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Rendiconti Lincei

, 5:385 | Cite as

Analisi at microscopio confocale laser della plasticitàdelle spine dendritiche

  • Michele di Papa
Neurobiologia
  • 58 Downloads

Riassunto

Si descrive un metodo originale con l’uso del microscopio confocale laser a scansione ed il colorante fluorescente Dil, per studiare la morfologia delle spine dendritiche in culture di neuroni ippocampali di ratto. È stato studiato lo sviluppo in cultura dalla prima alla quarta settimana, e la risposta ad uno stimolo funzionale, condizionando il medium con Tetrodotossina (TTX) o Picrotossina (PTX). La densità delle spine aumenta di 2,7 volte tra la prima e la terza settimana, mentre la lunghezza media cala da 1.6 a 1.1 am. Il rapporto tra il diametro della testa delle spine ed il diametro del collo aumenta, determinando teste maggiori alla terza settimana. La TTX aumenta la lunghezza delle spine, ne diminuisce la densità. La PTX esplica effetto opposto, la forma non cambia. Si ipotizza ehe la densità delle spine costituisce una funzione dei processi di crescita e di in-vecchiamento, e non delle afferenze. La formazione delle spine sembrerebbe indipendente e precedente la sinaptogenesi ehe a sua volta determinerebbe la forma delle spine durante la maturazione. La densità delle spine sarebbe direttamente, e la lunghezza inversamente, correlata aU’attività neuronale.

A confocal laser scanning microscope approach to dendritic spine plasticity

Abstract

We describe an original method using confocal laser scanning microscopy (CLSM) and the fluorescent marker Dil, to monitor dendritic spine morphology in cultures of rat hippocampal neurons. We performed a developmental analysis from the first to the fourth week in culture, and a functional study conditioning the culture medium with Tetrodotoxin (TTX) or Picrotoxin (PTX). The density of spines increased 2.7 fold between 1 and 3 weeks, while the mean length of the spines decreased from 1.6 to 1.1 am. The ratio between the diameter of the spine head and the diameter of the spine neck increased, resulting in larger spine heads at 3 weeks. The TTX increased spine length and decreased spine density. PTX had the opposite effect. We hypothesize that spine density is a growth and aging related process, and not related to input. Spine formation seems to preceed and be independent of synaptogenesis which is in turn involved in spine shaping during maturation. Spine density seems to be directly and spine length inversely related to neuronal firing rate.

Key words

Dendritic spine Confocal microscopy Dil Picrotoxin Tetrodotoxin 

References

  1. Amaral D. G., 1987.Memory: Anatomical organization of candidate brain regions. In: J. M. Brookhart, V. B. Mountcastle (eds.),Handbook of Physiology: The nervous system, v. Higher junctions of the nervous system. Am. Physiol. Soc, 5th ed., Bethesda: 211-294.Google Scholar
  2. Benes F. M., Vincent S. L., 1991.Changes in dendritic spine morphology in response to increased availability of monoamines in rat medial prefrontal cortex. Synapse, 9: 235–237.CrossRefGoogle Scholar
  3. Braitenberg V., Scnuz A., 1991a.Dendritic spines. In: V. Braitenberg, A. Schuz (eds.),Anatomy of the Cortex: Statistics and Geometry. Springer-Verlag, Berlin: 113–118.Google Scholar
  4. Braitenberg V., Schuz A., 1991b.Postnatal changes, possibly due to learning, in the guinea pig cortex. In: V. Braitenberg, A. Schuz (eds.),Anatomy of the Cortex: Statistics and Geometry. Springer-Verlag, Berlin: 131–139.Google Scholar
  5. Brock J. W., Prasad C, 1992.Alterations in dendritic spine density in the rat brain associated with protein malnutrition Dev. Brain Res., 66: 266–269.CrossRefGoogle Scholar
  6. Carlsson K, Aslund N., 1987.Confocal Imaging for 3-D digital microscopy. Applied Optics, 26: 3232–3238.CrossRefGoogle Scholar
  7. Cooper M. W., Smith S. J., 1992.A real time analysis of growth cone-target cell interactions during the formation of stable contacts between hippocampal neurons in culture. J. Neurobiol., 23: 814–828.CrossRefGoogle Scholar
  8. Cox I. J., Sheppard C. J. R., 1983.Digital image processing of confocal images. Image and Vision Computing, 1: 52–56.CrossRefGoogle Scholar
  9. Fifkova E., Anderson C. L., 1981.Stimulation-induced changes in dimension of stalks of dendritic spines in the dentate molecular layer. Exp. Neurol., 7: 621–627.CrossRefGoogle Scholar
  10. Ginty D. D., Bading H., Greenberg M. E., 1992.Trans-synaptic regulation of gene expression. Curr. Opin. Neurobiol., 2: 312–316.CrossRefGoogle Scholar
  11. Guthrie P. B., Segal M., Kater S. B., 1991.Independent regulation of calcium revealed by imaging dendritic spines. Nature, 354: 76–80.CrossRefGoogle Scholar
  12. Harris K. M., Jensen F. E., Tsao B., 1992.Three-dimensional structure of dendritic spines and synapses in rat hippocampus (CA1) at postnatal day 15 and adult ages: implications from the maturation of synaptic physiology and long-term potentiation. J. Neurosci., 12: 2685–2705.Google Scholar
  13. Harris K. M., Rosenberg P. A., 1993.Localization of synapses in rat cortical cultures. Neuroscience, 53: 495–508.CrossRefGoogle Scholar
  14. Hosokawa T., Bliss T. V. P., Fine A., 1992.Persistence of individual dendritic spines in living brain slices. NeuroReport, 3: 477–480.CrossRefGoogle Scholar
  15. Koch C, Zador A., 1993.The function of dendritic spines: devices subserving biochemical rather than electrical compartmentalization. J. Neurosci., 13: 413–442.Google Scholar
  16. Lisman J. E., Harris K. M., 1993.Quantal analysis and synaptic anatomy-integrating two views of hippocampal plasticity. TINS, 16: 141–147.Google Scholar
  17. Muller M., Gahwtler B. H., Rietschin L., Thompson S. M., 1993.Reversible loss of dendritic spines and altered excitability after chronic epilepsy in hippocampal slice cultures. Proc. Natl. Acad. Sci. USA, 90: 257–261.CrossRefGoogle Scholar
  18. Papa M, Pellicano M. P., Welzl H., Sadile A. G., 1993.Distributed changes in c-Fos and c-Jun immunoreac-tivity in the rat brain associated with arousal and habituation to novelty. Brain Res. Bull., 32: 509–515.CrossRefGoogle Scholar
  19. Papa M., Bundman M. C, Greenberger V., Segal M., 1994.Development and morphology of dendritic spines in primary cultures of hippocampal neurons. J. Neurosci, in press.Google Scholar
  20. Peters A., Kaiserman-Abramof I. R., 1970.The small pyramidal neuron of the rat cerebral cortex. The perikaryon, dendrites and spines. J. Anat., 127: 321–356.CrossRefGoogle Scholar
  21. Peters A., Palay S. L., Webster H.De F., 1991.The Vine Structure of the Nervous System: The Neurons and Supporting Cells. 3th ed., Oxford University Press, New York.Google Scholar
  22. Popov V. I., Bocharova L. S., 1992.Hibernation-induced structural changes in synaptic contacts between mossy fibres and hippocampal pyramidal neurons. Neuroscience, 48: 53–62.CrossRefGoogle Scholar
  23. Ramon y Cajal S., 1893.Neue Darstellung vom Histologischen Bau des Centralnervensystems. Archiv für Anatomie und Entwickelungsgeschichte. Anatomische Abteilung des Archives für Anatomie und Physiologie, 319-428.Google Scholar
  24. Ramony Cajal S., 1909.Histologie du Systeme Nerveux de l’Homme & des Vertèbres. Vol.1.L’Azoulay (transi.). Maloine, Paris. Republished in 1952. Instituto Ramon y Cajal, Madrid.Google Scholar
  25. Ray J., Peterson D. A., Schinstine M., Gage F. H., 1993.Proliferation, differentiation, and long-term culture of primary hippocampal neurons. Proc. Natl. Acad. Sci. USA, 90: 3602–3606.CrossRefGoogle Scholar
  26. Segal M., Manor D., 1992.Confocal microscopic imaging of [Ca 2+ ]i in cultured rat hippocampal neurons following exposure to n-methyl-d-aspartate. J. Physiology, 448: 655–676.Google Scholar
  27. Spacer J., 1985.Three-dimensional analysis of dendritic spines. II. Spine apparatus and other cytoplasmic components. Anat. Embryol., 171: 235- 243.CrossRefGoogle Scholar
  28. Stevens J. K., Trogadis J., 1984.Computer assisted reconstruction from serial electron micrographs. Annu. Rev. Neurobiol, 5: 341–369.Google Scholar
  29. Tanzi E., 1893.Fatti e le induzioni nell’odierna istologia del sistema nervoso. Riv. Sper. Freniatr. Med. Leg. Alienazioni Ment., 19: 419–472.Google Scholar
  30. Wickens J., 1988.Electrically coupled but chemically isolated synapses: dendritic spines and calcium in a rule for synaptic modification. Progr. in Neurobiol, 31: 507–528.CrossRefGoogle Scholar
  31. Wilson C. J., Groves P. M., Kitai S. T., Linder J. C, 1983.Three-dimensional structure of dendritic spine in the rat neostriatum. J. Neurosci., 3: 383–398.Google Scholar
  32. Woolley C. S., Gould E., Frankfurt M., McEwen B. S., 1990.Naturally occurring fluctuation in dendritic spine density on adult hippocampal pyramidal neurons. J. Neurosci., 10: 4035–4039.Google Scholar
  33. Zador A., Koch C., Brown T. H., 1990.Biophysical model of a Hebbian synapse. Proc. Natl. Acad. Sci. USA, 87: 6718–6722.CrossRefGoogle Scholar
  34. Zola-Morgan S., Squire L. R., 1993.Neuroanatomy of Memory. Annu. Rev. Neurosci., 16: 547–563.CrossRefGoogle Scholar

Copyright information

© Accademia nazionale dei Lincei 1994

Authors and Affiliations

  • Michele di Papa
    • 1
  1. 1.Istituto di Anatomia Umana Normale Facoltà di MedicinaChirurgia LT Universitédegli Studi di Napoli Via L. ArmanniNapoli

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