PROJECT SUMMARY Excessive intake of a diet rich in fats during pregnancy gives rise to offspring that are prone to overeating dietary fat and becoming obese. This generational effect of prenatal programming of ingestive behavior results in offspring that are prone to becoming obese. This proneness in offspring is attributed to an increase in the genesis of orexigenic enkephalin neurons in the hypothalamus. High levels and expression of hypothalamic enkephalin is a stimulator of high-fat diet intake. One potential mechanism for this change involves the chemokine CXCL12, which has been shown to be altered by the intake of a high-fat diet. Maternal CXCL12, similar to a high-fat diet, can also stimulate enkephalin levels and ingestive behavior in offspring. Currently, it is unknown how prenatal high-fat diet exerts its affects through CXCL12 to change the hypothalamic architecture in developing embryos. The goal of this proposal in using a rat model is pilot whether the reduction of maternal CXCL12 levels could prevent offspring brain changes. Aim 1a will examine the effects of CXCL12-neutralizing antibody paired with maternal HFD ingestion on changes in offspring hypothalamus. Utilizing the novel spatial gene transcriptome technique, immunofluorescence histology labeling of neurons and neuroglia in conjunction with markers of genesis and orexigenic neuropeptides will be performed. Afterwards, discrete regions of the hypothalamus will be barcoded, and the mRNA (resulting cDNA) will be collected for RNAseq. The results will provide unique location-based transcriptome changes overlaid by fluorescently labeled cell types. Aim 1b will test these same offspring for behavioral and weight changes that align with obesity-prone rats. Daily caloric intake and weight change will be tracked and behavioral tests to measure anxiety-like behavior, a marker for hyperphagia, will also be examined. These results will further support the hypothesis that HFD-induced increase in maternal CXCL12 plays a role in producing hyperphagic offspring and provide new higher resolution insight into neurogenesis events in offspring brain.