Acquisition of spontaneous electrical activity during embryonic development of gonadotropin-releasing hormone-3 neurons located in the terminal nerve of transgenic zebrafish (Danio rerio)
General and Comparative Endocrinology
There are multiple populations of gonadotropin-releasing hormone (GnRH) neurons that have distinct physiological and behavioral functions. Teleost fish have a population of GnRH3 neurons located in the terminal nerve (TN) associated with the olfactory bulb that is thought to play a neuromodulatory role in multiple physiological systems, including olfactory, visual, and reproductive. We used transgenic zebrafish in which the GnRH3 promoter drives expression of a green fluorescent protein to identify GnRH3 neurons during development in live embryos. Unlike with hypophysiotropic GnRH neurons of zebrafish, TN-GnRH3 neurons are of neural crest origin and are one of the first populations of GnRH neurons to develop in the early embryo. Using a combination of optical imaging and electrophysiology, we showed that during the first 3 days post-fertilization, TN-GnRH3 neurons increase in number, extend neural projections, move in association with tissue expansion, and acquire an adult-pattern of spontaneous action potential firing. Early during development, about half of the neurons were quiescent/non-firing. Later, at 3 days post-fertilization, there was an increase in the proportion of neurons showing action potential firing and an increase in the number of neurons that showed an adult-like tonic or beating pattern of action potential firing with a firing frequency similar to that seen in adult TN-GnRH3 neurons. This study represents the first neurophysiological investigation of developing GnRH neurons in live embryos – an important advancement in understanding their potential non-reproductive roles during embryogenesis.
Siddharth Ramakrishnan, Wenjau Lee, Sammy Navarre, David J. Kozlowski, Nancy L. Wayne, Acquisition of spontaneous electrical activity during embryonic development of gonadotropin-releasing hormone-3 neurons located in the terminal nerve of transgenic zebrafish (Danio rerio), General and Comparative Endocrinology, Volume 168, Issue 3, 15 September 2010, Pages 401-407, ISSN 0016-6480, 10.1016/j.ygcen.2010.05.009. (http://www.sciencedirect.com/science/article/pii/S0016648010002145) Keywords: Action potential; Electrophysiology; Embryo; Teleost
Fig. 1. Images of GnRH3:EMD neurons in zebrafish embryos. (A and B) Confocal images of GnRH:EMD neurons in the olfactory region of live embryos. (A) 27 hpf; (B) 47 hpf (same embryo). Scale bar is 20 μm. (C–E) Immunohistochemical (IHC) verification that EMD-expressing neurons in the olfactory region contain immunoreactive (ir) GnRH peptide. (C) EMD fluorescence; (D) irGnRH peptide; (E) merged images of EMD and irGnRH. Scale bar is 50 μm. (F and G) IHC control tissue in the absence of primary anti-GnRH antibody showing specificity of red fluorescence (i.e., irGnRH peptide). (F) GFP fluorescence; (G) IHC experiment in the absence of primary antibody. Scale bar is 10 μm. (H and I) Verification that neurons targeted for whole-cell electrophysiology are EMD-containing neurons. (H) Brightfield image of the intact brain in the olfactory region in the live embryo. (I) EMD image (same field of view as in H) showing a string of GnRH3:EMD neurons in the olfactory region. EMD diffuses into the tip of the electrode (marked by arrow) during whole-cell recording (I).
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Fig. 2. Passive displacement of GnRH3:EMD neurons in the live, intact embryo at 2 dpf, concomitant with tissue expansion. (A) Merged fluorescence and brightfield confocal images (single optical slice) from the same field of view at different times during development. Shown are representative images of the right upper quadrant of an embryo head over time (same embryo as shown in panel B insert). A box was positioned around the right GnRH3:EMD neuron cluster and a black dot was positioned at the center of the opening of the right nare at 52 hpf. The position of the box and dot were kept constant, relative to the field of view in subsequent images, revealing displacement of the GnRH3:EMD cluster and nare over this time period. Black arrows point to the edge of the opening of the nare. (B) Displacement of GnRH3:EMD clusters from 52 to 63 hpf (mean ± SEM; four clusters from two embryos). Y-axis refers to distance moved relative to the location of clusters at 52 hpf. Displacement in an anterior direction is shown by the black squares; displacement in a lateral direction is shown by the open circles. The insert shows the entire head of the embryo (projection of the z stack) at 52 hpf; arrows point to the bilateral TN-GnRH3:EMD neuron clusters.
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Fig. 3. Whole-cell current clamp recordings from TN-GnRH3:EMD neurons from 2 and 3 dpf transgenic embryos. Shown are excerpts from longer recording sessions. Inserts from the top traces of 2 and 3 dpf embryos show representative details from the recordings. Note that the x and y axes are different between 2 and 3 dpf.
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