Psilocybin-associated microRNA changes in the pig brain: Establishing an nCounter pipeline for cross-species microRNA quantification.

Novel treatment strategies are needed to address treatment resistance and delayed onset of action in psychiatry. Psilocybin has shown promise as a treatment for major depressive disorder, but the molecular changes associated with its effects remain incompletely understood. In parallel, microRNAs (miRNAs) have been implicated in depression and in responses to antidepressant interventions. Here, adolescent female pigs were used to explore miRNA expression profiles in the prefrontal cortex (PFC) and hippocampus (HIP) 1 day and 7 days after a single dose of psilocybin. We established a conservative NanoString nCounter workflow for pig tissue using the Human v3b miRNA Assay panel, cross-species reannotation based on complete human-pig sequence conservation, and evaluation of multiple normalization strategies. Using this pipeline, 12 dysregulated miRNAs were identified in the PFC and two in the HIP 1 day after psilocybin administration, whereas four dysregulated miRNAs were detected in the HIP after 7 days. Nine of the 18 miRNAs identified across the two regions have previously been linked to depression-related phenotypes. miR-212-3p and miR-107 showed the most robust acute regulation in the PFC across all four normalization approaches. These findings indicate that psilocybin administration is associated with region- and time-dependent miRNA changes in the pig brain and support the utility of this conservative nCounter pipeline for cross-species miRNA profiling. The study is exploratory and does not establish causality or direct antidepressant mechanisms.