665. CELLULAR MECHANISMS OF NICOTINE CRAVING

Abstract Background Tobacco-related diseases result in approximately 8 million premature deaths worldwide each year. Most smokers attempting to quit relapse within 48 h, when peak craving is experienced. Cellular mechanisms governing the risk of relapse in abstinent smokers are poorly understood. Aims & Objectives This study first aimed to establish conditions under which intravenous nicotine self-administration precipitated craving-related behaviors in rats and mice during abstinence. Then, we sought to identify brain regions in which neural activity was modified during nicotine abstinence using unbiased whole-brain activity mapping procedures. Finally, we used single-cell and spatial transcriptomics and single-cell connectomic mapping to investigate the cellular and circuit-level mechanisms of nicotine craving. Method Rats and mice consumed nicotine via intravenous self-infusions. Whole-brain c-Fos mapping, functional magnetic resonance imaging (fMRI), and in vivo electrophysiological recordings were used to identify brain regions in which neural activity was modified by nicotine self-administration. Multiplexed Analysis of Projections by Sequencing (MAPseq) and slice electrophysiological recordings were used to define the projection profiles of neurons. High-resolution spatial transcriptomics and single-nuclei RNA sequencing (snRNA-seq) were used to characterize the molecular identity of relevant neurons and nicotine-induced alterations in their transcriptional profiles. DREADD-based chemogenetics was used to modify neural activity in animals. Results We found that self-administration of amounts of nicotine sufficient to elicit intense craving during abstinence inhibits neural activity in the triangular septal nucleus (TSn), a largely unexplored limbic structure. MAPseq-based connectomic profiling showed that TSn neurons project to brain regions involved in memory and motivation, including the medial habenula (mHb). Spatial and single-nuclei transcriptomics revealed that mHb-projecting TSn neurons are remodeled by nicotine, with gene programs involved in excitatory synaptic transmission downregulated by the drug. Accordingly, slice physiology showed that nicotine induced profound deficits in excitatory synaptic transmission in the TSn-mHb circuit. Finally, we show that lesioning or chemogenetically silencing the TSn-mHb circuit exacerbates craving-like nicotine seeking during abstinence, while stimulating the TSn-mHb circuit attenuated nicotine craving. Discussion & Conclusions Our findings identify the TSn as a critical regulator of nicotine craving during abstinence. Using whole-brain activity mapping, transcriptomics, and functional assays, we demonstrate that nicotine suppresses excitatory synaptic transmission in the TSn-mHb circuit, a previously unrecognized mechanism of drug craving. Disrupting this pathway exacerbates nicotine-seeking behavior, while its activation mitigates craving. These results highlight the TSn-mHb circuit as a potential therapeutic target for nicotine dependence and relapse prevention.