Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP (original) (raw)
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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-
Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system
Nature, 2007
Detailed analysis of neuronal network architecture requires the development of new methods. Here we present strategies to visualize synaptic circuits by genetically labelling neurons with multiple, distinct colours. In Brainbow transgenes, Cre/lox recombination is used to create a stochastic choice of expression between three or more fluorescent proteins (XFPs). Integration of tandem Brainbow copies in transgenic mice yielded combinatorial XFP expression, and thus many colours, thereby providing a way to distinguish adjacent neurons and visualize other cellular interactions. As a demonstration, we reconstructed hundreds of neighbouring axons and multiple synaptic contacts in one small volume of a cerebellar lobe exhibiting approximately 90 colours. The expression in some lines also allowed us to map glial territories and follow glial cells and neurons over time in vivo. The ability of the Brainbow system to label uniquely many individual cells within a population may facilitate the analysis of neuronal circuitry on a large scale.
Brain Research, 2010
Transgenic mouse lines in which a fluorescent protein is constitutively expressed under the Thy1 gene promoter have become important models in cell biology and pathology studies of specific neuronal populations. As a result of positional insertion and/or copy number effects on the transgene, the populations expressing the fluorescent protein (eYFP+) vary markedly among the different mice lines. However, identification of the eYFP+ subpopulations has remained sketchy and fragmentary even for the most widely used lines such as Thy1-eYFP-H mice (Feng, G.
Imaging High-Resolution Structure of GFP-Expressing Neurons in Neocortex In Vivo
Learning & Memory, 2000
To detect subtle changes in neuronal morphology in response to changes in experience, one must image neurons at high resolution in vivo over time scales of minutes to days. We accomplished this by infecting postmitotic neurons in rat and mouse barrel cortex with a Sindbis virus carrying the gene for enhanced green fluorescent protein. Visualized with 2-photon excitation laser scanning microscopy, infected neurons showed bright fluorescence that was distributed homogeneously throughout the cell, including axonal and dendritic arbors. Single dendritic spines could routinely be resolved and their morphological dynamics visualized. Viral infection and imaging were achieved throughout postnatal development up to early adulthood (P 8-30), although the viral efficiency of infection decreased with age. This relatively noninvasive method for fluorescent labeling and imaging of neurons allows the study of morphological dynamics of neocortical neurons and their circuits in vivo.
European Journal of Neuroscience, 2001
The cerebellar Purkinje cell has been the focus of numerous studies involving the analysis of development and information processing in the nervous system. Purkinje cells represent less than 0.1% of the total cell content of the cerebellum. To facilitate studies of molecules that are expressed in such a small proportion of neurons, we have established procedures for the puri®cation of these cells. Transgenic mice were developed in which the expression of green¯uorescent protein (GFP) was controlled by the L7 promoter. In adult cerebellum, GFP¯uorescence was only detected in Purkinje cells, where it ®lled dendrites, soma and axons. GFP¯uorescence was detected in Purkinje cells as early as embryonic day 17 and increased during development in vivo and in dissociated cerebellar culture. Mirroring endogenous L7 expression, high levels of GFP were observed in retinal rod bipolar cells. Lower levels of GFP were seen in olfactory periglomerular cells, neurons in the interpeduncular nucleus, and superior colliculus neurons. Cerebella from transgenic mice were dissociated by mild enzymatic treatment and Purkinje cells were isolated by¯uorescence-activated cell sorting (FACS). By selecting optimal parameters, a fraction of viable Purkinje cells that was 94% pure was obtained. These results indicate that FACS is a powerful tool for isolating Purkinje cells from postnatal L7-GFP transgenic mice. GFP-positive neurons will also be useful in the real-time observation of dendritic morphogenesis and axonal outgrowth during development, or after neuronal activity in vitro.
2017
Investigation of the neuronalconnectionshave been conducted over a long span of time. Debate between researchers about contiguity or continuity of the nerve elements resulted in a new era in the research of neuronal pathways. The issue has been resolved when synaptic connections were discovered by the electron microscope. This made it possible to use two types of tract tracing methods. First non-transsynaptic and later trans-synaptic tracers were applied. The formerone is suitable to demonstrate direct neuronal connections; the latterare able to describe multisynaptic neuronal circuits.Development of trans-synaptic neurotropic viruses expressing reporter molecules was a great step in this research. GFP, a natural fluorescent protein was discovered in jellyfishmore than 50 years ago by Shimomura. Later it was found that GFP fluorescence wasstable, species-independent and could be monitored noninvasively using the techniques of fluorescence microscopy and flow cytometry. Later it was ...
Isolation and characterization of neural precursor cells from theSox1-GFP reporter mouse
European Journal of Neuroscience, 2005
We have made use of a reporter mouse line in which enhanced green fluorescence protein (GFP) is inserted into the Sox1 locus. We show that the GFP reporter is coexpressed with the Sox1 protein as well as with other known markers for neural stem and progenitor cells, and can be used to identify and isolate these cells by fluorescence-activated cell sorting (FACS) from the developing or adult brain and from neurosphere cultures. All neurosphere-forming cells with the capacity for multipotency and self-renewal reside in the Sox1-GFP-expressing population. Thus, the Sox1-GFP reporter system is highly useful for identification, isolation and characterization of neural stem and progenitor cells, as well as for the validation of alternative means for isolating neural stem and progenitor cells. Further, transplantation experiments show that Sox1-GFP cells isolated from the foetal brain give rise to neurons and glia in vivo, and that many of the neurons display phenotypic characteristics appropriate for the developing brain region from which the Sox1-GFP precursors were derived. On the other hand, Sox1-GFP cells isolated from the adult subventricular zone or expanded neurosphere cultures gave rise almost exclusively to glial cells following transplantation. Thus, not all Sox1-GFP cells possess the same capacity for neuronal differentiation in vivo.
Frontiers in molecular neuroscience, 2012
In the "GFP reconstitution across synaptic partners" (GRASP) method, non-fluorescent fragments of GFP are expressed in two different neurons; the fragments self-assemble at synapses between the two to form a fluorophore. GRASP has proven useful for light microscopic identification of synapses in two invertebrate species, Caenorhabditis elegans and Drosophila melanogaster, but has not yet been applied to vertebrates. Here, we describe GRASP constructs that function in mammalian cells and implement a transgenic strategy in which a Cre-dependent gene switch leads to expression of the two fragments in mutually exclusive neuronal subsets in mice. Using a transgenic line that expresses Cre selectively in rod photoreceptors, we demonstrate labeling of synapses in the outer plexiform layer of the retina. Labeling is specific, in that synapses made by rods remain labeled for at least 6 months whereas nearby synapses made by intercalated cone photoreceptors on many of the same inter...
European Journal of Neuroscience, 2007
The first postmitotic neurons in the developing neocortex establish the preplate layer. These early-born neurons have a significant influence on the circuitry of the developing cortex. However, the exact timing and trajectory of their projections, between cortical hemispheres and intra- and extra-cortical regions, remain unresolved. Here, we describe the creation of a transgenic mouse using a 1.3 kb golli promoter element of the myelin basic protein gene to target expression of a τ–green fluorescent protein (GFP) fusion protein in the cell bodies and processes of pioneer cortical neurons. During embryonic and early neonatal development, the timing and patterning of process extension from these neurons was examined. Analysis of τ-GFP fluorescent fibers revealed that progression of early labeled projections was interrupted unexpectedly by transient pauses at the corticostriatal and telencephalic–diencephalic boundaries before invading the thalamus just prior to birth. After birth the pioneering projections differentially invaded the thalamus, excluding some nuclei, e.g. medial and lateral geniculate, until postnatal days 10–14. Early labeled projections were also found to cross to the contralateral hemisphere as well as to the superior colliculus. These results indicate that early corticothalamic projections appear to pause before invading specific subcortical regions during development, that there is developmental regulation of innervation of individual thalamic nuclei, and that these early-generated neurons also establish early projections to commissural and subcortical targets.
European Journal of Neuroscience, 2007
The first postmitotic neurons in the developing neocortex establish the preplate layer. These early-born neurons have a significant influence on the circuitry of the developing cortex. However, the exact timing and trajectory of their projections, between cortical hemispheres and intra-and extra-cortical regions, remain unresolved. Here, we describe the creation of a transgenic mouse using a 1.3 kb golli promoter element of the myelin basic protein gene to target expression of a s-green fluorescent protein (GFP) fusion protein in the cell bodies and processes of pioneer cortical neurons. During embryonic and early neonatal development, the timing and patterning of process extension from these neurons was examined. Analysis of s-GFP fluorescent fibers revealed that progression of early labeled projections was interrupted unexpectedly by transient pauses at the corticostriatal and telencephalicdiencephalic boundaries before invading the thalamus just prior to birth. After birth the pioneering projections differentially invaded the thalamus, excluding some nuclei, e.g. medial and lateral geniculate, until postnatal days 10-14. Early labeled projections were also found to cross to the contralateral hemisphere as well as to the superior colliculus. These results indicate that early corticothalamic projections appear to pause before invading specific subcortical regions during development, that there is developmental regulation of innervation of individual thalamic nuclei, and that these early-generated neurons also establish early projections to commissural and subcortical targets.