Purification of Purkinje cells by fluorescence-activated cell sorting from transgenic mice that express green fluorescent protein (original) (raw)
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Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP
Neuron, 2000
The first report on GFP expression in heterologous cells illustrated its use as a vital stain for neurons (Chalfie Washington University School of Medicine St. Louis, Missouri 63110 et al., 1994). Since that time, neuroscience has been one of the greatest beneficiaries of GFP technology, and GFP has been used to facilitate the study of neuronal development and plasticity in transgenic worms, flies, fish, and mice (for example, Dynes and Ngai, 1998; Mur-Summary ray et al., 1998; van den Pol and Ghosh, 1998; Knobel et al., 1999; Zito et al., 1999; Rodriguez et al., 1999; We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed Higashijima et al., 2000)
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.
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-
Neuroscience, 2004
The development of the dendritic tree of a neuron is a complex process which is thought to be regulated strongly by signals from afferent fibers. In particular the synaptic activity of afferent fibers and activity-dependent signaling by neurotrophic factors are thought to affect dendritic growth. We have studied Purkinje cell dendritic arbor development in organotypic cultures under suppression of glutamate-mediated excitatory neurotransmission, achieved with multiple combinations of blockers of glutamate receptors. Despite the presence of either single receptor blockers or combinations of blockers predicted to fully suppress glutamate-mediated excitatory neurotransmission Purkinje cell dendritic arbors developed similar to those of control cultures. Furthermore, Purkinje cell dendritic arbors in organotypic cultures from brain-derived neurotrophic factor (BDNF) knockout mice or after pharmacological blockade of trk-receptors also developed in a way similar to control cultures. Our results demonstrate that during the stage of rapid dendritic arbor growth signals from afferent fibers are of minor importance for Purkinje cell dendritic development because a seemingly normal Purkinje cell dendritic tree developed in the absence of excitatory neurotransmission and BDNF signaling. Our results suggest that many aspects of Purkinje cell dendritic development can be achieved by an intrinsic growth program.
Developmental Brain Research, 1991
The early stages in the formation of Purkinje cell dendritic arbors have been analyzed using the horseradish peroxidase (HRP) 'in vitro' axonal tracing method, from embryonic day 19 (E19) to postnatal day 6 (P6). These stages comprise the transition from the bipolar Purkinje cell, at the end of its migration, to the phase of stellate cell with disoriented dendrites. Postmigratory Purkinje cells in the cortical plate exhibit poorly elaborated bipolar shapes, here named 'simple-fusiform' cells. They constitute the vast majority of labeled cells up to P0, and thereafter they decrease in number until P4. As a result of continuous outgrowth of new primary dendrites emerging from the apical pole but also from the basal and lateral aspects of the cell bodies, the Purkinje cells enter the 'complex-fusiform' phase, which peaks by P1 and slowly disappears by P6. The disappearance of 'complex-fusiform' cells is the result of an intense regressive process with resorption or retraction of the long dendrites that reaches a maximum by P3. We have called this stage: the Purkinje cell with 'regressive-atrophic' dendrites. This regression marks the initiation of the phase of the stellate cell, characterized by the explosive outgrowth of shorter perisomatic protrusions emerging in all directions. By P6, almost all the labeled Purkinje cells have attained this phase. The ultrastructural study of the labeled Purkinje cells has revealed that the transient dendrites of the fusiform cells have all the cytologic features of mature dendrites, particularly cytoskeletal elements (microtubules) and free polyribosomes. More importantly, axon terminals of unknown origin establish a few, constantly present, mature-like synaptic contacts on the dendritic shafts and spinous protrusions from P0, the earliest studied age. Their frequency increases on the Purkinje cells which enter the phase of stellate cell. Our results emphasize that the transformation of bipolar postmigratory Purkinje cells into the stellate cell stage results from a complex cascade of alternating creative and destructive processes, taking place in parallel with the formation and regression of mature synaptic contacts, between the remodelling dendritic arbors and unidentified afferent inputs. Purkinje cells, in all the different transitional stages, are present side by side in the same folial regions, at least until P4, and receive a similar contingent of synaptic input. This indicates that the dendritic remodelling is not driven by the synaptic inputs, but obeys either neural interactions that lead Purkinje cells to assume their monocellular layer configuration, or an internal clock depending on the Purkinje cell birthdate, or an interplay between these two kinds of mechanisms.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1993
Vertebrate sensory neurons have a pseudo-unipolar morphology; their somata are covered by satellite cells and lack dendrites or synaptic contacts. However, when neonatal rat sensory neurons from the nodose ganglia develop in culture in absence of satellite cells and with NGF, they form synapses among themselves. In this study, we investigated whether neonatal rat nodose neurons express dendrites under the same culture conditions. We show by Lucifer yellow injection that nodose neurons remain typically unipolar when cocultured with their ganglionic satellite cells. However, when these neurons are cultured without satellite cells, virtually all neurons acquire a multipolar morphology. Moreover, when NGF is added to satellite cell-free cultures, several neurons extend dendrites; these processes stain positively for microtubule-associated protein-2. NGF induces a 17-fold increase in dendritic outgrowth after 3 weeks but has little effect on axon number. In addition, we find that the abi...
Stem Cells, 2009
Neural stem or progenitor cells (NSC/NPCs) able to generate the different neuron and glial cell types of the cerebellum have been isolated in vitro, but their identity and location in the intact cerebellum are unclear. Here, we use inducible Cre recombination in GFAPCreERT2 mice to irreversibly activate reporter gene expression at P2 (postnatal day 2), P5, and P12 in cells with GFAP (glial fibrillary acidic protein) promoter activity and analyze the fate of genetically tagged cells in vivo. We show that cells tagged at P2-P5 with β-galactosidase or enhanced green fluorescent proteins reporter genes generate at least 30% of basket and stellate GABAergic interneurons in the molecular layer (ML) and that they lose their neurogenic potential by P12, after which they generate only glia. Tagged cells in the cerebellar white matter (WM) were initially GFAP/S100β+ and expressed the NSC/NPCs proteins LeX, Musashi1, and Sox2 in vivo. One week after tagging, reporter+ cells in the WM upregulat...
Development, 2003
Neurotrophins are candidate molecules for regulating dendritogenesis. We report here on dendritic growth of rat visual cortex pyramidal and interneurons overexpressing `brain-derived neurotrophic factor' BDNF and`neurotrophin 4/5' NT4/5. Neurons in organotypic cultures were transfected with plasmids encoding either `enhanced green fluorescent protein' EGFP,BDNF/EGFP or NT4/5/EGFP either at the day of birth with analysis at 5 days in vitro, or at 5 days in vitro with analysis at 10 days in vitro.In pyramidal neurons, both TrkB ligands increased dendritic length and number of segments without affecting maximum branch order and number of primary dendrites. In the early time window, only infragranular neurons were responsive. Neurons in layers II/III became responsive to NT4/5, but not BDNF,during the later time window. BDNF and NT4/5 transfectants at 10 days in vitro had still significantly shorter dendrites than adult pyramidal neurons,suggesting a massive growth spurt aft...
Cellular and Molecular Neurobiology, 2010
D6 is a promoter/enhancer of the mDach1 gene that is involved in the development of the neocortex and hippocampus. It is expressed by proliferating neural stem/ progenitor cells (NSPCs) of the cortex at early stages of neurogenesis. The differentiation potential of NSPCs isolated from embryonic day 12 mouse embryos, in which the expression of green fluorescent protein (GFP) is driven by the D6 promoter/enhancer, has been studied in vitro and after transplantation into the intact adult rat brain as well as into the site of a photochemical lesion. The electrophysiological properties of D6/GFP-derived cells were studied using the whole-cell patch-clamp technique, and immunohistochemical analyses were carried out. D6/GFPderived neurospheres expressed markers of radial glia and gave rise predominantly to immature neurons and GFAPpositive cells during in vitro differentiation. One week after transplantation into the intact brain or into the site of a photochemical lesion, transplanted cells expressed only neuronal markers. D6/GFP-derived neurons were characterised by the expression of tetrodotoxin-sensitive Na ?currents and K A -and K DR currents sensitive to 4-aminopyridine. They were able to fire repetitive action potentials and responded to the application of GABA. Our results indicate that after transplantation into the site of a photochemical lesion, D6/GFP-derived NSPCs survive and differentiate into neurons, and their membrane properties are comparable to those transplanted into the non-injured cortex. Therefore, region-specific D6/GFP-derived NSPCs represent a promising tool for studying neurogenesis and cell replacement in a damaged cellular environment.