Impact of bisphenol A on the developing GnRH3 neural system and locomotor behavior in Japanese medaka

Published: Neurotoxicology 10.1016/j.neuro.2015.12.003
Inagaki, T., Smith, N.*, Lee, E.K.*, Ramakrishnan, S., Low dose exposure to Bisphenol A alters development of Gonadotropin-Releasing Hormone3 neurons and larval locomotor behavior in Japanese Medaka, Neurotoxicology,2015 10.1016/j.neuro.2015.12.003

*T. INAGAKI1,2, E. K. LEE2, N.L. SMITH, S. RAMAKRISHNAN1,2; 1Biol., 2Neurosci. program, Univ. of Puget Sound, Tacoma, WA

Bisphenol A (BPA), a xenoestrogen, disrupts normal brain function and behavior mediated by neuroendocrine systems. Gonadotropin-releasing hormone (GnRH) neurons in the brain play a crucial role in reproductive physiology and behavior in most vertebrates. In fish, three types of GnRH neurons, GnRH1 at the preoptic area (POA), GnRH2 at the midbrain, and GnRH3 at the terminal nerve (TN), have been identified. While it is known that GnRH3 neurons project widely throughout the brain and spinal cord, and that neuromodulatory roles in reproductive behavior have been suggested, their functions remain unclear. Aside from reproduction, recent studies have also alluded to a role for GnRH in locomotion, with GnRH receptors found in the spinal cord. Here we examine effects of chronic exposure to low dose BPA through development on GnRH3 neuronal systems, and locomotor activity in transgenic Japanese medaka with GnRH3 neurons tagged with green fluorescent protein (GFP). Fertilized medaka eggs were collected daily, and placed individually in 12- or 24-well plates. Embryos in BPA groups were chronically exposed to 200 ng/ml of BPA starting on 1 day post- fertilization (dpf), through hatch until 10 days post hatch (20 dpf). To assess effects of chronic BPA exposure on GnRH3 neurons, brain images were taken from live embryos in eggs at 1, 2, 3 and 4 dpf, and intensity of GnRH3-GFP neurons were analyzed using an upright epifluorescence microscope.


At 3 dpf, GnRH3-GFP neurons in BPA-treated embryos showed 23.7% increased fluorescence in the terminal nerve population and 14.3% reduced intensity in the trigeminal population when compared to vehicle treated control embryos. Hatched larvae were kept in glass petridishes with continued exposure to 200 ng/ml BPA. Locomotor activity of larvae was observed at 20 dpf (~10 days post hatch) and analyzed using Noldus Ethovision XT. Head-growth, body sizes, times to hatching were also measured. We found that BPA exposed larvae (n=12 each group) showed significantly decreased locomotory activity both in total distance covered (88.36 ± 28 mm vs. 176.2±14.6 mm controls, p<0.012) and velocity of movement (2.53 ±0.6 mm/s vs. 4.74± 0.72 mm controls p<0.013). There were no significance differences in head-growth, body sizes and times to hatching between BPA and the control groups. This study will allow us to understand BPA effects on GnRH3 neuron development and how it may affect locomotion. (Supported by NSF Career Award 1253126 to SR).