Microglia and Adolescent Susceptibility to Developing an Alcohol Use Disorder

Bardo Lab Bardo - overall supervision of behavioral aspects of the project, data interpretation and manuscript preparation. Chandler- assist Peng with animal model, slice rat brains, run autoradiographic assays for PK-11195 binding, conduct autoradiographic image analysis, overall supervision of behavioral experiments, assist in surgeries, data analysis, graphics and manuscript preparation. Denehy- slice rat brains, assist in surgeries, conduct behavioral experiments on regular weekdays. 1. Finalize the piloted 2 bottle choice experimental protocol and help in the transfer of the model to the Nixon Lab at UT. Self-administration (2 bottle choice): binge treatment (3) x test delay (2) x sex (2), for 12 groups, with each group n=10. 2. Perfect delay discounting experiments and begin running 6 groups of rats exposed to ethanol or control diet (males v females, adolescent v adult exposure). Delay discounting: binge treatment (3) x test delay (2) x sex (2), for 12 groups, with each group n=10. 3. Cued go/no-go studies if time and funding permits. Pauly Lab Pauly - oversee the autoradiography ([3H]PK-11195) studies including the design, analysis, and interpretation of those data as well as preparation of the manuscripts. Hopkins . slice rat brains, oversee the autoradiography studies, assist with tissue harvesting as needed. Autoradiography [binge treatment (3) x sacrifice time point (4) x age (2) x sex (2)] 150 rats completed in Year 1 wil be sliced for binding. The plan is to slice 2 representative animals from each alcohol withdrawal time point into 7 sets of adjacent sections that can be used for various binding assays. For the first group of animals, we should collect as many brain sections as we can, from approximately +3.6 Bregma to -10 Bregma. Then we will run a screening assay on one set of tissue from these animals to see the pattern of microglial activation and then decide on how to slice the remaining tissue. The rest of the tissue will be sliced into 6 sets of sections, with the start/stop point to be determined. Peng Lab Peng . Oversight of the project, budgets and all progress reports to UT. Continue to oversee the performance of the AUD model for Pauly and Bardo labs as well as her own component. Schmidt . assist with animal model, microglia isolation and culturing 1. Determine the adolescentfs susceptibility to alcohol-induced effects on microglia via flow cytometry and gene effect. [binge treatment (3) x sacrifice time point (4) x age (2) x sex (2)] Perform AUD model, isolate microglia/macrophages and assess surface marker expression for activation state (phenotype) via flow cytometry and cytokines and growth factors via RT-PCR. 2. Examine whether microglia isolated from EtOH-exposed adolescent brains are primed ex vivo. (binge treatment x sacrifice time point x age x sex) Adolescent and adult rats will be exposed to 2-day binge alcohol and sacrificed at the time point of peak activation indicated in autoradiography and above studies (T2 likely) and the latest time point with evidence of activation in adolescents (e.g. T28 days). Microglia will be isolated by using Percoll density gradient centrifugation and cultured and exposed to LPS (concentration response) for 16-24 h. Cell culture supernatant will be collected for protein expression of the pro-inflammatory cytokines (namely TNFƒ¿, IL-6, and IL-1s). Cells will be lysed in Trizol for RNA extraction and real time RT-PCR for gene expression of pro-inflammatory cytokines.