Single nucleus transcriptomic analysis of rat nucleus accumbens reveals cell type-specific patterns of gene expression associated with volitional morphine intake

Abstract

Opioid exposure is known to cause transcriptomic changes in the nucleus accumbens (NAc). However, no studies to date have investigated cell type-specific transcriptomic changes associated with volitional opioid taking. Here, we use single nucleus RNA sequencing (snRNAseq) to comprehensively characterize cell type-specific alterations of the NAc transcriptome in rats self-administering morphine. One cohort of male Brown Norway rats was injected with acute morphine (10 mg/kg, i.p.) or saline. A second cohort of rats was allowed to self-administer intravenous morphine (1.0 mg/kg/infusion) for 10 consecutive days. Each morphine-experienced rat was paired with a yoked saline control rat. snRNAseq libraries were generated from NAc punches and used to identify cell type-specific gene expression changes associated with volitional morphine taking. We identified 1106 differentially expressed genes (DEGs) in the acute morphine group, compared to 2453 DEGs in the morphine self-administration group, across 27 distinct cell clusters. Importantly, we identified 1329 DEGs that were specific to morphine self-administration. DEGs were identified in novel clusters of astrocytes, oligodendrocytes, and D1R- and D2R-expressing medium spiny neurons in the NAc. Cell type-specific DEGs included Rgs9, Celf5, Oprm1, and Pde10a. Upregulation of Rgs9 and Celf5 in D2R-expressing neurons was validated by RNAscope. Approximately 85% of all oligodendrocyte DEGs, nearly all of which were associated with morphine taking, were identified in two subtypes. Bioinformatic analyses identified cell type-specific upstream regulatory mechanisms of the observed transcriptome alterations and downstream signaling pathways, including both novel and previously identified molecular pathways. These findings show that volitional morphine taking is associated with distinct cell type-specific transcriptomic changes in the rat NAc and highlight specific striatal cell populations and novel molecular substrates that could be targeted to reduce compulsive opioid taking.

Functional genomic mechanisms of opioid action and opioid use disorder: a systematic review of animal models and human studies

“…This therefore uncovers a hierarchy in transcriptional sensitivity to opioids among NAc cell-types, and extend on the previous investigation of microglia[24]. More recently, Reiner et al [36] used single-nucleus RNA-Seq (snRNA-seq, 10x Genomics 3’ gene expression assay) to analyze a larger number of nuclei (190,030) and genes (>2000) in the rat NAc, also following acute morphine injection (10 mg/kg, ip). DEG mostly affected neurons and oligodendrocytes, followed by astrocytes and microglia, reminiscent of the former study.…”

“…In other words, there are shared genetic determinants for brain adaptations triggered by acute or chronic exposure. A recent single-cell RNA-sequencing (scRNA-Seq) study [36] provides further evidence that these genetic determinants translate into at least partially similar transcriptional plasticity during acute or chronic opioid exposure (see 3. High cellular resolution approaches ).…”

“…We next directly confronted results from these 2 single-cell studies regarding acute morphine exposure. Both used Seurat [122] and dimensionality reduction (t-SNE [40] or UMAP [36]) to identify clusters, which were then assigned to cell-types using external data. We first compared marker genes reported to define those clusters, when available (Avey: 11/29 clusters; Reiner: 11/19 clusters).…”

Unraveling Psychiatric Disorders through Neural Single-Cell Transcriptomics Approaches

“…A single intraperitoneal injection of morphine in mice led to the detection of oligodendrocyte-specific morphine-dependent differential gene expression ( Cdkn1a , Phactr3 , and Sgk1 ) in myelin-forming OL and mature OL in the NAc [ 6 ]. Our previous work also demonstrated transcriptional changes in OL between acute morphine exposure and repeated morphine self-administration in rats [ 54 ]. Besides OL, astrocytes and D1R- and D2R-expressing MSNs also had altered transcriptomes in the rat NAc [ 54 ].…”

“…Our previous work also demonstrated transcriptional changes in OL between acute morphine exposure and repeated morphine self-administration in rats [ 54 ]. Besides OL, astrocytes and D1R- and D2R-expressing MSNs also had altered transcriptomes in the rat NAc [ 54 ]. Importantly, this work differentiated between cell type-specific transcriptional alterations that were associated with acute opioid exposure, repeated opioid self-administration, and the act of volitional opioid taking.…”