Social familiarity affects mating preference among various vertebrates. Here, we show that visual contact of a potential mating partner before mating (visual familiarization) enhances female preference for the familiarized male, but not for an unfamiliarized male, in medaka fish. Terminal-nerve gonadotropin-releasing hormone 3 (TN-GnRH3) neurons, an extrahypothalamic neuromodulatory system, function as a gate for activating mating preferences based on familiarity. Basal levels of TN-GnRH3 neuronal activity suppress female receptivity for any male (default mode). Visual familiarization facilitates TN-GnRH3 neuron activity (preference mode), which correlates with female preference for the familiarized male. GnRH3 peptides, which are synthesized specifically in TN-GnRH3 neurons, are required for the mode-switching via self-facilitation. Our study demonstrates the central neural mechanisms underlying the regulation of medaka female mating preference based on visual social familiarity.

BackgroundGenetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain.Methodology/Principal FindingsTo establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0–1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2–3 dpf embyos compared with 0–1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish.Conclusions/SignificanceWe established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.

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Adult medaka fish (Oryzias latipes) exhibit complex social behaviors that depend mainly on visual cues from conspecifics. The ontogeny of visually-mediated social behaviors from larval/juvenile to adult medaka fish, however, is unknown. In the present study, we established a simple behavioral paradigm to evaluate the swimming proximity to conspecifics based on visual cues in an inter-individual interaction of two medaka fish throughout life. When two fish were placed separately in a cylindrical tank with a concentric transparent wall, the two fish maintained close proximity to each other. A normal fish inside the tank maintained proximity to an optic nerve-cut fish outside of the tank, while the converse was not true. This behavioral paradigm enabled us to quantify visually-induced motivation of a single fish inside the tank. The proximity was detected from larval/juvenile to adult fish. Larval fish, however, maintained close proximity not only to conspecifics, but also to heterospecifics. As the growth stage increased, the degree of proximity to heterospecifics decreased, suggesting that shoaling preferences toward conspecifics and/or visual ability to recognize conspecifics is refined and established according to the growth stage. Furthermore, the proximity of adult female fish was affected by their reproductive status and social familiarity. Only before spawning, adult females maintained closer proximity to familiar males rather than to unfamiliar males, suggesting that proximity was affected by familiarity in a female-specific manner. This simple behavioral paradigm will contribute to our understanding of the neural basis of the development of visually-mediated social behavior using medaka fish.

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