Repeated TLR7 activation induces cell type- and brain region-specific transcriptome changes in male mice

Alcohol consumption triggers neuroinflammation, potentially creating a feed-forward loop that increases drinking. Previous studies have shown that activation of the toll-like receptor 7 (TLR7) leads to escalated drinking. In this study, we aim to identify cell type-specific transcriptomic patterns underlying TLR7-induced neuroinflammation, potentially leading to escalated drinking. Therefore, male C57BL/6J mice were treated with the selective TLR7 agonist R848 every-other day for 20 days in total. After 10 treatment-free days, half of the cohort underwent two bottle-choice drinking; the other half was sacrificed and brains were collected for single-nucleus RNA-Sequencing (snRNA-Seq) in the medial prefrontal cortex (mPFC) and central amygdala (AMG). The AMG showed a greater number of differentially expressed genes (DEGs), primarily in inhibitory and excitatory neurons. Among glial cells, AMG astrocytes exhibited the greatest number of DEGs, which were involved in blood-brain barrier (BBB) regulation (e.g., Cldn5, Mecom, Nrg1), a finding supported by secondary validation using Xenium in situ spatial transcriptomics on AMG-containing sections. BBB-regulatory genes, including those in the Wnt signaling pathway (e.g., Notch3, Top2a, Aldoc), were altered across multiple cell types in both regions. Together with the alterations observed in neurons, these findings suggest that repeated TLR7 activation induces persistent BBB and neuronal dysregulation, potentially leading to TLR7-induced escalated drinking.