Abstract
Understanding development of gonadotropin-releasing hormone (GnRH) neuronal circuits is fundamental to our understanding of reproduction, but not yet well understood. Most studies have been focused on GnRH neurons located in the hypothalamus and preoptic area (POA), which directly regulate the pituitary-gonadal axis. In zebrafish (Danio rerio), two forms of GnRH have been identified: GnRH2 and GnRH3. GnRH3 neurons in this species plays two roles: hypophysiotropic and neuromodulatory, depending on their location. GnRH3 neurons in the ventral telencephalon, POA, and hypothalamus control pituitary-gonadal function; in other areas (e.g., terminal nerve), they are neuromodulatory and without direct action on reproduction. To investigate the biology of GnRH neurons, a stable line of transgenic zebrafish was generated in which the GnRH3 promoter drives expression of a bright variant of green fluorescent protein (Emerald GFP, or EMD). This provides unprecedented sensitivity in detecting and imaging GnRH3 neurons during early embryogenesis in the transparent embryo. Using timelapse confocal imaging to monitor the time course of GnRH3:EMD expression in the live embryo, we describe the emergence and development of GnRH3 neurons in the olfactory region, hypothalamus, POA, and trigeminal ganglion. By 50 h post fertilization, these diverse groups of GnRH3 neurons project broadly in the central and peripheral nervous systems and make anatomical connections with each other. Immunohistochemistry of synaptic vesicle protein 2 (a marker of synaptic transmission) in this transgenic model suggests synaptic formation is occurring during early development of the GnRH3 neural network. Electrophysiology reveals early emergence of responsiveness to the stimulatory effects of kisspeptin in terminal nerve GnRH3 neurons. Overall, our findings reveal that the GnRH3 neuronal system is comprised of multiple populations of neurons as a complicated network.
Highlights
Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of fertility
It was confirmed that neurons in the terminal nerve/olfactory region, hypothalamus, preoptic area (POA), ventral telencephalon, and trigeminal ganglion express endogenous GnRH3 [11, 19]
Failure to show endogenous GnRH3 mRNA may be due to these neurons showing such low expression of message that it is undetectable using in situ hybridization, or that the transgene is being ectopically expressed in these retina neurons
Summary
Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of fertility. There is the curious and under-appreciated observation that GnRH3 is expressed in the trigeminal ganglion of the peripheral nervous system of medaka and zebrafish embryos [10, 11] This peripheral population of GnRH3 neurons, along with that in the terminal nerve associated with the olfactory region, play neuromodulatory functions that are not directly related to reproduction. We used a stable line of transgenic zebrafish generated by our laboratory, in which the GnRH3 promoter drives expression of a bright variant of green fluorescent protein (Emerald GFP, or EMD) This provides unprecedented sensitivity in detecting and imaging GnRH3 neurons in the live, intact embryo [19]. We began testing the hypotheses that: [1] Multiple populations of GnRH3 neurons develop connectivity with each other; [2] Kisspeptin regulates spontaneous action potential firing of GnRH3 neurons during embryogenesis; and [3] GnRH3 neurons exhibit the potential for synaptic communication during early embryonic development
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