Cell cycle restriction by histone H2AX limits proliferation of adult neural stem cells

Abstract

Adult neural stem cell proliferation is dynamic and has the potential for massive self-renewal yet undergoes limited cell division in vivo. Here, we report an epigenetic mechanism regulating proliferation and self-renewal. The recruitment of the PI3K-related kinase signaling pathway and histone H2AX phosphorylation following GABA_A receptor activation limits subventricular zone proliferation. As a result, NSC self-renewal and niche size is dynamic and can be directly modulated in both directions pharmacologically or by genetically targeting H2AX activation. Surprisingly, changes in proliferation have long-lasting consequences on stem cell numbers, niche size, and neuronal output. These results establish a mechanism that continuously limits proliferation and demonstrates its impact on adult neurogenesis. Such homeostatic suppression of NSC proliferation may contribute to the limited self-repair capacity of the damaged brain.

Neural stem cells (NSCs) reside in discrete germinal regions of the adult brain and self-renew to generate neurons throughout life. Within the largest neurogenic region, the subventricular zone (SVZ) of the lateral ventricles, NSCs (also referred to as type B cells) are localized to a germinal zone that also contains progeny cells with more restricted potential. Type B cells display ultrastructural and molecular characteristics of astroglial cells, including the expression of GFAP and GLAST (1, 2). The lineage from NSCs to neurons includes the immediate progeny, referred to as transit amplifying cells, which in turn generate young neurons, neuroblasts, that migrate to the olfactory bulb (OB), where they integrate as local interneurons (3). Following damage to the brain, such as stroke, SVZ NSCs have also been shown to migrate to the site of injury, to differentiate into neurons and functionally integrate into the brain parenchyma (4, 5).

Adult neurogenesis is dynamically regulated throughout life and is affected by aging, environmental challenges, exercise, stress, and neuropathological conditions (6, 7). The net extent of neurogenesis is the result of several processes including stem cell proliferation, differentiation, and the survival of newly generated cells. Consistently, the number of neurons migrating to selected brain regions, differentiating into neurons, and integrating into normal neuronal circuitry is correlated to, and possibly limited by, SVZ proliferation (8). Because NSCs are relatively quiescent whereas progenitor cells are actively proliferating, a small increase in stem cell numbers might be sufficient to lead to large differences in neuronal output. Elevated neurogenesis during neuropathological conditions is associated with enhanced stem and progenitor self-renewal (4, 8), and furthermore, depletion of the transit amplifying progenitors and neuroblasts in the SVZ leads to a marked increase in stem cell proliferation (9), suggesting that the relatively slow endogenous proliferation of adult NSCs is controlled by a homeostatic mechanism such that, when there are few stem and progenitor cells in the niche, they proliferate faster than if there are many cells. Hence, adult NCSs are endowed with enhanced self-renewal and cellular output under certain circumstances, possibly as a result of disinhibition of stem cell self-renewal.

GABA signaling represents one possible mechanism that regulates adult neurogenesis. Depolarizing neuroblasts in the SVZ stem cell niche nonsynaptically activate GABA_A receptors (GABA_ARs) in GFAP⁺ SVZ cells. The application of GABA_AR antagonist to slices containing the SVZ for 18 h leads to an increase in proliferating cells as visualized by BrdU incorporation (10). Surprisingly, GABA has also been shown to induce an epigenetic signal by PI3 kinase-related kinases (PIKKs) ATM- and ATR-dependent phosphorylation of the histone variant H2AX at Ser-139 (γH2AX), resulting in a reduced rate of proliferation in embryonic stem cells (11). Here, we asked whether a ligand-activated epigenetic mark of histone H2AX plays a critical role in stem cell self-renewal, niche size, and neurogenesis in the adult brain.

Results

SVZ NSCs Express Components Required to Respond to GABA and Display GABA_AR-Dependent Proliferation via H2AX in Vitro.

We developed a simple strategy using GFAP-GFP transgenic mice to identify SVZ cell types as previously described, based on microdissecting the SVZ and amplifying and analyzing primary neurospheres in vitro, which allowed identification of GFAP⁺ relatively quiescent stem cell clones (GFP^high) versus transit amplifying cell clones (GFP⁻) (12). Consistent with adult NSCs, GFP^high neurospheres appearing after 4 d in SVZ cultures expressed S100β, Sox2, SSEA1, and nestin (Fig. S1A). mRNA of all necessary components for functional GABA_ARs were found in GFP^high neurospheres, including at least one α, β, and γ subunit, with the β3 subunit being the predominant β subunit present, compared with the broader expression pattern seen in GFP⁻ neurospheres (Fig. S1B). mRNAs for the GABA synthesizing enzymes GAD65 and GAD67 were also detected (Fig. S1B), and immunohistochemical staining confirmed the expression of GAD65/67, GABA_AR β3, and GABA in these primary GFP^high neurospheres (Fig. S1C).

We next tested if activation or inhibition of GABA_AR signaling, by using the receptor agonist muscimol or antagonist bicuculline respectively, could have direct effects on proliferation of primary GFP⁺ NSCs. Dissociated cells from microdissected SVZs were placed in culture and stem cell clones displayed GABA_AR-dependent incorporation of the BrdU analogue 5-ethynyl-2′deoxyuridine (EdU) along with opposing, but also GABA_AR-dependent, regulation of H2AX phosphorylation (Fig. 1A). The number of dividing cells was markedly affected in SVZ clones by GABA_AR signaling following acute (2.5 h) treatment, such that muscimol decreased and bicuculline increased numbers of dividing cells (Fig. 1B). This effect was dependent on H2AX, as it was completely abolished in H2AX-deficient cells established from H2AX-null mutant mice (Fig. 1B). Each SVZ generated an average of 241 ± 49 (SD) stem cell clones, and GABA_AR activation did not have any effect on maintenance of multipotency or differentiation, as a similar number of clones appeared when SVZ cells were grown in the presence of muscimol (247 ± 57; Fig. S1D). However, muscimol (4 d) led to a marked reduction of clone size (Fig. S1E), suggesting a sustained suppression of proliferation. Taken together, these in vitro studies demonstrate that NSC numbers can be modulated in both directions by GABA_AR signaling in a manner dependent on the presence of histone H2AX.

Fig. 1.

Induction of H2AX phosphorylation in NSCs and the SVZ following GABA_AR signaling activation. (A) γH2AX immunostaining and EdU incorporation (30 min) in control (Ctr)-, muscimol (Mus)-, and bicuculline (Bic)-treated clones (2.5 h). (B) EdU incorporation (30 min) in clones grown from H2AX WT and H2AX-null mutant (KO) mice following single acute (2.5 h) treatment (n = 4). (C) γH2AX immunohistochemistry in the SVZ following acute (1.5 h) i.p. muscimol or bicuculline treatment. (D) γH2AX immunohistochemistry in cells positive for GFAP (arrows) or Tuj1 (arrowheads) in SVZ following acute (1.5 h) treatment. LV, lateral ventricle; CPu, caudate putamen striatum. (Scale bars: A, 20 μm; C, 50 μm; D, 10 μm.)

H2AX Is Necessary for GABA_AR-Dependent Modulation of Proliferation of NCSs and Progenitors Within the SVZ Niche.

To investigate the role of GABA_AR signaling via histone H2AX modifications in vivo, muscimol and bicuculline were administered acutely (1.5 h) to adult mice followed by immunohistochemistry or SVZ microdissection for Western blot analysis. Muscimol rapidly increased γH2AX immunohistochemistry in cells adjacent to the lateral wall of the lateral ventricle but was not significantly noted in neurons or glia in other regions of the brain (Fig. 1C). Western blot analysis confirmed that muscimol increased γH2AX levels and ATM/ATR substrate phosphorylation—the kinases that phosphorylate H2AX (13)—an effect specifically seen in the SVZ niche, as no effects were seen in striatum/caudate putamen (Fig. S2). Muscimol-induced H2AX phosphorylation (γH2AX) in the SVZ was localized to some GFAP⁺ SVZ cells as well as some tuj1⁺ neuroblasts (Fig. 1D). Thus, activation of GABA_AR induces kinases of the DNA damage response pathway as well as phosphorylation of H2AX specifically in cells of the SVZ niche, demonstrating the potential for a unique epigenetic, H2AX-dependent mechanism of proliferation regulation in this cell population.

If indeed H2AX represents an endogenous mechanism of tonic inhibition of stem cell proliferation, then in H2AX-deficient mice, there should be an overall increase of Ki67⁺ mitotically active cells (14) and an increase of BrdU incorporating cells in the SVZ. In accordance with this expectation, an increase in Ki67⁺ and BrdU⁺ cells per SVZ was observed in H2AX-deficient mice (Fig. 2 A and B). We then examined if the effects of this epigenetic mechanism can be acutely enhanced or reduced in vivo by examining the putative role of GABA_AR signaling via H2AX modification on proliferation in the SVZ by using BrdU (2 h) incorporation, in conjunction with acute (2.5 h) administration of GABA_AR antagonist and agonist. BrdU incorporation in the SVZ was significantly decreased by muscimol and increased by bicuculline (Fig. 2 C and D). The requirement of H2AX for GABA_AR regulation of SVZ proliferation was confirmed in H2AX-deficient mice, as the effect of agonist and antagonist was abolished in mice lacking H2AX (Fig. 2 E and F). We also addressed which cell types in the niche respond to GABA_AR signaling. Examination of in vivo effects of muscimol and bicuculline on adult GFAP⁺ SVZ stem cells revealed a rapid reduction (approximately twofold) or increase of BrdU⁺GFAP⁺ double-labeled cells identified by confocal microscopy stack analysis following muscimol and bicuculline, respectively (Fig. 2G and Fig. S3A). BrdU incorporation overall was significantly affected in the SVZ and comparison of the number of BrdU⁺GFAP⁺ double-positive cells to the total number of BrdU⁺ cells per SVZ revealed that the effects were not limited to GFAP⁺ stem cells (Fig. S3B), indicating effects also on type C transit amplifying progenitors. Similar results were also achieved by analyzing BrdU⁺GFP⁺ nuclei from GFAP-GFP transgenic mice in which double-positive nuclei (Fig. S3C) were quantified per SVZ following acute (2.5 h) muscimol or bicuculline treatment in conjunction with a BrdU pulse (2 h). The results revealed a decreased or increased number of BrdU⁺GFP⁺ cells in the SVZ, respectively (Fig. 2H and Fig. S3D). Hence, these data are consistent with the in vitro effects on NSCs and suggest a physiological role of H2AX phosphorylation for regulating stem cell proliferation in the adult SVZ niche. Taken together, these findings support the conjecture of an endogenous restriction of proliferation in the SVZ niche mediated by modifications of histone H2AX, which is established by GABA_AR signaling in the adult brain.

Fig. 2.

SVZ NSCs display H2AX-dependent proliferation, which can be modulated in response to GABA_AR signaling. (A) Quantification of Ki67⁺ cells in H2AX WT and KO mice (n = 6). (B) Number of BrdU⁺ cells in H2AX WT and KO mice following 2 h BrdU pulse (n = 4). (C) Representative WT SVZ segments following acute muscimol or bicuculline treatment (2.5 h) with BrdU (2 h); arrows indicate some BrdU⁺Ki67⁺ cells. (D) Quantification of SVZ BrdU LI (percentage BrdU⁺ normalized to Ki67⁺ per SVZ section) following acute treatments in WT (n = 6–7). (E) Representative H2AX KO SVZ segments following acute (2.5 h) muscimol or bicuculline treatment with BrdU (2 h); arrows indicate some BrdU⁺Ki67⁺ cells. (F) Quantification of SVZ BrdU LI following acute treatments in H2AX KO (n = 4). (G) Quantification of SVZ BrdU⁺GFAP⁺ cells following acute muscimol or bicuculline treatment with BrdU (n = 4). (H) Confocal stacks of BrdU immunohistochemistry in SVZ of GFAP-GFP mice following treatment. Arrowheads indicate GFP⁺BrdU⁺ nuclei. LV, lateral ventricle. (Scale bars: C and E, 20 μm; H, 10 μm.)

Long-Term Modulation of GABA_AR Signaling Has Consequences for NSC Numbers and Niche Size.

We next wanted to examine if sustained pharmacological modulation in vivo over a period of 4 d still affects proliferation and whether this results in changes in stem cell numbers and niche size (i.e., self-renewal). For this purpose, we administered muscimol or bicuculline at doses lower than those that lead to overt behavioral response during the 4-d period (Fig. 3A). Ki67 labeling following continuous muscimol or bicuculline treatment was significantly decreased or increased in the SVZ, respectively (Fig. 3B and Fig. S4A). This effect was largely dependent on H2AX, as only minor effects were seen in H2AX-deficient mice (Fig. 3B). Bicuculline induced corresponding changes of cells in G2/M marked by phosphorylated histone H3 (pHis3) compared with muscimol treatment (Fig. S4B), as well as an increase in the number of migrating neuroblasts as seen by an increase of the number of doublecortin⁺ (DCX⁺) cells 4 d after continuous bicuculline compared with control and muscimol treatment (Fig. S4 C and D). An increase in NSC numbers and niche size as a result of increased proliferation throughout the 4-d period was suggested by an increase and decrease in SVZ GFAP immunoreactivity following bicuculline and muscimol administration, respectively (Fig. 3 C and D). To directly examine the total pool of the NSC population, we genetically labeled NSCs by using GLAST-CreERT2;CAG-GFP mice (15, 16). Treatment with bicuculline for 8 d was combined with 5 d tamoxifen administration (Fig. 3E), and the number of GFP⁺ cells (i.e., total NSC pool) as well as GFP⁺Ki67⁺ cells (i.e., proliferating NSCs) in the SVZ were increased 4 wk later (Fig. 3 F and G and Fig. S4E). These data suggest that the increased proliferation of NSCs in the SVZ during continuous treatment leads to an increase in the number of NSCs and niche size.

Fig. 3.

Long-term modulation of GABA_AR signaling has consequences for proliferation and numbers of NSCs. (A) Timeline for chronic treatment. (B) Quantification of Ki67⁺ cells per SVZ from chronically treated H2AX WT and KO mice (n = 3–5). (C) Representative confocal stack of GFAP immunostaining in SVZs of 4-d treated mice. TOPRO-3 marks nuclei. (D) Quantification of GFAP immunoreactivity by integrated density analysis following chronic treatments (n = 4). (E) Timeline showing experimental design using GLASTcreERT2;CAG-GFP mice in F and G (Tx, tamoxifen). (F) Immunohistochemistry of GFP and Ki67 in a representative segment of SVZ at 36 d. Arrows indicate some GFP⁺Ki67⁺ cells. (G) Quantification of GFP⁺ cells per SVZ (n = 3–4). (H) Schematic timeline showing experimental design for I–K. (I) Quantification of BrdU⁺ LRCs at 33 d (n = 4). (J) Immunohistochemistry of OB BrdU⁺ and NeuN⁺ neurons at 33 d in the GCL. (K) Quantification of OB BrdU⁺ cells in at 33 d (n = 4). (Scale bars: C and F, 20 μm; J, 100 μm.)

Increases of SVZ NSCs by Long-Term Interference of GABA_AR Signaling Results in Sustained Effects on NSC Numbers and Neuronal Output.

A number of studies have indicated that stem cells can be identified within the SVZ as a result of their ability to retain BrdU as a result of their slow rate of cell division, and that the differentiated BrdU labeled progenies migrate out of the SVZ (9, 17–20). To examine long-term changes, we staggered chronic treatment (8 d agonist/antagonist) with an 8-d BrdU pulse with a 4-d overlap in between, and analyzed such label-retaining cells (LRCs) 3 wk later (Fig. 3H). Muscimol led to a marked and persistent reduction of LRCs whereas bicuculline caused a significant increase in LRCs that persisted to 3 wk after the end of the treatment (Fig. 3I). To address whether this increase represented a recruitment of quiescent stem cells, we administered BrdU and treatment sequentially for 8 d each, followed by a 3-wk chase period without any treatment (Fig. S5A). We then examined the ability of GABA_AR to lead to long-term changes in the number of prelabeled LRC cells in the niche. LRCs significantly increased 3 wk after termination of bicuculline treatment compared with muscimol treatment (Fig. S5B). The effect of bicuculline was comparable to the effect on LRCs in the staggered protocol (Fig. 3 H and I), suggesting that bicuculline primarily increases the rate of division of already cycling stem cells with limited recruitment of quiescent stem cells. This conclusion was confirmed by quantifying mitotically active (i.e., Ki67⁺) and mitotically inactive (i.e., quiescent, Ki67⁻) LRCs in the staggered experimental paradigm (Fig. S5C). The proportional changes in these populations were consistent following muscimol or bicuculline treatment despite the total number of NSCs being decreased or increased, respectively (Fig. S5 D and E). Thus, our data suggest that GABA_AR signaling increases the rate of self-renewal, resulting in long-term changes of stem cell numbers of the SVZ niche extending well beyond the end of the treatment period.

SVZ progenitors differentiate into neuroblasts, which migrate through the rostral migratory stream to the OB and differentiate into neurons (3). We tested whether the persistent changes of stem cell numbers with agonist and antagonist leads to changes in cellular output from the SVZ to the OB. Following 8 d treatment and 3 wk chase (Fig. 3H), the resulting number of BrdU⁺ cells was analyzed in the granule cell layer (GCL) of the OB. Muscimol decreased, whereas bicuculline increased, neuronal output (Fig. 3 J and K and Fig. S5F), correlating in magnitude with the effects seen on LRCs. Because BrdU also labels transit amplifying cells, we next examined if the effects on neurogenesis are a direct consequence of effects specifically on SVZ NSCs cells. For this purpose, we examined the OB of the treated GLAST-CreERT2;CAG-GFP mice (Fig. 3E; control experiment in Fig. S6). Quantification of both GFP⁺ and GFP⁺NeuN⁺ cells in the GCL of the OB revealed a marked increase following bicuculline treatment (Fig. 4 A–C and Fig. S7 A and B). This increase in GFP⁺NeuN⁺ cells with bicuculline (Fig. 4C) is comparable to the increase seen in the staggered protocol (Fig. 3K), confirming that the increased neurogenesis is primarily a result of effects of bicuculline acting on putative NSCs, rather than transit amplifying cells. These results also confirm a direct relation between stem cell niche size and neuronal output and that the extent of neurogenesis in the adult brain can be modulated by targeting the identified signaling pathway in the present study.

Fig. 4.

H2AX-dependent changes in niche size results in sustained effects on NSC numbers and neuronal output. (A) Timeline showing experimental design for lineage tracing using GLASTcreERT2;CAG-GFP mice in B and C. (B) Representative immunohistochemistry of OB GFP⁺NeuN⁺ at 36 d in the GCL. (C) Quantification of OB GCL GFP⁺NeuN⁺ cells at 36 d (n = 3–4). (D) Timeline showing experimental design for BrdU chase experiment in H2AX-null mice in E–G. (E) LRCs in H2AX WT and KO mice at 29 d (n = 8). (F) Representative immunohistochemistry of OB GCL BrdU⁺ and NeuN⁺ neurons of H2AX WT and KO at 29 d. (G) Quantification of BrdU⁺ cells in OB GCL of H2AX WT versus KO mice at 29 d (n = 4). (Scale bars, 100 μm.)

Suppression of NSC Self-Renewal by H2AX in Vivo Is a Limiting Factor for Neurogenesis.

Finally, we examined the physiological role of H2AX on NSC numbers and neuronal output. Consistent with the increased proliferative capacity in mice lacking H2AX by BrdU and Ki67 immunohistochemistry (Fig. 2 A and B), we found that administration of BrdU for 8 d followed by a 3-wk chase period without BrdU (Fig. 4D) leads to significantly increased number of LRCs in the absence of H2AX (Fig. 4E). Furthermore, we also found significantly more BrdU-labeled cells in the OB (Fig. 4 F and G and Fig. S8). These results demonstrate a physiological role of H2AX in a mechanism that restricts LRC numbers, which may have direct consequences on neurogenesis and neuronal output.

Discussion

NSCs are maintained by self-renewal throughout life in the adult brain, but the precise molecular mechanisms regulating stem cell pool size have not been fully elucidated. The stem cell niche is believed to be reserved as a specialized local microenvironment that has greater potential for self-renewal than is seen in the unchallenged brain. Neurogenesis in the adult brain actively contributes to odor discrimination and cognitive performance, and the age-related declines of these performances correlate with decreases in neurogenesis (21, 22). It is therefore critical to understand the mechanism controlling NSC numbers and its relation to neurogenesis and integration of new neurons. Our data show that the H2AX signaling pathway represents one mechanism restricting NSC proliferation under physiological conditions. When it has been disinhibited genetically or pharmacologically, larger stem cell numbers and increased niche size with greater cellular output is observed.

Previous work has demonstrated that the multipotent cell identity is maintained by suppression of lineage commitment to neuronal or glial fates, thereby preventing a premature depletion of the stem cell pool size (23–27). Accordingly, several diffusible and membrane-attached factors that regulate stem cell numbers have been identified (28). Most notably, Notch signaling regulates stem cell numbers (29) and can increase proliferation in the SVZ by maintaining expression of the multi-/pluripotency transcription factor Sox2, which regulates expression of Sonic hedgehog, which is required for survival and normal proliferation (29–32). Although activation of Notch modulates cell cycle time (33), it also leads to repression of proneural gene expression and maintenance, specifically of the NSC (34–36). In keeping, enhanced Notch signaling by the vascular niche factor pigment epithelium-derived factor diverts asymmetrical division to production of two self-renewing NSCs in the adult brain (37). An absence of Notch signaling in mice results in a transient increase of proliferation and subsequent depletion caused by premature differentiation into neurons (38). By using several different approaches, we show that epigenetic modification of histone H2AX also participates in determining NSC proliferation in a ligand-dependent manner. In these experiments, GABA_AR activation leads to activation of ATM/ATR PIKKs and phosphorylation of H2AX in the SVZ. Although between 1% and 15% of the cellular histone H2A is represented by the variant histone H2AX, activation of H2AX was selectively observed in the SVZ NSCs and its immediate progenitors in the SVZ niche.

The effects of GABA_AR activation on stem cell proliferation are dependent on H2AX given that the effects were abolished in vitro and in vivo in H2AX-deficient mice. Furthermore, the increased number of proliferating NSCs as well as LRCs in histone H2AX-deficient mice places this signaling pathway as a physiological regulator. This conclusion is consistent with robust short- as well as long-term effects on NSCs by pharmacologically blocking endogenous GABA_AR activation. It is notable that the increased neuronal output observed in this study does not seem to be paralleled by a depletion of the stem cells. On the contrary, our data unexpectedly support that it appears as a consequence of increased stem cell niche size, and hence may represent a unique mechanism from previously studied processes regulating NSC proliferation. This conclusion is based on the observed increase of GFAP immunoreactivity, analysis of cell numbers labeled in GLAST-CreERT2;CAG-GFP mice following treatment, and the increased number of proliferating cells in H2AX-null mutant mice. However, identification of new unique markers that would allow independent quantification of the NSC population would be desirable to validate this controversial conclusion.

Our results provide evidence of several unexpected general properties of the adult NSC germinal zone. First, the data on BrdU labeling of NSCs and transit amplifying cells as well as GLAST-CreERT2;CAG-GFP genetic cell tracing labeling only the GFAP⁺ NSC population proposes that changes in NSC pool size have direct consequences on neuronal output. Hence, although deposition of new neurons into the brain also depends on survival of newborn progenies and differentiation of neurons, an increase in stem cell numbers is sufficient in itself to increase neuronal output, at least in the normal young undamaged brain. Second, the NSC pool is subjected to a continuous dynamic regulation. We find that this regulation involves a restriction in stem cell niche size by epigenetic mechanisms limiting the rate of proliferation, which has consequences on self-renewal. GABA can be released via phasic release through Ca²⁺-dependent vesicular exocytosis from neurons and by tonic release, independent of action potentials and vesicles, by permeation through the Bestrophin 1 anion channel in neurons and glia (39). Neuroblasts within the germinal zone release GABA tonically, which acts on GFAP⁺ progenitors in the SVZ, negatively affecting proliferation (10, 40, 41). Bestrophin 1 gates GABA already at very low (i.e., resting) intracellular Ca²⁺ concentrations (100 nM) but increases release with increasing Ca²⁺ concentrations (39). Therefore, the concentration of ambient GABA in the SVZ could directly reflect cell numbers (i.e., at resting membrane, the tonic release would result in a homeostatic control of SVZ niche size) but would also be greatly affected by altering intracellular Ca²⁺ concentration in cell populations releasing GABA. Hence, a GABA-dependent homeostatic feedback inhibition controlling self-renewal and stem cell niche size in the adult brain and its disinhibition of the epigenetic marks of histone H2AX may contribute to the observed elevation of NSC proliferation and fast repopulation of immediate progenitors and neuroblasts from NSCs following their depletion (19).

Another unexpected finding is that H2AX-dependent mechanisms also lead to long-term alterations in NSC proliferation and neuronal output. Administration of GABA_AR agonist or antagonist over a period of several days resulted in sustained changes for several weeks after the end of treatment, as seen in genetically marked NSCs (i.e., LRCs) in GFAP immunoreactivity and in the number of pHis3⁺, Ki67⁺, and DCX⁺ cells. Therefore, the decreases seen following agonist treatment and increases following antagonist treatment after an 8-d treatment seem not to return to normal levels after 3 wk of recovery. The long-term effect of the H2AX signaling pathway on NSC proliferation and neuronal output could be a result of the slow cell division, and limited differentiation, of NSCs and the resulting delayed adjustment of cell numbers. Nevertheless, the clear increase in NSC numbers several weeks after the end of treatment could be a consequence of the nature of the signaling pathway. Activation of H2AX by DNA damage, such as double strand breaks or replication fork stalling, triggers temporary cell cycle arrest, but enforced DNA damage response activation can also lead to a state of irreversible growth arrest, i.e., replicative cellular senescence, at least during transformation induced by oncogene activation (42, 43). The long-lasting effect on NSCs observed in the present study is particularly relevant given that the GABA_AR is the pharmacological target for many drugs used clinically to treat, for example, anxiety disorders and epilepsy. Furthermore, the new-generation hypnotic drugs including zopiclone are commonly used as treatment for insomnia to promote sleep by enhancing the GABA_AR activity (44). Our results highlight important implications of therapeutic agents targeting the GABA_AR on self-renewal and neuronal output in the brain, but also identifies a mechanism amenable for pharmacological intervention that can result in increases of NSC numbers and neuronal output.

Materials and Methods

Animals and Tissue Preparation.

For primary clonal growth, the SVZ region was microdissected and dissociated. Male C57BL/6, GFAP-GFP, or H2AX WT and null (i.e., KO) mice between 8 and 12 wk of age were used. Acute in vivo treatments involved i.p. administration of muscimol (6 mg/kg) or bicuculline (4 mg/kg) whereas chronic treatments involved muscimol and bicuculline (4 mg/kg) as indicated twice daily. Tamoxifen (Sigma) dissolved in corn oil was administered by twice daily 1-mg i.p. injections over 5 d (accumulated dose of 10 mg).

Immunohistochemistry, Quantification, and Statistical Analysis.

Immunohistochemistry was performed on 14-μm coronal sections and quantified by counting immunopositive cells from at least six entire lengths of the lateral wall of the lateral ventricle (SVZ) and averaged for each animal replicate. BrdU labeling index (LI) was calculated as the percentage of BrdU⁺ normalized to total Ki67⁺ cells per ventricle length (SVZ). Statistical analysis was performed using GraphPad Prism software; Mann–Whitney or unpaired two-tailed tests were consistently used, with an α level of 0.05 for all statistical analysis. *P < 0.05, **P < 0.01, and ***P < 0.001.