Sea Urchin Embryos and Larvae as Biosensors for Neurotoxicants

Gennady A. Buznikov1, Theodore A. Slotkin2, Jean M. Lauder3

1 N.K. Koltzov Institute of Developmental Biology, Moscow, 2 Duke University Medical Center, Durham, North Carolina, 3 University of North Carolina School of Medicine, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 1.6
DOI:  10.1002/0471140856.tx0106s16
Online Posting Date:  August, 2003
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Sea urchin embryos and larvae provide an inexpensive high‐throughput system for determining developmental actions of neuropharmacologic agents or environmental neurotoxins in both applied and basic biologic contexts. The use of this system for the testing of chlorpyrifos, 1‐nicotine, lipophilic amides of choline, and ritanserin is described in detail.

Keywords: sea urchin embryos and larvae; chlorpyrifos; 1‐nicotine; ritanserin

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Maintaining and Handling Adult Sea Urchins
  • Basic Protocol 2: Harvesting Sea Urchin Gametes
  • Basic Protocol 3: Fertilizing and Incubating Sea Urchin Eggs
  • Basic Protocol 4: Embryonic and Larval Development of Sea Urchins
  • Protocols for Testing Neurotoxicants
  • Basic Protocol 5: Testing of Chlorpyrifos on Sea Urchin Embryos and Larvae
  • Alternate Protocol 1: Testing of L‐Nicotine on Sea Urchin Embryos and Larvae
  • Alternate Protocol 2: Testing of Arachidonoylcholine and Docosahexaenoylcholine on Sea Urchin Embryos and Larvae
  • Alternate Protocol 3: Testing of Ritanserin on Sea Urchin Embryos and Larvae
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Maintaining and Handling Adult Sea Urchins

  Materials
  • Sea urchin embryos and larvae, stages 1a or 1b, 3 or 4, 10, 11, 13 (Tables 1.6.1 and 1.6.2; Figs. and ; see protocol 4)
  • ASW (see recipe) or commercially available artificial sea water (e.g., Instant Ocean; Aquarium Systems)
  • 20 mM chlorpyrifos (cpf; Chem Service) in 100% (v/v) methanol, store up to 1 to 2 weeks at −20°C
  • Potential rescue compound, such as the polyenoic fatty acid derivatives (see recipe) dimethylaminoethyl arachidonate (AA‐DMAE) and dimethylaminoethyl docosahexaenoate (DHA‐DMAE), or other substance of interest
  • Digital imaging system, including microscope (e.g., Leica), digital color videocamera (e.g., Spot RT; Diagnostic Instruments), and computer with appropriate software
  • 12‐ or 24‐well tissue culture plates

Basic Protocol 2: Harvesting Sea Urchin Gametes

  • 20 mM L‐nicotine bitartrate in 100% (v/v) methanol, store in dark up to 1 to 2 weeks at 4°C
  • Potential rescue compound, such as:
    • 20 mM D‐tubocurarine chloride (Sigma), store up to 1 week at −20°C
    • 20 mM imechine (Latoxan), store up to 1 to 2 weeks at 4°C
    • 20 mM QX‐222 (Astra Pharmaceuticals), store up to 1 to 2 weeks at 4°C
    • 20 mM 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine) (H‐7; Sigma), store for several days at −20°C
  • CFASW (see recipe)
  • 2 mM PMA (see recipe)

Basic Protocol 3: Fertilizing and Incubating Sea Urchin Eggs

  • Polyenoic fatty acid derivatives (see recipe), including:
    • Arachidonoylcholine (AA‐choline)
    • Docosahexaenoylcholine (DHA‐choline)

Basic Protocol 4: Embryonic and Larval Development of Sea Urchins

  • 20 mM ritanserin (Sigma) in methanol, store up to 2 days at 20°C
  • 20 mM serotonin hydrochloride (5‐HT), store in dark up to 24 hr at 4°C
  • Polyenoic fatty acid derivatives (see recipe), such as the following serotonin agonists:
    • Serotonamide of arachidonic acid (arachidonoyl serotonin; AA‐5‐HT)
    • Serotonamide of docosahexaenoic acid (DHA‐5‐HT)
    • Serotonamide of eicosapentaenoic acid (EPA‐5‐HT)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Bezuglov, V.V., Zinchenko, G.N., Nikitina, L.A., and Buznikov, G.A. 2001. Arachidonoylcholine and N,N‐dimethylaminoethyl arachidonate are new cholinergic compounds. Bioorg. Khim. 27:227‐230.
   Bottger, S.A. and McClintock, J.B. 2001. The effects of organic and inorganic phosphates on fertilization and early development in the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 129:307‐315.
   Buznikov, G.A. 1990. Neurotransmitters in Embryogenesis. Harwood Academic Publishers, Chur, Switzerland.
   Buznikov, G.A. and Podmarev, V.I. 1990. The sea urchins Strongylocentrotus droebachiensis, S. nudus, and S. intermedius. In Animal Species for Developmental Studies, Vol. 1. Invertebrates (T.A. Dettlaff and S.G. Vassetzky, eds.) pp. 253‐285. Plenum, New York and London.
   Buznikov, G.A., Shmukler, Y.B., and Lauder, J.M. 1996. From oocyte to neuron: Do neurotransmitters function in the same way throughout development? Cell. Mol. Neurobiol. 16:533‐559.
   Buznikov, G.A., Jokanovic, M., Kovacevic, N., and Rakic, L.J. 1997a. Sea urchin embryos and larvae as biosensors for screening and detailed study of pharmacologically active substances. Arch. Toxicol. Kinet. Xenobiot. Metab. 5:393‐400.
   Buznikov, G.A., Koikov, L.N., Shmukler, Y.B., and Whitaker, M.J. 1997b. Nicotine antagonists (piperidines and quinuclidines) reduce the susceptibility of early sea urchin embryos to agents evoking calcium shock. Gen. Pharmacol. 29:49‐53.
   Buznikov, G.A., Marshak, T.L., Malchenko, L.A., Nikitina, L.A., Shmukler, Y.B., Buznikov, A.G., Rakic, L.J., and Whitaker, M.J. 1998. Serotonin and acetylcholine modulate the sensitivity of early sea urchin embryos to protein kinase C activators. Comp. Biochem. Physiol. A 120:457‐462.
   Buznikov, G.A., Lambert, H.W., and Lauder, J.M. 2001a. Serotonin and serotonin‐like substances as regulators of early embryogenesis and morphogenesis. Cell Tissue Res. 305:177‐186.
   Buznikov, G.A., Nikitina, L.A., Bezuglov, V.V., Lauder, J.M., Padilla, S., and Slotkin, T.A. 2001b. An invertebrate model of the developmental neurotoxicity of insecticides: Effects of chlorpyrifos and dieldrin in sea urchin embryos and larvae. Environ. Health Persp. 109:651‐661.
   Buznikov, G.A., Bezuglov, V.V., Nikitina, L.A., Slotkin, T.A., and Lauder, J.M. 2001c. Cholinergic regulation of sea urchin embryonic and larval development. Ross. Fiziol. Zh. Im. I. M. Sechenova 87:1548‐1556.
   Cameron, R.A., Mahairas, G., Rast, J.P., Martinez, P., Biondi, T.R., Swartzell, S., Wallace, J.C., Poutska, A.J., Livingston, B.T., Wray, G.A., Ettensohn, C.A., Lehrach, H., Britten, R.J., Davidson, E.H., and Hood, L. 2000. A sea urchin genome project: Sequence scan, virtual map, and additional resources. Proc. Natl. Acad. Sci. U.S.A. 97:9514‐9518.
   Czihak, G. (ed.) 1975. The Sea Urchin Embryo: Biochemistry and Morphogenesis. Springer, New York.
   Dettlaff, T.A. and Dettlaff, A.A. 1961. On relative dimensionless characteristics of development duration in embryology. Arch. Biol. 72:1‐16.
   Dinnel, P.A., Link, J.M., Stober, Q.J., Letourneau, M.W., and Roberts, W.F. 1989. Comparative sensitivity of sea urchin sperm bioassay to metals and pesticides. Arch. Environ. Contam. Toxicol. 18:748‐755.
   Falugi, C. 1993. Localization and possible role of molecules associated with the cholinergic system during “non‐nervous” developmental events. Eur. J. Histochem. 37:287‐294.
   Harvey, E.B. 1956. The American Arbacia and Other Sea Urchins. Princeton University Press, Princeton, N.J.
   Ivonnet, P.I. and Chambers, P.L. 1997. Nicotinic acetylcholine receptors of neuronal type occur in plasma membrane of sea urchin eggs. Zygote 5:277‐287.
   Slotkin, T.A. 1998. Fetal nicotine or cocaine exposure: Which one is worse? J. Pharmacol. Exp. Ther. 285:931‐945.
   Slotkin, T.A. 1999. Developmental cholinotoxicants: Nicotine and chlorpyrifos. Environ. Health Persp. 10(Suppl. 1):71‐80.
   Stephens, R.E. 1972. Studies on the development of the sea urchin Strongylocentrotus droebachiensis. I. Ecology and normal development. Biol. Bull. 142:132‐144.
Internet Resources
   http://www.stanford.edu/group/Urchin
  Describes all procedures related to maintaining adult sea urchins and obtaining and handling sea urchin embryos and larvae.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library