Single-nucleus RNA Sequencing Reveals Transcriptome Characters In The Prefrontal Cortex Of Mice Induced By Methamphetamine

Background: Methamphetamine(METH)is one of the most widely used addictive substances in China.Chronic methamphetamine abuse could cause compulsive drug seeking behavior and induce psychosis and cognitive dysfunction.Due to the complexity of cell types,connectivity and interaction of brain,the mechanisms of METH abuse and disorders have been unclear.Single-nucleus RNA sequence(sn-RNA seq)is an approach for massive single nucleus transcriptome analysis,being applied to identify cell subtypes in different brain regions,seek cell-specific gene markers and reveal the cell types and functions which involved in nervous system diseases.Researchers have already used this technique in morphine and cocaine treated animals to search for the alterations in transcriptomes and functions of different cell types in some brain regions.Chronic METH treatment could lead to structure damage,abnormal gene translation and promote neural adaptation in prefrontal cortex(PFC),as well as METH use disorders exacerbated.However,the transcriptional alterations of different types of cells in PFC and how they are involved in METH addiction and use disorders are still unclear.Objective: To explore the effects of chronic methamphetamine treatment on gene transcriptomes of different types of cells in mouse prefrontal cortex,and to provide clues for METH addiction and METH use disorders.Methods: After the 14-days intraperitoneal injection of(5mg/kg/d)methamphetamine,the mice were sacrificed and PFC was homogenized.Single nucleus were obtained by gradient Iodixanol medium solution.Illumina Hiseq platform were used for gene analysis with 2×150 bp double end high throughput sequencing model.Cells which expressed200-6000 numbers of genes and the ratio of UMI in mitochondrial and nucleus were under20% were included in this study.The variable genes were identified by calculating average expression and standardized variance of genes in all cells.PCA was used to calculate the similarities of different cells and UMAP analysis was used to identify a set of Clusters and evaluate the alterations of transcriptomes in different Clusters.The canonical and specific molecular marker genes of mouse prefrontal cortex were used to classify nuclei into different cell types.Analyzing the functions of variable genes in the Clusters which had significant differences in the transcriptomes between two groups by GO and KEGG analysis.The morphology of myelin sheath and its mitochondria were observed by transmission electron microscopy.Results: 20698 cells are included in the research,among which there are 9876 in METH group and 10822 in control group.33 Clusters are identified by PCA and UMAP analysis,of which 6 cell types are classified through gene annotation,including neurons(Rbfox3+)(Cluster 1,3,5-13,15-18,21-25),astrocytes(Gja1+)(Cluster 20),microgila(Cx3cr1+)(Cluster 4),mature oligodendrocytes(Mog+)(Cluster 0,2),oligodendrocyte progenitor cells(Pdgfra+)(Cluster 19)and newly formed oligodendrocytes(Bmp4+)(Cluster 28).Compare with the control group,the transcriptomes of Cluster 5(neurons),Cluster 4(microglia)and Cluster 0,2(mature oligodendrocytes)alter significantly in METH group.Several subtypes of neurons are observed after further analysis.Firstly there are Clusters defined as excitatory neurons(Neurod6+/Slc17a7+/Nrn1+)and inhibitory neurons(Neurod6+/Slc17a7+/Nrn1+).Then excitatory neurons were classified to L2/3(Cux2+),L4(Rorb+),L5(Etv1+)and L6(Syt6+/Ctgf+)neurons according to the six layers’ neuron projection.And the inhibitory neurons are classified by different originations,including Sst+ neurons from medial ganglionic eminence(MGE)and Vip+neurons from caudal ganglionic eminence(CGE).Among all the cell types,the transcriptomes of oligodendrocytes and microglia alter significantly.And the GO and KEGG analysis are performed for those variable genes in those Clusters.The results of GO analysis show that the variable genes of mature oligodendrocytes are related to ATP metabolism process,nucleoside triphosphate metabolism process,cytochrome-c oxidase activity,ATPase coupled ion transmembrane transport activity,ATPase activity,mitochondrial membrane composition and myelin sheath pathways.The variable genes of microglia are enriched in ubiquitin-like protein ligase binding,G protein-coupled receptor binding and monovalent inorganic cation transmembrane transporter activity related pathways.And the inflammation associated genes of microglia significantly increase in METH group.The results of KEGG analysis show that variable genes are enriched in neurodegenerative diseases related pathways,such as Huntington’s disease,Alzheimer’s disease and Parkinson’s disease.The results of transmission electron microscopy showed that the myelin sheath was damaged after chronic treatment with methamphetamine,and mitochondria of mature oligoendrocytes and myelin sheath were swollen,with the disrupted cristae.Conclusions: The transcriptomes of PFC mature oligodendrocytes and microglia alter significantly in METH chronic treated mice,which suggests the essential roles of mature oligodendrocytes and microglia in METH use disorders.