Single-cell profiling of glial cells from the mouse amygdala under opioid dependent and withdrawal states

Abstract

The cycle of substance use disorder (SUD) leading to dependence is a complex process involving multiple neurocircuitries and brain regions. The amygdala is the core brain region that is involved in processing withdrawal and anxiety and depressive-like behaviors. However, the transcriptional changes in each cell type within the amygdala during SUD remains unknown. Here, we performed single-cell RNA sequencing and classified all cell types in the mouse amygdala. We particularly focused on gene expression changes in glial cells under dependent state and compared to either naive or withdrawal state. Our data revealed distinct changes in key biological processes, such as gene expression, immune response, inflammation, synaptic transmission, and mitochondrial respiration. Significant differences were unraveled in the transcriptional profiles between dependence and withdrawal states. This report is the first single-cell RNA sequencing dataset from the amygdala under opioid dependence and withdrawal conditions, providing unique insights in understanding brain alterations during SUD.

Keywords: Neuroscience; Transcriptomics.

Graphical abstract

Figure 1

Identification of cell types in mouse amygdala (A) Schematic of the experimental workflow. Naive: naive mice; Dep: mice under morphine dependence condition; With: mice under morphine withdrawal condition (see details in Figure S1A). (B) UMAP (Uniform Manifold Approximation and Projection) plot showing the clustering of 77,957 cells (27141 from Naive, 27275 from Dep, 23541 from With) based on transcriptome. ASC, (astrocytes, Gja1⁺), OPC (oligodendrocyte progenitor cells, Pdgfra⁺), MG (microglial, Tmem119⁺), EC (endothelial cells, Cldn5⁺), NEUR (neurons, Syt1⁺), OLG (oligodendrocytes, Mobp⁺ or Cldn11⁺), MAC (macrophages, Pf4⁺), PC (pericytes, Vtn⁺), NFOLG (newly formed oligodendrocytes, Enpp6⁺), VSMC (vascular smooth muscle cells, Acta2⁺), DC (dendritic cells, Cd74⁺), EPC (ependymocytes, Ccdc153⁺), NSC (neural stem cells, Thbs4⁺), ARP (astrocyte-restricted precursors, Cd44⁺), NRP (neuronal-restricted precursors, Top2a⁺), T CELLS (T cells, Cd3d⁺), and NEUT (neutrophils, S100a9⁺). (C) UMAP plots of 9 cell populations showing the expression of cell type-specific/enriched marker genes. (D) Violin plot showing the expression of well-known cell type-specific/enriched marker genes in 17 cell clusters. (E) Bar plots showing the number of cells, number of detected genes in each cluster, the average number of features (genes) and UMI counts in each cluster. See also Figures S1–S8, Tables S1, S2, S3, S6, and S8.

Figure 2

Differentially expressed genes under morphine dependence and withdrawal conditions (A and B) Strip charts showing the logarithmic fold changes (log2FC) of all detected genes (dots) in 17 clusters. Genes in colored dots are significantly changed (FDR < 0.05 and FC > 10%) comparing Dep to Naive (A), or With to Dep (B). (C, and D) Bar plots showing the number of significantly downregulated (Down) or upregulated (Up) genes in 17 clusters, comparing Dep to Naive (C), or With to Dep (D). (E and F) Volcano plots showing the log2FC and -log10(FDR) of detected genes in MG, comparing Dep to Naive (E), or With to Dep (F). Significantly downregulated genes are dots in blue, upregulated genes are in red and genes in black are not significantly changed. (G) Dot plot showing the overlap (dots in red) of DEG in Dep vs. Naive (dots in green) with DEG in With vs. Dep (dots in blue) in MG. (H and I) Heatmaps showing the log2FC of heat shock protein expressions comparing Dep to Naive (H), or With to Dep (I) in 17 clusters. ∗ FDR < 0.05, ∗∗ FDR < 0.01, ∗∗∗ FDR < 0.001. See also Figures S5 and S6, Tables S4, S5, and S7.

Figure 3

Validation of single-cell RNA sequencing data in microglia cells (A) Violin plot overlaid with dot plot showing the expression levels of Dnaja1 in our scRNA-seq data of MG population. (B) Violin plot overlaid with boxplot showing the quantification of the RNAscope data. Data represent median expression of Dnaja1 (number of mRNA puncta) in Tmem119⁺ MG cells (n = 249 cells from 5 Naive mice, n = 280 cells from 5 Dep mice, n = 288 cells from 5 With mice). ∗∗∗∗ p value < 0.0001 by Mann-Whitney U-test. (C) Representative RNAscope images of mouse amygdala showing the Dnaja1 mRNA puncta in Tmem119⁺ MG cells. Dotted lines outline the area of each cell that was considered for quantification. Scale bars, 5 μm. (D) Violin plot overlaid with dot plot showing the expression levels of Ccl2 in our scRNA-seq data of MG population. (E) Violin plot overlaid with boxplot showing the quantification of the RNAscope data. Data represent median expression of Ccl2 (number of mRNA puncta) in Tmem119⁺ MG cells (n = 291 cells from 5 Naive mice, n = 303 cells from 5 Dep mice, n = 313 cells from 5 With mice). ∗∗∗∗ p value < 0.0001 by Mann-Whitney U-test. (F) Representative RNAscope images of mouse amygdala showing the Ccl2 mRNA puncta in Tmem119⁺ MG cells. Scale bars, 5 μm. (G) qPCR analysis of Tnf, Il1beta, and Tlr2 in MG cells isolated from the amygdala of Naive, Dep and With mice. p values were analyzed by two tailed Student’s t test. Data represent mean $\pm$ SEM of independent triplicate measurements. (H) Scatterplot of the fold changes of the DEGs in the bulk RNA-seq dataset (isolated microglia cells) and scRNA-seq dataset (MG population). Linear regression is depicted with the blue line. Pearson’s correlation coefficient and p value are shown in the plot. See also Figure S9.

Figure 4

Changes in biological pathways and processes under morphine dependence and withdrawal conditions (A and B) Enrichment maps of significant pathways (p value < 0.05 and FDR q value < 0.15) in MG comparing Dep to Naive (A), or With to Dep (B). Normalized enrichment scores (NES) were calculated for each pathway by GSEA. The networks were created using EnrichmentMap Cytoscape application. Pathways are shown as nodes which are colored by the corresponding NES, and edges represent the number of genes overlapping between two pathways. Clusters of nodes were labeled using the AutoAnnotate Cytoscape application to identify major biological themes. Positive NES (red nodes) indicate upregulation, while negative NES (blue nodes) indicate downregulation. (C) Heatmap of NES showing a subset of significant pathways (p value < 0.05 and FDR q value < 0.25) in 17 cell types comparing Dep to Naive (left), or With to Dep (right). Positive NES indicates upregulation, negative NES indicates downregulation and white indicates no significant change. See also Tables S9 and S10.

Figure 5

Changes in immune/inflammation related pathways and genes under morphine dependence and withdrawal conditions (A) Heatmap of NES showing a subset of immune/inflammation related significant pathways (p value < 0.05 and FDR q value < 0.25) in 8 cell types comparing Dep to Naive (left), or With to Dep (right). Positive NES indicates upregulation, negative NES indicates downregulation, and white indicates no significant change. (B) Heatmap showing the log2FC of inflammation related gene expressions comparing Dep to Naive in MG, MAC, DC, EC. (C and D) Heatmaps showing the log2FC of inflammation related gene expressions comparing With to Dep in MG, MAC, DC, EC (C), and in EPC, NFOLG, OLG, NEUR (D). ∗ FDR < 0.05, ∗∗ FDR < 0.01, ∗∗∗ FDR <0.001. See also Tables S9 and S10.

Figure 6

Changes in cell-cell interactions with chronic morphine treatment Ligands or receptors in the denoted cell type are represented by the nodes which are colored by the log2FC. Node borders indicate the FDR value of the DEG analysis. Edges represent ligand-receptor interactions. Edge color indicates the sum of scaled differential expression magnitudes from the ligand node and receptor node. The Figures have been filtered so that the top 75 edges representing the most differentially expressed node pairs are shown. The comparisons were between Dep and Naive samples. (A) Ligands expressed in MG with receptors expressed in NEUR. (B) Ligands expressed in NEUR with receptors expressed in MG. (C) Ligands expressed in MG with receptors expressed in EC. (D) Ligands expressed in EC with receptors expressed in MG. See also Figure S10.

Figure 7

Changes in cell-cell interactions during morphine withdrawal The comparisons were between With and Dep samples. (A) Ligands expressed in MG with receptors expressed in EC. (B) Ligands expressed in EC with receptors expressed in MG. (C) Ligands expressed in OLG with receptors expressed in NEUR. (D) Ligands expressed in EPC with receptors expressed in NEUR. See also Figure S11.