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Also, we evaluated the effects of those protocols on hippocampal parvalbumin PV content and the possible connection between the alterations in certain parts of hippocampal GABAergic system and behavioral patterns. Both ND and exercise protocols induced increase in testosterone, dihydrotestosterone and estradiol blood levels. Our results confirmed anxiogenic effects of ND observed in open field OF test decrease in the locomotor activity, as well as in frequency and cumulative duration in the centre zone and in elevated plus maze EPM test decrease in frequency and cumulative duration in open arms, and total exploratory activity , that were accompanied with a mild decrease in the number of PV interneurons in hippocampus.
Chronic exercise protocol induced significant increase in hippocampal PV neurons dentate gyrus and CA1 region , followed by anxiolytic-like behavioral changes, observed in both OF and EPM increase in all estimated parameters , and in evoked beam-walking test increase in time to cross the beam , compared to ND treated animals.
The applied dose of ND was sufficient to attenuate beneficial effects of exercise in rats by means of decreased exercise-induced anxiolytic effect, as well as to reverse exercise-induced augmentation in number of PV immunoreactive neurons in hippocampus. Our results implicate the possibility that alterations in hippocampal PV interneurons i. Selakovic D, Joksimovic J, Zaletel I, Puskas N, Matovic M, Rosic G The opposite effects of nandrolone decanoate and exercise on anxiety levels in rats may involve alterations in hippocampal parvalbumin—positive interneurons.
July 31, ; Accepted: November 28, ; Published: This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. All relevant data are within the paper and its Supporting Information file. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The authors have declared that no competing interests exist. Anabolic androgenic steroids AASs comprise a large class of synthetic compounds made up of testosterone and its derivatives. AASs have an important role in the treatment of various chronic diseases [ 1 ]. Top athletes use AASs in order to improve physical performance [ 2 ]. In the last few decades, the abuse of AASs has been widely spread among the adolescent males [ 3 ], and became a problem even among non-athletes, representing a public-health concern.
Increased prevalence of behavioral disorders, including unprovoked aggression and violence, has been reported in AASs abusers [ 4 ]. Long-term AASs abusers are characterized by high level of anxiety and extreme mood-swings [ 5 ].
Studies performed on animals also reported AASs modulation of anxiety behavior. Results obtained from animal experiments are controversial. Some authors reported anxiolytic-like effects [ 6 ], while other studies showed anxiogenic effects of AASs in rats [ 7 ]. However, it should be emphasized that some of those studies were performed on different species, with different classes, protocols and doses of AASs.
Beneficial effects of exercise on physical performance are well known. Reports for the impact of exercise on cognitive and emotional aspects of behavior are much more recent [ 8 ].
The behavioral effects of exercise depend on various features, such as training length acute vs. It has been shown that certain types of exercise protocols mild or moderate exercise have anxiolytic and antidepressant effects that influence the management of stress [ 10 ], while anxiogenic outcome was observed following high intensity exercise [ 11 ].
Also, chronic physical activity induced behavioral changes in animals [ 12 ], such as anxiolytic effects in rats [ 13 ] and anxiogenic effects in mice [ 14 ], depending on the type of exercise protocol.
Simultaneous application of AASs and chronic exercise showed contradictory results, possibly due to different protocols both for exercise and AASs administration. However, the results of numerous studies confirmed the attenuation of beneficial effects of exercise after AAS administration in rats [ 15 ]. The hippocampus is a structure that has a key role in cognitive and emotional processes.
Hippocampal formation has two main groups of neurons: GABA interneurons are widely distributed in several regions of brain and have a major role in modulating local noradrenergic, dopaminergic, serotonergic and glutamatergic neuronal circuitry. GABAergic dysfunction has been reported to be associated with depressive symptoms [ 17 ], mood disorders [ 18 ], bipolar disorder [ 19 ] and post-traumatic stress disorder [ 20 ].
Hippocampal GABAergic neurons, according to specific immunoreactivity, are divided into subpopulations: Parvalbumin PV belongs to the group of calcium-binding proteins and it is specific for vertebrates [ 21 ]. PV-positive neurons are widely distributed cell population that is present in different parts of the central nervous system, with a respectable number in hippocampus [ 22 ]. Since hippocampus plays one of the key roles in mood modulation and may be involved in control of some specific behavioral patterns [ 23 ], alterations in hippocampal PV content have been proposed as a possible explanation for exercise-induced behavioral changes.
It has been reported that behavioral alterations induced by various types of exercise protocols were accompanied with specific modification in hippocampal parvalbumin expression [ 24 — 26 ]. Although, there is no literature data concerning the alterations in hippocampal PV interneurons following AASs administration and its connection to behavioral alterations, the results that showed testosterone propionate-induced changes in spine density on neurons in the limbic system, including hippocampus, and their excitability [ 27 ], made good connection to the possible influence of AASs on hippocampal GABAergic system.
Since it has been reported that both various exercise protocols and AASs administration in different doses induced significant changes in sex hormone levels, and therefore can affect the neurogenesis in various brain regions, including hippocampus [ 28 ], it seems relevant to evaluate the specific effects of altered sex hormone levels on hippocampal PV content and its possible behavioral manifestations, as a start point for investigation of numerous AASs actions in generating of mood alterations that should be followed by more extensive research, as presented by Troakes and Ingram [ 29 ].
Considering the fact that nandrolone decanoate ND is one of the most commonly used AAS, the aim of this study was to evaluate the effects of chronic ND administration and exercise swimming protocol on behavioral changes in rats by means of specific behavioral tests, as well as on hippocampal PV content.
Serum testosterone T , dihydrotestosterone DHT and estradiol E2 were determined in order to quantify the effects of chronic AAS treatment and exercise protocols by means of the impact on sex hormone levels in blood. Additionally, we planned to estimate the possible connection between the alterations in certain parts of hippocampal GABAergic system and behavioral patterns following chronic ND abuse and exercise protocols. The animals had free access to food and water.
The experimental groups were as follows: The supraphysiological dose of ND was used in order to mimic the doses for heavy human AAS abusers [ 33 , 34 ]. The exercise protocol was performed following the adaptive period 20 minutes of swimming per day for one week in order to reduce water-induced stress [ 35 ].
The duration of the swimming trial was defined on the basis of a previous report as the protocol sufficient to induce immunohistochemical alterations in certain brain regions, such as hippocampus and prefrontal cortex that are reported to be involved in behavior alterationsin rats [ 36 ].
Since the swimming is an inherited behavior pattern among rodents [ 37 ], this protocol was used as an exercise model of endurance training. In order to eliminate the difference between exercise and non-exercise groups caused by water immersion, rats from sedentary control and ND groups were placed in the same water tank 7 cm water depth for short time 2 minutes each day of the training protocol, also in groups of 3—5 animals in order to maintain the same social context of swimming training.
The experimenter was present during the whole swimming protocol monitoring the rats. After the swimming session rats were towel dried and placed in a clean cage. The same-housed animals were tested on the same day, starting at approximately 9 a. All three behavioral tests were performed one by one for all investigated groups in a following order: Inter-trial-interval of approximately 15 minutes between the two consecutive tests was allowed in order to avoid minimize the cumulative effects of the repeated anxiety-provoking testing.
During the behavioral testing, mazes were cleaned following the trial for each animal to remove possible interfering scents. After the completion of behavioral tasks approximately at 1 p. Brains were removed for histological analysis. The open field OF paradigm was originally introduced as a measure of emotional behavior, but it is also a suitable test for the evaluation of general motor activity in animal models [ 15 ].
The maze consisted of black wood square arena 60x60x30 cm. The rats were placed in the centre of the arena and spontaneous exploration activity was recorded during five minutes. The following parameters were scored: The elevated plus maze EPM test is used for the estimation of anxious-like behavior.
EPM consisted of two opposite open 50x20 cm and two opposite enclosed arms 50x20x30 cm , elevated cm from the floor. Each rat was placed in the centre of the elevated plus maze facing the open arm, and was allowed 5 minutes for free exploration.
The following parameters were estimated: Those parameters are considered as indicators of anxiety level [ 39 , 40 ]. In order to estimate the overall exploratory activity in EPM test, we introduced a new parameter that included both patterns of exploratory activity observed in the EPM test the number of rearings and the number of head-dippings , since they are taking place in different zones of EPM closed and open arms, respectively in different time intervals, and presented it as total exploratory activity—TEA episodes the sum of the numbers of rearings and head-dippings.
Evoked beam-walking EBW test was performed in order to estimate emotional reactivity of animals by means of anxiety-provoking pattern effects on the performance in previously recorded beam-walking test [ 41 ]. Test was performed using apparatus consisted of white wooden box with the hole, as an escape box, for motivating the animal to cross the beam and the stainless steel beam x3x2 cm fixed between the base of the escape box cm above the floor and a vertical stainless steel pole 60 cm above the floor.
Rats were pre-trained to cross the beam four trials were performed with 15 minutes intervals. At the start of the trial, the rat was placed at the end of the beam opposite to the escape box and the time to cross the beam was recorded.
Fifteen minutes after the first recording, the rats were placed in the same starting position and the experimenter started tapping approximately every second with metal stick at the base of pole, while the rat traversed the beam anxiety-provoking pattern , until the rat reached the escape box [ 42 ].
The results were expressed as percentage of shortening the time to cross the beam between trials. OF and EPM tests were recorded by digital camera, mounted above mazes at the appropriate height. Serum samples were assessed for total testosterone and estradiol levels by Elecsys analyzer using the method of electrochemiluminescence immunoassay ECLIA. The sensitivities of the assays for testosterone and estradiol were 0.
Inter- and intra-assay coefficients of variance for testosterone and estradiol were 3. The sensitivity of the assay for dihydrotestosterone was 6.
Inter- and intra-assay coefficients of variance for dihydrotestosterone were 5. Slices were incubated in primary antibody—mouse monoclonal anti-PV 1: The number of immunoreactive neurons was always obtained on the dorsal hippocampus level of section was 3.
Parameters obtained in behavioral tests were initially submitted to Levene's test for homogeneity of variance and to Shapiro-Wilk test of normality.
Pearson's coefficient of correlation was used to analyze relationships between parameters obtained in behavioral tests and histological data, and simple linear regression analyses were performed. Statistical analysis was performed with SPSS version There was no significant alteration in the percentage of time moving following any of applied protocols.
However, beneficial effect of exercise was also manifested in combined group by reversing the ND induced reduction of exploratory activity to the control values. As shown in Fig 3 , neither exercise nor ND protocol induced significant change in the reduction of time to cross the beam compared to control data in S1 Table , section C.
As shown in Fig 4B , statistical analysis of PV immunoreactive neurons showed that neither exercise nor ND protocol induced significant change in the number of these cells in CA1 region compared to control group. A Distribution in the control group. B Number of PV immunoreactive neurons in hippocampal regions.
The main findings in this study are that both chronic swimming training and chronic exposure to ND had a significant influence on behavioral patterns in rats. Also, all three applied protocols were sufficient to induce alterations in the sex hormone profile Table 1.
It has been reported that prolonged ND treatment resulted in decrease of serum T in rats [ 44 — 46 ], and mice [ 47 ]. However, the elevation of serum T level observed in this study is in line with numerous reports that confirmed T levels increase following both acute [ 48 ] and chronic [ 49 — 52 ] AASs ND and other testosterone derivatives administration in rats.
The elevation of T in rat serum, as observed in this study, could be explained by increase in T production due to stimulation of endogenous production in Leydig cells [ 53 ].
The other explanation for this level of T serum increase could be found in the possibility that either endogenous and exogenous testosterone or testosterone like substances were measured [ 49 , 54 ].
Simultaneously, chronic exposure to AAS resulted in increase in E2 serum levels, as observed in our study, was also previously reported in rats [ 49 , 55 ].