Alterations in hippocampal calcineurin protein levels are associated with the impairing effects of nandrolone on dorsal and ventral hippocampus-dependent memory in rats

Abstract

Background

Nandrolone decanoate (ND) exposure has been associated with impairments in hippocampal-dependent learning and memory, potentially involving alterations in intracellular calcium signaling and Calcineurin protein expression. Given the distinct functional contributions of the dorsal hippocampus (dHPC) and ventral hippocampus (vHPC) to cognitive and emotional processes, this study investigated whether ND-induced behavioral changes are associated with alterations in Calcineurin protein levels in these hippocampal subregions.

Materials and methods

Twenty-seven male rats were randomly assigned to three groups (n = 9 per group) and received intraperitoneal injections of ND (5 or 15 mg/kg) or sesame oil as the vehicle control every third day for 22 days. Behavioral assessments included the Morris water maze (MWM) for spatial learning and memory, the open field test (OFT) for locomotor activity and anxiety-like behavior, and the passive avoidance test (PAT) for aversive memory. Following completion of behavioral testing, the animals were deeply anesthetized, and the brains were removed. The hippocampus was dissected from the right hemisphere and further separated into dorsal and ventral regions according to standard stereotaxic coordinates relative to Bregma. Tissue samples were homogenized, and Calcineurin protein levels in the dHPC and vHPC were quantified using a commercial ELISA kit according to the manufacturer’s instructions.

Results

ND administration impaired both spatial and avoidance memory and was associated with increased Calcineurin protein levels in the dHPC and vHPC. These findings suggest an association between increased Calcineurin protein levels and Nandrolone-induced cognitive deficits in both hippocampal subregions. Additionally, Nandrolone did not significantly alter locomotor activity but reduced anxiety-like behavior in rats.

Conclusions

These findings suggest that ND-associated learning and memory impairments are accompanied by alterations in Calcineurin protein levels in both the dHPC and vHPC. The region-dependent effects of ND on Calcineurin may contribute to the differential vulnerability of hippocampal subregions to ND exposure and provide further insight into the potential molecular mechanisms underlying the cognitive effects of anabolic-androgenic steroid exposure.

Introduction

The hippocampus, a critical component of the limbic system, plays an essential role in learning and memory processes [1]. In rodents, the hippocampus is divided into at least two regions: the dorsal hippocampus (dHPC) and the ventral hippocampus (vHPC). It exhibits functional and molecular heterogeneity along its dorsal–ventral axis. The dorsal hippocampus, located near the septum beneath the cerebral cortex, is primarily involved in cognitive processing, such as spatial learning. In contrast, the ventral hippocampus, which extends into the temporal lobe, is more closely connected to the amygdala and prefrontal cortex and is involved in emotional regulation, including anxiety and fear [2, 3]. Molecular analyses have confirmed these regional differences [4, 5].

Nandrolone decanoate (ND) is among the most commonly abused anabolic-androgenic steroids (AAS) [6]. Its misuse has been associated with alterations in hippocampal function, particularly in learning and memory [7–10]. ND influences hippocampal activity through both genomic and non-genomic mechanisms. The latter involve rapid signaling cascades that elevate intracellular calcium and modulate protein kinases and phosphatases [11, 12]. The balance between phosphorylation and dephosphorylation is critical for synaptic plasticity and memory formation [13] .Among calcium-dependent regulators, Calcineurin, a serine/threonine phosphatase, is particularly important. It is highly expressed in the brain, activated by intracellular calcium, and functions as a negative modulator of hippocampal-dependent memory [14, 15]. Upregulation of Calcineurin impairs memory, whereas inhibition of Calcineurin improves it [16, 17]. Previous studies suggest that ND enhances Calcineurin protein levels, potentially via Ca²⁺ entry through L-type calcium channels [17–19]. Recent studies from our group propose that increased Calcineurin activity levels, driven by rises in intracellular calcium concentration, may represent a cellular mechanism through which ND affects memory [12, 20].

Given the distinct roles of dHPC and vHPC in learning and memory, the present study aimed to determine whether ND-induced impairments in spatial and avoidance memory are associated with region-specific changes in Calcineurin protein levels. To our knowledge, this is the first study to simultaneously evaluate Calcineurin protein levels in dHPC and vHPC in parallel with behavioral outcomes.

Materials and methods

Animal and treatment

Male Wistar rats (PND 70 ± 2, 180–200 g) were obtained from Kermanshah University of Medical Sciences (KUMS) and kept in standard housing conditions under controlled temperature (22 ± 2 °C), with a 12-hour light-dark cycle (lights on at 7:00am). A total of 27 male rats were randomly distributed into 3 experimental groups, and each group contained 9 rats. Animals were housed in Plexiglass cages (4–5 rat in each cage) and given free access to water and standard chow. During the experiment rats were subjected to intraperitoneal (i.p.) injections with ND decanoate (Caspian, Iran), 5 mg/kg, 15 mg/kg, or sesame oil every third day for 22 days (8 injections) (Fig. 1). The ND dosage and treatment schedule was selected based on established rodent models of anabolic-androgenic steroid abuse and represents supraphysiological exposure in rats. Although direct interspecies dose conversion is not linear, these doses have been reported to induce anabolic, endocrine, and cardiovascular alterations in rodents comparable to those observed in human AAS abusers, supporting their translational relevance for modeling high-dose AAS exposure [7, 8, 21, 22]. The injections were given at 08:00 AM and experiments were carried out between 09:00 AM and 13:00 PM.

Fig. 1.

Time line of experimental protocols used to this study (see Materials and methods for details)

All experiments were conducted using a double-blind manner to minimize bias and ensure the validity of the results. For blinding, animals were assigned coded IDs rather than treatment labels. The experimenter conducting behavioral tasks did not know which treatment each animal received. Tissue samples were labeled with codes, so the analyst performing the biochemical analysis was also unaware of the treatment groups. The treatment key was only revealed after statistical analysis is complete. Besides, the person conducting the behavioral experiments was different from the person conducting the biochemical experiments, and the statistical analysis of the data was also performed by a third person.

Behavioral assessments

Behavioural data was recorded and analyzed by a video camera connected to the MazeRouter V3.1 video tracking system (Neuroscience and behavioral equipment’s, Iran).

Morris water maze (MWM)

Spatial learning and memory were assessed in the MWM [23]. The circular pool (180 cm diameter, 60 cm height) was filled with water (22 ± 2 °C) to a depth of 35 cm. The pool was divided into 4 quadrants and a hidden platform (10 cm diameter) was submerged 1 cm under the water surface in the target (southwest) quadrant. The platform position remained constant over acquisition days. Rats underwent four acquisition trials per day for four consecutive days (from experimental days 22 to 25). At the beginning of each acquisition trial, an animal was placed facing the pool wall with random starting positions and quadrants, in random order (designated north, east, south and west). Each rat was allowed a maximum of 60 s to find the platform. At the end of each trial, the rat was allowed to stay on the platform for 30 s to recognize the platform well. If the rat failed to find the platform within 60 s it was gently guided there by hand. Escape latency was recorded as the mean time to locate the platform.

A single probe trial was conducted without the platform for 60s and the time spent in the target quadrant was recorded [24, 25]. Twenty-four hours after the last acquisition trial, on day 5 (On experimental day 26) the platform was removed and the animals were allowed to start from the northeast quadrant. The 4-trial visible-platform task was conducted 30 min after the probe test to assess sensorimotor and visual abilities independent of spatial memory [26]. For this task, the platform was positioned 1 cm above the water surface and covered with aluminum foil to make it clearly visible. During each trial, the time required for the animal to reach the platform (escape latency) was recorded (see Fig. 1).

Open field test (OFT)

Locomotor activity and anxiety-like behavior were measured in an open field arena (80 × 80 × 40 cm) (On experimental day 27). Each rat was placed in the center and allowed to explore for 300 s while being video recorded. Ambulation frequency (squares crossed) and time spent in the center vs. periphery were analyzed [27] (see Fig. 1).

Passive avoidance test (PAT)

To assess Passive avoidance behavior, the inhibitory avoidance apparatus (shuttle box) was used. The shuttle box consists of one light and one dark chambers (Plexiglass) of the same size (20 × 20 × 30 cm3) separated by a transportable guillotine door. The floor of the dark chamber was made of stainless-steel bars (0.5 cm diameter) separated by a distance of 1 cm. On training day (On experimental day 28; see Fig. 1), for adaptation to the apparatus, 30 min before training, rats were placed in the lighted chamber and were allowed to explore for 30 s, and then guillotine door was raised. After the entering of the rats to the dark chamber, guillotine door was lowered and the rats remained there for 30 s. Following the habituation of all animals, each rat was placed in the light compartment of the apparatus facing away from the door and 10 s later the guillotine door was raised. The crossover latency was recorded with a chronometer when the animal had placed all four paws into the dark compartment and immediately the door was closed behind it. If the animal waited more than 60 s to enter the dark compartment, it was eliminated from the experiments. 10 s after entry into the dark compartment triggered a 0.7 mA foot shock (3 s) the rat was then removed from the apparatus and 2 min later; the process was repeated. Training was terminated when the rat remained in the light compartment for 120 s. All animals were trained with a maximum of two trials.

After 24 h (On experimental day 29), a retrieval test was conducted. Each animal was gently placed in the light chamber and after 10 s the guillotine door was raised. Step-through latency (STL) into the dark compartment was recorded (up to 300 s). The test session ended when the animal entered the dark compartment or remained in the light compartment for 300 s. During this session no electric shock was applied [28–30].

Behavioral testing was performed in a fixed sequence for all groups to minimize variability associated with task order and handling. The order of testing was as follows: the MWM, OFT, and PAT. In the MWM, starting positions and entry quadrants were randomized across trials to prevent positional bias. For the OFT and PAT, rats were tested individually, with the daily testing order randomized across groups to minimize time-of-day and handling effects.

The behavioral assessments were conducted sequentially on experimental days 26–29, separated by approximately 24 h. Although this interval is commonly used in behavioral studies to minimize acute stress and fatigue effects, we cannot completely exclude the possibility that prior testing experience, stress exposure, or learning from earlier tasks may have influenced performance in subsequent assessments.

Biochemical analysis

dHPC and vHPC tissue preparation

On the following day of behavioral tests (On experimental day 30), animals were decapitated under deep anesthesia with ketamine (100 mg/kg) and xylazine (25 mg/kg). The brain was rapidly extracted from the skull, submerged in phosphate buffered saline at 4 °C. Hemispheres were separated, and the right hippocampus was carefully isolated along its septotemporal axis by removing overlying cortex. dHPC was collected from ~ − 2.0 to − 4.0 mm relative to Bregma, and vHPC from levels posterior to ~ − 5.0 mm; the intermediate (medial) region ( ~ − 4.0 to − 5.0 mm) was excluded to prevent cross-contamination. Dissection cuts were made perpendicular to the longitudinal axis, and accuracy was confirmed with stereotaxic atlas plates [3, 31, 32] (Fig. 2). Tissue from both dHPC and vHPC was quickly transferred to a nitrogen tank in special containers.

Fig. 2.

Schematic representation of dorsal, intermediate, and ventral hippocampal subdivisions in the rat

Calcineurin protein levels assay

In the present study, Calcineurin was quantified using a commercial sandwich ELISA kit (Cat. No.: MBS3809497, MyBioSource, USA), which specifically detects total Calcineurin protein content and does not provide information regarding its enzymatic (phosphatase) activity or functional state.

Total protein concentration in each sample was first determined using the Bradford assay (Cat. No. DB0017; DNAbiotech, Iran). Subsequently, all samples were normalized to an equal total protein concentration to ensure comparability across groups. Under these standardized conditions, Calcineurin measurements obtained by ELISA reflect relative differences in protein abundance independent of variations in total protein content among samples.

For sample preparation, hippocampal tissues were homogenized in ice-cold phosphate-buffered saline (PBS, pH 7.4) supplemented with a protease inhibitor cocktail. Homogenates were centrifuged at 12,000 × g for 15 min at 4 °C, and the resulting supernatants were collected. After normalization, total Calcineurin protein levels were quantified according to the manufacturer’s instructions. All ELISA measurements were performed in duplicate, and each experimental group consisted of five independent biological replicates. The mean of technical duplicates was used for statistical analysis.

Statistical analysis

All statistical analyses were performed using GraphPad Prism Software, version 9. The normality of residual distributions was assessed using the Kolmogorov-Smirnov and D’Agostino-Pearson tests. All datasets satisfied the normality assumption; therefore, parametric analyses were applied throughout the study. Data were analyzed using one-way ANOVA or repeated-measures two-way ANOVA, as appropriate, followed by Tukey’s multiple comparisons post hoc test. Results are presented as mean ± standard error of the mean (SEM). A p-value < 0.05 was considered statistically significant.

Results

Effect of ND exposure on spatial ability

To evaluate hippocampus-dependent learning and memory, escape latency to find the hidden platform during four days of acquisition trials and the time spent in the target quadrant on the fifth day (probe trial) were measured. Repeated-measures ANOVA revealed a significant main effect of Day (training) across all groups (day factor: F _{(2.068, 49.63}) = 22.68, P < 0.001, η²p = 0.49), indicating that the animals successfully learned the task over the four acquisition days. Between-group comparisons revealed no significant differences among the control and ND (5 and 15 mg/kg) groups during the four-day acquisition trials (day × treatment: F _{(4.136, 49.63)} = 0.98, P = 0.44, η²p = 0.08; Fig. 3A). Moreover, the traveled distance decreased during acquisition phase from Day 1 to Day 4 (day factor: F _{(2.003, 48.08)} = 34.01, P < 0.001, η²p = 0.59). Between-group comparisons revealed no significant differences among the control and ND (5 and 15 mg/kg) groups during the acquisition phase (day × treatment: F _{(4.007,48.08)} = 0.64, P = 0.69, η²p = 0.05; Fig. 3B).

Fig. 3.

ND exposure does not affect spatial learning performance in adult male rats. In the MWM, no significant differences were observed between ND-treated and control groups in escape latency (A) or traveled distance to locate the hidden platform during acquisition trials (B). Insets show the mean escape latency and mean traveled distance across the 4-day training period. Swimming speed during training was also not significantly different between groups (C). Data are presented as mean ± SEM (n = 9 rats/group)

The insets showed that there were no significant differences in the mean values of escape latency (F _{(2, 24)} = 1.23, P = 0.30, η² = 0.09; Fig. 3A) and traveled distance (F _{(2, 24)} = 0.74, P = 0.48, η² = 0.06; Fig. 3B) during the four-day acquisition trials among experimental groups.

Notably, there was no significant difference in swimming speed across all experimental groups during the acquisition trials, indicating that ND administration did not affect general locomotor activity in this task (F _(2,24) = 0.65, P = 0. 52, η² = 0.05; Fig. 3C).

The probe trial was performed on the fifth day in a single trial without the platform. Time spent in the target quadrant across different groups was compared using one-way ANOVA. Data analysis revealed significant differences among the experimental groups (F _(2,24) = 3.89, P = 0.03, η² = 0.24; Fig. 4A). Tukey post hoc analysis showed that the ND-treated group (15 mg/kg) spent less time in the target quadrant than the control group. These results indicate that spatial memory was impaired by ND administration.

Fig. 4.

Spatial memory performance in the MWM following ND exposure in adult male rats. In the MWM probe trial, ND-treated rats spent significantly less time in the target quadrant compared to controls (A), a measure commonly interpreted as reduced Spatial memory performance. In the visible platform test, escape latency did not differ significantly between groups (B), indicating no effect on visual or motor function. Data are presented as mean ± SEM (n = 9 rats/group). *p < 0.05 indicates a significant difference ND and control groups

For the visible-platform task, average performance was calculated for each rat as mean escape latency. No significant differences in escape latency were observed among groups (F _(2,24) = 0.82, P = 0.44, η² = 0.06; Fig. 4B). Because this task places minimal demands on spatial learning and relies primarily on visual, motor, and procedural abilities, the absence of group differences suggests that these functions were broadly comparable across groups. Thus, performance in the visible-platform task does not indicate any major sensorimotor or visual impairments that could account for differences observed in the spatial learning tasks, although subtle deficits cannot be completely excluded.

Effect of ND exposure on locomotor activity and anxiety-like behavior

Locomotor activity of the rats in the OFT was evaluated using one-way ANOVA followed by Tukey post hoc analysis. As shown in Fig. 5A, there were no significant differences in locomotor activity between the ND (5 and 15 mg/kg) and control groups (F _(2,24) = 0.54; P = 0.58, η² = 0.04), indicating that ND had no significant effect on general locomotion. Another parameter, time spent in the center of the open field, was also assessed as an index of anxiety-like behavior, where increased time in the center reflects reduced anxiety. One-way ANOVA revealed significant differences among the groups in time spent in the center of the open field (F _(2,24) = 5.00; P = 0.01, η² = 0.29; Fig. 5B). Tukey post hoc analysis showed that rats treated with ND 15 mg/kg spent significantly more time in the center compared to the control group (p = 0.04). These results suggest that ND administration decreased anxiety-like behavior in rats.

Fig. 5.

Effects of ND exposure on passive avoidance learning and memory in adult male rats. In the PAT, ND-treated rats showed a significantly reduced step-through latency to enter the dark chamber compared to controls (A), a measure commonly interpreted as reduced passive avoidance memory. Time spent in the dark chamber was higher in the ND-treated group; however, this difference did not reach statistical significance (B). Data are presented as mean ± SEM (n = 9 rats/group). *p < 0.05 indicates a significant difference between ND and control groups

Effect of ND exposure on passive avoidance ability

During the acquisition phase of the passive avoidance task, all groups successfully reached the acquisition criterion during training, and no significant differences in performance were observed between groups (adaptation: F _(2,24) = 0.45, P = 0. 63, η² = 0.04; trial 1: F _(2,24) = 0.13, P = 0. 87, η² = 0.01; Table 1). None of the rats entered the dark chamber when the training procedure was repeated after 2 min (trial 2), indicating intact learning of the shock–context association. However, during the 24-hour retention test, ND-treated rats exhibited a significantly reduced step-through latency. Figure 6 shows the effects of ND administration (5 and 15 mg/kg) on passive avoidance memory. One-way ANOVA revealed significant differences among the experimental groups in step-through latency (STL) to enter the dark chamber (F _(2,24) = 3.49, P = 0. 04, η² = 0.23; Fig. 6A). Tukey post hoc analysis showed that the step-through latency to enter the dark chamber was significantly lower in the ND 15 mg/kg group compared to the control group (p = 0.03). In addition, one-way ANOVA revealed no significant differences among the groups in the average time spent in the dark chamber (F _(2,24) = 1.87, P = 0.17, η² = 0.14; Fig. 6B).

Table 1.

The data of latency to enter the dark chamber (s) for the training phase

Group	Adaptation	Trial 1	Trial 2
Control	13.22 ± 2.98	6.66 ± 1.01	120 ± 0
ND 5 mg/kg	11.89 ± 2.63	5.88 ± 1.06	120 ± 0
ND 15 mg/kg	15.67 ± 2.84	6.11 ± 1.14	120 ± 0

The values are mean ±S.E.M. There was no significant difference between experimental groups

Fig. 6.

Effects of ND exposure on locomotor activity and anxiety-like behavior in adult male rats. ND exposure doesn’t have a significant effect on locomotor activity of the rats (A). Rats treated with ND (15 mg/kg) spent significantly more time in the center of the open field compared to controls (B), a measure commonly interpreted as reduced anxiety-like behavior. Data are presented as mean ± SEM (n = 9 rats/group). *p < 0.05 indicates a significant difference between ND and control groups

These results indicate that passive avoidance memory was impaired by ND administration.

Reduced step-through latency in passive avoidance task could reflect altered shock sensitivity rather than memory. However, since all animals successfully met the acquisition criterion during training, a major deficit in shock perception or nociceptive responsiveness is unlikely. However, nociceptive thresholds were not directly measured, and thus subtle differences in pain sensitivity cannot be entirely excluded as a contributing factor to the observed retention latencies.

Effects of ND exposure on calcineurin protein levels in dHPC and vHPC

Changes in Calcineurin protein levels in the dHPC and vHPC were measured to investigate whether the observed alterations in spatial and passive avoidance learning and memory following ND administration were associated with changes in Calcineurin protein levels. For Calcineurin, a two-way repeated-measures ANOVA was performed with Treatment as the between-subject factor and Region (Dorsal vs. Ventral) as the within-subject factor. The analysis revealed significant main effects of Treatment (F _(2,24) = 97.00, P < 0.0001, ηp² = 0.890) and Region (F _(1,24) = 309.2, P < 0.0001, ηp² = 0.928). Importantly, a significant Treatment × Region interaction was also detected (F _(2,24) = 31.38, P < 0.0001, ηp² = 0.724), indicating that the effect of Treatment differed significantly between the Dorsal and Ventral regions.

Discussion

The present study focused on the effects of ND on hippocampus-dependent learning and memory and its association with Calcineurin protein levels in the dHPC and vHPC. We initially investigated whether ND affects spatial and avoidance learning and memory in a similar manner. We then examined Calcineurin protein levels in the dHPC and vHPC to better understand the relationship between ND’s effects on spatial and avoidance abilities and alterations in Calcineurin protein levels. The principal findings demonstrate that high-dose ND (15 mg/kg) impaired both spatial and avoidance memory, while increasing Calcineurin protein levels in both hippocampal regions. These findings extend previous evidence that anabolic-androgenic steroid (AAS) exposure disrupts cognitive function and identify Calcineurin signaling as a potential molecular mechanism underlying these effects.

Previous studies have reported that ND impairs hippocampus-dependent learning and memory [10–34]. Our findings are consistent with these reports and further demonstrate that both spatial memory and avoidance memory are vulnerable to chronic ND exposure. By examining Calcineurin protein expression in parallel with behavior, the present study provides additional insight into the molecular changes associated with ND-induced cognitive dysfunction. Notably, memory deficits were observed only in the 15 mg/kg group, whereas the 5 mg/kg dose produced no significant behavioral impairment. This dose-dependent pattern suggests that cognitive dysfunction emerges only after a threshold level of AAS exposure is reached.

In contrast to the behavioral data, Calcineurin protein levels were elevated in both ND-treated groups. This apparent dissociation between biochemical and behavioral outcomes may indicate that increased Calcineurin expression alone is not sufficient to impair memory. Rather, cognitive deficits may require a greater magnitude of Calcineurin signaling, increased enzymatic activity, or the engagement of additional downstream mechanisms that occur only at higher ND doses. It is also possible that compensatory plasticity mechanisms preserve behavioral performance in the 5 mg/kg group despite detectable molecular alterations. Therefore, the increase in Calcineurin protein expression observed at the lower dose may represent an early neurobiological response to ND exposure that precedes measurable cognitive impairment.

This interpretation is consistent with evidence that changes in molecular signaling pathways can occur before the appearance of behavioral deficits. Importantly, the present study measured total Calcineurin protein levels rather than phosphatase activity. Because Calcineurin function is regulated by intracellular calcium, calmodulin binding, and post-translational modifications, protein abundance may not directly reflect functional activation. Future studies assessing Calcineurin activity and downstream signaling targets will be necessary to determine whether the behavioral impairments observed at 15 mg/kg are associated with greater functional activation of the enzyme. The observed increase in Calcineurin expression is nevertheless biologically relevant because Calcineurin is a well-established negative regulator of synaptic plasticity.

The hippocampus exhibits functional specialization along its dorsal-ventral axis, with dorsal regions contributing predominantly to spatial processing and ventral regions playing a greater role in emotional and avoidance-related behaviors [3, 33, 35]. Consistent with this organization, ND-induced deficits were observed in behavioral tasks that engage both hippocampal domains. Notably, Calcineurin protein levels increased in both dHPC and vHPC, suggesting that ND-induced molecular alterations are not restricted to a single hippocampal subregion. These findings support the possibility that chronic ND exposure broadly disrupts hippocampal signaling pathways involved in multiple forms of memory. The mechanism by which ND increases Calcineurin remains to be fully established. Previous studies indicate that androgens can exert rapid nongenomic effects through membrane-associated androgen receptors, leading to enhanced calcium influx through L-type calcium channels [36–38]. Increased intracellular calcium may activate the Ca²⁺/calmodulin-dependent phosphatase Calcineurin, a well-established negative regulator of synaptic plasticity ([17, 20, 39–40]). Calcineurin activation promotes processes such as NMDA receptor internalization and synaptic weakening, which can impair learning and memory. The present findings are consistent with this model and, together with our previous observation that Calcineurin inhibition attenuates ND-induced memory deficits [12], further support a role for Calcineurin signaling in the cognitive effects of ND.

While some anxiolytic-like effects have been observed, the preponderance of evidence indicates that supraphysiological ND exposure is associated with disruptions in cognitive processing, including impairments in passive avoidance learning, likely through androgen receptor–mediated modulation of key neurotransmitter systems and synaptic plasticity mechanisms [8, 20]. Because inhibitory avoidance performance depends on both fear-related inhibition and memory of the aversive experience, the reduced step-through latency observed after high-dose ND administration may partially reflect its anxiolytic effects. Although anxiety-like behavior was reduced in the open field, ND also impaired hippocampus-dependent memory in the MWM probe trial, where anxiety has a minimal influence on performance. The reduced time spent in the target quadrant in the absence of locomotor or sensory deficits supports a genuine impairment in spatial memory retention rather than a generalized motivational or emotional effect. However, the concurrent impairment in MWM probe performance and the marked increase in hippocampal Calcineurin protein levels support the conclusion that ND also disrupts hippocampus-dependent memory processes. Thus, the intersection of anxiolysis and memory impairment reflects an underlying neuroendocrine modulation of limbic circuits, with androgen receptor signaling and downstream neurotransmitter systems likely contributing to these outcomes.

Although some behavioral effects were modest in magnitude, the corresponding effect sizes were comparable to those reported in previous studies of chronic anabolic-androgenic steroid exposure and were accompanied by pronounced increases in hippocampal Calcineurin protein levels, supporting their biological relevance.

A limitation of the present study is the exclusive use of male rats. This choice was made to maintain consistency with our previous cohort studies using the same experimental design; however, the results may not generalize to females, in whom gonadal hormones could differentially interact with ND to influence anxiety, hippocampal signaling, and memory. Future studies should address potential sex-specific effects.

Taken together, these findings suggest that ND is associated with impairments in both spatial and avoidance memory, alongside increased Calcineurin protein levels in the dHPC and vHPC. Calcineurin protein levels were quantified ex vivo from regionally dissected hippocampal tissue following completion of behavioral testing; therefore, individual-level correlations between biochemical measures and behavioral performance were not assessed, and interpretation is limited by the integrative, circuit-level nature of behavior. Nonetheless, the concurrent observation of elevated Calcineurin protein levels in both hippocampal regions and selective impairments in spatial and avoidance memory is consistent with a potential association between Calcineurin signaling and ND-related cognitive deficits. These findings point to Calcineurin as a candidate contributor to the adverse cognitive effects of AAS exposure in dorsal and ventral hippocampal regions, while not establishing a direct causal role (Fig. 7).

Fig. 7.

Effects of ND exposure on Calcineurin protein levels in the dHPC and vHPC of adult male rats. Calcineurin protein levels were significantly increased in both the dHPC and vHPC following ND exposure. Data are presented as mean ± SEM (n = 9 rats/group). **p < 0.01 and ****p < 0.001 indicate significant differences between ND and control groups

Author contributions

R. S., S. A. and F. Z. conducted the study, contributed to data collection, data analysis and manuscript Preparation; N. Y. and F. M. helped design the study and manuscript preparation.

Funding

This work was supported by the Razi University of Kermanshah. The sponsor was not involved in the research.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All procedures of current study were conducted in accordance with the “National Institutes of Health Guide for the Care and Use of Laboratory Animals” and approved by the local Animal Ethics Committee of Razi University, under the assigned approval number (IR.RAZI.AEC.1403.057). Moreover, this study was carried out in compliance with the ARRIVE guidelines.

Competing interests

The authors declare no competing interests.