Testosterone prevents but not reverses anhedonia in middle-aged males and lacks an effect on stress vulnerability in young adults

Hormones and Behavior 61 (2012) 623–630

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Testosterone prevents but not reverses anhedonia in middle-aged males and lacks an effect on stress vulnerability in young adults José Jaime Herrera-Pérez a, b, Lucía Martínez-Mota a, Roberto Chavira c, Alonso Fernández-Guasti b,⁎ a b c

Laboratorio de Farmacología Conductual, Dirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñíz”, México City, Mexico Departamento de Farmacobiología, Centro de Investigación y de Estudios, Avanzados del IPN, México City, Mexico Laboratorio de Hormonas Esteroides, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México City, Mexico

a r t i c l e

i n f o

Article history: Received 4 October 2011 Revised 7 February 2012 Accepted 11 February 2012 Available online 20 February 2012 Keywords: Aging Anhedonia Chronic mild stress Resilience Testosterone Antidepressant-like effect

a b s t r a c t Middle-aged male rats are more vulnerable than young adult ones to develop anhedonia when exposed to chronic mild stress (CMS). Clinical studies support the idea that in aged subjects the low testosterone (T) levels are related with their higher stress vulnerability and that this hormone possesses antidepressantlike actions. In this study we evaluated the role of gonadal hormones — mainly T — on the depressive-like behavior of middle-aged and young adult male rats submitted to CMS. In middle-aged rats we analyzed the effect of T restitution (at the levels of young adult animals) given 3 weeks before (experiment 1) or 3 weeks after (experiment 2) anhedonia development (indicated by a reduction in sucrose solution intake). T restitution before CMS effectively prevented anhedonia but failed to reverse it once installed. In young adult rats we studied if orchidectomy increased stress vulnerability and found that it failed to modify sucrose intake. These results indicate a stress-dependent differential effect of T in middle-aged rats an age differential role of gonadal hormones on the vulnerability to develop anhedonia. The results suggest that T is a resilience factor in middle-aged but not in young adult males. © 2012 Elsevier Inc. All rights reserved.

Introduction Depression is the most common psychiatric disease in the elderly with a prevalence ranging from 22 to 46% in patients over 65 years old (Lebowitz et al., 1997). This disorder is characterized by anhedonia (incapacity to experience pleasure), feelings of sadness, depressed mood and guilt (American Psychiatric Association, 2000). It is estimated that by the year 2020, depression will be the second cause of disability and mortality worldwide (Murray and Lopez, 1997). In the USA, the highest rate of suicides associated to depression is found in men older than 65 years and this rate doubles at around 85 years of age (Reynolds and Kupfer, 1999). These features make depression in late life a major public concern that acquires more importance as world life expectancy increases (United Nations, 2010). Aging is a complex process (Lamberts et al., 1997; Smith et al., 2005) that involves changes in various neuroendocrine systems that have been associated with depression. There are numerous reports describing that aged men, in contrast with young, can develop depression when exposed to everyday life stress (Bogdan and Pizzagalli, 2006; Millom and Davis, 1999; Plotsky et al., 1998) and that as aging advances there is a steady reduction in testosterone (T) levels as a result of an alteration of the hypothalamus–pituitary–gonadal (HPG) axis (Lamberts

⁎ Corresponding author. Fax: + 52 55 5483 2863. E-mail address: [email protected] (A. Fernández-Guasti). 0018-506X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2012.02.015

et al., 1997). Interestingly, such reduction in T levels (b2.5 ng/ml) in old men predicts a higher incidence of depressive illness (Shores et al., 2005). The relevance of T on vulnerability to depression is supported by several clinical studies in aged or young adult hypogonadal men; in this line, it has been shown that in men aged 50–89 the severity of depression (indicated by high scores in the Beck Depression Inventory) increases with age and is inversely related to bioavailable T (BarretConnor et al., 1999). There are also evidences showing that, in hypogonadal men (22–62 years old), low serum T levels are related with negative mood symptoms and these correlations disappeared after T replacement therapy (Wang et al., 1996). Accordingly, Shores et al. (2004) found that T treated hypogonadal patients (mean age 65 years) were 2 to 3 times less susceptible to develop depression than untreated patients. Furthermore, a study done to examine the effectiveness of T replacement as an antidepressant treatment shows that aged (more than 50 years old) eugonadal men with late-life depression (first episode after 45 years of age) responded better to T treatment than those with early-life first-episode depression (Perry et al., 2002). These data suggest that the levels of T in males are inversely related to depression vulnerability, and that T restitution could reverse this pathology; however, there is not a direct study demonstrating this relationship. Chronic mild stress (CMS) is an animal model used to study the neurobiological bases of depression (Willner, 1997). This model simulates anhedonia, a core symptom of depression, which is reflected as a decrease in sucrose solution consumption produced by the chronic

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exposure to mild stressors (Willner, 1997, 2005; Willner et al., 1987). It has been consistently observed that the stress-dependent sucroseintake reduction is specifically reversed by antidepressants and steroid hormones with antidepressant properties (Montgomery et al., 2001; Romano-Torres and Fernández-Guasti, 2010; Willner, 1997). In a previous study we found that, after CMS, a higher percentage — 73% — of middle-aged rats (13–15 months) developed anhedonia as compared with only a 35% of young adult ones (3–5 months). In addition, middle-aged male rats presented around a fourth of the basal levels of T and estradiol than young adult animals (HerreraPérez et al., 2008). These data, as clinical studies, suggest that the increased susceptibility to develop anhedonia is inversely related to the levels of male gonadal hormones. This study was aimed to evaluate the role of gonadal hormones — mainly T — on the depressive-like behavior of middle-aged and young adult male rats submitted to the CMS. In middle-aged rats we analyzed the effect of T restitution (at the levels found in young adult animals) given before (experiment 1) and after (experiment 2) anhedonia development. Results showed that T replacement prevented, but not reversed, the anhedonic action of CMS. On these bases, in a third experiment we studied in young adult rats if the withdrawal of gonadal hormones — by orchidectomy — increased stress vulnerability and found that this surgery failed to reduce sucrose intake. Materials and methods Animals Adult young and middle-aged (3–5 and 12–15 months, respectively) male Wistar rats were obtained from the Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñiz”. Animals were individually housed in small cages (35 × 25 × 15 cm) one week before starting the experiment and maintained on a 12:12 h dark–light inverted cycle (lights off at 10:00 h), with free access to water and food, under controlled temperature and humidity. These conditions varied according to requirements of the CMS procedure. Animal management was done following the general principles of laboratory animal care (NIH publication 85-23, 1985). All experimental procedures were performed in accordance with the Mexican official norm for animal care and handling (NOM-062-ZOO-1999) and approved by the Ethical Committee of the “CINVESTAV-IPN” and Instituto Nacional de Psiquiatría “Ramón de la Fuente Muñiz”. All efforts were made to minimize the number of animals used and their suffering. Testosterone restitution of middle-aged rats To restore the T levels of middle-aged animals to those found in young adults we used T-containing pellets, which were prepared with polydimethyl silicone tubes (Silastic Rx 50, Dow Corning, ID: 1.57 mm, OD: 3.18 mm). The tubes, of 1 cm long, were filled with T propionate (~9 mg, Sigma Chemicals) and their ends were sealed with pure silicone. These pellets were individually placed in the rats' cervical region under tribromoethanol (200 mg/kg, SigmaAldrich) anesthesia. The incision was sutured and cleaned with antiseptic solution and the animals returned to their home cages. In order to determine the time-course of hormone delivery of these T pellets in vivo, groups (n = 4–5) of unstressed middle-aged animals were sacrificed by decapitation at different intervals (3, 8, 15, 21 and 29 days) after the T pellet placement; then their trunk blood was collected in cold tubes for determining T serum levels, as described below. Once established the suitability of the T restitution method, another group of middle-aged males received T pellets and was left undisturbed for one week before starting the sucrose consumption training.

Thereafter these animals were subjected to the CMS as described below. Proper controls were included (see experiment 1 in Fig. 1). To evaluate the antidepressant-like effect of T, a group of anhedonic middle-aged rats (i.e. after 3 weeks of CMS exposure) received a T pellet. In parallel, an unstressed control group of rats received the pellet 3 weeks after baseline sucrose consumption determination (see experiment 2 in Fig. 1). Orchidectomy in young adult rats Young adult males were orchidectomized under anesthesia with tribromoethanol (200 mg/kg, Sigma-Aldrich). Briefly, a single midline incision was made in the low abdominal area to expose the testes; the vas deferens was bilaterally ligated and the testes were removed. The muscle and skin were sutured and the ventral area was cleaned with an antiseptic solution. Animals were returned to their home cages following surgery and left one week of recovery before sucrose consumption training (see experiment 3 in Fig. 1). In order to evaluate the T levels of orchidectomized young rats, a group was left undisturbed for three weeks after surgery and then sacrificed. Chronic mild stress model Sucrose consumption training Rats were allowed to adapt to the taste of a palatable sucrose solution (1%) for two consecutive weeks. During this period, 1 h every day, a bottle containing sucrose solution was presented to the rats at the beginning of the dark phase (10:00 h). After these two weeks, the baseline sucrose consumption was determined. For this purpose, the rats were water and food deprived for 20 h and thereafter presented with two bottles for 1 h: one containing sucrose solution (1%) and the other tap water (to control for non specific liquid intake changes). Fluid (sucrose solution or tap water) consumption was calculated by weighing the bottles before and after the test. In the CMS paradigm the animals were exposed, for 3–7 weeks, to several stressors: white noise (~90 dB), overcrowding (2–3 animals per cage), continuous light, soiled cage (250 ml water spilled into bedding), stroboscopic light (300 flashes/min), 45° cage tilt along the vertical axis and water deprivation. The stressors' schedule followed in this study (Table 1) has been reported to induce anhedonia in young adult and middle-aged rats (Herrera-Pérez et al., 2008). The males of the unstressed groups were maintained for three weeks without stress, but under comparable handling and storage conditions than the stressed animals. In all groups, the rats' sucrose solution and tap water consumption was determined weekly after a 20 h period of water and food deprivation (TEST, Table 1 for the stressed groups). This TEST consisted of an hour exposure to two bottles: one containing sucrose solution and the other containing tap water. The anhedonic state generated on the rats by the CMS was reflected as a reduction in their sucrose consumption of at least 2 g (Herrera-Pérez et al., 2008). Classification of the animals as anhedonic or no anhedonic was done after 3 weeks of CMS, on the basis of their weekly-evaluated sucrose solution intake. To avoid changes determined by differences in baseline sucrose-intake levels, individual sucrose consumption data were expressed as relative sucrose intake, which was calculated by dividing the sucrose intake at a given time between the baseline sucrose consumption. Experimental design Fig. 1 shows the time flow for the different experimental manipulations. Experiment 1: preventive effect of testosterone restitution on stress vulnerability of middle-aged rats. Middle-aged male rats (intact or with T restitution) were divided into two groups, control condition or exposed to CMS, matched for similar baseline sucrose consumption. In

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Fig. 1. Time flow of experimental manipulations. Experiment 1: effect of T restitution on stress vulnerability of middle-aged male rats. Experiment 2: antidepressant-like effect of T restitution in middle-aged rats. Experiment 3: effect of orchidectomy on stress vulnerability of young adult rats.

this experiment, middle-aged rats (n= 12) received a T replacement and three weeks later were subjected to the CMS. The weekly evaluation of its respective unstressed control group began three weeks after T replacement (n= 9). As control for treatment, a group of intact middle-aged rats was exposed to stress (n= 13) while others were unstressed (n= 12) (see experiment 1 in Fig. 1). The effect of T replacement is indicated by differences in the proportion of anhedonic rats and by the changes in weekly sucrose intake. Experiment 2: antidepressant-like effect of testosterone restitution in middle-aged rats. In this experiment we evaluated the antidepressantlike effect of T, that is, this steroid was administered once the animals showed a drastic decrease in sucrose intake or anhedonia. A group of 13 anhedonic middle-aged rats were selected after 3 weeks of CMS exposure: 6 of them received a T pellet while the remaining 7 did not. After this manipulation, these animals were maintained under CMS for other three consecutive weeks. Simultaneously, 8 intact middleaged rats were kept undisturbed (control condition) and their sucrose

Table 1 Chronic mild stress schedule. Time (hours) 7:00–8:00 8:00–9:00 9:00–10:00 10:00–11:00 11:00–12:00 12:00–13:00 13:00–14:00 14:00–15:00 15:00–16:00 16:00–17:00 17:00–7:00

First WED

THR

Baseline

WN WN WN

O O O O O/CL SC/CL SC/CL

FRI

SAT

SC/CL SC/CL CL SL SL SL SL SL

WD WD WD WN WN WN WN

WD WD

SUN

MON

TUE

Every subsequent WED FD/WD FD/WD FD/WD TEST

SL SL SL SL SL O/CL

O/CL CT/CL CT/CL CT/CL CT/CL CT CT FD/WD FD/WD FD/WD FD/WD

WN WN WN

WN: white noise (~90 dB), O: overcrowding (2–3 rats per cage), CL: continuous light, SC: soiled cage (250 ml water spilled into bedding), SL: stroboscopic light (300 flashes/min), WD: water deprivation, CT: cage tilt (45°), FD: food deprivation.

consumption was weekly evaluated for 6 weeks; meanwhile, 8 unstressed middle-aged rats received a T pellet implant 3 weeks after baseline sucrose consumption determination. These groups served as unstressed controls. The antidepressant-like effect of T is indicated by variations in sucrose solution intake. Experiment 3: effect of orchidectomy on stress vulnerability of young adult rats. Young adults (intact or orchidectomized) were used in this experiment and, as in previous experiments, were divided into two main groups following the aforementioned control and CMS conditions. A pool of young adult orchidectomized animals was subdivided into two groups: subjected to the CMS (n = 8) or left undisturbed (n = 6). In these groups, their weekly evaluation of sucrose intake began three weeks after removing the testes. Intact young adult male rats served as controls and were also subdivided into two groups: untressed (n = 8) and stressed (n = 8). The effect of orchidectomy is indicated by differences in the proportion of anhedonic subjects and as changes in the weekly sucrose intake. In experiments 1 and 3, the rats that were classified as non anhedonic after three weeks of CMS were stressed for four additional weeks in order to detect a probable late development of anhedonia. The levels of T were measured in T-replaced middle-aged, orchidectomized young adult rats. In experiment 1 the animals were sacrificed after 10 weeks of T-replacement, while in experiment 2 the animals were euthanized three weeks after the T-replacement. In experiment 3 two groups of rats were sacrificed at three or 10 weeks after orchidectomy; this procedures was done to be certain that young adult orchidectomized males possessed very low levels of T throughout the tests. These measurements were done in unstressed rats since we previously showed that stress importantly altered the levels of gonadal hormones (Martínez-Mota, et al., 2011). Intact aged-matched animals were used as controls. Testosterone serum level measurements The blood was centrifuged (4000 rpm for 25 min at 4 °C) to obtain serum samples that were stored at −20 °C. Total serum T

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concentrations were measured by radioimmunoassay using a commercial kit (TKTT1, Diagnostic Product Corporation). The procedure used antibody-coated tubes in which 125I-labeled T competed with free T in the sample for antibody sites. T total quantity (ng/ml) was determined using a calibration curve. The inter- and intra-assay variabilities were: 7.89 and 7.23%.

The T levels of unstressed middle-aged rats which received hormone restitution as preventive treatment (experiment 1) were 4.263 ± 0.396 ng/ml (n = 6) and those of control rats with hormone restitution given as antidepressant treatment (experiment 2) were 3.450 ± 0.533 (n = 6). When comparing these values with those of intact young adult rats no differences were found (one way ANOVA: F2,16 = 0.284, p = 0.757).

Statistics Sucrose solution intake was analyzed by a three-way analyses of variance (ANOVA) including the factors: treatment (T-replacement or gonadectomy), stress and time. When this ANOVA indicated significant differences, the data were further compared by two-way repeated measures analyses of variance (RM ANOVA), including the factor treatment and time or stress and time, and by the Tukey test as post hoc. The proportion of anhedonic/no anhedonic rats was evaluated using the Fisher exact test. T level data were analyzed using a one way ANOVA followed by the Tukey test. The statistical comparisons were carried out using the Sigma Plot software, version 11. A value of P b 0.05 was considered as statistically significant. Results Testosterone pellets are adequate for hormone restitution of middle-aged rats Fig. 2 shows T measurements at different intervals after the pellet placement in middle-aged rats. Three days after the T pellet placement, the hormone levels in middle-aged males rose from 1.02 ± 0.35 ng/ml (n = 7) to 7.02 ± 0.90 ng/ml (n = 5), a higher value than that found in young intact animals: 3.97 ± 1.09 ng/ml, (n = 7; dashed lines); however, 8 days after placing the T pellet, the hormone concentration in middle-aged rats was in the range of the levels found in young adult rats: 5.54 ± 0.43 ng/ml (n = 5) and was maintained stable throughout days 15: 4.72 ± 0.8 ng/ml (n = 4), 21: 4.63 ± 0.81 ng/ml (n = 4) and 29: 3.75 ± 0.12 ng/ml (n = 4). This data analysis using a one way ANOVA revealed significant differences among these measurements (F6,35 = 6.938, p b 001); post hoc analysis indicated that intact middle-aged animals had less serum T levels than intact young rats (p b 0.05) or middle-aged animals with T replacement (p b 0.05 in all cases). Serum T levels of middle-aged rats with hormone restitution were similar to those found in intact young animals, at all intervals after the pellet placement (one way ANOVA: F5,23 = 2.078, p = 0.105). These results indicate that this method of T restitution in middle-aged rats produces levels of this hormone similar to those of young adults for at least 29 days. After this time the pellet was replaced the times required to complete the length of the behavioral experiment.

Fig. 2. Serum T levels in middle-aged rats after several days of pellet placement. T levels of young rats (mean ± 1 SEM, n = 7) are represented within dashed lines. Data are expressed as means ± SEM (n = 4–5 for each group). Tukey test: Tukey test: *P b 0.05 compared to T levels at day 0.

Experiment 1: preventive effect of testosterone restitution on stress vulnerability of middle-aged rats Testosterone given before CMS prevents anhedonia. Fig. 3 shows the effect of CMS on relative sucrose intake of middle-aged animals: intact (full symbols) or with T restitution before stress exposure (empty symbols). The comparison of these data using a three-way ANOVA indicated an effect of T restitution (F1,168 = 6.210, p = 0.014) and of the interaction of T restitution and stress (F1,168 = 10.668, p = 0.001). This preventive effect of T was also evidenced by comparing the relative sucrose intake of rats with (Fig. 3, empty squares) or without (Fig. 3, full squares) T restitution and exposed to CMS through a two-way RM ANOVA, that indicated differences by T restitution (F1,23 = 10.529, p = 0.004) and by the interaction of T restitution with time (F3,69 = 6.106, p b 0.001). Post hoc comparisons revealed that after 2 and 3 weeks of CMS (p b 0.001 in both cases), relative sucrose intake of rats treated with T was higher than that of animals without hormone replacement. Fig. 3 (full symbols) shows that intact middle-aged animals reduced their sucrose consumption when exposed to mild stressors; meanwhile the sucrose intake of the paired unstressed group remained unchanged. The two-way RM ANOVA showed significant differences determined by stress (F1,23 = 5.638, p = 0.026) and interaction of stress and time (F3,69 = 3.144, p = 0.031) but not by time alone (F3,69 = 1.023, p = 0.388). Proportion analysis of sucrose intake indicated that 9 out of 13 intact middle-aged rats exposed to CMS developed anhedonia. Fig. 3 (empty symbols) also shows the temporal course of relative sucrose intake in middle-aged rats that received T restitution. The relative sucrose consumption of middle-aged rats replaced with T remained unaltered, similarly as in the unstressed group (two way RM ANOVA: for stress, F1,19 = 0.563, p = 0.362; for time, F3,57 = 2.418, p = 0.076; and interaction of both factors, F3,57 = 1.135, p = 0.343). Proportion analysis of sucrose intake showed that only 2 out of 12 stressed middle-aged rats with T restitution developed anhedonia. This proportion of anhedonic rats did not change even after four weeks more of stress (data not shown).

Fig. 3. Preventive effect of T on the anhedonic (reduction in sucrose intake) effects of stress. Relative sucrose solution intake of middle-aged intact (full symbols) or T treated (empty symbols) rats exposed to CMS (squares) or maintained unstressed (circles). Data are expressed as means ± SEM. Tukey test: *P b 0.05, **P b 0.01 compared to unstress intact group; ###P b 0.001 compared to stress non-T implanted group.

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The comparison of the proportion of anhedonic animals between the T-replaced and non-replaced middle-aged rats showed a significant (Fisher exact test, p = 0.015) lower percentage of anhedonic animals in the T-restored group. Experiment 2: antidepressant-like effect of testosterone restitution in middle-aged rats Testosterone restitution did not reverse anhedonia in middle-aged rats. Fig. 4 shows the relative sucrose intake of rats with T restitution as antidepressant treatment (empty symbols) and in those animals that did not receive T treatment (full symbols). The three-way ANOVA indicated only a main effect of stress (F1,100 = 48.042, p b 0.001) and no differences by T-treatment (F1,100 = 1.209, p = 0.274). The relative sucrose consumption of non T treated intact anhedonic middle-aged rats maintained under stress and of its respective unstressed group did not change along the experiment (full symbols, Fig. 4). The analysis of these data using a two-way RM ANOVA, confirmed only a main effect of stress (F1,39 = 13.003, p = 0.003). Fig. 4 (empty symbols) also shows that the relative sucrose intake of anhedonic-stressed middle aged rats did not change after T replacement. Data analysis through a two-way RM ANOVA indicated only a significant effect of stress (F1,12 = 8.824, p = 0.012). Finally, the comparison of the sucrose intake of anhedonic stressed animals with (empty squares) or without (full squares) T restitution indicated no differences determined by T (F1,11 = 2.622, p = 0.134), time (F3,33 = 2.260, p = 0.100) or by the interaction between these factors (F3,33 = 0.966, p = 0.420). Experiment 3. Effect of orchidectomy on stress vulnerability of young adult rats Orchidectomy did not increase the vulnerability of young adults to the CMS. T serum levels of both groups of orchidectomized young rats (three or 10 weeks after surgery, n = 6 each one) were inferior to the kit's detection limit (0.2 ng/ml); the one-way ANOVA indicated that these hormonal levels were significantly lower (F2,16 = 11.332, p b 0.001) than those found in intact young adult rats (n = 7, p = 0.003 for both cases). Fig. 5 shows the relative sucrose intake of intact (full symbols) and orchidectomized (empty symbols) young rats exposed to CMS, or maintained without stress. The three-way ANOVA indicated no differences among the groups. Intact young rats exposed to stress (Fig. 5, full symbols), as those unstressed, did not reduce their sucrose intake along the experiment.

Fig. 4. Absence of antidepressant-like effect of T restitution in middle-aged rats. Full symbols indicate the relative sucrose intake of intact unstressed (circles) or anhedonic (squares) middle-aged rats. Empty symbols indicate the relative sucrose intake of middle-aged rats that received T restitution after 3 weeks of control condition (circles) or after anhedonia development (after 3 weeks of CMS, squares). Data are expressed as means ± SEM. Tukey test: *P b 0.05, **P b 0.01 compared to unstress intact group; #P b 0.05, ##P b 0.01 compared to unstress T restitution group.

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Similarly, orchidectomized young adult rats (Fig. 5, empty symbols) did not change their sucrose consumption when exposed to the CMS, showing a relative sucrose intake similar to that found in the orchidectomized unstressed group. The relative sucrose intake remained unchanged even after four extra weeks of stress exposition (data not shown). The proportion analysis revealed that after three weeks of stress none of the stressed animals (intact or orchidectomized) can be considered as anhedonic. In all groups the relative tap water consumption remained unchanged (data not shown), as evidenced by a lack of statistical significant differences in this parameter after three-way ANOVAs for each experiment (data not shown). Discussion In the present study we found a stress-dependent differential effect of T in middle-aged rats: T restitution before CMS prevented anhedonia but was unable to reverse it once established, indicating a lack of antidepressant-like effect. We also found an age differential role of gonadal hormones on the vulnerability to stress: in middleaged rats, T replacement offered resistance to CMS, while in young adults, orchidectomy did not increase their vulnerability to develop anhedonia. In the CMS paradigm, changes in sucrose solution intake may result after non-specific alterations in total liquid consumption (caused by stress or antidepressant treatment) that can erroneously be interpreted as anhedonia or its reversal (Willner, 1997; Willner et al, 1987). As described above, we did not find changes in simple water intake, indicating that the reduced sucrose solution intake specifically reflects anhedonia. Testosterone restitution prevents but not reverses anhedonia in middleaged rats exposed to CMS T restitution — through silastic pellets — has shown to effectively increase T levels, improved sexual behavior in castrated young males (McGinnis and Dreifuss, 1989) and bone mineral density in middle-aged rats (Vanderschueren et al., 1992). In the current study we demonstrated that T propionate silastic pellets implants to middle-aged rats produce hormone levels similar to those detected in young adults. The concentration of T in middle-aged rats after 3 days of pellet placement was marginally higher than that of young adult rats (Fig. 2), possibly by a sum between the T released from the pellet and the endogenous production. Because the release of the hormone from the pellet is continuous, endogenous T production is abolished through a negative feedback loop.

Fig. 5. Lack of changes in sucrose intake in young adult rats after orchidectomy. Relative sucrose solution intake of young intact (full symbols) or orchidectomized (empty symbols) rats exposed to CMS (squares) and those maintained unstressed (circles). Data are expressed as means ± SEM.

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Regarding stress vulnerability, we found that T restitution to middle-aged rats, three weeks before CMS, prevents anhedonia. These data imply that in these animals T is an important factor that favors resiliency, which is defined as the capacity of an organism to successfully adapt to adversity (Fossion and Linkowski, 2007) by regulating the negative effects of stress (Wagnild, 2003). Thus, high physiological levels of T (similar to those of young adults) in middle-aged rats are crucial to prevent the effects of CMS. This finding is in line with others showing that the reduction in T was inversely related with a depressive-like behavior in the tail suppression test in the senescence accelerated prone mouse 10, an animal model of aging (Egashira et al., 2010). Furthermore, T and other androgens were able to reduce the depressive-like-behavior of aged mice when evaluated in the forced swimming test (Frye and Walf, 2009). Present results also are in agreement with clinical studies showing an inverse relationship between T levels and depression incidence in aged men (Joshi et al., 2010; Shores et al., 2005) and a reduction of depressive symptoms after T replacement to hypogonadal young (Shores et al., 2004; Wang et al., 1996) or aged men (Shores et al., 2009) and eugonadal men older than 50 years (Perry et al., 2002). As T prevented stress vulnerability, we evaluated if T restitution would reverse anhedonia in middle-aged rats. We found that T treatment to stressed animals with once installed anhedonia had no antidepressant-like effects. Contrary to these data, a study using aged intact mice showed that T and other androgens produced antidepressant-like effects in the forced swimming test (Frye and Walf, 2009), this difference can be explained by the species used (mice vs. rats), type of stress (acute vs. chronic) and T treatment regime (acute-injection vs. chronic–silastic pellets). However, the present results are in line with previous studies showing that T subchronic administration to orchidectomized young adult rats did not reduce immobility in the forced swimming test (Martínez-Mota and Fernández-Guasti, 2004) and that neither four weeks of T restitution to middle-aged orchidectomized rats nor T acute treatment to middle-aged intact rats were effective in restoring the immobility age-related increase in forced swimming (Frye et al., 2010). These authors also found that such depressive-like behavior in middle-aged rats was reduced after acute treatment with 3 α androstanediol, a metabolite of T. Thus, it seems important to evaluate this metabolite in middle-aged rats exposed to the CMS. It has also been described — mainly in females — that estradiol administration or its synthetic analogs produce antidepressant-like effects (Dhir and Kulkarni, 2008; Estrada-Camarena et al., 2003; Walf and Frye, 2009), considering that middle-aged rats have lower estradiol levels than young adults (Herrera-Pérez et al., 2008) it is feasible that restitution with this hormone reverses anhedonia. The lack of antidepressantlike actions of T also contrasts with several clinical reports indicating that T has antidepressant effects (Perry et al., 2002; Pope et al., 2003; Wang et al., 1996); however, in these studies the relationship between T and depression may be veiled by several factors (Carnahan and Perry, 2004). From these results it is clear that, in middle-aged rats, T restitution is useful when administered before stress and lacks an action once anhedonia is established. A putative explanation for this differential T action can be related to the opposite effects of this androgen or its metabolites, including estradiol, and corticosterone on neuroplasticity and neuroprotection (Gould et al., 2000; Pittenger and Duman, 2008; Smith et al., 2005); that is, when T is administrated to rats without stress, when costicosterone levels are not elevated (Herrera-Pérez et al., 2008), the androgen promotes neuroprotection and neuroplasticity (Cavus and Duman, 2003; Cooke, 2006; Janowsky, 2006; MacLusky et al., 2006; Tanapat et al., 1999); these actions, in turn, allow the establishment of several physiological alternatives to stress adaptation. On the contrary, when animals are exposed to stress, corticosterone levels increases (Bachis et al., 2008; Grippo et al., 2005; Konkle et al., 2003; Zheng et al., 2006), deteriorating synaptic plasticity and neurogenesis (Gould et al., 2000; Pittenger and Duman, 2008), and eventually leading

to depressive-like behaviors (Vollmayr et al., 2007). Under these conditions T is unable to reverse anhedonia. Differential effect of gonadal hormones on hedonic state of male rats according to age It has been postulated that orchidectomy impairs the regulation of affective-like behaviors in several animal models (Bernardi et al., 1989; Bitran et al., 1993; Edinger and Frye, 2005; Frye and Edinger, 2004). In animal models of anxiety such as the open field, elevated plus maze and the burying behavior tests, orchidectomized young adult rats (3–4 months age) exhibited an increase in anxiety-like behaviors (Edinger and Frye, 2005; Fernández-Guasti and MartínezMota, 2003; Frye and Edinger, 2004). However, we previously reported that orchidectomy of young adults did not modify the depressive-like behavior in the forced swimming test (MartínezMota and Fernández-Guasti, 2004). In agreement, the current study shows that orchidectomy also did not reduce sucrose solution intake in young rats under CMS. In contrast, Bonilla-Jaime et al. (2010) found that 60 days after castration, there was an increase in immobility in the forced swimming test, suggesting that the time after orchidectomy may be a relevant factor to develop depressive-like behaviors. In our experiments, young males did not develop anhedonia even 10 weeks after castration (we began to stress the animals three weeks after surgery and were maintained under this condition for seven consecutive weeks) supporting the idea that gonadal hormones are not essential for resilience in young adult animals. Accordingly, treatment with T to orchidectomized young adult rats failed to prevent the impact of acute stress on sexual behavior (Retana-Márquez et al., 2003) or to produce an antidepressant-like effect in the forced swimming test (Martínez-Mota and Fernández-Guasti, 2004). Contrary to our results, recently it was demonstrated that orchidectomy of young adult rats exposed to CMS for 21 days produce an increase in passive behaviors (immobility and maintenance movements) in the forced swimming test, as compared with sham operated animals, suggesting that testicular hormones confer resiliency to chronic stress (Wainwright et al., 2011). This difference could be explained by the variation in the animal model used and highlights the importance of using several paradigms when studying the participation of hormonal factors on resilience. The absence of an effect of orchidectomy on CMS vulnerability in young adult rats contrasts with some clinical studies revealing a higher incidence or severity of depression in patients with low T levels (Barret-Connor et al., 1999; Burris et al., 1992; Seidman and Walsh, 1999) and is in agreement with others that fail to find such relationship (Levitt and Joffe, 1988; Rubin et al., 1989). This inconsistency could be due to the inclusion of different populations (young and aged, healthy and ill, etc.), to the measurement of free or total T in different fluids (saliva, urine or blood) and to the different timing in phlebotomy for hormone measurements (Margolese, 2000; Zarrouf et al., 2009). Thus, these methodological variations could veil the influence of T on depression (Carnahan and Perry, 2004). Moreover, it is possible that alleviation of depressive symptoms in young hypogonadal men after T restitution could be secondary to alleviation of other symptoms related to hypogonadism, such as sexual impairment or fatigue (Zarrouf et al., 2009). The ineffectiveness of orchidectomy to modify young rats' vulnerability to mild stress also contrasts with the important role of T in middle-aged rats' resiliency. This age differential effect could be explained considering Lipsitz and Goldberger's hypothesis (1992). According to them, the animal's physiological functions are characterized by a complex interaction of many control mechanisms that allow adaptation to unpredictable changes of life. A theoretical advantage of this complexity in biological systems is the ability to synchronize its networks to adequately compensate alterations in one component of the system, keeping its ability to adapt to stressors (Lipsitz and Goldberger, 1992; reviewed in Smith et al., 2005). It is suggested that

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aging is characterized by a progressive loss of complexity and dynamic range of physiological function, which impairs the individual's ability to adapt to stress (Villancourt and Newell, 2002); in line, our previous study reported that middle-aged rats were more vulnerable to CMS than young adult animals (Herrera-Pérez et al., 2008). According to this hypothesis, aging is associated to a reduction in the number, size and/or neuron arborization in the hippocampus and prefrontal cortex (Markham and Juraska, 2002; Markham et al., 2005; Wong et al., 2000), that could impair central nervous system (CNS) function, making it more vulnerable to stress. This loss of neuronal complexity can be attributed, at least partly, to the age-dependent T reduction since this hormone and its metabolite, estradiol, are able to modulate neuroplasticity in the CNS (Cavus and Duman, 2003; Cooke, 2006; Janowsky, 2006; MacLusky et al., 2006; Tanapat et al., 1999) and to regulate neurotransmission systems in brain regions involved in learning, memory, emotion, motivation and cognition (Meyers et al., 2010; Zheng, 2009). Furthermore these hormones are able to inhibit the hypothalamus–pituitary– adrenal (HPA) axis activity (Viau, 2002) and the stress-induced neurodegeneration, primarily in hippocampus (Elder et al., 2006; Vollmayr et al., 2007) which is involved in depression and in the HPA axis regulation. All these observations indicate that maintaining an adequate level of T favors neuronal complexity networks and stress response in aged animals. These data, taken together, suggest that T restitution in middle-aged rats improve the response to stress and the interaction among different neurotransmitter systems; implying that T could increase the complexity of biological systems at least in two ways: a) by adding a functional component to the system and b) by favoring the interaction of other components. Thus, the T-dependent increase in physiological complexity could be reflected as a higher probability for an aged organism to adapt to stressful events. Contrary to aged rats, the lack of effect of testicular hormones depletion on young rats' vulnerability to CMS may be explained by the presence of physiological alternatives or resilience factors, inherent to the high complexity of young biological systems, which could compensate this absence allowing stress adaptation. Such factors may include other hormones (growth hormone, insulin-like growth factor I — IGF-I), neurotrophins (such as BDNF) and neurotransmitters (dopamine, serotonin and noradrenaline) (Charney, 2004) which are higher in young adults as compared with the aged (reviewed in Smith et al., 2005). Conclusion This study shows that in middle-aged animals, in contrast to young adults, T is an important resilience factor that prevents the development of anhedonia but it is unable to reverse it once installed. These findings highlight the importance of maintaining high levels of T in aged males, which could interfere with the development of psychopathologies as depression. Acknowledgments This work was partially financed by INPRFM, project number 3370.1 and J. H-P received a fellowship (130160) from CONACyT. References American Psychiatric Association, 2000. Diagnostic and Statistical Manual for Mental Disorders, 4th edition. American Psychiatric Press, Inc., Washington, D.C. Bachis, A., Cruz, M.I., Nosheny, R.L., Mocchetti, I., 2008. Chronic unpredictable stress promotes neuronal apoptosis in prefrontal cortex. Neurosci. Lett. 442, 104–108. Barret-Connor, E., Von Mühlen, D.G., Kritz-Silverstein, D., 1999. Bioavailable testosterone and depressed mood in older men: the Rancho San Bernardo study. J. Clin. Endocrinol. Metab. 84, 573–577. Bernardi, M., Genedani, S., Tagliavini, S., Bertolini, A., 1989. Effect of castration and testosterone in experimental models of depression in mice. Behav. Neurosci. 103, 1148–1150. Bitran, D., Kellogg, C.K., Hilvers, R.J., 1993. Treatment with an anabolic–androgenic steroid affects anxiety related behaviour and alters sensitivity of cortical GABAA receptors in the rat. Horm. Behav. 27, 568–583.

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