Average rotarod training latency Chr# 5 rs13478110 (9741228) with right flanking marker rs13478092(3595407) and left marker rs3718776 (150393227). This was mapped in 300 + (b6x129)F2 mice.
640 Diversity Outbred (DO) mice were exposed to 4 weeks of intermittent ethanol access (IEA) via 3-bottle choice (H20, 15% EtOH, 30% EtOH). 200 prefrontal cortex (PFC) samples were sent for RNA-seq. After QC and GBRS alignment, Pearson correlations were calculated for all genes with variant-stabilized transcript counts >1 and week four mean ethanol consumption. This gene set provides the list of genes for which FDR-corrected p-values < 0.05 and their Pearson correlation scores.
640 Diversity Outbred (DO) mice were exposed to 4 weeks of intermittent ethanol access (IEA) via 3-bottle choice (H20, 15% EtOH, 30% EtOH). 200 prefrontal cortex (PFC) samples were sent for RNA-seq. After QC and GBRS alignment, Pearson correlations were calculated for all genes with variant-stabilized transcript counts >1 and week four mean ethanol preference over water. This gene set provides the list of genes for which FDR-corrected p-values < 0.05 and their Pearson correlation scores.
Drug Naïve DO mice were tested for open field, light dark, hole board, novelty place preference before collecting the striatum. RNA-Seq data was analyzed with WGCNA using a soft thresholding power of 3 selected using the WGCNA scale-free topology R2 threshold of 0.9, signed network with a minimum module size of 30, correlation type is bicor, used numeric labels.
Chronic alcohol abuse alters the molecular structure and function of brain cells. Recent work suggests adaptations made by glial cells, such as astrocytes and microglia, regulate physiological and behavioral changes associated with addiction. Defining how alcohol dependence alters the transcriptome of different cell types is critical for developing the mechanistic hypotheses necessary for a nuanced understanding of cellular signaling in the alcohol-dependent brain. We performed RnA-sequencing on total homogenate and glial cell populations isolated from mouse prefrontal cortex (pfc) following chronic intermittent ethanol vapor exposure (cie). compared with total homogenate, we observed unique and robust gene expression changes in astrocytes and microglia in response to cie. Gene co-expression network analysis revealed biological pathways and hub genes associated with cie in astrocytes and microglia that may regulate alcohol-dependent phenotypes. Astrocyte identity and synaptic calcium signaling genes were enriched in alcohol-associated astrocyte networks, while tGf-β signaling and inflammatory response genes were disrupted by CIE treatment in microglia gene networks. Genes related to innate immune signaling, specifically interferon pathways, were consistently up-regulated across cie-exposed astrocytes, microglia, and total homogenate pfc tissue. This study illuminates the cell-specific effects of chronic alcohol exposure and provides novel molecular targets for studying alcohol dependence.
Authors:
Emma K Erickson, Yuri A Blednov, R Adron Harris, R Dayne Mayfield
Alcohol transcriptome changes in mice microglia log2FC
Description:
Microglia are fundamentally important immune cells within the central nervous system (CNS) that respond to environmental challenges to maintain normal physiological processes. Alterations in steady-state cellular function and over-activation of microglia can facilitate the initiation and progression of neuropathological conditions such as Alzheimer’s disease, Multiple Sclerosis, and Major Depressive Disorder. Alcohol consumption disrupts signaling pathways including both innate and adaptive immune responses that are necessary for CNS homeostasis. Coordinate expression of these genes is not ascertained from an admixture of CNS cell-types, underscoring the importance of examining isolated cellular populations to reveal systematic gene expression changes arising from mature microglia. Unbiased RNA-Seq profiling was used to identify gene expression changes in isolated prefrontal cortical microglia in response to recurring bouts of voluntary alcohol drinking behavior. The voluntary ethanol paradigm utilizes long-term consumption ethanol that results in escalated alcohol intake and altered cortical plasticity that is seen in humans. Gene coexpression analysis identified a coordinately regulated group of genes, unique to microglia, that collectively are associated with alcohol consumption. Genes within this group are involved in toll-like receptor signaling and transforming growth factor beta signaling. Network connectivity of this group identified Siglech as a putative hub gene and highlighted the potential importance of proteases in the microglial response to chronic ethanol. In conclusion, we identified a distinctive microglial gene expression signature for neuroimmune responses related to alcohol consumption that provides valuable insight into microglia-specific changes underlying the development of substance abuse, and possibly other CNS disorders.
Authors:
Gizelle M McCarthy, Sean P Farris, Yuri A Blednov, R Adron Harris, R Dayne Mayfield
Alcohol transcriptome changes in mice microglia total homogenate log2FC
Description:
Microglia are fundamentally important immune cells within the central nervous system (CNS) that respond to environmental challenges to maintain normal physiological processes. Alterations in steady-state cellular function and over-activation of microglia can facilitate the initiation and progression of neuropathological conditions such as Alzheimer’s disease, Multiple Sclerosis, and Major Depressive Disorder. Alcohol consumption disrupts signaling pathways including both innate and adaptive immune responses that are necessary for CNS homeostasis. Coordinate expression of these genes is not ascertained from an admixture of CNS cell-types, underscoring the importance of examining isolated cellular populations to reveal systematic gene expression changes arising from mature microglia. Unbiased RNA-Seq profiling was used to identify gene expression changes in isolated prefrontal cortical microglia in response to recurring bouts of voluntary alcohol drinking behavior. The voluntary ethanol paradigm utilizes long-term consumption ethanol that results in escalated alcohol intake and altered cortical plasticity that is seen in humans. Gene coexpression analysis identified a coordinately regulated group of genes, unique to microglia, that collectively are associated with alcohol consumption. Genes within this group are involved in toll-like receptor signaling and transforming growth factor beta signaling. Network connectivity of this group identified Siglech as a putative hub gene and highlighted the potential importance of proteases in the microglial response to chronic ethanol. In conclusion, we identified a distinctive microglial gene expression signature for neuroimmune responses related to alcohol consumption that provides valuable insight into microglia-specific changes underlying the development of substance abuse, and possibly other CNS disorders.
Authors:
Gizelle M McCarthy, Sean P Farris, Yuri A Blednov, R Adron Harris, R Dayne Mayfield
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
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
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
Analysis using RNA-seq of FACS-purified oligodendrocytes revealed a large cohort of morphine-regulated genes. In addition, to investigate cell-type-specific opioid responses, we performed single-cell RNA sequencing (scRNA-seq) of the nucleus accumbens of mice following acute morphine treatment. Differential expression analysis uncovered unique morphine-dependent transcriptional responses by oligodendrocytes and astrocytes.
Authors:
Denis Avey, Sumithra Sankararaman, Aldrin K Y Yim, Ruteja Barve, Jeffrey Milbrandt, Robi D Mitra
Differential gene expression in nucleus accumbens somatostatin interneurons_cocaine_mice_logFC
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
DEG female mouse forebrain 3-tri morphine vs saline_pvalue
Description:
To examine forebrain transcriptomic changes that might elucidate mechanisms of withdrawal, delayed development, and any long-term behavior changes, we generated transcriptomic signatures following our “3-trimester” exposure model (3-Tri). In addition, we also examined transcriptomes from animals that received opioids only during the gestational period (PND1) or only during the last trimester from PND 1–14 (PND 14). We sought to determine whether transcriptomic signatures vary based on the window of exposure, perhaps contributing to the discrepancies in the literature regarding acute and long-term outcomes. Brains were dissected from PND 1 pups 6 h after discovery. Brains were dissected from post-natal exposure only (PND 14) or 3-trimester exposure (3-tri) 6 h after the last morphine or saline injection. The number of animals per group was similar (N = 5–7 animals, male and female C57Bl/6NTac mice), and the quality controls, library construction and sequence parameters were also identical across all groups. Libraries were sequenced on a NovaSeq 6000 at a depth of 30 million total reads/sample using paired-end sequencing of 150 base pairs (PE150), to a depth of 30 million total reads/sample. Reads were then mapped to the mouse reference genome (Mus Musculus, GRCm38/mm10) using HISAT2 (version 2.2.1), and duplicated fragments were removed using Picard MarkDuplicates. Differential expression analysis between two conditions (e.g., Morphine and Saline) was performed in R (version 4.1.1) with DESeq2 (v1.32.0) package. Genes were assigned by the authors as differentially expressed if the (adjusted) (nominal) p-value < 0.05. All genes/scores are presented here.
Authors:
Amelia D Dunn, Shivon A Robinson, Chiso Nwokafor, Molly Estill, Julia Ferrante, Li Shen, Crystal O Lemchi, Jordi Creus-Muncunill, Angie Ramirez, Juliet Mengaziol, Julia K Brynildsen, Mark Leggas, Jamie Horn, Michelle E Ehrlich, Julie A Blendy
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