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Cent. Eur. J. Biol. • 7(4) • 2012 • 596-602 DOI: 10.2478/s11535-012-0046-z

Central European Journal of Biology

The effect of rapid and depot and on spatial performance in water maze

Research Article

Julius Hodosy1,2,*, Daniela Ostatnikova2, Vladimír Riljak3, Jaromir Myslivecek3, Peter Celec1,4,5

1Institute of Molecular Biomedicine, Comenius University, Faculty of , 811 08 Bratislava, 2Institute of Physiology, Comenius University, Faculty of Medicine, 811 08 Bratislava, Slovakia 3Charles University, 128 00 Prague, 4Institute of Pathophysiology, Comenius University, Faculty of Medicine, 811 08 Bratislava, Slovakia 5Department of Molecular Biology, Comenius University, 842 15 Bratislava, Slovakia Received 02 March 2012; Accepted 21 March 2012

Abstract: Men and women differ in some cognitive functions including spatial abilities. These differences seem to be affected by sex , but the results are controversial. The aim of this work is to describe the effects of rapid or depot testosterone and estradiol on spatial memory in rats. Thirty-two adult male Wistar rats were divided into 6 groups. Five groups were gonadectomized, and one group was left as control. Castrated groups received sterile , testosterone isobutyras, testosterone propionate, estradiol dipropionate or . We evaluated spatial performance (escape latency, overall improvement, and time in the quadrant after platform removal) of the rats in a spatial water maze. Animals receiving exogenous sex steroids showed higher plasma concentrations of the particular hormones. Experimental groups improved during the acquisition spatial trials in the water maze. No significant differences between the groups during probe trial were found. In overall improvement, the testosterone depot and estradiol depot groups showed less improvement in comparison to the control groups (P<0.05). No differences in respect to administered hormones were found in corresponding receptor gene expression in hippocampus. In conclusion, exogenous testosterone affects spatial memory of adult castrated males.

Keywords: Water maze • Spatial performance • Testosterone • Estradiol • Hippocampus • Rats © Versita Sp. z o.o.

1. Introduction of receptor types. So, particular brain regions can be targets for the actions of different classes of steroids. Although there is no major difference in performance in For example, receptors are thinly distributed current standardized intelligence tests between females in the neocortex of the rat, but are prevalent in preoptic and males, there are described sex differences in and hypothalamic areas and the anterior pituitary. Also particular subtypes of the tests [1]. These have been some neurons express receptors for more than one reported in certain cognitive tasks for many years, and . it appears that testosterone (TST) and other Authors reported testosterone modified spatial play a crucial role [2]. Spatial learning and memory have abilities resulting from animal research [3] and clinical been one of the major topics of interest since the late studies of individuals exposed prenatally to unusual seventies. The effect of sex hormones is determined hormone environments. Congenital adrenal hyperplasia by the location of the receptors, especially in the results in women with enhanced spatial performance [4], brain. Different areas of the adult brain have different as does a lifelong deficiency in -releasing steroid receptor patterns with overlapping distributions hormone (GnRH) in men [5].

* E-mail: [email protected] 596 J. Hodosy et al.

Conversion of testosterone to estradiol plays an 2.1 Surgery important role in sexual differentiation of spatial memory. After two weeks of acclimatization (12-weeks old To support this hypothesis, experiments concerning animals), five groups of rats underwent castration the prevention of TST conversion to estradiol were surgery under general anesthesia (ketamine 100 mg/kg needed. In their experiments, Williams and Meck [6] + xylazine 10 mg/kg in the same syringe, applied uncovered that early postnatal treatment of male rats intramuscularly). Castration was performed through by aromatase inhibitors (e.g. , ), a small incision in the scrotum, where both testes decreased their spatial orientation ability. On the other and epididymis were ligated with absorbable suture hand, there is some evidence that hormones and excised. The skin was sutured in two layers with (and not estradiol) are responsible for active formation absorbable silk size 4-0. All animals were allowed of sexually dimorphic telencephalic structures. two weeks for recovery after the surgery and before Fitch and Denennberg administrated testosterone hormone supplementation. A control group was left propionate to female rats during the first four days unchanged. after birth, resulting in the corpus callosum thickening to that of an adult male rat, whereas administration of 2.2 Hormonal supplementation synthetic () had no effect Two weeks after castration, hormonal supplementation [7]. was initiated and group assignment was as follows: Sex hormones can also influence the behavior of 1. The control group (CTRL), supplemented with sterile an individual in the environment, their motor activity olive oil intramuscularly each day, n=6. and pain sensitiveness. Spatial orientation and memory 2. The control castrated group (GDX), treated with abilities belong to the higher nervous functions, which sterile olive oil intramuscularly each day, n=5. are affected by sex hormones. Activation influence of 3. Depot testosterone (GDX+Td; testosterone sex hormones modulates these functions by altering isobutyras, 25 mg, Biotika, Slovenská Ľupča, hormonal levels in various stages of life beginning in Slovakia) - 2,5 mg/kg every 4 days, n=5 juvenility until caducity. It also depends on biological 4. Rapid testosterone (GDX+Tr; testosterone rhythms (ultradian, infradian annual) of sex hormones. propionate, Sigma Aldrich, St. Luis, USA) – 625 mg/kg The hippocampus, a key structure for memory function each day, n=6 [8], has quite a high concentration of androgen receptors 5. Depot estradiol (GDX+Ed; estradiol benzoate, [9]. In adult male rats, an androgen manipulation Sigma Aldrich, St. Luis, USA) – 200 mg/kg every influence on spontaneous and glutamate-induced 4 days, n=5 activity in CA 1 pyramidal cells had been observed [10]. 6. Rapid estradiol (GDX+Er; estradiol dipropionate, Whether the changes in receptor concentration plays a Biotika, Slovenská Ľupča) – 50 mg/kg each day, role has yet to be determined. n=5 The aim of our study was to evaluate whether The supplemented doses were administered for castration and subsequent hormonal supplementation another 21 days into the muscle alternating injection affect performance in the spatial water task in adult rats, sites between two hind limbs in order to allow recovery i.e., describe the activation effect of testosterone and from the injury. The rats receiving supplementation the subsequent molecular pathway in accordance to of depot forms every 4 days, received sterile olive oil administration of sex hormone form. during the times when they did not receive hormonal supplementation.

2. Experimental Procedures 2.3 Modified Morris water maze 2.3.1 Apparatus Thirty two adult 10-week old male Wistar rats (Dobrá We used the modified water maze that consists of a circular Voda Breeding Laboratories, Slovakia) were obtained pool (135 cm in diameter, 60 cm in height) filled with water for this experiment. Upon delivery, animals were (40 cm). Water temperature was maintained at 25±1°C. allowed two weeks for acclimatization. Animals were The maze was virtually divided into four quadrants and kept in separate cages in a controlled environment on the wall of each quadrant, one geometrical figure for (temperature 22ºC, humidity 50%) with a 12:12 light- orientation as an external cue was placed. Hidden 0.5 cm dark cycle, light period starting at 8:00 pm and ad beneath the surface of the water was a circular transparent libitum access to water and food pellets. The research platform (10 cm in diameter). The animal was recorded by on animals was approved by the ethical committee of camera above the pool. The experiment took place during the Faculty of Medicine, Comenius University. the dark phase of the light cycle.

597 The effect of rapid and depot testosterone and estradiol on spatial performance in water maze

2.3.2 Procedure subsequently transcribed into cDNA using Ready-To- On each of 4 consecutive days, rats received one block Go You-Prime First-Strand Beads (GE Healthcare). of acquisition phases, four trials in a block, during which qPCR was performed using TaqMan probes (Applied the platform remained hidden 0.5 cm beneath the surface Biosystems, probe numbers Rn00560747_m1 (AR); of the water in a constant location (quadrant). A different Rn00664737_m1 (Eralpha); Rn00562610_m1 (Erbeta); starting quadrant was used each trial. A trial consisted with Roche qPCR Mastermix (Roche) using the following of a swim followed by a 30-second rest on the platform. protocol: 2 min at 50°C, 10 min at 95°C followed by 50 15 s Any rat that did not find the platform within 60 seconds cycles at 95°C and 1min at 60°C in a final volume 10 μL. was gently guided to it by an experimenter. Escape latency (time to find the platform) was calculated. Daily 2.5 Statistical analysis difference/average swimming times and speed (working Data from Morris water maze were analyzed using memory – improvement during trials in one block) were One-way ANOVA with LSD post-hoc test. Microsoft calculated for each group. On the day 5, one probe trial Excel 2007, XLstatistics 5.51 and SPSS for Windows was performed by removing the platform. The duration v16.0 software were used for the analysis. P<0.05 of the trial was 60 seconds and time spent in the was considered significant. Data is presented as mean platform quadrant was recorded. The starting position ± standard error of the mean. for all animals was the quadrant opposite the platform. In addition, overall improvement was calculated as a difference between average swimming times on the first 3. Results and last day. The hormonal administration was successful, as proved 2.4 Additional analysis by hormonal assay (Figure 1; *P<0.05). GDX group When all animals finished their trials, they were sacrificed. after two weeks proved to have significantly lower was taken for hormonal analysis. Testosterone levels of testosterone in plasma, when compared to and estradiol levels were measured by DRG ELISA CTRL group (Figure 1A; P<0.05). On the other hand, commercial kits for measuring testosterone or estradiol both testosterone groups proved to have levels of (9% inter-assay, 3% intra-assay variability). Moreover, testosterone, approximately 6-fold higher than the the brain tissue was frozen in dry ice for examination of CTRL group (Figure 1A; P<0.05). Similarly, estradiol androgen and expression in the CA1 concentration in plasma was significantly increased in region of the hippocampus. Total mRNA was isolated both estradiol groups, whereas gonadectomy alone did using a chloroform-isopropanol (RNA Bee, TelTest, TX, not influence estradiol levels (Figure 1B; P<0.05). USA) method according to manufacturer instructions. RNA In the water maze task, all rats did not differ in yield and integrity was evaluated spectrophotometrically swim speed, suggesting no differences in swimming at A1=260 nm and A1/A2=260/280 nm, respectively. capabilities. All groups performed similarly during Samples with A1/A2 between 1.6 and 1.9 were used acquisition. In 4 days of acquisition trials, escape for downstream procedures. Purified mRNA was latencies showed a significant main effect of day,

Figure 1. Sex hormone levels in plasma. A. shows levels of testosterone in plasma in all groups; B. Estradiol levels in plasma in all groups. Data presented as + SEM, * denotes P<0.05 when compared to control group.

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Figure 2. Performance in water spatial task A. General improvement of animals within acquisition trials. All animals improved during days, however no significant difference was found between groups. B. Probe trial results. No significant changes in platform quadrant were found. C. Performance in spatial task expressed as overall improvement. Both groups of animals receiving depot forms of hormones performed worse than control group. All other groups had tendency to perform better than control, however this finding was statistically insignificant. Data presented as mean + SEM, * denotes P<0.05 when compared to control group.

F(3.042)=20.00, P<0.0001 (Figure 2A). There was no were found, although some statistically insignificant significant day x condition interaction for escape latency. trends were observed (Figure 3). During the probe trial, all groups spent equal time in the quadrant where the platform was previously hidden (Figure 2B, M±SEM, CTRL=17.51±1.93 s; 4. Discussion GDX=17.32±2.62 s; GDX+Td=13.82±3.10 s; GDX+Tr=16.31±2.14 s; GDX+Ed=18.70±1.45 s; First of all, our results confirmed that testosterone GDX+Er=21.69±5.12 s). When calculating overall administration increased plasma testosterone levels improvement as a difference in average escape in experimental animals. It did not matter whether latency during the first day and last day, we found testosterone was administered as a rapid or a depot significantly impaired improvement in GDX-Td and form. Moreover, castration decreased levels of GDX-Ed groups when compared to the control circulating total testosterone. Similarly, administration group. The difference in escape latency time of estradiol increased plasma levels of estradiol in was approximately halved in GDX+Td group and both estradiol receiving groups. Surprisingly, the reduced by 1/3 in GDX+Ed group (P<0.05). On the increase in plasma estradiol after administration of contrary, GDX alone and both rapid acting hormone depot estradiol was significantly lower in comparison groups seemed to have a trend to positively affect to administration of a rapid form. This could be improvement in spatial task (Figure 2C). partially explained by pharmacokinetic properties of Receptor expression analysis in the hippocampus is the drugs, where depot form is more lipophilic thus shown in Figure 3. No significant changes in expression distributing more in the tissues and less in plasma. of androgen, estrogen-alpha and estrogen- receptors On the other hand, rapid form of estradiol, being a

599 The effect of rapid and depot testosterone and estradiol on spatial performance in water maze

Figure 3. Expression of individual receptors in CA1 hippocampal area, no differences reaching statistical significance were observed, however A. Androgen receptor expression showed trend to be increased in GDX alone group, as well as in both groups receiving estradiol. B. Estrogen-alpha receptor expression had trend to increase in a group receiving depot estradiol. C. Estrogen-beta receptor expression on contrary had tendency to increase in GDX alone and estradiol depot group. RFU – relative fluorescence units. Data presented as mean + SEM.

less lipophilic molecule, predominantly distributes in systematic review [14]. In these experiments, there plasma. This is an interesting finding because different were no statistically significant differences between the preparation of drugs with different groups in the reference memory, mainly due to high and compartmentalization in organisms were not inter-individual differences and relatively low number considered as important in other spatial ability of animals in each group. studies. This may also be the reason why there are However, some differences in solving the water contradictory results when dealing with administration spatial task indicate effects of gonadal hormone of testosterone and spatial abilities in rats [11]. administration. Even though the administration of The main outcome of previous studies was that testosterone or estradiol in this study of adult male rats sex differences are associated with sex steroids. The did not reveal significant differences in water maze importance of prenatal testosterone on later spatial performance among these groups, by the fourth day, abilities was shown for rats [12] and for humans [13]. both groups receiving depot form of sex hormones Nevertheless, administration of androgens in adulthood appeared to have the tendency to perform worse. leads to several controversial results. The mechanism Overall improving analysis showed that testosterone of action of testosterone or gonadal hormones for groups improved significantly worse when compared spatial memory is believed to be either through to the control groups, as well as the estradiol depot cytoplasmic (long-term) or membrane receptors (short- group. This is consistent with previous studies term). The short-term effects of testosterone on brain performed in humans, where better visual-spatial function including spatial abilities are called activation performance in men with lower testosterone was found effects. However, these rapid effects are not generally [15]. Administration of depot testosterone resulted accepted and described as inconsistent in a recent in impaired overall improvement as an indicator of

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working memory during acquisition. This might point Castration of male rats impairs spatial memory, towards partial androgen receptor-mediated action of while supplementation with testosterone improves testosterone on spatial abilities in adult rats. In castrated spatial memory according to some authors [19]. rats, the administration of estradiol in the depot form Another study on castrated male rats showed impaired resulted in a similar overall impairment of latency time working memory, but an improved reference memory differences as administration of testosterone did. This [20]. In intact animals, on the contrary, testosterone might be interpreted as an indication that the action worsens spatial memory similarly to estradiol, while of testosterone on working memory is mediated by anastrozol – an inhibitor of aromatase decreases estrogen receptors. However, such definitive proof latency times to find the platform [21]. This is in of this hypothesis cannot be drawn from our results. contrast to our previously published results that proved Further studies on the effects of testosterone on negative effects of anastrozol on performance in Morris spatial abilities using estrogen receptor inhibitors water maze [22]. One research group pointed towards like and estrogen receptor knock-out mice differences between systemic and local cerebral are needed. Even though, a review by Spencer [16] concentrations of testosterone. The study showed that supports this thesis. local injections of TST, but not systemic testosterone Endogenous testosterone levels are associated manipulation, affected cognitive abilities [23]. A dose- with spatial abilities. However, the association is dependent increase in latency times was shown in far from being clear. Studies found testosterone male rats injected with testosterone into the amygdala to improve spatial abilities in hypogonadal men or [24] and into the hippocampus [9]. Interestingly, in patients with Alzheimer disease [17]. Androgen levels the same experiment similar effects were shown decline with aging. Androgens may exert anti-anxiety for flutamide – an androgen receptor antagonist. and cognitive-enhancing effects. Nevertheless, it is Comparable confusing results were achieved in an not clear, which androgens have anxiolytic-like and/or experiment with administration of testosterone and mnemonic effects and what the underlying mechanism anisomycin – a protein synthesis inhibitor [25]. Different of action is. Osborne et al. [18] showed that 3a-diol pharmacokinetics and perhaps dynamics may also decreased anxiety-like behavior in the elevated plus explain the differences in learning between depot and maze and light/dark box and concurrently increased rapid forms of sex hormones. cognition in water maze tasks, while 3b-diol improved In conclusion, our experiments show that cognition in water maze tasks of gonadectomized rats, exogenous testosterone impairs spatial memory of but had no effects on anxiety behavior, compared to castrated males supplemented with depot forms of oil vehicle or androsterone. Both 3a-diol and 3b-diol testosterone. Molecular mechanisms of testosterone are actually metabolites of testosterone acting through effects seem to be partially mediated through androgen intracellular estrogen receptor b [18], suggesting that receptor and partially through estrogen receptors the androgen-mediated spatial cognition changes are after conversion to estradiol via aromatase in different due to estrogen receptors. This finding is in consensus brain areas. Similar effects on spatial cognition were with our results, where on one hand, testosterone found in castrated adult males supplemented with administration impaired overall improving in water maze depot estradiol. Further studies taking into account performance and on the other hand, administration of the different pharmacokinetic profiles of various forms estrogen had the same effect. This would suggest that of applied sex steroids should uncover the detailed estrogen receptors really play a crucial role in spatial underlying molecular mechanism including the type memory. Although in consensus about the role of of receptors needed to mediate the effects of sex estrogen receptors on spatial abilities, our results are steroids in various brain regions and on various brain in contrast to those of Osborne et al. [18], where the functions. authors showed enhancement of the spatial cognition, whereas in our experiment there was impairment of spatial abilities. This could be explained by the fact Acknowledgements that even though the rats were gonadectomized, Osborne et al. used androgen supplementation at This study has been supported by a grant from normal levels, whereas in our experiment the levels of Slovak Research and Development Agency, supplemented testosterone and estradiol were rather No.: APVV-0753-10. The authors would like to thank supraphysiologic. According to our former study, men the Institute of Pharmacology of Slovak Academy of with higher levels of testosterone perform worse in Sciences for help with behavioral analysis and allowing spatial tasks [15]. the use of their tracking software.

601 The effect of rapid and depot testosterone and estradiol on spatial performance in water maze

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