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 three 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 three 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 advance in understanding their potential non-reproductive roles during embryogenesis.
Light-microscopic and immunoblot immunochemical procedures were used to study the distribution and biochemical characteristics of neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) in the suprachiasmatic nuclei (SCN) of the adult Siberian hamster. In the adult brain PSA-NCAM is located in regions capable of undergoing morphological rearrangements and thus is generally considered to be an indicator of neural plasticity. Immunostaining for PSA in the Siberian hamster SCN (using a monoclonal antibody against the α2,8-linked PSA of NCAM) was evident throughout the rostrocaudal axis of the SCN, with the most intense reaction in the ventrolater-al region. Immunoreactivity was present in the neuropil, which delineated groups of cells with unstained cytoplasm. Many of the SCN cells were aggregated into cords or clusters. The optic chiasm was free from label, except for short processes apparently extending from the densely stained neuropil in the ventrolateral SCN. Immunoreactivity was abolished by preincubating sections in an endoneuraminidase (endo-N) or by preincubation of the primary antibody with PSA-NCAM. Immunostaining of the nonsialylated NCAM polypeptide was also limited to the neuropil, but this was more diffuse and less regionally specific than PSA staining. The ventral SCN exhibited the most intense labeling for NCAM. Immunoblot analyses revealed the immunoreactive PSA-NCAM as a broad band migrating between apparent molecular weights in the range of 150-300 kD, which was ablated by treatment of SCN tissue extract with endo-N. This electrophoretic migration pattern of PSA-NCAM from the SCN region was similar to that seen in several other brain regions. The immunoreactive NCAM polypeptides appeared as three major bands, at apparent molecular weights of 120, 140 and 180 kD. Removal of PSA groups from PSA-NCAM with endo-N markedly increased the intensity of the 180-kD and, to a lesser extent, the 140-kD bands, implicating these NCAM isoforms as carriers of PSA. These results confirm the expression of PSA-NCAM in the adult SCN, which is evidence that cellular elements of the circadian pacemaker system may undergo morphological rearrangements.
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