RFRP3 affects GnRH3 neuron development and locomotion

RFRP-3 Affects Gnrh3 Neuronal Development And Larval Social Behavior In The Teleost Oryzias latipes

Sean Stackhouse, Hannah Martens, Siddharth Ramakrishnan

While the role of Gonadotropin releasing hormone (GnRH) neurons in sexual behavior is well studied, their regulation of social behavior is poorly understood. In this research, we examined the effect of early embryonic exposure of R-F-amide Related Protein3 (RFRP-3), a GnRH antagonist, on social behavior in the teleost medaka (Oryzias latipes). We used the mirror approaching behavior, previously demonstrated as shoaling, a basic social behavior found in many teleosts. Using a transgenic line of medaka with GnRH3-neurons tagged with the green fluorescent protein (GFP), we examined the effects of embryonic exposure to RFRP-3 on both GnRH3 neuron GFP intensity and effects on larval social behavior. Unlike the preoptic area GnRH1 neurons (associated with reproduction) or the midbrain GnRH2 neurons (involved in metabolism), the role of the GnRH3 system associated with the terminal nerve (TN) is poorly understood. This work aims to further investigate the role of the GnRH3 neuronal system and its putative role in modulating social behavior.

Transgenic medaka embryos were treated with 1µM of RFRP-3 from day 0 until hatch. GnRH3-GFP intensity in the TN and trigeminal (TG) areas were recorded using an epifluorescent microscope on 2-4 days post fertilization. Social behavior of larvae was tested at 3 weeks post hatch in a 10cm x 8cm enclosure with 2cm of normal fish water. A fish larva was placed in the chamber under the following conditions with a/an i) opaque wall; ii) mirror; iii) mirror on one side and a live fish separated by a clear barrier on the other; iv) opaque wall on one side and a live fish separated by a clear barrier on the other. Both RFRP-3 exposed and vehicle treated control fish were exposed to each condition. For tracking, the tank was divided into three equal segments, the mirror/wall region, center region, and a non-mirror/live-fish region. Fish behavior was recorded for 3 minutes after starting the trial and analyzed using the Noldus EthoVision software.

RFRP-3 treated fish showed no significant difference in GnRH3-GFP fluorescence intensity at 3dpf but showed 56% increased intensity at 4dpf (n=12, p<0.01) in the TN and 36% increase the TG (n=12, p<0.05). While there was no overall difference between motion between control and RFRP-3 treated medaka, treated fish were found to spend 70% more time near the mirror under condition (ii) as compared to control fish (p<0.05). We suggest that RFRP-3 exposure may be affecting the GnRH3 neuron development, which could then lead to altered social behavior.