Augmentation of Ca(2+) signaling in astrocytic endfeet in the latent phase of temporal lobe epilepsy

Abstract

Astrocytic endfeet are specialized cell compartments whose important homeostatic roles depend on their enrichment of water and ion channels anchored by the dystrophin associated protein complex (DAPC). This protein complex is known to disassemble in patients with mesial temporal lobe epilepsy and in the latent phase of experimental epilepsies. The mechanistic underpinning of this disassembly is an obvious target of future therapies, but remains unresolved. Here we show in a kainate model of temporal lobe epilepsy that astrocytic endfeet display an enhanced stimulation-evoked Ca(2+) signal that outlast the Ca(2+) signal in the cell bodies. While the amplitude of this Ca(2+) signal is reduced following group I/II metabotropic receptor (mGluR) blockade, the duration is sustained. Based on previous studies it has been hypothesized that the molecular disassembly in astrocytic endfeet is caused by dystrophin cleavage mediated by Ca(2+) dependent proteases. Using a newly developed genetically encoded Ca(2+) sensor, the present study bolsters this hypothesis by demonstrating long-lasting, enhanced stimulation-evoked Ca(2+) signals in astrocytic endfeet.

Keywords: GCaMP; astrocytes; dystrophin; glia; kainate; perivascular.

Figure 1

Experimental design. (A) rAAV-GFAP-GCaMP5E virus was injected into both hippocampi 2–3 weeks prior to unilateral, intracortical kainate injection. Acute hippocampal slices were prepared and imaged at 1, 3 and 7 days post kainate injection. (B) Immunofluorescence with green fluorescent protein (GFP) antibodies (green) showed robust GGaMP5E expression in GFAP immunopositive (red) astrocytes. The vascular endothelium was labeled with CD31 antibodies (blue). Arrow: astrocyte soma, double arrow: astrocyte process, arrowhead: endfoot. or, stratum oriens; pyr, stratum pyramidale; rad, stratum radiatum. Scale bars, 50 µm and 20 µm (boxed motif expanded in inset).

Figure 2

Two-photon imaging of stimulation-evoked astrocytic Ca²⁺ signals in adult mouse hippocampal slices 1, 3 and 7 days after intracortical kainate injection. (A) Standard deviation (SD) images of GCaMP5E fluorescence intensities from 1 Hz time-lapse recording 1 day after kainate injection. GCaMP5E fluorescent astrocytic somata (s), processes (p), and endfeet (e) are indicated. The fluorescence traces from the astrocytic compartments indicated in the images are shown below. Scale bar, 20 µm. (B) Amplitudes, duration, latency and rise rate of Schaffer collateral/commissural fiber (Scc) stimulation (20 Hz, 10 s) evoked GCaMP5E fluorescence transients in astrocytic somata, processes and endfeet in control (non-injected side; 20 mice, 22 slices), 1 day (11 mice, 16 slices), 3 days (3 mice, 6 slices) and 7 days (6 mice, 9 slices) after kainate injection. Lower panel shows average fEPSP for the different time points. Values are mean ± s.e.m. Asterisk, P < 0.05. Double asterisk, P < 0.0001.

Figure 3

Effect of mGluR antagonists on the stimulation evoked astrocytic Ca²⁺ signals in the kainate injected side. (A) Amplitude, duration, latency and rise rate of Ca²⁺ signals in different astrocytic territories before (−) and after (+) administration of the mGluR I/II antagonist MCPG. (B) Representative fEPSP traces before (CTRL; blue) and after (red) MCPG administration and mean fEPSP amplitudes. (C) As in (A), but with MPEP instead of MCPG. (D) As in (B), but with MPEP instead of MCPG. Values are mean ± s.e.m. Asterisk, P < 0.05. Double asterisk, P < 0.0001.