GABAA Increases Calcium in Subventricular Zone Astrocyte-Like Cells Through L- and T-Type Voltage-Gated Calcium Channels

In the adult neurogenic subventricular zone (SVZ), the behavior of astrocyte-like cells and some of their functions depend on changes in intracellular Ca2+ levels and tonic GABAA receptor activation. However, it is unknown whether, and if so how, GABAA receptor activity regulates intracellular Ca2+ dynamics in SVZ astrocytes. To monitor Ca2+ activity selectively in astrocyte-like cells, we used two lines of transgenic mice expressing either GFP fused to a Gq-coupled receptor or DsRed under the human glial fibrillary acidic protein (hGFAP) promoter. GABAA receptor activation induced Ca2+ increases in 40–50% of SVZ astrocytes. GABAA-induced Ca2+ increases were prevented with nifedipine and mibefradil, blockers of L- and T-type voltage-gated calcium channels (VGCC). The L-type Ca2+ channel activator BayK 8644 increased the percentage of GABAA-responding astrocyte-like cells to 75%, suggesting that the majority of SVZ astrocytes express functional VGCCs. SVZ astrocytes also displayed spontaneous Ca2+ activity, the frequency of which was regulated by tonic GABAA receptor activation. These data support a role for ambient GABA in tonically regulating intracellular Ca2+ dynamics through GABAA receptors and VGCC in a subpopulation of astrocyte-like cells in the postnatal SVZ.


INTRODUCTION
Neurogenesis persists in two regions of the adult brain, the SVZ along the lateral ventricle and the subgranular zone in the hippocampal dentate gyrus (Lledo et al., 2006;Zhao et al., 2008). The SVZ contains a mosaic of cell types including neuroblasts ensheathed by cells with astrocytic features such as glial fi brillary acidic protein (GFAP) expression. A subpopulation of these GFAP-expressing cells (also called astrocyte-like cells or SVZ astrocytes) generates intermediate progenitors called transit amplifying cells. The latter generate neuroblasts that differentiate into interneurons in the olfactory bulb. Previous studies have shown that the distinct steps of neurogenesis (i.e. migration, proliferation and differentiation) are infl uenced by local molecules. These molecules differentially affect intracellular Ca 2+ dynamics and regulate Ca 2+ -dependent intracellular pathways (Bordey, 2006;Pathania et al., 2010). One such local molecule is the amino acid γ-aminobutyric acid (GABA) that is synthesized and released by SVZ neuroblasts as shown using immunohistochemistry for glutamic acid decarboxylase (GAD) (the enzyme that catalyzes the decarboxylation of glutamate to GABA) and GABA, and electrophysiology to show functional release from neuroblasts (Stewart et al., 2002;Nguyen et al., 2003;Bolteus and Bordey, 2004;De Marchis et al., 2004;Liu et al., 2005;Gascon et al., 2006;Platel et al., 2008).
GABA acts through specifi c receptors, GABA A receptors, which are expressed in both SVZ neuroblasts and astrocytes (Stewart et al., 2002;Nguyen et al., 2003;Wang et al., 2003b;Bolteus and Bordey, 2004;Liu et al., 2005). In developing systems, the GABA A receptor is thought to regulate the behavior of immature cells through depolarization leading to the canonical activation of VGCCs and GABA A increases calcium in subventricular zone astrocyte-like cells through L-and T-type voltage-gated calcium channels equipped with an Olympus FluoView 1000 confocal microscope and a water-immersion Nomarski phase-contrast and fl uorescence 60× objective (N.A. 0.9).
Whole-cell patch clamp recordings were obtained as previously described (Wang et al., 2003a,b;Bolteus and Bordey, 2004;Liu et al., 2006). Pipettes had resistances of 6-8 MΩ when fi lled with an intracellular solution containing the following: 110 mM KCl, 1.0 mM CaCl 2 , 10 mM EGTA, 10 mM HEPES, 50 µM Alexa Fluor 488 dye and an ATP-regenerating solution that included 4 mM K 2 ATP, 20 mM K 2phosphocreatine, 50 U/ml creatine phosphokinase, and 6 mM MgCl 2 . The pH and the osmolarity were adjusted to 7.2 and 290 mOsm, respectively. The liquid junction potential (∼4 mV) was not corrected. Whole-cell recordings were performed using an Axopatch 200B amplifi er, and current signals were low-pass fi ltered at 2-5 kHz and digitized on-line at 5-20 kHz using a Digidata 1320 digitizing board (Axon Instruments, Foster City, CA, USA). Recorded cells were held at −60 mV. Voltage steps were applied from −100 to +100 mV by 20 mV increment. Capacitive and leak currents were not subtracted.

CALCIUM IMAGING
SVZ cells were loaded by pressure application of Fluo-4 AM (100 µM in aCSF, 0.4% Pluronic acid F-127, Invitrogen). We observed no differences in data obtained with either hGFAP-DsRed or hGFAP-MrgA1:GFP mice regarding the pharmacology of muscimol responses; we therefore pooled the data. Images were acquired every 2 s with FluoView acquisition software. In acute slices from hGFAP-MrgA1:GFP mice, Fluo-4 AM-astrocyte-like cells were distinguished from other SVZ cells due to their enhanced green fl uorescence on their cell membrane. In addition, application of a selective peptide agonist of MrgA1 receptors (that has no endogenous receptors) was routinely used to induce Ca 2+ responses selectively in SVZ astrocytes to identify them at the end of the experiments. ROIs were placed on SVZ cells that responded to the peptide during a 10-20 s application period.
For spontaneous movies, images were acquired every 2 s (0.5 Hz) for 10 min in each condition with 5 min of wash-in or wash-out between each movie. F 0 (i.e. baseline) and F are the mean fl uorescence intensities measured throughout all of the regions of interest (ROIs) and in each ROI, respectively. A change in fl uorescence was considered to be a Ca 2+ increase if it was >15% F/F 0 increase. Intracellular Ca 2+ changes were calculated using Calsignal (Platel et al., 2007) and Clampfi t 10. F/F 0 was detected with Calsignal and traces were exported into Clampfi t for peak analysis using the threshold detection function. For peak analysis, the baseline for each ROI trace was manually adjusted to zero. In addition, traces from control and drug-treated movies were concatenated and the same threshold for peak detection was used. ROIs were designated as "responding" if the cell was responsive at least 50% of muscimol applications in order to perform subsequent pharmacology. "Nonresponsive" cells were cells that displayed no increase in F/F 0.
Data are expressed as mean ± standard error. Statistical analysis used a two-tailed t-test except where noted.

PHARMACOLOGY
Ca 2+ imaging experiments were performed in the presence of the GABA B blocker CGP 52432 (1 µM) and blockers of glutamate receptors including D-APV (50 µM) for NMDA receptors, NBQX

ANIMALS
Experiments were performed in several lines of transgenic mice: (1) Mice expressing DsRed under the human GFAP promoter (hGFAP-DsRed mice) were produced by the co-authors, N.A. Jensen and J.V. Nielsen . To generate the hGFAP-DsRed transgene, the pDsRed2-1 plasmid (Clontech) was initially modifi ed to introduce a PacI site downstream of the SV40 polyadenylation signal, by fi lling in an Afl II site. Subsequently, the hGFAP promoter (Brenner et al., 1994) was cloned into the BglII and SalI sites, before a rabbit β-globin intron was inserted into the BamHI sites between the hGFAP promoter and the DsRed coding sequence. For microinjection, the transgene was excised from the plasmid backbone by digestion with BglII and PacI, gel-purifi ed and microinjected, at a concentration of 6 ng/µl, into the pronucleus of fertilized B6D2F1 mouse eggs as previously described . Transgenic hGFAP-DsRed mice were identifi ed by PCR with primers: 5′-TCTGGGCACAGTGACCTCAGTG and 5′-GGGACATCTTCCCATTCTAAAC.
(2) hGFAP-DsRed mice were crossed with homozygote mice carrying GFP under the doublecortin promoter (DCX-GFP mice, FVB/N strain, a gift from Dr. R. Miller, University of Chicago, originally from Gensat) to generate hGFAP-DsRed/DCX-GFP mice. These mice were used in 25% of the experiments instead of hGFAP-DsRed mice. (3) hGFAP-tTA/TetO-MrgA1:GFP mice (called hGFAP-MrgA1:GFP mice) were a gift from Dr. Ken McCarthy (University of North Carolina at Chapel Hill). In the absence of doxycycline, astrocytes express MrgA1 receptors fused to GFP (Fiacco et al., 2007). All experimental protocols were approved by the Institutional Animal Care and Use Committees of Yale School of Medicine and by the Danish National Animal Care and Use Committee. Mice were used between postnatal day (P) 20 and P42.

CHARACTERIZATION OF DSRED-POSITIVE CELLS IN THE SVZ OF HGFAP-DSRED MICE
We fi rst verifi ed that DsRed is expressed in astrocyte-like cells of the SVZ. We immunostained for GFAP (blue) in sections from hGFAP-DsRed/DCX-GFP mice (Figures 1A,B). We observed bright DsRed-positive cells, which were GFAP-positive (arrows in Figure 1B), and some faint DsRed cells, which were GFAP-negative and DCX-GFP-positive (arrowhead in Figure 1B). This result was confi rmed by staining for both GFAP and DCX in sections from hGFAP-DsRed mice ( Figure S1 in Supplementary Material). DsRedpositive cells occasionally stained positive for the transit amplifying cell marker epidermal growth factor receptor (EGFR, Figure S1B in Supplementary Material, arrowhead). The known half-life of DsRed (∼4 days) allows it to persist at a lower level in daughter cells. These fi ndings are thus in agreement with GFAP-positive cells (i.e. SVZ astrocytes) being neural progenitors that generate EGFR-cells and neuroblasts (Doetsch et al., 2002;Platel et al., 2009).
We next performed patch clamp recordings of DsRed-fl uorescent cells in acute slices where cells with different fl uorescence intensity could be observed (Figures 1C,D). Every bright DsRed-fl uorescent cell recorded had a hyperpolarized resting potential (mean of −80.8 ± 1.0 mV), low input resistance (57.4 ± 9.1 MΩ, n = 7), and a linear current-voltage relationship following depolarizing steps (from −160 to +100 mV, Figures 1E,F) from a holding potential of −80 mV. Thus, bright DsRed-fl uorescent have characteristics of thus tested the effects of three VGCC blockers on the percentage of SVZ astrocytes displaying muscimol-induced Ca 2+ increases in slices from both hGFAP-DsRed and hGFAP-MrgA1:GFP mice. VGCCs are grouped into three families: (1) the high-voltage activated dihydropyridine (DHP)-sensitive channels (L-type, Ca V 1.x), (2) the high-voltage activated DHP-insensitive channels (P-, N-and R-type, Ca V 2.x), and (3) the low-voltage activated channels (T-type, Ca V 3.x). We tested the following three blockers: Nickel (Ni 2+ , 100 µM) for R-type Ca V 2.3 and T-type Ca V 3.2 channels (high sensitivity), and for other T-type channels (low-sensitivity); the DHP antagonist nifedipine (10 µM) for L-type channels, and mibefradil (10 µM) for T-type channels. Ni 2+ signifi cantly decreased the percentage of muscimol-responding astrocytes but by only 30 ± 9.4% (n = 50 cells, six slices, Figures 4E,F). Nifedipine and mibefradil signifi cantly decreased the percentage of muscimol-responding astrocytes by 61 ± 10% Figures 4A,B and 48 ± 4%, respectively (n = 217 cells, six slices, and 78 cells, four slices, respectively, Figures 4E,F). When used together, these three VGCC blockers abolished muscimol responses in 90 ± 4% of the responding SVZ astrocytes (Figures 4E,F). Of the cells that continue to respond, none of these drugs had any effect on the amplitude or area of the Ca 2+ responses ( Figure 4G). These data suggest that GABA A -induced Ca 2+ responses in astrocyte-like cells of the SVZ are predominately mediated by Ca 2+ infl ux through L-and T-type VGCCs.
As illustrated in Figures 3D and 4E, 40-50% of SVZ astrocytes display Ca 2+ increases in response to GABA A receptor activation. Considering that nearly all SVZ astrocytes tested have been reported to display functional GABA A currents (Liu et al., 2005), the lack of Ca 2+ responses could be due to either the absence of functional VGCCs or the fact that GABA A depolarization does not reach VGCC threshold. We thus tested the L-type VGCC activator BayK 8644. In the presence of BayK 8644, there was a signifi cant increase (to 190% of control) in the % of cells responding to muscimol (from ∼35% to 75%, Figures 4C,D,E,F). These data suggest that most astrocyte-like cells express DHP-sensitive VGCCs. BayK 8644 also increased the area, but not the amplitude, of muscimol-induced Ca 2+ increases (Figure 4G), suggesting that BayK 3644 prolonged Ca 2+ responses due to GABA A receptor activation.
Considering that increases in intracellular Ca 2+ can further trigger Ca 2+ increases from intracellular stores, we tested 2-APB, a blocker of inositol-1,4,5-trisphosphate receptors (IP3) and IP3-sensitive intracellular stores, among other channels at 100 µM (Bootman et al., 2002;Peppiatt et al., 2003). Bath application of 2-APB resulted in a 30 ± 9% decrease in the number of muscimol-responding astrocytes, but this decrease was not signifi cant (Figures 4E,F, t-test, p > 0.05). Nevertheless, when analyzing cells that continue to respond after drug application, 2-APB signifi cantly reduced the amplitude and area of muscimol-induced Ca 2+ transients, while nifedipine and other VGCC blockers did not ( Figure 4G). These data suggest that Ca 2+ release from intracellular stores contributes to the increase in intracellular Ca 2+ concentration following GABA A depolarizationinduced Ca 2+ infl ux through VGCCs.

AMBIENT GABA CONTROLS THE FREQUENCY OF BASELINE CA 2+ ACTIVITY IN SVZ ASTROCYTES
We previously reported that 60% of SVZ astrocytes recorded with the patch clamp technique displayed a tonic current due to GABA A receptor activation (Liu et al., 2005). We thus examined whether astrocytes of the SVZ (Liu et al., 2006). By contrast, every recorded, faint DsRed-fl uorescent cell displayed characteristics of neuroblasts (Wang et al., 2003a;Bolteus and Bordey, 2004). These characteristics include high input resistance (mean of 4.4 ± 1.3 GΩ), depolarized zero-current resting potentials (−42.7 ± 8.7 mV, n = 5), and the presence of voltage-dependent outward currents (Figures 1E,F). These data suggest that bright and faint DsRed-fl uorescent cells can be unambiguously identifi ed as astrocyte-like cells and neuroblasts, respectively, in acute slices.

GFP-POSITIVE CELLS IN THE SVZ OF HGFAP-MRGA1:GFP MICE ARE ASTROCYTE-LIKE CELLS
One important limitation of the hGFAP-DsRed mice for performing Ca 2+ imaging is the fact that not every astrocyte-like cell is DsRedfl uorescent (Figures 1A,B). This is more apparent in sections from hGFAP-DsRed mice crossed with hGFAP-GFP mice. While some DsRed-fl uorescent cells are GFP-positive (arrows), not all GFPpositive cells are DsRed-fl uorescent (arrowheads, Figures 2A-C). We thus acquired transgenic mice that express an exogenous Gq-protein coupled receptor (called Mas-related gene A1, MrgA1) in GFAP-expressing cells (i.e. astrocytes). MrgA1 has no endogenous ligand in the brain (Fiacco et al., 2007). The MrgA1 receptor fused to GFP was targeted to astrocytes using the inducible tet-off system. Mice expressing the tetracycline transactivator (tTA) under the human GFAP promoter were crossed to mice in which the MrgA1:GFP receptor was transcribed using the tet-off (tetO) minimal promoter. In the SVZ of hGFAP-tTA/tetO-MrgA1:GFP mice (referred henceforth as hGFAP-MrgA1:GFP mice), GFP displays a membrane expression selectively in all astrocyte-like cells (GFAP+ cells, red) but not in neuroblasts (DCX+ cells, blue, Figures 2D,E). In addition, SVZ astrocytes loaded with the Ca 2+ indicator dye Fluo-4 AM can be further identifi ed with a peptide agonist for MrgA1 receptors that does not bind endogenous receptors in the brain. Pressure application of phe-leu-arg-phe amide peptide (FLRFa, 50 µM, 10-20 s) induced Ca 2+ increases in GFP-decorated cells, i.e. astrocyte-like cells (Figures 2F,G).

GABA A RECEPTOR ACTIVATION LEADS TO CA 2+ INCREASES IN SVZ ASTROCYTES THROUGH VGCCS
We previously reported that astrocyte-like cells express functional GABA A receptors using patch clamp recordings (Liu et al., 2005). However, it remained unknown whether these receptors led to Ca 2+ increases in these cells. We pressure loaded slices with Fluo-4 AM in slices from hGFAP-DsRed and hGFAP-MrgA1:GFP mice (Figures 3A,E,  respectively). Application of the GABA A receptor agonist muscimol (50 µM, 5 s) increased Ca 2+ in 38.1 ± 4.8% and 51.3 ± 3.5% of SVZ astrocytes from hGFAP-DsRed and hGFAP-MrgA1:GFP mice (106 cells, n = 17 slices and 396 cells, 16 slices, respectively, Figures 3B,D,F). Experiments were performed in the presence of glutamate and GABA B receptor blockers (see Materials and Methods). GABA A -induced Ca 2+ responses were abolished by either bicuculline or picrotoxin (50 µM), two GABA A receptor antagonists, in 94 ± 4.67% of the muscimolresponding cells (27 cells, n = 5 slices total, Figures 3C,G and 4E,F).
GABA A receptor activation is known to depolarize parenchymal astrocytes (Barakat and Bordey, 2002;Bekar and Walz, 2002). Following GABA A receptor-induced depolarization, intracellular Ca 2+ increases in immature cells are also thought to result from the canonical activation of VGCCs (Owens and Kriegstein, 2002;Meier et al., 2008). We  Figure 5D). To test the effect of drugs on Ca 2+ activity, a 10-min recording was following by a 5-min drug wash-in and an additional 10-min recording in the presence of the drug. With no drug application (control movies), spontaneous Ca 2+ activity was ambient GABA exerted a tonic regulation of Ca 2+ dynamics in SVZ astrocytes. Recordings were performed in the presence of blockers of GABA B and glutamate receptors, as described in the Methods. We found that 77.8% of astrocyte-like cells displayed baseline Ca 2+ activity in the form of Ca 2+ transients at an average frequency of 0.252 Hz stable over time. Following analysis of Ca 2+ frequency in the fi rst and second 10-min periods, we found that 26.3 ± 4.8% and 19.0 ± 2.5% of the cells displayed a non-signifi cant increase and decrease in the frequency of Ca 2+ transient to 117.0 ± 6.3% and 81.7 ± 6.8% of control, respectively (n = 47 cells, three slices). Spontaneous Ca 2+ activity was eliminated following 15-20 min of 2-APB application (data not shown), as expected, since regenerative Ca 2+ transients have been shown to involve Ca 2+ from internal stores that rely on the activation of IP3 receptors in various cell types (D'Andrea et al., 1993;Ciapa et al., 1994;Liu et al., 2001;Bellamy, 2006). When recorded in the presence of bicuculline (Bic) under baseline conditions, wash-out of bicuculline had two distinct effects on SVZ astrocytes. In 79 ± 3% of the astrocyte-like cells bicuculline wash-out resulted in a signifi cant increase in the frequency of Ca 2+ transients to 216 ± 39% of control (from 0.154 to 0.36 Hz, n = 90 cells, Figures 5A,B, p < 0.01). In addition, in 18 ± 6% of the SVZ astrocytes bicuculline wash-out resulted in a signifi cant decrease in the frequency of Ca 2+ transients to 72.9 ± 4.6% of control (from 0.45 to 0.34 Hz, n = 15/90 cells, Figure 5B). Wash-out of SR-95531 (gabazine, 100 nM), a non-competitive GABA A receptor antagonist, had similar effects (data not shown). Gabazine wash-out increased and decreased the frequency of Ca 2+ transients in 41% and 27% of the SVZ astrocytes, respectively (n = 3 slices, data not shown). Bath application of bicuculline had the opposite effects to that of bicuculline wash-out, as expected. Bicuculline wash-in signifi cantly decreased the frequency of Ca 2+ transients to 60 ± 6% of control in 46 ± 8% of astrocyte-like cells (from 0.252 to 0.175 Hz, n = 185 cells) and increased the frequency to 223 ± 32% of control in 31 ± 3% of the cells in the same slices (from 0.10 to 0.25 Hz, n = 185 cells, four slices, Figures 5C,D, p < 0.05).

DISCUSSION
In this study we fi rst report the use of two transgenic mouse lines to perform Ca 2+ imaging in astrocyte-like cells of the SVZ. Without these mice, identifying Fluo-4 AM-loaded astrocytes among other SVZ cells was not feasible. hGFAP-DsRed mice are the most practical mouse line for performing Ca 2+ imaging with the green fl uorescent dye Fluo-4 AM since DsRed fl uorescence is readily distinguishable from Fluo-4 fl uorescence. However, in these mice not every SVZ astrocyte fl uoresces red, and it is unclear whether a sub-population is targeted. In addition, the long half-life of DsRed could be an issue for positive identifi cation. Newly born neuroblasts are DsRed fl uorescent but are dimmer than astrocytes. The hGFAP-MrgA1:GFP mouse line appears to be the best model to study Ca 2+ activity in SVZ astrocytes. GFP being fused to the MrgA1 receptor highlights the cell membrane and appears brighter than cytoplasmic Fluo-4 AM-loading. In addition, and perhaps more importantly, SVZ astrocytes can be selectively identifi ed following increases in intracellular Ca 2+ using the FLRFa peptide. In our system bath loading of the Ca 2+ indicator Fluo-4 AM resulted in surface labeling of neuroblasts (data not shown). In the SVZ, cells are densely packed, thus limiting dye diffusion inside the tissue. In addition, neuroblasts tend to "bulge out" of the slice surface while astrocytes remain anchored inside the tissue as previously reported (Wang et al., 2003a). Instead, we used pressure loading inside the tissue resulting in preferential loading of SVZ astrocytes. With these experiments, we recommend using hGFAP-MrgA1:GFP mice to study Ca 2+ activity in astrocyte-like cells of the SVZ in future studies. Next, our pharmacological analyses suggest that the GABA Amediated Ca 2+ increases arise from Ca 2+ infl ux through L-and Ttype VGCCs that are then amplifi ed by Ca 2+ -induced Ca 2+ release from internal stores possibly involving IP3 receptors. This pathway is well-described in other neuronal and non-neuronal cell populations, and implies that GABA A receptor activation results in membrane depolarization that, in turn, activates VGCCs. Indeed, astrocytes are known to be depolarized by GABA A receptor activation resulting in GABA A -induced Ca 2+ increases (Bekar and Walz, 2002;Meier et al., 2008). GABA A depolarization is likely due to high intracellular Cl − in SVZ astrocytes, although expression of the Cl − importing transporter Na + /K + /2Cl − (NKCC1) and importantly the absence of the exporting Cl − transporter K + /2Cl − (KCC2) have not been examined in SVZ cells.
Considering the low-activation threshold of T-type VGCCs (as low as −70 mV) (Nilius et al., 2006), it is easily conceivable that these VGCCs can be opened by GABA A -induced depolarization based on the published biophysical properties of SVZ astrocytes. Indeed, GABA A currents (100 µM GABA) range from −20 to −400 pA in gap-junction coupled SVZ astrocytes held at −84 mV (Liu et al., 2005). The current has a mean of −60 pA in SVZ astrocytes recorded in the presence of a gap junction channel blocker (unpublished data). These cells were recorded with an intracellular solution containing near-physiological chloride concentration. SVZ astrocytes' mean input resistance is 250 MΩ (range from 50 to 500 MΩ) and their mean resting potential is −85 mV ranging from −73 to −95 mV (Liu et al., 2006). Thus, a 60-pA GABA A -current in a 250-MΩ astrocyte would result in a 15-mV depolarization, which is suffi cient to reach the threshold for T-type VGCC activation.
L-type VGCCs are high-voltage activated ranging between −50 and −40 mV (Lacinova, 2005). Around ∼40-50% of total astrocytes are muscimol-responsive in control conditions (from Figure 3E). Of those, ∼40% are nifedipine-sensitive (see Figure 4F). Therefore, the population of astrocytes that reach the threshold for activation by L-type VGCCs comes to ∼16-20% of all SVZ astrocytes. Similarly, SVZ astrocytes with higher input resistances (above 250 MΩ) correspond to ∼40% of the total SVZ astrocytes (see Figure 6 in Liu et al., 2006). Of those with higher input resistance, 20% also have FIGURE 4 | Muscimol responses in SVZ astrocytes are inhibited by L-and T-type Ca 2+ channel blockers. (A,C) Confocal images of Fluo-4 AM-loaded sagittal slices from hGFAP-MrgA1:GFP mice in control and in the presence of nifedipine in (A) (10 µM, an L-type Ca 2+ channel blocker) or BayK in (C) (10 µM, a L-type Ca 2+ channel enhancer). The red circles indicate muscimol-responsive SVZ astrocytes; the white circles indicate non-responsive cells. More cells respond to muscimol in the presence of BayK (indicated by red circles) while nifedipine silences cells. (B) Representative muscimol-induced Ca 2+ responses from a "responding" cell (red circle) and subsequent block by nifedipine. (D) Representative muscimol-induced Ca 2+ responses after bath application of BayK from a previously "non-responding" cell (white circle). Red bar: 10 s muscimol (20 µM). (E) Bar graphs showing the % of cells responding to muscimol from total ROI analyzed. Bic: bicuculline. (F) Bar graphs of the % of active cells responding after drug treatment. (G) Bar graphs of % of control of amplitude and area from cells that continued to respond after drug treatment. NS = not signifi cant, * = p < 0.05. resting potentials more depolarized than −80 mV, both of which would be required to reach L-type VGCC activation threshold. In this population, a current of 120 pA or less will be suffi cient to reach the activation threshold of L-type VGCCs.
In the non-responding SVZ astrocytes, it is possible that either these cells do not express functional VGCCs or that GABA Adepolarization did not reach the activation threshold for VGCCs. The fact that in the presence of BayK 8644, three-quarters of the SVZ astrocytes responded to muscimol suggests that the majority of, and perhaps all, astrocyte-like cells expresses DHP-sensitive VGCCs. These data suggest that GABA A -depolarization in non-responding astrocyte-like cells does not reach the threshold to open VGCCs.
A question associated with the above data is whether there is enough GABA in the ambient milieu to activate GABA A receptors and modulate Ca 2+ activity. GABA is synthesized and released by neuroblasts (Stewart et al., 2002;Nguyen et al., 2003;Bolteus and Bordey, 2004;De Marchis et al., 2004;Liu et al., 2005;Gascon et al., 2006) that may provide a tonic versus a phasic release of GABA (for review and discussion see Bordey, 2007). There are no known phasic or synaptic sources of GABA in the SVZ. It was suggested that ambient GABA levels may be in the µM range in the SVZ (Bolteus and Bordey, 2004;Bolteus et al., 2005). GABA levels are expected to fl uctuate over time because neuroblasts migrate, thus changing the microenvironment of astrocyte-like cells. GABA A receptors in astrocyte-like cells have a reported EC 50 for GABA of 15 µM and are thus expected to be tonically activated by low µM ambient GABA (Liu et al., 2005). Indeed, based on published data, GABA A receptors are tonically activated in 60% of the astrocyte-like cells (Liu et al., 2005). It was shown that bicuculline or picrotoxin applications induced a small shift in the current baseline that would correspond to a small depolarization of astrocyte-like cells.
Consistent with the predictions discussed above, we report that astrocyte-like cells of the SVZ display spontaneous Ca 2+ transients, the activity of which is tonically regulated by ambient GABA acting on GABA A receptors. In particular, GABA A receptor inhibition revealed that tonic GABA A activation had two effects on SVZ astrocyte-like cells, dividing SVZ astrocytes into two functionally distinct populations. In one subpopulation of these cells, tonic GABA A receptor activation increased the frequency of Ca 2+ transients while it decreased it in the other subpopulation. This latter GABA A action may result from a shunting effect of GABA A conductance in astrocyte-like cells. In future work, it will be important to understand how Ca 2+ transients are generated in SVZ astrocytes.
In conclusion, tonic regulation of Ca 2+ activity by ambient GABA could have major implications for the behavior of the Ca 2+ -dependent release of diffusible molecules from astrocytes such as ATP (Striedinger et al., 2007). Considering that GABA A receptor activation has been shown to limit SVZ astrocyte proliferation (Liu et al., 2005), it remains to be determined whether GABA A 's effect on proliferation involves L-and/or T-type VGCCs.