GABA(A) Increases Calcium in Subventricular Zone Astrocyte-Like Cells Through L- and T-Type Voltage-Gated Calcium Channels

Abstract

In the adult neurogenic subventricular zone (SVZ), the behavior of astrocyte-like cells and some of their functions depend on changes in intracellular Ca(2+) levels and tonic GABA(A) receptor activation. However, it is unknown whether, and if so how, GABA(A) receptor activity regulates intracellular Ca(2+) dynamics in SVZ astrocytes. To monitor Ca(2+) 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. GABA(A) receptor activation induced Ca(2+) increases in 40-50% of SVZ astrocytes. GABA(A)-induced Ca(2+) increases were prevented with nifedipine and mibefradil, blockers of L- and T-type voltage-gated calcium channels (VGCC). The L-type Ca(2+) channel activator BayK 8644 increased the percentage of GABA(A)-responding astrocyte-like cells to 75%, suggesting that the majority of SVZ astrocytes express functional VGCCs. SVZ astrocytes also displayed spontaneous Ca(2+) activity, the frequency of which was regulated by tonic GABA(A) receptor activation. These data support a role for ambient GABA in tonically regulating intracellular Ca(2+) dynamics through GABA(A) receptors and VGCC in a subpopulation of astrocyte-like cells in the postnatal SVZ.

Keywords: GABA; GABA receptors; astrocyte; neurogenesis; progenitor cells; proliferation; stem cells; subventricular zone.

Figure 1

Characterization of hGFAP-DsRed-fluorescent cells in the SVZ. (A) Confocal images (one optical section) of GFAP immunostaining (blue) in the SVZ contained in a section from a hGFAP-DsRed/DCX-GFP mouse (P30). (B) Higher power photographs of the staining shown in the white square in (A). The arrows point to GFAP-positive DsRed-fluorescent cells while the arrowhead points to GFAP-negative, GFP-fluorescent neuroblasts. (C and D) Photographs of Alexa fluor 488-filled bright (C) and faint (D) red cells during patch clamp recording in acute slices from hGFAP-DsRed mice. Scale bar: 15 μm. (E) Traces of whole cell currents obtained from the cells shown in (C, bright red) and (D, faint red). The cells in (C) and (D) display the current profiles of a GFAP-progenitor and a neuroblast, respectively. (F) Mean current–voltage relationships (measured at the end of the voltage steps, arrows in E) of bright cells (red filled circles, n = 6) and faint cells (blue open circles, n = 5) give reversal potentials of −81 and −43 mV, respectively.

Figure 2

Characterization of hGFAP-tTA-MrgA1:GFP mice in the SVZ. (A–C) Confocal z-section of a slice from a hGFAP-tTA-MrgA1:GFP/hGFAP-DsRed mouse. The arrows point to SVZ astrocytes that are both DsRed and GFP-fluroescent, while the arrowheads point to astrocytes that are only GFP-positive. Scale bar = 30 μm. (D) A confocal z-stack (four images, spaced by 1.5 μm) of a slice from a hGFAP-tTA-MrgA1:GFP mouse. Astrocytes express the MrgA1:GFP. GFP signal colocalized with GFAP (red) but does not colocalize with neuroblast marker DCX (blue). Scale bar = 30 μm (E) Higher power image of region indicated by a white box in (D). Scale bar = 10 μm. (F) Confocal images of Fluo-AM-loaded slice from a hGFAP-tTA-MrgA1:GFP mouse before and during peptide FLRFa application. Regions of interest (ROIs) are indicated by colored circles. Scale bar = 10 μm (G) Traces showing F/F₀ from ROIs in (F), analyzed in CalSignal. CC = corpus callosum, Str = striatum.

Figure 3

GABA_A receptors regulate Ca²⁺ dynamics in SVZ astrocytes. (A) Z-stack confocal image (six images spaced by 1.5 μm) of Fluo-4 AM loaded SVZ cells in a coronal slice from a hGFAP-DsRed mouse. Scale bar = 10 μm. (B) Confocal image of the same cells shown in the white square in (A) under control conditions, during and following muscimol application. The arrow points to the DsRed-fluorescent cell, which responded to muscimol. (C) Representative muscimol-induced Ca²⁺ responses under control and in the presence of bicuculline (a GABA_A receptor blocker). The red trace represents the average of the individual gray traces (n = 7 cells). (D) Box plots of the percentage of GFAP-progenitors responding to muscimol in hGFAP-DsRed or hGFAP-MrgA1:GFP mice (n = 9 and 17 slices, respectively). Box: SEM, bar: median, diamonds: individual slice values. (E) Confocal image (one optical section) of Fluo-4 AM loaded SVZ cells (green) in a sagittal slice from a hGFAP-MrgA1:GFP mouse. Second panel shows SVZ cells responding to muscimol. The last panel is an overlay of images from responses to FLRFa peptide (orange, indicating SVZ astrocytes) and muscimol (green). Scale bar = 10 μm. (F) Confocal image of the same cells shown in the white square in (E) under control conditions, during and following muscimol application. The arrow points to the MrgA1:GFP-fluorescent, FLRFa-responsive cell, which also responded to muscimol. (G) Representative muscimol-induced Ca²⁺ responses under control and in the presence of bicuculline in cells from hGFAP-MrgA1:GFP slices. The red trace represents the average of the individual gray traces (n = 8 cells).

Figure 4

Muscimol responses in SVZ astrocytes are inhibited by L- and T-type Ca²⁺ 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 Ca²⁺ channel blocker) or BayK in (C) (10 μM, a L-type Ca²⁺ 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 Ca²⁺ responses from a “responding” cell (red circle) and subsequent block by nifedipine. (D) Representative muscimol-induced Ca²⁺ 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 significant, * = p < 0.05.

Figure 5

Spontaneous Ca²⁺ activity in SVZ astrocytes are sensitive to GABA_A receptor inhibition. (A,C) Representative traces of spontaneous Ca²⁺ activity from SVZ astrocytes before and after bicuculine (50 μM) wash-out (A) or wash-in (C). (B) Bar graphs showing that 79% of SVZ astrocytes show an increased frequency to 220% of control during bicuculline wash-out. (D) Bar graphs showing that during bicuculline wash-I, 40% of SVZ astrocytes show a decreased 60% in frequency to compared to control. *indicates statistical significance.