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Role of gonadal hormones in anxiety and fear memory formation and inhibition in male mice
Physiology & Behavior 105 (2012) 1168–1174
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Role of gonadal hormones in anxiety and fear memory formation and inhibition in male mice Carmel M. McDermott a, Dana Liu a, Laura A. Schrader a, b,⁎ a b
Dept. of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118 Neuroscience Program, Tulane University, New Orleans, LA 70118
a r t i c l e
i n f o
Article history: Received 1 September 2011 Received in revised form 16 December 2011 Accepted 16 December 2011 Keywords: Fear conditioning Extinction Castration Elevated Plus Maze Open Field Assay Puberty Hippocampus
a b s t r a c t Recent research investigating Pavlovian fear conditioning and fear extinction has elucidated the neurocircuitry involved in acquisition and inhibition of fear responses. Modulatory factors that may underlie individual differences in fear acquisition and inhibition, however, are not well understood. Testosterone is known to affect anxiety-like behavior and cognitive processing. In this study, we hypothesized that castration would increase anxiety and reduce memory for contextual fear conditioning in an age-dependent manner. In addition, castration would reduce the rate of extinction to context, as high levels of testosterone correlate with reduced PTSD-like symptoms. We compared behaviors in male mice that were castrated at one of two different time points, either before puberty (at 4 weeks) or after puberty (at 10 weeks) to sham-operated control mice. The behaviors investigated included: anxiety, cued and contextual fear conditioning, and extinction of the fear memory. An interaction of hormone status and age and a significant effect of age were measured in the elevated plus maze, a measure of anxiety. Castration caused a significant reduction of contextual fear memory, but no effect on cued fear memory. There was no significant effect of castration on extinction. Interestingly, a significant effect of age of the mouse at the time of testing was observed on extinction. These results suggest that endogenous androgens during puberty are important for anxiety and fear memory formation. In addition, these results define a late post-adolescent developmental time point for changes in anxiety and fear extinction. © 2012 Elsevier Inc. All rights reserved.
1. Introduction 1.1. Human studies Depression and anxiety-related disorders afflict men at approximately half the rate at which they affect women [1]. Convincing evidence suggests that testosterone and/or its metabolites play a protective role against these disorders, and low levels of testosterone are correlated with increased affective disorders. For example, hypogonadal men, who suffer from lower than normal levels of testosterone, are more likely to suffer from anxiety and depression disorders, and treatment with testosterone alleviates these symptoms [2-4]. In addition, bioavailable testosterone levels undergo a gradual decrease during aging, and this decrease is correlated to an increase in the incidence of depression-related disorders [5]. Gender differences are also seen in the processing of traumatic episodes. A meta-analysis study suggests that even when exposure to
⁎ Corresponding author at: 2000 Percival Stern Hall, 6400 Freret St, New Orleans, LA 70118. Tel.: + 1 504 862 3139. E-mail address: [email protected] (L.A. Schrader). 0031-9384/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2011.12.016
the potentially traumatic event is controlled for, men tend to develop posttraumatic stress disorder (PTSD) less frequently and of a less severe nature than women [6]. Therefore, further investigation into the role of testosterone in anxiety, fear memory formation and inhibition is warranted. 1.2. Puberty and the role of androgens Puberty is a developmental time point in adolescence where sex hormone levels increase dramatically [7], and changes in behavior occur. In humans, adolescence is characterized by increased impulsivity, risk taking behavior and susceptibility to various neuropsychiatric disorders [8,9]. The effects of androgens during puberty are important mechanisms to modulate various behaviors. For example, low testosterone levels in young males were related to increased levels of anxiety, depression and attention problems [10]. Data from animal studies show that the levels of sexual [11] and social behaviors [12,13] are reduced in males castrated before puberty compared to males castrated after puberty. In addition, neuron and circuit structure can undergo dramatic changes during puberty in limbic areas of the brain [14-16], suggesting the function of these areas is impacted by puberty.
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1.3. Fear memory and extinction A number of studies have examined the role of testosterone in fear memory. In 55 day old male rats, removal of endogenous testosterone by castration resulted in a trend toward reduced memory for hippocampus-dependent contextual fear conditioning, an effect that was reversed by administration of testosterone and its metabolites, but did not affect cued fear conditioning [17]. In older rats, no effect of castration was seen in memory formation for contextual fear conditioning [18]. Administration of 3α-diol, a metabolite of testosterone, enhanced inhibitory avoidance and contextual fear memory in aged (24 months) male intact mice, increased inhibitory avoidance in young (12 weeks) intact mice, and increased contextual fear conditioning in young GDX mice. Interestingly, no effect on cued fear conditioning in any of the groups was observed [19]. Testosterone administration to young female mice also enhanced contextual fear memory [20]. These studies suggest that a developmental profile of the influence of gonadal hormones on hippocampus-dependent fear memory may exist, but this has not yet been investigated. Extinction is the process by which a fear memory is inhibited. Existing theories suggest that fear extinction involves a combination of new associative learning processes and decreased responsiveness to the fear-eliciting stimulus [21]. Little is known about the role of testosterone or its metabolites in extinction; however developmental aspects of extinction have been investigated. Adolescent rats (35 days) exhibited impaired cue extinction retention compared to adults and younger animals [22,23]. In addition, early adolescent (4 week) C57 male mice showed enhanced cued fear memory relative to adults, but a similar extinction rate to the associated cue as adult animals [24]. These differences may be due to the activational effects of hormone levels at different ages or organizational effects of the hormone surge during puberty. Therefore, the role of androgens in fear memory formation during postnatal development, particularly during puberty, warrants further study. This study investigated the effects of gonadal steroids on anxiety, fear memory and extinction in male mice that were castrated either before puberty or in adulthood. Based on previous literature, we hypothesized that castration would increase anxiety in both ages and reduce memory for contextual fear conditioning. In addition, castration would reduce the rate of extinction to context in both young and adult mice, as high levels of testosterone correlate with reduced PTSD-like symptoms. Our results show a significant age and hormone status interaction in anxiety-related behavior measured on the elevated plus maze. In addition, castration significantly reduced contextual fear memory. Contrary to our hypothesis, we observed no effect of castration on extinction at either age. Interestingly, however, young mice, 8 weeks at time of testing, did exhibit enhanced fear extinction compared to older animals, 14 weeks at time of testing. These studies define a role for gonadal hormones during puberty in regulation of fear behaviors and a late developmental time point for fear extinction. 2. Methods Subjects were male 129svE mice from Charles Rivers. Young animals (n = 20) arrived at 3 weeks and adult animals (n = 18) arrived at 9 weeks. All experiments were performed within the guidelines of the Tulane University Institutional Animal Care and Use Committee and the US Army Medical Research and Materiel Command Animal Care and Use Review Office (ACURO). Mice were housed in groups of three or four on a 12 h light–dark cycle with access to food and water ad libitum.
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were performed at either 4 weeks (pre-adolescent) or 10 weeks of age (adult). Orchidectomy was performed through a single transverse incision across the end of the scrotum. The testicles were removed and a ligature was used around the vas deferens and spermatic vessels to prevent hemorrhage. Sham animals received similar incisions with no testicle removal. Buprenorphine (0.03 mg/kg) was injected at the time of surgery for pain relief. Mice were allowed to recover for 2 weeks before any behavioral testing was performed. The effectiveness of castration was determined after behavior upon sacrifice, and bulbospongiosus muscles were dissected from the penile bulb and immediately weighed. 2.2. Behavior 2.2.1. Anxiety - Open Field Analysis of anxiety behavior commenced two weeks after surgery. Two tests of anxiety behavior were assessed: open field (Weiss et al., 2000) and elevated plus maze (EPM) [52,53]. These tests are based on a rodents’ proclivity to avoid well-lit, open spaces. Less anxious animals will spend more time in the center of the open field and in the open arms of the elevated plus maze. Open field was performed 14 days after surgery. A single mouse was placed in the center of a white, Plexiglas chamber measuring 43 cm in length x 43 cm in width x 18 cm in height, under well-lit conditions. The animal explored the novel environment for 15 minutes and movements were monitored by a camera interfaced with a tracking system (US HVS Image 2100 Tracking System). The area was divided into 16 virtual squares (10.75 × 10.75 cm) by the program, and the middle four squares were defined as the center area. The Plexiglas chamber was wiped clean with 70% ethanol between trials. 2.2.2. Elevated Plus Maze The elevated plus-maze (EPM) was performed 17 days after surgery. EPM consisted of four arms (5 cm in width x 30 cm in length) arranged perpendicularly in a plus shape and elevated 38 cm above the floor and placed under well-lit conditions. The entire maze was tan and two arms were enclosed by 15.5 cm opaque Plexiglas walls and two arms were open. Each animal was placed in the center of the EPM facing a closed arm and allowed to move freely for 5 minutes. Behavior was monitored by a camera interfaced with the tracking system. The EPM was wiped clean with 70% ethanol between trials. 2.2.3. Fear Conditioning Mice were 3 weeks post-surgery at the time of fear conditioning. Fear conditioning was performed using a computer-controlled, sound-attenuated, conditioning chamber (29 × 19 × 25 cm, Med Associates). On the day of the fear conditioning training, the mouse was exposed to the chamber for the first time and allowed to explore the chamber. At 120 seconds a 30 second tone (white noise) commenced and the tone co-terminated with one 2 s electrical foot shock (0.5 mA) through a stainless steel grid floor. After the foot shock, the mouse was allowed to explore the context for an additional 150 seconds. The behavior of the mouse was measured by a digital infrared video camera mounted in front of the chamber. 2.2.4. Context-dependent freezing 24 hours after fear conditioning the mouse was returned to the chamber to measure context-dependent fear conditioning. The mouse was placed in the chamber for 300 seconds and the percentage of freezing was analyzed using Video Freeze Software. The chamber was wiped clean with 70% ethanol between animals.
2.1. Gonadectomy Mice were anesthetized by intraperitoneal injection of a ketamine (80 mg/kg) and xylazine (8 mg/kg) mixture. Gonadectomies
2.2.5. Cue-dependent freezing 48 hours after initial fear conditioning mice were returned to the chamber. The context was altered with plastic floors and alternative
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context shape. Vanilla scent was added in the context for alternative olfactory cues. After 150 seconds they were exposed to the tone for 150 seconds and the percentage of freezing was measured before and during tone exposure. The chamber was wiped clean with isopropyl alcohol between animals. 2.2.6. Shock Threshold and Fear Extinction At 4 weeks post-surgery mice were placed in the fear conditioning chamber and allowed to explore for 30 s before being exposed to a series of foot shocks. The foot shocks lasted 2 s and ranged in intensity from 0.1 to 0.7 mA. The behavior of the animals was recorded and responses were scored in order to determine differences in responses to increasing shock sensitivities. 48 hours after shock threshold, mice were returned to the chamber for 5 min to monitor freezing (day 1 of extinction). This was repeated for 4 days and the degree of freezing was plotted to monitor extinction of fear.
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3.1. Anxiety Since anxiety levels can modulate memory, the anxiety levels in the young and adult males, castrated and sham-operated were examined. Anxiety behavior was assayed using the open field and elevated plus maze as described in methods. A two-way ANOVA (hormone status x age) showed that there was no significant interaction (F1,32 = 0.31; p = 0.58), effect of castration (F1,32 = 2.89; p = 0.1) or age (F1,32 = 0.36; p = 0.55) in the amount of time spent in the center of the open field (Fig. 1A). The percent time spent in the open arms of the EPM was also evaluated (Fig. 1B). There was a significant interaction of hormone status and age (F1,32 = 5.45; p = 0.03), no significant main effect of hormone status (F1,32 = 0.54; p = 0.47), but a main effect of age (F1,32 = 4.4; p = 0.04). The number of entries into the open arms as a percentage of total number of arm entries was also evaluated (data not shown). No significant interaction (F1,32 = 0.22; p = 0.64), effect of castration (F1,32 = 0.09; p = 0.77) or age (F1,32 = 0.35; p = 0.56) was observed, suggesting that the groups entered the arms of the maze to a similar extent. Surprisingly, there was an effect of age in the distance traveled in the open field and EPM (data not shown). Young males explored the open field significantly less than adults (F1,32 = 9.28; p = 0.005). No significant interaction (F1,32 = 0.05; p = 0.83) or effect of castration (F1,32 = 0.052; p = 0.82) was observed. There was also a trend toward reduced distance traveled in the elevated plus in the young compared to the adult (F1,32 = 3.7; p = 0.06), but no significant interaction (F1,32 = 0.014; p = 0.9), or effect of castration (F1,32 = 0.04; p = 0.84) was observed. These results suggest that castration had no significant effect on anxiety in either the young or adult males, but
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The goal of this study was to determine the effect of gonadal hormones on anxiety, fear conditioning and extinction in male mice that were castrated at two different time points, before puberty and as adults. In order to accomplish this, the source of endogenous androgens was removed by castration in males and the behavioral results from sham-operated intact and castrated males were compared. The effectiveness of castration was confirmed in animals after behavioral studies were completed. Castration causes shrinkage of the bulbospongiosus muscles (BSM) [25]. Castrations were confirmed by comparison of BSM mass in sham and castrated animals. Average BSM weight in adult castrated males (n = 8; 42.5 ± 1.4 mg) was significantly decreased compared to adult sham males (n = 9, 58.3 ± 2.6 mg; p b 0.01, unpaired t-test). One adult castrated male was removed from the study for ineffective castration. BSM weight in young castrated animals was also significantly decreased (n = 10; 27.8 ± 3.0 mg, p b 0.05) compared to sham-operated intact males (n = 10; 37.9 ± 3.1 mg).
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Fig. 1. Results of assessment of the effect of castration before and after puberty in males on anxiety behaviors. A. Bar graph showing the percentage of time spent in the center of the field in the sham-operated and mice that were castrated before puberty (left) and after puberty (right). B. Bar graph showing the results of the percent time spent in the open arms of the EPM in sham-operated male mice or mice castrated before puberty (left) and after puberty (right). A significant interaction of age and hormone status and a main effect of age were observed (*, p b 0.05).
that there was a significant interaction and effect of age in anxiety measured on the EPM.
3.2. Fear Conditioning Animals were trained on day 1 of fear conditioning with a mild training paradigm, consisting of 120 seconds habituation to a chamber, followed by a 30 second auditory cue co-terminating with a single 0.5 mA foot shock. Animals remained in the chamber for another 150 seconds. Animals were tested for contextual memory 24 hours after training. There was no significant interaction (F1,31 = 0.31; p = 0.58), a significant effect of hormone status (F1,31 = 4.0; p = 0.05), but not age (F1,31 = 1.17; p = 0.29) on contextual fear conditioning memory (Fig. 2A). Since we saw effects on path length in the anxiety measures, we analyzed average motion during nonfreezing episodes in the contextual test. We observed no significant interaction (F1,31 = 0.01; p = 0.93), effect of hormone status (F1,31 = 0.44; p = 0.51) or age (F1,31 = 0.07; p = 0.79). One adult sham animal was removed from this analysis due to technical difficulties with the equipment. These results suggest a significant effect of castration on contextual fear conditioning, but no effect on motion during non-freezing episodes. The cued test was performed 48 hours after training. Importantly, very little freezing was observed in the modified context before introduction of the cue (Fig. 2B). There was no significant interaction
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two-way repeated measures ANOVA (repeated measures on stimulation intensity). There was no significant interaction (F8,126 = 1.6; p = 0.14) or effect of castration (F1,126 = 0.98; p = 0.32) in the adult animals (Fig. 3A). As expected, there was a significant within subjects effect of stimulation intensity (F8,126 = 100; p b 0.0001). In the young animals, there was no significant interaction (F8,162 = 0.86; p = 0.55), but there was an effect of castration (F 1,162 = 7.3, p = 0.008). A significant effect of intensity was also observed (F8,162 = 153.3; p b 0.0001; Fig. 3B). These data suggest that castration increased the response to an aversive stimulus only in the young males.
3.3. Extinction
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We investigated the extinction of the fear response to context 48 hours after the foot shock response assay. Freezing was assessed during repeated exposure to the context for 5 minutes administered over 4 days. To assay extinction learning, we reasoned that a threshold of freezing on day 1 must be reached in order to be adequately trained to assay extinction. We set a threshold at 45% freezing for the first day of extinction training. Two young castrated males froze less than 40% on first day; therefore these animals were eliminated from the extinction data. We first determined the effect of castration in the adult males (Fig. 4A). Two-way repeated measures ANOVA (repeated measures on extinction trial) revealed no significant interaction (F3,56 =1.1; p=0.37), effect of castration (F1,42 =1.01; p=0.32), but a significant effect of trial (F3,56 =11.29; pb 0.001; Fig. 4B). Similarly, in the young males there was no significant interaction (F3,64 =0.14; p=0.93) or effect of castration (F1,64 =2.0, p=0.16), but a significant effect of trial as expected (F3,64 =17.4; pb 0.0001). Since there was no significant effect of
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(F1,32 = 0.35; p = 0.56), effect of age (F1,32 = 0.96; p = 0.35), or hormone status (F1,32 = 3.72; p = 0.06) in the modified context before the tone, suggesting the animals were not generalizing the context. No significant interaction (F1,32 = 0.68; p = 0.41), effect of hormone status (F1,32 = 1.79; p = 0.19) or age (F1,32 = 0.22; p = 0.64) was observed for freezing during the cue (Fig. 2C). Together, these results suggest that castration significantly decreases contextual fear memory but not cued memory in young and adult males. The effect of age and castration on foot shock response was also tested. The response to an increasing intensity of foot shock was measured [54] and compared between hormone status groups in the individual ages (Fig. 3A, B). The data in each age group were analyzed by
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Fig. 2. Contextual and cued fear conditioning results from animals castrated before and after puberty. A. Bar graph showing the amount of time spent freezing in response to the context 24 hours after training in animals castrated before puberty (left) and after puberty (right). Castration significantly reduced contextual fear memory formation compared to sham animals (*, p b 0.05). B. Amount of freezing observed in novel context before commencement of cue (white noise tone) in cued test. There were no significant differences observed in the amount of freezing to the novel context. C. Bar graph showing the amount of time spent freezing in response to the cue (white noise tone) 48 hours after the training from mice castrated before puberty (left) and after puberty (right).
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Stimulus intensity (mA) Fig. 3. Results of measurement of sensitivity to foot shock intensity. Response to increasing intensity of foot shock measured as in methods, in mice castrated after puberty (A) and mice castrated before puberty (B). No effect of castration was observed in adult mice, however, a significant of castration was observed in the young males.
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and in adulthood. We report an interaction of age and hormone status on anxiety in the EPM, with a significant main effect of age. In addition, we observed an effect of hormone status on contextual fear conditioning. Studies in adult animals demonstrated no effect or subtle differences between sham and castrated animals, consistent with our results. In interpretation of our data, it is also important to consider a recent study that found that shipping stress during the peripubertal/adolescent period reduced the effects of sex hormones on sex behavior and caused hypoactivity of the hypothalamo-pituitary axis [26]. While our adult animals were 10 weeks (post-pubescent) at the time of shipping, our young animals were 4 weeks at the time of shipping. The behavior of the young animals in particular and their response to the castration may have been affected by the shipping stressor.
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The results from the EPM indicate a significant interaction of age and hormone status and a main effect of age. Studies from rodents confirm a differential role for testosterone in anxiety behavior in young and adult animals. Both mice and rats demonstrate an increase in anxiety behavior after castration as adults [12, 13]. Similar to our results, neonatal castration reduced measures of anxiety in the adult [27]. Previous studies showed that castration in adult mice, on the other hand, increased the amount of time spent in the perimeter of the open field, an indication of increased anxiety, however there was no significant effect in the elevated zero maze [28]. Another study reported that castration in adult rats caused a decrease in the amount of time spent in the open arms of elevated plus [29]. Administration of testosterone or its metabolites was anxiolytic in young GDX mice [14, 15], aged intact mice [15], and GDX rats [13], but had no overall effect on anxious behavior in young intact mice [15]. Therefore, our results of a significant interaction of age and hormonal status are consistent with previous literature that castration before puberty reduced anxiety and after puberty increased anxiety. One possible explanation for this result, however, could be that young males explored both the open field and EPM less, as there was a trend toward reduced path length of the young males in the elevated plus and significantly lower path length of the young males in the open field. Entries into the open arms, however, were not significantly different. This suggests that while the young animals traveled less distance, they entered the open arms an equivalent number of times as the adult animals.
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Days Fig. 4. The effect of castration on extinction training. Animals were tested for foot shock sensitivity and then 48 hours later received extinction training, consisting of context exposure with no US, for 5 minutes a day over a 4 day period. A. Results from extinction training in males castrated after puberty. B. Results from four days of extinction training in mice castrated before puberty. Castration before puberty had no significant effect on extinction at either age. C. Comparison of extinction rate in young compared to adult mice. Young male mice exhibited rapid extinction to context compared to adult male mice.
castration, the sham-intact and castrated groups were combined from each age into young and adult groups (Fig. 4C). Interestingly, a significant effect of age emerged. A significant interaction (F3,96 =7.66; pb 0.0001), effect of age (F1,96 =10.27; p=0.05) and trial (F3,96 =93.51; pb 0.0001) was observed. Together these results suggest that while both the young and adult animals extinguished the fear, no effect of castration was observed in either the young or adult animals on the rate of extinction. 4. Discussion In this study, we investigated the role of castration at two ages, before puberty to determine the effects of testosterone during puberty,
4.2. Fear Conditioning The findings of this study showed a significant effect of castration in the contextual fear memory formation test. This result suggests that the presence of testosterone may result in functional changes during puberty or activational effects after puberty that can affect fear memory, particularly hippocampus-dependent contextual fear memory formation. Other studies examined the effects of castration in fear conditioning in rats. One study in adult rats (approximately 130 days at castration) showed no effect of castration on cued and contextual fear conditioning in rats that were trained over several days [18], while another study showed no effect on cued fear conditioning with a trend toward a significant reduction of freezing in contextual fear conditioning in younger (55 days at time of castration) male rats [17]. This reduction in fear conditioning memory was reversed by testosterone and testosterone metabolite replacement [17] but not estradiol [19]. It is generally considered that contextual memory is dependent on the hippocampus and amygdala, while cued memory is only dependent on the amygdala [30]. Together with the current study, these data suggest that androgens enhance hippocampus-dependent fear memory formation and have no effect on amygdala-dependent cued fear memory.
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4.3. Fear Extinction We observed no significant influence of castration on the rate of extinction of the contextual memory in the mice castrated either pre-or post-puberty. There were no significant effects of castration before puberty in the young adult and no effect of castration in adults on the rate of extinction. The young males, regardless of hormone state, extinguished the contextual fear memory significantly faster than the adult males. In agreement with our results, a previous study also showed no effect of castration on extinction of the contextual fear in adult animals [18]. These results suggest that androgens, either during puberty or in adulthood, do not play a role in extinction of fear memory. We did, however, observe a significant effect of age, regardless of hormone state, on extinction of the contextual fear. In this study, young males, 8 weeks at the time of testing, extinguished fear of the context significantly faster than adult animals, 14 weeks at the time of testing, independent of hormone status. This result suggests the presence of a post-adolescent developmental time point that is important for fear memory inhibition. Other studies have shown a developmental regulation of extinction of the cued memory, with adolescent males exhibiting a reduction in extinction retention [22], and a delay in the rate of extinction in 4 week old mice compared to 6 or 8 week mice. This is in contrast to our results showing enhanced extinction of the contextual fear memory in the young mice. Previous studies have suggested that the mechanisms of extinction in young (weanling) mice differ from the mechanisms in the adult mice. It is thought that in young animals, the fear memory is basically forgotten, while in adult animals, a new memory of the conditioned stimulus as non-aversive is formed [31]. This forgetting is proposed as a mechanism of extinction in young rats, as young rats do not show renewal or reinstatement of extinguished fear [31-33]. Our results suggest modulation at an additional, later developmental time point for the extinction of the contextual memory. Furthermore, we suggest that gonadal hormones during puberty do not mediate the effect on the extinction rate, since castration before puberty had no effect on extinction rate. 4.4. Shock Sensitivity Another factor that can affect the fear memory formation in these experiments is differences in the sensation of the foot shock. Previous studies have shown that testosterone can modulate pain responses. Endogenous or testosterone replacement had analgesic effects in a tail-flick experiment [29]. In our study, there was no significant effect of castration in response to increasing stimulus intensity in the adult, suggesting no modulation of the response to foot shock by testosterone. In the young mice, however, castration significantly increased the response to foot shock. This is in contrast to an effect of castration to decrease fear memory formation; therefore differences in shock sensitivity cannot explain the castration-dependent difference in contextual fear memory formation or in the rates of extinction between young and adult mice as both groups started out with similar levels of freezing. 4.5. Neural substrates The neural substrates and circuitry involved in fear conditioning and extinction processes have been detailed. The amygdala is known to be critical for fear learning, while context-associated fear learning also involves the hippocampus [30]. Fear extinction is thought to be a form of inhibitory learning and is considered to be a process distinct from fear learning. Three brain areas and their interconnecting circuitry are highly implicated in the extinction circuitry, including the hippocampus [34], the amygdala [35], and the medial prefrontal cortex [36]. Therefore, modulation of plasticity in these
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areas or the connections between the areas can modulate fear conditioning and/or fear extinction. Since we observed effects on contextual modulation of fear memory in our studies, testosterone- or age-mediated changes in the hippocampus likely play a role in our effects. Cued fear conditioning was unaffected; therefore, we do not believe that the locus of the effects is the amygdala. A recent paper showed that extinction modulates expression of insulin growth factor-2 (IGF2) in the hippocampus, and IGF2 infusion regulates extinction [37]. One of the cellular targets of this IGF signaling is neurogenesis. Neurogenesis is approximately twice as high in prepubertal males compared to adults [16, 38-41], and this reduction of neurogenesis in adulthood is not dependent on androgens [16]. Since we observed an effect of age, but not castration in the extinction process, changes in neurogenesis are a possible explanation for the effects on extinction, further studies are necessary to examine this issue. Furthermore, the pyramidal cells of the hippocampus are richly endowed with androgen receptors and manipulation of testosterone levels directly affects spine synapse density in the CA1 region [42], providing another possible mechanism for modulation of contextual fear memory in our experiments. Together, these data suggest that the function of the hippocampus, but not the amygdala, in fear memory formation is modulated by androgens. Furthermore, extinction of the contextual fear memory is not affected by androgens, but is reduced in the adult. This differential effect of castration and age on fear memory formation and extinction, respectively suggests different mechanisms are involved in regulation of each process. Since we observed a late developmental difference in extinction in male mice, the developmental difference that we observed on extinction may also occur in the prefrontal cortex. It is well recognized that the prefrontal cortex continues to develop into late adolescence. In humans the volume of gray matter in the frontal cortex increases until around adolescence and then decreases between adolescence and young adulthood [43-45]. This reduction in gray matter during adolescence is thought to be caused by neuronal loss [46]. Concurrent with these anatomical changes are changes in the ability to perform prefrontal cortex-dependent tasks during adolescence [47,48]. The effects of gonadal hormones on other prefrontal tasks appear to be fairly complex. Castration and/or hormone replacement significantly impaired performance on a range of frontal lobe-dependent tasks. Castration in adult male rats, increased the time required to reach a training criterion in the T-maze delayed alternation paradigm, a task that is sensitive to lesions of the prefrontal cortex [49]. Kritzer et al (2007) found more selective effects of castration on prefrontal function [50]. Castration adversely affected acquisition in a response alternation task and a light/dark discrimination task requiring behavioral flexibility. These effects were eliminated by supplementation with testosterone propionate but not estradiol. This suggests that aromatization of testosterone to estradiol is not necessary for this task. On the other hand, other tasks examining prefrontal functions, such as motivation and response withholding, were sensitive to castration. The effects were alleviated by supplementation with testosterone propionate or estradiol, suggesting aromatization is necessary for these tasks. Other tasks examining impulsivity and reversal learning were insensitive to castration. Using a delayed match to position version of the T-maze, Gibbs (2005) observed no differences between gonadectomized and intact rats [51], although by using longer delays with supraphysiologic doses of testosterone propionate, an effect became evident. Thus, the literature suggests that gonadal hormones affect prefrontal function but in a highly selective manner. In conclusion, we saw an overall effect of castration on contextual fear conditioning. Furthermore, we observed developmental effects on extinction, in that young males showed an increased extinction rate. Extinction in sham animals was similar to castrated animals at each age, suggesting that testosterone is not a mechanism of the developmental change, and another mechanism underlies the switch
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to delayed extinction. While we saw no significant effect of castration in the extinction studies, the developmental differences that we observed are important for consideration in treatment of PTSD and anxiety disorders across the lifespan. Acknowledgements This study was funded by DOD Award #08-1-0590. The authors thank Dr. Shari Birnbaum for comments and discussion on earlier versions of the manuscript. References [1] Pigott TA. Anxiety disorders in women. Psychiatr Clin North Am 2003;26(3): 621–72 vi-vii. [2] Kaminetsky JC. Benefits of a new testosterone gel formulation for hypogonadal men. Clin Cornerstone 2005;7(Suppl 4):S8–S12. [3] Veras AB, Nardi AE. The complex relationship between hypogonadism and major depression in a young male. Prog Neuropsychopharmacol Biol Psychiatry 2010;34(2):421–2. [4] Cooper MA, Ritchie EC. Testosterone replacement therapy for anxiety. Am J Psychiatry 2000;157(11):1884. [5] Barrett-Connor E, Von Muhlen DG, Kritz-Silverstein D. Bioavailable testosterone and depressed mood in older men: the Rancho Bernardo Study. J Clin Endocrinol Metab 1999;84(2):573–7. [6] Tolin DF, Foa EB. Sex differences in trauma and posttraumatic stress disorder: a quantitative review of 25 years of research. Psychol Bull 2006;132(6):959–92. [7] McEwen BS. Gonadal steroids and brain development. Biol Reprod 1980;22(1): 43–8. [8] Spear LP. The adolescent brain and age-related behavioral manifestations. Neurosci Biobehav Rev 2000;24(4):417–63. [9] Spear LP. Adolescence and the trajectory of alcohol use: introduction to part VI. Ann N Y Acad Sci 2004;1021:202–5. [10] Granger DA, et al. Salivary testosterone diurnal variation and psychopathology in adolescent males and females: individual differences and developmental effects. Dev Psychopathol 2003;15(2):431–49. [11] Schulz KM, et al. Gonadal hormones masculinize and defeminize reproductive behaviors during puberty in the male Syrian hamster. Horm Behav 2004;45(4): 242–9. [12] Schulz KM, Sisk CL. Pubertal hormones, the adolescent brain, and the maturation of social behaviors: Lessons from the Syrian hamster. Mol Cell Endocrinol 2006;254–255:120–6. [13] Schulz KM, et al. Testicular hormone exposure during adolescence organizes flank-marking behavior and vasopressin receptor binding in the lateral septum. Horm Behav 2006;50(3):477–83. [14] Zehr JL, et al. Dendritic pruning of the medial amygdala during pubertal development of the male Syrian hamster. J Neurobiol 2006;66(6):578–90. [15] Zehr JL, et al. Adolescent development of neuron structure in dentate gyrus granule cells of male Syrian hamsters. Dev Neurobiol 2008;68(14):1517–26. [16] Ho A, et al. The pubertal-related decline in cellular proliferation and neurogenesis in the dentate gyrus of male rats is independent of the pubertal rise in gonadal hormones. Dev Neurobiol 2011, doi:10.1002/dneu.20987. [17] Edinger KL, Lee B, Frye CA. Mnemonic effects of testosterone and its 5alphareduced metabolites in the conditioned fear and inhibitory avoidance tasks. Pharmacol Biochem Behav 2004;78(3):559–68. [18] Anagnostaras SG, et al. Testicular hormones do not regulate sexually dimorphic Pavlovian fear conditioning or perforant-path long-term potentiation in adult male rats. Behav Brain Res 1998;92(1):1–9. [19] Frye CA, Edinger K, Sumida K. Androgen administration to aged male mice increases anti-anxiety behavior and enhances cognitive performance. Neuropsychopharmacology 2008;33(5):1049–61. [20] Agis-Balboa RC, et al. Enhanced fear responses in mice treated with anabolic androgenic steroids. Neuroreport 2009;20(6):617–21. [21] Myers KM, Davis M. Mechanisms of fear extinction. Mol Psychiatry 2007;12(2): 120–50. [22] McCallum J, Kim JH, Richardson R. Impaired extinction retention in adolescent rats: effects of D-cycloserine. Neuropsychopharmacology 2010;35(10): 2134–42. [23] Kim JH, Richardson R. Extinction in preweanling rats does not involve NMDA receptors. Neurobiol Learn Mem 2010;94(2):176–82.
[24] Hefner K, Holmes A. Ontogeny of fear-, anxiety- and depression-related behavior across adolescence in C57BL/6J mice. Behav Brain Res 2007;176(2):210–5. [25] Wainman P, Shipounoff GC. The effects of castration and testosterone propionate on the striated perinal musculature in the rat. Endocrinology 1941;29:975–8. [26] Laroche J, et al. Reduced behavioral response to gonadal hormones in mice shipped during the peripubertal/adolescent period. Endocrinology 2009;150(5): 2351–8. [27] Zuloaga DG, Jordan CL, Breedlove SM. The Organizational Role of Testicular Hormones and the Androgen Receptor in Anxiety-Related Behaviors and Sensorimotor Gating in Rats. Endocrinology 2011;152(4):1572–81. [28] Benice TS, Raber J. Dihydrotestosterone modulates spatial working-memory performance in male mice. J Neurochem 2009;110(3):902–11. [29] Frye CA, Seliga AM. Testosterone increases analgesia, anxiolysis, and cognitive performance of male rats. Cogn Affect Behav Neurosci 2001;1(4):371–81. [30] Phillips RG, LeDoux JE. Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav Neurosci 1992;106(2):274–85. [31] Yap CS, Richardson R. Extinction in the developing rat: an examination of renewal effects. Dev Psychobiol 2007;49(6):565–75. [32] Kim JH, Richardson R. Immediate post-reminder injection of gamma-amino butyric acid (GABA) agonist midazolam attenuates reactivation of forgotten fear in the infant rat. Behav Neurosci 2007;121(6):1328–32. [33] Kim JH, Richardson R. A developmental dissociation of context and GABA effects on extinguished fear in rats. Behav Neurosci 2007;121(1):131–9. [34] Bouton ME, et al. Contextual and temporal modulation of extinction: behavioral and biological mechanisms. Biol Psychiatry 2006;60(4):352–60. [35] Barad M, Gean PW, Lutz B. The role of the amygdala in the extinction of conditioned fear. Biol Psychiatry 2006;60(4):322–8. [36] Quirk GJ, Garcia R, Gonzalez-Lima F. Prefrontal mechanisms in extinction of conditioned fear. Biol Psychiatry 2006;60(4):337–43. [37] Agis-Balboa RC, et al. A hippocampal insulin-growth factor 2 pathway regulates the extinction of fear memories. EMBO J 2011;30(19):4071–83. [38] McDonald HY, Wojtowicz JM. Dynamics of neurogenesis in the dentate gyrus of adult rats. Neurosci Lett 2005;385(1):70–5. [39] Heine VM, et al. Prominent decline of newborn cell proliferation, differentiation, and apoptosis in the aging dentate gyrus, in absence of an age-related hypothalamuspituitary-adrenal axis activation. Neurobiol Aging 2004;25(3):361–75. [40] He J, Crews FT. Neurogenesis decreases during brain maturation from adolescence to adulthood. Pharmacol Biochem Behav 2007;86(2):327–33. [41] Cowen DS, et al. Age-dependent decline in hippocampal neurogenesis is not altered by chronic treatment with fluoxetine. Brain Res 2008;1228:14–9. [42] Leranth C, Petnehazy O, MacLusky NJ. Gonadal hormones affect spine synaptic density in the CA1 hippocampal subfield of male rats. J Neurosci 2003;23(5): 1588–92. [43] Jernigan TL, et al. Maturation of human cerebrum observed in vivo during adolescence. Brain 1991;114(Pt 5):2037–49. [44] Giedd JN, et al. Brain development during childhood and adolescence: a longitudinal MRI study. Nat Neurosci 1999;2(10):861–3. [45] Sowell ER, et al. In vivo evidence for post-adolescent brain maturation in frontal and striatal regions. Nat Neurosci 1999;2(10):859–61. [46] Markham JA, Morris JR, Juraska JM. Neuron number decreases in the rat ventral, but not dorsal, medial prefrontal cortex between adolescence and adulthood. Neuroscience 2007;144(3):961–8. [47] Casey BJ, Giedd JN, Thomas KM. Structural and functional brain development and its relation to cognitive development. Biol Psychol 2000;54(1–3):241–57. [48] Sowell ER, et al. Improved memory functioning and frontal lobe maturation between childhood and adolescence: a structural MRI study. J Int Neuropsychol Soc 2001;7(3):312–22. [49] Kritzer MF, et al. Gonadectomy impairs T-maze acquisition in adult male rats. Horm Behav 2001;39(2):167–74. [50] Kritzer MF, et al. Effects of gonadectomy on performance in operant tasks measuring prefrontal cortical function in adult male rats. Horm Behav 2007;51(2): 183–94. [51] Gibbs RB. Testosterone and estradiol produce different effects on cognitive performance in male rats. Horm Behav 2005;48(3):268–77. [52] Kulkarni SK, Sharma AC. Elevated plus-maze: a novel psychobehavioral tool to measure anxiety in rodents. Methods Find Exp Clin Pharmacol 1991;13(8):573–7. [53] Itoh J, Nabeshima T, Kameyama T. Utility of an elevated plus-maze for dissociation of amnesic and behavioral effects of drugs in mice. Eur J Pharmacol 1991;194(1): 71–6. [54] Alexander JC, et al. The role of calsenilin/DREAM/KChIP3 in contextual fear conditioning. Learn Mem 2009;16(3):167–77.
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Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
Role of gonadal hormones in anxiety and fear memory formation and inhibition in male mice Carmel M. McDermott a, Dana Liu a, Laura A. Schrader a, b,⁎ a b
Dept. of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118 Neuroscience Program, Tulane University, New Orleans, LA 70118
a r t i c l e
i n f o
Article history: Received 1 September 2011 Received in revised form 16 December 2011 Accepted 16 December 2011 Keywords: Fear conditioning Extinction Castration Elevated Plus Maze Open Field Assay Puberty Hippocampus
a b s t r a c t Recent research investigating Pavlovian fear conditioning and fear extinction has elucidated the neurocircuitry involved in acquisition and inhibition of fear responses. Modulatory factors that may underlie individual differences in fear acquisition and inhibition, however, are not well understood. Testosterone is known to affect anxiety-like behavior and cognitive processing. In this study, we hypothesized that castration would increase anxiety and reduce memory for contextual fear conditioning in an age-dependent manner. In addition, castration would reduce the rate of extinction to context, as high levels of testosterone correlate with reduced PTSD-like symptoms. We compared behaviors in male mice that were castrated at one of two different time points, either before puberty (at 4 weeks) or after puberty (at 10 weeks) to sham-operated control mice. The behaviors investigated included: anxiety, cued and contextual fear conditioning, and extinction of the fear memory. An interaction of hormone status and age and a significant effect of age were measured in the elevated plus maze, a measure of anxiety. Castration caused a significant reduction of contextual fear memory, but no effect on cued fear memory. There was no significant effect of castration on extinction. Interestingly, a significant effect of age of the mouse at the time of testing was observed on extinction. These results suggest that endogenous androgens during puberty are important for anxiety and fear memory formation. In addition, these results define a late post-adolescent developmental time point for changes in anxiety and fear extinction. © 2012 Elsevier Inc. All rights reserved.
1. Introduction 1.1. Human studies Depression and anxiety-related disorders afflict men at approximately half the rate at which they affect women [1]. Convincing evidence suggests that testosterone and/or its metabolites play a protective role against these disorders, and low levels of testosterone are correlated with increased affective disorders. For example, hypogonadal men, who suffer from lower than normal levels of testosterone, are more likely to suffer from anxiety and depression disorders, and treatment with testosterone alleviates these symptoms [2-4]. In addition, bioavailable testosterone levels undergo a gradual decrease during aging, and this decrease is correlated to an increase in the incidence of depression-related disorders [5]. Gender differences are also seen in the processing of traumatic episodes. A meta-analysis study suggests that even when exposure to
⁎ Corresponding author at: 2000 Percival Stern Hall, 6400 Freret St, New Orleans, LA 70118. Tel.: + 1 504 862 3139. E-mail address: [email protected] (L.A. Schrader). 0031-9384/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2011.12.016
the potentially traumatic event is controlled for, men tend to develop posttraumatic stress disorder (PTSD) less frequently and of a less severe nature than women [6]. Therefore, further investigation into the role of testosterone in anxiety, fear memory formation and inhibition is warranted. 1.2. Puberty and the role of androgens Puberty is a developmental time point in adolescence where sex hormone levels increase dramatically [7], and changes in behavior occur. In humans, adolescence is characterized by increased impulsivity, risk taking behavior and susceptibility to various neuropsychiatric disorders [8,9]. The effects of androgens during puberty are important mechanisms to modulate various behaviors. For example, low testosterone levels in young males were related to increased levels of anxiety, depression and attention problems [10]. Data from animal studies show that the levels of sexual [11] and social behaviors [12,13] are reduced in males castrated before puberty compared to males castrated after puberty. In addition, neuron and circuit structure can undergo dramatic changes during puberty in limbic areas of the brain [14-16], suggesting the function of these areas is impacted by puberty.
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1.3. Fear memory and extinction A number of studies have examined the role of testosterone in fear memory. In 55 day old male rats, removal of endogenous testosterone by castration resulted in a trend toward reduced memory for hippocampus-dependent contextual fear conditioning, an effect that was reversed by administration of testosterone and its metabolites, but did not affect cued fear conditioning [17]. In older rats, no effect of castration was seen in memory formation for contextual fear conditioning [18]. Administration of 3α-diol, a metabolite of testosterone, enhanced inhibitory avoidance and contextual fear memory in aged (24 months) male intact mice, increased inhibitory avoidance in young (12 weeks) intact mice, and increased contextual fear conditioning in young GDX mice. Interestingly, no effect on cued fear conditioning in any of the groups was observed [19]. Testosterone administration to young female mice also enhanced contextual fear memory [20]. These studies suggest that a developmental profile of the influence of gonadal hormones on hippocampus-dependent fear memory may exist, but this has not yet been investigated. Extinction is the process by which a fear memory is inhibited. Existing theories suggest that fear extinction involves a combination of new associative learning processes and decreased responsiveness to the fear-eliciting stimulus [21]. Little is known about the role of testosterone or its metabolites in extinction; however developmental aspects of extinction have been investigated. Adolescent rats (35 days) exhibited impaired cue extinction retention compared to adults and younger animals [22,23]. In addition, early adolescent (4 week) C57 male mice showed enhanced cued fear memory relative to adults, but a similar extinction rate to the associated cue as adult animals [24]. These differences may be due to the activational effects of hormone levels at different ages or organizational effects of the hormone surge during puberty. Therefore, the role of androgens in fear memory formation during postnatal development, particularly during puberty, warrants further study. This study investigated the effects of gonadal steroids on anxiety, fear memory and extinction in male mice that were castrated either before puberty or in adulthood. Based on previous literature, we hypothesized that castration would increase anxiety in both ages and reduce memory for contextual fear conditioning. In addition, castration would reduce the rate of extinction to context in both young and adult mice, as high levels of testosterone correlate with reduced PTSD-like symptoms. Our results show a significant age and hormone status interaction in anxiety-related behavior measured on the elevated plus maze. In addition, castration significantly reduced contextual fear memory. Contrary to our hypothesis, we observed no effect of castration on extinction at either age. Interestingly, however, young mice, 8 weeks at time of testing, did exhibit enhanced fear extinction compared to older animals, 14 weeks at time of testing. These studies define a role for gonadal hormones during puberty in regulation of fear behaviors and a late developmental time point for fear extinction. 2. Methods Subjects were male 129svE mice from Charles Rivers. Young animals (n = 20) arrived at 3 weeks and adult animals (n = 18) arrived at 9 weeks. All experiments were performed within the guidelines of the Tulane University Institutional Animal Care and Use Committee and the US Army Medical Research and Materiel Command Animal Care and Use Review Office (ACURO). Mice were housed in groups of three or four on a 12 h light–dark cycle with access to food and water ad libitum.
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were performed at either 4 weeks (pre-adolescent) or 10 weeks of age (adult). Orchidectomy was performed through a single transverse incision across the end of the scrotum. The testicles were removed and a ligature was used around the vas deferens and spermatic vessels to prevent hemorrhage. Sham animals received similar incisions with no testicle removal. Buprenorphine (0.03 mg/kg) was injected at the time of surgery for pain relief. Mice were allowed to recover for 2 weeks before any behavioral testing was performed. The effectiveness of castration was determined after behavior upon sacrifice, and bulbospongiosus muscles were dissected from the penile bulb and immediately weighed. 2.2. Behavior 2.2.1. Anxiety - Open Field Analysis of anxiety behavior commenced two weeks after surgery. Two tests of anxiety behavior were assessed: open field (Weiss et al., 2000) and elevated plus maze (EPM) [52,53]. These tests are based on a rodents’ proclivity to avoid well-lit, open spaces. Less anxious animals will spend more time in the center of the open field and in the open arms of the elevated plus maze. Open field was performed 14 days after surgery. A single mouse was placed in the center of a white, Plexiglas chamber measuring 43 cm in length x 43 cm in width x 18 cm in height, under well-lit conditions. The animal explored the novel environment for 15 minutes and movements were monitored by a camera interfaced with a tracking system (US HVS Image 2100 Tracking System). The area was divided into 16 virtual squares (10.75 × 10.75 cm) by the program, and the middle four squares were defined as the center area. The Plexiglas chamber was wiped clean with 70% ethanol between trials. 2.2.2. Elevated Plus Maze The elevated plus-maze (EPM) was performed 17 days after surgery. EPM consisted of four arms (5 cm in width x 30 cm in length) arranged perpendicularly in a plus shape and elevated 38 cm above the floor and placed under well-lit conditions. The entire maze was tan and two arms were enclosed by 15.5 cm opaque Plexiglas walls and two arms were open. Each animal was placed in the center of the EPM facing a closed arm and allowed to move freely for 5 minutes. Behavior was monitored by a camera interfaced with the tracking system. The EPM was wiped clean with 70% ethanol between trials. 2.2.3. Fear Conditioning Mice were 3 weeks post-surgery at the time of fear conditioning. Fear conditioning was performed using a computer-controlled, sound-attenuated, conditioning chamber (29 × 19 × 25 cm, Med Associates). On the day of the fear conditioning training, the mouse was exposed to the chamber for the first time and allowed to explore the chamber. At 120 seconds a 30 second tone (white noise) commenced and the tone co-terminated with one 2 s electrical foot shock (0.5 mA) through a stainless steel grid floor. After the foot shock, the mouse was allowed to explore the context for an additional 150 seconds. The behavior of the mouse was measured by a digital infrared video camera mounted in front of the chamber. 2.2.4. Context-dependent freezing 24 hours after fear conditioning the mouse was returned to the chamber to measure context-dependent fear conditioning. The mouse was placed in the chamber for 300 seconds and the percentage of freezing was analyzed using Video Freeze Software. The chamber was wiped clean with 70% ethanol between animals.
2.1. Gonadectomy Mice were anesthetized by intraperitoneal injection of a ketamine (80 mg/kg) and xylazine (8 mg/kg) mixture. Gonadectomies
2.2.5. Cue-dependent freezing 48 hours after initial fear conditioning mice were returned to the chamber. The context was altered with plastic floors and alternative
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context shape. Vanilla scent was added in the context for alternative olfactory cues. After 150 seconds they were exposed to the tone for 150 seconds and the percentage of freezing was measured before and during tone exposure. The chamber was wiped clean with isopropyl alcohol between animals. 2.2.6. Shock Threshold and Fear Extinction At 4 weeks post-surgery mice were placed in the fear conditioning chamber and allowed to explore for 30 s before being exposed to a series of foot shocks. The foot shocks lasted 2 s and ranged in intensity from 0.1 to 0.7 mA. The behavior of the animals was recorded and responses were scored in order to determine differences in responses to increasing shock sensitivities. 48 hours after shock threshold, mice were returned to the chamber for 5 min to monitor freezing (day 1 of extinction). This was repeated for 4 days and the degree of freezing was plotted to monitor extinction of fear.
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3.1. Anxiety Since anxiety levels can modulate memory, the anxiety levels in the young and adult males, castrated and sham-operated were examined. Anxiety behavior was assayed using the open field and elevated plus maze as described in methods. A two-way ANOVA (hormone status x age) showed that there was no significant interaction (F1,32 = 0.31; p = 0.58), effect of castration (F1,32 = 2.89; p = 0.1) or age (F1,32 = 0.36; p = 0.55) in the amount of time spent in the center of the open field (Fig. 1A). The percent time spent in the open arms of the EPM was also evaluated (Fig. 1B). There was a significant interaction of hormone status and age (F1,32 = 5.45; p = 0.03), no significant main effect of hormone status (F1,32 = 0.54; p = 0.47), but a main effect of age (F1,32 = 4.4; p = 0.04). The number of entries into the open arms as a percentage of total number of arm entries was also evaluated (data not shown). No significant interaction (F1,32 = 0.22; p = 0.64), effect of castration (F1,32 = 0.09; p = 0.77) or age (F1,32 = 0.35; p = 0.56) was observed, suggesting that the groups entered the arms of the maze to a similar extent. Surprisingly, there was an effect of age in the distance traveled in the open field and EPM (data not shown). Young males explored the open field significantly less than adults (F1,32 = 9.28; p = 0.005). No significant interaction (F1,32 = 0.05; p = 0.83) or effect of castration (F1,32 = 0.052; p = 0.82) was observed. There was also a trend toward reduced distance traveled in the elevated plus in the young compared to the adult (F1,32 = 3.7; p = 0.06), but no significant interaction (F1,32 = 0.014; p = 0.9), or effect of castration (F1,32 = 0.04; p = 0.84) was observed. These results suggest that castration had no significant effect on anxiety in either the young or adult males, but
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The goal of this study was to determine the effect of gonadal hormones on anxiety, fear conditioning and extinction in male mice that were castrated at two different time points, before puberty and as adults. In order to accomplish this, the source of endogenous androgens was removed by castration in males and the behavioral results from sham-operated intact and castrated males were compared. The effectiveness of castration was confirmed in animals after behavioral studies were completed. Castration causes shrinkage of the bulbospongiosus muscles (BSM) [25]. Castrations were confirmed by comparison of BSM mass in sham and castrated animals. Average BSM weight in adult castrated males (n = 8; 42.5 ± 1.4 mg) was significantly decreased compared to adult sham males (n = 9, 58.3 ± 2.6 mg; p b 0.01, unpaired t-test). One adult castrated male was removed from the study for ineffective castration. BSM weight in young castrated animals was also significantly decreased (n = 10; 27.8 ± 3.0 mg, p b 0.05) compared to sham-operated intact males (n = 10; 37.9 ± 3.1 mg).
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Fig. 1. Results of assessment of the effect of castration before and after puberty in males on anxiety behaviors. A. Bar graph showing the percentage of time spent in the center of the field in the sham-operated and mice that were castrated before puberty (left) and after puberty (right). B. Bar graph showing the results of the percent time spent in the open arms of the EPM in sham-operated male mice or mice castrated before puberty (left) and after puberty (right). A significant interaction of age and hormone status and a main effect of age were observed (*, p b 0.05).
that there was a significant interaction and effect of age in anxiety measured on the EPM.
3.2. Fear Conditioning Animals were trained on day 1 of fear conditioning with a mild training paradigm, consisting of 120 seconds habituation to a chamber, followed by a 30 second auditory cue co-terminating with a single 0.5 mA foot shock. Animals remained in the chamber for another 150 seconds. Animals were tested for contextual memory 24 hours after training. There was no significant interaction (F1,31 = 0.31; p = 0.58), a significant effect of hormone status (F1,31 = 4.0; p = 0.05), but not age (F1,31 = 1.17; p = 0.29) on contextual fear conditioning memory (Fig. 2A). Since we saw effects on path length in the anxiety measures, we analyzed average motion during nonfreezing episodes in the contextual test. We observed no significant interaction (F1,31 = 0.01; p = 0.93), effect of hormone status (F1,31 = 0.44; p = 0.51) or age (F1,31 = 0.07; p = 0.79). One adult sham animal was removed from this analysis due to technical difficulties with the equipment. These results suggest a significant effect of castration on contextual fear conditioning, but no effect on motion during non-freezing episodes. The cued test was performed 48 hours after training. Importantly, very little freezing was observed in the modified context before introduction of the cue (Fig. 2B). There was no significant interaction
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two-way repeated measures ANOVA (repeated measures on stimulation intensity). There was no significant interaction (F8,126 = 1.6; p = 0.14) or effect of castration (F1,126 = 0.98; p = 0.32) in the adult animals (Fig. 3A). As expected, there was a significant within subjects effect of stimulation intensity (F8,126 = 100; p b 0.0001). In the young animals, there was no significant interaction (F8,162 = 0.86; p = 0.55), but there was an effect of castration (F 1,162 = 7.3, p = 0.008). A significant effect of intensity was also observed (F8,162 = 153.3; p b 0.0001; Fig. 3B). These data suggest that castration increased the response to an aversive stimulus only in the young males.
3.3. Extinction
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We investigated the extinction of the fear response to context 48 hours after the foot shock response assay. Freezing was assessed during repeated exposure to the context for 5 minutes administered over 4 days. To assay extinction learning, we reasoned that a threshold of freezing on day 1 must be reached in order to be adequately trained to assay extinction. We set a threshold at 45% freezing for the first day of extinction training. Two young castrated males froze less than 40% on first day; therefore these animals were eliminated from the extinction data. We first determined the effect of castration in the adult males (Fig. 4A). Two-way repeated measures ANOVA (repeated measures on extinction trial) revealed no significant interaction (F3,56 =1.1; p=0.37), effect of castration (F1,42 =1.01; p=0.32), but a significant effect of trial (F3,56 =11.29; pb 0.001; Fig. 4B). Similarly, in the young males there was no significant interaction (F3,64 =0.14; p=0.93) or effect of castration (F1,64 =2.0, p=0.16), but a significant effect of trial as expected (F3,64 =17.4; pb 0.0001). Since there was no significant effect of
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(F1,32 = 0.35; p = 0.56), effect of age (F1,32 = 0.96; p = 0.35), or hormone status (F1,32 = 3.72; p = 0.06) in the modified context before the tone, suggesting the animals were not generalizing the context. No significant interaction (F1,32 = 0.68; p = 0.41), effect of hormone status (F1,32 = 1.79; p = 0.19) or age (F1,32 = 0.22; p = 0.64) was observed for freezing during the cue (Fig. 2C). Together, these results suggest that castration significantly decreases contextual fear memory but not cued memory in young and adult males. The effect of age and castration on foot shock response was also tested. The response to an increasing intensity of foot shock was measured [54] and compared between hormone status groups in the individual ages (Fig. 3A, B). The data in each age group were analyzed by
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Fig. 2. Contextual and cued fear conditioning results from animals castrated before and after puberty. A. Bar graph showing the amount of time spent freezing in response to the context 24 hours after training in animals castrated before puberty (left) and after puberty (right). Castration significantly reduced contextual fear memory formation compared to sham animals (*, p b 0.05). B. Amount of freezing observed in novel context before commencement of cue (white noise tone) in cued test. There were no significant differences observed in the amount of freezing to the novel context. C. Bar graph showing the amount of time spent freezing in response to the cue (white noise tone) 48 hours after the training from mice castrated before puberty (left) and after puberty (right).
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and in adulthood. We report an interaction of age and hormone status on anxiety in the EPM, with a significant main effect of age. In addition, we observed an effect of hormone status on contextual fear conditioning. Studies in adult animals demonstrated no effect or subtle differences between sham and castrated animals, consistent with our results. In interpretation of our data, it is also important to consider a recent study that found that shipping stress during the peripubertal/adolescent period reduced the effects of sex hormones on sex behavior and caused hypoactivity of the hypothalamo-pituitary axis [26]. While our adult animals were 10 weeks (post-pubescent) at the time of shipping, our young animals were 4 weeks at the time of shipping. The behavior of the young animals in particular and their response to the castration may have been affected by the shipping stressor.
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The results from the EPM indicate a significant interaction of age and hormone status and a main effect of age. Studies from rodents confirm a differential role for testosterone in anxiety behavior in young and adult animals. Both mice and rats demonstrate an increase in anxiety behavior after castration as adults [12, 13]. Similar to our results, neonatal castration reduced measures of anxiety in the adult [27]. Previous studies showed that castration in adult mice, on the other hand, increased the amount of time spent in the perimeter of the open field, an indication of increased anxiety, however there was no significant effect in the elevated zero maze [28]. Another study reported that castration in adult rats caused a decrease in the amount of time spent in the open arms of elevated plus [29]. Administration of testosterone or its metabolites was anxiolytic in young GDX mice [14, 15], aged intact mice [15], and GDX rats [13], but had no overall effect on anxious behavior in young intact mice [15]. Therefore, our results of a significant interaction of age and hormonal status are consistent with previous literature that castration before puberty reduced anxiety and after puberty increased anxiety. One possible explanation for this result, however, could be that young males explored both the open field and EPM less, as there was a trend toward reduced path length of the young males in the elevated plus and significantly lower path length of the young males in the open field. Entries into the open arms, however, were not significantly different. This suggests that while the young animals traveled less distance, they entered the open arms an equivalent number of times as the adult animals.
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Days Fig. 4. The effect of castration on extinction training. Animals were tested for foot shock sensitivity and then 48 hours later received extinction training, consisting of context exposure with no US, for 5 minutes a day over a 4 day period. A. Results from extinction training in males castrated after puberty. B. Results from four days of extinction training in mice castrated before puberty. Castration before puberty had no significant effect on extinction at either age. C. Comparison of extinction rate in young compared to adult mice. Young male mice exhibited rapid extinction to context compared to adult male mice.
castration, the sham-intact and castrated groups were combined from each age into young and adult groups (Fig. 4C). Interestingly, a significant effect of age emerged. A significant interaction (F3,96 =7.66; pb 0.0001), effect of age (F1,96 =10.27; p=0.05) and trial (F3,96 =93.51; pb 0.0001) was observed. Together these results suggest that while both the young and adult animals extinguished the fear, no effect of castration was observed in either the young or adult animals on the rate of extinction. 4. Discussion In this study, we investigated the role of castration at two ages, before puberty to determine the effects of testosterone during puberty,
4.2. Fear Conditioning The findings of this study showed a significant effect of castration in the contextual fear memory formation test. This result suggests that the presence of testosterone may result in functional changes during puberty or activational effects after puberty that can affect fear memory, particularly hippocampus-dependent contextual fear memory formation. Other studies examined the effects of castration in fear conditioning in rats. One study in adult rats (approximately 130 days at castration) showed no effect of castration on cued and contextual fear conditioning in rats that were trained over several days [18], while another study showed no effect on cued fear conditioning with a trend toward a significant reduction of freezing in contextual fear conditioning in younger (55 days at time of castration) male rats [17]. This reduction in fear conditioning memory was reversed by testosterone and testosterone metabolite replacement [17] but not estradiol [19]. It is generally considered that contextual memory is dependent on the hippocampus and amygdala, while cued memory is only dependent on the amygdala [30]. Together with the current study, these data suggest that androgens enhance hippocampus-dependent fear memory formation and have no effect on amygdala-dependent cued fear memory.
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4.3. Fear Extinction We observed no significant influence of castration on the rate of extinction of the contextual memory in the mice castrated either pre-or post-puberty. There were no significant effects of castration before puberty in the young adult and no effect of castration in adults on the rate of extinction. The young males, regardless of hormone state, extinguished the contextual fear memory significantly faster than the adult males. In agreement with our results, a previous study also showed no effect of castration on extinction of the contextual fear in adult animals [18]. These results suggest that androgens, either during puberty or in adulthood, do not play a role in extinction of fear memory. We did, however, observe a significant effect of age, regardless of hormone state, on extinction of the contextual fear. In this study, young males, 8 weeks at the time of testing, extinguished fear of the context significantly faster than adult animals, 14 weeks at the time of testing, independent of hormone status. This result suggests the presence of a post-adolescent developmental time point that is important for fear memory inhibition. Other studies have shown a developmental regulation of extinction of the cued memory, with adolescent males exhibiting a reduction in extinction retention [22], and a delay in the rate of extinction in 4 week old mice compared to 6 or 8 week mice. This is in contrast to our results showing enhanced extinction of the contextual fear memory in the young mice. Previous studies have suggested that the mechanisms of extinction in young (weanling) mice differ from the mechanisms in the adult mice. It is thought that in young animals, the fear memory is basically forgotten, while in adult animals, a new memory of the conditioned stimulus as non-aversive is formed [31]. This forgetting is proposed as a mechanism of extinction in young rats, as young rats do not show renewal or reinstatement of extinguished fear [31-33]. Our results suggest modulation at an additional, later developmental time point for the extinction of the contextual memory. Furthermore, we suggest that gonadal hormones during puberty do not mediate the effect on the extinction rate, since castration before puberty had no effect on extinction rate. 4.4. Shock Sensitivity Another factor that can affect the fear memory formation in these experiments is differences in the sensation of the foot shock. Previous studies have shown that testosterone can modulate pain responses. Endogenous or testosterone replacement had analgesic effects in a tail-flick experiment [29]. In our study, there was no significant effect of castration in response to increasing stimulus intensity in the adult, suggesting no modulation of the response to foot shock by testosterone. In the young mice, however, castration significantly increased the response to foot shock. This is in contrast to an effect of castration to decrease fear memory formation; therefore differences in shock sensitivity cannot explain the castration-dependent difference in contextual fear memory formation or in the rates of extinction between young and adult mice as both groups started out with similar levels of freezing. 4.5. Neural substrates The neural substrates and circuitry involved in fear conditioning and extinction processes have been detailed. The amygdala is known to be critical for fear learning, while context-associated fear learning also involves the hippocampus [30]. Fear extinction is thought to be a form of inhibitory learning and is considered to be a process distinct from fear learning. Three brain areas and their interconnecting circuitry are highly implicated in the extinction circuitry, including the hippocampus [34], the amygdala [35], and the medial prefrontal cortex [36]. Therefore, modulation of plasticity in these
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areas or the connections between the areas can modulate fear conditioning and/or fear extinction. Since we observed effects on contextual modulation of fear memory in our studies, testosterone- or age-mediated changes in the hippocampus likely play a role in our effects. Cued fear conditioning was unaffected; therefore, we do not believe that the locus of the effects is the amygdala. A recent paper showed that extinction modulates expression of insulin growth factor-2 (IGF2) in the hippocampus, and IGF2 infusion regulates extinction [37]. One of the cellular targets of this IGF signaling is neurogenesis. Neurogenesis is approximately twice as high in prepubertal males compared to adults [16, 38-41], and this reduction of neurogenesis in adulthood is not dependent on androgens [16]. Since we observed an effect of age, but not castration in the extinction process, changes in neurogenesis are a possible explanation for the effects on extinction, further studies are necessary to examine this issue. Furthermore, the pyramidal cells of the hippocampus are richly endowed with androgen receptors and manipulation of testosterone levels directly affects spine synapse density in the CA1 region [42], providing another possible mechanism for modulation of contextual fear memory in our experiments. Together, these data suggest that the function of the hippocampus, but not the amygdala, in fear memory formation is modulated by androgens. Furthermore, extinction of the contextual fear memory is not affected by androgens, but is reduced in the adult. This differential effect of castration and age on fear memory formation and extinction, respectively suggests different mechanisms are involved in regulation of each process. Since we observed a late developmental difference in extinction in male mice, the developmental difference that we observed on extinction may also occur in the prefrontal cortex. It is well recognized that the prefrontal cortex continues to develop into late adolescence. In humans the volume of gray matter in the frontal cortex increases until around adolescence and then decreases between adolescence and young adulthood [43-45]. This reduction in gray matter during adolescence is thought to be caused by neuronal loss [46]. Concurrent with these anatomical changes are changes in the ability to perform prefrontal cortex-dependent tasks during adolescence [47,48]. The effects of gonadal hormones on other prefrontal tasks appear to be fairly complex. Castration and/or hormone replacement significantly impaired performance on a range of frontal lobe-dependent tasks. Castration in adult male rats, increased the time required to reach a training criterion in the T-maze delayed alternation paradigm, a task that is sensitive to lesions of the prefrontal cortex [49]. Kritzer et al (2007) found more selective effects of castration on prefrontal function [50]. Castration adversely affected acquisition in a response alternation task and a light/dark discrimination task requiring behavioral flexibility. These effects were eliminated by supplementation with testosterone propionate but not estradiol. This suggests that aromatization of testosterone to estradiol is not necessary for this task. On the other hand, other tasks examining prefrontal functions, such as motivation and response withholding, were sensitive to castration. The effects were alleviated by supplementation with testosterone propionate or estradiol, suggesting aromatization is necessary for these tasks. Other tasks examining impulsivity and reversal learning were insensitive to castration. Using a delayed match to position version of the T-maze, Gibbs (2005) observed no differences between gonadectomized and intact rats [51], although by using longer delays with supraphysiologic doses of testosterone propionate, an effect became evident. Thus, the literature suggests that gonadal hormones affect prefrontal function but in a highly selective manner. In conclusion, we saw an overall effect of castration on contextual fear conditioning. Furthermore, we observed developmental effects on extinction, in that young males showed an increased extinction rate. Extinction in sham animals was similar to castrated animals at each age, suggesting that testosterone is not a mechanism of the developmental change, and another mechanism underlies the switch
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