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Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons.

Belachew S, Chittajallu R, Aguirre AA, Yuan X, Kirby M, Anderson S, Gallo V - J. Cell Biol. (2003)

Bottom Line: The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming.We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs.These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuroscience Research, Children's Research Institute, Children's National Medical Center, Washington, DC 20010-2970, USA.

ABSTRACT
Neurogenesis is known to persist in the adult mammalian central nervous system (CNS). The identity of the cells that generate new neurons in the postnatal CNS has become a crucial but elusive issue. Using a transgenic mouse, we show that NG2 proteoglycan-positive progenitor cells that express the 2',3'-cyclic nucleotide 3'-phosphodiesterase gene display a multipotent phenotype in vitro and generate electrically excitable neurons, as well as astrocytes and oligodendrocytes. The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming. We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs. These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

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A subset of postnatal NeuN+ hippocampal neurons express low levels of CNP-GFP and are electrically excitable. (A–F) 0.5-μm thick single plane confocal scanning images representing two different levels (levels of planes in A–C and D–F are separated by 4 μm) of the same field of CA3 region of hippocampus from P6 CNP-GFP transgenic mice revealed that some cells were NeuN (red)-immunoreactive and expressed GFP fluorescence (A–C, insets of high magnification of a single cell). Distinct microscopic planes (A–C and D–F) within the same cells (C and F, arrowheads) showed a diffuse colocalization of the two signals, providing a more accurate demonstration of NeuN/CNP-GFP coexpression. Most of these NeuN+/CNP-GFP+ cells were located in the stratum radiatum, but sparse NeuN+/CNP-GFP+ cells were also found in the pyramidal layer (G–I, arrowhead). (G–I) Z-series (10 μm thick) confocal scanning image centered on the dashed area of (F) at higher magnification. Lower levels of GFP fluorescence were detected in all NeuN+/CNP-GFP+ cells (C, F, and G–I, arrowheads), as compared with CNP-GFP+ cells from the same field (G–I, star), that were NeuN−. Cells that expressed low levels of GFP that were NeuN− were also found (G–I, arrow). (J) Quantitative image analysis of GFP fluorescence intensity (linear arbitrary scale from 0 to 250 U; paired columns represent incremental intervals of 20 arbitrary fluorescence units) revealed a bimodal distribution, demonstrating that the average GFP fluorescence of NeuN+/CNP-GFP+ neurons (red) was 3.5-fold lower than that of NeuN−/CNP-GFP+ cells (green; total cells counted = 252, two independent experiments, equal number of cells analyzed for each population). Electrophysiological recordings were performed in weakly CNP-GFP+ cells from the CA1 and CA3 pyramidal layer of P3–P8 hippocampal slices (K–L). Upper parts of K–L represent single examples of fluorescence images showing the neuron-like arborized morphology of two different CNP-GFP+ cells recorded in CA3 and visualized after filling with a rhodamine-coupled dye. Lower parts of K–L display repetitive action potentials elicited by depolarizing the same cells with electrotonic current pulses (step size = 10–30 pA, step duration = 300 ms). (M–O) Representative example of a biocytin-filled (arrow in M) recorded cell in the CA3 stratum radiatum area, showing that cells that expressed low levels of GFP and that were able to propagate action potentials were consistently NeuN+ (arrow in N and O) by post-hoc immunostaining. Bars: 50 μm (A–F), 33 μm (G–I), and 60 μm (M–O).
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fig6: A subset of postnatal NeuN+ hippocampal neurons express low levels of CNP-GFP and are electrically excitable. (A–F) 0.5-μm thick single plane confocal scanning images representing two different levels (levels of planes in A–C and D–F are separated by 4 μm) of the same field of CA3 region of hippocampus from P6 CNP-GFP transgenic mice revealed that some cells were NeuN (red)-immunoreactive and expressed GFP fluorescence (A–C, insets of high magnification of a single cell). Distinct microscopic planes (A–C and D–F) within the same cells (C and F, arrowheads) showed a diffuse colocalization of the two signals, providing a more accurate demonstration of NeuN/CNP-GFP coexpression. Most of these NeuN+/CNP-GFP+ cells were located in the stratum radiatum, but sparse NeuN+/CNP-GFP+ cells were also found in the pyramidal layer (G–I, arrowhead). (G–I) Z-series (10 μm thick) confocal scanning image centered on the dashed area of (F) at higher magnification. Lower levels of GFP fluorescence were detected in all NeuN+/CNP-GFP+ cells (C, F, and G–I, arrowheads), as compared with CNP-GFP+ cells from the same field (G–I, star), that were NeuN−. Cells that expressed low levels of GFP that were NeuN− were also found (G–I, arrow). (J) Quantitative image analysis of GFP fluorescence intensity (linear arbitrary scale from 0 to 250 U; paired columns represent incremental intervals of 20 arbitrary fluorescence units) revealed a bimodal distribution, demonstrating that the average GFP fluorescence of NeuN+/CNP-GFP+ neurons (red) was 3.5-fold lower than that of NeuN−/CNP-GFP+ cells (green; total cells counted = 252, two independent experiments, equal number of cells analyzed for each population). Electrophysiological recordings were performed in weakly CNP-GFP+ cells from the CA1 and CA3 pyramidal layer of P3–P8 hippocampal slices (K–L). Upper parts of K–L represent single examples of fluorescence images showing the neuron-like arborized morphology of two different CNP-GFP+ cells recorded in CA3 and visualized after filling with a rhodamine-coupled dye. Lower parts of K–L display repetitive action potentials elicited by depolarizing the same cells with electrotonic current pulses (step size = 10–30 pA, step duration = 300 ms). (M–O) Representative example of a biocytin-filled (arrow in M) recorded cell in the CA3 stratum radiatum area, showing that cells that expressed low levels of GFP and that were able to propagate action potentials were consistently NeuN+ (arrow in N and O) by post-hoc immunostaining. Bars: 50 μm (A–F), 33 μm (G–I), and 60 μm (M–O).

Mentions: To determine whether highly proliferative postnatal CNP-GFP–expressing cells were also able to generate neurons or astrocytes in vivo, we investigated the existence of intermediate stages of neuronal and astroglial differentiation (i.e., expressing low levels of GFP) in CNS germinative areas. We were unable to find even a single cell expressing both GFAP and GFP between P2 and P10, either in the hippocampus or in the SVZ (unpublished data). Conversely, we found that numerous postnatal CNP-GFP+ cells were also NeuN+ in the hippocampus (Fig. 6 , A–I). Hippocampal NeuN+/CNP-GFP+ committed neurons all expressed low levels of GFP (Fig. 6 J), and were mostly distributed at P6 within stratum radiatum and lucidum of CA1 and CA3 (Fig. 6, A–F; Fig. 6, C and F, arrowheads), and in the molecular layer and hilar region of the dentate gyrus. Scattered NeuN+/CNP-GFP+ neurons were also observed in the CA1 (unpublished data) and CA3 (Fig. 6, A–F) pyramidal layers and in the granule layer of the dentate gyrus. Postnatal NeuN+/CNP-GFP+ hippocampal neurons were identified in two different CNP-GFP transgenic lines (unpublished data).


Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons.

Belachew S, Chittajallu R, Aguirre AA, Yuan X, Kirby M, Anderson S, Gallo V - J. Cell Biol. (2003)

A subset of postnatal NeuN+ hippocampal neurons express low levels of CNP-GFP and are electrically excitable. (A–F) 0.5-μm thick single plane confocal scanning images representing two different levels (levels of planes in A–C and D–F are separated by 4 μm) of the same field of CA3 region of hippocampus from P6 CNP-GFP transgenic mice revealed that some cells were NeuN (red)-immunoreactive and expressed GFP fluorescence (A–C, insets of high magnification of a single cell). Distinct microscopic planes (A–C and D–F) within the same cells (C and F, arrowheads) showed a diffuse colocalization of the two signals, providing a more accurate demonstration of NeuN/CNP-GFP coexpression. Most of these NeuN+/CNP-GFP+ cells were located in the stratum radiatum, but sparse NeuN+/CNP-GFP+ cells were also found in the pyramidal layer (G–I, arrowhead). (G–I) Z-series (10 μm thick) confocal scanning image centered on the dashed area of (F) at higher magnification. Lower levels of GFP fluorescence were detected in all NeuN+/CNP-GFP+ cells (C, F, and G–I, arrowheads), as compared with CNP-GFP+ cells from the same field (G–I, star), that were NeuN−. Cells that expressed low levels of GFP that were NeuN− were also found (G–I, arrow). (J) Quantitative image analysis of GFP fluorescence intensity (linear arbitrary scale from 0 to 250 U; paired columns represent incremental intervals of 20 arbitrary fluorescence units) revealed a bimodal distribution, demonstrating that the average GFP fluorescence of NeuN+/CNP-GFP+ neurons (red) was 3.5-fold lower than that of NeuN−/CNP-GFP+ cells (green; total cells counted = 252, two independent experiments, equal number of cells analyzed for each population). Electrophysiological recordings were performed in weakly CNP-GFP+ cells from the CA1 and CA3 pyramidal layer of P3–P8 hippocampal slices (K–L). Upper parts of K–L represent single examples of fluorescence images showing the neuron-like arborized morphology of two different CNP-GFP+ cells recorded in CA3 and visualized after filling with a rhodamine-coupled dye. Lower parts of K–L display repetitive action potentials elicited by depolarizing the same cells with electrotonic current pulses (step size = 10–30 pA, step duration = 300 ms). (M–O) Representative example of a biocytin-filled (arrow in M) recorded cell in the CA3 stratum radiatum area, showing that cells that expressed low levels of GFP and that were able to propagate action potentials were consistently NeuN+ (arrow in N and O) by post-hoc immunostaining. Bars: 50 μm (A–F), 33 μm (G–I), and 60 μm (M–O).
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fig6: A subset of postnatal NeuN+ hippocampal neurons express low levels of CNP-GFP and are electrically excitable. (A–F) 0.5-μm thick single plane confocal scanning images representing two different levels (levels of planes in A–C and D–F are separated by 4 μm) of the same field of CA3 region of hippocampus from P6 CNP-GFP transgenic mice revealed that some cells were NeuN (red)-immunoreactive and expressed GFP fluorescence (A–C, insets of high magnification of a single cell). Distinct microscopic planes (A–C and D–F) within the same cells (C and F, arrowheads) showed a diffuse colocalization of the two signals, providing a more accurate demonstration of NeuN/CNP-GFP coexpression. Most of these NeuN+/CNP-GFP+ cells were located in the stratum radiatum, but sparse NeuN+/CNP-GFP+ cells were also found in the pyramidal layer (G–I, arrowhead). (G–I) Z-series (10 μm thick) confocal scanning image centered on the dashed area of (F) at higher magnification. Lower levels of GFP fluorescence were detected in all NeuN+/CNP-GFP+ cells (C, F, and G–I, arrowheads), as compared with CNP-GFP+ cells from the same field (G–I, star), that were NeuN−. Cells that expressed low levels of GFP that were NeuN− were also found (G–I, arrow). (J) Quantitative image analysis of GFP fluorescence intensity (linear arbitrary scale from 0 to 250 U; paired columns represent incremental intervals of 20 arbitrary fluorescence units) revealed a bimodal distribution, demonstrating that the average GFP fluorescence of NeuN+/CNP-GFP+ neurons (red) was 3.5-fold lower than that of NeuN−/CNP-GFP+ cells (green; total cells counted = 252, two independent experiments, equal number of cells analyzed for each population). Electrophysiological recordings were performed in weakly CNP-GFP+ cells from the CA1 and CA3 pyramidal layer of P3–P8 hippocampal slices (K–L). Upper parts of K–L represent single examples of fluorescence images showing the neuron-like arborized morphology of two different CNP-GFP+ cells recorded in CA3 and visualized after filling with a rhodamine-coupled dye. Lower parts of K–L display repetitive action potentials elicited by depolarizing the same cells with electrotonic current pulses (step size = 10–30 pA, step duration = 300 ms). (M–O) Representative example of a biocytin-filled (arrow in M) recorded cell in the CA3 stratum radiatum area, showing that cells that expressed low levels of GFP and that were able to propagate action potentials were consistently NeuN+ (arrow in N and O) by post-hoc immunostaining. Bars: 50 μm (A–F), 33 μm (G–I), and 60 μm (M–O).
Mentions: To determine whether highly proliferative postnatal CNP-GFP–expressing cells were also able to generate neurons or astrocytes in vivo, we investigated the existence of intermediate stages of neuronal and astroglial differentiation (i.e., expressing low levels of GFP) in CNS germinative areas. We were unable to find even a single cell expressing both GFAP and GFP between P2 and P10, either in the hippocampus or in the SVZ (unpublished data). Conversely, we found that numerous postnatal CNP-GFP+ cells were also NeuN+ in the hippocampus (Fig. 6 , A–I). Hippocampal NeuN+/CNP-GFP+ committed neurons all expressed low levels of GFP (Fig. 6 J), and were mostly distributed at P6 within stratum radiatum and lucidum of CA1 and CA3 (Fig. 6, A–F; Fig. 6, C and F, arrowheads), and in the molecular layer and hilar region of the dentate gyrus. Scattered NeuN+/CNP-GFP+ neurons were also observed in the CA1 (unpublished data) and CA3 (Fig. 6, A–F) pyramidal layers and in the granule layer of the dentate gyrus. Postnatal NeuN+/CNP-GFP+ hippocampal neurons were identified in two different CNP-GFP transgenic lines (unpublished data).

Bottom Line: The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming.We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs.These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuroscience Research, Children's Research Institute, Children's National Medical Center, Washington, DC 20010-2970, USA.

ABSTRACT
Neurogenesis is known to persist in the adult mammalian central nervous system (CNS). The identity of the cells that generate new neurons in the postnatal CNS has become a crucial but elusive issue. Using a transgenic mouse, we show that NG2 proteoglycan-positive progenitor cells that express the 2',3'-cyclic nucleotide 3'-phosphodiesterase gene display a multipotent phenotype in vitro and generate electrically excitable neurons, as well as astrocytes and oligodendrocytes. The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming. We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs. These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

Show MeSH
Related in: MedlinePlus