Directing Astroglia from the Cerebral Cortex into Subtype Specific Functional Neurons methods
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Genetic Fate Mapping Demonstrates Reprogramming of Postnatal Cortical Astroglia into Glutamatergic Neurons
Perforated patch clamp recordings of these fate-mapped astroglia-derived cells reprogrammed by Neurog2 revealed their functional neuronal identity as these cells fired APs following step-current injection in current clamp ( n = 8; ). In the next set of experiments, we assessed whether neurons derived from fate-mapped astroglia could give rise to functional glutamatergic autapses ( ). Step-depolarisation of GFP/DsRed-double-positive neurons at 0.05 Hz evoked a sequence of both autaptic and polysynaptic components (2 out of 8 cells recorded) consistent with the excitatory nature of the recorded neurons (average age of the cells: 18.1±2.2 DPI;, insets), while at higher stimulation frequency (1 Hz) the autaptic component with a short decay time typical of glutamatergic synaptic transmission could be observed in isolation ( ). Consistent with their glutamatergic nature,...
Genetic Fate Mapping Demonstrates Reprogramming of Postnatal Cortical Astroglia into Glutamatergic Neurons
(A) Fluorescence micrograph depicting a GFP-positive neuron derived from a fate-mapped astroglia, prepared from postnatal cortex of tamoxifen-induced GLAST::CreERT2/Z/EG mice, 14 d after transduction with Neurog2-DsRed. The inset shows control untransduced GFP-positive astroglia. (B) DsRed expression in the same cell indicating forced expression of Neurog2. (C) Step-current injection into the cell shown in (A) and (B) results in repetitive firing of action potentials. (D-E) Fluorescence micrographs depicting another GFP-positive neuron, derived from a fate-mapped astroglia, following Neurog2-induced reprogramming 19 d after transduction. (F) Step-depolarisation at 1 Hz of the neuron shown in (D) and (E) evokes an autaptic response exhibiting a short decay time (90%-10%) typical of glutamatergic synaptic transmission (5.2 ms). The inset shows a single response evoked at 0.0...
Dlx2 Directs Postnatal Cortical Astroglia towards Acquiring a GABAergic Identity
(A) Fluorescence micrograph depicts a GFP-positive neuron derived from a fate-mapped astroglia, prepared from postnatal cortex of tamoxifen induced GLAST::CreERT2/Z/EG mice that has been reprogrammed by forced expression of Dlx2. (A') DsRed expression in the same cell at 20 DPI indicates prior reprogramming by Dlx2. (A") Step-current injection in current clamp in the cell shown in (A) and (A') results in a single action potential. (B) Fluorescence micrograph depicts a GFP-positive neuron derived from a fate-mapped astroglia, prepared from postnatal cortex of tamoxifen induced GLAST::CreERT2/Z/EG mice that has been reprogrammed by forced expression of Dlx2. (B') DsRed expression in the same cell at 17 DPI indicates prior reprogramming by Dlx2. (B") Step-current injection in current clamp in the cell shown in (B) and (B') results in repetitive firing of action potentials. No...
Dlx2 Directs Postnatal Cortical Astroglia towards Acquiring a GABAergic Identity
Next we assessed whether some of these relatively immature neurons derived from Dlx2-reprogrammed astroglia may nevertheless establish functional autaptic or synaptic connections. We first performed immunocytochemistry for vGluT1 and for vGaT, the latter known to be expressed in synaptic vesicles located in presynaptic terminals of GABAergic neurons. In sharp contrast to reprogramming by Neurog2, astroglia-derived neurons reprogrammed by Dlx2 were devoid of vGluT1 immunoreactivity (unpublished data), but some of them (33.7%±3.6% at 22.0±0.6 DPI, n = 339 DsRed-positive neurons counted; n = 3 independent experiments) were found to exhibit labelling of vGaT-positive puncta outlining both their soma and their processes ( ). In addition, a small subset of DsRed-positive neurons exhibited GAD67 immunoreactivity ( '). These findings therefore suggest that...
Neurosphere Cells Derived from the Adult Injured Cerebral Cortex Can Be Reprogrammed into Synapse-Forming Functional...
To examine the extent of plasticity of glial cells derived from the adult lesioned cortex we also tested as a proof-of-principle experiment whether adult lesioned cortex-derived neurosphere cells could also be directed by forced expression of Dlx2 towards MAP2-expressing neurons ( '). However, Dlx2 reprogrammed neurons were rather few and fragile due to their rather small soma size, thus hampering extensive electrophysiological analysis. Nevertheless we could record from one cell shown in, where step-depolarisation evoked an autaptic response that was blocked by bicuculline, indicating the development of functional GABAergic connections ( '). These data show that even adult cells isolated from the injured cortex and expanded as neurospheres can be instructed by forced expression of Neurog2 or Dlx2 to generate mature neurons able to establish functional glutamatergic or GABAergic conn...
Reprogramming of Adult Injured Cortex-Derived Neurosphere Cells into Functional Synapse-Forming Neurons
The successful reprogramming of astroglia following expansion under neurosphere conditions also encouraged us to investigate whether neurosphere cells derived from the adult cerebral cortex after injury can be similarly directed towards neurogenesis. Thus, we assessed here whether adult cortex-derived neurosphere cells also can be driven towards fully functional neurons following forced expression of Neurog2 or Dlx2. Indeed, virtually all the cells expressing Neurog2 acquired a neuronal identity as revealed by MAP2 staining and their ability to fire APs. Moreover, we also provide evidence by vGluT1 immunoreactivity that the neurons derived from these lesion cortex-derived neurosphere cells undergo a subtype specification similar to reprogrammed postnatal astroglia and differentiate into glutamatergic neurons. Consistent with the development of functional synapses by Neurog2-reprogramm...
Materials and Methods
Experiments were conducted either on C57BL/6J mice or transgenic GLAST::CreERT2/Z/EG double heterozygous mice. Briefly, heterozygous GLAST::CreERT2 mice were crossed with the Z/EG reporter mouse line to generate double heterozygous mutants. The activation of the tamoxifen-inducible form of the Cre recombinase was done as follows: From postnatal day 2, i.e. at the peak of cortical astrogliogenesis, until sacrifice at postnatal day 7, tamoxifen (20 mg/mL, dissolved in corn oil, Sigma-Aldrich, Munich, Germany) was administered as described by Mori et al. to mothers and mice pups thus received tamoxifen via the milk from their mother during lactation. Using the same mouse line as in this study, Mori et al. found that by recombination already at E18 virtually all fate-mapped cells give rise to glia, indicating that at that stage GLAST expressing cells have lost their intrinsic neurogenic...
Cell Cultures of Astroglia from the Postnatal Cerebral Cortex
For culturing postnatal astroglia we followed the procedure described previously by Heins et al. (2002). After removal of the meninges, grey matter tissue from P5-P7 cerebral cortex of C57BL/6J or GLAST::CreERT2/Z/EG mice was dissected and dissociated mechanically. Subsequently, cells were centrifuged for 5 min at 1,000 rpm, re-suspended, and plated in a medium consisting of DMEM/F12 (Gibco), 3.5 mM glucose (Sigma), 10% fetal calf serum (Gibco), 5% horse serum (Gibco), penicillin/streptomycin (Gibco), and supplemented with B27 (Gibco), 10 ng/mL epidermal growth factor (EGF, Roche), and 10 ng/mL fibroblast growth factor 2 (FGF2, Roche). Contaminating oligodendrocyte precursor cells were removed by brusquely shaking the culture flasks several times. Cells were passaged after 1 wk using trypsin/EDTA (Gibco) and plated on poly-D-lysine (Sigma-Aldrich, Munich, Germany) glass coated...
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Perforated patch clamp recordings of these fate-mapped astroglia-derived cells reprogrammed by Neurog2 revealed their functional neuronal identity as these cells fired APs following step-current injection in current clamp ( n = 8; ). In the next set of experiments, we assessed whether neurons derived from fate-mapped astroglia could give rise to functional glutamatergic autapses ( ). Step-depolarisation of GFP/DsRed-double-positive neurons at 0.05 Hz evoked a sequence of both autaptic and polysynaptic components (2 out of 8 cells recorded) consistent with the excitatory nature of the recorded neurons (average age of the cells: 18.1±2.2 DPI;, insets), while at higher stimulation frequency (1 Hz) the autaptic component with a short decay time typical of glutamatergic synaptic transmission could be observed in isolation ( ). Consistent with their glutamatergic nature, fate-mapped astroglia reprogrammed by forced expression of Neurog2 also exhibited a dense labelling of vGluT1-positive puncta ( ). These data clearly demonstrate that Neurog2 instructs fate-mapped astroglia from the postnatal cerebral cortex to acquire a glutamatergic identity.
(A) Fluorescence micrograph depicting a GFP-positive neuron derived from a fate-mapped astroglia, prepared from postnatal cortex of tamoxifen-induced GLAST::CreERT2/Z/EG mice, 14 d after transduction with Neurog2-DsRed. The inset shows control untransduced GFP-positive astroglia. (B) DsRed expression in the same cell indicating forced expression of Neurog2. (C) Step-current injection into the cell shown in (A) and (B) results in repetitive firing of action potentials. (D-E) Fluorescence micrographs depicting another GFP-positive neuron, derived from a fate-mapped astroglia, following Neurog2-induced reprogramming 19 d after transduction. (F) Step-depolarisation at 1 Hz of the neuron shown in (D) and (E) evokes an autaptic response exhibiting a short decay time (90%-10%) typical of glutamatergic synaptic transmission (5.2 ms). The inset shows a single response evoked at 0.05 Hz revealing both an autaptic and a polysynaptic response (asterisk) due to recruitment of other neurons in the cultured network, indicating the excitatory nature of the fate-mapped reprogrammed neuron. (G-H) Another example of a GFP-positive neuron derived from a fate-mapped astroglia follo...
(A) Fluorescence micrograph depicts a GFP-positive neuron derived from a fate-mapped astroglia, prepared from postnatal cortex of tamoxifen induced GLAST::CreERT2/Z/EG mice that has been reprogrammed by forced expression of Dlx2. (A') DsRed expression in the same cell at 20 DPI indicates prior reprogramming by Dlx2. (A") Step-current injection in current clamp in the cell shown in (A) and (A') results in a single action potential. (B) Fluorescence micrograph depicts a GFP-positive neuron derived from a fate-mapped astroglia, prepared from postnatal cortex of tamoxifen induced GLAST::CreERT2/Z/EG mice that has been reprogrammed by forced expression of Dlx2. (B') DsRed expression in the same cell at 17 DPI indicates prior reprogramming by Dlx2. (B") Step-current injection in current clamp in the cell shown in (B) and (B') results in repetitive firing of action potentials. Note the high action potential firing rate undergoing little adaptation (approximately 80 Hz). (C-C") Traces show recordings from a Dlx2-reprogrammed cell classified as a low-threshold burst-spiking neuron. (C) Depolarizing current injection at a holding potential of -62 mV resulted...
Next we assessed whether some of these relatively immature neurons derived from Dlx2-reprogrammed astroglia may nevertheless establish functional autaptic or synaptic connections. We first performed immunocytochemistry for vGluT1 and for vGaT, the latter known to be expressed in synaptic vesicles located in presynaptic terminals of GABAergic neurons. In sharp contrast to reprogramming by Neurog2, astroglia-derived neurons reprogrammed by Dlx2 were devoid of vGluT1 immunoreactivity (unpublished data), but some of them (33.7%±3.6% at 22.0±0.6 DPI, n = 339 DsRed-positive neurons counted; n = 3 independent experiments) were found to exhibit labelling of vGaT-positive puncta outlining both their soma and their processes ( ). In addition, a small subset of DsRed-positive neurons exhibited GAD67 immunoreactivity ( '). These findings therefore suggest that Dlx2 induces a GABAergic identity in the reprogrammed astroglia. Consistent with an interneuron phenotype, we could also record in 9 out of 33 neurons spontaneous synaptic currents exhibiting a slow decay time, characteristic of GABAergic synaptic events ( ). Finally, in few cases, step-depolarisati...
To examine the extent of plasticity of glial cells derived from the adult lesioned cortex we also tested as a proof-of-principle experiment whether adult lesioned cortex-derived neurosphere cells could also be directed by forced expression of Dlx2 towards MAP2-expressing neurons ( '). However, Dlx2 reprogrammed neurons were rather few and fragile due to their rather small soma size, thus hampering extensive electrophysiological analysis. Nevertheless we could record from one cell shown in, where step-depolarisation evoked an autaptic response that was blocked by bicuculline, indicating the development of functional GABAergic connections ( '). These data show that even adult cells isolated from the injured cortex and expanded as neurospheres can be instructed by forced expression of Neurog2 or Dlx2 to generate mature neurons able to establish functional glutamatergic or GABAergic connections, respectively.
The successful reprogramming of astroglia following expansion under neurosphere conditions also encouraged us to investigate whether neurosphere cells derived from the adult cerebral cortex after injury can be similarly directed towards neurogenesis. Thus, we assessed here whether adult cortex-derived neurosphere cells also can be driven towards fully functional neurons following forced expression of Neurog2 or Dlx2. Indeed, virtually all the cells expressing Neurog2 acquired a neuronal identity as revealed by MAP2 staining and their ability to fire APs. Moreover, we also provide evidence by vGluT1 immunoreactivity that the neurons derived from these lesion cortex-derived neurosphere cells undergo a subtype specification similar to reprogrammed postnatal astroglia and differentiate into glutamatergic neurons. Consistent with the development of functional synapses by Neurog2-reprogrammed adult cortex-derived neurosphere cells, we could record spontaneous glutamatergic events in these cultures. Conversely, following forced expression of Dlx2, we could observe the generation of functional GABAergic neurons from adult cortex-derived neurosphere cells, indicating that the same dichot...
Experiments were conducted either on C57BL/6J mice or transgenic GLAST::CreERT2/Z/EG double heterozygous mice. Briefly, heterozygous GLAST::CreERT2 mice were crossed with the Z/EG reporter mouse line to generate double heterozygous mutants. The activation of the tamoxifen-inducible form of the Cre recombinase was done as follows: From postnatal day 2, i.e. at the peak of cortical astrogliogenesis, until sacrifice at postnatal day 7, tamoxifen (20 mg/mL, dissolved in corn oil, Sigma-Aldrich, Munich, Germany) was administered as described by Mori et al. to mothers and mice pups thus received tamoxifen via the milk from their mother during lactation. Using the same mouse line as in this study, Mori et al. found that by recombination already at E18 virtually all fate-mapped cells give rise to glia, indicating that at that stage GLAST expressing cells have lost their intrinsic neurogenic potential.
For culturing postnatal astroglia we followed the procedure described previously by Heins et al. (2002). After removal of the meninges, grey matter tissue from P5-P7 cerebral cortex of C57BL/6J or GLAST::CreERT2/Z/EG mice was dissected and dissociated mechanically. Subsequently, cells were centrifuged for 5 min at 1,000 rpm, re-suspended, and plated in a medium consisting of DMEM/F12 (Gibco), 3.5 mM glucose (Sigma), 10% fetal calf serum (Gibco), 5% horse serum (Gibco), penicillin/streptomycin (Gibco), and supplemented with B27 (Gibco), 10 ng/mL epidermal growth factor (EGF, Roche), and 10 ng/mL fibroblast growth factor 2 (FGF2, Roche). Contaminating oligodendrocyte precursor cells were removed by brusquely shaking the culture flasks several times. Cells were passaged after 1 wk using trypsin/EDTA (Gibco) and plated on poly-D-lysine (Sigma-Aldrich, Munich, Germany) glass coated coverslips at a density of 60,000 cells per coverslip (in 24-well plates; BD Biosciences, Erembodegem, Belgium) in the same medium. The vast majority of the cells (>90%) in these cultures were positive for glial fibrillary acidic protein (GFAP) as previously described.
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