Submission Date

4-24-2023

Document Type

Paper- Restricted to Campus Access

Department

Neuroscience

Adviser

Carlita Favero

Committee Member

Johannes Karreth

Committee Member

Ryan Walvoord

Department Chair

Carlita Favero

Project Description

Fetal Alcohol Spectrum Disorders (FASDs) is a group of disorders caused by prenatal alcohol exposure (PAE) that causes physical, behavioral, and sensory issues amongst others in infants. FASDs’ impacts on behavior related to reward-seeking, attention, movement, and addiction can be attributed to dopaminergic circuits in the brain, particularly the mesocortical and mesolimbic pathways. Thus, these pathways, originating in the ventral tegmental area (VTA) and passing through the subpallium, are a source of interest for our lab. Previous research in the Favero Lab has focused on the impacts of PAE on dopaminergic axon development. This research shifts from the focus on axons to the surrounding microglia that are responsible for axon guidance in developing embryos, especially in the subpallium. A particular molecule of interest is fractalkine, which is an essential chemokine that communicates between the neurons and microglia to facilitate microglial activation. For this project, pregnant dams were introduced to ethanol, at a binge level of PAE, or air vapor exposures between embryonic days 7.5 and 14.5. At E18.5, the embryos were c-sectioned and sex genotyped to find one male and one female in each litter. Their brains were cryo-embedded and sectioned in a coronal orientation for analysis. Iba1 staining using immunohistochemistry labeled for microglia in the subpallium. Several months of this project were dedicated to troubleshooting immunohistochemistry techniques in both fluorescence and biotin secondary staining methods. Ultimately, diaminobenzidine (DAB) staining as biotin secondary was used for imaging. Images were taken using compound microscopy and analyzed using ImageJ, specifically the “skeleton” plugin and its associated 2D skeleton analysis feature. Because of the blind procedure, the samples collected were all air-exposed (control) litters. These litters were analyzed for sex differences and differences between fractalkine receptor knockout mice and wild type. No significant differences were found. Future research will expand on the current data set and use the protocols established in this work to look at the ethanol-treated litters. More research will also include other variables such as developmental age and chronological differences in microglial morphology.

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