GABAA Receptor Signaling Induces Osmotic Swelling and Cell Cycle Activation of Neonatal Prominin+ Precursors§

Authors

  • Tiziana Cesetti,

    1. Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Heidelberg, Germany
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  • Tatiana Fila,

    1. Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Heidelberg, Germany
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  • Kirsten Obernier,

    1. Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Heidelberg, Germany
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  • C. Peter Bengtson,

    1. Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Heidelberg, Germany
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  • Yuting Li,

    1. Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Heidelberg, Germany
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  • Claudia Mandl,

    1. Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Heidelberg, Germany
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  • Gabriele Hölzl-Wenig,

    1. Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Heidelberg, Germany
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  • Francesca Ciccolini

    Corresponding author
    1. Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Heidelberg, Germany
    • Francesca Ciccolini, Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Im Neuenheimer Feld 345, Heidelberg 69120, Germany
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    • Telephone: 49-6221-548696; Fax: 49-6221-546700


  • Author contributions: T.C.: collection of data and analysis, data interpretation, manuscript writing; T.F.: collection of data and analysis, data interpretation, manuscript writing; K.O.: collection of data and analysis; C.P.B.: collection of data and analysis, data interpretation, final approval of manuscript; Y.L.: collection of data and analysis; C.M.: collection of the data; G.H.-W.: collection of the data; F.C.: conception and design, data analysis and interpretation, manuscript writing. T.C. and T.F. contributed equally to this article.

  • Disclosure of potential conflicts of interest is found at the end of this article.

  • §

    First published online in STEM CELLSEXPRESS December 9, 2010.

Abstract

Signal-regulated changes in cell size affect cell division and survival and therefore are central to tissue morphogenesis and homeostasis. In this respect, GABA receptors (GABAARs) are of particular interest because allowing anions flow across the cell membrane modulates the osmolyte flux and the cell volume. Therefore, we have here investigated the hypothesis that GABA may regulate neural stem cell proliferation by inducing cell size changes. We found that, besides neuroblasts, also neural precursors in the neonatal murine subependymal zone sense GABA via GABAARs. However, unlike in neuroblasts, where it induced depolarization-mediated [Ca2+]i increase, GABAARs activation in precursors caused hyperpolarization. This resulted in osmotic swelling and increased surface expression of epidermal growth factor receptors (EGFRs). Furthermore, activation of GABAARs signaling in vitro in the presence of EGF modified the expression of the cell cycle regulators, phosphatase and tensin homolog and cyclin D1, increasing the pool of cycling precursors without modifying cell cycle length. A similar effect was observed on treatment with diazepam. We also demonstrate that GABA and diazepam responsive precursors represent prominin+ stem cells. Finally, we show that as in in vitro also in in vivo a short administration of diazepam promotes EGFR expression in prominin+ stem cells causing activation and cell cycle entry. Thus, our data indicate that endogenous GABA is a part of a regulatory mechanism of size and cell cycle entry of neonatal stem cells. Our results also have potential implications for the therapeutic practices that involve exposure to GABAARs modulators during neurodevelopment. STEM CELLS 2011;29:307–319

INTRODUCTION

Water transport in the brain is essential for the maintenance of intracerebral pressure and is associated with the choroid plexus, the specialized glia cells forming the brain-blood barrier, and the ependymal lining of the ventricle. The exchange of water in the brain is regulated by transport mechanisms such as solute cotransport and signal-regulated movement of ions across the cell membrane [1]. In this respect, GABA is of particular interest because by regulating the membrane permeability to anions via activation of GABAA receptors (GABAARs), it controls osmolyte flux, volume, and acidity of the cell.

GABA is the main inhibitory neurotransmitter in the adult brain and modulators of GABAergic transmission are widely used for therapeutic purposes. GABA also displays a trophic function from early stages of neurodevelopment [2]. In the embryonic ventricular zone, activation of GABAAR increases the proliferation of radial glia precursors [3]. However, the opposite effect has been reported for secondary progenitors located in the embryonic subventricular zone [3], where GABAAR activation reduces DNA synthesis/mitosis possibly via depolarization-mediated Ca2+ entry [4]. GABA signaling exerts a complex regulation also in the adult subependymal zone (SEZ) where neuroblasts and glial fibrillary acidic protein (GFAP)-expressing cells, which include stem cells as well as astrocytes, regulate ambient GABA levels, by mediating its nonsynaptic release and uptake, respectively [5]. In turn, GABAAR activation downregulates neuroblast proliferation and migration [6–8]. Activation of GABAAR also inhibits the proliferation of GFAP-expressing cells in the adult niche [5]. However, the cellular mechanisms underlying these effects are still unclear.

Embryonic radial glia and postnatal stem cells are in contact with both the cerebrospinal fluid and the blood vessels [9]. Therefore, it is conceivable that they are endowed with specific mechanisms to regulate water flux. We have here investigated the possible involvement of GABA in this process and the correlation with stem cell proliferation. Our results show that both in vitro and in vivo GABAAR activation promotes cell cycle entry of prominin+ stem cells via Cl influx, hyperpolarization-mediated osmotic swelling, and overexpression of epidermal growth factor receptor (EGFR) at the cell surface.

MATERIALS AND METHODS

Tissue Dissection and Cell Culture

Neonatal C57B6 mice (between P4 and P9) were decapitated, in accordance with the local ethical guidelines for the care and use of laboratory animals (Karlsruhe, Germany). The SEZ of the lateral ventricle was dissected and dissociated cells were processed for sorting either immediately or after being plated overnight at a density of 105 cells per ml in NS-A (Euroclone, Eching, Germany) medium supplemented with 10 ng/ml fibroblast growth factor (FGF)-2 (R&D System, Wiesbaden-Nordenstadt, Germany) as already described [10]. Proliferation medium contained NS-A or Neurobasal (NBA; Invitrogen, Darmstadt, Germany) media, 2% B27, 2 mM L-glutamine, penicillin 100 U/ml, streptomycin 100 mg/ml, FGF-2 10 ng/ml, and EGF 20 ng/ml. See Supporting Information Methods for a detailed list of the drugs used.

FACS and Clonal Analysis

Cells were incubated with primary PSANCAM antibody together with a fluorescently labeled secondary antibody (Molecular Probes, Darmstadt, Germany) or with prominin-PE and then stained with EGF-Alexa (20 ng/ml) as previously described [11, 12]. For clonal analysis, single cells were plated by FACS-automated cell deposition in each well of 96-well plates (Nunc, Wiesbaden, Germany) in NSA proliferation medium as described before [13]. Changes in forward scattering (FSC) reflected variations in the osmolarity of the medium (Supporting Information Fig. S6) and were visualized using FACSDiva 6.1.3 and OriginLab 8. FACS histograms illustrating the frequency distribution of FSC were fitted with OriginLab eight by single Gaussian functions whose mean, SD, and adjusted R2 were calculated. The statistical significance of the variations between control and treated groups was calculated using Student's t test.

Diazepam Injection

P7 mice were injected with diazepam (3 μg/g of body weight) or vehicle alone (phosphate-buffered saline [PBS]) twice with an interval of 12 hours between each injection and sacrificed 12 hours after the last injection.

Organotypic Slice Preparation and Infection

Brains embedded in low-melting point agarose gel (4% in PBS) were cut into 300-μm thick coronal sections containing the SEZ using a vibratome (CU65 Cooling Unit & HM650V Vibratome, Microm, Walldorf, Germany) in iced slicing medium (in mM: Sucrose, 150; NaCl, 40; KCl, 4; MgCl2, 7; NaH2PO4, 1.25; CaCl2, 0.5; glucose, 10; NaHCO3, 26; gassed with 95% O2 and 5% CO2). Slices were cultured as described previously [13]. Slices were infected by pipetting 1 μl viral dilution onto the lateral ventricle and analyzed 2 days later. Lentiviruses were prepared as described previously [12].

Immunocytochemistry

Cells were fixed and immunostained as previously described [14]. Primary antibodies were detected using Alexa 488-, Alexa 555-, or Cy3-conjugated secondary antibodies (Molecular Probes). As indicated, the primary antibody was incubated without permeabilization on live or fixed cells for GABAAR β-chain and EGFR staining, respectively. 4′,6-Diamidine-2′-phenylindole-dihydrochloride (DAPI) 1:1,000 (Roche) was used for nuclear counterstaining. Immunopositive cells were quantified from at least three independent experiments by analyzing an average of 100 cells across multiple fields with a conventional fluorescence microscope (DMIRBE microscope; Leica, Germany).

Immunohistofluorescence

P7 mice were deeply anesthetized with an intraperitoneal injection (2 ml/kg body weight) of sodium-pentobarbital (Narcoren, Merial, Germany) and perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4. Brains were postfixed overnight in 4% PFA at 4°C and transferred into a 30% sucrose solution. After embedding in 4% low-melting agarose, 40-μm thick coronal vibratome sections were cut and processed for immunohistochemistry as previously described [13]. Image acquisition and analysis were done as described previously [12].

Live Imaging

For Ca2+ imaging, the cells were loaded at room temperature (RT) for 20 minutes with 2–4 μM Fluo3-AM (Molecular Probes) and imaged in Ringer's solution as previously described [10]. For imaging with a potentiometric dye, the cells were stained at RT for 10 minutes with 75 nM DiBAC4 [3] (Molecular Probes) in Ringer's solution and imaged in the same solution. See Supporting Information Methods for a detailed protocol.

Patch Clamp

Whole-cell patch-clamp recordings and fluorescence imaging were performed at RT as previously described [15]. See Supporting Information Methods for a detailed protocol.

Reverse Transcription Polymerase Chain Reaction

For each sample, a total of 25000 PS+/EGFRhigh cells were sorted directly in lysis buffer and processed for total RNA purification (RNeasy Micro Kit Quiagen). Total RNA was reversely transcribed using Oligo-dT primers and M-MLV reverse transcriptase, RNase H Minus (all from Promega, Mannheim, Germany). Amplified DNA was visualized by ethidium bromide-staining. Amplification of GAPDH was used as an internal control, total brain extract was used as a positive control, and H2O was used as a negative control.

Quantitative Reverse Transcription Polymerase Chain Reaction

Cells (>10,000 cells) were sorted into lysis buffer and the total RNA was extracted with RNeasy Micro Kit (Qiagen, Hilden, Germany). Total RNA was reversely transcribed as described earlier. See Supporting Information Methods for a detailed protocol.

RESULTS

Expression of GABA and GABA Receptors in Precursors and Neuroblasts

We first investigated the presence and function of GABAARs in neonatal SEZ precursors. To this end, we purified cells expressing Poli-Sialated Neural Cell Adhesion Molecule (PSANCAM) and either low (PS+/EGFRlow) or high levels (PS+/EGFRhigh) of EGFR from the neonatal SEZ by flow cytometry. Using this approach, we have recently shown that they represent respectively neuroblasts and precursors, the latter consisting of transit-amplifying precursors (TAPs) and activated stem cells [12]. Reverse transcription polymerase chain reaction analysis of the total mRNA purified from the two populations (primers list in Supporting Information Table 1) revealed a similar pattern of cDNA amplification products that included all the GABAAR subunits, except α6, α2, and δ subunits, and the GABABR and GABACR subunits (Supporting Information Fig. S1a and Supporting Information Table S2).

On immunohistofluorescence with a pan GABAAR β antibody a strong fluorescent signal was mainly observed within the cells lining the lateral ventricle (Fig. 1D and Supporting Information Fig. S2, arrowheads) and in PSANCAM+ cells at the basal border of the SEZ (Supporting Information Fig. S2). A weaker immunoreactivity was also observed in the medial SEZ, where most EGFR+ cells coexpressed the GABAAR β subunit (71.61% ± 1.66%, n = 4; arrow in Fig. 1D). These cells, as the majority of sorted neuroblasts and precursors (Supporting Information Fig. S1d), also coexpressed GABA and its synthesizing enzymes GAD65 and GAD67. However, the expression levels of GAD65 and GAD67 transcripts in PS+/EGFRlow cells were 13.72 ± 6.5-fold and 8.24 ± 3.2-fold higher than in PS+/EGFRhigh precursors, respectively (Supporting Information Fig. S1e). Furthermore, whereas neuroblasts displayed a strong immunoreactivity to the pan GABAAR β antibodies, precursors showed a much weaker and only punctated immunostaining (Fig. 2A), indicating that the levels of receptor expression differ between the two populations.

Figure 1.

Expression of GABAergic signaling components in situ. (A–D): Confocal photomicrographs of neonatal subependymal zone slices after double immunostaining as indicated. Immunolabeling in (D) was performed without permeabilization. Scale bar = 100 μm (lower magnification images [left]), 50 μm (higher magnification images [right] from the boxed area). Arrows and arrowheads show double- and single-immunopositive cells, respectively. Abbreviations: D, dorsal; EGFR, epidermal growth factor receptor; L, lateral; M, medial; V, ventral.

Figure 2.

GABAergic currents in neuroblasts and precursors. (A): Representative photomicrographs of PS+/EGFRhigh cells (left panel) and PS+/EGFRlow cells (right panel) fixed 1 day after sorting showing GABA receptors (GABAARs) β immunoreactivity (red) and 4′,6-diamidine-2′-phenylindole-dihydrochloride nuclear couterstaining (blue). Scale bar = 50 μm. (B): Representative differential interference contrast (DIC) photomicrographs of the indicated sorted populations. Scale bar = 20 μM. (C): Representative currents evoked by GABA (200 μM) in the indicated cell population cell types, as indicated (Vh = −70 mV). (D, E): Plots show the mean current density (D) and (E) the percentage of cells in each group responding to GABA. (F): Differential interference contrast (F1, F2) and fluorescence (F3, F4) photographs of eGFP+ cells in day in vitro 2 (DIV2) infected slices used for patch clamp recordings. Representative images of a large (F1, F3) and a small (F2, F4) cell are shown. Scale bar = 20 μM. (G): GABAergic currents in representative large (CM = 22 pF) and small (CM = 4.1 pF) eGFP+ cells as well as in an eGFP− neuron in the neighboring striatum (Vh = −70 mV). (H, I): Plots show the current density (H) and the percentage of cells in each group responding to GABA (I). Abbreviations: Cont, cell contacting neighboring cells; EGFR, epidermal growth factor receptor; GFP, green fluorescent protein; Isol, isolated; PS, Poli-Sialated Neural Cell Adhesion Molecule.

Acute Responses to GABAA Receptor Activation in Precursors and Neuroblasts

We next performed whole-cell patch-clamp experiments on both sorted populations to test whether such GABAARs were functional. In all neuroblasts tested, bath application of GABA (200 μM) elicited a large inward Cl current (Fig. 2C–2E and Supporting Information Fig. S3 and Supporting Information Table S3), with an EC50 of 31.4 ± 2.14 μM (Supporting Information Fig. S3c, S3d). The inward current was blocked by the selective GABAAR antagonist bicuculline (97% ± 0.6%) and similarly evoked by the agonist muscimol (25 μM; Supporting Information Fig. S3a). In contrast, only a few isolated precursors (24%) responded to GABA with an average current density 33 times smaller than in neuroblasts (Fig. 2C, 2D and Supporting Information Table S3). The percentage of cells responding to GABA was threefold higher (78%) in precursors contacting each other than in isolated cells, though their current density was comparable (Supporting Information Table S3 and Fig. 2D, 2E), suggesting that cell coupling may increase the proportion of responding cells [12]. Although we tested this possibility with the gap junction blockers, unspecific effects made this data uninterpretable. Meclofenamic acid, which is known to bind β2/3 GABAAR subunits [16], inhibited GABA currents even in neuroblasts, which are not electrically coupled [12], and carbenoxolone evoked an unspecific Ca2+ rise in live imaging experiments (data not shown).

We next measured the response to GABA in precursors in situ. We have previously shown that EGFR+ cells can be identified in telencephalic slices using a lentivirus-expressing enhanced green fluorescent protein (eGFP) under the control of the EGFR promoter (pEGFR-eGFP) [12]. Two days after infection “large” and “small” eGFP+ cells can be distinguished in the infected slices, which display functional characteristics of precursors and preneuroblasts, respectively [12]. On application of GABA all “small” eGFP+ cells tested responded. Instead only 15% of the “large” eGFP+ cells showed GABA currents (Fig. 2I and Supporting Information Table S4). Furthermore, the amplitude of the GABA current recorded from the latter cell group was fivefold smaller than the one recorded from the first (Fig. 2G and Supporting Information Table S4). Thus, the incidence of GABAAR function of large eGFP+ cells in situ was similar to that of precursors in vitro, both analyses revealing a GABA-responsive subgroup of precursors in the neonatal SEZ.

We further investigated the responses of PS+/EGFRhigh precursors and PS+/EGFRlow neuroblasts to GABA with Ca2+ imaging. In most isolated neuroblasts (66%, n = 44), GABA evoked a rapid increase in [Ca2+]i, as indicated by the increase in Fluo-3 fluorescence (60% ± 8%), that was further increased by a subsequent coapplication of a high concentration of K+ (HiK) (53% ± 5.5%; Fig. 3A). In contrast, GABA elicited a [Ca2+]i increase in only one of the 42 precursors tested (2.4%), and no responses to HiK were observed (Fig. 3B).

Figure 3.

GABA receptor (GABAAR)-mediated [Ca2+]i and membrane voltage responses. (A, B): Fluorescence changes (F/F0) in Fluo-3 AM-loaded PS+/EGFRlow (A) and PS+/EGFRhigh (B) cells measuring acute responses to GABA (200 μM) and 40 mM K+ (HiK) applications, as indicated by the horizontal bars. Traces from three representative cells in each cell group are shown. (C, D): Representative fluorescence changes (F/F0) on muscimol (25 μM) application in PS+/EGFRlow (C) and PS+/EGFRhigh (D) cells loaded with DiBCA4(3). (E): Graph representing the mean (±SEM) muscimol-induced changes in DiBCA4(3) fluorescence, as a percentage of baseline fluorescence (n = 78 for PS+/EGFRhigh and n = 97 for PS+/EGFRlow). Abbreviations: EGFR, epidermal growth factor receptor; HiK, high concentration of K+; PS, Poli-Sialated Neural Cell Adhesion Molecule.

To directly assess the effect of GABA on the membrane potential of precursors and neuroblasts, we used the potentiometric dye DiBC4 [3]. In agreement with the Ca2+ imaging, the majority of neuroblasts (89% n = 97; Fig. 3C, 3E) displayed a mean 25% increase in DiBC4 fluorescence on application of muscimol. In contrast in precursors, muscimol evoked a mean 25% decrease in DiBC4 fluorescence, reversible on wash out, suggesting a GABAAR-mediated hyperpolarization (Fig. 3D, 3E). Almost all (97%, n = 78) precursors displayed such response, reflecting the fact that in these cultures most cells are in contact with each other [12]. In summary, these data show that the majority of neuroblasts respond to muscimol with depolarization and Ca2+ influx. In contrast, when contacting neighboring precursor cells, almost all precursors undergo hyperpolarization and no intracellular Ca2+ elevation following GABAAR activation.

GABAAR Activation Induces Osmotic Swelling and Regulates EGFR Trafficking in Precursors

It has been shown that GABAAR-dependent movement of Cl across the cell membrane leads to an osmotic regulation of the cell size in embryonic stem (ES) cells and in glioma cells [17, 18]. As in cells undergoing osmotic changes the degree of light scattering varies inversely with the cell volume [19], we next investigated the effect of selective GABAAR activation on the properties of forward light scattering (FSC) of neuroblasts and precursors. For these experiments, we only used EGFR detection to purify precursors and neuroblasts, as most EGFRhigh and EGFRlow are also PS+ [12]. After dissection, dissociated SEZ cells were allowed to recover overnight. The next day they were stained and briefly exposed to muscimol (25 μM; 15 min at 37°C) or left untreated as control before FACS analysis. We found that muscimol induced a decrease of the FSC (Fig. 4A, n = 3), indicating osmotic swelling in EGFRhigh precursors but not in EGFRlow cells (Supporting Information Fig. S4a). This effect of muscimol was prevented by bicuculline cotreatment (data not shown). However, application of muscimol immediately after dissection did not significantly alter the FSC of either cell population (data not shown). This could be due to occlusion by the release of GABA during the dissection process. We, therefore, analyzed the FSC of SEZ cells that during the dissection, dissociation, and EGFR staining had been exposed to the GABAAR blocker bicuculline (50 μM, for 30 minutes). This treatment increased the FSC of EGFRhigh precursors (Fig. 4B) but not of the EGFRlow cells (Supporting Information Fig. S4b), indicating that activation of GABAARs by endogenous GABA, released during the dissection occludes the effect of muscimol on FSC at this time point.

Figure 4.

GABA receptor (GABAAR) activation induces osmotic swelling and regulates EGFR surface expression in precursor cells. (A, B): Representative FACS histograms illustrating the frequency distribution of FSC values of EGFRhigh cells. Curves were fitted by single Gaussian functions whose mean, SD, and adjusted R2 were calculated. (A): Analysis of FSC was done on cells 24 hours after isolation that were either exposed to muscimol for 15 minutes (red: mean = 0.97 × 105, SD = 0.20 × 105, R2 = 0.84, n = 2091) or left untreated as control (green: mean = 1.09 × 105, SD = 0.25 × 105, R2 = 0.94, n = 2091; p < 0.01, t test). (B): FSC was analyzed on freshly dissociated cells that had been treated with bicuculline during dissection and staining for 30 minutes (blue: mean = 1.06 × 105, SD = 0.20 × 105, R2 = .89, n = 2017) or left untreated as control (green: mean = 0.95 × 105, SD = 0.16 × 105, R2 = 0.91, n = 2017). (C): Quantitative analysis of the number of EGFRhigh cells in subependymal zone (SEZ) cells dissociated and stained in the presence of bicucculline normalized to untreated (control) cells. (D): Photomicrographs of SEZ-dissociated cells immunostained with EGFR antibodies and DAPI. Scale bar= 50 μm. Bicuculline was applied for 30 minutes and cells were fixed and stained 6 hours after plating. (E): Quantification of (D). Values are normalized to the control. (F, G): Quantification of the percentage cells, which displayed EGFR immunoreactivity on immunostaining with (“control p.”) or without (“control not p.”) permeabilization in control (F) and SEZ cells treated with bicuculline during dissection and for 6 hours after plating (G). Cell numbers in (E), (F), and (G) were quantified with a conventional microscope as the sum of viable nuclei from at least 10 microscopic fields (n = 3; *, p < .05). (H): Quantification of EGFRhigh cells in SEZ cells dissected in normal sucrose solution or in solution where Cl was substituted with NO3− and treated with bicuculline (30 minutes) or left untreated (control). Values are normalized to the control (n = 3; *, p < 0.05). Abbreviations: EGFR, epidermal growth factor receptor; FSC, forward scattering.

The bicuculline treatment also reduced the percentage of EGFRhigh cells (Fig. 4C, n = 5), confirmed by immunocytochemistry with EGFR antibodies (Fig. 4D, 4E). This effect was not due to cell death as assessed by propidium iodide (PI) incorporation (Supporting Information Fig. S4e) and was not observed in EGFRlow cells (Supporting Information Fig. S4b–S4d). We next immunostained permeabilized and nonpermeabilized SEZ cells in culture that had been treated with bicuculline during dissection and plated in the presence of this antagonist for 6 hours or left untreated as control. Consistent with the FACS analysis, we found that bicuculline did not affect the proportion of cells expressing intracellular EGFR, but reduced the proportion of cells expressing EGFR at the cell surface (Fig. 4F, 4G). Finally, exposing the cells, either during dissection or after plating, to hyper or hypo-osmotic solution decreased and increased EGFR expression at the cell surface, respectively (Supporting Information Fig. S6). This shows that osmotic tension per se regulates cell surface expression of EGFR in stem cells. To investigate whether the Cl influx from GABAAR is directly involved in upregulating the cell surface expression of EGFR, we replaced most of the Cl in the dissection medium with the GABAAR impermeable NO3−, whereas cell dissociation and staining were still performed in medium containing standard Cl concentrations [20]. This transient decrease in the influx of Cl was enough to reduce the number of EGFRhigh cells in control but not in bicuculline-treated cells (Fig. 4H), indicating that Cl influx from activated GABAAR is directly involved in regulating the surface expression of EGFR. Taken together, our results indicate a direct link between GABAAR induced osmotic swelling and expression of EGFR at the cell surface.

GABAARs Activation Increases the Proliferation of EGFRHigh Cells In Vitro and In Vivo

We next investigated the effect of GABA signaling on precursor proliferation. We first analyzed this issue in vitro using clonal assays.

Exposure to bicuculline (50 μM, 30 minutes) during dissection and sorting significantly reduced the clone-forming capability of EGFRhigh cells (Fig. 5A). A similar but weaker effect when bicuculline was added to the medium after sorting during clone formation (Fig. 5B), likely reflecting the fact that when isolated only few precursors display functional GABAAR. Addition of bicuculline to the culture medium also reduced the total number of cells in bulk cultures of sorted EGFRhigh cells grown in vitro for 4 days, whereas a similar treatment with GABA (10 μM) produced the opposite effect (Fig. 5C). In conclusion, our in vitro experiments suggest that GABA enhances EGFRhigh precursor proliferation.

Figure 5.

GABAergic modulation of EGFRhigh cell proliferation in vitro and in vivo. (A, B): Quantification of clone formation in EGFRhigh cells treated with bicuculline normalized to untreated EGFRhigh cells (control). Bicuculline was present during dissection and sorting for a total of 30 minutes (A) or only after sorting during clone formation assay (10 days; [B]). (C): Number of viable cells (normalized to control) present 4 days after plating 3000 PS+/EGFRhigh cells per matrigel-coated coverslip in the indicated conditions. Cell counts were made after fixation and DAPI staining from at least 10 microscopic fields using wide-field fluorescence (n = 3). (D): Confocal photomicrographs of SEZ coronal slices from control and diazepam-injected mice double-immunostained as indicated. Scale bar = 100 μm. (E): Quantification of (D). (F, G): Quantification of number (F) and clonogenic activity (G) of EGFRhigh cells sorted from the SEZ vehicle (control) or diazepam-injected mice, represented as a percentage of the control. *, p < 0.05 compared with control. Abbreviations: EGFR, epidermal growth factor receptor.

Neuroblasts in the SEZ are sensitive to benzodiazepines [7, 8]. As we had found that PS+/EGFRhigh and PS+/EGFRlow cells express a similar range of GABAAR subunits (Supporting Information Fig. S2a), it was possible that also precursors displayed diazepam sensitive GABAARs. We, therefore, investigated the possibility that diazepam may affect GABAAR function and the proliferation of precursors. Exposure to diazepam (18 μM, 15 minutes) before sorting significantly increased the clonogenic capability of EGFRhigh cells (Supporting Information Fig. S5a) but not of EGFRlow cells (data not shown), an effect that was occluded if bicuculline was added to the dissection medium (Supporting Information Fig. S5b). Moreover, the clonogenic activity of EGFRhigh cells was increased by the addition of diazepam to the medium during clone formation and this effect was potentiated by the simultaneous presence of GABA (10 μM) and diazepam (Supporting Information Fig. S5c).

We next injected intraperitoneally neonatal (P7) mice with diazepam to enhance endogenous GABAAR activation. FACS analysis revealed that after 24 hours the SEZ of diazepam-injected animals contained significantly more EGFRhigh cells than the control counterpart (70%, n = 6; Fig. 5F). Furthermore, EGFRhigh cells isolated from diazepam-treated animals were significantly more clonogenic than control cells (30%, n = 6; Fig. 5G). In contrast, no difference was detected within EGFRlow cells (Supporting Information Fig. S5d, S5e). This data was confirmed by immunohistochemistry. In the SEZ of diazepam-treated animals, both the number of DAPI+ nuclei per region of interest and of EGFR+ cells, as percentage of the DAPI+ nuclei, were greater than in control animals (Fig. 5D). This increase was not due to a change in cell viability (Supporting Information Fig. S5f–S5h) but rather in cell proliferation, as revealed by the quantitative analysis of cycling (Ki67+) and mitotic (pHH3+) cells in control and treated mice (Fig. 5D, 5E). Diazepam treatment almost doubled the number of EGFR+/ Ki67+ and EGFR+/ pHH3+ double-positive cells and many of the extra mitotic cells were located in close proximity of the lateral ventricle (Fig. 5D, 5E). In addition, analysis of the ratio pHH3/Ki67 (0.29 ± 0.03 vs. 0.27 ± 0.07), revealed no change in cell cycle length, indicating that diazepam promoted cell cycle entry of EGFR+ precursors. Unlike in ES cells where GABAAR activation induces phosphorylation of histone H2AX [17], in the neonatal SEZ immunohistochemistry for phospho-histone-H2AX (γH2AX) did not reveal any differences between diazepam-treated and mice control mice (data not shown). Taken together, our results suggest that diazepam-mediated GABAAR activation positively modulates the proliferation of neural precursors both in vitro and in vivo.

GABAARs Activation Increases Proliferation of Prominin+ Precursors

It has been previously shown that clonogenic EGFRhigh cells include two different cell types: activated stem cells, that is, precursors expressing both high levels of EGFR and stem cell markers, and TAPs [11, 12, 21]. We next investigated whether diazepam affects the proliferation of stem cells or TAPs in vivo. Immunohistochemistry of fixed brain tissue revealed that diazepam injection not only increased EGFR expression but also the number of EGFR+/GFAP+ cells (Fig. 6A), indicating that enhanced GABAAR signaling leads to an increase in the number of activated stem cells. Interestingly, double-immunopositive cells were preferentially located at the apical border of the germinal region, suggesting that they may represent cells directly contacting the ventricular cavity. In the postnatal SEZ, prominin is expressed by multiciliated ependymal cells and stem cells displaying one short cilium protruding into the ventricular cavity [22–24]. We, therefore, used prominin expression to isolate cells contacting the lateral ventricle. Active cilia were observed exclusively within prominin+-sorted EGFRlow cells (about 35% of examined cells, n = 31), with the majority of these cells exhibiting several motile cilia (Supporting Information videos). Independent of EGFR expression, GFAP was also expressed in isolated prominin+ cells, (40.7% ± 7.9% of prominin+/EGFRhigh and 32.8% ± 7.7% of prominin+/EGFRlow cells were GFAP+; Fig. 6B). We next carried out clonal analysis to investigate the effect of diazepam on clonogenic SEZ precursors. Prominin+/EGFRhigh cells represented a small percentage (about 15%) of all EGFRhigh cells and had a clone-forming capability comparable with that of prominin/EGFRhigh (Supporting Information Table S5). Diazepam treatment significantly increased the number of prominin+/EGFRhigh but not of prominin/EGFRhigh cells and significantly enhanced the clonogenic capability of both populations (Supporting Information Table S5).

Figure 6.

Diazepam increases the number of activated stem cells. (A): Confocal photomicrographs of neonatal subependymal zone (SEZ) slices from vehicle (control; upper panels) and diazepam (lower panels)-injected P7 mice double-immunostained as indicated. Scale bar = 100 μm; inset 25 μm. (B): Photomicrograph showing GFAP immunoreactivity in prominin+/EGFRhigh cells fixed and immunostained 24 hours after sorting. Scale bar = 40 μm. (C): Photomicrographs showing GABAAR immunoreactivity in the indicated SEZ populations, fixed and immunostained after dissection and sorting. Scale bar = 50 μm. (D): Quantification of (C). Values are normalized to the number of DAPI-positive cells. (E, F): Representative currents evoked by GABA (200 μM; black) and blocked by bicuculline (50 μM; green) in prominin/EGFRlow (E) and prominin+/EGFRlow (F) cells. (G, H): Representative currents evoked by GABA (10 μM; black) and enhanced by diazepam (18 μM; red) in prominin/EGFRlow (G) and prominin+/EGFRlow (H) cells. (I, J): Summary plots representing the percentage of cells responding to GABA (200 μM; [I]) and the relative mean current density (J) in prominin/EGFRlow (n = 26), prominin+/EGFRlow (n = 52), and prominin+/EGFRhigh (n = 20) cells. Cells were sorted and recorded 1 day after plating. (K): Analysis of FSC in prominin+/EGFRhigh cells. Cells were treated with bicuculline for 30 minutes during tissue dissociation and then stained for prominin and EGFR before sorting. Note the rightward shift in the FSC distribution of bicuculline-treated cells (blue: mean = 1.70 × 105, SD = 0.45 × 105, R2 = 0.82, n = 2012) compared with control (green: mean = 1.07 × 105, SD = 0.53 × 105, R2 = .88, n = 2,012; p < 0.01). Abbreviations: EGFR, epidermal growth factor receptor; FSC, forward scattering; GFAP, glial fibrillary acidic protein.

Consistent with these observations, immunostaining of sorted SEZ cells with antibodies to the GABAARs β revealed immunoreactivity in prominin+ cells, although to a lesser extent than in prominin/EGFRlow cells, mostly representing neuroblasts [12] (Fig. 6C, 6D). Application of GABA (200 μM) evoked a large inward current sensitive to bicuculline in all prominin/EGFRlow cells tested (Fig. 6E). Instead, within prominin+ cells (both EGFRhigh and EGFRlow), only a fraction of the cells responded to GABA, with a much smaller current density (34 and 71 times, respectively; Fig. 6I, 6J), that was also blocked by bicuculline (Fig. 6F, n = 5/5). As for PS+/EGFRhigh cells (Fig. 2D, 2E), the contact with at least one neighboring cell increased the percentage of responding cells in both prominin+/EGFRhigh cells, from 40% (n = 10) to 80% (n = 10), and in prominin+/EGFRlow cells, from 60% (n = 35) to 100% (n = 17), without affecting the current density. Furthermore, in prominin/EGFRlow cells the current produced by a lower GABA concentration (10 μM; 19.9 ± 5.4 pA/pF) was increased by diazepam (18 μM) by 98.05% ± 17.6% (n = 20; Fig. 6G). When the same protocol was applied to prominin+/EGFRlow cells, a very small current (less than 10 pA) was detected on the application of 10 μM GABA only in some of the cells in contact (27%, n = 14), and the coapplication of diazepam either increased this current (119% ± 46%) or unmasked a current in cells not responding to GABA 10 μM (Fig. 6H). In total, 50% of the prominin+/EGFRlow cells in contact tested responded to diazepam (n = 14). In isolated cells, no response was observed to GABA 10 μM, but in 1 of 11 cells, the coapplication of diazepam unmasked a small current. Thus, despite differences in the amount of responding cells and in current density evoked by GABA, both neuroblasts and prominin+ cells are sensitive to diazepam. The presence of diazepam-sensitive GABAAR in prominin+ cells further supports the hypothesis that their endogenous activation may induce osmotic swelling and expression of EGFR in this cell population, thereby promoting their proliferation. To confirm this hypothesis, we analyzed the FSC of the four populations on blockade of the endogenous GABAAR activation. We found that bicuculline treatment induced a shift in the FSC only in prominin-expressing populations (prominin+/EGFRhigh and prominin+/EGFRlow), whereas independent of EGFR expression, no change was observed in prominin cells (Fig. 6K and data not shown).

Taken together, these results indicate that diazepam selectively promotes EGFR expression on prominin+ cells including activated stem cells.

GABAARs Activation Promotes the Cell Cycle Entry in Neonatal Precursors

It has been shown that an increase in cell size can regulate proliferation and in particular the progression through the G1 phase of the cell cycle [25]. The tumor suppressor phosphatase and tensin homolog (PTEN) plays a critical role in neural precursor proliferation and growth [26]. It regulates the G1-S transition by modulating the expression of cyclin D1 and p27 kip1 [27], and it negatively regulates the proliferation of embryonic [28, 29] and postnatal neural stem cells [30]. To investigate whether GABAAR activation could promote the cell cycle entry in precursors, sorted EGFRhigh and EGFRlow cells were plated on chamber slides and treated for 6 hours with muscimol (25 μM) or bicuculline (50 μM) and then processed for immunocytochemistry to analyze PTEN and cyclin D1 expression. We observed a significant reduction in the number of PTEN+ and an increase in the number of cyclin D1+ cells after muscimol treatment, whereas bicuculline produced the opposite effect (Fig. 7A–7C). In contrast, neither treatment affected PTEN or cyclin D1 expression when EGFRhigh cells were plated in the absence of EGF, in medium containing only FGF-2 (Fig. 7B, 7C). Similarly, no significant effect was observed when the cells were treated with bicuculline (50 μM, 30 minutes) only before sorting and fixed immediately after plating (data not shown). This indicates that EGFR signaling downstream of GABAAR activation is required for PTEN and cyclinD1 regulation. Consistently, both treatments did not affect either PTEN or cyclin D1 in EGFRlow cells (Supporting Information Fig. S4f, S4g).

Figure 7.

Diazepam increases EGFR+ cell proliferation by regulating PTEN/cyclinD expression. (A): Photomicrographs showing of PTEN and cyclin D1 immunoreactivity of EGFRhigh-sorted cells treated for 6 hours as indicated and immunostained. Scale bar = 50 μm; inset 15 μm. (B, C): Quantification of the number of EGFRhigh cells, which were PTEN (B) or cyclin D1 (C) immunopositive. Cells were treated as described in (A) and plated in E/F or in fibroblast growth factor (F) containing medium. Results are normalized to the relative untreated controls. (D, E): Confocal photomicrographs of neonatal subependymal zone slices obtained from vehicle (control) and diazepam-injected P7 mice after double immunostaining as indicated. Scale bar = 100 μm (large images); insets 25 μm. Higher magnification images of the boxed areas (E1, E2) illustrating the different intracellular localization of cyclin D are shown in (E). Abbreviations: ctrl, control; EGFR, epidermal growth factor receptor; E/F, epidermal growth factor + fibroblast growth factor; PTEN, phosphatase and tensin homolog.

To confirm that the activation of PTEN/cyclin D1 pathway occurs also in vivo on GABAARs activation, we performed immunohistochemistry for these two antigens on neonatal telencephalic slices obtained from control and diazepam-injected animals. This analysis revealed a significant decrease in the number of PTEN+ cells and an increase in the number of cyclin D1+ cells within the EGFR+ cell population in slices from diazepam-treated animals (Fig. 7D, 7E). Interestingly, almost all the cyclin D1+ cells observed were also EGFR+ and lined the lateral wall of the ventricle (Fig. 7D, 7E), suggesting a role for GABAAR-mediated signaling in promoting cell cycle entry of quiescent precursors in this region. In particular, we observed that GABAAR activation induced a shift in the localization of cyclin D1 from the cytoplasm to the nucleus both in vitro (Fig. 7A) and in vivo (Fig. 7E), in agreement with previous observations showing that PTEN regulates the intracellular localization of cyclin D1 [27].

Taken together, our data show that activation of GABAARs by endogenous GABA in vivo promotes the cell cycle entry of precursors lining the lateral ventricle. In particular, endogenous GABA increases the number of apical cells actively cycling via a mechanism involving EGFR and the regulation of PTEN/cyclin D1 expression.

DISCUSSION

This study provides evidence that in the neonatal SEZ GABAAR activation promotes EGFR cell surface expression and subsequent cell cycle entry of prominin+ precursors via a Ca2+-independent mechanism, which involves Cl hyperpolarization-mediated osmotic swelling and that benzodiazepine exerts a similar effect.

The specific effects of GABA on the stem cell population and the downstream intracellular mechanisms have not been elucidated [31]. This is also due to the difficulties of unambiguously identifying distinct precursor types.

In this study, we show that purified neonatal neuroblasts have large GABAergic currents carried by Cl with an affinity to GABA comparable with those already reported in postnatal SEZ cells [32, 33] and embryonic cortical newborn neurons [34] and that these receptors show benzodiazepine sensitivity. In comparison with neuroblasts, precursors display a lower incidence of GABAergic currents with a much smaller current density, nonetheless modulated by diazepam. The electrophysiological analysis correlates well with the quantitative difference in the expression of GABAAR between the different populations revealed by immunocytochemistry and is reminiscent of previous observations in the embryonic neocortex, where progenitors are known to display a much lower density of GABAARs than cortical neurons [34], suggesting that GABAAR function correlates with differentiation. However, our data indicate differences between precursor subgroups, whereby stem cells (prominin+) are more sensitive to GABA than TAPs. A possible explanation for this observation is that functional GABAAR expression in the latter is further hampered by the rapid proliferation characterizing this cell population.

Along with changes in the level of expression, we showed that GABAAR activation in precursors and neuroblasts leads to different physiological outcomes, that is, in neuroblasts it induces depolarization and a rise in Ca2+ levels, whereas in precursors it is hyperpolarizing and does not increase [Ca2+]i, suggesting that the Cl gradient is different in these two cell types [35]. This result further highlights the similarity between neonatal SEZ precursors and the embryonic neocortex, where the depolarizing effect of GABA along with increase in [Ca2+]i are primarily confined to cells undergoing neuronal differentiation [36]. Consistent with the opposite effects on membrane potential in the two cell types, osmotic swelling was not observed in neuroblasts but only in precursors, and in particular, in cells expressing prominin. This is consistent with our findings showing a greater expression of GABA signaling-associated molecules in the cells lining the lateral ventricle.

Prominin labels cells that contact and line the lateral ventricle, including ependymal cells that undergo phenotypic maturation during the first postnatal week [23]. One of the functions of these cells is to regulate exchange between the cerebrospinal and the interstitial fluid [1]. This involves both a specialized mechanism to regulate ion transport as well as the expression of selective water channels such as acquaporins [37–39]. In cells expressing these channels, the direction of the water flux is determined only by the osmotic gradient. Underscoring the importance of the process of water exchange in these populations, it was found that a lack of aquaporin 4, expressed in ependymal cells and in neurosphere precursors derived from adult stem cells, leads to a functional and structural breakdown of the ependyma and to an impairment of multiple aspects of neural precursor growth in vitro [40]. Therefore, our observations that GABAAR activation promotes osmotic swelling in prominin+ precursors suggest the possibility that GABA participates in the regulation of osmotic tension in this specialized subset of precursors. Within the glia lineage GABAAR is also highly expressed in astrocytes, which play a pivotal role in the regulation of water exchange in the brain. Interestingly, it has been recently shown that overexpression of diazepam-binding inhibitor, an endogenous-negative allosteric modulator of GABAARs, causes hydrocephalus [41].

A few reports have also shown osmotic swelling on GABAAR activation in other neural cells. For example, in cerebellar interneurons [42] and more recently in embryonic and peripheral neural crest stem cells [17] GABAAR-mediated osmotic tension induced Ca2+ influx and cell cycle arrest. These observations underscore how the consequence of the event of swelling are not stereotypical but strictly dependent on the cell type and its intrinsic properties.

We here found that on GABAA-R-induced osmotic swelling there is an increase of plasma membrane expression of EGFR. It is well known that tyrosine kinase receptors and specifically EGFR can be activated both by cell swelling or shrinkage [43, 44] and can modulate numerous intracellular signaling pathways, such as focal adhesion kinase, mitogen-activated protein (MAP) kinase (MAPK), or phosphoinositide-3 kinase (PI3K) [45, 46]. Our data indicate that EGFR signaling downstream of GABAARs promotes cell cycle entry of prominin+ precursors rather than a change in their cycle kinetics. Although the number of activated prominin+ precursors appears to be small, they may represent a source of a large number of proliferating and differentiating cells due to clonal expansion. Furthermore, as osmotic swelling may spread between coupled cells, this may amplify the response to GABA within the precursor pool. Indeed, our data show that GABAergic signaling is also increased by cell-cell contact, and this may explain the increase in the number of clonogenic prominin/EGFRhigh observed on diazepam injection.

CONCLUSION

In summary, our model proposes that endogenous GABAAR signaling activates prominin+ precursors in the neonatal SEZ via regulation of cell size and EGFR expression. This process may reflect the fact that prominin+ precursors are involved in the regulation of water exchange in the brain. In addition, we show that benzodiazepine treatment promotes precursor proliferation in the neonatal SEZ. These results may have potential implication for the therapy of neurological disorders involving administration of GABAARs modulators.

Acknowledgements

T.C., K.O., and F.C. acknowledge the Landesstiftung Baden-Württemberg for its support.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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