Mice treatment and result of behaviour tests.

(A) The construction mouse model of chronic methamphetamine abuse. (B) The track visualizations and heatmaps of Y maze in 2 and 4 weeks. (C) The track visualizations, heatmaps and the discrimination and preference index of 2 weeks NOR test. (D-E) The total exploration time and frequency of familiar or novel objects of 2 weeks NOR test. (F) The track visualizations, heatmaps and the discrimination and preference index of 4 weeks NOR test. (G-H) The total exploration time and frequency of familiar or novel objects of 4 weeks NOR test. Asterisks indicate statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). All data are presented as mean ± SEM.

Hippocampus cell clusters of saline and METH mice.

(A) The Umap-distributed plots showing 31 hippocampal cell clusters in saline and METH groups. (B) The average expression of known marker genes of hippocampal clusters. (C) The percentage of cells in hippocampal clusters in saline and METH groups. (D) The marker genes expression’s featureplots of astrocyte, endothelia, microglia and oligodendrocyte.

Differentally expressed genes(DEGs) induced by chronic METH exposure in hippocampal cell types.

(A) The manhattanplot showing the number of DEGs in each hippocampal cell types(METH v.s. saline), the red dots and number represented for up-regulated genes and the blue ones represented for down-regulated genes. (B) The upset plot showing the number of all of unique and some of shared genes of hippocampal cell types. (C-F) The biocprocess in GO terms enriched separately by up or down-regulated genes of astrocyte, endothelia, microglia and oligodendrocyte. Criteria for DEGs by Muscat: p_value < 0.05, abs(log2FC) > 0.25).

Hippocampal cell types crosstalk alterations induced by chronic METH treatment.

(A) The total number of cell communication interactions and the intensity of interactions in saline and METH groups. (B-C) The comparison of the number and the strength of interactions of each cell types between METH and saline groups. (D-E) The incoming and outgoing communication patterns of secreting cells of each groups and the inflection point parameter in clustering algorithm. (F) The identificaton of signaling networks with large (or small) differences according to the Euclidean distance in a shared two-dimensional space. (G) The comparison of the overall information flow of each signal path between two groups. (H) Heatmap showing the incoming, outgoing and all communicated informations’ comparison of each signal of each cell type.

The development direction of hippocampal neural stem cells and the regeneration of hippocampal nerve were analyzed in pseudo-time series.

(A, C) The pseudo temporal differentiation locus of neural stem cells, astrocytes and neuroblasts in saline(A) or METH(C) group. (B, D) Heatmap showing the expression of specific genes(rows) in subclusters (columns) along the maturation trajectory from neural stem cells to neuroblasts or astrocytes in saline(B) or METH(D) group, and GO or KEGG funtional enrichment of those genes. (E, F) Pseudotime expression graphs of 4 representative specific marker genes in these cell types showing the development tendency difference in saline(E) or METH(F) group.

Transcription factor(TF) analysis and function prediction.

(A-B) Heatmap of the average expression and the area under the curve (AUC) scores of TF motifs estimated per cell by SCENIC. Shown are top five differentially activated motifs in each cell type of saline and METH groups, respectively. (C) The upset plot showing the number of all of unique and some of shared TFs of hippocampal cell types. (D-G) Gene regulatory network analysis and function enrichment using top 10 of SCENIC identifies critical TFs of astrocytes, endothelia, microglia and oligodendrocytes. (H) Enrichment analysis of target genes predicted by TFs of neuronal development related cell types(Neuron from NSC, NSC and neuroblast).