance (ANOVA) followed by the post hoc Dunnett test. The criterion for statistical significance was p b 0.05 for all comparisons. In experiment 2, effect sizes (η2 for the one-way ANOVA and Cohen's d for the post hoc test) were calculated to evaluate magnitudes of effects of drug treatments on emission of 22-kHz calls (η2: large = 0.14, medium = 0.06, small = 0.01; Cohen's d: large = 0.80, medium = 0.50, small = 0.20). Results Experiment 1: Relationship between the emission of 22-kHz calls and circulating testosterone levels The total duration of air puff-induced 22-kHz calls was significantly affected by treatment (F2,37 = 6.65, p b 0.01). The post hoc test indicated that castrated rats with cholesterol implants exhibited significantly shorter 22-kHz calls than rats that had received a sham operation and cholesterol implants (p b 0.01). There was no significant difference between castrated rats with testosterone implants and rats that had received a sham operation and cholesterol implants (Fig. 1a). The total number of air puff-induced 22-kHz calls was also significantly affected by treatment (F2,37 = 4.48, p b 0.05). The post hoc test indicated that castrated rats with cholesterol implants exhibited significantly fewer 22-kHz calls than rats that had received sham operations and cholesterol implants (p b 0.05). There was no significant difference between castrated rats with testosterone implants and rats that had received sham operations and cholesterol implants (Fig. 1b). In contrast, the total duration of freezing and the total number of fecal boli produced were not significantly affected by treatment (freezing: F2,37 = 1.94, p = 0.14, fecal boli: F2,37 = 0.33, p = 0.72) (Fig. 1c, d). Plasma testosterone levels were significantly affected by treatment (F2,37 = 30.5, p b 0.01). The post hoc test indicated that castrated rats with cholesterol implants had significantly lower plasma testosterone levels (1.60 ± 0.11 × 10−2 ng/ml) than rats that had received a sham operation and cholesterol implants (2.42 ± 0.34 ng/ml) (p b 0.01). There was no difference between castrated rats with testosterone implants (2.11 ± 0.17 ng/ml) and rats that had received a sham operation and cholesterol implants. Experiment 2: Effects of sex hormone receptor antagonists on the emission of 22-kHz calls The total duration of air puff-induced 22-kHz calls was significantly affected by treatment (F3,54 = 3.08, p b 0.05). The effect size was large (η2 = 0.15). The post hoc test indicated that only rats pretreated with a binary mixture of flutamide and tamoxifen exhibited significantly shorter 22-kHz calls than control rats (p b 0.05) (Fig. 2a), and the effect size for paired comparisons was larger (Cohen's d = 1.10) than those of
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Fig. 1. The total duration of 22-kHz calls (a), the total number of 22-kHz calls (b), the total duration of freezing (c), and the total number of fecal boli (d) observed in male rats after they received an air puff stimulus in Experiment 1. Each subject received one of three types of pretreatment 3 weeks before the experiment: a sham operation with a cholesterol implant (Sham + C, n = 14), castration with a cholesterol implant (Cast + C, n = 12), castration with a testosterone implant (Cast + T, n = 14). Each bar represents the mean ± standard error; **p b 0.01, *p b 0.05.
other two paired comparisons (pretreatment with flutamide and control: Cohen's d = 0.27, pretreatment with tamoxifen and control: Cohen's d = 0.02). Although the total number of air puff-induced 22kHz calls was not significantly affected by treatment (F3,54 = 2.07, p = 0.12) and the effect size was medium (η2 = 0.10), the post hoc test indicated that rats pretreated with a binary mixture of flutamide and tamoxifen exhibited significantly fewer 22-kHz calls than control rats (p b 0.05) (Fig. 2b). The effect size for paired comparisons between the two was larger (Cohen's d = 0.86) than those of other two paired comparisons (pretreatment with flutamide and control: Cohen's d = 0.24, pretreatment with tamoxifen and control: Cohen's d = 0.15). The total duration of freezing and the total number of fecal boli produced were not significantly affected by treatment (freezing: F3,54 = 0.27, p = 0.85, fecal boli: F3,54 = 0.60, p = 0.61) (Fig. 2c, d). Discussion This study measured the role of testosterone in the emission of stress-induced 22-kHz calls in male rats using an air puff stimulus as a stressor. In Experiment 1, removal of testosterone by castration significantly reduced the rats' emission of air puff-induced 22-kHz calls, but the calls returned to pre-castration levels when castrated rats received testosterone implants. In Experiment 2, rats displayed significantly fewer air puff-induced 22-kHz calls when they were pretreated with both an androgen receptor antagonist and an estrogen receptor antagonist simultaneously, although pretreatment with only one of the
two drugs did not have a significant and effective effect. The results of this study show that testosterone in male rats has a positive effect on emission of stress-induced 22-kHz calls, and calling behavior may be regulated by the activation of both androgen and estrogen receptors. Although behavioral, physiological, neuroanatomical, and pharmacological studies indicate that 22-kHz calls are a valid measure of stress-induced negative affective states in rats (Sanchez, 2003), it is probable that the reduction in 22-kHz calls induced by low testosterone levels was not due to a reduction in stress responses in the castrated animals. Earlier studies have demonstrated that testosterone levels influence stress responses in rats. For example, rats show an increased acoustic startle reflex in the presence of bright ambient light, a behavior called light-enhanced startle (LES); LES is seen reliably in castrated male rats but not as reliably in gonadally intact male rats, and testosterone replacement attenuates the LES in the castrated rats (Toufexis et al., 2005). A footshock or exposure to a novel open field increases the incidence of a well-known hormonal stress response, the hypothalamic pituitary adrenal (HPA) response. The HPA response is greater in castrated rats than in intact rats, and testosterone treatment of castrated animals returns their post-stress display of the HPA response to intact levels (Handa et al., 1994). Testosterone had the opposite effect in these studies to its effect on stress-induced 22-kHz calls in our present study. Furthermore, low testosterone levels in our present study did not affect rats' freezing responses or defecation, both of which are well known to be typical behavioral stress responses in rats (Million et al., 2003; Rodgers, 1997). These findings suggest that the reduction
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Fig. 2. The total duration of 22-kHz calls (a), the total number of 22-kHz calls (b), the total duration of freezing (c), and the total number of fecal boli (d) observed in male rats after they received an air puff stimulus in Experiment 2. Each subject received one of four types of subcutaneous injection once a day for 7 consecutive days before the experiment: corn oil (Corn oil; n = 16), flutamide (FL, 50 mg/kg; n = 14), tamoxifen (TM, 1 mg/kg; n = 12), a binary mixture of flutamide and tamoxifen (FL + TM, 50 mg/kg and 1 mg/kg respectively; n = 16). Each bar represents the mean ± standard error; *p b 0.05.
in 22-kHz calls induced by low testosterone levels in this study was regulated by other mechanisms with little relation to the difference in testosterone-dependent stress responses in male rats. In the present study, we observed testosterone-dependent changes in the emission of 22-kHz calls that were not obviously correlated with typical stress responses (freezing and defecation) in male rats. One possible explanation of this is that stress-induced 22-kHz calls in male rats can be characterized not only as a simple stress response but also as a typical masculine behavior in rats. One observation that supports this suggestion is that dominant male rats in seminatural visible burrow systems are more likely than subordinate males to engage in the risk assessment component of sentinel behaviors (Blanchard et al., 1991). Their behavior may be related to the link between dominance and the emission of 22-kHz alarm calls, because dominant animals are more likely to be the first ones to detect danger (i.e., the existence of a predator) (Blanchard et al., 1991). Moreover, it is known that dominant–subordinate relationships can influence the emission of stress-induced 22-kHz calls in pair-housed male rats; the duration of 22-kHz calls may be associated with submissive behavior (Inagaki et al., 2005). These facts indicate that 22-kHz calls would possess an aspect of social biological function in contrast to stress-induced individual reactions such as the freezing behavior. Accordingly, it may be important to consider social hierarchical positions when using 22-kHz calls to evaluate negative affective states in male rats; however, future studies are of course necessary to confirm this because there could be complex interactions between steroid treatments and dominance status on 22-kHz calls.
This study shows that testosterone-dependent emission of stressinduced 22-kHz calls in male rats may be mediated via both testosterone and its metabolites, because testosterone can be aromatized to 17β-estradiol that binds to estrogen receptors, although testosterone does not bind to estrogen receptors in physiological concentrations (MacLusky et al., 1987). Similar hormonal mechanisms regulated by estrogen receptors have been reported in earlier studies that investigated the inhibitory effects of testosterone on HPA responses (Lund et al., 2006) and anxiety-related behaviors (Frye et al., 2008) in male rats. Other studies have demonstrated that the emission of 22-kHz calls is mediated by cholinergic projections to the hypothalamic preoptic area (Brudzynski, 1994; Brudzynski and Barnabi, 1996), which contains not only androgen and estrogen receptors but also the cytochrome P450 enzyme aromatase (Shughrue et al., 1997; Simerly et al., 1990; Zhao et al., 2007). Therefore, it is conceivable that complex mechanisms involving the interaction between two types of sex hormone ligands and receptors in the hypothalamus may be involved in testosteronedependent emission of stress-induced 22-kHz calls in male rats. On the other hand, there is also a possibility that the effects of two drugs may be attributable to an additive effect of one of two sex hormone receptors. Further studies are necessary to clarify this issue. In conclusion, the present study shows that testosterone may play an important role in the regulation of stress-induced 22-kHz calls in male rats. Recent studies have shown that 22-kHz calls are inextricably linked to negative affective states in rats (Brudzynski, 2009; Sanchez, 2003). Our data may provide new insight into the neurohormonal background of the negative affective vocalizations in rats. Further research in
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