Moderate Exercise Rewires Autophagy Genes and Eases Methamphetamine Withdrawal in Rats

Methamphetamine is one of the most damaging addictive substances known to neuroscience, and the process of withdrawing from it can leave the brain’s frontal cortex in a state of molecular disarray. A new experimental study published in BMC Pharmacology and Toxicology suggests that something as accessible as moderate-intensity continuous training, a steady form of aerobic exercise, may help steer that disarray back toward normal. Researchers led by Amir Hossein Haghighi of Hakim Sabzevari University in Iran, working with colleagues in Italy, found that rats given regular moderate treadmill-style exercise during a nine-week withdrawal period showed measurably better locomotor and exploratory behavior than sedentary withdrawing animals, and that these behavioral improvements were mirrored by meaningful changes in the expression of autophagy-related genes in the frontal cortex. The findings add to a growing body of evidence that structured physical activity is not merely a general tonic for the brain but can act on specific molecular pathways implicated in drug-induced neural injury.

The research team designed their experiment around a carefully staged protocol. Male rats received methamphetamine at a dose of 5 milligrams per kilogram per day for 21 consecutive days, a regimen sufficient to produce dependence-like neurochemical changes. After this exposure phase, the animals entered a nine-week withdrawal period. During withdrawal, one group of methamphetamine-exposed rats remained sedentary, while another group undertook moderate-intensity continuous training, commonly abbreviated as MICT. Control animals received saline instead of the drug. The researchers deliberately separated their statistical comparisons into phases: first comparing saline-treated animals with methamphetamine-exposed animals to establish what the drug itself did, then comparing methamphetamine-exposed sedentary rats with methamphetamine-exposed rats that exercised, to isolate the effect of training during withdrawal. This phased analytical approach allowed the team to distinguish drug effects from exercise effects rather than conflating the two.

Behaviorally, the consequences of methamphetamine exposure were stark. Using the open field test, a classic behavioral assay in which an animal is placed in an arena and its movements are tracked, the researchers found that methamphetamine-exposed rats traveled significantly shorter total distances, moved at lower velocities, and reared up on their hind legs less often than saline-treated controls. Rearing is generally interpreted as a marker of exploratory motivation and vertical activity, so its reduction signaled blunted curiosity and diminished spontaneous locomotion. Intriguingly, the methamphetamine-exposed animals showed a significant increase in grooming behavior, a repetitive self-directed action that in this context is often read as a stress-related or stereotyped response. Together, the pattern described animals that were less mobile, less exploratory, and more prone to repetitive behavior, a constellation consistent with the frontal cortical dysfunction that chronic stimulant exposure is known to produce.

To understand what was happening inside the frontal cortex, the team turned to real-time polymerase chain reaction, a technique that quantifies how actively specific genes are being transcribed into messenger RNA. They focused on a panel of seven genes: C/EBPβ, DDIT4, TSC-2, mTOR, LC3-II, Nrg-1, and EphB1. Several of these sit at the heart of the autophagy machinery, the cellular recycling system that engulfs damaged proteins and organelles and delivers them to lysosomes for degradation. LC3-II is a widely used molecular marker of autophagosome formation, while mTOR, the mechanistic target of rapamycin, is the master negative regulator of autophagy initiation. Upstream of mTOR, TSC-2 acts as a brake on mTOR signaling, and DDIT4, also known as REDD1, is a stress-induced protein that suppresses mTOR activity. C/EBPβ is a transcription factor that can drive the expression of autophagy genes under stress. Nrg-1 and EphB1, by contrast, are involved in neural development, synaptic signaling, and neuronal resilience.

The molecular readout after methamphetamine exposure told a coherent story of dysregulated autophagy signaling. Compared with saline controls, the methamphetamine-exposed rats showed significantly increased expression of C/EBPβ, DDIT4, TSC-2, and LC3-II in the frontal cortex, alongside significantly decreased expression of mTOR, Nrg-1, and EphB1. In plain terms, the drug appeared to push the autophagy system toward overactivation: the mTOR pathway, which normally keeps autophagy in check, was suppressed, while the upstream activators and the autophagosome marker LC3-II were elevated. At the same time, the reduced expression of Nrg-1 and EphB1 pointed to compromised support for synaptic maintenance and neuronal signaling. Because the frontal cortex governs executive function, decision-making, and behavioral inhibition, this molecular shift offers a plausible mechanistic bridge to the impaired locomotor and exploratory behavior observed in the open field.

The most striking results emerged when the researchers examined what happened after nine weeks of withdrawal, with or without exercise. In the sedentary methamphetamine-withdrawn group, many of the drug-induced alterations persisted. But in the group that performed moderate-intensity continuous training during withdrawal, the picture changed substantially. Exercise significantly increased total distance traveled and rearing behavior in the open field test, indicating restored locomotor activity and exploratory drive, and it significantly decreased grooming behavior, suggesting a reduction in the stress-linked repetitive responding that methamphetamine had amplified. These were not subtle statistical artifacts; the exercised animals behaved measurably more like healthy controls than their sedentary withdrawing counterparts did.

At the molecular level, the exercise intervention produced a pattern that was almost a mirror image of the drug’s effects. Moderate-intensity continuous training significantly reduced LC3-II expression, the marker of autophagosome formation that methamphetamine had driven upward, and it significantly increased the expression of mTOR, Nrg-1, and EphB1, the genes the drug had suppressed. In other words, exercise appeared to rebalance the autophagy signaling network, pulling it back from the overactivated state that chronic methamphetamine exposure had imposed, while simultaneously restoring the expression of genes that support synaptic integrity and neuronal communication. The authors interpret this as evidence that MICT attenuates the molecular impairments caused by methamphetamine in the frontal cortex, and that this molecular rebalancing accompanies, and may underpin, the behavioral recovery observed in the open field.

The significance of these findings lies partly in what they suggest about the biology of addiction recovery. Autophagy is a double-edged sword in the addicted brain: it is essential for clearing toxic protein aggregates and damaged mitochondria, but excessive or poorly regulated autophagic activity can contribute to neuronal dysfunction and cell death. Methamphetamine is known to generate oxidative stress, mitochondrial damage, and protein misfolding in dopaminergic and cortical neurons, and an overdriven autophagy response is one plausible downstream consequence. By restoring mTOR activity and reducing LC3-II, exercise may have recalibrated this system toward a healthier set point. Meanwhile, the recovery of Nrg-1 and EphB1 expression hints that exercise supports the synaptic and neurotrophic machinery that stimulant drugs erode, potentially facilitating the repair of cortical circuits involved in craving, impulse control, and reward processing.

The study also carries practical implications, though the authors are careful to frame them within the limits of an animal model. Exercise-based interventions are inexpensive, widely scalable, and largely free of the side effects that complicate pharmacological treatments for stimulant use disorder, for which no approved medications currently exist. If the mechanisms observed in rats translate even partially to humans, structured moderate aerobic exercise during early recovery could serve as an adjunct therapy that eases the behavioral disturbances of withdrawal while promoting molecular repair in the prefrontal regions most damaged by methamphetamine. Rehabilitation programs for people recovering from stimulant addiction increasingly incorporate physical activity, and this study provides a mechanistic rationale for that practice, linking a specific exercise intensity to specific gene expression changes in a specific brain region.

Caveats remain, as they always do in translational neuroscience. Rats are not people, the dose and duration of methamphetamine exposure in the laboratory do not perfectly recapitulate human patterns of use, and the open field test captures only a slice of the behavioral repertoire affected by addiction. The researchers also note that their work was conducted in compliance with the ARRIVE 2.0 guidelines and approved by the ethics committee of Hakim Sabzevari University, and that the study received no external funding. Future work will need to determine whether the autophagy-related gene changes persist after exercise stops, whether they correlate directly with neuronal survival and synaptic density, and whether other exercise modalities, such as high-intensity interval training, produce similar or different molecular signatures. For now, however, the study stands as a compelling demonstration that the brain’s recovery from methamphetamine injury is not a passive process, and that moderate, sustained exercise can actively participate in it, one gene and one behavior at a time.

Subject of Research: Effects of moderate-intensity aerobic exercise on autophagy-related gene expression and behavior in the frontal cortex during methamphetamine withdrawal in rats

Article Title: Moderate-intensity continuous training modulates the expression of autophagy-related genes in the frontal cortex and improves behavioral responses in rats during methamphetamine withdrawal

Article References: Haghighi, A. H., Izadimanesh, F., Hosseini-Kakhak, S. A., Asadi-Shekaari, M., Marefati, H., Shahrabadi, H., Iellamo, F., Farsetti, P., Bei, R., & Perrone, M. A. (2026). Moderate-intensity continuous training modulates the expression of autophagy-related genes in the frontal cortex and improves behavioral responses in rats during methamphetamine withdrawal. BMC Pharmacology and Toxicology. https://doi.org/10.1186/s40360-026-01237-6

Image Credits: AI Generated

DOI: 10.1186/s40360-026-01237-6

Keywords: methamphetamine, withdrawal, autophagy, moderate-intensity continuous training, frontal cortex, mTOR, LC3-II, open field test, exercise, neurorehabilitation, gene expression, rats