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Deficits in testosterone facilitate enhanced fear response
Psychoneuroendocrinology (2005) 30, 333–340
www.elsevier.com/locate/psyneuen
Deficits in testosterone facilitate enhanced fear response* Jean A. Kinga,b,*, Washington L. De Oliveiraa,b, Nihal Patela,b a
Department of Psychiatry, University of Massachusetts Medical School, Worcester, MA 01655, USA Center for Comparative NeuroImaging, University of Massachusetts Medical School, Worcester, MA 01655, USA
b
Received 16 February 2004; received in revised form 10 April 2004; accepted 30 September 2004
KEYWORDS Testosterone; Unconditioned fear response; Trimethylthiazoline (TMT); Freezing; Analgesia
Summary Testosterone has been implicated in many behaviors related to sexual and reproductive function, but its role in fear responses is unclear. Studies in both humans and animals have linked altered testosterone concentrations to externalizing behaviors like aggression and violence, but less to more internalizing behaviors like fear. Therefore, the current study was designed to investigate the effects of testosterone on innate fear response in male rats. TMT (2,5-dihydo-2,4,5trimethylthiazoline), a chemical extracted from fox feces, was used to elicit a fear response in the male rats with normal and diminished levels of testosterone. Behavioral indices such as in freezing response, and fear-induced analgesia were monitored in response to TMT. The results demonstrate that deficits in testosterone resulted in a significant increase in the freezing time and fear-induced analgesia. These studies suggest that testosterone decline may have a significant effect on increasing innate fear response and fear-induced enhancement of analgesia in male rats. Q 2004 Elsevier Ltd. All rights reserved.
1. Introduction Fear is a physiological response to a dangerous occurrence (Rosen and Schulkin, 1998), and is *
This publication was made possible by Grant Number R01 MH067096-01 from the National Institute of Mental Health. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIMH. * Corresponding author. Address: Center for Comparative NeuroImaging, Department of Psychiatry, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA. Tel.: C1 508 856 4979; fax: C1 508 856 8090. E-mail address: [email protected] (J.A. King).
essential for an organism’s survival. Extensive research investigating neuroanatomical regions that sub-serve the fear response has, through the utilization of conditioning paradigms, contributed to most of our current understanding of the mechanism of phobia (LeDoux, 2000). Phobias are the most prevalent form of anxiety disorders, affecting 15% of Americans. It is also the second most common psychiatric illness among men over 25 years of age (US Dept. of Health and Human Services, 1999). Sex differences in fear responses have been observed in rodents (Archer, 1975; Stock et al.,
0306-4530/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.psyneuen.2004.09.005
334 2001), domestic fowl (Jones, 1987), dogs (Goddard and Beilharz, 1984), sheep (Vandenheede and Bouissou, 1993), and man (Gray, 1987; Fredrikson et al., 1996). Women are more than twice as likely to suffer from phobias and four times more likely to have multiple phobias (Fredrikson et al., 1996), suggesting that the response to fear-inducing stimuli may be sexually dimorphic with females being more fearful than males (Gray, 1987). In addition, women have been shown to have more animal and situational phobias than men (Lindal and Stefansson, 1993; Fredrikson et al., 1996), suggesting that the potency of particular fearinducing stimuli may differ by gender. Although these observations lend support to the view that the hormonal milieu may be a contributing factor in the expression of fear and phobias, the role of gonadal hormones like testosterone, in modulation of the fear response remains under-explored and therefore not very well understood. Most studies investigating innate fear assess the response to different aspects of predator exposure or interaction. One of the most specific response measures is freezing or immobility, a state in which all movement ceases with the exception of those necessary for breathing (Godsil et al., 2000). Furthermore, when innate fear was studied in rats using the odor of a cat as the fear-inducing stimulus (Blanchard et al., 1989; Zangrossi and File, 1992), the researchers showed that exposure to cat odor resulted in a significant increase in plasma corticosterone concentrations and avoidance behavior, but freezing behavior occurred only briefly or not at all. Meanwhile, in a more recent approach, Wallace and Rosen (2000) demonstrated that 2,5-dihydro2,4,5-trimethylthiazoline (TMT), a sulfur-containing chemical extracted from fox feces, elicits a freezing response in laboratory-bred rats. Since, there is evidence indicating that TMT does not provoke fear responses in conditioned fear tests (Vernet-Maury et al., 1984; Wallace and Rosen, 2000), it was reasoned that TMT may be more selective for investigating unconditioned fear. Unlike fear-inducing stimuli such as cat fur odor, which has not been isolated and probably contains several components, TMT is a single molecule (Wallace and Rosen, 2000). Thus, using TMT as the fear-inducing stimulus, the present study was undertaken to investigate the effects of testosterone on the innate fear response of male rats and attempted to test the hypothesis that testosterone depletion may heighten innate fear response in these rats. Moreover, in order to assess further the potency of the fear reaction, nociceptive threshold was monitored subsequent to exposure to the fearinducing stimulus.
J.A. King et al.
2. Methods 2.1. Animals Adult male Sprague–Dawley (SD) rats (nZ30) were obtained from Harlan Sprague–Dawley Laboratories (Indianapolis, IN). At the beginning of the experiment, the rats weighed approximately 200 g. The animals were group-housed (2 per cage) according to their treatment conditions, normal, sham-operated or castrate. They were maintained in ambient temperature (22–24 8C) on a 12 h light/dark cycle (light on from 07:00 to 19:00). The rats had access to commercial pellet food and water ad libitum. All animals were acquired and cared for in accordance with guidelines published in the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication N. 80-23, revised 1996). This study was approved by the (International Animal Care and Use Committee, IACCUC) committee of the University of Massachusetts Medical School.
2.2. Surgery Male SD rats (nZ10 per group) were randomly assigned to one of three groups: normal (intact), castrated, or sham-operated. For surgery, the animals were anesthetized with 0.4 ml of ketamine (Ketaset; Fort Dodge Animal Health, Fort Dodge, IA) and 0.25 ml of medetomidine (Dormitor; Pfizer Animal Health, New York, NY). A 2 cm ventral incision was then made in the scrotum and the skin was retracted to expose the tunica. The tunica was pierced, and the aperture was stretched with blunt forceps. The testes were exposed by applying gentle pressure to the pelvic region. The spermatic artery was clamped and cauterized, and the testes were removed. The epididymis, vas deferens and ducts were replaced into the tunica. The animals were injected with 0.3 ml of atipamezole (Antisedan; Pfizer Animal Health, New York, NY) to reverse the anesthesia. Sham-operated animals were exposed to the entire procedure noted above with the exception of the removal of the testes.
2.3. Blood sampling and T-level measurement Blood samples were collected from the entire cohort of animals (nZ10/group) on baseline (day before surgery); days 7 and 21 (post-surgery) via a tail venous puncture. The blood samples were then centrifuged at 5000!g for 15 min and
Deficits in testosterone facilitate enhanced fear response plasma was collected. The plasma samples were stored at K80 8C until assays were performed. Plasma testosterone levels were determined with the aid of a RIA kit obtained from ICN Biomedicals, Inc. (catalog number 07-289102) Costa Mesa, CA. The specificity of the testosterone antiserum was 100% for testosterone, approximately 7% for 5 alpha-dihydrotestosterone and less than 0.10% for hydroxytestosterone, estrone, progesterone, and corticosterone.
2.4. Sexual interest test The animals were removed from their home cage and brought to a behavioral testing room adjacent to the holding area. Sexual interest monitoring was performed in the entire cohort of animals (nZ 10/group) on days 7 and 14 (post-surgery). A female rat in the estrous phase was introduced into a cage of the male rat in order to assess his sexual interest, which was measured by the number of times the male rat mounted the female rat.
2.5. Freezing time upon exposure to fox (TMT) scent Freezing time measurements were evaluated on days 7, 14, 21 and 28 post castration in the entire cohort of animals. The freezing response consisted of cessation of all movement except those necessary for breathing (Godsil et al., 2000). Male SD rats (castrated, normal, sham-operated (nZ10/group) were removed from their home cage and brought to an adjoining laboratory. Twenty-four hours prior to testing, each animal was placed in a clean Plexiglas cage identical to their home cage (absent bedding), as a means of acclimating to the novel environment. They were observed under this condition for 5 min. For testing, the rat was placed in an identical Plexiglas cage and the predator odor scent was introduced in the form of 2,5-dihydro-2,4,5-trimethylthiazoline (TMT). TMT was obtained from Phero Tech, Inc. (British Columbia, Canada). A swab containing 100 ml of the odor was placed on a Whatman filter paper and hung over the cage of the experimental group for 5 min. During the 5 min period, fear related behaviors were monitored, including the total freezing time.
2.6. Tail flick test All three groups of animals were subjected to a tail flick test 30 min prior to exposure to TMT (control period) and 30 min post exposure to TMT. During this test, animals were held firmly while their entire
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tail (100 mm from attached region) was submerged into a beaker of water heated at 50 8C, and the time it took the animals to flick their tails out of the water was noted and recorded.
2.7. Statistical analyses Within group differences across the days for blood testosterone levels, sexual interest, and freezing time were analyzed using a repeated measures analysis of variance (ANOVA). A one-way ANOVA was used to analyze the difference between the three groups for blood testosterone levels, sexual interest, freezing time and tailflick. P%0.05 was considered significant for all statistical tests, and all data are presented as meanGSEM.
3. Results 3.1. Blood T-level A repeated measures analysis of variance showed a significant decrease in blood T levels for all 3 groups (normal, sham-operated, and castrated) on days 7 and 21 (F(2, 23)Z12.905, p%0.0002) when compared to baseline. Circulating blood testosterone levels were not significantly different among the groups at baseline (F (2, 23)Z1.430, p%0.2597). By post-castration day 7, T-levels of castrated rats decreased significantly (F (2, 23)Z7.586, p%0.003) when compared to normal and sham-operated rats (Fig. 1). The blood testosterone level of castrated rats decreased even further by 21 days postcastration (F (2, 23)Z7.039, p%0.004) compared to normal and sham-operated rats (Fig. 1).
3.2. Sexual interest Castrates showed a significant difference in sexual interest (as measured by the number of mountings in the presence of an estrus female) (F (2, 23)Z 9.873, p%0.0008) across days 7, 14, and 21. Furthermore, castrates were significantly different than normal and sham-operated animals on day 7 (F (2, 23)Z8.257, p%0.002) (Fig. 2). Similarly, on day 14 post castration, these rats continued to show a significant decrease in sexual interest compared to normal and sham-operated (F(2, 23)Z6.746, p%0.004) (Fig. 2).
3.3. Freezing response A repeated measures analysis of variance showed that fear response measured by changes in
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Figure 1 Bar graph depicting mean blood testosterone levels (GSEM) for normal (stripped bar), sham-operated (black bar) and castrated (white bar) rats. T levels for castrated rats decreased notably to 0.015 ng/ml by day 21 postcastration. The *indicates a significant difference between the groups on day 7 (p%0.002) and day 21 (p%0.004).
the freezing time with exposure to TMT (for animals from all three conditions) were significantly different across days 7, 14, 21 and 28 (F(2,23)Z10.615, p%0.0005). But castrates showed no significant difference in freezing
response 7 days post-castration (F(2,23)Z1.34, p%0.28) when compared to normal and shamoperated. However, by 14 days post-surgery, castrated rats showed a significant increase in their freezing response as compared to normal
Figure 2 Mean number of mountings (GSEM) for all three groups on days 7 (black bar) and 14 (square bar). The * indicates a significant difference between the groups on day 7 (p%0.001) and day 14 (p%0.004).
Deficits in testosterone facilitate enhanced fear response
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Figure 3 Mean differences in freezing time (GSEM) during fox scent exposure for all rat groups on days 7 (black bar), 14 (square bar), 21 (patterned bar) and 28 (white bar). The * indicates a significant difference between the groups on days 14 (p%0.001), 21 (p%0.0001) and 28 (p%0.0001).
and sham-operated (F(2,23)Z9.267, p%0.001) (Fig. 3). This increase in the freezing response time of castrated rats was also seen on day 21 postcastration (Fig. 3) when compared to normal and sham-operated animals (F(2,23)Z28.445, p%0.0001). Similarly, the increase in freezing time in response to TMT persisted until day 28 post-castration (F(2,23)Z16.867, p%0.0001 (Fig. 3).
3.4. Tailflick There were significant differences between the groups (normal, sham-operated and castrated) on day 14 (F(2,23)Z13.71, p%0.0001) (Fig. 4). These significant differences in mean latencies between the groups persisted until one week later (F(2,23)Z10.335, p%0.0001) (Fig. 5).
4. Discussion The main objective of this study was to investigate the effects of testosterone deficiency on innate or unconditioned fear response of male rats and to determine if prior exposure to a fear-inducing stimulus altered their nociceptive threshold. Our results show that decreased circulating blood testosterone levels were accompanied by
an increase in fear response, as demonstrated by the significant increase in freezing time postexposure to TMT. However, this response is apparently reflective of the organization effects of sex steroids, like testosterone (Hebbard et al., 2003). Since, the present study demonstrated that although T levels were significantly diminished by day 7, it did not alter the freezing response in castrates until T-levels were chronically low. That is, the initial significant response to fear-inducing odor was observed on day 14 and persisted until 28 days post-castration (last time point assessed). Previous studies have investigated the role of testosterone in the fear response in other species, particularly ewes and heifers (Vandenheed and Bouissou, 1993; Boissy and Bouissou, 1994; Bouissou and Vanderheed, 1996). These researchers looked at the long-term effect (4–8 months post-treatment) of testosterone treatment. Their results indicated that increases in testosterone correlated with a reduction in fear, as manifested by decreases in trotting, immobilization and flinching in response to classically reported fear-inducing stimuli in sheep. These fear-inducing stimuli included isolation from conspecifics, surprise (novel red ball falling from the ceiling) and the presence of a human, thus supporting the organizational effect of testosterone on the fear response. In addition, previous research in mice has reported that
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Figure 4 Mean tail flick latencies (GSEM) of normal (stripped bar), sham-operated (black bar), and castrated rats (white bar) during pre and post exposure to fox scent on day 14. The * indicates a significant difference (p%0.0001) between the groups post fox scent.
an acute testosterone injection resulted in a decrease in unconditioned fear response to predator odor within 30 min (Domek et al., 1992). Taken together, these studies suggest that the impact of T-levels on the fear response may be
reflective of the duration of treatment. Nevertheless, one cannot disregard the possibility that the reported change in freezing associated with decrease T-levels maybe due to either direct or indirect alterations in HPG and HPA modulators
Figure 5 Mean tail flick latencies (GSEM) for normal (stripped bar), sham-operated (black bar), and castrated rats (white bar) during pre and post exposure to fox scent on day 21. The * indicates a significant difference (p%0.0001) between the groups post fox scent.
Deficits in testosterone facilitate enhanced fear response affected by T (Dafopoulos et al., 2004; Viau and Meaney, 2004). In addition, the fear responses noted in the presence of TMT may be dependent on the limited size of enclosure used for testing (Wallace and Rosen, 2000). Although the studies linking T to fear are limited, the connection between androgens and other behaviors, including aggression, dominance, and mood disorders are numerous. For example, in a study investigating the dose–response relationship among T levels, behavior and mood in eugonadal and hypogonadal men, O’Connor and colleagues (2002) reported that testosterone treatment led to a significant increase in verbal aggression, hostility, anger and irritability in hypogonadal subjects, but had no effect on T treated eugonadal subjects. This is supported by reports from other laboratories (Maras et al., 2003) correlating higher levels of testosterone with externalizing behavior, a type of overt aggression, in adolescent boys. Furthermore, it has been shown that anabolic steroids cause an increase in euphoria, irritability, mood swings, anger and hostility in healthy, non-athletic men (Su et al., 1993). In addition, there is also sparse support for the impact of T on internalizing behaviors. Reports have demonstrated that low levels of T are linked to depression in elderly males (Margolese, 2000). Recent studies using single photon emission-computed tomography has shown that testosterone replacement enhances blood perfusion in the midbrain and superior frontal gyrus in hypogonadal men (Azad et al., 2003). Similarly, alterations in blood flow to the brain have been linked independently to depression and fear in humans (Navarro et al., 2002; Wik et al., 1996). Taken together, these results lead to the speculation that decreased T levels may support decrease in brain perfusion. Another important finding of the current study is the apparent role of testosterone in modulating the analgesic response post-exposure to a fearinducing stimulus. Our data showed that 14 days post-castration, there was significant increase in tailflick latencies between castrated, normal and sham-operated rats after exposure to TMT. However, the increase seen in the castrates was significantly higher than the intact and sham operated groups. This suggests that the fear reaction in castrated rats triggered a significant increase in the nociceptive threshold beyond that seen in intact and sham-operated animals. The finding of a fear triggered increase in nociceptive threshold is supported by work in mice which demonstrated that fear-inducing stress increased nociceptive threshold (Suaudeau and Costentin,
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2000). In contrast, the tailflick response prior to the fear-inducing paradigm was not significantly different between the groups. In this regard, previous studies showed that short-term treatment with T increased tailflick latencies in T-treated gonadectomized male rats as compared to intact and gonadectomized rats (Forman et al., 1989; Frye and Seliga, 2001). Further studies examining a more systematic dose response curve and times between treatment and behavior may help clarify these differences. The present data on the analgesic effects postexposure to TMT explores a novel component of nociception in T-deficient animals. One possible reason for this heightened analgesic effect in castrated animals in response to TMT, may result from an enhanced release of endorphins (Suaudeau and Costentin, 2000) to fear stress. Previous studies using naltrexone, a prototypic opioid receptor antagonist, support the premise that stress-induced analgesia can be opioid mediated (Lapo et al., 2003). In summary, given the prominent effects of testosterone on a myriad of different behaviors, it is not surprising that testosterone may play a role in the fear response. Further studies on the involvement of neural pathways and brain mechanisms involved in hormonal substrates of the fear response are therefore warranted.
Acknowledgements We would like to thank Jeffrey Tenney and Victoria Rossi for their assistance with this project. This work was supported by National Institute of Mental Health Grant R01 MH067096 to J.A.K. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Mental Health.
References Archer, J., 1975. Rodent sex differences in emotional and related behaviour. Behav. Biol. 14, 451–479. Azad, N., Pitale, S., Barnes, W.E., Friedman, N., 2003. Testosterone treatment enhances regional brain perfusion in hypogonadal men. J. Clin. Endocr. Metab. 88, 3064–3068. Blanchard, R.J., Blanchard, D.C., Hori, K., 1989. An ethoexperimental approach to the study of defense, in: Blanchard, R.J., Brain, P.F., Blanchard, D.C., Parmigiani, S. (Eds.), Ethoexperimental approaches to the study of behavior. Kluwer Academic Press, Dordrecht, pp. 114–136. Boissy, A., Bouissou, M.F., 1994. Effect of androgen treatment on behavioral and physiological responses of heifers to feareliciting situations. Horm. Behav. 28, 66–83.
340 Bouissou, M.F., Vanderheed, M., 1996. Long-term effects of androgen treatment on fear reactions n ewes. Horm. Behav. 30, 93–99. Dafopoulos, K.C., Kotsovassilis, C.P., Milingos, S.D., Kallitsaris, A.T., Georgadakis, G.S., Sotiros, P.G., Messinis, I.E., 2004. FSH and LH responses to GnRH after ovariectomy in postmenopausal women. Clin. Endocri. 60, 120–124. Domek, D., Niekrasz, I., Garnica, A., Seane, T., 1992. The patent and rapid behavior-specific actions of androgens. Soc. Neurosci. Abstr. 18, 342, 814. Forman, L.J., Tinlge, V., Estilow, S., Cater, J., 1989. The response to analgesia testing is affected by gonadal steroids in the rat. Life Sci. 45, 447–454. Fredrikson, M., Annas, P., Fisher, H., Wik, G., 1996. Gender and age differences in the prevalence of specific fears and phobias. Behav. Res. Ther. 34, 33–39. Frye, C.A., Seliga, A.M., 2001. Testosterone increases analgesia, anxiolysis, and cognitive performance of male rats. Cogn. Affect. Behav. Neurosci. 1, 371–381. Goddard, M.E., Beilharz, R.G., 1984. A factor analysis of fearfulness in potential guide dogs. Appl. Anim. Behav. Sci. 12, 253–265. Godsil, B.P., Quinn, J.J., Fanselow, M.S., 2000. Body temperature as a conditional response measure for pavlovian fear conditioning. Learn. Mem. 7, 353–356. Gray, J.A., 1987. The psychology of fear and stress, second ed Cambridge University Press, Cambridge p. 442. Hebbard, P.C., King, R.R., Malsbury, C.W., Harley, C.W., 2003. Two organizational effects of pubertal testosterone in male rats: transient social memory and shift away from long-term potentiation following a tetanus in hippocampal CA1. Exp. Neurol. 182, 470–475. Jones, R.B., 1987. The assessment of fear in the domestic fowl, in: Zayam, R., Duncan, I.J.H. (Eds.), Cognitive aspects of social behaviour in the domestic fowl. Elsevier, Amsterdam, pp. 40–81. Lapo, I.B., Konarzewski, M., Sadowski, B., 2003. Effect of cold acclimation and repeated swimming on opioid and nonopioid swim stress-induced analgesia in selectively bred mice. Physiol. Behav. 78, 345–350. LeDoux, J.E., 2000. Cognitive–emotional interactions—listen to the brain, in: Lane, R., Nadel, L. (Eds.), Cognitive Neuroscience of Emotion. Oxford University Press, New York, pp. 129–155. Lindal, E., Stefansson, T., 1993. The lifetime prevalence of anxiety disorders in Iceland as estimated by the US National Institute of Mental Health Diagnostic Interview Schedule. Acta Psychiatr Scand 88, 29–34. Maras, A., Laucht, M., Gerdes, D., Wilhelm, C., Lewicka, S., Haack, D., Malisova, L., Schmidt, M.H., 2003. Association of
J.A. King et al. testosterone and dihydrotestosterone with externalizing behavior in adolescent boys and girls. Psychoneuroendocrinology 28, 932–940. Margolese, H.C., 2000. The male menopause and mood: testosterone decline and depression in the aging male—is there a link?. J. Geriatr. Psychiatry Neurol. 13, 93–101. Navarro, V., Gasto, C., Lomena, F., Mateos, J.J., Marcos, T., Portella, M.J., 2002. Normalization of frontal cerebral perfusion in remitted elderly major depression: a 12-month follow up SPECT study. Neuroimage 16, 781–787. O’Connor, D.B., Archer, J., Hair, W.M., Wu, F.C.W., 2002. Exogenous testosterone, aggression, and mood in eugonadal and hypogonadal men. Physiol. Behav. 75, 557–566. Rosen, J.B., Schulkin, J., 1998. From normal fear to pathological anxiety. Psychol. Rev. 105, 325–350. Stock, H.S., Caldarone, B., Abrahamsen, G., Mongeluzi, D., Wilson, M.A., Rosellini, R.A., 2001. Sex differences in relation to conditioned fear-induced enhancement of morphine analgesia. Physiol. Behav. 72, 439–447. Su, T.P., Pagliaro, M., Schmidt, P.J., Pickar, D., Wolkowitz, O., Rubinow, D.R., 1993. Neuropsychiatric effects of anabolic steroids in male normal volunteers. JAMA 269, 2760–2764. Suaudeau, C., Costentin, J., 2000. Long lasting increase in nociceptive threshold induced in mice by forced swimming: involvement of an endorphinergic mechanism. Stress 3, 221–227. US Department of Health and Human Services, 1999.Anon., 1999. Mental Health: A Report of the Surgeon General. US Department of Health and Human Services, Substance Abuse and Mental Health Services Administration, Center for Mental Health Services, National Institutes of Health, Rockville. Vandenheede, M., Bouissou, M.F., 1993. Effect of androgen treatment on fear reactions in ewes. Horm. Behav. 27, 435–448. Vernet-Maury, E., Polak, E.H., Demael, A., 1984. Structure– activity relationship of stress-inducing odorants in the rat. J. Chem. Ecol. 10, 1007–1008. Viau, V., Meaney, M.J., 2004. Testosterone-dependent variations in plasma and intrapituitary corticosteroid binding globulin and stress hypothalamic–pituitary–adrenal activity in the male rat. J. Endocrinol. 181, 223–231. Wallace, K.J., Rosen, J.B., 2000. Predator odor as unconditioned fear stimulus in rats: elicitation of freezing by trimethylthiazoline, a component of fox feces. Behav. Neurosci. 114, 912–922. Wik, G., Fredrikson, M., Fischer, H., 1996. Cerebral correlates of anticipated fear: a PET study of specific phobia. Int. J. Neurosci. 87, 267–276. Zangrossi Jr., H., File, S.E., 1992. Behavioral consequences in animal tests of anxiety and exploration of exposure to cat odor. Brain Res. Bull. 29, 381–388.
www.elsevier.com/locate/psyneuen
Deficits in testosterone facilitate enhanced fear response* Jean A. Kinga,b,*, Washington L. De Oliveiraa,b, Nihal Patela,b a
Department of Psychiatry, University of Massachusetts Medical School, Worcester, MA 01655, USA Center for Comparative NeuroImaging, University of Massachusetts Medical School, Worcester, MA 01655, USA
b
Received 16 February 2004; received in revised form 10 April 2004; accepted 30 September 2004
KEYWORDS Testosterone; Unconditioned fear response; Trimethylthiazoline (TMT); Freezing; Analgesia
Summary Testosterone has been implicated in many behaviors related to sexual and reproductive function, but its role in fear responses is unclear. Studies in both humans and animals have linked altered testosterone concentrations to externalizing behaviors like aggression and violence, but less to more internalizing behaviors like fear. Therefore, the current study was designed to investigate the effects of testosterone on innate fear response in male rats. TMT (2,5-dihydo-2,4,5trimethylthiazoline), a chemical extracted from fox feces, was used to elicit a fear response in the male rats with normal and diminished levels of testosterone. Behavioral indices such as in freezing response, and fear-induced analgesia were monitored in response to TMT. The results demonstrate that deficits in testosterone resulted in a significant increase in the freezing time and fear-induced analgesia. These studies suggest that testosterone decline may have a significant effect on increasing innate fear response and fear-induced enhancement of analgesia in male rats. Q 2004 Elsevier Ltd. All rights reserved.
1. Introduction Fear is a physiological response to a dangerous occurrence (Rosen and Schulkin, 1998), and is *
This publication was made possible by Grant Number R01 MH067096-01 from the National Institute of Mental Health. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIMH. * Corresponding author. Address: Center for Comparative NeuroImaging, Department of Psychiatry, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA. Tel.: C1 508 856 4979; fax: C1 508 856 8090. E-mail address: [email protected] (J.A. King).
essential for an organism’s survival. Extensive research investigating neuroanatomical regions that sub-serve the fear response has, through the utilization of conditioning paradigms, contributed to most of our current understanding of the mechanism of phobia (LeDoux, 2000). Phobias are the most prevalent form of anxiety disorders, affecting 15% of Americans. It is also the second most common psychiatric illness among men over 25 years of age (US Dept. of Health and Human Services, 1999). Sex differences in fear responses have been observed in rodents (Archer, 1975; Stock et al.,
0306-4530/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.psyneuen.2004.09.005
334 2001), domestic fowl (Jones, 1987), dogs (Goddard and Beilharz, 1984), sheep (Vandenheede and Bouissou, 1993), and man (Gray, 1987; Fredrikson et al., 1996). Women are more than twice as likely to suffer from phobias and four times more likely to have multiple phobias (Fredrikson et al., 1996), suggesting that the response to fear-inducing stimuli may be sexually dimorphic with females being more fearful than males (Gray, 1987). In addition, women have been shown to have more animal and situational phobias than men (Lindal and Stefansson, 1993; Fredrikson et al., 1996), suggesting that the potency of particular fearinducing stimuli may differ by gender. Although these observations lend support to the view that the hormonal milieu may be a contributing factor in the expression of fear and phobias, the role of gonadal hormones like testosterone, in modulation of the fear response remains under-explored and therefore not very well understood. Most studies investigating innate fear assess the response to different aspects of predator exposure or interaction. One of the most specific response measures is freezing or immobility, a state in which all movement ceases with the exception of those necessary for breathing (Godsil et al., 2000). Furthermore, when innate fear was studied in rats using the odor of a cat as the fear-inducing stimulus (Blanchard et al., 1989; Zangrossi and File, 1992), the researchers showed that exposure to cat odor resulted in a significant increase in plasma corticosterone concentrations and avoidance behavior, but freezing behavior occurred only briefly or not at all. Meanwhile, in a more recent approach, Wallace and Rosen (2000) demonstrated that 2,5-dihydro2,4,5-trimethylthiazoline (TMT), a sulfur-containing chemical extracted from fox feces, elicits a freezing response in laboratory-bred rats. Since, there is evidence indicating that TMT does not provoke fear responses in conditioned fear tests (Vernet-Maury et al., 1984; Wallace and Rosen, 2000), it was reasoned that TMT may be more selective for investigating unconditioned fear. Unlike fear-inducing stimuli such as cat fur odor, which has not been isolated and probably contains several components, TMT is a single molecule (Wallace and Rosen, 2000). Thus, using TMT as the fear-inducing stimulus, the present study was undertaken to investigate the effects of testosterone on the innate fear response of male rats and attempted to test the hypothesis that testosterone depletion may heighten innate fear response in these rats. Moreover, in order to assess further the potency of the fear reaction, nociceptive threshold was monitored subsequent to exposure to the fearinducing stimulus.
J.A. King et al.
2. Methods 2.1. Animals Adult male Sprague–Dawley (SD) rats (nZ30) were obtained from Harlan Sprague–Dawley Laboratories (Indianapolis, IN). At the beginning of the experiment, the rats weighed approximately 200 g. The animals were group-housed (2 per cage) according to their treatment conditions, normal, sham-operated or castrate. They were maintained in ambient temperature (22–24 8C) on a 12 h light/dark cycle (light on from 07:00 to 19:00). The rats had access to commercial pellet food and water ad libitum. All animals were acquired and cared for in accordance with guidelines published in the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication N. 80-23, revised 1996). This study was approved by the (International Animal Care and Use Committee, IACCUC) committee of the University of Massachusetts Medical School.
2.2. Surgery Male SD rats (nZ10 per group) were randomly assigned to one of three groups: normal (intact), castrated, or sham-operated. For surgery, the animals were anesthetized with 0.4 ml of ketamine (Ketaset; Fort Dodge Animal Health, Fort Dodge, IA) and 0.25 ml of medetomidine (Dormitor; Pfizer Animal Health, New York, NY). A 2 cm ventral incision was then made in the scrotum and the skin was retracted to expose the tunica. The tunica was pierced, and the aperture was stretched with blunt forceps. The testes were exposed by applying gentle pressure to the pelvic region. The spermatic artery was clamped and cauterized, and the testes were removed. The epididymis, vas deferens and ducts were replaced into the tunica. The animals were injected with 0.3 ml of atipamezole (Antisedan; Pfizer Animal Health, New York, NY) to reverse the anesthesia. Sham-operated animals were exposed to the entire procedure noted above with the exception of the removal of the testes.
2.3. Blood sampling and T-level measurement Blood samples were collected from the entire cohort of animals (nZ10/group) on baseline (day before surgery); days 7 and 21 (post-surgery) via a tail venous puncture. The blood samples were then centrifuged at 5000!g for 15 min and
Deficits in testosterone facilitate enhanced fear response plasma was collected. The plasma samples were stored at K80 8C until assays were performed. Plasma testosterone levels were determined with the aid of a RIA kit obtained from ICN Biomedicals, Inc. (catalog number 07-289102) Costa Mesa, CA. The specificity of the testosterone antiserum was 100% for testosterone, approximately 7% for 5 alpha-dihydrotestosterone and less than 0.10% for hydroxytestosterone, estrone, progesterone, and corticosterone.
2.4. Sexual interest test The animals were removed from their home cage and brought to a behavioral testing room adjacent to the holding area. Sexual interest monitoring was performed in the entire cohort of animals (nZ 10/group) on days 7 and 14 (post-surgery). A female rat in the estrous phase was introduced into a cage of the male rat in order to assess his sexual interest, which was measured by the number of times the male rat mounted the female rat.
2.5. Freezing time upon exposure to fox (TMT) scent Freezing time measurements were evaluated on days 7, 14, 21 and 28 post castration in the entire cohort of animals. The freezing response consisted of cessation of all movement except those necessary for breathing (Godsil et al., 2000). Male SD rats (castrated, normal, sham-operated (nZ10/group) were removed from their home cage and brought to an adjoining laboratory. Twenty-four hours prior to testing, each animal was placed in a clean Plexiglas cage identical to their home cage (absent bedding), as a means of acclimating to the novel environment. They were observed under this condition for 5 min. For testing, the rat was placed in an identical Plexiglas cage and the predator odor scent was introduced in the form of 2,5-dihydro-2,4,5-trimethylthiazoline (TMT). TMT was obtained from Phero Tech, Inc. (British Columbia, Canada). A swab containing 100 ml of the odor was placed on a Whatman filter paper and hung over the cage of the experimental group for 5 min. During the 5 min period, fear related behaviors were monitored, including the total freezing time.
2.6. Tail flick test All three groups of animals were subjected to a tail flick test 30 min prior to exposure to TMT (control period) and 30 min post exposure to TMT. During this test, animals were held firmly while their entire
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tail (100 mm from attached region) was submerged into a beaker of water heated at 50 8C, and the time it took the animals to flick their tails out of the water was noted and recorded.
2.7. Statistical analyses Within group differences across the days for blood testosterone levels, sexual interest, and freezing time were analyzed using a repeated measures analysis of variance (ANOVA). A one-way ANOVA was used to analyze the difference between the three groups for blood testosterone levels, sexual interest, freezing time and tailflick. P%0.05 was considered significant for all statistical tests, and all data are presented as meanGSEM.
3. Results 3.1. Blood T-level A repeated measures analysis of variance showed a significant decrease in blood T levels for all 3 groups (normal, sham-operated, and castrated) on days 7 and 21 (F(2, 23)Z12.905, p%0.0002) when compared to baseline. Circulating blood testosterone levels were not significantly different among the groups at baseline (F (2, 23)Z1.430, p%0.2597). By post-castration day 7, T-levels of castrated rats decreased significantly (F (2, 23)Z7.586, p%0.003) when compared to normal and sham-operated rats (Fig. 1). The blood testosterone level of castrated rats decreased even further by 21 days postcastration (F (2, 23)Z7.039, p%0.004) compared to normal and sham-operated rats (Fig. 1).
3.2. Sexual interest Castrates showed a significant difference in sexual interest (as measured by the number of mountings in the presence of an estrus female) (F (2, 23)Z 9.873, p%0.0008) across days 7, 14, and 21. Furthermore, castrates were significantly different than normal and sham-operated animals on day 7 (F (2, 23)Z8.257, p%0.002) (Fig. 2). Similarly, on day 14 post castration, these rats continued to show a significant decrease in sexual interest compared to normal and sham-operated (F(2, 23)Z6.746, p%0.004) (Fig. 2).
3.3. Freezing response A repeated measures analysis of variance showed that fear response measured by changes in
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Figure 1 Bar graph depicting mean blood testosterone levels (GSEM) for normal (stripped bar), sham-operated (black bar) and castrated (white bar) rats. T levels for castrated rats decreased notably to 0.015 ng/ml by day 21 postcastration. The *indicates a significant difference between the groups on day 7 (p%0.002) and day 21 (p%0.004).
the freezing time with exposure to TMT (for animals from all three conditions) were significantly different across days 7, 14, 21 and 28 (F(2,23)Z10.615, p%0.0005). But castrates showed no significant difference in freezing
response 7 days post-castration (F(2,23)Z1.34, p%0.28) when compared to normal and shamoperated. However, by 14 days post-surgery, castrated rats showed a significant increase in their freezing response as compared to normal
Figure 2 Mean number of mountings (GSEM) for all three groups on days 7 (black bar) and 14 (square bar). The * indicates a significant difference between the groups on day 7 (p%0.001) and day 14 (p%0.004).
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Figure 3 Mean differences in freezing time (GSEM) during fox scent exposure for all rat groups on days 7 (black bar), 14 (square bar), 21 (patterned bar) and 28 (white bar). The * indicates a significant difference between the groups on days 14 (p%0.001), 21 (p%0.0001) and 28 (p%0.0001).
and sham-operated (F(2,23)Z9.267, p%0.001) (Fig. 3). This increase in the freezing response time of castrated rats was also seen on day 21 postcastration (Fig. 3) when compared to normal and sham-operated animals (F(2,23)Z28.445, p%0.0001). Similarly, the increase in freezing time in response to TMT persisted until day 28 post-castration (F(2,23)Z16.867, p%0.0001 (Fig. 3).
3.4. Tailflick There were significant differences between the groups (normal, sham-operated and castrated) on day 14 (F(2,23)Z13.71, p%0.0001) (Fig. 4). These significant differences in mean latencies between the groups persisted until one week later (F(2,23)Z10.335, p%0.0001) (Fig. 5).
4. Discussion The main objective of this study was to investigate the effects of testosterone deficiency on innate or unconditioned fear response of male rats and to determine if prior exposure to a fear-inducing stimulus altered their nociceptive threshold. Our results show that decreased circulating blood testosterone levels were accompanied by
an increase in fear response, as demonstrated by the significant increase in freezing time postexposure to TMT. However, this response is apparently reflective of the organization effects of sex steroids, like testosterone (Hebbard et al., 2003). Since, the present study demonstrated that although T levels were significantly diminished by day 7, it did not alter the freezing response in castrates until T-levels were chronically low. That is, the initial significant response to fear-inducing odor was observed on day 14 and persisted until 28 days post-castration (last time point assessed). Previous studies have investigated the role of testosterone in the fear response in other species, particularly ewes and heifers (Vandenheed and Bouissou, 1993; Boissy and Bouissou, 1994; Bouissou and Vanderheed, 1996). These researchers looked at the long-term effect (4–8 months post-treatment) of testosterone treatment. Their results indicated that increases in testosterone correlated with a reduction in fear, as manifested by decreases in trotting, immobilization and flinching in response to classically reported fear-inducing stimuli in sheep. These fear-inducing stimuli included isolation from conspecifics, surprise (novel red ball falling from the ceiling) and the presence of a human, thus supporting the organizational effect of testosterone on the fear response. In addition, previous research in mice has reported that
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Figure 4 Mean tail flick latencies (GSEM) of normal (stripped bar), sham-operated (black bar), and castrated rats (white bar) during pre and post exposure to fox scent on day 14. The * indicates a significant difference (p%0.0001) between the groups post fox scent.
an acute testosterone injection resulted in a decrease in unconditioned fear response to predator odor within 30 min (Domek et al., 1992). Taken together, these studies suggest that the impact of T-levels on the fear response may be
reflective of the duration of treatment. Nevertheless, one cannot disregard the possibility that the reported change in freezing associated with decrease T-levels maybe due to either direct or indirect alterations in HPG and HPA modulators
Figure 5 Mean tail flick latencies (GSEM) for normal (stripped bar), sham-operated (black bar), and castrated rats (white bar) during pre and post exposure to fox scent on day 21. The * indicates a significant difference (p%0.0001) between the groups post fox scent.
Deficits in testosterone facilitate enhanced fear response affected by T (Dafopoulos et al., 2004; Viau and Meaney, 2004). In addition, the fear responses noted in the presence of TMT may be dependent on the limited size of enclosure used for testing (Wallace and Rosen, 2000). Although the studies linking T to fear are limited, the connection between androgens and other behaviors, including aggression, dominance, and mood disorders are numerous. For example, in a study investigating the dose–response relationship among T levels, behavior and mood in eugonadal and hypogonadal men, O’Connor and colleagues (2002) reported that testosterone treatment led to a significant increase in verbal aggression, hostility, anger and irritability in hypogonadal subjects, but had no effect on T treated eugonadal subjects. This is supported by reports from other laboratories (Maras et al., 2003) correlating higher levels of testosterone with externalizing behavior, a type of overt aggression, in adolescent boys. Furthermore, it has been shown that anabolic steroids cause an increase in euphoria, irritability, mood swings, anger and hostility in healthy, non-athletic men (Su et al., 1993). In addition, there is also sparse support for the impact of T on internalizing behaviors. Reports have demonstrated that low levels of T are linked to depression in elderly males (Margolese, 2000). Recent studies using single photon emission-computed tomography has shown that testosterone replacement enhances blood perfusion in the midbrain and superior frontal gyrus in hypogonadal men (Azad et al., 2003). Similarly, alterations in blood flow to the brain have been linked independently to depression and fear in humans (Navarro et al., 2002; Wik et al., 1996). Taken together, these results lead to the speculation that decreased T levels may support decrease in brain perfusion. Another important finding of the current study is the apparent role of testosterone in modulating the analgesic response post-exposure to a fearinducing stimulus. Our data showed that 14 days post-castration, there was significant increase in tailflick latencies between castrated, normal and sham-operated rats after exposure to TMT. However, the increase seen in the castrates was significantly higher than the intact and sham operated groups. This suggests that the fear reaction in castrated rats triggered a significant increase in the nociceptive threshold beyond that seen in intact and sham-operated animals. The finding of a fear triggered increase in nociceptive threshold is supported by work in mice which demonstrated that fear-inducing stress increased nociceptive threshold (Suaudeau and Costentin,
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2000). In contrast, the tailflick response prior to the fear-inducing paradigm was not significantly different between the groups. In this regard, previous studies showed that short-term treatment with T increased tailflick latencies in T-treated gonadectomized male rats as compared to intact and gonadectomized rats (Forman et al., 1989; Frye and Seliga, 2001). Further studies examining a more systematic dose response curve and times between treatment and behavior may help clarify these differences. The present data on the analgesic effects postexposure to TMT explores a novel component of nociception in T-deficient animals. One possible reason for this heightened analgesic effect in castrated animals in response to TMT, may result from an enhanced release of endorphins (Suaudeau and Costentin, 2000) to fear stress. Previous studies using naltrexone, a prototypic opioid receptor antagonist, support the premise that stress-induced analgesia can be opioid mediated (Lapo et al., 2003). In summary, given the prominent effects of testosterone on a myriad of different behaviors, it is not surprising that testosterone may play a role in the fear response. Further studies on the involvement of neural pathways and brain mechanisms involved in hormonal substrates of the fear response are therefore warranted.
Acknowledgements We would like to thank Jeffrey Tenney and Victoria Rossi for their assistance with this project. This work was supported by National Institute of Mental Health Grant R01 MH067096 to J.A.K. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Mental Health.
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