Abstract
Nandrolone, is an anabolic androgenic steroid (AAS) used by adolescents and young adults. Supraphysiologic doses of AAS are correlated with dysfunctions in anxiety and reward. This study examined whether exposure to nandrolone before puberty altered anxiety- and addictive-like behaviors. Dopamine type 2 receptors (D2DR) in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) were also analyzed. Beginning on day 28 and ending on day 37, male rats received a daily injection of nandrolone decanoate (20 mg/kg) and were subsequently evaluated for anxiety. Their locomotor response (sensitization) and preference (conditioned place preference (CPP) to cocaine (15 mg/kg) were also assessed. Nandrolone reduced anxiety and ambulation, accelerated the development of sensitization to cocaine, and reduced CPP to cocaine by 27%. Expression of D2DR in the NAc and the PFC of males was increased by nandrolone, whereas treatment with cocaine reduced accumbal D2DR. We hypothesize that nandrolone accelerated the development of the neural circuitry that participates in behavioral sensitization and reduced the rewarding properties of cocaine. The observed increase in accumbal D2DR may have potentially mediated the reduction in anxiety and ambulation and hastened the maturation of the neural circuitry responsible for the sensitized response to cocaine.
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Introduction
Anabolic-androgenic steroids (AAS), derivatives of the gonadal hormone testosterone (T), were developed in the 1950 s to treat men with hypogonadism and delayed puberty1. The term androgenic comes from their masculinizing properties, anabolic from their promotion of metabolic processes, such as enhanced protein synthesis and erythropoiesis2. AAS provide athletes with an edge in training and competition, by increasing muscle strength and endurance and promoting competitive behaviors3. Before the 1990 s, the use of AAS was circumscribed mainly to athletes during training and before competitions4. However, during the last two decades, young men have been using AAS all year round to improve their physical appearance4,5.The use of AAS as self-medication is also on the rise among the female-to-male transgender population6.
In the USA, about 3–4 million people have used AAS; of those, approximately 1 million have developed dependence7. The worldwide prevalence of AAS use is 3.3%, which is higher for men (6.4%) than for women (1.6%)8. AAS users report administering doses that can be more than 100 times the physiological dose9. This is of significant concern because AAS can have deleterious side effects, particularly on the cardiovascular system. AAS can also cause hepatic toxicity, decrease fertility, alter secondary sexual characteristics10,11 and increase the prevalence of psychiatric disorders12,13,14. Some symptoms are the result of exposure to AAS (mania, aggression, risk-taking behaviors, irritability), whereas others result from AAS withdrawal (depression, loss of libido, suicidal thoughts, hypersomnia)15,16. There is considerable variability in the display and range of these symptoms. Fortunately, in very few people, these symptoms are disabling.
Androgens, including AAS, have rewarding properties17,18,19 and may contribute to the development of substance abuse and dependency to other drugs7,20. They also render brain substrates of the reward system more susceptible to the rewarding effects of several drugs of abuse such as cocaine, amphetamine, alcohol and opioids21,22. Approximately 32% of subjects using AAS will develop a dependency to AAS7, this risk is higher for women and adolescents of both sexes23,24. Several studies report a higher prevalence of drug abuse, particularly cocaine, in AAS users than in the general population25,26. Owing to their widespread non-medical use and associated adverse effects, anabolic-androgenic steroids (AAS) were reclassified as Schedule III controlled substances by the U.S. Drug Enforcement Administration (DEA) in 2004. Nonetheless, AAS do not have a direct psychoactive effect and although evidence indicates AAS have addictive potential, there is currently no specific diagnostic criteria for AAS substance use disorder.
Among the AAS, nandrolone (19-nortestosterone), in its long-lasting ester form of nandrolone decanoate (ND), is the AAS most widely used worldwide27. The androgenic activity of this compound is lower than that of dihydrotestosterone (DHT); in contrast, its anabolic properties are higher than those of T28making it attractive for abuse by male and female athletes. Nandrolone has a higher affinity for the androgen receptor (AR) than T and is less susceptible to degradation by the 17 beta-hydroxysteroid dehydrogenase enzyme, which increases its appeal. Furthermore, although nandrolone and T can be reduced to DHT in target tissue containing the enzyme 5 alpha-reductase, the binding of the enzyme to nandrolone is weaker than that to T29,30. Also, the binding of nandrolone to AR is weaker than that of DHT. This explains the more potent effects of nandrolone compared to T on target tissues without 5 alpha reductase activity and the weaker effect on tissues with a high 5 alpha reductase activity, resulting in greater anabolic/androgenic properties31,32.
Adolescents, compared to children and adults, show the highest incidence of risk-taking behavior and experimentation with drugs of abuse33. The use of AAS by adolescents is alarming because they can increase the behavioral response to other drugs of abuse (cross-sensitize) such as fenproporex and cocaine34,35,36. Cocaine, one of the main drugs used by people who abuse AAS37 shares many of the harmful side effects on the cardiovascular system38. It also promotes risk-taking behavior39 aggravating the detrimental health effects of AAS.
Drugs of abuse exert their behavioral and addictive effects by acting on regions of the brain associated with decision-making, motivation, and reward, such as the prefrontal cortex (PFC) and nucleus accumbens (NAc)40. Dopaminergic receptors in these brain areas participate in regulating addictive behaviors41,42. Decreased D2-like dopamine receptor (D2DR) expression is associated with increased novelty-seeking and risk-taking behaviors, traits associated with addiction43,44. Manipulating striatal D2DR can alter the response to drugs of abuse, such as cocaine. Mice lacking striatal presynaptic D2DR show increased sensitivity to the locomotor activating effects of cocaine45 while rats with increased D2DR sensitivity show enhanced cocaine self-administration46.
Insults during adolescence affect brain development and interfere with the maturation of higher brain functions such as learning and memory. Our hypothesis was that AAS during adolescence reorganizes dopaminergic circuitry resulting in learning and memory deficits and increased risk taking and addictive behaviors. This study investigated whether exposure to nandrolone before puberty affected anxiety-like behaviors, as well as the behavioral response to cocaine. The nucleus accumbens and prefrontal cortex were studied to determine whether nandrolone and/or cocaine induce changes in the D2DR population of these brain substrates. We used the rat as our animal model since they are susceptible to AAS, their physiological responses are similar to that of humans, their brain undergoes synaptic remodeling with sex steroids, and they exhibit risk taking behaviors similar to humans. They also are one of the lowest steps in the evolutionary scale of laboratory animals.
Materials and methods
Animals
All the experiments were designed to minimize the number of animals used for each experiment. The significance level (alpha) was set at 0.05 and the power at 0.80, which means there was an 80% chance of detecting a true effect, while maintaining a 5% probability of a Type I error. The behavioral sensitization protocol was used to determine the sample size.
Pregnant Sprague Dawley rats were purchased from Charles Rivers Laboratories (Willmington, MA, USA). Dams were housed in pairs, with water and Purina® rat chow provided ad libitum. They were kept in a temperature and humidity-controlled room, in a light-dark cycle with lights off at 5 PM (12 L:12D). After parturition, pups were cross-fostered, and each dam was housed with their litter of 8–10 pups. The litter was half male and half female. The experimenter did not keep track of the origin of each pup. The day of weaning (day 23), animals were separated from the dam and males and females housed separately in groups of 2–3 per cage. Although male and female progeny were included in the study, this manuscript only reports results obtained from males. We are currently preparing a manuscript discussing the data obtained with the female population. The experimental protocol was designed and approved prior to the initiation of the study. All animal experiments were revised and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Puerto Rico Medical Sciences Campus (Protocol# 1140215) and adhere to USDA, NIH, AAALAC and ARRIVE guidelines. All the methods used are in accordance with NIH, AAALAC and ARRIVE guidelines and were followed as described in the UPR MSC IACUC-approved protocol (#1140215).
Animals were monitored daily for erratic behavior, such as a reduction in 15–20% body weight in a week, or the inability to eat or drink for a period of 24 h. No animals in the study displayed any of the above behaviors, which were the humane endpoints established for this study.
After the behavioral tests were concluded, animals were euthanized in a separate room by decapitation in a guillotine followed by rapid freezing of the brain in dry ice. Euthanasia was carried out by decapitation, a method acceptable by the American Veterinary Medical Association when its use is required by the experimental design and approved by IACUC (AVMA Guidelines, 2020). For the determination of dopaminergic receptors this method is necessary to avoid artifacts that may occur following stress or administration of an anesthetic47. Personnel performing decapitations were properly trained and had experience in this procedure.
Drugs and chemicals
The AAS used in this study was 4-estren-17beta-ol-3-one decanoate (nandrolone decanoate) (Steraloids, Inc., Newport, RI, USA), dissolved in sesame oil. Sesame oil was used as vehicle because of its stability against oxidation and its common use as a solvent for steroids hormones. Although it contains minute amounts of phytosterols, this is true for other commonly used solvents such as peanut and corn oils48,49. Nonethless all of our control comparisons were with sesame oil-treated rats to avoid any confound that may be attributed to the use of sesame oil. Nandrolone was administered subcutaneously (s.c.) at a dose of 20 mg/kg/day. Doses between 500 and 2000 mg/week have been reported by users of AAS50. The dose of 20 mg/kg/day in rats is equivalent to a human dose of 1350 mg/wk in a 60 kg man, similar to the supraphysiological doses used by AAS users50,51,52. Cocaine-HCl (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.9% sterile saline and administered intraperitoneally (i.p.) at a dose of 15 mg/kg. The dose of 15 mg/kg of cocaine has been used extensively in our laboratory and has been proven to be effective in inducing behavioral sensitization and Conditioned Place Preference (CPP) to cocaine. We have found that higher doses (30 mg/kg) can induce tolerance, and lower doses are not as effective in a context-free behavioral sensitization paradigm36,53.
Nandrolone treatment
On postnatal day 28 (PN-28), rats were weighed and randomly distributed into two groups that for 10 consecutive days received daily injections of nandrolone (ND) (20 mg/kg) or of sesame oil (Oil). Days 28 to 37 are within the conservative range for adolescence that spans days 28 to 4254 in rats. We used two cohorts of rats of 40 individuals each for a total of 80 rats. To minimize potential confounders and variability when conducting behavioral studies, each session had representatives of each control and experimental group.
The first cohort of rats was treated first with nandrolone (n = 20 oil, n = 20 nandrolone) and then at day 39, all 40 rats were tested in the open field. This cohort was further subdivided into the following groups to be tested for behavioral sensitization: saline/oil (n = 10); cocaine/oil (n = 10), saline/nandrolone (n = 10) and cocaine nandrolone (n = 10). The test took place from days 40 to 62. They were euthanized on day 63.
Our second cohort (n = 40) was also treated with nandrolone (n = 20 oil, n = 20 nandrolone) and then on day 38, all 40 rats were tested in the Elevated Plus Maze (EPM). From days 40 to 53 this cohort was further subdivided into the following groups: saline/oil (n = 10), cocaine/oil (n = 10), saline/nandrolone (n = 10), cocaine/nandrolone (n = 10) and tested for conditioned place preference (CPP) to cocaine. This last group was euthanized on day 54.
Elevated plus maze
The EPM is a frequently used paradigm to measure anxiety-related behaviors55. Rats previously exposed to anxiogenic drugs decrease the time spent in the open arms of the maze, while anxiolytic drugs increase the time spent in open arms56.
Our testing apparatus consisted of a plus-shaped custom-made apparatus with two 50 cm open arms and two 50 cm enclosed arms without a roof. The apparatus stands at a height of 70 cm from the ground. The open arms had a 1 cm ledge; the floors were lined with rugged plastic to avoid slipping. An infrared video camera was placed in the center above the maze. The camera was connected to a computer containing the ANY-MazeTM software. At the beginning of the test, the rats were placed at the junction of the open and closed arms and the video tracking system was activated. The software automatically recorded the number of entries into the open and closed arms, as well as the time spent in each arm. The entry into an arm was defined as the time point when more than 95% of the rat is in the arm. This was considered to be time zero. The test ended after 5 min. The amount of time spent in the closed and open arms and the number of entries into the open and closed arms were measured. The more time spent in closed arms, the higher the anxiety.
Open field test
The OFT is an assay of locomotor activity that is used to measure anxiety, exploratory and risk-taking behavior, and thigmotaxis57,58,59. Anxiolytic drugs increase the time rats spend in the center area60. Locomotor activity chambers from Versamax™ were used to measure Open Field Behavior. These chambers are made of clear acrylic (42 cm × 42 cm × 30 cm) with 16 equally spaced (2.5 cm) infrared beams across the length and width of the cage at a height of 2 cm (horizontal beams). An additional set of 16 infrared beams were located at the height of 10 cm (stereotyped activity). All beams were connected to a Data Analyzer that sent information to a personal computer.
The animals were placed in the activity chambers and allowed to roam freely for 10 min. Breaking of infrared beams determined the position of the rats in the activity chamber. The amount of time spent in the center of the chamber versus in the periphery was compared, as well as the total distance traveled. Animals that spent less time in the center of the chamber were classified as more anxious and as showing less risk-taking behavior when compared to their counterpart controls (oil treated).
Locomotor activity
Horizontal and stereotyped activity was measured with an automated animal activity cage system (Versamax™; AccuScan Instruments, Columbus, Ohio) (see description above). This system differentiates between horizontal, stereotyped, or rearing activity based on sequential breaking of different horizontal beams (Horizontal), the same beams (Stereotypies) or vertical beams (Rearing). Stereotyped behavior refers to repetitive motor responses of seemingly unknown function that arise in certain contexts, such as after psychostimulant administration61,62.
Activity was measured in an isolated room with low illumination. Animals were habituated to the chamber for 1 h, 1 day prior to injections (Day 39). On days 40, 44, 52, and 62, rats were placed for 30 min in the chambers, and basal locomotor activity was recorded. The animals then received a saline or cocaine injection and locomotor activity was recorded for 60 additional minutes. On days 41–43, animals received a daily injection of 0.9% saline or cocaine (15 mg/kg) in their home cages. During days 45–51 and 53–61, animals remained undisturbed in their home cages (Fig. 1). Animals were sacrificed the day after the last behavioral test at 63 days of age.
Behavioral sensitization (A) and CPP (B) protocols. A Five days after rats were weaned from their mother, they received a daily oil or nandrolone injection for ten consecutive days (PN-28 to 37). For the behavioral sensitization experiments, rats were habituated to the locomotor activity chamber for 1 h (2 days post-nandrolone treatment, PN-39). The data obtained from the first ten minutes of habituation (Day 39 and Day 40 was used as the data for the open field test (Fig. 2B and C). From days 40 through 44, on day 52, and on day 62, rats were injected with saline or with cocaine (15 mg/kg). Rats were injected in the locomotor activity chamber (days 40,44, 52, and 62) or in their home cage (days 41, 42, and 43) (see the context of injection). From days 45 through 51 and from days 53 through day 61, rats remained undisturbed in their home cages. B. Five days after rats were weaned from their mother, they received a daily oil or nandrolone injection for ten consecutive days (PN-28 to 37). On day 38, a second group of rats were tested in the elevated plus maze (EPM). These same rats were used for the CPP experiments. To determine the rat’s preference for a particular CPP chamber, rats were allowed to roam freely through both chambers for 3 days (Days 40–42: pre-conditioning). The amount of time spent in each chamber was calculated to determine which side it preferred. For the following 10 days (days 43–52, conditioning days), rats were injected daily, alternating between saline and cocaine (15 mg/kg) injections. Rats received cocaine in the non-preferred chamber and saline in the preferred chamber. Saline animals received saline in both chambers. After the injection, rats were confined for 30 min in the chamber where they received the injection. On the last day (day 53: post-conditioning day), the animals were placed in the activity chamber and allowed to roam freely between the two chambers for 30 min. The amount of time spent in each chamber was recorded and compared to that spent in pre-conditioning.
Conditioned place preference
Cocaine-induced CPP was measured using Versamax™ activity chambers (described above). Each chamber was divided into 2 smaller chambers. For the pre- and post-conditioning sessions, the chambers were separated by an acrylic wall that had an opening; during the conditioning phase, the wall was replaced by a solid acrylic wall that separated the two chambers. Each chamber had different visual and tactile cues. During preconditioning, animals were placed in the CPP apparatus for 3 consecutive days and allowed to roam between both chambers for 15 min. The amount of time spent in each chamber was recorded. The conditioning phase consisted of alternating injections of saline and cocaine for 10 days. Saline was injected in the preferred chamber, and cocaine was injected in the non-preferred chamber with 24 h of separation between injections. The rats were confined for 30 min to the chamber where they received the injection. During postconditioning, the animals were placed in the activity chamber and allowed to roam between the 2 chambers for 15 min (Fig. 1). The time spent in each chamber during pre- and post-conditioning was compared. Rats that showed a significant increase in the time spent in the chamber where they received cocaine displayed conditioned place preference. This method has been validated by many laboratories63,64.
Western blots
Western blots were used to quantify D2DR levels in mPFC and NAc. The protein concentration of the samples was determined using the BioRad Protein Assay method (BioRad Laboratories, Hercules, CA, USA). The molecular weight standards used for all Western blots were the BioRad Precision Plus Protein™ All Blue Standards (#1610373). For our gels, 20 µg of protein were mixed in SDS/-mercaptoethanol, vortexed and heated at 95 °C for 7 min prior to separation by 10% SDS-PAGE (BioRad Laboratories). Following electrophoresis, proteins were transferred to a 0.2 μm nitrocellulose membrane using Trans-Blot Turbo (BioRad Laboratories). Nonspecific binding to the membrane was blocked by incubation in Odyssey blocking buffer for 60 min at room temperature. This was followed by overnight incubation at 4 °C with a D2 receptor antibody (1:200; Santa Cruz Biotech, Santa Cruz, CA, USA, #sc-5303) and a B-Actin antibody (1:2500; Abcam, MA, #ab8227) dissolved in Odyssey Blocking Buffer. The D2DR receptor antibody used was a mouse monoclonal antibody raised against amino acids 1–50 that has been validated by various researchers65,66,67. The next day, the membranes were washed 3X in TRIS buffered saline and polysorbate 20 (PBS-T). After washing, the membranes were incubated for one hour in IRDye 680RD goat anti-rabbit (1:15000; LI-COR, Lincoln, NE, USA, #926-68071) and IRDye 800CW goat anti-mouse (1:15000; LICOR, Lincoln, NE, USA, #926-32210). Proteins were detected using the Odyssey CLx infrared imaging system (excitation/emission filters at 700 nm/800 nm range, LI-COR Biosciences, Lincoln, NE, USA). The optical density of the D2 receptors of each sample was obtained using Odyssey software (LI-COR Biosciences), normalized against the background, and then corrected against their own levels of B-Actin. The Western blots images were not subjected to image editing or processing by editing software with the exception of cropping the images for publication purposes. We have included the replicates of the Western blots as part of the Supplementary Information.
Statistical analyses
All data were analyzed using GraphPad Prism version 9.00 for Windows (GraphPad Software, San Diego, California, USA). An unpaired t-test was used to compare two groups, a two-way ANOVA was used to compare more than two groups, and repeated measures MANOVA was used to analyze repeated measures.
To evaluate the effects of nandrolone and cocaine on behavioral sensitization, a linear mixed-effects model was applied to average locomotor activity measured between 30 and 60 min across three exposure days. The model included nandrolone and cocaine as between-subject factors, day as a within-subject factor, and subject ID as a random effect to account for repeated measures. Fixed effects and their interactions were tested, including the three-way interaction between nandrolone, cocaine, and day. Statistical significance was assessed using Wald z-tests, and parameter estimates were used to interpret main effects and interaction terms related to sensitization patterns. In addition, the time course of each group was analyzed separately using repeated measures (RM) ANOVA with days (40, 44, 52, and 62) and minutes (35–90) as repeated factors. Tukey multiple comparisons were used for post hoc analysis to compare locomotor and stereotyped activity over time.
CPP was analyzed by repeated measures MANOVA, using pre- and postconditioning as repeated factors. A two-way ANOVA was used to compare the expression of D2DR in the PFC and the NAc between the groups. The results of the statistical analysis are included as supplemental material. Data are presented as the mean ± standard error of the mean (SEM). A p-value of less than 0.05 (p < 0.05) was considered statistically significant.
Results
Open field and basal locomotor activity
Rats treated with nandrolone spent more time in the center of the open field and ambulated less than oil-treated rats (Fig. 2A and B, respectively). Data were analyzed with Student’s T test—Fig. 2A: t = 2.361, df = 36, p = 0.0264; Fig. 2B: t = 5.174, df = 36, p < 0.0001. The decrease in distance traveled corresponds to the first 10 min of habituation (sensitization protocol-Day 39). A decrease in total horizontal activity was also observed during the first 10 min in the habituation portion on day 40 of the sensitization protocol (Fig. 2C; Student’s T-test: t = 2.152, df = 18, p < 0.0453). This difference was not observed on subsequent test days (days 44, 52, and 62).
Open field activity of prepubertal rats exposed to nandrolone. Prepubertal males were injected daily with nandrolone (20 mg/kg) or sesame oil from PN 28–37. On days 39 and 40, rats were tested for open-field behavior (see Fig. 1A). Rats were placed in the activity cage, and the time spent in the center of the field, as well as the distance traveled, was recorded for 10 min using the Accuscan Versamax monitoring system. Rats treated with nandrolone (20 mg/kg) spent more time in the center of the open field (A) and traveled less distance on days 0 (B) and 1 (C) than oil-treated rats. Data are presented as mean ± SEM (n = 10) and were analyzed with a Student’s t-test. Asterisks represent a significant difference compared to the oil group.
Elevated plus maze
Nandrolone administration decreased the time spent in the closed arms of the EPM compared to rats that received oil (Fig. 3A: Student T-test: t = 2.623, df = 18, p = 0.0173). Additionally, nandrolone decreased the total number of arms entered, which is considered an indication of decreased locomotor activity (Fig. 3B: Student’s T-test: t = 3.28, df = 18, p = 0.0041).
Results from the EPM test of prepubertal rats treated with nandrolone. Male rats were injected daily with nandrolone (20 mg/kg) or sesame oil from PN 28–37 and tested in the elevated plus maze on day 38. Rats were placed at the junction between the open and closed arms, and behavioral activity was recorded for 5 min using the ANY-MazeÔ tracking software. Rats treated with nandrolone spent more time in the open arms (A), an indication of decreased anxiety. In addition, rats that received nandrolone had fewer total entries than oil-treated rats (B), an indication of reduced ambulation. Data are presented as mean ± SEM (n = 10) and analyzed with a Student t-test. Asterisks represent a significant difference compared to oil-treated rats.
Behavioral sensitization
To assess the influence of nandrolone on cocaine-induced behavioral sensitization, a mixed-effects analysis was conducted using average activity levels from 30 to 60 min across repeated exposure days. Cocaine significantly increased locomotor activity compared to controls (β = 1156.97, p = 0.004), with a progressive enhancement across days, as shown by significant interactions on Day 13 (β = 1650.71, p = 0.004) and Day 23 (β = 2698.76, p < 0.001), indicating robust sensitization (see Table 1 of supplementary material).
Nandrolone alone did not significantly affect activity (β = − 142.06, p = 0.657), but a significant three-way interaction with cocaine and Day 5 (β = 2003.57, p = 0.013) showed that nandrolone enhanced cocaine-induced activity early in the exposure period, i.e. they displayed behavioral sensitization (Fig. 4D and E) (see Table 1 of supplementary material). This effect was not significant on later days (Day 13: p = 0.399; Day 23: p = 0.899), suggesting that nandrolone may accelerate the onset of sensitization to cocaine without affecting its long-term magnitude. Of the 12 cocaine-induced locomotor activity time points measured during the 60 min after injection, 8 were higher in rats who received nandrolone-cocaine (Fig. 4C vs. 4D). This pattern was maintained on day 52: 10 of 12 cocaine-induced locomotor activity time points for rats treated with nandrolone-cocaine were higher than those on day 40 (Fig. 4D).
Cocaine-induced locomotor activity of rats exposed to nandrolone during postnatal days 28–37. Cocaine-induced locomotor activity of prepubertal rats injected daily from PN 28–37 with nandrolone (20 mg/kg) or sesame oil and tested for behavioral sensitization to cocaine from PN 40–62. A and B Timecourse of locomotor activity of saline (A) and nandrolone (B) treated males. No differences between oil and nandrolone-treated males were observed. C. A significant increase in cocaine-induced locomotor activity was observed when comparing the timecourses of day 40 vs. day 52 and day 40 vs. day 62 in Oil-treated males. Thus, these males displayed behavioral sensitization by day 52. D. A significant increase in cocaine-induced locomotor activity was observed when comparing the timecourses of day 40 vs. day 44 vs. day 52 and day 62 in nandrolone-treated males. These results suggest that nandrolone accelerates the maturation of the neural circuitry that regulates behavioral sensitization. Data are presented as mean ± SEM and analyzed with Repeated Measures ANOVA using Tukey’s multiple comparison for post-hoc analysis. E. Rats that received nandrolone show a robust increase in cocaine-induced locomotor activity by day 44, an increase that is maintained after two withdrawal periods. In contrast, it is at day 52 that oil-treated rats show a difference that is further increased by day 62. Data are presented as mean ± SEM (n = 10). Repeated Measures Two-Way ANOVA: F(3,27) = 4.173, p = 0.0150. Oil-Coc: Day 40 vs. Day 44, p = 0.9968; Day 40 vs. Day 52, p = 0.0288; Day 40 vs. Day 62, p = 0.0001. ND-Coc: Day 40 vs. Day 44, p = 0.0011; Day 40 vs. Day 52, p = < 0.0001; Day 40 vs. Day 62, p = 0.0002. Oil-Coc vs. ND- Coc: Day 40, p = 0.8684; Day 44, p = 0.0002; Day 52, p = 0.0230; Day 62 p = 0.9209. P < 0.05 was considered statistically significant on Day 40 vs. Day 44, Day 52, and Day 62. (*): significantly different within groups; (#): significantly different between groups (Oil-Cocaine vs. Nandrolone-Cocaine).
In comparison, oil-cocaine-treated rats did not show differences in cocaine-induced locomotor activity between days 40 and 44 in any of the 12 measured time points and showed differences in only 5 of the 12 time points comparing day 40 with day 52 (Fig. 4C), reiterating that rats treated with nandrolone-cocaine displayed behavioral sensitization earlier (day 44) than oil-cocaine treated rats (day 52) (Fig. 4D: Two-way RM ANOVA, days, F (2.45, 22.08) = 13.03; p < 0.0001). No significant differences were observed between days in oil-saline and nandrolone-saline groups (Fig. 4A-B). Similar results were obtained with stereotyped activity (Fig. 5A-E: Two-way RM ANOVA, Days, F(2.58, 23.23) = 10.03, p = 0.0003). Additionally, the cocaine-induced locomotor activity of the rats treated with nandrolone was greater than that of the rats treated with oil at 9 of the 12 time points measured on day 44.
Cocaine-induced stereotyped activity of rats exposed to nandrolone during postnatal days 28–37. Cocaine-induced stereotyped activity of prepubertal rats injected daily from PN 28–37 with nandrolone (20 mg/kg) or sesame oil and tested for behavioral sensitization to cocaine from PN 40–62. A and B. Timecourse of stereotyped activity of saline (A) and nandrolone (B) treated males. No differences between oil and nandrolone-treated males were observed. C. A significant increase in cocaine-induced stereotyped activity was observed when comparing the timecourses of day 40 vs. day 52 and day 40 vs. day 62 in Oil-treated males. Thus, saline males displayed behavioral sensitization by day 52. D. A significant increase in cocaine-induced locomotor activity was observed when comparing the timecourses of day 40 vs. day 44 vs. day 52 and day 62 in nandrolone-treated males. As we observed with total horizontal activity, these results indicate that nandrolone accelerates the development of behavioral sensitization. Data are presented as mean ± SEM (n = 10) and analyzed with Repeated Measures of Two-Way ANOVA using Tukey’s multiple comparisons for post hoc analysis. E. Rats that received nandrolone show a robust increase in cocaine-induced stereotyped activity by day 44, an increase that is maintained after two withdrawal periods. In contrast, it is at day 52 that oil-treated rats show a difference that is further increased by day 62. Data are presented as mean ± SEM. Repeated Measures Two-Way ANOVA: F(3,27) = 6.746, p = 0.0015. Oil-Coc: Day 40 vs. Day 44, p = 0.5042; Day 40 vs. Day 52, p = 0.015; Day 40 vs. Day 62, p < 0.0001. ND-Coc: Day 40 vs. Day 44, p < 0.0001; Day 40 vs. 52, p = < 0.0001; Day 40 vs. Day 62, p < 0.0002. Oil-Coc vs. ND- Coc: Day 40, p = 0.9224; Day 44, p < 0.0001; Day 52, p = 0.0281; Day 62 p = 0.9999. P < 0.05 was considered statistically significant on Day 40 vs. Day 44, Day 52, and Day 62. (*): significantly different within groups; (#): significantly different between groups (Oil-Cocaine vs. Nandrolone-Cocaine).
Western blot analysis of D2DR in mPFC (sensitization brains)
Our results indicate that cocaine decreased D2DR in mPFC (Fig. 6A and C). This effect was not altered by preexposure to nandrolone (Fig. 6A and C): One-way ANOVA, Tukey multiple comparisons, F (3, 12) = 110.5, < 0.0001; Oil-Sal vs. ND-Sal, p = 0.1559; Oil-Sal vs. Oil-Coc, p < 0.0001; Oil-Sal vs. ND-Coc, p < 0.0001; ND-Sal vs. Oil-Coc, p < 0.0001; ND-Sal vs. ND-Coc, p < 0.0001; Oil-Coc vs. ND-Coc, p = 0.9909.
D2DR in the mPFC and NAc of rats exposed to nandrolone during postnatal days 28–37 and tested for behavioral sensitization. A and C: Nandrolone did not affect D2DR expression in the mPFC. However, a decrease in D2DR expression in the mPFC was observed in animals treated with cocaine. B and D: In contrast, nandrolone treatment increased D2DR expression in the NAc and, similar to what was observed in the PFC, cocaine reduced D2DR in the NAc. Data are presented as mean ± SEM (n = 4). Representative western blots of D2DR and B-Actin in the mPFC (A) and NAc (B) of rats exposed to nandrolone during postnatal days 28–37, tested for behavioral sensitization and sacrificed on PN 63. Data were analyzed using One-Way ANOVA. Western blots images were cropped for publishing purposes; replicates are included as Supplementary Material.
Western blot analysis of D2DR in NAc (sensitization brains)
Rats treated with nandrolone showed an increase in D2DR expression in NAc (Fig. 6B and D). Cocaine treatment decreased the expression of D2DR in this brain area; this decrease was more pronounced in animals that received nandrolone (Fig. 6B and D: One-way ANOVA, Tukey’s multiple comparisons, F(3, 12) = 84.90, < 0.0001; Oil-Sal vs. ND-Sal, p = 0.0002; Oil-Sal vs. Oil-Coc, p = 0.0339; Oil-Sal vs. ND-Coc, p < 0.0001; ND-Sal vs. Oil-Coc, p < 0.0001; ND-Sal vs. ND-Coc, p < 0.0001; Oil-Coc vs. ND-Coc, p = 0.0002.
Conditioned place preference
All animals showed CPP to cocaine. However, nandrolone decreased the time spent in the chamber associated with cocaine (Fig. 7). Rats treated with oil-cocaine showed more robust CPP to cocaine than rats treated with nandrolone-cocaine (Fig. 7). During postconditioning, oil-treated rats spent 70% of their time on the side associated with cocaine vs. 31% during the preconditioning phase. On the contrary, rats treated with nandrolone spent 48% of their time during post-conditioning in the chamber associated with the drug versus 30% during preconditioning. Data were analyzed with a two-way ANOVA, with pre- and post-conditioning as repeated measures = F(1, 62) = 28.22, p < 0.0001, and cocaine as independent factor. Cocaine effect: F(3,62) = 21.97, p < 0.0001; Oil-Coc pre vs. postconditioning, p < 0.0001; ND-Coc pre vs. postconditioning, p = 0.0397; Oil-Coc post vs. ND-Coc postconditioning, p = 0.0035.
Conditioned Place Preference to cocaine of rats exposed to nandrolone during postnatal days 28–37. Male rats were injected daily from PN 28–37 with nandrolone (20 mg/kg) or sesame oil and tested for CPP to cocaine from PN 40–53. Nandrolone-treated males showed a decrease in the time spent in the chamber associated with cocaine compared to oil-treated males. During the postconditioning test, nandrolone-treated rats spent 48% of their time in the chamber associated with cocaine compared to 70% spent by oil-treated males. Rats injected with saline did not show a change in the time spent in the chamber where they were injected with saline. Although oil and nandrolone-treated males were conditioned to cocaine, conditioning was more robust in Oil-treated males. Data are presented as mean ± SEM (n = 8) and analyzed by a Two Way ANOVA (See supplementary material for detailed statistical analysis).
Western blot analysis for D2DR in mPFC (CPP brains)
Rats that received cocaine had decreased D2DR in PFC compared to saline-treated rats (Fig. 8A and C). However, the brains of the males treated with nandrolone used in the CPP experiments had more D2DR in the PFC than the oil-treated rats (Fig. 8A and C). The difference between these two experiments is that the animals used for the CPP experiments were killed on day 53, and those used for the sensitization experiments were sacrificed 11 days later, on day 64. Additionally, for the sensitization studies, rats received 7 cocaine injections (days 40–44, day 52, and day 62), while, for the CPP experiments, rats received 5 cocaine injections (days 43, 45, 47, 49, and 51). Figure 8C: One-way ANOVA, Tukey multiple comparisons, F(3, 12) = 28.78, < 0.0001; Oil-Sal vs. ND-Sal, p = 0.1752; Oil-Sal vs. ND-Coc, p = 0.0013; Oil-Sal vs. ND-Coc, p = 0.0007; ND-Sal vs. Oil-Coc, p < 0.0001; ND-Sal vs. ND-Coc, p < 0.0001; Oil-Coc vs. ND-Coc, p = 0.9825.
D2DR in the mPFC and NAc of rats exposed to nandrolone during postnatal days 28–37 and tested for CPP. Representative western blots of D2DR and B-Actin in the mPFC (A) and NAc (B) of rats exposed to nandrolone during postnatal days 28–37, tested for CPP and sacrificed on PN 53. B and D: Nandrolone increased D2DR expression in the mPFC and NAc of prepubertal rats. Fig. 6B and D: In contrast, cocaine decreased D2DR expression in both brain areas (mPFC and NAc), similar to what was observed in brains obtained from animals that were tested for behavioral sensitization and killed at a later age, day 64 (Fig. 6B and D). Data are presented as mean ± SEM (n = 4) and analyzed using One-Way ANOVA. Western blots images were cropped for publishing purposes; replicates are included as Supplementary Material.
Western blot analysis for D2DR in NAc (CPP brains)
The results obtained from the brains of animals sensitized to cocaine (Fig. 8B and D) are similar to those obtained with rats that were conditioned to cocaine; rats treated with nandrolone showed increased expression of D2DR in NAc. In contrast, cocaine treatment decreased the expression of D2DR in this area of the brain. Figure 8D: One-way ANOVA, Tukey’s multiple comparisons, F (3, 12) = 45.87, < 0.0001; Oil-Sal vs. ND-Sal, p = 0.0316; Oil-Sal vs. Oil-Coc, p < 0.0001; Oil-Sal vs. ND-Coc, p = 0.0001; ND-Sal vs. Oil-Coc, p < 0.0001; ND-Sal vs. ND-Coc, p < 0.0001; Oil-Coc vs. ND-Coc, p = 0.6828.
Discussion
Summary of results
Prepubertal males treated with nandrolone (PN 28–37) showed reduced anxiety and basal locomotor activity compared to oil-treated males. In contrast, nandrolone increased risk-taking behavior, augmented the locomotor response to cocaine after 5 injections, and accelerated the development of cocaine sensitization in young animals (i.e., they required less cocaine injections than oil-treated males). Nandrolone also attenuated the CPP to cocaine. Changes in D2DR in NAc may partially mediate these behavioral changes: an increase in D2DR was observed in the NAc of males treated with nandrolone, while those treated with cocaine or with nandrolone and cocaine had less D2DR in NAc and PFC.
Anxiety and risk-taking behaviors
The use of AAS is associated with adverse effects on emotion, cognition, and rewarding behaviors68. Most of the effects of AAS on anxiety appear after cessation of use, which may be at the end of an AAS cycle or after longer withdrawal periods69. Additionally, people with symptoms of depression or withdrawal are more likely to revert to the use anabolic steroids or other drugs of abuse70.
Children born to mothers with polycystic ovarian syndrome, which results in increased androgen exposure during fetal development, have a higher prevalence of psychiatric disorders, such as depression and anxiety71. Additional studies in rodents confirm that exposure to excessive androgens during prenatal development increases the number of offspring that subsequently display anxiety behaviors72. In addition, several psychiatric disorders like anxiety, depression, mood disorders, psychosis, and substance abuse tend to manifest themselves close to puberty73.
Very few studies have examined the effects of AAS administered prepubertally on emotional behaviors. Our study found that preexposure to nandrolone decreased anxiety-like behaviors. Nandrolone-treated rats showed a decrease in the time spent in the closed arms of an EPM and an increase in the time spent in the center of an open field compared to oil-treated rats. An increase in time spent in the center of an open field is also associated with increased risk-taking behavior, as rats that venture more into the center of an open field are at greater risk of being detected by predators.
Previous studies show that the administration of nandrolone (15 mg/kg) to adult rats decreased anxiety, as measured in the EPM74. Decreased anxiety following androgen administration has also been reported by others75,76,77,78. However, it is not unequivocally established whether AAS have anxiolytic properties since several studies with adult male rats79 or with adolescent rats tested as adults80,81 report that AAS exert anxiogenic effects.
The difference between studies investigating the effect of AAS on anxiety can vary depending on the type of androgen administered, the age of exposure, the duration of treatment, and the dose administered. These last studies used mainly an aromatizable form of androgen, and although the doses used were smaller (5 mg/kg vs. 20 mg/kg), as well as the total dose administered (60, 150, or 175 mg/kg vs. 200 mg/kg) the duration of treatment was longer (30–35 days vs. 10 days). Additionally, our current study is unique in that nandrolone was administered prior to puberty (PN 28–37), and the effects on anxiety and risk-taking behaviors were evaluated the day after the last injection (days 38 and 39) and not after a withdrawal period.
A correlation between high-risk behaviors in adolescents and testosterone has also been established. For example, a positive correlation was found between salivary testosterone and nucleus accumbens activation in response to a monetary reward in adolescent boys aged 10 to 1682. Not only do testosterone levels correlate positively with receiving a financial reward, but AAS users also engage more frequently in other risky behaviors, such as gambling.
Locomotor activity
Rats treated with nandrolone showed a decrease in the distance traveled in an open field and a reduction in the total number of arm entries when tested in an EPM, indicative of lower ambulation. Lower locomotor activity of males treated with nandrolone was also evident during the first 10 min of habituation (day 39) and on day 40 of the sensitization trial (Fig. 3C). This effect of nandrolone on locomotor activity appears to be related to novelty-induced exploration since it was not observed after further testing (days 44, 52 and 62).
Most studies agree that androgens decrease ambulation. In a similar study, prepubertal males treated with nandrolone (15 mg/kg) for 30 days showed reduced locomotor activity in an open field and EPM83. In adults, nandrolone is reported to decrease activity in the running wheel84in an open field79,85 and in the EPM86. Nonetheless, there are some studies that fail to find an effect87 and others that report a decrease in cocaine-induced locomotor activity88 after nandrolone withdrawal.
The mechanism by which nandrolone decreases locomotor activity is unclear. It has previously been reported that testosterone administration to prepubertal rats modulates the expression of enzymes that participate in dopaminergic metabolism in brain regions, such as the substantia nigra, that regulate locomotion and affect89. Others report that the addictive properties of AAS may be exerted through the endogenous opioid system that in turn stimulate dopaminergic centers in the brain90.
AAS and cocaine
Several studies indicate that AAS users are more likely to develop a dependency on other drugs of abuse7,91. These results must be interpreted with caution since, in some cases, AAS users abused other drugs before using AAS. In addition, studies in adult rodents investigating the role of androgens in modulating the response to drugs of abuse have provided conflicting reports.
Prior studies from our laboratory and of others indicate that removal of the primary source of androgens by gonadectomy increases the response to psychostimulants36,92,93,94,95although not all studies have obtained similar results96,97,98,99,100,101. Furthermore, testosterone administration to adult males is reported to decrease the acute locomotor response to cocaine and amphetamine in gonadectomized36,95 and gonadally intact drug-naive males92,102. Indeed, our laboratory has found that testosterone is necessary for adult male rats to develop and express sensitization to cocaine36.
Previous studies indicate that prepubertal male rats do not become sensitized to cocaine at this early age103,104,105. In fact, our control rats did not show sensitization until after a withdrawal period and re-exposure to cocaine. Oil-cocaine rats showed sensitization when they were 52 days of age. At this time, they had received 5 daily cocaine injections, a 7-day withdrawal period, and a cocaine challenge. Sensitization in these oil-treated animals became more robust after a second withdrawal period and re-exposure to cocaine when they were 62 days of age. On the contrary, we found that exposure to nandrolone during days 28–37 accelerated the development of sensitization, which was evident after 5 cocaine injections, when they were 44 days of age.
We also observed that cocaine-induced hyperactivity was maintained for a longer period of time in nandrolone-treated males (day 52). It is not clear whether this effect can be attributed to changes in cocaine metabolism. Cocaine and nandrolone are metabolized mainly in the liver, cocaine by esterases and cytochrome P450 enzymes, while nandrolone metabolism occurs mainly by 5α-reductase and 3α- and 3β-hydroxysteroid dehydrogenase enzymes. Therefore, it is not surprising that pharmacokinetic interactions have been observed between the compounds. Synergistic effects on the cardiovascular system106 and seizures107as well as altered dopaminergic and serotonergic outflow88have been reported. Unfortunately, although areas of the mesocorticolimbic pathway are rich in androgen receptors108very few studies have investigated if androgens alter the metabolism of cocaine109,110. Another possibility is that exposure to androgens during the prepubertal period accelerates the maturation of some components of the mesocorticolimbic circuitry essential for the display of behavioral sensitization. For example, the cytochrome P450 system is involved in the metabolism of androgens and cocaine111 therefore, both drugs can interact pharmacokinetically and affect the individual response of each drug. Furthermore, treatment with the antiandrogen flutamide decreases plasma levels of cocaine and its main metabolites, suggesting that testosterone may enhance the effects of cocaine112as we have previously reported36.
Our data also indicate that prior exposure to AAS increased the behavioral response to cocaine, a process known as cross-sensitization. Cross-sensitization occurs with previous exposure between the same drug and between different drugs of abuse113. The mechanisms that mediate cross-sensitization are not fully understood. Evidence suggests that dopamine is the substrate involved in this process114. Studies found that androgens like testosterone induce cross-sensitization to cocaine in prepubertal but not in adult male rats35.
One scenario may be that AAS promotes cross-sensitization by activating corticotropin-releasing hormone (CRH) gene expression. The gene that codes for CRH contains androgen and estrogen response elements that modulate expression of CRH115,116. Indeed, we have recently reported that altered corticotropin-releasing hormone receptor 1 (CRF-R1) sensitivity may lead to the observed DA hyperresponsiveness observed in socially isolated adolescent rats117. In addition, increases in dopamine, particularly in the medio-striatal brain region, is implicated in processing reward value, as well in mediating stereotyped behavior that results from psychostimulant administration118.
CPP
All rats tested between days 40 and 53 days developed CPP to cocaine. In addition, nandrolone-cocaine treated rats spent less time in the cocaine-associated chamber during the postconditioning day compared to oil-cocaine treated rats. The increase in time spent in the chamber associated with cocaine was 124% in oil-cocaine rats compared to 57% in those pretreated with nandrolone. Thus, although both groups of rats showed CPP to cocaine, CPP was lower in rats that previously received nandrolone.
Our studies agree with those of others that report a decrease in CPP to other drugs of abuse, such as cannabinoids119 and opioids120after AAS administration. Furthermore, exposure to nandrolone (15 mg/kg) during PN 40–53 decreased sucrose consumption 15 days after withdrawal77. However, there are studies that indicate that withdrawal from nandrolone increases cannabinoids121 and alcohol122 intake. Therefore, there is no consensus on whether prior exposure to nandrolone results in an enhancement or an aversion to other drugs of abuse or rewarding stimuli such as sucrose. However, there is evidence that the addictive properties of AAS can be exerted through the endogenous opioid system, which in turn stimulates the dopaminergic centers of the brain90.
The CPP paradigm involves several cognition components, such as acquisition, retrieval, and extinction of spatial and contextual memories123,124,125. Several lines of evidence link the use of AAS with cognitive dysfunction126 and altered decision-making when studied in paradigms such as the operant discounting task126,127,128,129.
We cannot discard the possibility that during the CPP test, the reduced time spent in the chamber associated with cocaine is due to a decrease in motivation. However, the increased sensitized locomotor response to cocaine displayed on day 44 by animals that received nandrolone evokes uncertainty about this possibility. Sensitization has been defined as the successive increase in locomotor hyperactivity elicited by repeated administration of psychostimulants. It involves neuroadaptations in the mesocorticolimbic system that contribute to changes in the motivational circuitry underlying craving and relapse130,131,132. Since many investigators relate sensitization to increased motivation, and animals treated with nandrolone showed increased sensitization, we are inclined to favor changes in the cognitive and non-rewarding aspects of CPP17.
The effects of testosterone on cognition ranges from neuroprotective to inducing severe executive dysfunction depending on the dose134. Very high or very low plasma concentrations of testosterone (like those found at the tails of a normal distribution curve) are associated with impaired cognitive function126,135,136,137. For example, patients with prostate cancer treated with androgen deprivation therapy136 or elderly men with low testosterone137perform poorer on visuomotor tasks. Similarly, men that have abused AAS for several years show deficits when tested on several cognitive tasks, particularly those related to visuospatial memory138. Data in animals yield similar results. Performance on the Morris Water Maze, a hippocampal-dependent spatial learning and memory test is impaired in AAS-treated rats135,139.
Additionally, the rewarding properties of AAS are evident in previous studies that find that rodents will self-administer AAS orally and intracranially17,19an effect that disappears with the administration of an androgen receptor antagonist133.
AAS and D2DR
After the conclusion of our behavioral studies, we investigated whether the D2DR receptors in NAc and mPFC were affected by prior treatment with nandrolone, cocaine, or both. Separate groups of animals were used for the above-mentioned experiments. The rats used for CPP received 5 injections of cocaine (15 mg/kg), one every other day for 10 days, and were killed 24 h after the last injection of cocaine. Rats used for the sensitization experiments received 7 cocaine injections (15 mg/kg) during a 23-day period: 5 daily injections, a 7-day withdrawal period, 1 challenge injection, a 9-day withdrawal period, a second challenge injection, and were euthanized 24 h after the last cocaine injection. Thus, the sensitization group received two additional cocaine injections and underwent two drug-free periods of 7 and 9 days prior to euthanasia.
D2DR in the NAc of rats used for the sensitization experiments
Rats treated with nandrolone (days 28–37) had a higher concentration of D2DR in the NAc on the day of euthanasia (days 54 and 63) compared to oil-saline rats. In contrast, all the groups that received cocaine had lower levels of D2DR compared to oil saline rats. Interestingly, groups that were injected with nandrolone (days 28–37) and used for sensitization experiments (euthanized on day 63) had the lowest levels of D2DR in the NAc of all groups.
Behavioral sensitization has two phases: initiation and expression. The initiation comprises rapid neural effects that induce behavioral sensitization; the expression has long-term consequences114. Initiation of behavioral sensitization does not require activation of dopamine receptors140however, its expression does62,141,142. Most studies agree that the NAc, although not essential for the development of locomotor sensitization to cocaine, is necessary for its expression143. Unfortunately, the role of each dopaminergic receptor subtype in the process of sensitization is still not entirely clear.
Some studies report that D2DR in the NAc does not affect cocaine-induced locomotor activity or behavioral sensitization41,62,144. However, other studies attest to D2DR modulation of cocaine-induced sensitization143. For example, blocking D2DR receptors in the NAc significantly decreased cocaine-induced locomotor activity145,146,147 and abolished cocaine-induced sensitization146. Furthermore, deletion of D2DR in medium spiny neurons (MSN) of the NAc in mice results in decreased cocaine-induced locomotor activity113. In contrast, experiments using the conditional mutant mice “autodrd2KO”, which is characterized by a lack of D2DR auto receptors (those in dopamine neurons and terminals), indicate that sensitivity to cocaine is enhanced in these animals45. Mice lacking D2DR auto receptors also show enhanced CPP as well as hyperlocomotion (without altering dopamine transporters function)45.
These data argue that the process by which D2DRs in the NAc modulate the response to cocaine can vary, depending on whether these receptors are in dopaminergic terminals or GABAergic MSN in the NAc. This may partially explain several conflicting reports. It is possible that the observed decrease in D2DR in the NAc in the current study occurred mainly in dopaminergic terminals. Repeated exposure to cocaine during sensitization would transiently result in increased dopamine (DA), which, in turn, would induce down-regulation of D2DR. D2DRs have a higher affinity for DA than D1DR148,149. This would result in less dopaminergic autoinhibition and greater, or extended, DA release, resulting in an exacerbated locomotor response to cocaine, as we observed in the current study. It is also possible that DA, by modulating AMPA trafficking, contributes to the enhancement of sensitization150. However, more studies are needed to determine the cell type or terminal where the decrease in D2DR occurred.
Additionally, nandrolone treatment increased D2DR in NAc. This group of animals showed the highest locomotor activity in response to cocaine and developed sensitization earlier than oil-treated rats (after 5 cocaine injections). These data are in agreement with previous studies that show that D2DR availability predicts future drug seeking behavior99.
Androgens modulate D2DR
Androgens can modulate dopamine receptors in the mesocorticolimbic circuitry89,151,152,153. Previous research found that administering nandrolone for two weeks at low doses (1 and 5 mg/kg) increased D2DR in the NAc core and shell of male rats but found that a higher dose (15 mg/kg) had no effect154. The authors of this last study did not state the age or weight of the rats. In our study, the rats were 28 days old when they received the first injection of nandrolone, and the dose used was 20 mg/kg. Thus, we cannot determine whether the difference between these two studies is dose-related or age-related.
Nandrolone can also alter DA metabolism155. A decrease in the activity of the dopamine-metabolizing enzymes monoamine oxidase A and B has been reported after nandrolone treatment156,157. Furthermore, levels of tyrosine hydroxylase in the substantia nigra increase prior to puberty, coinciding with the increase in testosterone89.
The Akil group, among others, have selectively bred rats according to their initial response to a novel environment and classified them as high responders (HR) and low responders (LR)158,159,160,161. These two lines also show differences in behavioral traits relevant to addiction, with HR displaying a greater amount of drug taken, persistence of drug-seeking, and drug-induced locomotor activity162. They also differ in the neural substrates that regulate addictive behaviors. Like the males treated with nandrolone in this study, HRs have higher D2DR in the NAc core compared to LR158. HRs also have higher levels of fibroblast growth factor 2 (FGF2) and lower D1DR levels in NAc. Interestingly, previous studies show that testosterone increases plasma levels of FGF2 and of Insulin Growth Factor163 and mRNA expression of FGF2 in vitro.164 Dysregulation of neurotrophic factors like FGF2 is involved in increased vulnerability to drugs132,136. Indeed, drugs such as amphetamines and cocaine modulate the expression of FGF2161,165. This may explain the synergistic effect of nandrolone and cocaine in decreasing D2DR in the NAc.
Prefrontal cortex
The mPFC plays an important role in addictive behaviors such as decision-making, memory retrieval, and cocaine-seeking behaviors and is necessary for the induction of sensitization to cocaine166,167,168,169. It contains a distinct population of glutamatergic pyramidal neurons that project to the striatum and other subcortical regions that express D1DR and D2DR170. Dopaminergic modulation of glutamatergic function contributes to reward, salience, attention, and working memory171,172,173,174 Recent evidence indicates that DA modulates ensemble activity, facilitating and strengthening information processing in the PFC173.
The maturation of the PFC circuitry continues after puberty, and dopaminergic innervation from the VTA to the PFC increases gradually until day 60175. At approximately this age, males have acquired adult testosterone plasma levels and the ability to display male sexual behavior176. An increase in dendritic spine density after androgen or estrogen administration suggests that gonadal steroids play a role in PFC function177. Interestingly, on day 40, an increase in D2DR receptors in the PFC is observed, coinciding with the activation of the HPG axis in male rats178. It is possible that in our current study, nandrolone administration from day 28 to 37 accelerated the maturation of the motivation circuitry in the PFC, which in turn could be responsible for the expression of sensitization at a younger age.
Data availability
All relevant data concerning the study are contained within this paper.
References
Basaria, S., Wahlstrom, J. T. & Dobs, A. S. Clinical review 138: Anabolic-androgenic steroid therapy in the treatment of chronic diseases. J. Clin. Endocrinol. Metab. 86, 5108–5117 (2001).
Kochakian, C. D. & Welder, A. A. Anabolic-androgenic steroids: in cell culture. Vitr Cell. Dev. Biol. - Anim. 29, 433–438 (1993).
Bahrke, M. S., Yesalis, C. E. & Wright, J. E. Psychological and behavioural effects of endogenous testosterone and anabolic-Androgenic steroids. An update. Sport Med. 22, 367–390 (1996).
Denham, B. E. Effects of mass communication on attitudes toward anabolic steroids: An analysis of high school seniors. J. Drug Issues. 36, 809–829 (2006).
Kindlundh, A. M. S., Isacson, D. G. L., Berglund, L. & Nyberg, F. Factors associated with adolescent use of doping agents: anabolic-androgenic steroids. Addiction 94, 543–553 (1999).
Metastasio, A., Negri, A., Martinotti, G. & Corazza, O. Transitioning bodies. The case of self-prescribing sexual hormones in gender affirmation in individuals attending psychiatric services. Brain Sci. 8, 1–10 (2018).
Pope, H. G. et al. The lifetime prevalence of anabolic-androgenic steroid use and dependence in americans: current best estimates. Am. J. Addict. 23, 371–377 (2014).
Sagoe, D., Molde, H., Andreassen, C. S., Torsheim, T. & Pallesen, S. The global epidemiology of anabolic-androgenic steroid use: A meta-analysis and meta-regression analysis. Ann. Epidemiol. 24, 383–398 (2014).
Brower, K., Eliopulos, A., Catlin, H., Beresford, P. & Blow, C. Evidence for physical and psychological dependence on anabolic androgenic steroids in eight weight lifters. Am. J. Psychiatry. 147, 510–512 (1990).
Parkinson, A. B. & Evans, N. A. Anabolic androgenic steroids: A survey of 500 users. Med. Sci. Sports Exerc. 38, 644–651 (2006).
van Amsterdam, J., Opperhuizen, A. & Hartgens, F. Adverse health effects of anabolic-androgenic steroids. Regul. Toxicol. Pharmacol. 57, 117–123 (2010).
Pope, H. G. et al. Adverse health consequences of performance-enhancing drugs: an endocrine society scientific statement. Endocr. Rev. 35, 341–375 (2014).
Oberlander, J. G. & Henderson, L. P. The Sturm und Drang of anabolic steroid use: angst, anxiety, and aggression. Trends Neurosci. 35, 382–392 (2012).
Onakomaiya, M. M. & Henderson, L. P. Mad men, women and steroid cocktails: A review of the impact of sex and other factors on anabolic androgenic steroids effects on affective behaviors. Psychopharmacol. (Berl). 233, 549–569 (2016).
Pagonis, T. A., Angelopoulos, N. V., Koukoulis, G. N. & Hadjichristodoulou, C. S. Psychiatric side effects induced by supraphysiological doses of combinations of anabolic steroids correlate to the severity of abuse. Eur. Psychiatry. 21, 551–562 (2006).
Pope, H. G. & Katz, D. L. Affective and psychotic symptoms associated with anabolic steroid use. Am. J. Psychiatry. 145, 487–490 (1988).
Johnson, L. R. & Wood, R. I. Oral testosterone self-administration in male hamsters. Neuroendocrinology 73, 285–292 (2001).
Wood, R. I. Oral testosterone self-administration in male hamsters: Dose-response, voluntary exercise, and individual differences. Horm. Behav. 41, 247–258 (2002).
Wood, R. I. Reinforcing aspects of androgens. Physiol. Behav. 83, 279–289 (2004).
Brower, K. J., Blow, F. C., Beresford, T. P. & Fuelling, C. Anabolic-androgenic steroid dependence. J. Clin. Psychiatry. 50, 31–33 (1989).
Busardo, F. et al. The impact of nandrolone decanoate on the central nervous system. Curr. Neuropharmacol. 13, 122–131 (2015).
Scarth, M., Havnes, I. A. & Bjørnebekk, A. Anabolic-androgenic steroid use disorder: case for recognition as a substance use disorder with specific diagnostic criteria. Br. J. Psychiatry 1–5. https://doi.org/10.1192/bjp.2025.73 (2025).
Kanayama, G., Hudson, J. I. & Pope, H. G. Long-term psychiatric and medical consequences of anabolic-androgenic steroid abuse: A looming public health concern? Drug Alcohol Depend. 98, 1–12 (2008).
Penatti, C. A. A., Oberlander, J. G., Davis, M. C., Porter, D. M. & Henderson, L. P. Chronic exposure to anabolic androgenic steroids alters activity and synaptic function in neuroendocrine control regions of the female mouse. Neuropharmacology 61, 653–664 (2011).
DuRant, R. H., Rickert, V. I., Ashworth, C. S., Newman, C. & Slavens, G. Use of multiple drugs among adolescents who use anabolic steroids. N Engl. J. Med. 328, 922–926 (1993).
Kindlundh, A., Lindblom, J., Bergström, L., Wikberg, J. & Nyberg, F. The anabolic-androgenic steroid nandrolone decanoate affects the density of dopamine receptors in the male rat brain. Eur. J. Neurosci. 13, 291–296 (2001).
Lood, Y., Eklund, A., Garle, M. & Ahlner, J. Anabolic androgenic steroids in Police cases in Sweden 1999–2009. Forensic Sci. Int. 219, 199–204 (2012).
Saartok, T., Dahlberg, E. & Gustafsson, J. Å. Relative binding affinity of anabolic-androgenic steroids: comparison of the binding to the androgen receptors in skeletal muscle and in prostate, as well as to sex hormone-binding Globulin. Endocrinology 114, 2100–2106 (1984).
Bergink, E. W., Janssen, P. S. L., Turpun, E. W. & Van Der Vies, J. Comparison of the receptor binding properties of nandrolone and testosterone under in vitro and in vivo conditions. J. Steroid Biochem. 22, 831–836 (1985).
Kicman, A. T. Pharmacology of anabolic steroids. Br. J. Pharmacol. 154, 502–521 (2008).
Tenniswood, M., Bird, C. E. & Clark, A. F. The role of androgen metabolism in the control of androgen action in the rat prostate. Mol. Cell. Endocrinol. 27, 89–96 (1982).
van der Vies, J. Implications of basic Pharmacology in the therapy with esters of nandrolone. Acta Endocrinol. (Copenh). 110, S38–S44 (1985).
Substance Abuse and Mental Health Services Administration (SAMHSA). Key substance use and mental health indicators in the United States: Results from the 2023 National Survey on Drug Use and Health. HHS Publ. No. PEP23-07-01-006, NSDUH Ser. H-58, 1–75. https://www.samhsa.gov/data/ (2023).
Conceic, C. Q. et al. Behavioral cross-sensitization between testosterone and Fenproporex in adolescent and adult rats. Brazilian J. Med. Biol. Res. 51, 5–9 (2018).
Engi, S. A., Cruz, F. C., Crestani, C. C. & Planeta, C. S. Cross-sensitization between testosterone and cocaine in adolescent and adult rats. Int. J. Dev. Neurosci. 46, 33–37 (2015).
Menéndez-Delmestre, R. & Segarra, A. C. Testosterone is essential for cocaine sensitization in male rats. Physiol. Behav. 102, 96–104 (2011).
Durant, R. H. Use of multiple drugs among adolescents who use anabolic steroids. N. Engl. J. Med. (1993).
Afonso, L., Mohammad, T. & Thatai, D. Crack whips the heart: A review of the cardiovascular toxicity of cocaine. Am. J. Cardiol. 100, 1040–1043 (2007).
Ersche, K. D. et al. Abnormal structure of frontostriatal brain systems is associated with aspects of impulsivity and compulsivity in cocaine dependence. Brain 134, 2013–2024 (2011).
Baker, D. A. et al. Neuroadaptations in cystine-glutamate exchange underlie cocaine relapse. Nat. Neurosci. 6, 743–749 (2003).
Beyer, C. E. & Steketee, J. D. Cocaine sensitization: modulation by dopamine D2 receptors. Cereb. Cortex. 12, 526–535 (2002).
Steketee, J. D. & Walsh, T. J. Repeated injections of sulpiride into the medial prefrontal cortex induces sensitization to cocaine in rats. Psychopharmacol. (Berl). 179, 753–760 (2005).
Goldstein, R. Z. & Volkow, N. D. Drug addiction and its underlying neurobiological basis: neuroimaging evidence for the involvement of the frontal cortex. Am. J. Psychiatry 159, 1642–1652 (2002).
Koob, G. F. & Volkow, N. D. Neurocircuitry of addiction. Neuropsychopharmacology 35, 217–238 (2010).
Bello, E. P. et al. Cocaine supersensitivity and enhanced motivation for reward in mice lacking dopamine D2 autoreceptors. Nat. Neurosci. 14, 1033–1038 (2011).
Marinelli, M. & White, F. J. Enhanced vulnerability to cocaine self-administration is associated with elevated impulse activity of midbrain dopamine neurons. J. Neurosci. 20, 8876–8885 (2000).
Bhathena, S. J. Comparison of effects of decapitation and anesthesia on metabolic and hormonal parameters in Sprague-Dawley rats. Life Sci. 50, 1649–1655 (1992).
Moreau, R. A. et al. Phytosterols and their derivatives: structural diversity, distribution, metabolism, analysis, and health-promoting uses. Prog Lipid Res. 70, 35–61 (2018).
Ostlund, R. E., Racette, S. B., Okeke, A. & Stenson, W. F. Phytosterols that are naturally present in commercial corn oil significantly reduce cholesterol absorption in humans. Am. J. Clin. Nutr. 75, 1000–1004 (2002).
Bonnecaze, A. K., O’Connor, T. & Aloi, J. A. Characteristics and attitudes of men using anabolic androgenic steroids (AAS): A survey of 2385 men. Am. J. Mens Health 14, (2020).
Nair, A. & Jacob, S. A simple practice guide for dose conversion between animals and human. J. Basic. Clin. Pharm. 7, 27 (2016).
Clark, A. S. & Henderson, L. P. Behavioral and physiological responses to anabolic-androgenic steroids. Neurosci. Biobehav Rev. 27, 413–436 (2003).
Segarra, A. C. et al. Estrogen receptors mediate estradiol’s effect on sensitization and CPP to cocaine in female rats: role of contextual cues. Horm. Behav. 65, 77–87 (2014).
Spear, L. P. The adolescent brain and age-related behavioral manifestations. Neurosci. Biobehav. Rev. 24, (2000).
Pellow, S., Chopin, P., File, S. E. & Briley, M. Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J. Neurosci. Methods. 14, 149–167 (1985).
Biedermann, S. V. et al. An elevated plus-maze in mixed reality for studying human anxiety-related behavior. BMC Biol. 15, 1–13 (2017).
Löfgren, M., Johansson, I. M., Meyerson, B., Turkmen, S. & Bäckström, T. Withdrawal effects from progesterone and estradiol relate to individual risk-taking and explorative behavior in female rats. Physiol. Behav. 96, 91–97 (2009).
Momeni, S., Sharif, M., Ågren, G. & Roman, E. Individual differences in risk-related behaviors and voluntary alcohol intake in outbred Wistar rats. Behav. Pharmacol. 25, 206–215 (2014).
Prut, L., Belzung, C., Rabelias, U. F. & Psychobiologie, E. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: A review. Eur. J. Pharmacol. 463, 3–33 (2003).
Treit, D. & Fundytus, M. Thigmotaxis as a test for anxiolytic activity in rats. Pharmacol. Biochem. Behav. 31, 959–962 (1988).
Anagnostaras, S. G. & Robinson, T. E. Sensitization to the psychomotor stimulant effects of amphetamine: modulation by associative learning. Behav. Neurosci. 110, 1397–1414 (1996).
Vanderschuren, L. J. & Kalivas, P. W. Alterations in dopaminergic and glutamatergic transmission in the induction and expression of behavioral sensitization: a critical review of preclinical studies. Psychopharmacol. (Berl). 151, 99–120 (2000).
Prus, A. J., James, J. R. & Rosecrans, J. A. Conditioned Place Preference. Methods Behav. Anal. Neurosci. http://www.ncbi.nlm.nih.gov/pubmed/17208653 (2009).
Carey, R. J., Damianopoulos, E. N. & Shanahan, A. B. Cocaine conditioned behavior: a cocaine memory trace or an anti-habituation effect. Pharmacol. Biochem. Behav. 90, 625–631 (2008).
Soares-Cunha, C. et al. Activation of D2 dopamine receptor-expressing neurons in the nucleus accumbens increases motivation. Nat. Commun. 7, 1–11 (2016).
Selley, D. E. et al. Attenuated dopamine receptor signaling in nucleus accumbens core in a rat model of chemically-induced neuropathy. Neuropharmacology 166, 107935 (2020).
Morales-Mulia, S., Magdaleno-Madrigal, V. M., Nicolini, H., Genis-Mendoza, A. & Morales-Mulia, M. Orexin-A up-regulates dopamine D2 receptor and mRNA in the nucleus accumbens shell. Mol. Biol. Rep. 47, 9689–9697 (2020).
Grönbladh, A., Nylander, E. & Hallberg, M. The neurobiology and addiction potential of anabolic androgenic steroids and the effects of growth hormone. Brain Res. Bull. 126, 127–137 (2016).
Pope, H. G., Kouri, E. M., Powell, K. F., Campbell, C. & Katz, D. L. Anabolic-androgenic steroid use among 133 prisoners. Compr. Psychiatry. 37, 322–327 (1996).
Malone, D. A., Dimeff, R. J., Lombardo, J. A. & Sample, R. H. Psychiatric effects and psychoactive substance use in anabolic-androgenic steroid users. Clin. J. Sport Med. 5, 25–31 (1995).
Dokras, A., Clifton, S., Futterweit, W. & Wild, R. Increased prevalence of anxiety symptoms in women with polycystic ovary syndrome: systematic review and meta-analysis. Fertil. Steril. 97, 225–230e2 (2012).
Hu, M. et al. Maternal testosterone exposure increases anxiety-like behavior and impacts the limbic system in the offspring. Proc. Natl. Acad. Sci. U S A. 112, 14348–14353 (2015).
Paus, T., Keshavan, M. & Giedd, J. N. Why do many psychiatric disorders emerge during adolescence? Nat. Rev. Neurosci. 9, 947–957 (2008).
Kouvelas, D. et al. Nandrolone abuse decreases anxiety and impairs memory in rats via central androgenic receptors. Int. J. Neuropsychopharmacol. 11, 925–934 (2008).
Aikey, J. L., Nyby, J. G., Anmuth, D. M. & James, P. J. Testosterone rapidly reduces anxiety in male house mice (Mus musculus). Horm. Behav. 42, 448–460 (2002).
Bitran, D., Kellogg, C. K. & Hilvers, R. J. Treatment with an anabolic-androgenic steroid affects anxiety-related behavior and alters the sensitivity of cortical GABAA receptors in the rat. Horm. Behav. 27, 568–583 (1993).
Rainer, Q. et al. Chronic nandrolone decanoate exposure during adolescence affects emotional behavior and monoaminergic neurotransmission in adulthood. Neuropharmacology 83, 79–88 (2014).
Edinger, K. L. & Frye, C. A. Intrahippocampal administration of an androgen receptor antagonist, flutamide, can increase anxiety-like behavior in intact and DHT-replaced male rats. Horm. Behav. 50, 216–222 (2006).
Rosic, G., Joksimovic, J., Selakovic, D., Milovanovic, D. & Jakovljevic, V. Anxiogenic effects of chronic exposure to nandrolone decanoate (ND) at Supraphysiological dose in rats: A brief report. Neuroendocrinol. Lett. 35, 703–710 (2014).
Ricci, L. A., Morrison, T. R. & Melloni, R. H. Serotonin modulates anxiety-like behaviors during withdrawal from adolescent anabolic-androgenic steroid exposure in Syrian hamsters. Horm. Behav. 62, 569–578 (2012).
Morrison, T. R., Ricci, L. A. & Melloni, R. H. Dopamine D2 receptors act upstream of AVP in the latero-anterior hypothalamus to modulate adolescent anabolic/androgenic steroid-induced aggression in Syrian hamsters. Behav. Neurosci. 129, 197–204 (2015).
Op De MacKs, Z. A. et al. Testosterone levels correspond with increased ventral striatum activation in response to monetary rewards in adolescents. Dev. Cogn. Neurosci. 1, 506–516 (2011).
El-Shamarka, M. E. S., Sayed, R. H., Assaf, N., Zeidan, H. M. & Hashish, A. F. Combined neurotoxic effects of cannabis and nandrolone decanoate in adolescent male rats. Neurotoxicology 76, 114–125 (2020).
Keleta, Y. B., Lumia, A. R., Anderson, G. M. & McGinnis, M. Y. Behavioral effects of pubertal anabolic androgenic steroid exposure in male rats with low serotonin. Brain Res. 1132, 129–138 (2007).
Joksimovic, J. et al. The role of neuropeptide-y in nandrolone decanoate-induced Attenuation of antidepressant effect of exercise. PLoS One. 12, 1–16 (2017).
Minkin, D. M., Meyer, M. E. & van Haaren, F. Behavioral effects of long-term administration of an anabolic steroid in intact and castrated male Wistar rats. Pharmacol. Biochem. Behav. 44, 959–963 (1993).
Salvador, A., Moya-Albiol, L., Martínez-Sanchis, S. & Simón, V. M. Lack of effects of anabolic-androgenic steroids on locomotor activity in intact male mice. Percept. Mot. Skills. 88, 319–328 (1999).
Kurling-Kailanto, S., Kankaanpää, A. & Seppälä, T. Subchronic nandrolone administration reduces cocaine-induced dopamine and 5-hydroxytryptamine outflow in the rat nucleus accumbens. Psychopharmacol. (Berl). 209, 271–281 (2010).
Purves-Tyson, T. D. et al. Testosterone regulation of sex steroid-related mRNAs and dopamine-related mRNAs in adolescent male rat substantia Nigra. BMC Neurosci. 13, 1 (2012).
Bontempi, L. & Bonci, A. µ-Opioid receptor-induced synaptic plasticity in dopamine neurons mediates the rewarding properties of anabolic androgenic steroids. Sci. Signal. 13, 1–12 (2020).
Mȩdraś, M., Brona, A. & Jóźków, P. The central effects of Androgenic-Anabolic steroid use. J. Addict. Med. 12, 184–192 (2018).
Beatty, W. W., Dodge, A. M. & Traylor, K. L. Stereotyped behavior elicited by amphetamine in the rat: influences of the testes. Pharmacol. Biochem. Behav. 16, 565–568 (1982).
Dluzen, D. E., Green, M. A. & Ramirez, V. D. The effect of hormonal condition on dose-dependent amphetamine-stimulated behaviors in the male rat. Horm. Behav. 20, 1–6 (1986).
Forgie, M. L. & Stewart, J. Sex differences in the locomotor-activating effects of amphetamine: role of Circulating testosterone in adulthood. Physiol. Behav. 55, 639–644 (1994).
Purves-Tyson, T. D. et al. Testosterone attenuates and the selective Estrogen receptor modulator, raloxifene, potentiates amphetamine-induced locomotion in male rats. Horm. Behav. 70, 73–84 (2015).
Caine, S. B. et al. Effect of gonadectomy and gonadal hormone replacement on cocaine self-administration in female and male rats. Neuropsychopharmacology 29, 929–942 (2004).
Chen, R., Osterhaus, G., McKerchar, T. & Fowler, S. C. The role of exogenous testosterone in cocaine-induced behavioral sensitization and plasmalemmal or vesicular dopamine uptake in castrated rats. Neurosci. Lett. 351, 161–164 (2003).
Chin, J. et al. Quiñones-Jenab, V. Endogenous gonadal hormones modulate behavioral and neurochemical responses to acute and chronic cocaine administration. Brain Res. 945, 123–130 (2002).
Haney, M., Castanon, N., Cador, M., Le Moal, M. & Mormède, P. Cocaine sensitivity in Roman high and low avoidance rats is modulated by sex and gonadal hormone status. Brain Res. 645, 179–185 (1994).
Harrod, S. B., Mactutus, C. F., Browning, C. E., Welch, M. & Booze, R. M. Home cage observations following acute and repeated IV cocaine in intact and gonadectomized rats. Neurotoxicol Teratol. 27, 891–896 (2005).
Jackson, L. R., Robinson, T. E. & Becker, J. B. Sex differences and hormonal influences on acquisition of cocaine self-administration in rats. Neuropsychopharmacology 31, 129–138 (2006).
Long, S. F., Dennis, L. A., Russell, R. K., Benson, K. A. & Wilson, M. C. Testosterone implantation reduces the motor effects of cocaine. Behav. Pharmacol. 5(1), 103–106 (1994).
Kabbaj, M., Isgor, C., Watson, S. J. & Akil, H. Stress during adolescence alters behavioral sensitization to amphetamine. Neuroscience 113, 395–400 (2002).
Lepsch, L. B. et al. Exposure to chronic stress increases the locomotor response to cocaine and the basal levels of corticosterone in adolescent rats. Addict. Biol. 10, 251–256 (2005).
Trzcińska, M., Bergh, J., DeLeon, K., Stellar, J. R. & Melloni, R. H. Social stress does not alter the expression of sensitization to cocaine. Physiol. Behav. 76, 457–463 (2002).
Tseng, Y. Cardiovascular toxicities of nandrolone and cocaine in spontaneously hypertensive rats. Fundam. Appl. Toxicol. 22(1), 113–121 (1994).
Long, S. F., Wilson, M. C. & Davis, W. M. The effects of nandrolone decanoate on cocaine-induced kindling in male rats. Neuropharmacology 39, 2442–2447 (2000).
Tobiansky, D. J. et al. Androgen regulation of the mesocorticolimbic system and executive function. Front. Endocrinol. (Lausanne). 9, 279 (2018).
Bowman, B. P. et al. Effects of sex and gonadectomy on cocaine metabolism in the rat. J. Pharmacol. Exp. Ther. 290, 1316–1323 (1999).
Lukas, S. E. et al. Sex differences in plasma cocaine levels and subjective effects after acute cocaine administration in human volunteers. Psychopharmacol. (Berl). 125, 346–354 (1996).
McDonnell, A. M. & Dang, C. H. Basic review of the cytochrome p450 system. J. Adv. Pract. Oncol. 4, 263–268 (2013).
Yamamoto, R. T. et al. Antiandrogen pretreatment alters cocaine pharmacokinetics in men. J. Addict. Med. 1, 198–204 (2007).
Smith, R. J., Lobo, M. K., Spencer, S. & Kalivas, P. W. Cocaine-induced adaptations in D1 and D2 accumbens projection neurons (a dichotomy not necessarily synonymous with direct and indirect pathways). Curr. Opin. Neurobiol. 23, 546–552 (2013).
Kalivas, P. W. & Stewart, J. Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity. Brain Res. Rev. 16, 223–244 (1991).
Bao, A. M. et al. F. A direct androgenic involvement in the expression of human corticotropin-releasing hormone. Mol. Psychiatry. 11, 567–576 (2006).
Bao, A. M. & Swaab, D. F. Gender difference in age-related number of corticotropin-releasing hormone-expressing neurons in the human hypothalamic paraventricular nucleus and the role of sex hormones. Neuroendocrinology 85, 27–36 (2007).
Novoa, J. et al. Social isolation of adolescent male rats increases anxiety and K+-induced dopamine release in the nucleus accumbens: role of CRF-R1. Eur. J. Neurosci. 54, 4888–4905 (2021).
Blum, K. et al. Dopamine dysregulation in reward and autism spectrum disorder. Brain Sci. 14(7), 33 (2024).
Célérier, E. et al. Influence of the anabolic-androgenic steroid nandrolone on cannabinoid dependence. Neuropharmacology 50, 788–806 (2006).
Célérier, E. et al. Effects of nandrolone on acute morphine responses, tolerance and dependence in mice. Eur. J. Pharmacol. 465, 69–81 (2003).
Struik, D. et al. The anabolic steroid nandrolone alters cannabinoid self-administration and brain CB1 receptor density and function. Pharmacol. Res. 115, 209–217 (2017).
Johansson, P., Hallberg, M., Kindlundh, A. & Nyberg, F. The effect on opioid peptides in the rat brain, after chronic treatment with the anabolic androgenic steroid, nandrolone decanoate. Brain Res. Bull. 51, 413–418 (2000).
Anagnostaras, S. G., Maren, S. & Fanselow, M. S. Temporally graded retrograde amnesia of contextual fear after hippocampal damage in rats: Within-subjects examination. J. Neurosci. 19, 1106–1114 (1999).
Gould, T. J. & Leach, P. T. Cellular, molecular, and genetic substrates underlying the impact of nicotine on learning. Neurobiol. Learn. Mem. 107, 108–132 (2014).
Riedel, W. J., Klaassen, T., Deutz, N. E. P., Van Someren, A. & Van Praag, H. M. Tryptophan depletion in normal volunteers produces selective impairment in memory consolidation. Psychopharmacol. (Berl). 141, 362–369 (1999).
Wallin, K. G., Alves, J. M. & Wood, R. I. Anabolic–androgenic steroids and decision making: probability and effort discounting in male rats. Psychoneuroendocrinology 57, 84–92 (2015).
Cooper, S. E., Goings, S. P., Kim, J. Y. & Wood, R. I. Testosterone enhances risk tolerance without altering motor impulsivity in male rats. Psychoneuroendocrinology 40, 201–212 (2014).
Wallin-Miller, K. G., Kreutz, F., Li, G. & Wood, R. I. Anabolic-androgenic steroids (AAS) increase sensitivity to uncertainty by Inhibition of dopamine D1 and D2 receptors. Psychopharmacol. (Berl). 235, 959–969 (2018).
Wood, R. I. et al. Roid Rage in rats? Testosterone effects on aggressive motivation, impulsivity and tyrosine hydroxylase. Physiol. Behav. 110–111, 6–12 (2013).
Chefer, V. I. et al. Endogenous κ-opioid receptor systems regulate mesoaccumbal dopamine dynamics and vulnerability to cocaine. J. Neurosci. 25, 5029–5037 (2005).
Kumar, A. et al. J. Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity in striatum. Neuron 48, 303–314 (2005).
Thomas, M. J., Kalivas, P. W. & Shaham, Y. Neuroplasticity in the mesolimbic dopamine system and cocaine addiction. Br. J. Pharmacol. 154, 327–342 (2008).
Peters, K. D. & Wood, R. I. Androgen dependence in hamsters: overdose, tolerance, and potential opioidergic mechanisms. Neuroscience 130, 971–981 (2005).
Hauger, L. E., Westlye, L. T. & Bjørnebekk, A. Anabolic androgenic steroid dependence is associated with executive dysfunction. Drug Alcohol Depend. 208, 107874 (2020).
Magnusson, K. et al. Nandrolone decanoate administration elevates hippocampal prodynorphin mRNA expression and impairs Morris water maze performance in male rats. Neurosci. Lett. 467, 189–193 (2009).
McGinty, J. F., Whitfield, T. W. & Berglind, W. J. Brain-derived neurotrophic factor and cocaine addiction. Brain Res. 1314, 183–193 (2010).
Warren, M. F., Serby, M. J. & Roane, D. M. The effects of testosterone on cognition in elderly men: a review. CNS Spectr. 13, 887–897 (2008).
Kanayama, G., Kean, J., Hudson, J. I. & Pope, H. G. Cognitive deficits in long-term anabolic-androgenic steroid users. Drug Alcohol Depend. 130, 208–214 (2013).
Tanehkar, F. et al. Voluntary exercise does not ameliorate Spatial learning and memory deficits induced by chronic administration of nandrolone decanoate in rats. Horm. Behav. 63, 158–165 (2013).
White, F. J., Joshi, A., Koeltzow, T. E. & Hu, X. T. Dopamine receptor antagonists fail to prevent induction of cocaine sensitization. Neuropsychopharmacology 18, 26–40 (1998).
Steketee, J. D. & Kalivas, P. W. Drug wanting: behavioral sensitization and relapse to drug-seeking behavior. Pharmacol. Rev. 63, 348–365 (2011).
Thomas, M. J., Beurrier, C., Bonci, A. & Malenka, R. C. Long-term depression in the nucleus accumbens: A neural correlate of behavioral sensitization to cocaine. Nat. Neurosci. 4, 1217–1223 (2001).
Di Chiara, G. The role of dopamine in drug abuse viewed from the perspective of its role in motivation. Drug Alcohol Depend. 38, 95–137 (1995).
Mattingly, B. A., Hart, T. C., Lim, K. & Perkins, C. Selective antagonism of dopamine D1 and D2 receptors does not block the development of behavioral sensitization to cocaine. Psychopharmacol. (Berl). 114, 239–242 (1994).
Baker, D. A., Khroyan, T. V., O’Dell, L. E., Fuchs, R. A. & Neisewander, J. L. Differential effects of intra-accumbens sulpiride on cocaine-induced locomotion and conditioned place preference. J. Pharmacol. Exp. Ther. 279, 392–401 (1996).
Manvich, D. F. et al. Selective D2 and D3 receptor antagonists oppositely modulate cocaine responses in mice via distinct postsynaptic mechanisms in nucleus accumbens. Neuropsychopharmacology 44, 1445–1455 (2019).
Neisewander, J. L., Fuchs, R. A., O’Dell, L. E. & Khroyan, T. V. Effects of SCH-23390 on dopamine D1 receptor occupancy and locomotion produced by intraaccumbens cocaine infusion. Synapse 30, 194–204 (1998).
Dreyer, J. K., Herrik, K. F., Berg, R. W. & Hounsgaard, J. D. Influence of phasic and tonic dopamine release on receptor activation. J. Neurosci. 30, 14273–14283 (2010).
Rice, M. E. & Cragg, S. J. Dopamine spillover after quantal release: rethinking dopamine transmission in the nigrostriatal pathway. Brain Res. Rev. 58, 303–313 (2008).
Boudreau, A. C. & Wolf, M. E. Behavioral sensitization to cocaine is associated with increased AMPA receptor surface expression in the nucleus accumbens. J. Neurosci. 25, 9144–9151 (2005).
Aubele, T. & Kritzer, M. F. Androgen influence on prefrontal dopamine systems in adult male rats: localization of cognate intracellular receptors in medial prefrontal projections to the ventral tegmental area and effects of gonadectomy and hormone replacement on glutamate-stimulated. Cereb. Cortex. 22, 1799–1812 (2012).
Bertozzi, G. et al. The role of anabolic androgenic steroids in disruption of the physiological function in discrete areas of the central nervous system. Mol. Neurobiol. 55, 5548–5556 (2018).
Tobiansky, D. J. et al. Testosterone and corticosterone in the mesocorticolimbic system of male rats: effects of gonadectomy and caloric restriction. Endocrinology 159, 450–464 (2018).
Kindlundh, A. M. S., Lindblom, J., Bergström, L., Wikberg, J. E. S. & Nyberg, F. The anabolic-androgenic steroid nandrolone decanoate affects the density of dopamine receptors in the male rat brain. Eur. J. Neurosci. 13, 291–296 (2001).
Zotti, M. et al. Chronic nandrolone administration induces dysfunction of the reward pathway in rats. Steroids 79, 7–13 (2014).
Birgner, C., Kindlundh-Högberg, A. M. S., Nyberg, F. & Bergström, L. Altered extracellular levels of DOPAC and HVA in the rat nucleus accumbens shell in response to sub-chronic nandrolone administration and a subsequent amphetamine challenge. Neurosci. Lett. 412, 168–172 (2007).
Birgner, C. et al. The anabolic androgenic steroid nandrolone decanoate affects mRNA expression of dopaminergic but not serotonergic receptors. Brain Res. 1240, 221–228 (2008).
Aydin, C., Frohmader, K., Emery, M., Blandino, P. & Akil, H. Chronic stress in adolescence differentially affects cocaine vulnerability in adulthood in a selectively bred rat model of individual differences: role of accumbal dopamine signaling. Stress https://doi.org/10.1080/10253890.2020.1790520 (2020).
Birt, I. A. et al. Genetic liability for internalizing versus externalizing behavior manifests in the developing and adult hippocampus: insight from a Meta-analysis of transcriptional profiling studies in a selectively bred rat model. Biol. Psychiatry. 89, 339–355 (2021).
Clinton, S. M. et al. Neonatal fibroblast growth factor treatment enhances cocaine sensitization. Pharmacol. Biochem. Behav. 103, 6–17 (2012).
Turner, C. A. et al. Neonatal FGF2 alters cocaine self-administration in the adult rat. Pharmacol. Biochem. Behav. 92, 100–104 (2009).
Flagel, S. B. et al. Genetic background and epigenetic modifications in the core of the nucleus accumbens predict addiction-like behavior in a rat model. Proc. Natl. Acad. Sci. U. S. A. 113, E2861–E2870 (2016).
Ghanim, H. et al. Effect of testosterone on FGF2, MRF4, and myostatin in hypogonadotropic hypogonadism: relevance to muscle growth. J. Clin. Endocrinol. Metab. 104, 2094–2102 (2019).
Saito, H., Kasayama, S., Kouhara, H., Matsumoto, K. & Sato, B. Up-regulation of fibroblast growth factor (FGF) receptor mRNA levels by basic FGF or testosterone in androgen-sensitive mouse mammary tumor cells. Biochem. Biophys. Res. Commun. 174, 136–141 (1991).
Mueller, D., Chapman, C. A. & Stewart, J. Amphetamine induces dendritic growth in ventral tegmental area dopaminergic neurons in vivo via basic fibroblast growth factor. Neuroscience 137, 727–735 (2006).
Cador, M., Bjijou, Y., Cailhol, S. & Stinus, L. D-Amphetamine-induced behavioral sensitization: implication of a glutamatergic medial prefrontal cortex-ventral tegmental area innervation. Neuroscience 94, 705–721 (1999).
Li, Y. et al. Both glutamate receptor antagonists and prefrontal cortex lesions prevent induction of cocaine sensitization and associated neuroadaptations. Synapse 34, 169–180 (1999).
Dalley, J. W. et al. Cortical cholinergic function and deficits in visual attentional performance in rats following 192 IgG-saporin-induced lesions of the medial prefrontal cortex. Cereb. Cortex. 14, 922–932 (2004).
Moorman, D. E., James, M. H., McGlinchey, E. M. & Aston-Jones, G. Differential roles of medial prefrontal subregions in the regulation of drug seeking. Brain Res. 1628, 130–146 (2015).
Gaspar, P., Bloch, B. & Le Moine, C. D1 and D2 receptor gene expression in the rat frontal cortex: cellular localization in different classes of efferent neurons. Eur. J. Neurosci. 7, 1050–1063 (1995).
Brozoski, T. J., Brown, R. M., Rosvold, H. E. & Goldman, P. S. Cognitive deficit caused by regional depletion of dopamine in prefrontal cortex of rhesus monkey. Sci. (80-). 205, 929–932 (1979).
Chudasama, Y. & Robbins, T. W. Dopaminergic modulation of visual attention and working memory in the rodent prefrontal cortex. Neuropsychopharmacology 29, 1628–1636 (2004).
Lohani, S., Martig, A. K., Deisseroth, K., Witten, I. B. & Moghaddam, B. Dopamine modulation of prefrontal cortex activity is manifold and operates at multiple Temporal and Spatial scales. Cell. Rep. 27, 99–114e6 (2019).
Yokel, R. A. & Wise, R. A. Increased lever pressing for amphetamine after Pimozide in rats: implications for a dopamine theory of reward. Sci. (80-). 187, 547–549 (1975).
Salas, J., Scherrer, J. F., Lustman, P. J. & Schneider, F. D. Racial differences in the association between nonmedical prescription opioid use, abuse/dependence, and major depression. Subst. Abus. 37, 25–30 (2016).
Segarra, A. C. & Strand, F. L. Perinatal administration of nicotine alters subsequent sexual behavior and testosterone levels of male rats. Brain Res. 480, 151–159 (1989).
Hajszan, T., MacLusky, N. J., Johansen, J. A., Jordan, C. L. & Leranth, C. Effects of androgens and estradiol on spine synapse formation in the prefrontal cortex of normal and testicular feminization mutant male rats. Endocrinology 148, 1963–1967 (2007).
Andersen, S. L., Thompson, A. P., Krenzel, E. & Teicher, M. H. Pubertal changes in gonadal hormones do not underlie adolescent dopamine receptor overproduction. Psychoneuroendocrinology 27, 683–691 (2002).
Acknowledgements
The authors would like to thank Lidalee Silva, Erick Quintana, and Bethzaida Suarez for their technical assistance and Dr Sehwan Jong for his statistical analysis assistance. This work was funded by the Office of International Science and Engineering (OISE) of NSF through the Partnerships for Research and Education (PIRE) Program (OISE-#1545803).
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J.A.F.A. and C.R.Q. participated in designing the experiments, data collection, analysis, writing and editing the paper. I.G.S.M., A.M.M., A.G.S., E.U.P.C. and R.J.T.R. participated in data collection. A.C.S. designed the study, oversaw project, data analysis, discussion and wrote the paper. All authors reviewed the manuscript.
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Freire-Arvelo, J.A., Rivero, C.J., Santiago-Marrero, I.G. et al. Nandrolone alters the behavioral response to cocaine as well as striatal and cortical dopamine receptors of prepubertal male rats. Sci Rep 15, 33205 (2025). https://doi.org/10.1038/s41598-025-17890-6
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DOI: https://doi.org/10.1038/s41598-025-17890-6
