Correlative fluorescence and electron microscopy of biocytin-filled neurons with a preservation of the postsynaptic ultrastructure.

Abstract : Several techniques enable to inject intracellularly neurons with dyes and to use light and electron microscopy to correlate the physiological data with the morphological properties of the neuron. However, the ultrastructure of the neuron is usually obscured by the injected dye thus notably precluding the analysis of the postsynaptic specialisation and that of the other organelles. To overcome this problem, we have developed a technique based on fluorophore- and ultra small gold-conjugated streptavidins. We report, that this method facilitates the identification of intracellular organelles of the biocytin-filled neuron and of postsynaptic densities. This method is valid for the study of early postnatal neurons that are particularly refractory to this type of analysis. The procedure introduced here consists of the following steps: (1) injection of biocytin into the neuron by a patch-clamp pipette, (2) aldehyde fixation, (3) reaction with a fluorophore-conjugated streptavidin, (4) analysis with a fluorescence microscope, (5) formation of avidin-biotin complexes (ABC), (6) reaction with an ultra small gold-conjugated streptavidin, (7) silver enhancement of gold, (8) postfixation with osmium tetroxide and embedding in resin, (9) ultrathin sectioning and analysis with an electron microscope. Using this method, we show that in early postnatal hippocampal neurons, that have been injected with biocytine, it is possible to determine the morphology of the dendritic and axonal trees (including very thin details such as spines and filopodia) and to identify the localisation of the symmetric and asymmetric synapses on dendrites of the injected neuron.
Type de document :
Article dans une revue
Journal of Neuroscience Methods, Elsevier, 2002, 117 (1), pp.81-5
Liste complète des métadonnées

Littérature citée [3 références]  Voir  Masquer  Télécharger

http://www.hal.inserm.fr/inserm-00484848
Contributeur : Roman Tyzio <>
Soumis le : mercredi 19 mai 2010 - 12:30:19
Dernière modification le : lundi 19 janvier 2015 - 11:49:49
Document(s) archivé(s) le : jeudi 1 décembre 2016 - 01:50:51

Fichier

 Accès restreint
Fichier visible le : jamais

Connectez-vous pour demander l'accès au fichier

Identifiants

  • HAL Id : inserm-00484848, version 1
  • PUBMED : 12084567

Collections

Citation

Youri Morozov, Ilgam Khalilov, Yehezkel Ben-Ari, Alfonso Represa. Correlative fluorescence and electron microscopy of biocytin-filled neurons with a preservation of the postsynaptic ultrastructure.. Journal of Neuroscience Methods, Elsevier, 2002, 117 (1), pp.81-5. 〈inserm-00484848〉

Partager

Métriques

Consultations de la notice

47