1. INTRODUCTION:

Stress negatively impacts the quality of human life and increases the risk of cardiovascular disorders, psychological disorders and infectious diseases. Adolescents in particular suffer from heightened stress due to the various physical, developmental and psychosocial changes that occur simultaneously^(1,2). Interestingly, studies have shown that adolescents are more susceptible to stress-induced emotional and cognitive dysfunctions due to their extensive neurobiological changes^(3,4), which may explain the increased prevalence of psychiatric disorders in adolescence⁽⁵⁾. Social bullying is one the most common forms of psychosocial stress which is increasingly considered as a predisposing factor for psychiatric disorders. Bullying and social stress can be modeled in animals using sensory contact model-induced social defeat paradigm where evoked confrontation between two animals of the same species results in the ultimate emergence of a dominant and a subordinate (socially defeated) at the end of the social interaction^(6,7). Chronic exposure to social confrontations especially during adolescence has been shown to result in long-lasting psychosomatic disturbances and behavioral pathologies ^(8,9,10). Therefore, the sensory contact model-induced-chronic social defeat could be useful in investigating the effect of potential psychotropic drugs in simulation of clinical conditions.

Testosterone, the male sex hormone, plays an important role in cognitive function and behavior through its genomic and nongenomic effects on the brains^(11,12). Several lines of evidence support anxiolytic and antidepressant effects of testosterone where anxiety and depression are more prevalent in women and in ageing men with declining testosterone levels^(11,13). Studies in humans and animals have shown that the supplementation of testosterone could result in the improvement of anxiety^(14,15) and depressive behavior ^(16,17). Furthermore, cognition and memory have been shown to decline with aging together with the testosterone levels⁽¹²⁾. Observational studies have found a positive relationship between testosterone and spatial abilities where men outperform women^(18,19). Clinical studies in ageing men⁽²⁰⁾ and women⁽²¹⁾; and in Alzheimer’s disease patients⁽²²⁾ have shown positive effects of testosterone on spatial and verbal memory. However, inconsistent and contradictory studies suggest that the effect of testosterone might be dose-related^(12,23). In addition, testosterone metabolism could significantly contribute to the complexity of its actions and makes it difficult to maintain long-term physiologic steady serum levels after exogenous testosterone administration. Testosterone undecanoate was used in our study as it was found to be superior to other testosterone release preparations and represented a convenient and effective tool for testosterone administration in experimental animals⁽²⁴⁾. Given its well-known role in learning and memory⁽²⁵⁾, cAMP-response-element-binding protein (CREB), the transcription factor, will be used as a marker for the molecular alterations underlying the potential effects of testosterone on social defeat-induced behaviors. CREB has been shown to play a role in depressive and anxiety disorders⁽²⁵⁾. Studies have demonstrated region-specific effects of CREB where elevated levels of CREB in the hippocampus are associated with enhanced memory and reduced depressive behaviors^(26,27,28), while elevated levels of CREB in the nucleus accumbens produced opposite effects^(29,30). Also, disruption of CREB function in the nucleus accumbens has been shown to result in anxiety-like behaviors⁽³⁰⁾, while induction of CREB function in the amygdala produced similar behavioral effects⁽³¹⁾. The aim of the present work was to investigate the possible effects of testosterone undecanoate on behavior and cognitive functions in adolescent male mice exposed to chronic social defeat induced by SCM.

2. MATERIALS AND METHODS:

2.1. Animals:

80 Weaned male Swiss albino mice (22 days) were brought to our facility, caged in standard plastic cages (4-6 per cage) under standard laboratory conditions and left undisturbed for 7 consecutive days for adaptation. Dedicated efforts were made to minimize animal suffering and the number of animals used in accordance with the Guidelines for Animal Experiments of Faculty of Pharmacy, the British University in Egypt. Mice were isolated for 5 days to abolish any social effects (D29-D34).

2.2. Sensory contact model (SCM):

This model was established according to Kudryavtseva and co-workers (2014)⁽³²⁾. Pairs of animals of similar weight were placed in stainless steel cages (28x14x10 cm) divided by a clear perforated Plexiglas partition that allows visual and olfactory but not physical contact. Pairs were left undisturbed for 2 days for adaptation to the housing conditions and sensory contact. Every day (10:00-11:00 am), the partition was removed for 10 min to allow physical interaction for 12 consecutive days (D36-D48). One of the partners clearly dominates the other within 2 or 3 daily encounters. During the tests, one animal was seen to attack, bite, and chase the other who only displayed defensive behavior (sideways, upright postures, withdrawal, lying on the back or freezing). Aggressive confrontations were discontinued if the aggression has lasted more than 3 minutes. After every session, the winner remained resident in their compartments while defeated mouse was paired with the winning member in a new unfamiliar cage.

2.3. Experimental Design:

defeated mice are randomly assigned to control group (vehicle), testosterone undecanoate (100mg/Kg) or testosterone undecanoate (500mg/Kg) given intramuscular (i.m.) once for 45 days (D48-D93) administered 2 hours after the last SCM session. An additional group consists of mice isolated for 5 days served as normal undefeated control.

2.4. Drugs:

commercially available Nebido® (Testosterone undecanoate 1000 mg/4 ml, solution for injection) was diluted in sterile castor oil and used for i.m. injection.

2.5. Behavioral assessment

2.5.1. Open-field test (OFT):

The open field used was a black Plexiglas box (40×40×30 cm). Test was performed under dim light conditions (10 lux). Animal was placed in the center of the arena and videotaped for 5 min., the open field was wiped thoroughly after each session using 70% propanol ⁽³³⁾. Videos were evaluated using Any Maze® video tracking software (Stoelting co.) for the following parameters: total distance travelled, time spent in the center of the arena and total immobility time.

2.5.2. Novel object recognition (NOR):

The test was performed in the open field apparatus described previously. Each mouse was habituated to the arena for a 10 min session followed by a 5 min familiarization session in which mouse was allowed to explore 2 identical objects placed in a fixed position⁽³⁴⁾, 24 hours later, mouse was subjected to a 3 min test phase in which one of the familiar objects was replaced by a novel one⁽³⁵⁾. The position of the novel object was counterbalanced between subjects. Both familiarization and test sessions were videotaped and assessed for the total exploratory time of familiar and novel objects, defined as directing the nose toward the object at a distance less than or equal to 2 cm. The preference index was calculated as follow: (time spent exploring the novel object - time spent exploring the familiar object)/time exploring both novel and identical objects⁽³⁶⁾.

2.5.3. Elevated plus maze test (EPM):

The apparatus was established according to Bourin et al., 2007⁽³⁷⁾. In brief, the apparatus consisted of an elevated (40cm) central platform (5×5cm) with two opposite open arms (5×30cm) and 2 opposite closed ones (5×30×15cm) Animal was placed on the central platform and videotaped for minutes. Videos were evaluated for number of entries and time spent in either open or closed arms.

2.5.4. Morris water maze (MWM):

The test was done according to Vorhees and Williams, 2006⁽³⁸⁾. Apparatus: a round pool (diameter: 95cm, height: 35cm) was filled to a depth of 32 cm with water. A suitable amount of non-toxic water-based tempura paint was used to make water opaque. Water temperature was maintained at 25±0.5°C. The escape platform was an 8 cm diameter glass cylinder with a rough surface, placed in a fixed position in the northwest quadrant of the pool 1cm below water surface. The platform remained in the same position throughout the all learning sessions.

Procedure:

-Day 1 visible platform: water level in the pool was adjusted to 1 cm lower than the platform. A small plastic flag was fixed on the platform to increase visibility. In the beginning of the trial, the mouse was placed on the platform for 20 sec. to allow orientation to external cues. Mice were then gently lowered into the pool facing the wall at one of quadrants other than that containing the platform. After the mouse was released, the researcher step back from the pool to a designated position, serving as an additional distal visual cue. If the mouse reached the platform within 60, it was immediately removed from the pool. If the platform was not located after 60 s of swimming, the mouse was gently guided to the platform and allowed to stand on it for additional 20s for further orientation to external cues. After removal from the pool, mice were dried using a paper towels and placed in a warming cage before being returned to the home cage.

Days 2-5 hidden platform: mice received five trials, 60s each, daily for 5 consecutive days with an inter-trial interval of 60s. In each trial, mouse was placed into the pool, facing the wall at one of four possible starting points: North, East, South or West. The starting point for each trial was randomly determined with the conditions that two successive trials never began from the same position and that each mouse use all possible start locations in each day. The escape latency was recorded when the mouse reached the platform. If the mouse failed to escape within 60 s, the trial was recorded as an escape failure.

Day 6 Probe: a probe test was performed 24 h after the last training session aiming to examine spatial reference memory. The platform was removed from the pool and the mouse was introduced to the pool and allowed to swim for 60s. The time spent in the northwest (quadrant) platform was recorded.

2.6. Quantitative Real Time PCR (qRT-PCR):

RNA was isolated from the frontal cortex and hippocampus of the mice after microdissection of the brain as described previously⁽³⁹⁾. Once the frontal cortex or the hippocampus were isolated from the animal, they were snap-frozen in liquid nitrogen and stored in -80^oC for further processing. RNA was extracted from either a lobe of the frontal cortex or the hippocampus using Trizol Reagent (Invitrogen, USA) as per instruction of the manufacturer. Isolated RNA was quantified spectrophotometrically using UV-Vis Spectrophotometer Quawell q5000 (Quawell, USA) and RNA purity was detected using 260/280 ratio. RNA was treated with RNAse-Free DNAse (Thermo Scientific, USA) to remove any contaminating genomic DNA. Then, cDNA was prepared from DNAse treated RNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Gene expression was done by qRT-PCR using gene specific primers (sequence of the primers used are given in table-1) and Maxima SYBR Green qPCR Master Mix, 2X (Thermo Scientific, USA). All qRT-PCR experiments were done on StepOnePlus Real time PCR system (Applied Biosystems, USA). The relative RNA expression was calculated from threshold cycle (Ct) detected by the instrument using the formula 2-ΔCt., relative to β-actin as the house keeping gene.

Table-1: Primers sequences used in qRT-PCR experiment:

Gene

Forward Primer

Reverse Primer

β-actin

5'-CTTGCTCTG

GGCCTCGTC-3’

5'-GGCTGTATTC

CCCTCCATC-3'

CREB

5′-ACTGTAACGGT

GCCAACTCC-3′

5′-GAATGGTAGTAC

CCGGCTGA-3′

2.7. Statistical analyses:

Data obtained from behavioral tests were expressed as mean ± confidence interval 95% (CI 95%), while the data of the qRT-PCR was expressed as means ± standard error of mean (SEM). Multiple comparisons among groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey test as a post-hoc test, data obtained from MWM was analyzed using two way ANOVA for time and treatment factors. The probability level less than 0.05 was considered to indicate statistical significance. All Statistical procedures were performed using IBM SPSS version 17 computer package, USA. Graphs were sketched using GraphPad Prism version 5 software (GraphPad Software Inc., USA).

3. RESULTS:

3.1. Effects of sensory contact model (social defeat) on mice:

SCM significantly reduced distance traveled and average speed in the open field test compared to negative control group (F (3, 28) = 23.14, P<0.0001 and F (3, 28) = 24.01, P<0.0001, respectively, Fig. 1A and 1B). Moreover, SCM significantly reduced the time spent in the center compared to the negative control, while time spent in the corner and immobility time where significantly higher (F (3, 28) = 7.23, P=0.0010 and F (3, 28) = 13.97, P<0.0001 and F (3, 28) = 7.882, P=0.0006, respectively, Fig. 1C, 1D and 1E). In addition, SCM significantly reduced preference index of defeated group in the NOR test compared to negative control group, indicating a deteriorated retention memory (F (3, 28) = 14.16, P<0.0001, Fig. 2). Time spent in the open arms in the EPM test was significantly reduced in the socially defeated group when compared to the negative control group, indicating a high level of anxiety induced by SCM (F (3, 28) = 42.67, P<0.0001, Fig. 3A). In Morris water maze test (MWM), socially defeated mice expressed poor learning ability throughout the training days compared to the negative control group indicated by the escape time (Fig. 4A). While time in the target quadrant was significantly reduced by SCM when compared to the negative control group (F (3, 28) = 17.4, P<0.0001. Fig. 4B) indicating a deteriorating retention and spatial memory. Finally, qRT-PCR analysis showed a slight reduction in the frontal cortex relative mRNA expression of CREB in the defeated group compared to negative control group (Fig. 5A). However, CREB mRNA expression level was significantly reduced in the hippocampus of defeated group when compared to negative control group (Fig. 5B).

3.2. Effects of testosterone undecanoate treatment:

In the open field test, testosterone undecanoate (500 mg/kg) (TU 500) treatment showed a significant increase in both distance traveled and average speed compared to the defeated group, while testosterone undecanoate (100 mg/kg) (TU 100) did not have any significant effect (Fig. 1A and 1B, respectively). However, TU 100 treatment did have an anxiolytic effect indicated by the significantly increased time spent in the center (F (3, 28) = 7.23, P=0.0010, Fig. 1C) and the significantly reduced time spent in the corner (F (3, 28) = 13.97, P<0.0001, Fig. 1D) and the significantly reduced immobility time when compared to the defeated group (F (3, 28) = 7.882, P=0.0006, Fig. 1E). TU 500 showed a significant decrease in the immobility time (Fig. 1E), while the change in both time spent in the center and corners was not significant when compared to the defeated group (Fig. 1C and 1D). Moreover, both TU 100 and TU 500 did improve the retention memory as indicated by the significant increase in the preference index compared to the defeated group in the NOR test (F (3, 28) = 14.16, P<0.0001, Fig. 2). In the EPM test, both TU 100 and TU 500 did show an anxiolytic effect through a significant increase in the time spent in the open arms and a significant reduction in the time spent in the closed arms compared to the defeated group (F (3, 28) = 42.67, P<0.0001 and F (3, 28) = 26.56, P<0.0001, Fig. 3A and 3B, respectively). In MWM, both TU 100 and TU 500 showed a significant improvement in learning memory as indicated by a significant reduction in the escape time throughout the training days (Fig. 4A) as well as a significant increase in the time spent in the target quadrant in the probe day (F (3, 28) = 17.4, P<0.0001, Fig. 4B) with TU 500 showing a greater improvement in learning memory.

Although TU 100 did show an increase in the relative mRNA expression of CREB in both frontal cortex and hippocampus regions (Fig. 5A and 5B), the increase was not significant compared to the defeated group. Interestingly, TU 500 treatment showed a significant increase in CREB relative mRNA expression in the prefrontal cortex region compared to both defeated and negative control groups (Fig. 5A). However, the increase in CREB mRNA level in the hippocampus region induced by TU 500, was not significant (Fig. 5B).

4. DISCUSSION:

Several studies have demonstrated effects of testosterone on brain chemistry and behavior⁽¹²⁾. However, the results were confounding probably owing to the variability in dosing, timing, route of administration, gender and state of gonads which makes maintaining long-term physiologic steady serum levels after testosterone administration in experimental animals a major challenge⁽¹²⁾. According to Callies et al, a single injection of testosterone undecanoate (TU) (100mg/kg) could yield physiological testosterone levels in orchidectomized rats for a minimum of four weeks, while a single injection of high dose TU (500mg/kg) could induce supraphysiological testosterone levels for up to six weeks in non-orchidectomized rats⁽²⁴⁾. Therefore, TU represents a convenient and effective tool for testosterone administration in experimental animals as it was found to be superior to other testosterone release preparations. We set out to investigate the effects of exogenous testosterone undecanoate (100mg/kg and 500mg/kg i.m. injection) on socially defeated male mice with a view to testing the hypothesis that exogenous testosterone modulates the influence of sensory contact model or social defeat on behavior and brain CREB mRNA level.

Endogenous or exogenous testosterone has been reported to influence several behavioral traits⁽⁴⁰⁾ through its genomic as well as its non-genomic effects⁽⁴¹⁾. In this study, administration of testosterone resulted in a significant increase in both distance traveled and average speed compared to the defeated group in the open field test. These effects could point to cerebral cortical stimulation since studies have shown that testosterone supplementation particularly within the adolescence period may regulate dopamine neurotransmission⁽⁴²⁾. The resulting locomotor hyperactivity might be a consequence of increased dopaminergic transmission in the nucleus accumbens which has been shown to be associated with increased locomotor activity ⁽⁴³⁾. Testosterone has also been reported to regulate GABAergic neurotransmission⁽⁴⁴⁾. However, the exact mechanism of testosterone interactions with the various neurotransmission pathways remains to be defined. Testosterone has been shown to affect learning and cognitive behavior⁽¹²⁾where numerous studies have demonstrated the gender-related effects of testosterone on memory⁽¹¹⁾as testosterone supplementation in castrated male animals improved their spatial memory compared to that of females⁽⁴⁵⁾. Also some human and animal studies have reported that testosterone could improve spatial working memory^(46,47). Few studies have also assessed age-related differences in response among males which could probably correlate with declining testosterone levels with ageing⁽¹²⁾. In this study, both TU 100 and TU 500 did improve the retention memory as indicated by the significant increase in the preference index compared to the defeated group in the NOR test. In MWM, both TU 100 and TU 500 showed a significant improvement in learning memory as indicated by a significant reduction in the escape time throughout the training days as well as a significant increase in the time spent in the target quadrant in the probe day. Our results are in line with those reported by Spritzer et al. 2011, where testosterone had positive effects on spatial learning and memory which were dependent on the duration of testosterone replacement and the nature of the spatial task⁽⁴⁸⁾. Although the mechanisms underlying testosterone effects on spatial memory are not well-defined, pyramidal spine density in the hippocampus⁽⁴⁹⁾ and increased pyramidal cell activity in the amygdala as well as improved memory have been associated with increased testosterone levels⁽⁴⁷⁾.In line with the reported effects of testosterone on anxiety⁽¹²⁾, we found that TU 100 treatment did have an anxiolytic effect indicated by the significantly increased time spent in the center, reduced time spent in the corner and reduced immobility time when compared to the defeated group in the open field test, while TU 500 showed a significant decrease in the immobility time when compared to the defeated group. Moreover, in the EPM test, both TU 100 and TU 500 did show an anxiolytic effect through a significant increase in the time spent in the open arms and a significant reduction in the time spent in the closed arms compared to the defeated group. The effects of endogenous and exogenous testosterone on anxiety-related behaviors in rodents and humans have been investigated in numerous studies including reported anxiolytic effects of testosterone administration in women⁽¹⁵⁾ and anxiolysis in the EPM⁽¹⁴⁾ and the marble-burying behavior tests in animals⁽⁵⁰⁾. However, nandrolone, a synthetic testosterone derivative, has been reported to induce anxiogenic effects in male rats⁽⁵¹⁾, suggesting the ability of testosterone to increase or decrease anxiety through different mechanisms that remain to be elucidated.

Cyclic AMP (cAMP)-response element binding protein (CREB) is an activity-dependent transcription factor that has a pivotal role in synaptic plasticity and learning and memory^(25,52). The role of CREB in memory has been demonstrated by several lines of evidence including deficits in hippocampal long-term potentiation (LTP) and memory deficits in mutant mice with diminished CREB activity^(53,54), and promotion of memory consolidation through upregulation of CREB activity ^(55,56). Furthermore, CREB is reportedly involved in depression pathogenesis and treatment as evidenced by its upregulated hippocampal expression following chronic antidepressant treatment⁽⁵⁷⁾, in addition, alterations in CREB activity within the nucleus accumbens has been shown to mediate responses to rewarding and aversive stimuli^(30,58). In our study, chronic social defeat did significantly reduce mRNA expression of CREB in the hippocampus region, which has been associated with reduced preference index of defeated group in the NOR test compared to negative control group, indicating a deteriorated retention memory. In Morris water maze test (MWM), socially defeated mice expressed poor learning ability throughout the training days indicated by the reduced escape time and reduced time spent in the target quadrant when compared to the negative control group indicating a deteriorating retention and spatial memory. On the other hand, TU treatment slightly increased the mRNA expression of CREB in the hippocampus region, although not significant, but TU 100 and TU 500 did improve the retention memory as indicated by the significant increase in the preference index compared to the defeated group in the NOR test along with a significant improvement in learning memory as indicated by a significant reduction in the escape time throughout the training days and increase in the time spent in the target quadrant in the probe day in MWM test. This could signify a potential role of CREB in mediating TU effects on memory improvement. CREB has also been shown to regulate anxiety as evidenced by elevated anxiety levels in mutant mice with CREB ablation^(59,60) and mice expressing dominant negative form of CREB in the nucleus accumbens ⁽³⁰⁾. Furthermore, anxiolytic treatment or CREB overexpression in the nucleus accumbens resulted in diminished anxiety behavior ⁽⁶¹⁾. In our study, TU 500 treatment showed a significant increase in CREB relative mRNA expression in the prefrontal cortex region compared to both defeated and negative control groups, respectively. The increased CREB mRNA expression after TU 500 treatment could be seen in the reduced anxiety level manifested by the increased time in the open arm in the EPM test and increased time spent in the center zone in the OF test. Therefore, disturbances of CREB pathways could be underlying the behavioral alterations which also seem to be relevant to humans where rare genomic variants of patients with bipolar disorder and co-morbid anxiety have been linked to CREB pathways ⁽⁶²⁾.

CONCLUSION:

Exogenous testosterone administration results in age-related neurobehavioral responses after exposure to chronic social defeat; however, more studies are needed to clarify the mechanisms that underlie them.

Figure (2): Effect of testosterone on preference index of socially defeated mice in the NOR.

Both dose levels of testosterone significantly increased preference index as compared to both defeated and high dose groups. Data expressed as mean± SEM (n=8). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. ***: p˂0.001, ****: p˂ 0.0001, compared to defeated group.

Figure (3): Effect of testosterone on the behavior of socially defeated mice in the elevated plus maze.

Both dose levels of testosterone increased time in the open arms and correspondingly decreased time in closed ones. However, low dose group spent longer time in the open arms compared to the high dose. Data expressed as mean± SEM (n=8). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. *: p˂0.05, **: p˂0.01, ****: p˂0.0001 compared to defeated group.

Figure (4): Effect of testosterone on the performance of socially defeated mice in the Morris water maze. Socially defeated mice expressed poor learning ability throughout the training days while high dose testosterone animals rapidly recognized the escape platform compared to low dose ones.

In the probe test, both dose levels equally enhanced memory of the socially defeated animals. Data expressed as mean± SEM (n=8). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Figure (4A): *: sig. diff. from control, #: sig. diff. from defeated, $: sig. diff. from low dose. Figure (4B): *: ***: p˂ 0.001, ****: p˂0.0001, compared to defeated group.

Figure (5): Relative expression level of CREB mRNA in A) frontal cortex (FC) and B) hippocampus (HC).

Defeated group showed a significant decrease in expression levels in HC but not FC. TU 100 showed a slight increase in CREB expression in both FC and HC. However, TU 500 induced a significant CREB mRNA expression in the FC as compared to both negative control and defeated group. a: significant different from negative control at p<0.05, b: significant different from defeated group at p<0.05.

AUTHOR CONTRIBUTIONS:

Sayed, M. and Ibrahim, M. conceived this work, Sayed, M. and Ibrahim, M. designed it, Kamal, M. and Sayed, M. acquired data, Kamal, M. and Ibrahim, M. analyzed data and interpreted data together with Tikamdas, R. and Sayed, M., Tikamdas R, and Sayed, M. drafted the work; all authors critically revised it and approved the final version.

CONFLICT OF INTEREST STATEMENT:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Received on 22.03.2020 Modified on 06.05.2020

Research J. Pharm. and Tech. 2020; 13(12):6041-6049.

DOI: 10.5958/0974-360X.2020.01053.7