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 nuclear accumbens (NA) and amygdale (Amyg) of inbred alcohol-preferring mice were examined for differential gene expression and it findings suggest that changes in gene expression in the ACB of iP rats are associated with the reinforcing effects of ethanol (EtOH). This gene set comprises 201 genes that were significantly expressed in the NA during the experiment.
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 paraclique using a bicor was used with a correlation coefficient threshold of |0.5| (unsigned), minimum seed clique size of 5, minimum finished paraclique size of 10, proportional glom factor of 0.2 for paraclique construction.
Ethanol Induced Hypothermia Chr# 7 rs13479153(25722935) with right flanking marker rs3700068(4187548) and left marker rs3716088(140189839). This was mapped in 300 + (b6x129)F2 mice.
Average rotarod training latency Chr# 7 mCV23423763(68111945) with right flanking marker rs3700068(4187548) and left marker rs3663988(146505067). This was mapped in 300 + (b6x129)F2 mice.
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
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.
Alcohol interaction of dependence and MG depletion the medial prefrontal cortex q-value
Description:
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.
H3 dopaminylation at glutamine 5 (H3Q5dop) plays a critical role in heroin-mediated transcriptional plasticity in midbrain regions, particularly the VTA. In rats undergoing abstinence from heroin self-administration (SA), we found acute and persistent accumulation of H3Q5dop in VTA. Attenuation of H3Q5dop during abstinence induced persistent changes in gene expression programs associated with neuronal signaling and dopaminergic function in heroin abstinence and led to reduced heroin-seeking behavior. Interestingly, the observed changes in molecular pathways after heroin SA showed significant yet reversed overlap with the same genes altered in cocaine SA.
Authors:
Sasha L Fulton, Swarup Mitra, Ashley E Lepack, Jennifer A Martin, Andrew F Stewart, Jacob Converse, Mason Hochstetler, David M Dietz, Ian Maze
Alcohol dependence in the medial prefrontal cortex q-value
Description:
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.
Alcohol transcriptome changes in mice microglia total homogenate p-value
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
Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that act as ligand-activated transcription factors. Although prescribed for dyslipidemia and type-II diabetes, PPAR agonists also possess anti-addictive characteristics. PPAR agonists decrease ethanol consumption and reduce withdrawal severity and susceptibility to stress-induced relapse in rodents. However, the cellular and molecular mechanisms facilitating these properties have yet to be investigated. We tested three PPAR agonists in a continuous access two-bottle choice (2BC) drinking paradigm and found that tesaglitazar (PPARα/γ; 1.5 mg/kg) and fenofibrate (PPARα; 150 mg/kg) decreased ethanol consumption in male C57BL/6J mice while bezafibrate (PPARα/γ/β; 75 mg/kg) did not. We hypothesized that changes in brain gene expression following fenofibrate and tesaglitazar treatment lead to reduced ethanol drinking. We studied unbiased genomic profiles in areas of the brain known to be important for ethanol dependence, the prefrontal cortex (PFC) and amygdala, and also profiled gene expression in liver. Genomic profiles from the non-effective bezafibrate treatment were used to filter out genes not associated with ethanol consumption. Because PPAR agonists are anti-inflammatory, they would be expected to target microglia and astrocytes. Surprisingly, PPAR agonists produced a strong neuronal signature in mouse brain, and fenofibrate and tesaglitazar (but not bezafibrate) targeted a subset of GABAergic interneurons in the amygdala. Weighted gene co-expression network analysis (WGCNA) revealed co-expression of treatment-significant genes. Functional annotation of these gene networks suggested that PPAR agonists might act via neuropeptide and dopaminergic signaling pathways in the amygdala. Our results reveal gene targets through which PPAR agonists can affect alcohol consumption behavior.
Authors:
Laura B Ferguson, Dana Most, Yuri A Blednov, R Adron Harris
Chronic cocaine - Cocaine-paired (conditioned place preference) vs. Control (saline or cocaine-non-paired) DNA microarray All genes on microarray presented After the pre-conditioning phase where animals were allowed access to either compartment for 15 minutes for 4 consecutive days, the conditioning phase for the cocaine-paired groups and cocaine non-paired groups began, consisting of eight subsequent daily sessions. For both groups, cocaine (10 mg / kg) or saline injections were administered on alternate days. For the cocaine-paired groups, rats were immediately placed in one of the two compartments for 30 min with the door in place restricting a z transformation followed by z test and anova followed by Student-Newman-Keuls' post hoc test. Gene expression profile was assessed 24 h after the last conditioning session that corresponded to 48 h after last cocaine exposure, when drug has been eliminated from the body and transient transcriptional changes are likely to be minimal. Therefore, changes in gene expression at this time-point are likely to reflect longer lasting adaptations that may account for maintenance of cocaine-induced memories. The complete lists of normalized gene expression values for the hippocampus of saline-treated, cocaine non-paired and cocaine-paired groups are presented. Analyses revealed that 214 transcripts were differentially regulated in the hippocampus of cocaine-paired rats vs. non-paired and saline-treated controls. Cocaine-induced conditioned place preference caused significant increases in the expression of 151 genes and caused decreases in the expression of 63 genes. (NIF Table ID 130.1 [83])
Authors:
Krasnova IN, Li SM, Wood WH, McCoy MT, Prabhu VV, Becker KG, Katz JL, Cadet JL
Chronic cocaine - Cocaine-paired (conditioned place preference) vs. Control (saline or cocaine-non-paired) DNA microarray All genes on microarray presented After the pre-conditioning phase where animals were allowed access to either compartment for 15 minutes for 4 consecutive days, the conditioning phase for the cocaine-paired groups and cocaine non-paired groups began, consisting of eight subsequent daily sessions. For both groups, cocaine (10 mg / kg) or saline injections were administered on alternate days. For the cocaine-paired groups, rats were immediately placed in one of the two compartments for 30 min with the door in place restricting a z transformation followed by z test and anova followed by Student-Newman-Keuls' post hoc test. Gene expression profile was assessed 24 h after the last conditioning session that corresponded to 48 h after last cocaine exposure, when drug has been eliminated from the body and transient transcriptional changes are likely to be minimal. Therefore, changes in gene expression at this time-point are likely to reflect longer lasting adaptations that may account for maintenance of cocaine-induced memories. The complete lists of normalized gene expression values for the frontal cortex of saline-treated, cocaine non-paired and cocaine-paired groups are presented. Differences in the expression of 39 transcripts in the frontal cortex were related to the conditioned place preference paradigm. These include increases in the level of 22 genes and decreases in 17 genes. (NIF Table ID 130.3 [83.5])
Authors:
Krasnova IN, Li SM, Wood WH, McCoy MT, Prabhu VV, Becker KG, Katz JL, Cadet JL
Differentially expressed geens in the central amygdala (CeA) of male Sprague-Dawley rats (300-350 g prior to surgery, 325-375 g at start of self-administration) on day 2 following methamphetamine withdrawal. Gene Expression was evaluated via RNA-seq. Data taken from Supplementary Table S2. Values presented are adjusted p-values. Data available from GEO with accession number GSE111243."
Authors:
Hannah M Cates, Xuan Li, Immanuel Purushothaman, Pamela J Kennedy, Li Shen, Yavin Shaham, Eric J Nestler
Differentially expressed geens in the central amygdala (CeA) of male Sprague-Dawley rats (300-350 g prior to surgery, 325-375 g at start of self-administration) on day 35 following methamphetamine withdrawal. Gene Expression was evaluated via RNA-seq. Data taken from Supplementary Table S2. Values presented are adjusted p-values. Data available from GEO with accession number GSE111243."
Authors:
Hannah M Cates, Xuan Li, Immanuel Purushothaman, Pamela J Kennedy, Li Shen, Yavin Shaham, Eric J Nestler
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