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In-vivo RGB marking and multicolour single-cell tracking in the adult brain.

Gomez-Nicola D, Riecken K, Fehse B, Perry VH - Sci Rep (2014)

Bottom Line: We hypothesised that RGB marking, the tagging of individual cells with unique hues resulting from simultaneous expression of the three basic colours red, green and blue, provides a convenient toolbox for the study of the CNS anatomy at the single-cell level.RGB marking also enabled us to track the spatial and temporal fate of neural stem cells in the adult brain.The application of different viral envelopes and promoters provided a useful approach to track the generation of neurons vs. glial cells at the neurogenic niche, allowing the identification of the prominent generation of new astrocytes to the striatum.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biological Sciences, University of Southampton, Southampton, United Kingdom.

ABSTRACT
In neuroscience it is a technical challenge to identify and follow the temporal and spatial distribution of cells as they differentiate. We hypothesised that RGB marking, the tagging of individual cells with unique hues resulting from simultaneous expression of the three basic colours red, green and blue, provides a convenient toolbox for the study of the CNS anatomy at the single-cell level. Using γ-retroviral and lentiviral vector sets we describe for the first time the in-vivo multicolour RGB marking of neurons in the adult brain. RGB marking also enabled us to track the spatial and temporal fate of neural stem cells in the adult brain. The application of different viral envelopes and promoters provided a useful approach to track the generation of neurons vs. glial cells at the neurogenic niche, allowing the identification of the prominent generation of new astrocytes to the striatum. Multicolour RGB marking could serve as a universal and reproducible method to study and manipulate the CNS at the single-cell level, in both health and disease.

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RGB tracing of hippocampal neurogenesis.RGB marking by the intraparenchymal (hilus) administration of ecotropic SFFV γ-retroviral (Eco-SFFV-RV) vectors (see experimental scheme, top). (A) RGB-marked new neurons in the dentate gyrus, 6 weeks after the intraparenchymal delivery of Eco-SFFV-RV. (B) Sholl analysis of the complexity of the dendritic arborisation of newly generated neurons, represented as mean number of intersections at a given distance from soma (µm). (C) Detail of the distal segments of the dendrites of RGB-marked new neurons (in A). High-power magnification of single-marked dendritic distal segments (D, black and white), used to analyse dendritic spine density (E; spines/µm). (F) Detail of the synaptic boutons of RGB-marked new granule neurons, projecting to the CA3 layer. High-power magnification of single-marked synaptic boutons (G, black and white), used to analyse bouton complexity and maturation, as %boutons/size class (H). (A–H) Fluorescence is shown in red (mCherry), green (Venus) and blue (Cerulean). All images were analysed using confocal microscopy. ML, molecular layer; GCL, granule cell layer; HIL, hilus; SL CA3, stratum lucidum CA3; PYR CA3, stratum pyramidale CA3. Scale bar: A, 100 µm; C, F, 20 µm; D, G, 5 µm.
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f4: RGB tracing of hippocampal neurogenesis.RGB marking by the intraparenchymal (hilus) administration of ecotropic SFFV γ-retroviral (Eco-SFFV-RV) vectors (see experimental scheme, top). (A) RGB-marked new neurons in the dentate gyrus, 6 weeks after the intraparenchymal delivery of Eco-SFFV-RV. (B) Sholl analysis of the complexity of the dendritic arborisation of newly generated neurons, represented as mean number of intersections at a given distance from soma (µm). (C) Detail of the distal segments of the dendrites of RGB-marked new neurons (in A). High-power magnification of single-marked dendritic distal segments (D, black and white), used to analyse dendritic spine density (E; spines/µm). (F) Detail of the synaptic boutons of RGB-marked new granule neurons, projecting to the CA3 layer. High-power magnification of single-marked synaptic boutons (G, black and white), used to analyse bouton complexity and maturation, as %boutons/size class (H). (A–H) Fluorescence is shown in red (mCherry), green (Venus) and blue (Cerulean). All images were analysed using confocal microscopy. ML, molecular layer; GCL, granule cell layer; HIL, hilus; SL CA3, stratum lucidum CA3; PYR CA3, stratum pyramidale CA3. Scale bar: A, 100 µm; C, F, 20 µm; D, G, 5 µm.

Mentions: We next aimed at targeting the neural stem cell pool in the subgranular layer of the dentate gyrus (DG), in order to achieve RGB tracing of newly generated granule cells (Figure 4). We used a γ-retroviral approach, to specifically target the dividing cell pool and label proliferating neural stem cells without marking mature granule cells. A first approach using VSVG-SFFV-RV did not deliver optimal transduction rates of the neurogenic pool at the DG, therefore reducing the generation of mixed colours and also not offering optimal expression strength to analyse the cells by fluorescence. We optimised the vectors by using the mouse ecotropic envelope (Eco-SFFV-RV), previously described to be effective in the transduction of neural stem cells6. Eco-SFFV-RV vectors facilitated effective and reproducible RGB labelling of newly generated granule cells, six weeks after the injection (Figure 4a). We could identify individually labelled granule cells, easily distinguishable based on their distinct colours, as illustrated by different 3D rendering methods (Supplementary Movie 1). Expression of the three fluorescent proteins (mCherry, Venus, Cerulean) did not influence the differentiation of new granule cells as evidenced by their morphological similarity: there are no statistically significant differences in the Sholl analysis (Figure 4b). The combinatorial expression of the three fluorescent proteins also permitted RGB marking of the dendritic compartment in the molecular layer (ML; Figure 4c) and the dendritic spines (Figure 4d) whose density was not influenced by the expression of the different fluorescent proteins, showing non-significant statistical differences (Figure 4e). Similarly, RGB marking of newly generated granule cells permitted the identification of their axonal compartment (mossy fibers), projecting to the CA3 area of the hippocampus (Figure 4f). Tracing of dentate neurogenesis with Eco-SFFV-RV allowed the identification of RGB-coded synaptic boutons at the CA3 layer, which showed no morphological differences related to the expression of the three fluorescent tags (Figure 4h).


In-vivo RGB marking and multicolour single-cell tracking in the adult brain.

Gomez-Nicola D, Riecken K, Fehse B, Perry VH - Sci Rep (2014)

RGB tracing of hippocampal neurogenesis.RGB marking by the intraparenchymal (hilus) administration of ecotropic SFFV γ-retroviral (Eco-SFFV-RV) vectors (see experimental scheme, top). (A) RGB-marked new neurons in the dentate gyrus, 6 weeks after the intraparenchymal delivery of Eco-SFFV-RV. (B) Sholl analysis of the complexity of the dendritic arborisation of newly generated neurons, represented as mean number of intersections at a given distance from soma (µm). (C) Detail of the distal segments of the dendrites of RGB-marked new neurons (in A). High-power magnification of single-marked dendritic distal segments (D, black and white), used to analyse dendritic spine density (E; spines/µm). (F) Detail of the synaptic boutons of RGB-marked new granule neurons, projecting to the CA3 layer. High-power magnification of single-marked synaptic boutons (G, black and white), used to analyse bouton complexity and maturation, as %boutons/size class (H). (A–H) Fluorescence is shown in red (mCherry), green (Venus) and blue (Cerulean). All images were analysed using confocal microscopy. ML, molecular layer; GCL, granule cell layer; HIL, hilus; SL CA3, stratum lucidum CA3; PYR CA3, stratum pyramidale CA3. Scale bar: A, 100 µm; C, F, 20 µm; D, G, 5 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4273606&req=5

f4: RGB tracing of hippocampal neurogenesis.RGB marking by the intraparenchymal (hilus) administration of ecotropic SFFV γ-retroviral (Eco-SFFV-RV) vectors (see experimental scheme, top). (A) RGB-marked new neurons in the dentate gyrus, 6 weeks after the intraparenchymal delivery of Eco-SFFV-RV. (B) Sholl analysis of the complexity of the dendritic arborisation of newly generated neurons, represented as mean number of intersections at a given distance from soma (µm). (C) Detail of the distal segments of the dendrites of RGB-marked new neurons (in A). High-power magnification of single-marked dendritic distal segments (D, black and white), used to analyse dendritic spine density (E; spines/µm). (F) Detail of the synaptic boutons of RGB-marked new granule neurons, projecting to the CA3 layer. High-power magnification of single-marked synaptic boutons (G, black and white), used to analyse bouton complexity and maturation, as %boutons/size class (H). (A–H) Fluorescence is shown in red (mCherry), green (Venus) and blue (Cerulean). All images were analysed using confocal microscopy. ML, molecular layer; GCL, granule cell layer; HIL, hilus; SL CA3, stratum lucidum CA3; PYR CA3, stratum pyramidale CA3. Scale bar: A, 100 µm; C, F, 20 µm; D, G, 5 µm.
Mentions: We next aimed at targeting the neural stem cell pool in the subgranular layer of the dentate gyrus (DG), in order to achieve RGB tracing of newly generated granule cells (Figure 4). We used a γ-retroviral approach, to specifically target the dividing cell pool and label proliferating neural stem cells without marking mature granule cells. A first approach using VSVG-SFFV-RV did not deliver optimal transduction rates of the neurogenic pool at the DG, therefore reducing the generation of mixed colours and also not offering optimal expression strength to analyse the cells by fluorescence. We optimised the vectors by using the mouse ecotropic envelope (Eco-SFFV-RV), previously described to be effective in the transduction of neural stem cells6. Eco-SFFV-RV vectors facilitated effective and reproducible RGB labelling of newly generated granule cells, six weeks after the injection (Figure 4a). We could identify individually labelled granule cells, easily distinguishable based on their distinct colours, as illustrated by different 3D rendering methods (Supplementary Movie 1). Expression of the three fluorescent proteins (mCherry, Venus, Cerulean) did not influence the differentiation of new granule cells as evidenced by their morphological similarity: there are no statistically significant differences in the Sholl analysis (Figure 4b). The combinatorial expression of the three fluorescent proteins also permitted RGB marking of the dendritic compartment in the molecular layer (ML; Figure 4c) and the dendritic spines (Figure 4d) whose density was not influenced by the expression of the different fluorescent proteins, showing non-significant statistical differences (Figure 4e). Similarly, RGB marking of newly generated granule cells permitted the identification of their axonal compartment (mossy fibers), projecting to the CA3 area of the hippocampus (Figure 4f). Tracing of dentate neurogenesis with Eco-SFFV-RV allowed the identification of RGB-coded synaptic boutons at the CA3 layer, which showed no morphological differences related to the expression of the three fluorescent tags (Figure 4h).

Bottom Line: We hypothesised that RGB marking, the tagging of individual cells with unique hues resulting from simultaneous expression of the three basic colours red, green and blue, provides a convenient toolbox for the study of the CNS anatomy at the single-cell level.RGB marking also enabled us to track the spatial and temporal fate of neural stem cells in the adult brain.The application of different viral envelopes and promoters provided a useful approach to track the generation of neurons vs. glial cells at the neurogenic niche, allowing the identification of the prominent generation of new astrocytes to the striatum.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biological Sciences, University of Southampton, Southampton, United Kingdom.

ABSTRACT
In neuroscience it is a technical challenge to identify and follow the temporal and spatial distribution of cells as they differentiate. We hypothesised that RGB marking, the tagging of individual cells with unique hues resulting from simultaneous expression of the three basic colours red, green and blue, provides a convenient toolbox for the study of the CNS anatomy at the single-cell level. Using γ-retroviral and lentiviral vector sets we describe for the first time the in-vivo multicolour RGB marking of neurons in the adult brain. RGB marking also enabled us to track the spatial and temporal fate of neural stem cells in the adult brain. The application of different viral envelopes and promoters provided a useful approach to track the generation of neurons vs. glial cells at the neurogenic niche, allowing the identification of the prominent generation of new astrocytes to the striatum. Multicolour RGB marking could serve as a universal and reproducible method to study and manipulate the CNS at the single-cell level, in both health and disease.

Show MeSH