Journal of Neurochemistry

Volume 62, Issue 4 p. 1586-1595

Subcellular Fractionation on Percoll Gradient of Mossy Fiber Synaptosomes: Morphological and Biochemical Characterization in Control and Degranulated Rat Hippocampus

Philippe Taupin,

Philippe Taupin

INSERM U 29, Paris, France

Search for more papers by this author

Sylvie Zini,

Sylvie Zini

INSERM U 29, Paris, France

Search for more papers by this author

François Cesselin,

François Cesselin

NSERM U 288, Paris, France

Search for more papers by this author

Yezekiel Ben-Ari,

Yezekiel Ben-Ari

INSERM U 29, Paris, France

Search for more papers by this author

Marie-Paule Roisin,

Corresponding Author

Marie-Paule Roisin

INSERM U 29, Paris, France

Address correspondence to Dr. P. Taupin and reprint requests to Dr. M.-P. Roisin at INSERM U 29, 123 Boulevard de Port-Royal, 75014 Paris, France.Search for more papers by this author

Philippe Taupin,

Philippe Taupin

INSERM U 29, Paris, France

Search for more papers by this author

Sylvie Zini,

Sylvie Zini

INSERM U 29, Paris, France

Search for more papers by this author

François Cesselin,

François Cesselin

NSERM U 288, Paris, France

Search for more papers by this author

Yezekiel Ben-Ari,

Yezekiel Ben-Ari

INSERM U 29, Paris, France

Search for more papers by this author

Marie-Paule Roisin,

Corresponding Author

Marie-Paule Roisin

INSERM U 29, Paris, France

Address correspondence to Dr. P. Taupin and reprint requests to Dr. M.-P. Roisin at INSERM U 29, 123 Boulevard de Port-Royal, 75014 Paris, France.Search for more papers by this author

First published: April 1994

https://doi.org/10.1046/j.1471-4159.1994.62041586.x

Citations: 42

Read the full text

About

PDF

Tools

Share a link

Email
Facebook
Twitter
LinkedIn
Reddit
Wechat

Abstract

Abstract: A method for preparation of hippocampal mossy fiber synaptosomes directly from the postnuclear pellet is presented. This method represents an adaption of that previously described for the isolation of synaptosomes by centrifugation through Percoll gradients directly from the supernatant fraction. We have characterized by electron microscopy two fractions, PII and PIII, enriched in mossy fiber synaptosomes; fraction PIII had 75% mossy fiber synaptosomes with well-preserved morphology (large size 3 μm, complex morphology, high synaptic vesicle density, multisynapses), whereas fraction PII contained 12%. These fractions were enriched in lactate dehydrogenase activity indicating that the integrity of synaptosomes was preserved. Compared with the other synaptosomal fractions, these fractions showed greater levels of dynorphin A (1–8) immunoreactivity and endogenous zinc, which are particularly concentrated in hippocampal mossy fiber terminals. Furthermore, we prepared synaptosomes from adult hippocampus after neonatal irradiation, which destroys the majority of granule cells and associated mossy fibers. The levels of dynorphin and zinc decreased by 88 and 70% in fraction PII and by 95 and 90%, respectively, in PIII. These results suggest that the rapid Percoll procedure is convenient for the purification of mossy fiber synaptosomes.

References

Altman J. and Das G. D. (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. Comp. Neural. 124, 319–336.
10.1002/cne.901240303
CASPubMedWeb of Science®Google Scholar
Amaral D. G., and Dent J. A. (1981) Development of the mossy fibers and their expansions. J. Comp. Neural. 195, 51–86.
10.1002/cne.901950106
CASPubMedWeb of Science®Google Scholar
Aniksztejn L., Charton G., and Ben-Ari Y. (1987) Selective release of endogenous zinc from the hippocampal mossy fiber in situ. Brain Res. 404, 58–64.
10.1016/0006-8993(87)91355-2
CASPubMedWeb of Science®Google Scholar
Bayer S. A. (1980) Development of the hippocampal region in the rat. I. Neurogenesis examined with 3H-thymidine autoradiography. J. Comp. Neural. 190, 87–114.
10.1002/cne.901900107
PubMedWeb of Science®Google Scholar
Bayer S. A., and Altman J. (1975) Radiation-induced interference with postnatal hippocampal cytogenesis in rats and its longterm effects on the acquisition of neurons and glia. J. Comp. Neural. 163, 1–20.
10.1002/cne.901630102
PubMedWeb of Science®Google Scholar
Ben-Ari Y. and Represa A. (1990) Brief seizure episodes induce long-term potentiation and mossy fibre sprouting in the hippocampus. Trends Neurosci. 13, 312–318.
10.1016/0166-2236(90)90135-W
CASPubMedWeb of Science®Google Scholar
Blackstad T. W., and Kjaerheim A. (1961) Special axo-dendritic synapses in the hippocampal cortex: electron and light microscopic studies on the layer of mossy fibers. J. Comp. Neural. 117, 133–159.
10.1002/cne.901170202
PubMedWeb of Science®Google Scholar
Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
10.1016/0003-2697(76)90527-3
CASPubMedWeb of Science®Google Scholar
Braitenberg V. and Schttz A. (1983) Some anatomical comments on the hippocampus, in Neurobiology of the Hippocampus ( W. Seifert, ed), pp. 21–37. Academic Press, New York .

Google Scholar
Chavkin C., Bakhit C., Weber E., and Bloom F. E. (1983) Relative contents and concomitant release of prodynorphin neoendorphin-derived peptides in rat hippocampus. Proc. Nad. Acad. Sci. USA 80, 7669–7673.
10.1073/pnas.80.24.7669
CASPubMedWeb of Science®Google Scholar
Cook T. M., and Crutcher K. A. (1988) Lesion-induced CA1 mossy fibers in the rat represent a neoinnervation. Exp. Brain Res. 70, 433–436.
10.1007/BF00248368
CASPubMedWeb of Science®Google Scholar
Cotman C. W., Ratman J. A., Ganong A. H., and Perkins M. N. (1986) Effects of excitatory amino acid antagonists on evoked and spontaneous excitatory potentials in guinea-pig hippocampus. J. Physiol. (Lond.) 378, 403–415.
10.1113/jphysiol.1986.sp016227
CASPubMedWeb of Science®Google Scholar
Crawford I. and Connor J. (1973) Localization and release of glutamic acid in relation to the hippocampal mossy fiber pathway. Nature 244, 442–443.
10.1038/244442a0
CASPubMedWeb of Science®Google Scholar
Crawford I. L., and Connor J. D. (1972) Zinc in maturing rat brain: hippocampal concentration and localization. J. Neurochem. 19, 1451–1458.
10.1111/j.1471-4159.1972.tb05088.x
CASPubMedWeb of Science®Google Scholar
Crawford I. L., and Harris N. F. (1984) Distribution and accumulation of zinc in whole brain and subcellular fractions of hippocampal homogenates, in The Neurobiology of Zinc, Pan A: Physiochemistry, Anatomy, and Techniques ( C. J. Frederickson, G. A. Howell, and E. J. Kasarskis, eds), pp. 157–171. Alan R. Liss, New York .

Google Scholar
De Robertis E., De Iraldi A. P., Rodriguez G., and Gomez J. (1961) On the isolation of nerve endings and synaptic vesicles. J. Biophys. Biochem. Cytol. 9, 229–235.
10.1083/jcb.9.1.229
PubMedWeb of Science®Google Scholar
Dunkley P. R., Jarvie P. E., Heath J. W., Kidd G. J., and Rostas J. A. P. (1985) A new rapid procedure for purifying synaptosomes on Percoll gradients. Neurosci. Lett. Suppl. 19, S. 58.

Google Scholar
Dunkley P. R., Jarvie P. E., Heath J. W., Kidd G. J., and Rostas J. A. P. (1986) A rapid method for isolation of synaptosomes on Percoll gradients. Brain Res. 372, 115–129.
10.1016/0006-8993(86)91464-2
CASPubMedWeb of Science®Google Scholar
Dunkley P. R., Heath J. W., Harisson S. M., Jarvie P. E., Glenfield P. J., and Rostas J. A. P. (1988) A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions. Brain Res. 441, 59–71.
10.1016/0006-8993(88)91383-2
CASPubMedWeb of Science®Google Scholar
Frederickson C. J., Klitenick M. A., Manton W. L., and Kirkpatrick J. B. (1983) Cytoarchitectonic distribution of zinc in the hippocampus of man and rat. Brain Res. 273, 335–339.
10.1016/0006-8993(83)90858-2
CASPubMedWeb of Science®Google Scholar
Gaarskjaer F. B. (1986) The organization and development of the hippocampal mossy fiber system. Brain Res. Rev. 11, 335–357.
10.1016/0165-0173(86)90004-4
Web of Science®Google Scholar
Gaiarsa J. L., Beaudoin M., and Ben-Ari Y. (1992) Effect of neonatal degranulation on the morphological development of rat CA3 pyramidal neurons: inductive role of mossy fibers on the formation of thorny excrescences. J. Comp. Neural. 321, 612–625.
10.1002/cne.903210408
CASPubMedWeb of Science®Google Scholar
Goldschmidt R. B., and Steward O. (1980) Preferential neurotoxicity of colchicine for granule cells of the dentate gyrus of the adult rat. Proc. Natl. Acad. Sci. USA 77, 3047–3051.
10.1073/pnas.77.5.3047
CASPubMedWeb of Science®Google Scholar
Gray E. G., and Whittaker V. P. (1962) The isolation of nerve endings from brain: an electron microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat. (Lond.) 96, 79–88.

PubMedGoogle Scholar
Hajos F. (1975) An improved method for the preparation of synaptosomal fractions in high purity. Brain Res. 93, 485–489.
10.1016/0006-8993(75)90186-9
CASPubMedWeb of Science®Google Scholar
Haug F. (1967) Electron microscopical localization of the zinc in hippocampal mossy fiber synapses by a modified sulphide silver procedure. Histochimie 8, 355–368.
10.1007/BF00401978
CASPubMedWeb of Science®Google Scholar
Hicks S. P., and D'Amoto C. J. (1966) Effects of ionizing radiation on mammalian development, in Advances in Teratology, ( D. H. M. Woolam, ed), pp. 195–250. Logos Press, London .

Google Scholar
Hoffman D. W., and Zamir N. (1984) Localization and quantitation of dynorphin B in the rat hippocampus. Brain Res. 324, 354–357.
10.1016/0006-8993(84)90048-9
CASPubMedWeb of Science®Google Scholar
Ibata Y. and Otsuka N. (1969) Electron microscopic demonstration of zinc in the hippocampal formation using sulphide silver technique. J. Histochem. Cytochem. 17, 171–175.
10.1177/17.3.171
CASPubMedWeb of Science®Google Scholar
Israel M. and Whittaker V. P. (1965) The isolation of mossy fibre endings from the granular layer of the cerebrellar cortex. Experientia XXI 15, 325–326.
10.1007/BF02144693
Web of Science®Google Scholar
Marchbanks R. M. (1967) Osmotically sensitive potassium and sodium compartments of synaptosomes. J. Biochem. 104, 148–157.

CASPubMedWeb of Science®Google Scholar
McGinty J. F., Henriksen S. J., Goldstein A., Terenius L., and Bloom F. E. (1983) Dynorphin is contained within hippocampal mossy fibers: immunochemical alterations after kainic acid administration and colchicine induced neurotoxicity. Proc. Natl. Acad. Sci. USA 80, 589–593.
10.1073/pnas.80.2.589
CASPubMedWeb of Science®Google Scholar
Morgan I. G. (1976) Synaptosomes and cell separation. Neuroscience 1, 159–165.
10.1016/0306-4522(76)90072-5
CASPubMedWeb of Science®Google Scholar
Nagy A. and Delgado-Escueta A. V. (1984) Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll). J. Neurochem. 43, 1114–1123.
10.1111/j.1471-4159.1984.tb12851.x
CASPubMedWeb of Science®Google Scholar
Niklowitz W. (1966) Elektronenmikroskopische untersuchungen am ammonshorn. Z. Zellforsch. 75, 485–500.
10.1007/BF00341507
CASPubMedWeb of Science®Google Scholar
Ottersen O. P., and Storm-Mathisen J. (1984) Neurons containing or accumulating transmitter amino acids, in Handbook of Chemical Neuroanatomy, Vol. 3, Part II: Classical Transmitters and Transmitter Receptors in the CNS ( A. Bjorklund, T. Hokfelt, and M. J. Kuhar, eds), pp. 141–246. Elsevier, Amsterdam .

Google Scholar
Represa A. and Ben-Ari Y. (1992) Kindling is associated with the formation of novel mossy fibre synapse in the CA3 region. Exp. Brain Res. 92, 69–78.
10.1007/BF00230384
PubMedWeb of Science®Google Scholar
Represa A., Tremblay E., and Ben-Ari Y. (1987) Aberrant growth of mossy fibers and enhanced kainic acid binding sites induced in rats by early hyperthyroidism. Brain Res. 423, 325–328.
10.1016/0006-8993(87)90856-0
PubMedWeb of Science®Google Scholar
Represa A., Dessi F., Beaudoin M., and Ben-Ari Y. (1991) Effects of neonatal γ ray irradiation on rat hippocampus—I. Postnatal maturation of hippocampal cells. Neuroscience 42, 137–150.
10.1016/0306-4522(91)90154-G
CASPubMedWeb of Science®Google Scholar
Robain O., Represa A., Jardin L., and Ben-Ari Y. (1989) Selective destruction of mossy fibers and granule cells with preservation of the GABAergic network in the inferior region of the rat hippocampus after colchicine treatment. J. Comp. Neurol. 285, 274–287.
10.1002/cne.902850209
CASPubMedWeb of Science®Google Scholar
Schlessinger A. R., Cowan W. M., and Gottlieb D. I. (1975) An autoradiographic study of the time of origin and the pattern of granule cells migration in the dentate gyrus of the rat. J. Comp. Neural. 159, 149–176.
10.1002/cne.901590202
Google Scholar
Sloviter R. S. (1982) A simplified Timm stain procedure compatible with formaldehyde fixation and routine paraffin embedding of rat brain. Brain Res. Bull. 8, 771–774.
10.1016/0361-9230(82)90104-6
CASPubMedWeb of Science®Google Scholar
Storm-Mathisen J., Leknes A. K., Bore A. T., Vaaland J. L., Edminson P., Haug F. S., and Ottersen O. P. (1983) First visualization of glutamate and GABA in neurones by immunochemistry. Nature 301, 517–520.
10.1038/301517a0
CASPubMedWeb of Science®Google Scholar
Terrian D. M., Johnston D., Claiborne B. J., Ansah-Yiadom R., Strittmatter W. J., and Rea M. A. (1988) Glutamate and dynorphin release from a subcellular fraction enriched in hippocampal mossy fiber synaptosomes. Brain Res. Bull. 21, 343–351.
10.1016/0361-9230(88)90146-3
CASPubMedWeb of Science®Google Scholar
Thorne B., Wonnacott S., and Dunkley P. R. (1991) Isolation of hippocampal synaptosomes on Percoll gradients: cholinergic markers and ligand binding sites. J. Neurochem. 56, 479–484.
10.1111/j.1471-4159.1991.tb08175.x
CASPubMedWeb of Science®Google Scholar
Vincent R. and Nadeau D. (1983) A micromethod for quantitation of cellular proteins in Percoll with the Coomassie brilliant blue dye-binding assay. Anal. Biochem. 135, 355–362.
10.1016/0003-2697(83)90696-6
CASPubMedWeb of Science®Google Scholar
Whittaker V. P. (1969) The synaptosome, in Handbook of Neurochemistry, Vol. 2. ( A. Lajtha, ed), pp. 327–364. Plenum Press, New York .
10.1007/978-1-4899-7321-4_14
Google Scholar

Citing Literature

Volume62, Issue4

April 1994

Pages 1586-1595

Subcellular Fractionation on Percoll Gradient of Mossy Fiber Synaptosomes: Morphological and Biochemical Characterization in Control and Degranulated Rat Hippocampus

Abstract

References

Citing Literature

References

Information

About Wiley Online Library

Help & Support

Opportunities

Connect with Wiley

Subcellular Fractionation on Percoll Gradient of Mossy Fiber Synaptosomes: Morphological and Biochemical Characterization in Control and Degranulated Rat Hippocampus

Abstract

References

Citing Literature

References

Related

Information