Targeting of green fluorescent protein to secretory granules in oxytocin magnocellular neurons and its secretion from neurohypophysial nerve terminals in transgenic mice.

Oxytocin (OT) is a hypothalamic nonapeptide that is synthesized as part of a larger precursor protein that also contains an approximately 10-kDa protein called neurophysin at its C-terminus. This precursor protein is trafficked through the regulated secretory pathway into secretory granules and then axonally transported to and secreted from nerve terminals in the neural lobe of the pituitary. In this paper, we show that the AI-03 transgene that contains enhanced green fluorescent protein (EGFP) fused to the end of the neurophysin at the C-terminus of the OT pre-prohormone, is expressed selectively in OT-magnocellular neurons and is trafficked to secretory granules in transgenic mice. The EGFP-containing secretory granules are then transported to OT-neurosecretory terminals in the neurohypophysis, where the EGFP fluorescence undergoes depolarization-induced calcium-dependent secretion. The endogenous fluorescence in the neural lobes is sufficiently intense to image secretory events in individual OT nerve terminals (neurosecretosomes) isolated from the posterior pituitaries in these transgenic mice.

[1]  R. Tsien,et al.  green fluorescent protein , 2020, Catalysis from A to Z.

[2]  F. Bree.  Trafficking of the Vasopressin and Oxytocin Prohormone Through the Regulated Secretory Pathway , 2001 .

[3]  H. Gainer,et al.  Cell-Specific Expression and Subcellular Localization of Neurophysin-CAT-Fusion Proteins Expressed from Oxytocin and Vasopressin Gene Promoter-Driven Constructs in Transgenic Mice , 2001, Experimental Neurology.

[4]  H. Gainer,et al.  Gene regulation in the magnocellular hypothalamo-neurohypophysial system. , 2001, Physiological reviews.

[5]  P. Seeburg,et al.  Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies , 2001, Progress in Neurobiology.

[6]  G. Feng,et al.  Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP , 2000, Neuron.

[7]  D. Murphy,et al.  Sorting of the vasopressin prohormone into the regulated secretory pathway , 2000, FEBS letters.

[8]  Ginns,et al.  Transgenic Expression of Green Fluorescent Protein in Mouse Oxytocin Neurones , 1999, Journal of neuroendocrinology.

[9]  H. Gainer,et al.  Single Cell Reverse Transcription-Polymerase Chain Reaction Analysis of Rat Supraoptic Magnocellular Neurons: Neuropeptide Phenotypes and High Voltage-Gated Calcium Channel Subtypes. , 1999, Endocrinology.

[10]  K Kusano,et al.  Quantitative analysis of oxytocin and vasopressin messenger ribonucleic acids in single magnocellular neurons isolated from supraoptic nucleus of rat hypothalamus. , 1999, Endocrinology.

[11]  Daniel F. Hanley,et al.  GABA- and Glutamate-Activated Channels in Green Fluorescent Protein-Tagged Gonadotropin-Releasing Hormone Neurons in Transgenic Mice , 1999, The Journal of Neuroscience.

[12]  H. Gainer,et al.  Effects of Osmotic Pressure and Brain‐Derived Neurotrophic Factor on the Survival of Postnatal Hypothalamic Oxytocinergic and Vasopressinergic Neurons in Dissociated Cell Culture , 1999, Journal of neuroendocrinology.

[13]  A. N. van den Pol,et al.  Selective Neuronal Expression of Green Fluorescent Protein with Cytomegalovirus Promoter Reveals Entire Neuronal Arbor in Transgenic Mice , 1998, The Journal of Neuroscience.

[14]  S. House,et al.  Stationary Organotypic Cultures of Oxytocin and Vasopressin Magnocellular Neurones from Rat and Mouse Hypothalamus , 1998, Journal of neuroendocrinology.

[15]  J. Burbach,et al.  Transgenic and Transcriptional Studies on Neurosecretory Cell Gene Expression , 1998, Cellular and Molecular Neurobiology.

[16]  Simon C Watkins,et al.  Neuronal Peptide Release Is Limited by Secretory Granule Mobility , 1997, Neuron.

[17]  P. Richard,et al.  L‐, N‐ and T‐ but neither P‐ nor Q‐type Ca2+ Channels Control Vasopressin‐Induced Ca2+ Influx in Magnocellular Vasopressin Neurones Isolated from the Rat Supraoptic Nucleus , 1997, The Journal of physiology.

[18]  G. Wang,et al.  Role of Q‐type Ca2+ Channels in Vasopressin Secretion From Neurohypophysial Terminals of the Rat , 1997, The Journal of physiology.

[19]  I. Robinson,et al.  Neurohypophyseal and fluid homeostasis in transgenic rats expressing a tagged rat vasopressin prepropeptide in hypothalamic neurons. , 1996, Endocrinology.

[20]  S. Radovick,et al.  Cell-specific Expression of the Human Gonadotropin-releasing Hormone Gene in Transgenic Animals* , 1996, The Journal of Biological Chemistry.

[21]  Bi-Cheng Wang,et al.  Crystal structure of the neurophysin—oxytocin complex , 1996, Nature Structural Biology.

[22]  H. Gerdes,et al.  Visualization of protein transport along the secretory pathway using green fluorescent protein , 1995, FEBS letters.

[23]  C. Taylor,et al.  Comparison of two microwave based antigen-retrieval solutions in unmasking epitopes in formalin-fixed tissue for immunostaining. , 1995, Anticancer research.

[24]  R. Weinberg,et al.  An osmium-free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry. , 1995, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[25]  D. Carter,et al.  Cell Specific Expression of a Vasopressin Transgene in Rats , 1994, Journal of neuroendocrinology.

[26]  E. Stuenkel,et al.  Intracellular calcium and vasopressin release of rat isolated neurohypophysial nerve endings. , 1993, The Journal of physiology.

[27]  J. Russell,et al.  Oxytocin and vasopressin release within the supraoptic and paraventricular nuclei of pregnant, parturient and lactating rats: A microdialysis study , 1993, Neuroscience.

[28]  H. Gainer,et al.  Ultrastructural immunolocalization of rat oxytocin-neurophysin in transgenic mice expressing the rat oxytocin gene , 1992, Brain Research.

[29]  R. Bicknell,et al.  Activation of κ-opioid receptors inhibits depolarisation-evoked exocytosis but not the rise in intracellular Ca2+ in secretory nerve terminals of the neurohypophysis , 1992, Brain Research.

[30]  A. Fatatis,et al.  Secretion from rat neurohypophysial nerve terminals (neurosecretosomes) rapidly inactivates despite continued elevation of intracellular Ca2+ , 1992, Brain Research.

[31]  J. Kiss,et al.  Coexpression of vasopressin and oxytocin in hypothalamic supraoptic neurons of lactating rats. , 1991, Endocrinology.

[32]  H. Gainer,et al.  Cell‐Specific Expression of the Rat Oxytocin Gene in Transgenic Mice , 1990, Journal of neuroendocrinology.

[33]  E. Stuenkel.  Effects of membrane depolarization on intracellular calcium in single nerve terminals , 1990, Brain Research.

[34]  J. Windle,et al.  Immortalization of hypothalamic GnRH by genetically targeted tumorigenesis , 1990, Neuron.

[35]  J. Windle,et al.  Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice. , 1990, Molecular endocrinology.

[36]  F S Fay,et al.  Intracellular calibration of the fluorescent calcium indicator Fura-2. , 1990, Cell calcium.

[37]  R. Bicknell,et al.  Functional κ-opioid receptors on oxytocin and vasopressin nerve terminals isolated from the rat neurohypophysis , 1988, Brain Research.

[38]  N. Falke.  Dynorphin (1–8) inhibits stimulated release of oxytoxin but not vasopressin from isolated neurosecretory endings of the rat neurohypophysis , 1988, Neuropeptides.

[39]  H. Gainer,et al.  Dynorphin A inhibits and naloxone increases the electrically stimulated release of oxytocin but not vasopressin from the terminals of the neural lobe. , 1988, Endocrinology.

[40]  G. Dayanithi,et al.  Requirements for hormone release from permeabilized nerve endings isolated from the rat neurohypophysis. , 1987, Journal of Physiology.

[41]  G. Dayanithi,et al.  Hormone release from isolated nerve endings of the rat neurohypophysis. , 1987, The Journal of physiology.

[42]  P. Seeburg,et al.  The hypogonadal mouse: reproductive functions restored by gene therapy. , 1986, Science.

[43]  I. Chaiken,et al.  Molecular properties of the oxytocin/bovine neurophysin biosynthetic precursor. Studies using a semisynthetic precursor. , 1985, The Journal of biological chemistry.

[44]  R. Tsien,et al.  A new generation of Ca2+ indicators with greatly improved fluorescence properties. , 1985, The Journal of biological chemistry.

[45]  H. Gainer,et al.  Neurophysin in the hypothalamo-neurohypophysial system. I. Production and characterization of monoclonal antibodies , 1985, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[46]  H. Gainer,et al.  Neurophysin in the hypothalamo-neurohypophysial system. II. Immunocytochemical studies of the ontogeny of oxytocinergic and vasopressinergic neurons , 1985, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[47]  H. Gainer,et al.  Onset of neurophysin self‐association upon neurophysin/neuropeptide hormone precursor biosynthesis , 1983, FEBS letters.

[48]  G. Leng,et al.  Endogenous opiates regulate oxytocin but not vasopressin secretion from the neurohypophysis , 1982, Nature.

[49]  H. Gainer,et al.  Synthesis, transport, and release of posterior pituitary hormones. , 1980, Science.

[50]  D. Swaab,et al.  The localization of oxytocin, vasopressin, somatostatin and luteinizing hormone releasing hormone in the rat neurohypophysis , 1979, Cell and Tissue Research.

[51]  D. Swaab,et al.  Specific immunoelectronmicroscopic localization of vasopressin and oxytocin in the neurohypophysis of the rat , 1977, Cell and Tissue Research.

[52]  V. Du Vigneaud,et al.  Purification of oxytocin and vasopressin by way of a protein complex. , 1958, The Journal of biological chemistry.

[53]  H. Gainer,et al.  Transgenic Models for Studies of Oxytocin and Vasopressin , 2001 .

[54]  F. Dudek,et al.  Genetic targeting of green fluorescent protein to gonadotropin-releasing hormone neurons: characterization of whole-cell electrophysiological properties and morphology. , 2000, Endocrinology.

[55]  K. Herrup.  Transgenic and ES cell chimeric mice as tools for the study of the nervous system , 1995 .

[56]  E. Breslow,et al.  Molecular, thermodynamic, and biological aspects of recognition and function in neurophysin-hormone systems: a model system for the analysis of protein-peptide interactions. , 1990, Advances in enzymology and related areas of molecular biology.

[57]  F. Dudek,et al.  Printed in U.S.A. Copyright © 2000 by The Endocrine Society Whole-Cell Recordings from Preoptic/Hypothalamic Slices Reveal Burst Firing in Gonadotropin-Releasing Hormone Neurons Identified with Green Fluorescent Protein in Transgenic Mice* , 2022 .