Molecularly-defined cell types within the septal complex and their role in opioid dependence and withdrawal

Abstract

Opioid withdrawal is an unpleasant experience and produces changes in pain, anxiety, depression, and general dysphoria. Because withdrawal is so intense, persons with opioid use disorder (OUD) often return to drug use to relieve this aversive state. Therefore, it is imperative to understand the neural substrates that are disrupted by opioid dependence and withdrawal. Repeated opioid exposure dysregulates neural circuitry involved in natural reward and aversion, thereby shaping a system that drives drug-seeking. Among limbic brain circuitry, the lateral septum (LS) is a forebrain area that gates motivated behaviors based on environmental and emotional contexts. While in vivo pharmacological studies have linked the LS to drug intake, withdrawal, and reinstatement of drug-seeking behavior, this molecularly heterogeneous region lacks clarity regarding the specific cell-types that are affected by long-term opioid exposure and consequently contribute to maladaptive behaviors during opioid withdrawal. To this end, we used single-nucleus RNA-sequencing (snRNAseq) to develop a transcriptional atlas of LS cell types perturbed during morphine dependence and withdrawal. We discovered that chronic morphine altered the transcriptional landscape of most LS neuronal cell classes, modulating genes involved in synaptic transmission and protein synthesis. Naloxone-precipitated withdrawal had the largest effect on two major cell types, one of which is a group of neurons expressing the gene for Neurotensin (Nts; LS-Nts neurons). Using 2-photon calcium imaging, we demonstrate that LS-Nts neurons in morphine-dependent mice remain more active during prolonged opioid withdrawal. Ex vivo electrophysiology revealed that enhanced glutamatergic drive onto LS-Nts neurons may underlie their hyperactivity in the absence of opioids. Finally, we showed that silencing these neurons via Cre-dependent expression of tetanus toxin light chain (TetTox) during opioid withdrawal exacerbates pain coping behaviors and alters sociability in a sexually-dimorphic manner. Together, these results suggest that LS-Nts neurons are a key neural substrate disrupted during opioid withdrawal and establish the LS as a crucial regulator of adaptive behaviors, specifically pertaining to OUD.