Nandrolone Raises Calcineurin in Rat Hippocampus Alongside Memory Deficits

The new study, published in Behavioral and Brain Functions, is the first to measure calcineurin protein levels in these two hippocampal subregions in parallel with behavioral outcomes after chronic steroid exposure, and it offers a candidate molecular thread connecting steroid abuse to cognitive dysfunction.

The research team, led by Romina Salmani and colleagues working across Kermanshah University of Medical Sciences and collaborating institutions, assigned twenty-seven male Wistar rats to three groups of nine. Two groups received intraperitoneal injections of nandrolone decanoate at 5 or 15 mg/kg, while a control group received sesame oil, the vehicle. Injections were given every third day for 22 days, a total of eight doses, a schedule the authors selected to model supraphysiological anabolic-androgenic steroid abuse in rodents. All experiments were conducted double-blind: animals carried coded identifiers rather than treatment labels, the behavioral tester did not know which compound each rat had received, tissue samples were coded for the biochemist, and a third person performed the statistical analysis with the treatment key revealed only afterward.

Behavioral testing began after the dosing period and followed a fixed sequence. In the Morris water maze, rats underwent four acquisition trials per day for four consecutive days, searching for a hidden platform submerged one centimeter beneath the water surface. All groups learned the task: repeated-measures analysis showed a significant effect of training day on escape latency and traveled distance, with no significant day-by-treatment interaction for either measure. Swimming speed during training also did not differ between groups, arguing against a gross locomotor explanation. The visible-platform task, run thirty minutes after the probe test, likewise showed no group differences in escape latency, indicating that visual, motor, and procedural abilities were broadly comparable, although the authors note that subtle deficits cannot be fully excluded.

The critical divergence appeared in the probe trial, when the platform was removed and memory for its location was tested. Rats treated with the 15 mg/kg dose spent significantly less time in the target quadrant than controls, a difference the authors interpret as impaired spatial memory retention. Because locomotion, vision, and acquisition-phase learning were all intact, the deficit points specifically to memory retrieval or retention rather than a general performance failure. Notably, the 5 mg/kg dose produced no significant behavioral impairment in this task, a dose-dependent pattern the authors suggest reflects a threshold beyond which cognitive dysfunction emerges after steroid exposure.

A parallel picture emerged in the passive avoidance test, which measures aversive memory using a light-dark shuttle box and a mild foot shock. All groups met the acquisition criterion during training, and none of the rats re-entered the dark chamber when training was repeated two minutes later, indicating intact learning of the shock-context association. But twenty-four hours later, at the retention test, rats given 15 mg/kg nandrolone showed a significantly reduced step-through latency to enter the dark compartment compared with controls. The authors acknowledge that reduced latency could in principle reflect altered shock sensitivity rather than memory, but they argue that a major perceptual or nociceptive deficit is unlikely given intact acquisition, while conceding that pain thresholds were not directly measured.

The open field test added an emotional dimension to the findings. General locomotor activity, measured as squares crossed, did not differ between nandrolone-treated and control groups. Yet rats receiving the high dose spent significantly more time in the center of the arena, a measure commonly interpreted as reduced anxiety-like behavior. This anxiolytic-like effect raises an interpretive question for the avoidance data, since inhibitory avoidance depends partly on fear-related inhibition. The authors counter that the Morris water maze probe deficit occurred in a task where anxiety has minimal influence, and that the combination of reduced target-quadrant time with normal locomotion and sensory function supports a genuine spatial memory impairment rather than a motivational or emotional artifact.

The biochemical core of the study came after behavioral testing, when animals were decapitated under deep anesthesia and the right hippocampus was dissected along its septotemporal axis. The dorsal hippocampus was collected from roughly two to four millimeters posterior to Bregma and the ventral hippocampus from levels posterior to about five millimeters, with the intermediate region excluded to prevent cross-contamination. Tissue was homogenized, normalized to equal total protein using a Bradford assay, and total calcineurin protein was quantified with a commercial sandwich ELISA run in duplicate. The authors emphasize that this kit detects total protein content and provides no information about enzymatic phosphatase activity or functional state, an important caveat when interpreting the molecular data.

Statistical analysis using two-way repeated-measures ANOVA, with treatment as the between-subject factor and hippocampal region as the within-subject factor, revealed significant main effects of both treatment and region, and, critically, a significant treatment-by-region interaction, indicating that the effect of nandrolone differed between dorsal and ventral subregions. Calcineurin protein levels were elevated in both regions in the nandrolone-treated groups. This creates an intriguing dissociation: at 5 mg/kg, calcineurin rose without behavioral impairment, while at 15 mg/kg both the molecular change and the memory deficits appeared. The authors propose that increased calcineurin expression alone may not be sufficient to disrupt memory, and that cognitive deficits may require a greater magnitude of signaling, functional enzyme activation, or additional downstream mechanisms that emerge only at higher doses. They interpret the lower-dose increase as a possible early neurobiological response that precedes measurable cognitive impairment.

The mechanistic framing rests on calcineurin’s established role as a calcium/calmodulin-dependent serine-threonine phosphatase and a negative regulator of hippocampal-dependent memory: upregulation impairs memory, whereas inhibition improves it. Nandrolone is known to act through both genomic and rapid non-genomic pathways, the latter involving elevated intracellular calcium, potentially via calcium entry through L-type calcium channels, which could activate calcineurin. Calcineurin activation promotes processes such as NMDA receptor internalization and synaptic weakening. The authors also cite their previous observation that calcineurin inhibition attenuates nandrolone-induced memory deficits, which together with the present findings supports, without proving, a causal role for calcineurin signaling in the cognitive effects of the steroid.

Several limitations frame the conclusions. Only male rats were studied, so the results may not generalize to females, in whom gonadal hormones could interact differently with the steroid. Calcineurin was measured ex vivo after behavioral testing, so individual-level correlations between protein levels and performance were not assessed. Behavioral tasks were administered sequentially about twenty-four hours apart, and the authors cannot exclude that prior testing experience influenced later performance. Protein abundance may not reflect enzymatic activation, and future work measuring calcineurin activity and downstream targets is needed. What the study does establish is a convergent pattern: chronic high-dose nandrolone in male rats is associated with impaired spatial memory retention and aversive memory, reduced anxiety-like behavior, unchanged locomotion, and elevated calcineurin protein in both dorsal and ventral hippocampus, positioning this phosphatase as a candidate molecular contributor to the cognitive costs of anabolic-androgenic steroid abuse.

Subject of Research: Associations between chronic nandrolone exposure, hippocampal calcineurin protein levels, and memory performance in male rats

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

Article References: Salmani, R., Alimohammadi, S., Zarei, F., Yousofvand, N., & Moradpour, F. (2026). Alterations in hippocampal calcineurin protein levels are associated with the impairing effects of nandrolone on dorsal and ventral hippocampus-dependent memory in rats. Behavioral and Brain Functions, 22(1), Article 43. https://doi.org/10.1186/s12993-026-00363-9

Image Credits: AI Generated

DOI: 10.1186/s12993-026-00363-9

Keywords: nandrolone decanoate, anabolic-androgenic steroids, hippocampus, calcineurin, spatial memory, passive avoidance, Morris water maze, dorsal hippocampus, ventral hippocampus, rats, anxiety-like behavior, ELISA