Abstinence from heroin self-administration increases dendritic spine head diameter and the proportion of mushroom spines in rat prelimbic cortical neurons projecting to the paraventricular thalamus

Relapse to opioid use remains the principal obstacle to recovery, often driven by enduring neuroadaptations that sustain vulnerability across abstinence. We have previously shown that the projection from the rat prelimbic cortex (PL) to the paraventricular thalamus (PVT) is functionally potentiated following abstinence from heroin self-administration and is necessary for cued heroin seeking, yet whether this pathway undergoes structural remodeling remains unknown. Using an intersectional viral strategy to selectively label PL→PVT projection neurons, we reconstructed apical tuft dendritic spines in three dimensions from rats following 14 days of home-cage abstinence from heroin self-administration. We found that abstinence significantly increased spine head diameter (d H ) and shifted the entire head-diameter distribution without altering overall spine density. Using an unsupervised model-based clustering to avoid arbitrary morphometric thresholds, we identified three distinct spine populations corresponding to classical thin, stubby, and mushroom categories. We found that abstinence reduced the density of thin-like spines and increased the density of mushroom-like spines. Notably, this accumulation of mature, mushroom-like spines positively correlated with the total amount of heroin consumed during self-administration. Overall, these findings identify the structural potentiation of excitatory inputs onto PL→PVT projection neurons as a fundamental component of the enduring circuit plasticity that sustains heroin seeking.