Microglia depletion and alcohol gene expression logFC
Description:
Alcohol abuse induces changes in microglia morphology and immune function, but whether microglia initiate or simply amplify the harmful effects of alcohol exposure is still a matter of debate. Here, we determine microglia function in acute and voluntary drinking behaviors using a colony-stimulating factor 1 receptor inhibitor (PLX5622). We show that microglia depletion does not alter the sedative or hypnotic effects of acute intoxication. Microglia depletion also does not change the escalation or maintenance of chronic voluntary alcohol consumption. Transcriptomic analysis revealed that although many immune genes have been implicated in alcohol abuse, down regulation of microglia genes does not necessitate changes in alcohol intake. Instead, microglia depletion and chronic alcohol result in compensatory upregulation of alcohol-responsive, reactive astrocyte genes, indicating astrocytes may play a role in regulation of these alcohol behaviors. Taken together, our behavioral and transcriptional data indicate that microglia are not theprimary effector cell responsible for regulation of acute and voluntary alcohol behaviors. Because microglia depletion did not regulate acute or voluntary alcohol behaviors, we hypothesized that these doses were insufficient to activate microglia and recruit them to an effector phenotype. Therefore, we used a model of repeated immune activation using polyinosinic:polycytidylic acid
We investigated the role of microglia in a mouse model of alcohol dependence using a colony stimulating factor 1 receptor inhibitor (PLX5622) to deplete microglia and a chronic intermittent ethanol vapor two-bottle choice drinking procedure. Additionally, we examined anxiety-like behavior during withdrawal. We then analyzed synaptic neuroadaptations in the central nucleus of the amygdala (CeA) and gene expression changes in the medial prefrontal cortex (mPFC) and CeA from the same animals used for behavioral studies.
The current study used two inbred mouse strains, C57BL/6 J and A/J, to investigate the genetics of behavioral responses to fentanyl. Mice were tested for conditioned place preference and fentanyl-induced locomotor activity. C57BL/6J mice formed a conditioned place preference to fentanyl injections and fentanyl increased their activity. Neither effect was noted in A/J mice. We conducted RNA-sequencing on the nucleus accumbens of mice used for fentanyl-induced locomotor activity. Surprisingly, we noted few differentially expressed genes using treatment as the main factor. However many genes differed between strains.
Authors:
Samuel J Harp, Mariangela Martini, Will Rosenow, Larry D Mesner, Hugh Johnson, Charles R Farber, Emilie F Rissman
Differential gene expression in nucleus accumbens somatostatin interneurons_cocaine_mice_pvalue
Description:
To characterize transcriptional alterations that cocaine induces in these cells, we perform cell type-specific RNA-sequencing on FACS-isolated nuclei of somatostatin interneurons and identified 1100 DETs enriched for processes related to neural plasticity. To profile the entire (non poly-A selected) transcriptome of NAc somatostatin interneurons, we generated a transgenic reporter line (SST-TLG498 mice) to label the nuclei of these cells with a modified form of EGFP that is retained in the nuclear membrane (EGFP-F)22, enabling their isolation from NAc dissections using FACS. We succeeded in FACS-isolating nuclei suitable for RNA-sequencing from individual SST-TLG498 mice. We proceeded with differential expression analysis of the RNA-sequencing data to identify differentially expressed transcripts (DETs) in NAc somatostatin interneurons in response to repeated cocaine exposure: 778 transcripts were upregulated by cocaine and 322 were downregulated.
Authors:
Efrain A Ribeiro, Marine Salery, Joseph R Scarpa, Erin S Calipari, Peter J Hamilton, Stacy M Ku, Hope Kronman, Immanuel Purushothaman, Barbara Juarez, Mitra Heshmati, Marie Doyle, Casey Lardner, Dominicka Burek, Ana Strat, Stephen Pirpinias, Ezekiell Mouzon, Ming-Hu Han, Rachael L Neve, Rosemary C Bagot, Andrew Kasarskis, Ja Wook Koo, Eric J Nestler
DEG effect of brain region 72hrs post-CIE (C57 and D2)_pvalue
Description:
Male C57BL/6J and DBA/2J mice (10 weeks old upon arrival) were purchased from the Jackson Laboratory and assigned to either the air control or CIE group (N = 1 per strain/ treatment). Treatment was coded as 0 for AIR and 1 for CIE, 0 for B6 and 1 for D2. Regions were collapsed into three groups based on the PCA clustering and coded as 0 for hippocampus (CA1 and CA3 regions), 1 for cortex (Prl, ILC, and VCX), and 2 for the remaining subcortical and limbic and mesolimbic tissue (VTA, NAc, NAs, DMS, CeA, and BST). The final data set included 11 brain regions and 87 samples. Mice were allowed to self-administer alcohol (15% v/v vs. water) for 2 h a day (5 days a week) 6 weeks prior to treatment in order to establish baseline consumption. Access to 15% alcohol versus water started 30 min prior to the start of the dark cycle. Following establishment of baseline drinking, two male mice representative of each strain were separated into two groups to be exposed to either weekly cycles of CIE exposure (CIE group) or air control (AIR group) exposure. Mice assigned to the CIE treatment group were exposed to alcohol vapor for 16 h a day followed by 8 h of withdrawal for 4 days. Following the fourth vapor exposure, mice were given a 72-h abstinence period before resuming ethanol intake in the home cage for 5 days. Mice in the AIR control treatment group were similarly treated but exposed only to air in the inhalation chambers. This pattern of CIE or air control exposure followed by 5 days of ethanol self-administration was repeated for four cycles. A fifth cycle of CIE (or air) exposure followed the fourth ethanol intake evaluation period, and brain tissue was collected 72 h after the last cycle ended. To capture expression patterns due to region, strain, and treatment or their interactions, we applied a linear model to the expression data: y ∼ Treatment + Strain + Tissue + Treatment*Strain + Treatment*Region (Supplemental Table 4).
Authors:
Megan K Mulligan, Khyobeni Mozhui, Ashutosh K Pandey, Maren L Smith, Suzhen Gong, Jesse Ingels, Michael F Miles, Marcelo F Lopez, Lu Lu, Robert W Williams
Sig. DEG mouse PFC excitatory neurons (cluster 11) P21 vs. P60_pvalue
Description:
We performed single cell RNA sequencing (scRNA-seq) to classify all neuron subtypes in prefrontal cortex (PFC) of adolescent (P21) (N = 4) and adult (P60) (N = 12) male C57BL/6 mice (strain mentioned but not explicit in publication) to characterize the transcriptional changes associated with this period (P21-P60). 12 independent biological replicates were used for each age. Each biological replicate was generated by pooling brain tissue from two mice (see methods for more info). To detect similar populations and identify corresponding cell clusters between the 10,646 P21 cells and the 11, 886 P60 PFC cells, we aligned the two scRNA-seq data sets in t-SNE by cross-correlation analysis (CCA)17 (Fig. (Fig.4a).4a). Using bootstrapped correlation, all clusters identified in the adult PFC are detected in the P21 PFC. Based on the expression of cell type-specific markers, the non-neuronal cells were clustered as: astrocytes (Gja1+), oligodendrocyte (Aspa+), newly formed (NF) oligodendrocytes (Bmp4+), oligodendrocyte precursors (OPC) (Pdgfra+), microglia (C1qa+) and endothelial cells (Flt1+) (Fig. 1c, d). The neurons express Snap25 and can be divided into excitatory (Slc17a7+) and inhibitory (Gad2+) neurons (Fig. 1c, d). We then analyzed transcriptional dynamics in each of the neuron subtypes between adolescence (P21) and adulthood (P60) in mouse. The differentially expressed genes between P21 and P60 cells for each cluster was performed using the “FindMarkers” function from the Seurat package using a likelihood ratio test and correcting for the number of detected unique molecular identifier (UMI) bias. Genelists contain significantly differentiated genes in each cell population cluster with fold change > 1.5 and p < 0.05.
Authors:
Aritra Bhattacherjee, Mohamed Nadhir Djekidel, Renchao Chen, Wenqiang Chen, Luis M Tuesta, Yi Zhang
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