Abstract
Here we discuss the useful properties of a preparation of isolated neurosecretory nerve terminals obtained from mammalian neurohypophyses (posterior pituitaries). These nerve terminals release the two neuropeptides, oxytocin and vasopressin, which are easily assayed by radioimmunoassay and/or ELISA. Depolarization-induced exocytosis is dependent on the same parameters as those regulating release from the whole, intact neurohypophysis. The isolated nerve terminals can be identified and also imaged for intracellular Ca2+ and a functional synapse can be reconstituted using their purified neurosecretory granules. Furthermore, some nerve terminals are large enough to allow the use of patch-clamp techniques and the monitoring of exocytosis by capacitance and amperometric measurements. We also present evidence which show that the isolated neurohypophysial nerve terminals represent a powerful tool for studying the mechanisms underlying depolarization–secretion coupling (D-SC).
Key words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lemos JR (2012) Magnocellular neurons in eLS. John Wiley & Sons, Ltd: Chichester. DOI: 10.1002/9780470015902.a0000176.pub2
Nordmann JJ, Dayanithi G, Lemos JR (1987) Isolated neurosecretory nerve endings as a tool for studying the mechanism of stimulus–secretion coupling. Biosci Rep 7:411–426
Morris JF, Pow DV (1993) New anatomical insights into the inputs from hypothalamic magnocellular neurons. In: North WG, Moses AM, Share L (eds) The neurohypophysis: a window on brain function, Annals of the New York Academy of Science. The New York Academy of Sciences, New York, pp 16–33
Knott TK, Dayanithi G, Coccia V, Custer EE, Lemos JR, Treistman SN (2000) Tolerance to acute ethanol inhibition of peptide hormone release in the isolated neurohypophysis. Alcohol Clin Exp Res 24:1077–1083
Wang XM, Lemos JR, Dayanithi G, Nordmann JJ, Treistman SN (1991) Ethanol reduces vasopressin release by inhibiting calcium currents in nerve terminals. Brain Res 551:338–341
Wang XM, Dayanithi G, Lemos JR, Nordmann JJ, Treistman SN (1991) Calcium currents and peptide release from neurohypophysial terminals are inhibited by ethanol. J Pharmacol Exp Ther 259:705–711
Marrero HG, Lemos JR (2003) Loose-patch clamp currents from the hypothalamo-neurohypophysial system of the rat. Pflugers Arch 446:702–713
Lemos JR, Nordmann JJ, Cooke IM, Stuenkel EL (1986) Single channels and ionic currents in peptidergic nerve terminals. Nature 319:410–412
Lemos JR, Nordmann JJ (1986) Ionic channels and hormone release from peptidergic nerve terminals. J Exp Biol 128:53–72
Wang XM, Treistman SN, Lemos JR (1991) Direct identification of individual vasopressin-containing nerve terminals of the rat neurohypophysis after ‘whole-cell’ patch-clamp recordings. Neurosci Lett 124:125–128
Custer EE, Ortiz-Miranda S, Knott TK et al (2007) Identification of the neuropeptide content of individual rat neurohypophysial terminals. J Neurosci Methods 163:226–234
De Crescenzo V, ZhuGe R, Velazquez-Marrero C et al (2004) Ca2+ syntillas, miniature ca2+ release events in terminals of hypothalamic neurons, are increased in frequency by depolarization in the absence of ca2+ influx. J Neurosci 24:1226–1235
Knott TK, Hussy N, Cuadra AE et al (2012) Atp appears to act via different receptors in terminals vs. somata of the hypothalamic neurohypophysial system. J Neuroendocrinol 24:681–689
Knott TK, Marrero HG, Fenton RA, Custer EE, Dobson JG Jr, Lemos JR (2007) Endogenous adenosine inhibits cns terminal Ca(2+) currents and exocytosis. J Cell Physiol 210:309–314
McNally JM, De Crescenzo V, Fogarty KE, Walsh JV, Lemos JR (2009) Individual calcium syntillas do not trigger spontaneous exocytosis from nerve terminals of the neurohypophysis. J Neurosci 29:14120–14126
McNally JM, Woodbury DJ, Lemos JR (2004) Syntaxin 1a drives fusion of large dense-core neurosecretory granules into a planar lipid bilayer. Cell Biochem Biophys 41:11–24
Yin Y, Dayanithi G, Lemos JR (2002) Ca(2+)-regulated, neurosecretory granule channel involved in release from neurohypophysial terminals. J Physiol 539:409–418
Nordmann JJ, Dayanithi G, Cazalis M (1986) Do opioid peptides modulate, at the level of the nerve endings, the release of neurohypophysial hormones? Exp Brain Res 61:560–566
Ueta Y, Dayanithi G, Fujihara H (2011) Hypothalamic vasopressin response to stress and various physiological stimuli: visualization in transgenic animal models. Horm Behav 59:221–226
Fenwick EM, Marty A, Neher E (1982) A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine. J Physiol 331:577–597
Kidokoro Y (1985) Electrophysiology of adrenal chromaffin cells. In: Poisner AM, Trifaro JM (eds) The electrophysiology of the secretory cell. Elsevier, New York
Maruyama Y, Peterson OH (1982) Single-channel currents in isolated patches of plasma membrane from basal surface of pancreatic acini. Nature 299:159–161
Hagiwara S, Byerly L (1983) The calcium channel. Trends Neurosci 6:189–193
Mason WT, Dyball RE (1986) Single ion channel activity in peptidergic nerve terminals of the isolated rat neurohypophysis related to stimulation of neural stalk axons. Brain Res 383:279–286
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100
Carbone E, Lux HD (1984) A low voltage-activated, fully inactivating Ca channel in vertebrate sensory neurones. Nature 310:501–502
Fox AP, Nowycky MC, Tsien RW (1987) Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurons. J Physiol 394:149–172
Dunlap K, Luebke JI, Turner TJ (1995) Exocytotic Ca2+ channels in mammalian central neurons. Trends Neurosci 18:89–98
Kawamoto EM, Vivar C, Camandola S (2012) Physiology and pathology of calcium signaling in the brain. Front Pharmacol 3:61
Byerly L, Yazejian B (1986) Intracellular factors for the maintenance of calcium currents in perfused neurones from the snail, Lymnea stagnalis. J Physiol 370:631–650
Wang X, Treistman SN, Wilson A, Nordmann JJ, Lemos JR (1993) Calcium channels and peptide release from neurosecretory terminals. News Physiol Sci 8:64–68
Rae J, Cooper K, Gates P, Watsky M (1991) Low access resistance perforated patch recordings using amphotericin b. J Neurosci Methods 37:15–26
Wang G, Dayanithi G, Custer EE, Lemos JR (2002) Adenosine inhibition via a(1) receptor of N-type Ca2+ current and peptide release from isolated neurohypophysial terminals of the rat. J Physiol 540:791–802
Wang G, Dayanithi G, Newcomb R, Lemos JR (1999) An R-type Ca2+ current in neurohypophysial terminals preferentially regulates oxytocin secretion. J Neurosci 19:9235–9241
Thorn PJ, Wang XM, Lemos JR (1991) A fast, transient K+ current in neurohypophysial nerve terminals of the rat. J Physiol 432:313–326
Wang G, Lemos JR (1992) Tetrandrine blocks a slow, large-conductance, Ca(2+)-activated potassium channel besides inhibiting a non-inactivating Ca2+ current in isolated nerve terminals of the rat neurohypophysis. Pflugers Arch 421:558–565
Wang G, Jiang MX, Coyne MD, Lemos JR (1993) Comparison of effects of tetrandrine on ionic channels of isolated rat neurohypophysial terminals and Y1 mouse adrenocortical tumor cells. Zhongguo Yao Li Xue Bao 14:101–106
Wang X, Treistman SN, Lemos JR (1992) Two types of high-threshold calcium currents inhibited by ω-conotoxin in nerve terminals of rat neurohypophysis. J Physiol 445:181–199
Lemos JR, Wang G (2000) Excitatory versus inhibitory modulation by ATP of neurohypophysial terminal activity in the rat. Exp Physiol 85:67s–74s
Knott TK, Velazquez-Marrero C, Lemos JR (2005) ATP elicits inward currents in isolated vasopressinergic neurohypophysial terminals via p2x2 and p2x3 receptors. Pflugers Arch 450:381–389
Treistman SN, Bayley H, Lemos JR, Wang XM, Nordmann JJ, Grant AJ (1991) Effects of ethanol on calcium channels, potassium channels, and vasopressin release. Ann N Y Acad Sci 625:249–263
Wang G, Dayanithi G, Kim S et al (1997) Role of Q-type Ca2+ channels in vasopressin secretion from neurohypophysial terminals of the rat. J Physiol 502(Pt 2):351–363
Edwards FA, Gibb AJ, Colquhoun D (1992) ATP receptor-mediated synaptic currents in the central nervous system. Nature 359:144–147
Silinsky EM, Gerzanich V, Vanner SM (1992) ATP mediates excitatory synaptic transmission in mammalian neurones. Br J Pharmacol 106:762–763
Ueno S, Harata N, Inoue K, Akaike N (1992) Atp-gated current in dissociated rat nucleus solitarii neurons. J Neurophysiol 68:778–785
Zimmermann H (1994) Signalling via ATP in the nervous system. Trends Neurosci 17:420–426
Sperlagh B, Mergl Z, Juranyi Z, Vizi ES, Makara GB (1999) Local regulation of vasopressin and oxytocin secretion by extracellular ATP in the isolated posterior lobe of the rat hypophysis. J Endocrinol 160:343–350
Troadec JD, Thirion S, Nicaise G, Lemos JR, Dayanithi G (1998) Atp-evoked increases in [Ca2+]i and peptide release from rat isolated neurohypophysial terminals via a P2X2 purinoceptor. J Physiol 511(Pt 1):89–103
Bobanovic LK, Royle SJ, Murrell-Lagnado RD (2002) P2X receptor trafficking in neurons is subunit specific. J Neurosci 22:4814–4824
Rubio ME, Soto F (2001) Distinct localization of P2X receptors at excitatory postsynaptic specializations. J Neurosci 21:641–653
Shibuya I, Tanaka K, Hattori Y et al (1999) Evidence that multiple P2X purinoceptors are functionally expressed in rat supraoptic neurones. J Physiol 514(Pt 2):351–367
Sperlágh B, Vizi ES, Wirkner K, Illes P (2006) P2X7 receptors in the nervous system. Prog Neurobiol 78:327–346
Sladek CD, Song Z (2012) Diverse roles of G-protein coupled receptors in the regulation of neurohypophyseal hormone secretion. J Neuroendocrinol 24:554–565
Gomes DA, Song Z, Stevens W, Sladek CD (2009) Sustained stimulation of vasopressin and oxytocin release by ATP and phenylephrine requires recruitment of desensitization-resistant P2X purinergic receptors. Am J Physiol Regul Integr Comp Physiol 297:R940–R949
Gordon GR, Baimoukhametova DV, Hewitt SA, Rajapaksha WR, Fisher TE, Bains JS (2005) Norepinephrine triggers release of glial ATP to increase postsynaptic efficacy. Nat Neurosci 8:1078–1086
Sawyer CH, Clifton DK (1980) Aminergic innervation of the hypothalamus. Fed Proc 39:2889–2895
Cuadra AE, Knott T, Custer E, Lemos JR (2013) P2X7 Receptor-Mediated Currents in Rat Hypothalamic Neurohypophysial System (HNS) Terminals. J Cell Physiol in press
Brethes D, Dayanithi G, Letellier L, Nordmann JJ (1987) Depolarization-induced Ca2+ increase in isolated neurosecretory nerve terminals measured with fura-2. Proc Natl Acad Sci USA 84:1439–1443
Stuenkel EL, Nordmann JJ (1993) Intracellular calcium and vasopressin release of rat isolated neurohypophysial nerve endings. J Physiol 468:335–355
Sasaki N, Dayanithi G, Shibuya I (2005) Ca2+ clearance mechanisms in neurohypophysial terminals of the rat. Cell Calcium 37:45–56
Berridge MJ (2006) Calcium microdomains: organization and function. Cell Calcium 40:405–412
De Crescenzo V, Fogarty KE, Zhuge R et al (2006) Dihydropyridine receptors and type 1 ryanodine receptors constitute the molecular machinery for voltage-induced Ca2+ release in nerve terminals. J Neurosci 26:7565–7574
ZhuGe R, DeCrescenzo V, Sorrentino V et al (2006) Syntillas release Ca2+ at a site different from the microdomain where exocytosis occurs in mouse chromaffin cells. Biophys J 90:2027–2037
Sun XP, Callamaras N, Marchant JS, Parker I (1998) A continuum of insp3-mediated elementary Ca2+ signalling events in Xenopus oocytes. J Physiol 509(Pt 1):67–80
ZhuGe R, Fogarty KE, Tuft RA, Lifshitz LM, Sayar K, Walsh JV Jr (2000) Dynamics of signaling between Ca(2+) sparks and Ca(2+)-activated K(+) channels studied with a novel image-based method for direct intracellular measurement of ryanodine receptor Ca(2+) current. J Gen Physiol 116:845–864
Kim KT, Koh DS, Hille B (2000) Loading of oxidizable transmitters into secretory vesicles permits carbon-fiber amperometry. J Neurosci 20:RC101
Marrero HG, Lemos JR (2010) Ionic conditions modulate stimulus-induced capacitance changes in isolated neurohypophysial terminals of the rat. J Physiol 588:287–300
Giovannucci DR, Stuenkel EL (1997) Regulation of secretory granule recruitment and exocytosis at rat nerohypophysial nerve endings. J Physiol 498:735–751
Cazalis M, Dayanithi G, Nordmann JJ (1987) Hormone release from isolated nerve endings of the rat neurohypophysis. J Physiol 390:55–70
Mueller P, Rudin DO, Tien HT, Wescott WC (1962) Reconstitution of cell membrane structure in vitro and its transformation into an excitable system. Nature 194:979–980
Woodbury DJ, Hall JE (1988) Role of channels in the fusion of vesicles with a planar bilayer. Biophys J 54:1053–1063
Woodbury DJ, Hall JE (1988) Vesicle-membrane fusion. Observation of simultaneous membrane incorporation and content release. Biophys J 54:345–349
Cohen FS, Niles WD, Akabas MH (1989) Fusion of phospholipid vesicles with a planar membrane depends on the membrane permeability of the solute used to create the osmotic pressure. J Gen Physiol 93:201–210
Woodbury DJ (1999) Nystatin/ergosterol method for reconstituting ion channels into planar lipid bilayers. Methods Enzymol 294:319–339
Woodbury DJ, Miller C (1990) Nystatin-induced liposome fusion. A versatile approach to ion channel reconstitution into planar bilayers. Biophys J 58:833–839
Jahn R, Fasshauer D (2012) Molecular machines governing exocytosis of synaptic vesicles. Nature 490:201–207
Rognlien KT, Woodbury DJ (2003) Reconstituting snare proteins into blms. In: Tein HT, Ottova-Leitmannova A (eds) Planar lipid bilayers (blm) and their applications. Elsevier Science, Amsterdam, pp 479–488
Woodbury DJ, Rognlien K (2000) The t-snare syntaxin is sufficient for spontaneous fusion of synaptic vesicles to planar membranes. Cell Biol Int 24:809–818
McNally JM, Woodbury DJ, Lemos JR (2003) Syntaxin 1a is sufficient for spontaneous fusion of both neurohypophysial and chromaffin dense core granules to a planar lipid bilayer. In: 43rd annual meeting of the American Society for Cell Biology, San Francisco
Liu Q, Chen B, Yankova M et al (2005) Presynaptic ryanodine receptors are required for normal quantal size at the Caenorhabditis elegans neuromuscular junction. J Neurosci 25:6745–6754
Fix M, Melia TJ, Jaiswal JK et al (2004) Imaging single membrane fusion events mediated by snare proteins. Proc Natl Acad Sci USA 101:7311–7316
Weber T, Zemelman BV, McNew JA et al (1998) Snarepins: minimal machinery for membrane fusion. Cell 92:759–772
Tucker WC, Weber T, Chapman ER (2004) Reconstitution of Ca2+ -regulated membrane fusion by synaptotagmin and snares. Science 304:435–438
Lee DE, LeW MG, Woodbury DJ (2012) Vesicle fusion to planar membranes is enhanced by cholesterol and low temperature. Chem Phys Lipids 166:45–54
Chow RH, von Ruden L, Neher E (1992) Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells. Nature 356:60–63
Zhou Z, Misler S, Chow RH (1996) Rapid fluctuations in transmitter release from single vesicles in bovine adrenal chromaffin cells. Biophys J 70:1543–1552
Chow RH, Rüden L (1995) Electrochemical detection of secretion from single cells. In: Sakmann BaN E (ed) Single channel recording. Plenum, New York, pp 245–275
Bowen ME, Weninger K, Brunger AT, Chu S (2004) Single molecule observation of liposome-bilayer fusion thermally induced by soluble N-ethyl maleimide sensitive-factor attachment protein receptors (snares). Biophys J 87:3569–3584
Liu TT, Tucker WC, Bhalla A, Chapman ER, Weisshaar JC (2005) Snare-driven, 25-millisecond vesicle fusion in vitro. Biophys J 89:2458–2472
Bredt DS, Hwang PM, Snyder SH (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347:768–770
Custer EE, Knott TK, Cuadra AE, Ortiz-Miranda S, Lemos JR (2012) P2X purinergic receptor knockout mice reveal endogenous ATP modulation of both avp and ot release from the intact neurohypophysis. J Neuroendocrinol 24:674–680
Zhang BJ, Kusano K, Zerfas P, Iacangelo A, Young WS III, Gainer H (2002) Targeting of green fluorescent protein to secretory granules in oxytocin magnocellular neurons and its secretion from neurohypophysial nerve terminals in transgenic mice. Endocrinology 143:1036–1046
LaBella FS, Sanwal M (1965) Isolation of nerve endings from the posterior pituitary gland. Electron microscopy of fractions obtained by centrifugation. J Cell Biol 25(Suppl):179–193
Lee CJ, Dayanithi G, Nordmann JJ, Lemos JR (1992) Possible role during exocytosis of a ca(2+)-activated channel in neurohypophysial granules. Neuron 8:335–342
Andreoli TE, Monahan M (1968) The interaction of polyene antibiotics with thin lipid membranes. J Gen Physiol 52:300–325
Cass A, Finkelstein A, Krespi V (1970) The ion permeability induced in thin lipid membranes by the polyene antibiotics nystatin and amphotericin b. J Gen Physiol 56:100–124
Woodbury DJ (1990) Vesicle-membrane fusion detected by simultaneous electrical and optical measurements. In: Proceedings of the twelfth annual international conference of the IEEE Engineering in Medicine and Biology Society 12(4):1747–1748
Woodbury DJ, McNally JM, Lemos JR (2007) Snare-induced fusion of liposomes to a planar bilayer. In: Leitmannova Liu A (ed) Planar lipid bilayers. Elsevier Press, London
Lemos JR, Nowycky MC (1989) Two types of calcium channels coexist in peptide-releasing vertebrate nerve terminals. Neuron 2:1419–1426
Ortiz-Miranda S, Dayanithi G, Velásquez-Marrero C, Custer E, Treistman SN, Lemos JR (2010) Differential modulation of N-type calcium channels by μ-opioid receptors in oxytocinergic vs. vasopressinergic neurohypophysial terminals. J Cell Physiol 225:276–288
Wang G, Thorn PJ, Lemos JR (1992) A novel large-conductance Ca(2+)-activated potassium channel and current in nerve terminals of the rat neurohypophysis. J Physiol 457:47–74
Wynne PM, Puig SI, Martin GE, Treistman SN (2009) Compartmentalized β subunit distribution determines characteristics and ethanol sensitivity of somatic, dendritic, and terminal large-conductance calcium-activated potassium channels in the rat central nervous system. J Pharmacol Exp Ther 329:978–986
Kilic G, Stolpe A, Lindau M (1996) A slowly activating voltage-dependent K + current in rat pituitary nerve terminals. J Physiol 497:711–725
Wilke RA, Ahern GP, Jackson MB (1998) Membrane excitability in the neurohypophysis. Adv Exp Med Biol 449:193–200
Ortiz-Miranda S, Dayanithi G, Custer E, Treistman SN, Lemos JR (2005) Micro-opioid receptor preferentially inhibits oxytocin release from neurohypophysial terminals by blocking R-type Ca2+ channels. J Neuroendocrinol 17:583–590
Rusin KI, Giovannucci DR, Stuenkel EL, Moises HC (1997) Κ-opioid receptor activation modulates Ca2+ currents and secretion in isolated neuroendocrine nerve terminals. J Neurosci 17(17):6565–6574
Hussy N, Bres V, Rochette M et al (2001) Osmoregulation of vasopressin secretion via activation of neurohypophysial nerve terminals glycine receptors by glial taurine. J Neurosci 21:7110–7116
Saito T, Dayanithi G, Saito J et al (2008) Chronic osmotic stimuli increase salusin-β-like immunoreactivity in the rat hypothalamo-neurohypophyseal system: possible involvement of salusin-β on [Ca2+]i increase and neurohypophyseal hormone release from the axon terminals. J Neuroendocrinol 20:207–219
Dayanithi G, Cazalis M, Nordmann JJ (1987) Relaxin affects the release of oxytocin and vasopressin from the neurohypophysis. Nature 325:813–816
Zhang SJ, Jackson MB (1995) GABAa receptor activation and the excitability of nerve terminals in the rat posterior pituitary. J Physiol 483(Pt 3):583–595
Wilke RA, Hsu S-F, Jackson MB (1998) Dopamine D4 receptor mediated inhibition of potassium current in neurohypophysial nerve terminals. J Pharmacol Exp Ther 284:542–548
Acknowledgements
Many thanks to the father of the “kikis,” Jean J. Nordmann, for developing and teaching us this wonderful preparation. We also wish to acknowledge seminal work in developing this preparation by Vincent Coccia, Govindan Dayanithi, Valerie DeCrescenzo, Alex Dopico, Thomas Knott, Joseph Lockhart, Karen Ocorr, Sonia Ortiz-Miranda, Mark Savage, Edward Stuenkel, Peter Thorn, Steven Treistman, Cristina Velazquez, Gang Wang, Xiaoming Wang, and Yong Yin. Supported by NIH grants: NS29470, DA10487 and NS063192 to J.R.L.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Lemos, J.R., McNally, J., Custer, E., Cuadra, A., Marrero, H., Woodbury, D. (2014). Isolated Neurohypophysial Terminals: Model for Depolarization–Secretion Coupling. In: Thorn, P. (eds) Exocytosis Methods. Neuromethods, vol 83. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-676-4_10
Download citation
DOI: https://doi.org/10.1007/978-1-62703-676-4_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-675-7
Online ISBN: 978-1-62703-676-4
eBook Packages: Springer Protocols