Conference Agenda

Symposium Proposals Genes, Brain and Behavior 2024

Abstract

Symposium Chair

Chair Name:
Rajani Maiya

Co-Chair Name (Optional):
Igor Ponomarev

Email:
rmaiya@lsuhsc.edu

Organization:
LSU Health Sciences Center, New Orleans

Department:
Physiology

Position:
Assistant Professor

Species:
Mouse

Chronic alcohol (ethanol) use alters brain gene expression and results in neuroadaptations that lead to tolerance and dependence. Alcohol’s effects on gene expression are diverse and cell-type specific. Recent advances in molecular, genomics, and bioinformatics approaches have allowed us to extract these cell-type specific alcohol-induced gene expression signatures. This symposium will share unpublished findings using a range of cutting-edge molecular techniques to uncover cell-type specific neuroadaptations caused by alcohol at the transcriptome and epigenome level in a variety of rodent models of excessive alcohol intake. Dr. Igor Ponomarev will discuss the role of brain microvasculature in the neuroimmune modulation of alcohol consumption in mice. He will present data on transcriptional signatures of neuroimmune activation and chronic drinking in enriched brain vasculature in high drinking FVB/NJ X C57BL/6J F1 hybrid mice. Dr. Amy Lasek will talk about the transcription factor STAT3 and its role in regulating binge alcohol consumption. Dr. Lasek will present new findings examining STAT3 signaling in the PFC and dorsal and ventral hippocampus (dHPC and vHPC) of mice after Drinking in the dark (DID) procedure. DID led to activation of STAT3 and increased expression of known and predicted STAT3 target genes. Experiments are currently underway to determine genome-wide transcriptional targets of STAT3 in the PFC and vHPC to identify cell-type specific STAT3 transcriptional signatures following chronic DID. Dr. Ethan Anderson will discuss G9a, a histone methyltransferase that is implicated in AUD. Alcohol reduces G9a in the NAc in mice and mimicking this reduction with an AAV-mediated shRNA knockdown blocks stress-potentiated alcohol drinking. Dr. Anderson will present new results showing that G9a acts selectively through NAc dynorphin (NAcDyn+) neurons but not NAc enkephalin-containing (NAcEnk+) neurons - to alter stress-potentiated drinking. Transcriptomic analysis revealed that NAc G9a alters genes are associated with excitability and suggest that NAc G9a modulates stress-potentiated drinking by altering the excitability of NAcDyn+ neurons. Dr. Maiya will discuss cell-type specific transcriptional signatures of social stress-escalated alcohol consumption in the paraventricular thalamus (PVT). Using a mouse model of social defeat stress (SDS)-induced escalation of alcohol consumption and a system for activity-dependent genetic tagging, Dr. Maiya examined the overlap between neural ensembles activated by SDS and alcohol consumption. The PVT was strongly activated by both SDS and alcohol and chemogenetic inhibition of SDS-activated cells in the PVT attenuated stress-escalated drinking. snRNASeq analysis of the PVT from SDS and alcohol-exposed mice revealed numerous cell-type specific neuroadaptations including alterations in transforming growth factor beta signaling that may drive stress-escalated drinking. The symposium is professionally balanced having two men and two women speakers with diverse backgrounds, and scientists at different career stages from different academic environments. Collectively, these investigators will present innovative approaches to discern cell-type specific genomic and epigenomic signatures of alcohol that can be harnessed to identify novel molecular targets within distinct cell-types for the treatment of alcohol use disorders (AUD). All speakers have agreed to participate in the symposia if accepted.

Speaker 1

Name:
Igor Ponomarev

Email:
Igor.Ponomarev@ttuhsc.edu

Organization:
Texas Tech University Health Sciences Center

Department:
Department of Pharmacology and Neuroscience

Position:
Associate Professor

Species:
Mouse

Speaker 2

Name:
Amy W. Lasek

Email:
Amy.Lasek@vcuhealth.org

Organization:
Virginia Commonwealth University

Department:
Department of Pharmacology and Toxicology

Position:
Professor

Species:
Mouse

Speaker 3

Name:
Ethan Anderson

Email:
eanderson1@lsu.edu

Organization:
Louisiana State University

Department:
Department of Comparative Biomedical Sciences

Position:
Assistant Professor

Species:
Mouse

Speaker 4

Name:
Rajani Maiya

Email:
rmaiya@lsuhsc.edu

Organization:
LSU Health Sciences Center, New Orleans

Department:
LSU Healthe Sciences Center

Position:
Assistant Professor

Species:
Mouse

AW Lasek, L Carvalho, and H Chen

STAT3 is a transcription factor activated by cytokine, chemokine, and growth factor receptor stimulation. It plays an important role in the innate immune response and is a key regulator of astrocyte reactivity. We previously demonstrated increased activating phosphorylation of STAT3 (pSTAT3) in rat hippocampal astrocytes after chronic ethanol exposure and withdrawal and in the postmortem hippocampus of human subjects with alcohol use disorder. Here, we measured pSTAT3 and its transcriptional targets in the prefrontal cortex (PFC) and ventral hippocampus (vHPC) of mice after chronic binge-like ethanol consumption. C57BL/6J mice underwent the drinking in the dark (DID) procedure for 4 h per day, 4 days per week for 6 weeks. Immediately following the final drinking session, pSTAT3 levels were higher in the PFC and vHPC of ethanol-drinking compared to water-drinking mice. We next identified genome wide pSTAT3 targets in the PFC and vHPC following chronic DID using the CUT&RUN assay. pSTAT3 differentially associated with ~2500 gene promoters in each brain region and sex following chronic drinking. Surprisingly, there was very little overlap in pSTAT3 differentially bound regions between brain regions and sexes. KEGG pathway analysis indicated that many of the enriched biological pathways were also different between brain regions and sexes. Finally, to determine the behavioral relevance of astrocyte-expressed STAT3, we tested inducible astrocyte-specific Stat3 knockout mice for ethanol consumption. Preliminary data indicates decreased binge-like ethanol consumption only in male astrocyte STAT3 knockout mice. These results show sex-specific roles for STAT3 in the regulation of gene expression and alcohol consumption.

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, USA

Funding Support: NIAAA U01AA020912 (INIA-Neuroimmune).

E.M. Anderson

Stress perpetuates the cycle of excessive alcohol drinking and contributes to the transition to an alcohol-use disorder (AUD). A common mechanism that regulates both stress-sensitivity and alcohol use is epigenetic regulation of gene transcription. One epigenetic modifier implicated in models of AUD is G9a, a histone methyltransferase that dimethylates lysine 9 on histone 3 (H3K9me2). We recently showed that alcohol decreases G9a in the nucleus accumbens (NAc). Mimicking this reduction of NAc G9a with an AAV-mediated shRNA knockdown decreases stress-potentiated alcohol drinking; however, the mechanism is not fully understood. Here we investigated if there is a cell-type specific effect of NAc G9a, how G9a alters the NAc transcriptome, and if this epigenetic modifier alters neuronal excitability. Since the dynorphin (Dyn) system plays a prominent role in stress/ethanol-related behaviors and dynorphin is present in a major subset of NAc neurons (NAc^Dyn+), we hypothesized that G9a acts selectively through (NAc^Dyn+) neurons - but not enkephalin-containing (NAc^Enk+) neurons - to alter stress-potentiated drinking. We tested this by injecting a novel cre-dependent AAV virus (AAV-DIO-shG9a) into the NAc of both dynorphin-cre and enkephalin-cre mice. These mice underwent 4 weeks of two-bottle choice drinking (baseline) and then were injected with the kappa opioid receptor agonist U50,488 (5mg/kg, i.p.) before drinking. Control Dyn-cre mice exhibited stress-potentiated drinking, but the G9a knockdown experimental mice did not, demonstrating an effect of G9a in NAc^Dyn+ neurons. In contrast, experimental Enk-cre mice did exhibit stress-potentiated drinking, suggesting no effect of G9a in NAc^Enk+. A transcriptomic analysis revealed that NAc G9a alters genes associated with excitability, so we next used slice physiology to measure changes in current-evoked spiking activity and rheobase. We found that a G9a knockdown led to an increase in NAc intrinsic excitability. Combined, these results suggest that the effects of NAc G9a on stress-potentiated drinking are mediated by altering the excitability of NAc^Dyn+ neurons. Thus, targeting G9a and/or NAc^Dyn+ neurons could help reduce escalated alcohol drinking in patients with AUD.

Louisiana State University, Department of Comparative Biomedical Sciences, Baton Rouge, LA, 70803

Supported by NIH grants K01 DA046513, R01 DA032708, and P50 AA10761.

E. Dore¹, E. Blaze¹, E. Garcia¹, F. Paliarin¹, N. Salem², A.W. Warden¹, R.D. Mayfield², and R. Maiya¹

Social stress is pervasive in humans, and its severity is associated with various neuropsychiatric conditions, including addiction. Social stress can lead to escalated alcohol consumption and increased risk of relapse to alcohol seeking in humans and animals. The biological underpinnings of this are not clear and must be elucidated to facilitate identification of therapeutic targets and inform therapeutic strategies. Using a system for activity-dependent genetic tagging and a mouse model for social defeat stress (SDS)-induced escalation of alcohol consumption, we examined the overlap between neurons activated by SDS and those activated by alcohol consumption in the same subjects. Using bigenic Fos-2A-Cre:Ai14 mice, we found that repeated SDS increased alcohol consumption in both male and female mice and led to robust neural activation in several brain regions compared to unstressed controls. Of these, the paraventricular thalamus (PVT) was particularly intriguing as it was also strongly activated by both SDS and alcohol exposure and there was a high degree of overlap (88%) between stress (expressing tdTomato) and alcohol activated (expressing cFos) cells. Chemogenetic inhibition of SDS-activated cells in the PVT attenuated stress-escalated alcohol consumption in both sexes. Single nuclei RNA sequencing (snRNASeq) of the PVT from male bigenic mice exposed to both stress and alcohol revealed five transcriptionally distinct neuronal subtypes within the PVT, PVT1-5. Of these, the highest number of stress and alcohol activated cells were found in PVT2. Differential gene expression (DEG) analysis revealed that majority of the DEGs were localized to cells activated by both stress and alcohol. Pathway and upstream regulatory element analysis of DEGs revealed that genes belonging to the transforming growth factor beta family were overrepresented suggesting a role for this pathway in stress and alcohol induced neuroadaptations within the PVT2 cell type. Future studies will examine the consequences of perturbing this pathway on social stress-escalated alcohol consumption.

Funding: AA030652-01 and LSUHSC startup funds

¹Department of Physiology, Louisiana State University Health Sciences Center, New Orleans, LA, 70112, USA and ² Waggoner Center for Alcohol and Addiction Research, Department of Neuroscience, The University of Texas at Austin, Austin, TX, 78712, USA.

Brent Kisby; Christian Bustamante; Sambantham Shanmugam; Isabel Castro-Piedras; Igor Ponomarev

Chronic high alcohol (ethanol) drinking is one of the characteristics of Alcohol Use Disorder (AUD). Toll-like receptor 3 (Tlr3) – dependent innate immune activation in rodents contributes to escalated ethanol consumption in a sex and genotype-dependent manner and our preliminary data suggested that these effects may be mediated by Tlr3 activation in microvasculature cell types. Micro-vessels consist of cell populations that form blood brain barrier (BBB), including endothelial cells (ECs), astrocyte end-feet, and pericytes. We hypothesize that Tlr3 activation-induced changes in gene expression in these cell types contribute to BBB dysfunction and escalated alcohol intake. The goal of this study was to determine the effects of Tlr3 activation and/or chronic alcohol drinking on gene expression in enriched brain microvasculature in high-drinking FVB/NJ X C57BL/6J F1 hybrid mice. Male mice were randomly assigned to receive repeated injections of Poly(I:C) (PIC), a Tlr3 agonist, or saline (9 injections total). Mice were allowed to choose between alcohol or water every other day (18 dinking sessions) and were assigned to one of four groups: saline/water (SW), saline/ethanol (SE), PIC/water (PW), and PIC/ethanol (PE). Brains were harvested 24 hours after the final alcohol session, micro-vessels from frontal cortex were purified using mechanical homogenization and density-gradient centrifugation, and RNA sequencing was performed to compare gene expression between groups. Tlr3 activation with PIC resulted in escalated ethanol consumption and we identified 1,588 genes differentially expressed (DEGs) between PE and SE groups at nominal p-value of less than 0.05 and 74 DEGs at 5% FDR. The top DEGs were Rsad2, H2-K1, Slc16a1, and Ifi44, which are implicated in the immune response and are cell type-specific to the brain microvasculature. Subsequent bioinformatics analysis utilized Weighted Gene Co-expression Network Analysis (WGCNA) to identify clusters (modules) of genes correlated with the effects of Tlr3 activation and/or ethanol consumption. We identified a total of 43 modules, many of which were enriched with cell type-specific genes, such as ECs and pericytes, and were associated with Tlr3 activation and escalated ethanol consumption. Taken together, these data suggest that vascular cell types are responsive to repeated Tlr3 activation and could contribute to excessive ethanol consumption.

¹Texas Tech University Health Sciences Center, Lubbock, TX 79430

Supported by AA027096 and AA028370.