BDE-47 and BDE-49 Inhibit Axonal Growth in Primary Rat Hippocampal Neuron-Glia 1 Co-Cultures via Ryanodine Receptor-Dependent Mechanisms 2

(PBDEs) are widespread environmental contaminants associated with adverse neurodevelopmental outcomes in children and preclinical models; however, the mechanisms by which PBDEs cause developmental neurotoxicity remain 30 speculative. The structural similarity between PBDEs and non-dioxin-like (NDL) polychlorinated 31 biphenyls (PCBs) suggests shared toxicological properties. Consistent with this, both NDL 32 PCBs and PBDEs have been shown to stabilize ryanodine receptors (RyRs) in the open 33 configuration. NDL PCB effects on RyR activity are causally linked to increased dendritic 34 arborization, but whether PBDEs similarly enhance dendritic growth is not known. In this study, 35 we quantified the effects of individual BDE congeners on not only dendritic but also axonal 36 growth since both are regulated by RyR-dependent mechanisms, and both are critical 37 determinants of neuronal connectivity. Primary neuronal-glial co-cultures dissociated from the 38 neonatal rat hippocampus were exposed to BDE-47 or BDE-49 in the culture medium at 39 concentrations ranging from 20 pM to 2 µM. At these concentrations, neither PBDE congener 40 altered dendritic arborization. In contrast, at concentrations ≥ 200 pM, both congeners delayed 41 neuronal polarization resulting in significant inhibition of axonal outgrowth during the first few 42 days in vitro . The axon inhibitory effects of these PBDE congeners occurred independent of 43 cytotoxicity, and were blocked by pharmacological antagonism of RyR or siRNA knockdown of 44 RyR2. These results demonstrate that the molecular and cellular mechanisms by which PBDEs 45 interfere with neurodevelopment overlap with but are distinct from those of NDL PCBs, and 46 suggest that altered patterns of neuronal connectivity may contribute to the developmental 47 neurotoxicity of PBDEs. Spontaneous Ca 2+ transients were measured in dissociated hippocampal cells cultured on 96-well plates Inc, Corning, NY). At DIV 2, cells were loaded with the Ca 2+ - dye Fluo-4 AM (4 µM; Invitrogen) in Locke’s buffer (8.6 mM 4-(2-hydroxyethyl)-1- acid, 5.6 mM KCl, 154 mM NaCl, 5.6 mM glucose, 1.0 mM MgCl 2 , 2.3 CaCl 2 , and 0.0001 mM glycine, pH 7.4) at 37°C for 30 min. Cultures were washed with Locke’s Buffer and transferred to the ImageXpress Micro XLS high content imaging system. The temperature was maintained at 37˚C throughout the recording period. Fluorescence was recorded every 15 s over the 26 min experiment. After 5 min of baseline recording, vehicle, 215 BDE-47 or BDE-49 was added to the cultures and data captured for an additional 20 min. At the conclusion of the 20 min recording, viable neurons were identified by adding 30 mM KCl to the medium to depolarize neurons; any neurons that did not respond with a transient Ca 2+ spike were not included in the subsequent analyses. The transient amplitude of Ca 2+ fluorescence was measured by normalizing peak change in Fluo-4 AM fluorescence (ΔF) to the baseline 220 fluorescence (F0) and presented as the mean ΔF/F0 per neuron. Area under the curve (AUC) measurements were calculated via the trapezoid method using Graphpad Prism version 4.0 (San Diego, CA). Baseline levels were held constant at zero and peaks less than 10% of total amplitude were excluded. Levels of calcium flux within each cell were collected throughout the experimental time from all treatments. Within each field of view, > 30 cells were identified for analysis; 1 field was imaged per well in 2 wells per experimental group. Statistical comparisons were made on mean values per group with cultures obtained from four independent dissections N=4 per group).