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. 2014 Aug 20;34(34):11228-32.
doi: 10.1523/JNEUROSCI.0015-14.2014.

Astroglial connexin43 hemichannels tune basal excitatory synaptic transmission

Oana Chever  1 , Chun-Yao Lee  1 , Nathalie Rouach  2
Affiliations

Astroglial connexin43 hemichannels tune basal excitatory synaptic transmission

Oana Chever et al. J Neurosci. .

Abstract

Fast exchange of extracellular signals between neurons and astrocytes is crucial for synaptic function. Over the last few decades, different pathways of astroglial release of neuroactive substances have been proposed to modulate neurotransmission. However, their involvement in physiological conditions is highly debated. Connexins, the gap junction forming proteins, are highly expressed in astrocytes and have recently been shown to scale synaptic transmission and plasticity. Interestingly, in addition to gap junction channels, the most abundant connexin (Cx) in astrocytes, Cx43, also forms hemichannels. While such channels are mostly active in pathological conditions, they have recently been shown to regulate cognitive function. However, whether astroglial Cx43 hemichannels are active in resting conditions and regulate basal synaptic transmission is unknown. Here we show that in basal conditions Cx43 forms functional hemichannels in astrocytes from mouse hippocampal slices. We furthermore demonstrate that the activity of astroglial Cx43 hemichannels in resting states regulates basal excitatory synaptic transmission of hippocampal CA1 pyramidal cells through ATP signaling. These data reveal Cx43 hemichannels as a novel astroglial release pathway at play in basal conditions, which tunes the moment-to-moment glutamatergic synaptic transmission.

Keywords: astrocytes; basal synaptic transmission; connexin43; hemichannel; hippocampus; physiology.

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Figures

Figure 1.
Figure 1.
Astroglial Cx43 HCs are open in basal conditions. A, Representative basal EtBr uptake (red) in stratum radiatum astrocytes labeled with GFAP (green) in hippocampal slices. Scale bar, 50 μm. B, Higher magnification of astroglial EtBr uptake (red) in basal conditions (Ct), and in the presence of CBX (200 μm), Gap26 (100 μm), or Gap26 scramble peptides (100 μm), applied 15 min prior and during EtBr uptake assay. Scale bar, 25 μm. C, Astrocytic EtBr uptake normalized to control conditions in slices from wild-type mice (WT) treated with CBX (200 μm, n = 7), Gap26 (100 μm, n = 7), Gap26 scramble (100 μm, n = 7) and 10Panx (400 μm, n = 5) peptides (left), and from astroglial conditional Cx43 knock-out mice (Cx43−/−) treated with CBX (200 μm, n = 4) or Gap26 (100 μm, n = 3; right). Asterisks indicate statistical significance performed on raw data (*p < 0.05, **p < 0.01, Student's paired t test).
Figure 2.
Figure 2.
Astroglial Cx43 HCs modulate basal excitatory synaptic transmission. A, B, Astroglial Cx43 HCs potentiate synaptic transmission. A, Quantification over time of EPSC amplitudes recorded in a representative CA1 pyramidal cell before and during Gap26 application (red). B, Sample traces and quantification of evoked EPSCs recorded in CA1 pyramidal neurons before [control (Ct), black] and during Gap26 application (Gap26, red, n = 9). C, D, Specificity of the Gap26 mimetic peptide. C, Sample traces and quantification of evoked EPSCs recorded in CA1 pyramidal cells before (black) and during (gray) the application of the Gap26 scramble peptide (n = 8). D, Sample traces and quantification of evoked EPSCs recorded in CA1 pyramidal neurons from astroglial conditional Cx43 knock-out mice (Cx43−/−) before (black) and during (red) the application of Gap26 (n = 17). Gap26 and scramble peptides were applied at 100 μm, and their effects were quantified after 10 min. Calibration: 20 pA, 20 ms. Asterisks indicate statistical significance (***p < 0.001, Student's paired t test).
Figure 3.
Figure 3.
Astroglial Cx43 HCs release ATP in basal conditions. A, Left, Schematic representation of the experimental design to measure quantitatively ATP release from hippocampal slices maintained in wells with oxygenated ACSF. Right, Quantification of luminescence detection of ATP release measured in 500 μl of ACSF containing three hippocampal slices in control conditions (Ct; black) and after Gap26 application (30 min; red; n = 4). B, Left, Schematic representation of dynamic extracellular ATP imaging in a hippocampal slice, using local delivery of luciferin-luciferase through a pipette located in CA1 stratum radiatum. Right, Quantification over time of luminescence detection of extracellular ATP levels before and during Gap26 application (red, n = 7). Calibration: 1 arbitrary unit, 1 min. Asterisks indicate statistical significance [Student's paired t test (A) or one-way repeated-measures ANOVA and Dunnett's post hoc test (B): **p < 0.01, ***p < 0.001].
Figure 4.
Figure 4.
ATP signaling mediates astrocytic Cx43 HC regulation of synaptic transmission. A, Endogenous extracellular ATP increases basal excitatory synaptic transmission through P2 receptors. Sample traces and quantification of evoked CA1 fEPSPs recorded before (Ct; black) and during application of ATP P2 receptor antagonists (RB2+PPADS; gray, n = 5). Calibration: 0.2 mV, 10 ms. B, C, Blockade of ATP P2 receptors occludes the effect of Cx43 HC inhibition on synaptic transmission. B, Quantification over time of EPSC amplitudes recorded in a representative CA1 pyramidal cell in the presence of ATP P2 receptor antagonists (RB2+PPADS preincubation; gray) before and during Gap26 application (red). C, Corresponding sample traces and quantification of averaged EPSC amplitudes (n = 6). Calibration: 40 pA, 40 ms. The effect of Gap26 (100 μm) and a mix of RB2 and PPADS (30 μm) was quantified after a 10–20 min application. Asterisks indicate statistical significance (**p < 0.01, Student's paired t test).
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