Correlative fluorescence and electron microscopy of biocytin-filled neurons with a preservation of the postsynaptic ultrastructure.
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.
Morozov
Youri
Y
INMED/INSERM U29, 163 Route de Luminy, BP 13, 13009 Marseille, France.
Khalilov
Ilgam
I
Ben-Ari
Yehezkel
Y
Represa
Alfonso
A
D000818:Animals; D000831:Animals, Newborn; D001369:Axons / Q000502:physiology / Q000648:ultrastructure; D003712:Dendrites / Q000502:physiology / Q000648:ultrastructure; D005404:Fixatives; D005456:Fluorescent Dyes; D006624:Hippocampus / Q000254:growth & development / Q000502:physiology / Q000648:ultrastructure*; D007150:Immunohistochemistry / Q000379:methods*; D007395:Interneurons / Q000502:physiology / Q000648:ultrastructure; D008239:Lysine / Q000031:analogs & derivatives*; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009445:Neuroanatomy / Q000379:methods*; D009474:Neurons / Q000502:physiology / Q000648:ultrastructure*; D009924:Organ Culture Techniques; D015388:Organelles / Q000502:physiology / Q000648:ultrastructure; D017966:Pyramidal Cells / Q000502:physiology / Q000648:ultrastructure; D051381:Rats; D019809:Streptavidin; D013570:Synaptic Membranes / Q000502:physiology / Q000648:ultrastructure; D016610:Tissue Embedding / Q000379:methods; D016707:Tissue Fixation / Q000379:methods
D005404:Fixatives; D005456:Fluorescent Dyes; D019809:Streptavidin; C013411:biocytin; D008239:Lysine