Acknowledgments This research is supported by NSF CAREER Award to SR. Authors thank Sean Stackhouse, Jessica L Dyck and Jessica M Evans for fish care. Transgenerational effects of Bisphenol A on embryogenesis, GnRH3 neural systems and locomotor behavior in Japanese medaka T. INAGAKI 1,2, N. L. SMITH 2,3, K.M.SHERVA 2,3, S. RAMAKRISHNAN 1,2 Dept. of Biology 1, Neuroscience program 2, Dept. of Chemistry/Biochemistry 3, University of Puget Sound, Tacoma, WA Introduction Methods Figure 1 Figure 3 Figure 2 Movement (20 dpf) Terminal Nerve (TN) GnRH3 neural population Trigeminal (TG) GnRH3 neural population Parental BPA exposure alters F2 fluorescence of GnRH3-GFP neurons BPA exposure in F1 generation decreased F2 locomotor activity at 20 dpf Figure 4 SV2 expression was reduced only in G1 larvae Speed (20 dpf) BPA exposure to F1 generation (parents) results in significant alternations in the development of GnRH3 neurons, embryonic mortality, time to hatch, synaptic connectivity and locomotor behavior of F2 generation. Transgenerational BPA effects were more potent in G1 fish whose parents were exposed to BPA for life-long. Some effects were different from BPA effects in F1 generation reported in the previous study. Timeline/Experimental Design  Testing: -GnRH3-GFP intensity -Survival rates -Time to hatch -Locomotor behavior -Synaptic connectivity 100 μm Discussion/Summary Bisphenol A (BPA) is a synthetic non-steroidal environmental chemical. Evidence suggests that chronic exposure to low-dose BPA may induce a variety of adverse effects in developing brain. Our previous study shows that BPA exposure (200 ng/ml) during embryogenesis disrupts normal development of Gonadotropin- Releasing-Hormone 3(GnRH3) neurons on 3 days post fertilization (dpf), alters physiological development, and decreases larval locomotor activity on 20 dpf. Yet, transgenerational impacts of BPA are not understood. W Sv2 expression (20 dpf) Survival rates Time to hatch 3 dpf GnRH3-GFPintensity 4 dpf GnRH3-GFPintensity Normalized Fluorescence at 3 dpf. n=15 (G0), 5 (G1), 8 (G2), 12 (G3) and 15 (G4). Normalized Fluorescence at 4 dpf. n=12 (G0), 15 (G1), 10 (G2), 18 (G3) and 11 (G4). Using a transgenic medaka fish model with GnRH3 neurons tagged with GFP, this study examined if BPA produces transgenerational alternations in GnRH3 neural systems, embryonic mortality, and larval locomotor behavior. Subjects were F2 transgenic Japanese medaka embryos/larvae that have never been exposed to BPA (F2), but their parents (F1) were exposed to BPA for different durations in their life span. General Methods GnRH3-GFP neurons at the terminal nerve (TN) and the trigeminal nerve (TG) were visualized from 3 and 4 dpf F2 embryos, and fluorescence intensity of each neural population was quantified. For behavior tests, 20 dpf F2 larvae were placed into the 6-well plates, and locomotor activity was tracked using the Noldus EthoVision. Immunohistchemical staining with SV2 antibody was performed at 20 dpf. Raw data were analyzed on ANOVAs and independent t-tests. Values are shown as mean± SEM. p<0.05, 2-tailed is considered as significance. 100 μm 75 μm 50 μm