PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jcellbiolHomeThe Rockefeller University PressEditorsContactInstructions for AuthorsThis issue
 
J Cell Biol. 1986 July 1; 103(1): 171–187.
PMCID: PMC2113786

Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures

Abstract

A prerequisite for many studies of neurons in culture is a means of determining their original identity. We needed such a technique to study the interactions in vitro between a class of spinal cord neurons, sympathetic preganglionic neurons, and their normal target, neurons from the sympathetic chain. Here, we describe how we use two highly fluorescent carbocyanine dyes, which differ in color but are otherwise similar, to identify neurons in culture. The long carbon chain carbocyanine dyes we use are lipid-soluble and so become incorporated into the plasma membrane. Neurons can be labeled either retrogradely or during dissociation. Some of the labeled membrane gradually becomes internalized and retains its fluorescence, allowing identification of cells for several weeks in culture. These dyes do not affect the survival, development, or basic physiological properties of neurons and do not spread detectably from labeled to unlabeled neurons. It seems likely that cells become retrogradely labeled mainly by lateral diffusion of dye in the plane of the membrane. If so, carbocyanine dyes may be most useful for retrograde labeling over relatively short distances. An additional feature of carbocyanine labeling is that neuronal processes are brightly fluorescent for the first few days in culture, presumably because dye rapidly diffuses into newly inserted membrane. We have used carbocyanine dyes to identify sympathetic preganglionic neurons in culture. Our results indicate that preganglionic neurons can survive in the absence of their target cells and that several aspects of their differentiation in the absence of target appear normal.

Full Text

The Full Text of this article is available as a PDF (2.7M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Axelrod D. Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization. Biophys J. 1979 Jun;26(3):557–573. [PubMed]
  • Axelrod D, Wight A, Webb W, Horwitz A. Influence of membrane lipids on acetylcholine receptor and lipid probe diffusion in cultured myotube membrane. Biochemistry. 1978 Aug 22;17(17):3604–3609. [PubMed]
  • Barde YA, Edgar D, Thoenen H. New neurotrophic factors. Annu Rev Physiol. 1983;45:601–612. [PubMed]
  • Bennett MR, Lai K, Nurcombe V. Identification of embryonic motoneurons in vitro: their survival is dependent on skeletal muscle. Brain Res. 1980 May 26;190(2):537–542. [PubMed]
  • Bentivoglio M, Kuypers HG, Catsman-Berrevoets CE, Dann O. Fuorescent retrograde neuronal labeling in rat by means of substances binding specifically to adenine-thymine rich DNA. Neurosci Lett. 1979 May;12(2-3):235–240. [PubMed]
  • Berg DK. New neuronal growth factors. Annu Rev Neurosci. 1984;7:149–170. [PubMed]
  • Betz W. Functional and non-functional contacts between ciliary neurones and muscle grown in vitro. J Physiol. 1976 Jan;254(1):75–86. [PubMed]
  • Bonhoeffer F, Huf J. Recognition of cell types by axonal growth cones in vitro. Nature. 1980 Nov 13;288(5787):162–164. [PubMed]
  • Bonhoeffer F, Huf J. Position-dependent properties of retinal axons and their growth cones. Nature. 315(6018):409–410. [PubMed]
  • Bray D. Surface movements during the growth of single explanted neurons. Proc Natl Acad Sci U S A. 1970 Apr;65(4):905–910. [PubMed]
  • Calof AL, Reichardt LF. Motoneurons purified by cell sorting respond to two distinct activities in myotube-conditioned medium. Dev Biol. 1984 Nov;106(1):194–210. [PubMed]
  • Chalazonitis A, Greene LA, Nirenberg M. Electrophysiological chracteristics of chick embryo sympathetic neurons in dissociated cell culture. Brain Res. 1974 Mar 22;68(2):235–252. [PubMed]
  • Chun LL, Patterson PH. Role of nerve growth factor in the development of rat sympathetic neurons in vitro. I. Survival, growth, and differentiation of catecholamine production. J Cell Biol. 1977 Dec;75(3):694–704. [PMC free article] [PubMed]
  • de Laat SW, van der Saag PT, Elson EL, Schlessinger J. Lateral diffusion of membrane lipids and proteins is increased specifically in neurites of differentiating neuroblastoma cells. Biochim Biophys Acta. 1979 Dec 4;558(2):247–250. [PubMed]
  • de Laat SW, van der Saag PT, Elson EL, Schlessinger J. Lateral diffusion of membrane lipids and proteins during the cell cycle of neuroblastoma cells. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1526–1528. [PubMed]
  • Dembowsky K, Czachurski J, Seller H. Morphology of sympathetic preganglionic neurons in the thoracic spinal cord of the cat: an intracellular horseradish peroxidase study. J Comp Neurol. 1985 Aug 22;238(4):453–465. [PubMed]
  • Denis-Donini S, Glowinski J, Prochiantz A. Glial heterogeneity may define the three-dimensional shape of mouse mesencephalic dopaminergic neurones. Nature. 1984 Feb 16;307(5952):641–643. [PubMed]
  • Derzko Z, Jacobson K. Comparative lateral diffusion of fluorescent lipid analogues in phospholipid multibilayers. Biochemistry. 1980 Dec 23;19(26):6050–6057. [PubMed]
  • Dragsten PR, Blumenthal R, Handler JS. Membrane asymmetry in epithelia: is the tight junction a barrier to diffusion in the plasma membrane? Nature. 1981 Dec 24;294(5843):718–722. [PubMed]
  • Dryer SE, Chiappinelli VA. An intracellular study of synaptic transmission and dendritic morphology in sympathetic neurons of the chick embryo. Brain Res. 1985 Sep;354(1):99–111. [PubMed]
  • Fallon JR. Preferential outgrowth of central nervous system neurites on astrocytes and Schwann cells as compared with nonglial cells in vitro. J Cell Biol. 1985 Jan;100(1):198–207. [PMC free article] [PubMed]
  • Feldman EL, Axelrod D, Schwartz M, Heacock AM, Agranoff BW. Studies on the localization of newly added membrane in growing neurites. J Neurobiol. 1981 Nov;12(6):591–598. [PubMed]
  • Greene LA. Quantitative in vitro studies on the nerve growth factor (NGF) requirement of neurons. I. Sympathetic neurons. Dev Biol. 1977 Jul 1;58(1):96–105. [PubMed]
  • Hamburger V. Cell death in the development of the lateral motor column of the chick embryo. J Comp Neurol. 1975 Apr 15;160(4):535–546. [PubMed]
  • Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. [PubMed]
  • Harper CG, Gonatas JO, Stieber A, Gonatas NK. In vivo uptake of wheat germ agglutinin-horseradish peroxidase conjugates into neuronal GERL and lysosomes. Brain Res. 1980 Apr 28;188(2):465–472. [PubMed]
  • Honig MG. The development of sensory projection patterns in embryonic chick hind limb. J Physiol. 1982 Sep;330:175–202. [PubMed]
  • Hume RI, Honig MG. Excitatory action of ATP on embryonic chick muscle. J Neurosci. 1986 Mar;6(3):681–690. [PubMed]
  • Jacobson K, Hou Y, Derzko Z, Wojcieszyn J, Organisciak D. Lipid lateral diffusion in the surface membrane of cells and in multibilayers formed from plasma membrane lipids. Biochemistry. 1981 Sep 1;20(18):5268–5275. [PubMed]
  • Katz LC, Burkhalter A, Dreyer WJ. Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex. Nature. 1984 Aug 9;310(5977):498–500. [PubMed]
  • Keizer K, Kuypers HG, Huisman AM, Dann O. Diamidino yellow dihydrochloride (DY . 2HCl); a new fluorescent retrograde neuronal tracer, which migrates only very slowly out of the cell. Exp Brain Res. 1983;51(2):179–191. [PubMed]
  • Klausner RD, Wolf DE. Selectivity of fluorescent lipid analogues for lipid domains. Biochemistry. 1980 Dec 23;19(26):6199–6203. [PubMed]
  • Ko CP, Burton H, Bunge RP. Synaptic transmission between rat spinal cord explants and dissociated superior cervical ganglion neurons in tissue culture. Brain Res. 1976 Dec 3;117(3):437–460. [PubMed]
  • Kriegstein AR, Dichter MA. Morphological classification of rat cortical neurons in cell culture. J Neurosci. 1983 Aug;3(8):1634–1647. [PubMed]
  • Landmesser L. Contractile and electrical responses of vagus-innervated frog sartorius muscles. J Physiol. 1971 Mar;213(3):707–725. [PubMed]
  • Landmesser L. The distribution of motoneurones supplying chick hind limb muscles. J Physiol. 1978 Nov;284:371–389. [PubMed]
  • Landmesser L. The development of motor projection patterns in the chick hind limb. J Physiol. 1978 Nov;284:391–414. [PubMed]
  • Leifer D, Lipton SA, Barnstable CJ, Masland RH. Monoclonal antibody to Thy-1 enhances regeneration of processes by rat retinal ganglion cells in culture. Science. 1984 Apr 20;224(4646):303–306. [PubMed]
  • Montecucco C, Pozzan T, Rink T. Dicarbocyanine fluorescent probes of membrane potential block lymphocyte capping, deplete cellular ATP and inhibit respiration of isolated mitochondria. Biochim Biophys Acta. 1979 Apr 19;552(3):552–557. [PubMed]
  • Mudge AW. Schwann cells induce morphological transformation of sensory neurones in vitro. Nature. 1984 May 24;309(5966):367–369. [PubMed]
  • Neff N, Decker C, Horwitz A. The kinetics of myoblast fusion. Exp Cell Res. 1984 Jul;153(1):25–31. [PubMed]
  • Nishi R, Berg DK. Dissociated ciliary ganglion neurons in vitro: survival and synapse formation. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5171–5175. [PubMed]
  • Oppenheim RW, Heaton MB. The retrograde transport of horseradish peroxidase from the developing limb of the chick embryo. Brain Res. 1975 Nov 14;98(2):291–302. [PubMed]
  • Oppenheim RW, Maderdrut JL, Wells DJ. Cell death of motoneurons in the chick embryo spinal cord. VI. Reduction of naturally occurring cell death in the thoracolumbar column of Terni by nerve growth factor. J Comp Neurol. 1982 Sep 10;210(2):174–189. [PubMed]
  • Pagano RE, Sleight RG. Defining lipid transport pathways in animal cells. Science. 1985 Sep 13;229(4718):1051–1057. [PubMed]
  • Patterson PH. Environmental determination of autonomic neurotransmitter functions. Annu Rev Neurosci. 1978;1:1–17. [PubMed]
  • Pfenninger KH, Johnson MP. Membrane biogenesis in the sprouting neuron. I. Selective transfer of newly synthesized phospholipid into the growing neurite. J Cell Biol. 1983 Oct;97(4):1038–1042. [PMC free article] [PubMed]
  • Pfenninger KH, Maylié-Pfenninger MF. Lectin labeling of sprouting neurons. II. Relative movement and appearance of glycoconjugates during plasmalemmal expansion. J Cell Biol. 1981 Jun;89(3):547–559. [PMC free article] [PubMed]
  • Simons TJ. Actions of a carbocyanine dye on calcium-dependent potassium transport in human red cell ghosts. J Physiol. 1979 Mar;288:481–507. [PubMed]
  • Sims PJ, Waggoner AS, Wang CH, Hoffman JF. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry. 1974 Jul 30;13(16):3315–3330. [PubMed]
  • Skirboll L, Hökfelt T, Norell G, Phillipson O, Kuypers HG, Bentivoglio M, Catsman-Berrevoets CE, Visser TJ, Steinbusch H, Verhofstad A, et al. A method for specific transmitter identification of retrogradely labeled neurons: immunofluorescence combined with fluorescence tracing. Brain Res. 1984 Dec;320(2-3):99–127. [PubMed]
  • Sleight RG, Pagano RE. Transbilayer movement of a fluorescent phosphatidylethanolamine analogue across the plasma membranes of cultured mammalian cells. J Biol Chem. 1985 Jan 25;260(2):1146–1154. [PubMed]
  • Sowers AE. Characterization of electric field-induced fusion in erythrocyte ghost membranes. J Cell Biol. 1984 Dec;99(6):1989–1996. [PMC free article] [PubMed]
  • Struck DK, Pagano RE. Insertion of fluorescent phospholipids into the plasma membrane of a mammalian cell. J Biol Chem. 1980 Jun 10;255(11):5404–5410. [PubMed]
  • Suzuki N. Anterograde fluorescent labeling of olfactory receptor neurons by Procion and Lucifer dyes. Brain Res. 1984 Oct 8;311(1):181–185. [PubMed]
  • Tanaka H, Obata K. Survival of HRP-labeled spinal motoneurons of chick embryo in tissue and cell cultures. Brain Res. 1983 Sep;285(3):390–395. [PubMed]
  • Thanos S, Bonhoeffer F. Investigations on the development and topographic order of retinotectal axons: anterograde and retrograde staining of axons and perikarya with rhodamine in vivo. J Comp Neurol. 1983 Oct 1;219(4):420–430. [PubMed]
  • Trojanowski JQ. Native and derivatized lectins for in vivo studies of neuronal connectivity and neuronal cell biology. J Neurosci Methods. 1983 Nov;9(3):185–204. [PubMed]
  • Waggoner A. Optical probes of membrane potential. J Membr Biol. 1976 Jun 30;27(4):317–334. [PubMed]
  • Wakade AR, Edgar D, Thoenen H. Both nerve growth factor and high K+ concentrations support the survival of chick embryo sympathetic neurons. Evidence for a common mechanism of action. Exp Cell Res. 1983 Apr 1;144(2):377–384. [PubMed]
  • Wigston DJ, Sanes JR. Selective reinnervation of intercostal muscles transplanted from different segmental levels to a common site. J Neurosci. 1985 May;5(5):1208–1221. [PubMed]
  • Wolf DE. Determination of the sidedness of carbocyanine dye labeling of membranes. Biochemistry. 1985 Jan 29;24(3):582–586. [PubMed]
  • Yoshikami D, Okun LM. Staining of living presynaptic nerve terminals with selective fluorescent dyes. Nature. 1984 Jul 5;310(5972):53–56. [PubMed]
  • Zidovetzki R, Yarden Y, Schlessinger J, Jovin TM. Rotational diffusion of epidermal growth factor complexed to cell surface receptors reflects rapid microaggregation and endocytosis of occupied receptors. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6981–6985. [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press