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bioRxiv · 10.64898/2025.12.16.694564

Methylphenidate exposure alters brain gene expression and induces transgenerational DNA methylation changes in Poecilia reticulata guppies

Alfaro, R. J.·De Serrano, A. R.·Sokolowski, D.·Hughes, K. A.·Rodd, F. H.·Anreiter, I.
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Abstract

Chronic exposure to stimulants is known to affect behavioral phenotypes and epigenetic profiles intergenerationally. We have previously shown that chronic methylphenidate hydrochloride (MPH) exposure in male guppies (Poecilia reticulata) induces persistent, paternally transmitted behavioral changes across multiple unexposed generations. Here, we investigated the underlying epigenetic signatures of this transgenerational behavioral inheritance. Building on our previous study, which used composite behavioral scores, we investigates the transgenerational inheritance patters of individual behaviors and found a robust, male-lineage-dependent increase in swimming across unexposed G2-G4 female offspring. Our molecular analysis showed that that chronic MPH exposure in G1 males alters brain gene expression, with 76 differentially expressed genes including genes with developmental and locomotory functions, and an over-representation of long non-coding RNAs. Furthermore, we found that unexposed G4 descendants from MPH-treated lineages exhibit widespread changes in brain DNA methylation, including genes related to developmental and swimming behavior. Interestingly, we found one lnRNA, LOC103476631, as both differentially expressed in G1 males and differentially methylated in G4 fish, providing a compelling candidate for ncRNA-directed DNA methylation as a mechanism for the observed transgenerational epigenetic inheritance of swimming behavior. Author SummaryDrugs used to treat attention-deficit/hyperactivity disorder, such as methylphenidate, are widely prescribed to children and adolescents during key stages of brain development. We previously showed that exposing male guppies to a low, chronic dose of methylphenidate changed anxiety-like behavior not only in the treated fish but also in several generations of their unexposed descendants. In this study, we asked how such long-lasting behavioral effects might be recorded in the brain. We first confirmed that a simple measure of swimming behavior remains altered in descendants of treated males across multiple generations. We then examined brains from the directly exposed fathers and from great-grand-offspring that were never exposed to the drug. In fathers, we found changes in the activity of genes, including many long non-coding RNAs, a class of genes that has been linked to transgenerational inheritance. In great-grand-offspring, we found significantly altered DNA methylation profiles, an epigenetic mark that has also been associated with transgenerational inheritance. Together, our findings suggest that developmental exposure to a commonly used stimulant can leave a stable molecular memory in the brain that persists across generations.

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Alfaro, R. J., De Serrano, A. R., Sokolowski, D., Hughes, K. A., Rodd, F. H., Anreiter, I.. 2025-12-18. Methylphenidate exposure alters brain gene expression and induces transgenerational DNA methylation changes in Poecilia reticulata guppies. https://doi.org/10.64898/2025.12.16.694564

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