Background and Purpose—Although in vitro studies suggest that non-neurogenic regions of the adult central nervous system potentially contain multipotent parenchymal progenitors, neurons are clearly not replaced in most brain regions after injury. Here, in a well-established model of mild transient brain ischemia, we explored Olig2 antagonism and Pax6 overexpression as potential avenues to redirect endogenous progenitors proliferating in situ toward a neuronal fate.
Methods—Retroviral vectors containing either Pax6 or a strong activator form of the repressor Olig2 (Olig2VP16), ie, a functionally dominant negative form of Olig2, were stereotaxically injected into the lateral striatum at 48 hours after 30 minutes middle cerebral artery occlusion (MCAo)/reperfusion.
Results—Retroviral modulation of fate determinants resulted in a significant number of infected cells differentiating into Doublecortin (DCX)-expressing immature neurons that were not observed after injection of a control virus. Whole-cell patch-clamp recordings in acute brain slices showed that the percentage of virus-infected cells with Na+ currents was increased by inhibition of the repressor function of Olig2 and by overexpression of Pax6. Furthermore, on retroviral transduction of fate determinants, we detected newly generated cells within the ischemic lesion that were capable of generating single action potentials and that received synaptic input.
Conclusions—Taken together, these data show that resident glia in the striatum can be reprogrammed toward functional neuronal differentiation following brain injury.
The transcription factors Olig2 and Pax6 play largely opposing roles in determining progenitor cell fate in neurosphere culture, as well as in adult neurogenesis in vivo.1–4 Following injury, an increase in the number of Olig2+ cells has been observed as a pan-lesion phenomenon throughout the brain.5–7 Repression of proneurogenic factors, such as Pax6, serves as a key mechanism underlying glial fate restriction by Olig2; using retroviral gene transfer, both Olig2 antagonism and overexpression of Pax6 have previously been shown to induce doublecortin (DCX)-expressing neuroblasts in a cortical stab wound lesion.6 This prompted us to examine the histopathologic and functional effects of retroviral gene transduction of neuronal fate determinants in the context of stroke.
Materials and Methods
Animals
All procedures were approved by an official committee. Nestin-GFP mice and GFAP-eGFP mice have been described in detail previously.8–10 129Sv mice aged 10 to 12 weeks and weighing 20 to 25 g were used for retroviral injections.
Surgical Procedures
Mice were anesthetized with 1.5% isofluorane and maintained in 1% isofluorane in 69% N20 and 30% O2 using a vaporizer. Ischemia was induced by 30 minutes filamentous middle cerebral artery occlusion (MCAo)/reperfusion as described in detail previously.9 For retroviral injections, mice were mounted onto a stereotaxic head holder (David Kopf Instruments) in the flat-skull position. Approximately 1 mm anterior and 1.5 mm lateral to bregma, a 1-μL, 30–gauge, gas-tight syringe (Hamilton) was inserted to a depth of 4 mm and retracted to a depth of 3 mm from the dural surface, and 1 μL of retroviral suspension was injected.
Histological Procedures and Imaging
Brains were perfusion-fixed with 4% paraformaldehyde and cut into 40-μm sections. Antibodies were diluted in Tris-buffered saline containing 0.1% Triton X-100 and 3% donkey serum. Primary antibodies were goat antidoublecortin (Santa Cruz Biotechnologies) 1:200, polyclonal guinea-pig anti-GFAP (AdvancedImmunoChemical) 1:1000, polyclonal rabbit anti-GFP (Abcam) 1:400, rabbit anti-Iba1 (Wako Chemicals) 1:500, mouse anti-NeuN (Chemicon) 1:100 rabbit anti-NG2 (Chemicon) 1:100, polyclonal rabbit anti-Sox2 (Chemicon) 1:500, and rabbit anti-Olig2 (DF308). FITC-, RhodX- or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were all used at a concentration of 1:250. Phenotypic analyses of GFP+ cells were performed using a spectral confocal microscope (Leica TCS SP2). Appropriate gain and black-level settings were determined on control tissues stained with secondary antibodies alone.
Electrophysiology
Acute striatal slices (150 μm thick) for in situ recordings were prepared as reported previously.9 The perfusion chamber was continuously perfused with bicarbonate-buffered bath solution composed of (in mmol/L): 134 NaCl, 2.5 KCl, 1.3 MgCl2, 2 CaCl2, 1.25 K2HPO4, 26 NaHCO3, and 10 D-glucose; this solution was equilibrated with 95% O2 and 5% CO2 at a rate of 1 to 2 mL/min to a final pH of 7.4. Drugs were applied via the bath. To minimize indirect neuronal effects induced by kainate application, 0.5 μm tetrodotoxin and 0.1 mmol/L CdCl2 was added to the bath solution to block voltage-gated sodium and calcium channels, respectively. Patch-clamp recordings were performed with an EPC-9/2 double patch-clamp amplifier in combination with TIDA software (HEKA) in voltage-clamp mode. Whole-cell recordings were obtained with pipettes filled with the standard pipette solution composed of (in mmol/L): 130 KCl, 2 MgCl2, 0.5 CaCl2, 2 Na-ATP, 5 EGTA, and 10 HEPES. The pH was adjusted to 7.3 with KOH. To confirm intracellular access, Alexa Fluor 594 (10 μg/mL, Molecular Probes, Invitrogen) was filled in the pipettes. Pipettes were pulled from borosilicate capillaries (inner diameter, 0.87 mm; outer diameter, 1.5 mm; open resistance, 6 to 8 MΩ; Hilgenberg) using a P-2000 laser-based pipette puller (Sutter Instrument Co). Experiments were performed at room temperature (21°C–25°C).
The ischemic striatum was identified using standard transmission optics (Zeiss Axioskop; ×5 Zeiss objective, numeric aperture: 0.15). Images were recorded with a charge-coupled device (CCD) camera (Variocam, PCO Computer Optics, Kelheim, Germany). GFP+ cells were identified by fluorescence optics (excitation at 488 nm using a monochromator, Polychrome IV, Till Photonics). The emitted light was collected at 530±10 nm with a CCD camera QuantiCam (b/w VGA, Phase; ×60 water-immersion Olympus objective, numeric aperture: 0.8). Alexa Fluor 594 fluorescence was detected at an excitation wavelength of 589 nm, and an emission at 616±4 nm.
Statistics
Values are presented as mean±SEM. Comparisons between groups were performed by means of one-way analysis of variance, with level of significance set at 0.05 and with two-tailed probability values.
Results
Olig2 Expression in Glial Cells With Complex Membrane Properties
First, we characterized Olig2 expression in our mild ischemia model using GFAP-eGFP transgenic mice. In uninjured striatum, two types of GFAP-eGFP+ cells were morphologically distinguishable: astrocytes with high-fluorescence intensity display ramified processes and show passive membrane currents with no apparent time and voltage dependence. Cells with lower-fluorescence intensity have round somata and few processes with little, if any, branching, and display voltage-gated complex currents.11 In agreement with reports from other brain regions, about half of all brightly-fluorescent cells in normal striatum showed GFAP immunoreactivity (85 of 150 randomly selected cells; 3 animals), whereas weakly-fluorescent cells did not. In contrast, most of the weakly-fluorescent GFAP-eGFP+ cells displayed colabeling with Olig2 (95 out of 100 cells analyzed; 3 animals), which was never detected in brightly-fluorescent cells (Figure 1A). The GFAP-eGFP cell population increased during the first week after 30 minutes MCAo both in the vicinity of the ischemic lesion and, to a lesser degree, within the lesion core (Figure 1B). GFAP-eGFP+ cells predominantly displayed the complex electrophysiological phenotype early after insult.11 GFAP-eGFP+ cells in ischemic striatum adopted a variety of hypertrophic morphologies (especially rod-shaped cells) with frequent nestin and NG2 coexpression (Figure 1 C, D). Consistent with their frequent NG2 immunoreactivity, 72% of GFAP-eGFP+ cells (150 cells; 3 animals) were also Olig2 immunoreactive at 4 days postevent (Figure 1E).
Figure 1. Olig2 expression in glia reacting to brain ischemia. A, Two populations of GFP+ cells (green) in normal striatum of GFAP-eGFP mice. Red: Olig2. B, GFAP-eGFP+ cells (green) are induced at 4 days after MCAo. GFAP protein: blue, nestin: red. C and D, Following MCAo, GFAP-eGFP cells assume reactive morphologies with frequent nestin (red in C) and NG2 (red in D) coexpression. E, Olig2 expression (red) in GFAP-eGFP+ cells in ischemic striatum at 4 days after MCAo. Note characteristic “rod-shaped” hypertrophic morphologies of GFAP-eGFP cells. F, Olig2 expression in nestin-GFP+ cells at 4 days after MCAo. As we have previously reported, nestin-GFP+ cells proliferate in situ in the ischemic lesion, show NG2 immunoreactivity and adopt a complex electrophysiological phenotype.9 Scale bar (in F) 30 μm in A, 235 μm in B, 37 μm in C, 27 μm in D, 29 μm in E, 38 μm in F.
Reactive glia can also be visualized by nestin promoter-GFP transgenic mice. Nestin-GFP+ cells were induced during the first week after MCAo and showed voltage-gated currents.9 Here, immunohistochemical analysis revealed Olig2 immunoreactivity in the majority (ie, 90% of 200 cells characterized in 3 animals) of nestin-GFP+ cells 4 days postlesion (Figure 1F). Thus, most glial cells reacting to injury as monitored here upregulated transcription factor Olig2, prompting analysis of its functional relevance.
Transduction of Pax6 or of a Dominant Negative Form of Olig2 Induces Early Neuronal Characteristics in Proliferating Resident Progenitors
Forty-eight hours postevent, retroviruses (titers: 106 to 107) containing either only GFP (CMMP), the potent neurogenic factor Pax6 and GFP (Pax6-IRES-GFP), or a fusion of Olig2 to the transactivator VP16 and GFP (Olig2VP16) were injected into the ischemic lateral striatum (day 3). The fusion protein of Olig2 with the activator VP16 antagonizes Olig2 function, as Olig2 acts normally as a repressor.1,6 Mice were euthanized on days 10 or 17 after MCAo, and the fate of GFP+ cells was analyzed (Table 1, Figure 2). The majority of GFP+ cells displayed NG2 immunoreactivity. Only a few GFP+ microglia and even fewer astroglial cells were observed after viral transduction. Independent of treatment group, Sox2 expression was rare in GFP+ cells. No DCX+/GFP+ cells were detected in the control condition. However, DCX+ cells emerged both after Olig2VP16 and Pax6 transduction (Table 1, Figure 2).
Table 1. Phenotypes of Virus-Infected Cells
Day
10
17
Vector
CMMP
Olig2
PAX6
CMMP
Olig2
PAX6
For each counting, 100–200 GFP+ cells from at least 3 different animals per condition were analyzed.
GFAP immunoreactivity (%)
4
10
3
7
11
4
Iba1 immunoreactivity (%)
13
16
5
20
11
3
NG2 immunoreactivity (%)
62
56
52
53
54
61
DCX immunoreactivity (%)
0
3
6
0
4
7
Sox2 immunoreactivity (%)
2
4
2
4
2
5
Figure 2. Major phenotypes of GFP+ cells after intralesional injection of retroviral vectors. A, Irrespective of condition and time point investigated, most GFP-labeled cells (green) displayed NG2 immunoreactivity (red). B, The percentage of retrovirus-infected microglia was low (Table 1), thus permitting further electrophysiological analysis of GFP+ macroglia. Insets show cell in boxed area in single channels (GFP: green; microglial antigen Iba1: red) with separate wavelengths. C, Both after Olig2-VP16-IRES-GFP and Pax6-IRES-GFP injection, DCX immunoreactivity (red) was detected in GFP+ cells within the lesion. Image of Olig2-VP16-IRES-GFP–transduced cell at 10 days after MCAo. GFAP: blue. D, GFAP expression (blue) was relatively rare in GFP+ cells. E, Independent of treatment group and time point investigated, stem/progenitor cell marker Sox2 (red) was widely expressed by GFAP+-reactive astrocytes (blue), whereas GFP-transduced cells mostly lacked Sox2 expression. Scale bar (in C) 13 μm in A, 60 μm in B, 15 μm in C, 35 μm in D and 13 μm in E.
Next, we analyzed the physiological properties of GFP+ cells in acute slices (Table 2). We selected for cells with a stable membrane potential between −50 and −85 mV (n=182). The membrane was clamped from the holding potential of −70 mV to hyper- and depolarizing potentials ranging from −170 to 100 mV (50 ms, 10 mV increments). Most GFP+ cells displayed complex membrane properties characterized by voltage-dependent, outwardly-rectifying K+ currents similar to those previously described for cells termed “glial precursors,” NG2 glia, or complex glial cells (Figure 3).11–13 A subset of these complex cells displayed rapidly-activating and inactivating inward currents elicited with depolarization followed by outward currents. The threshold for the activation of this inward current was about −40 mV and the time to peak was about 1 ms, indicating the presence of voltage-gated Na+ channels (Figure 3B). The percentage of cells with Na+ currents was increased after Olig2VP16 or Pax6 transduction.
Table 2. Electrophysiologic Results
Vector
10
17
CMMP
Olig2
PAX6
CMMP
Olig2
PAX6
*n=Number of GFP+ cells analyzed.
†P<0.05 compared with CMMP treatment.
‡P<0.01 compared with CMMP treatment.
Proportions of all transduced cells
n* (number of animals)
16 (5)
38 (7)
39 (6)
18 (6)
38 (5)
33 (6)
Microglia
31%
5%
0%
28%
10%
0%
Passive astrocytes
0%
29%
0%
5%
3%
3%
Complex cells without Na+ current
50%
26%
23%
28%
24%
15%
Complex cells with Na+ current
19%
40%
77%
39%
63%
82%
Characteristics of complex cells with Na+ current
Max. Na+ current Mean±SEM (pA)
168±51
226±38
200±26
125±41
157±22
318±55‡
Membrane Potential Mean±SEM (mV)
−59±2
−55±2
−66±1†
−56±3
−54±2
−66±1†
Membrane Resistance Mean±SEM (MΩ)
167±23
231±89
1927±235‡
393±181
215±36
1983±284‡
Neuronal properties of all transduced cells
Elicited single AP (n)
0% (11)
3% (36)
15% (39)
0% (13)
3% (33)
25% (32)
Spontaneous activity (n)
0% (10)
0% (30)
5% (36)
0% (13)
0% (33)
0% (31)
Figure 3. Most GFP+ cells show complex membrane properties. GFP fluorescence induced by Pax6 vector in cells in acute slices 17 days after MCAo (left). Membrane currents were recorded as described in the text (middle). Insets show beginning of depolarizing-voltage jumps in expanded time scale after correction for passive current components. On the right, the responses of these two cells to current injection (current clamp mode) are displayed. Note that in B as opposed to A, a Na+ current could be elicited by depolarizing voltage steps.
Control-virus infected complex cells failed to generate action potentials on membrane depolarization in current-clamp mode. By contrast, action potentials could be elicited after overexpression of Pax6 and, albeit in fewer cells, also after transduction of Olig2VP16 (current injections for 200 ms). Commonly, we detected a single action potential during the depolarizing pulse. However, in some cases, more than one action potential was observed. To record spontaneous postsynaptic currents, the membrane was clamped at −70 mV, and we analyzed 10 records for 10 seconds. Spontaneous current events were observed in two of 36 cells after Pax6 transduction (Figure 4). In line with immunohistological results, only relatively few microglia were transduced (Figure 5A). Even fewer GFP+ cells displayed passive membrane properties typical of classical astrocytes (Figure 5B).
Figure 4. Single action potentials and spontaneous postsynaptic currents after transduction of Pax6. GFP+ cell recorded 10 days after MCAo is shown on the upper left panel (A). Membrane currents (B) and membrane potential (C) were recorded as described in text. D, sample current traces are displayed from the cell described in A, B, and C, which show spontaneous inward currents while the membrane was clamped at −70 mV.
Figure 5. A subset of GFP+ cells displays membrane characteristics of activated microglia or classical astrocytes. A, Fluorescence image of GFP+ cell (17 days postevent) dialyzed with Alexa Fluor 594 during recording (left). Cell displays current pattern typical of activated microglia with delayed outward rectifying currents in addition to the inwardly rectifying currents with inactivation at hyperpolarizing potentials.28 B, Example of a cell expressing passive, voltage-independent currents characteristic of classical astrocytes 17 days after MCAo.
Discussion
The adult brain displays a narrow capacity for self repair. In vitro studies suggest that non-neurogenic regions may harbor multipotent parenchymal progenitors.14–16 However, neurons are clearly not replaced in most brain regions after injury. We have previously demonstrated that reactive glia characterized by nestin-GFP, NG2, or GFAP-eGFP expression become proliferative and express voltage-gated currents in ischemic striatum.9,11 Here, we show that in parallel, these cells also express Olig2, a key antineurogenic transcription factor implicated in proliferation and glial scar formation.6,17,18 Moreover, antagonizing the repressor function of Olig2 or overexpressing Pax6 by intraparenchymal injection of retroviral vectors permitted a small but significant number of infected cells to adopt an immature neuronal phenotype characterized by DCX expression. Similarly, the percentage of GFP+ cells with Na+ currents was increased by modulation of fate determinants. Furthermore, we were able to elicit single action potentials in GFP+ cells after overexpression of Pax6 and, albeit in fewer cells, also by interfering with Olig2 function. Finally, spontaneous postsynaptic currents were detectable in a small subset of transduced cells after overexpression of Pax6.
Retroviruses were injected at 48 hours after 30 minutes MCAo into the ischemic lateral striatum. In line with an earlier study, we did not detect DCX expression in the progeny of infected cells under control conditions.19 At both time points investigated, the majority of GFP+ progenitors expressed chondroitin sulfate proteoglycan NG2, which is also expressed by nestin-GFP+ cells after MCAo.9 These data are in line with recent findings showing that even after injury, cells labeled by Olig2::CreERTM-mediated recombination generate mostly NG2+ glia, few astrocytes, and no cells colabeling with neuroblast or neuronal antigens.29
Under both treatment conditions, cells capable of eliciting depolarization-induced single action potentials emerged. Importantly, in our earlier stroke studies using nestin-GFP and GFAP-eGFP reporter mice as well as in control virus-infected cells investigated here, we never detected complex cells capable of producing action potentials.9,11 Furthermore, the subgroup of NG2+ cells that generated action potentials and received synaptic input as previously described in white matter showed a particularly high vulnerability to hypoxia/ischemia.20 Taken together, it therefore seems improbable that a similar cell type is both present in lateral striatum under baseline conditions and induced to divide after ischemia. Physiological parameters indicate that DCX+ cells mature toward functional neuronal differentiation. However, we acknowledge that there was no relevant change in physiological characteristics between days 10 and 17 after MCAo. Furthermore, we did not detect virus-infected cells expressing neuronal marker NeuN. Clearly, many factors may represent a major impediment to the generation of fully mature neurons after brain ischemia, possibly the most important of which is chronic neuroinflammation.21
NG2+ O-2A progenitors are capable of firing single action potentials in vitro.22 NG2+ precursors in early postnatal mouse cortex have also been shown to generate a single immature action potential, possibly indicating that these cells are undergoing neuronal differentiation.23 Importantly, early postnatal NG2+ cells can be transformed into neurons under appropriate culture conditions.24 By contrast, NG2+ cells reacting to injury of the adult mammalian cortex do not give rise to multipotent neurospheres.16 The limited neurogenic response observed in a prior study6 and again here, may be caused by the fact that viral vectors predominantly target NG2+ progenitors. When early postnatal cortical astroglia are reprogrammed to adopt a neuronal fate, electrophysiological differentiation into a neuronal phenotype in vitro leads to repetitive spiking behavior after current injection; however they may also pass through a developmental stage characterized by firing single action potentials.25,30
Pax6-transduced cells stand out in displaying significantly more hyperpolarized membrane potentials, higher peak Na+ currents, and higher input resistances as compared with the other groups (Table 2). At the 17-day time point, the percentage of Pax6-IRES-GFP–transduced cells capable of producing an action potential reached 25%. Similarly, newly generated granule neurons in the adult hippocampus show high input resistances and, unlike mature granule neurons that generate trains of action potentials under current clamp, they predominantly fire single action potentials.26,27
In conclusion, our results provide additional proof-of-principle evidence that neurogenesis can be evoked in an injured, non-neurogenic region of adult mammalian central nervous system, such as in the spinal cord31, the cerebral cortex6 and the striatum (this work), and present for the first time functional physiological data on these regenerated neurons.
Acknowledgments
The authors wish to thank Annemarie Bunge for excellent technical assistance.
Sources of Funding
This work was supported by the VolkswagenStiftung (Lichtenberg Program to M.E.), Deutsche Forschungsgemeinschaft (SFB TR 43 to M.E.; SFBs 596 and 870 to M.G.), Bundesministerium für Bildung und Forschung (Center for Stroke Research Berlin; M.E.; Cell-Based Regenerative Strategies, Stem Cells in Reactive Gliosis, M.G.) and the European Union (ARISE and Eustroke, M.E.; EUTRACC, M.G.).
Disclosures
None.
Footnote
*G.K. and K.G. contributed equally to this work.
References
1.
Hack MA, Sugimori M, Lundberg C, Nakafuku M, Götz M. Regionalization and fate specification in neurospheres: the role of Olig2 and Pax6. Mol Cell Neurosci. 2004; 25: 664–678.
Hack MA, Saghatelyan A, de Chevigny A, Pfeifer A, Ashery-Padan R, Lledo PM, Götz M. Neuronal fate determinants of adult olfactory bulb neurogenesis. Nat Neurosci. 2005; 8: 865–872.
Colak D, Mori T, Brill MS, Pfeifer A, Falk S, Deng C, Monteiro R, Mummery C, Sommer L, Götz M. Adult neurogenesis requires Smad4-mediated bone morphogenic protein signaling in stem cells. J Neurosci. 2008; 28: 434–446.
Brill MS, Snapyan M, Wohlfrom H, Ninkovic J, Jawerka M, Mastick GS, Ashery-Padan R, Saghatelyan A, Berninger B, Götz M. A dlx2- and pax6-dependent transcriptional code for periglomerular neuron specification in the adult olfactory bulb. J Neurosci. 2008; 28: 6439–6452.
Fancy SP, Zhao C, Franklin RJ. Increased expression of Nkx2.2 and Olig2 identifies reactive oligodendrocyte progenitor cells responding to demyelination in the adult CNS. Mol Cell Neurosci. 2005; 27: 247–254.
Buffo A, Vosko MR, Ertürk D, Hamann GF, Jucker M, Rowitch D, Götz M. Expression pattern of the transcription factor Olig2 in response to brain injuries: implications for neuronal repair. Proc Natl Acad Sci USA. 2005; 102: 18183–18188.
Sontheimer H, Trotter J, Schachner M, Kettenmann H. Channel expression correlates with differentiation stage during the development of oligodendrocytes from their precursor cells in culture. Neuron. 1989; 2: 1135–1145.
Palmer TD, Markakis EA, Willhoite AR, Safar F, Gage FH. Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci. 1999; 19: 8487–8497.
Buffo A, Rite I, Tripathi P, Lepier A, Colak D, Horn AP, Mori T, Götz M. Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain. Proc Natl Acad Sci USA. 2008; 105: 3581–3586.
Chen Y, Miles DK, Hoang T, Shi J, Hurlock E, Kernie SG, Lu QR. The basic helix-loop-helix transcription factor olig2 is critical for reactive astrocyte proliferation after cortical injury. J Neurosci. 2008; 28: 10983–10989.
Yamashita T, Ninomiya M, Hernández Acosta P, García-Verdugo JM, Sunabori T, Sakaguchi M, Adachi K, Kojima T, Hirota Y, Kawase T, Araki N, Abe K, Okano H, Sawamoto K. Subventricular zone-derived neuroblasts migrate and differentiate into mature neurons in the post-stroke adult striatum. J Neurosci. 2007; 26: 6627–6636.
Káradóttir R, Hamilton NB, Bakiri Y, Attwell D. Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter. Nat Neurosci. 2008; 11: 450–456.
Chittajallu R, Aguirre A, Gallo V. NG2-positive cells in the mouse white and grey matter display distinct physiological properties. J Physiol. 2004; 561: 109–122.
Berninger B, Costa MR, Koch U, Schroeder T, Sutor B, Grothe B, Götz M. Functional properties of neurons derived from in vitro reprogrammed postnatal astroglia. J Neurosci. 2007; 27: 8654–8664.
Aguirre AA, Chittajallu R, Belachew S, Gallo V. NG2-expressing cells in the subventricular zone are type C-like cells and contribute to interneuron generation in the postnatal hippocampus. J Cell Biol. 2004; 165: 575–589.
Schmidt-Hieber C, Jonas P, Bischofberger J. Enhanced synaptic plasticity in newly generated granule cells of the adult hippocampus. Nature. 2004; 429: 184–187.
Lyons SA, Pastor A, Ohlemeyer C, Kann O, Wiegand F, Prass K, Knapp F, Kettenmann H, Dirnagl U. Distinct physiologic properties of microglia and blood-borne cells in rat brain slices after permanent middle cerebral artery occlusion. J Cereb Blood Flow Metab. 2000; 20: 1537–1549.
Dimou L, Simon C, Kirchhoff F, Takebayashi H, Götz M. Progeny of Olig2-expressing progenitors in the gray and white matter of the adult mouse cerebral cortex. J Neurosci. 2008; 28: 10434–10442.
Ohori Y, Yamamoto S, Nagao M, Sugimori M, Yamamoto N, Nakamura K, Nakafuku M. Growth factor treatment and genetic manipulation stimulate neurogenesis and oligodendrogenesis by endogenous neural progenitors in the injured adult spinal cord. J Neurosci. 2006; 26: 11948–11960.
eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.
Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.
From the Klinik and Poliklinik für Neurologie (G.K., K.G., M.E.), the Klinik und Hochschulambulanz für Psychiatrie und Psychotherapie (G.K.), and the Center for Stroke Research, (G.K., K.G., M.E.), Charité Universitätsmedizin Berlin, Berlin, Germany; Cellular Neurosciences (G.C., H.K.), Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany; Department of Physiological Genomics (A.B., M.G.), Institute of Physiology, Ludwig-Maximilians University, Munich, Germany; Institute for Stem Cell Research (A.B., M.G.), HelmholtzZentrum Munich, Germany.
From the Klinik and Poliklinik für Neurologie (G.K., K.G., M.E.), the Klinik und Hochschulambulanz für Psychiatrie und Psychotherapie (G.K.), and the Center for Stroke Research, (G.K., K.G., M.E.), Charité Universitätsmedizin Berlin, Berlin, Germany; Cellular Neurosciences (G.C., H.K.), Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany; Department of Physiological Genomics (A.B., M.G.), Institute of Physiology, Ludwig-Maximilians University, Munich, Germany; Institute for Stem Cell Research (A.B., M.G.), HelmholtzZentrum Munich, Germany.
From the Klinik and Poliklinik für Neurologie (G.K., K.G., M.E.), the Klinik und Hochschulambulanz für Psychiatrie und Psychotherapie (G.K.), and the Center for Stroke Research, (G.K., K.G., M.E.), Charité Universitätsmedizin Berlin, Berlin, Germany; Cellular Neurosciences (G.C., H.K.), Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany; Department of Physiological Genomics (A.B., M.G.), Institute of Physiology, Ludwig-Maximilians University, Munich, Germany; Institute for Stem Cell Research (A.B., M.G.), HelmholtzZentrum Munich, Germany.
From the Klinik and Poliklinik für Neurologie (G.K., K.G., M.E.), the Klinik und Hochschulambulanz für Psychiatrie und Psychotherapie (G.K.), and the Center for Stroke Research, (G.K., K.G., M.E.), Charité Universitätsmedizin Berlin, Berlin, Germany; Cellular Neurosciences (G.C., H.K.), Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany; Department of Physiological Genomics (A.B., M.G.), Institute of Physiology, Ludwig-Maximilians University, Munich, Germany; Institute for Stem Cell Research (A.B., M.G.), HelmholtzZentrum Munich, Germany.
From the Klinik and Poliklinik für Neurologie (G.K., K.G., M.E.), the Klinik und Hochschulambulanz für Psychiatrie und Psychotherapie (G.K.), and the Center for Stroke Research, (G.K., K.G., M.E.), Charité Universitätsmedizin Berlin, Berlin, Germany; Cellular Neurosciences (G.C., H.K.), Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany; Department of Physiological Genomics (A.B., M.G.), Institute of Physiology, Ludwig-Maximilians University, Munich, Germany; Institute for Stem Cell Research (A.B., M.G.), HelmholtzZentrum Munich, Germany.
From the Klinik and Poliklinik für Neurologie (G.K., K.G., M.E.), the Klinik und Hochschulambulanz für Psychiatrie und Psychotherapie (G.K.), and the Center for Stroke Research, (G.K., K.G., M.E.), Charité Universitätsmedizin Berlin, Berlin, Germany; Cellular Neurosciences (G.C., H.K.), Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany; Department of Physiological Genomics (A.B., M.G.), Institute of Physiology, Ludwig-Maximilians University, Munich, Germany; Institute for Stem Cell Research (A.B., M.G.), HelmholtzZentrum Munich, Germany.
From the Klinik and Poliklinik für Neurologie (G.K., K.G., M.E.), the Klinik und Hochschulambulanz für Psychiatrie und Psychotherapie (G.K.), and the Center for Stroke Research, (G.K., K.G., M.E.), Charité Universitätsmedizin Berlin, Berlin, Germany; Cellular Neurosciences (G.C., H.K.), Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany; Department of Physiological Genomics (A.B., M.G.), Institute of Physiology, Ludwig-Maximilians University, Munich, Germany; Institute for Stem Cell Research (A.B., M.G.), HelmholtzZentrum Munich, Germany.
Correspondence to Matthias Endres, MD, Klinik und Poliklinik für Neurologie, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany. E-mail [email protected]
Metrics & Citations
Metrics
Citations
Download Citations
If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Select your manager software from the list below and click Download.
Lina Hao,
Hongmei Jia,
Feifei Wei,
Junbo Zhang,
Jian Zhang,
Chunying Guo,
Liying Wang,
CDC42 Regulates the ERK Pathway to Improve Oxygen‒Glucose Deprivation/Reoxygenation-Induced Neural Oxidative Stress and Apoptosis, Molecular Neurobiology, (2025).https://doi.org/10.1007/s12035-025-04768-x
Molecular mechanism of METTL14-mediated m6A modification regulating microglial function post ischemic stroke, Brain Research Bulletin, 220, (111156), (2025).https://doi.org/10.1016/j.brainresbull.2024.111156
Vascular endothelial growth factor promotes transdifferentiation of astrocytes into neurons via activation of the
MAPK
/
Erk‐Pax6
signal pathway
, Glia, 71, 7, (1648-1666), (2023).https://doi.org/10.1002/glia.24361
Pregnenolone enhances the proliferation of mouse neural stem cells and promotes oligodendrogenesis, together with Sox10, and neurogenesis, along with Notch1 and Pax6, Neurochemistry International, 163, (105489), (2023).https://doi.org/10.1016/j.neuint.2023.105489
Identification of adeno-associated virus variants for gene transfer into human neural cell types by parallel capsid screening, Scientific Reports, 12, 1, (2022).https://doi.org/10.1038/s41598-022-12404-0
Ghrelin Regulates Expression of the Transcription Factor Pax6 in Hypoxic Brain Progenitor Cells and Neurons, Cells, 11, 5, (782), (2022).https://doi.org/10.3390/cells11050782
Region-specific distribution of Olig2-expressing astrocytes in adult mouse brain and spinal cord, Molecular Brain, 14, 1, (2021).https://doi.org/10.1186/s13041-021-00747-0
Modulation of Fate Determinants Olig2 and Pax6 in Resident Glia Evokes Spiking Neuroblasts in a Model of Mild Brain Ischemia
Stroke
Vol. 41
No. 12
Purchase access to this journal for 24 hours
Stroke
Vol. 41
No. 12
Restore your content access
Note: This functionality works only for purchases done as a guest. If you already have an account, log in to access the content to which you are entitled.
eLetters(0)
eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.
Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.