Usefulness of Fluorescent Proteins: Gfp, Dsred and Fruit Fluorescent Proteins Inidentifying Multisynaptic Neuronal Chains. History and Our Own Experience (original) (raw)
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International Journal of Molecular Sciences
One hundred and twenty-five years ago there was a lively discussion between Hungarian and Spanish neuroscientists on the nature of neural connections. The question was whether the neurofibrils run from one neuron to the next and connect neurons as a continuous network or the fibrils form an internal skeleton in the neurons and do not leave the cell; however, there is close contact between the neurons. About 50 years later, the invention of the electron microscope solved the problem. Close contacts between individual neurons were identified and named as synapses. In the following years, the need arose to explore distant connections between neuronal structures. Tracing techniques entered neuroscience. There are three major groups of tracers: (A) non-transsynaptic tracers used to find direct connections between two neuronal structures; (B) tracers passing gap junctions; (C) transsynaptic tracers passing synapses that are suitable to explore multineuronal circuits. According to the dire...
Use of fluorescent probes to follow membrane traffic in nerve terminals
Brazilian Journal of Medical and Biological Research, 1998
Optical tracers in conjunction with fluorescence microscopy have become widely used to follow the movement of synaptic vesicles in nerve terminals. The present review discusses the use of these optical methods to understand the regulation of exocytosis and endocytosis of synaptic vesicles. The maintenance of neurotransmission depends on the constant recycling of synaptic vesicles and important insights have been gained by visualization of vesicles with the vital dye FM1-43. A number of questions related to the control of recycling of synaptic vesicles by prolonged stimulation and the role of calcium to control membrane internalization are now being addressed. It is expected that optical monitoring of presynaptic activity coupled to appropriate genetic models will contribute to the understanding of membrane traffic in synaptic terminals.
Proceedings of the National Academy of Sciences, 2000
Physiological properties of central nervous system neurons infected with a pseudorabies virus were examined in vitro by using whole-cell patch-clamp techniques. A strain of pseudorabies virus (PRV 152) isogenic with the Bartha strain of PRV was constructed to express an enhanced green fluorescent protein (EGFP) from the human cytomegalovirus immediate early promoter. Unilateral PRV 152 injections into the vitreous body of the hamster eye transsynaptically infected a restricted set of retinorecipient neurons including neurons in the hypothalamic suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) of the thalamus. Retinorecipient SCN neurons were identified in tissue slices prepared for in vitro electrophysiological analysis by their expression of EGFP. At longer postinjection times, retinal ganglion cells in the contralateral eye also expressed EGFP, becoming infected after transsynaptic uptake and retrograde transport from infected retinorecipient neurons. Retinal ga...
Experimental Physiology, 2001
Using a genetically modified herpes simplex virus encoding green fluorescent protein we sought to establish if this viral modification could be used in transneuronal tracing studies of the sympathetic nervous system. The herpes simplex virus encoding green fluorescent protein was injected into the adrenal medulla of three hamsters and six rats. After a suitable survival period, neurones in the sympathetic intermediolateral cell column of the thoracolumbar spinal cord, rostra1 ventral medulla and paraventricular nucleus of the hypothalamus were clearly identified by the presence of a green fluorescence in the cytoplasm of the neurones of both species. Thus, herpes simplex virus encoding green fluorescent protein labelled chains of sympathetic neurones in the hamster and rat and therefore has the potential to be used in transneuronal tracing studies of autonomic pathways in these species. Experimental Physiology (2001) 86.6,695-702.
Labeling Neural Cells Using Adenoviral Gene Transfer of Membrane-Targeted GFP
Neuron, 1996
The structures of recombinant adenoviruses containing Kyoto University Faculty of Medicine Kyoto, 606 the wild-type and modified GFP proteins are schematically illustrated in . Infection of the recombinant Japan † Molecular Neurobiology Laboratory adenovirus carrying the wild-type GFP cDNA under the control of the strong and ubiquitous CAG promoter The Salk Institute La Jolla, California 92037 (Niwa et al., 1991; the virus clone AdV-CA-GFP) resulted in a green fluorescence in COS cells ( ). The fluorescence was preferentially localized to the nucleus, which could be detected as early as 12 hr after infection, Summary and continuously increased up to 48 hr after infection. The fluorescence was stable and resistant to formalde-We describe an experimental system to visualize the hyde fixation, as reported previously (Chalfie et al., soma and processes of mammalian neurons and glia 1994). However, when the AdV-CA-GFP virus was inin living and fixed preparations by using a recombinant fected into cultured cortical neurons, the fluorescence adenovirus vector to transfer the jellyfish green fluo-
Microscopy techniques and the study of synapses
2007
Microscope techniques have been largely underestimated as powerful tools for the study of cell function and with some exceptions mostly restricted to descriptive studies. The development of new techniques and methods as well as the development of more powerful image analysis software in the last decade finally has provided the conditions to use microscopy in much more extensive and powerful ways. The development of new imaging software has also resuscitated the interest in "old" techniques such as Golgi staining methods that have suddenly come back to the spot light due to the possibility to be combined with image analysis tools. Microscopy is no longer the way to only get a "pretty picture" to illustrate our papers, but also a very powerful tool to study brain function. We propose here different strategies and methodologies that can be applied to the study of synaptic development and function using fluorescence, brightfield and electron microscopy. The use of these techniques allows the study of parameters such as the expression of synaptic proteins, number and morphology of dendritic spines, and number, type and structure of synapses.
2014
This chapter addresses the basic applications of tract-tracing and preembedding immunoperoxidase and immunogold-silver labeling for transmission electron microscopy, focusing primarily on identifying the cellular and subcellular localization of proteins of relevance to neurotransmission and on defining synaptic connectivity within neuronal circuits. Information is provided regarding the use of preembedding immunoperoxidase and immunogold techniques to identify the cellular and subcellular localization of neuronal receptors and transporters. The chapter also describes in detail a triple-labeling approach designed by our laboratory for identifying synaptic inputs to neuronal cell populations defined both by their projection targets and by their transmitter phenotype. Protocols presented in the Appendix are designed to enable researchers trained in small animal surgery, immunocytochemistry, electron microscopy, and appropriate laboratory safety procedures to perform ultrastructural investigations similar to those described here.