Activation of calcineurin underlies altered trafficking of α2 subunit containing GABAA receptors during prolonged epileptiform activity

Abstract

Fast inhibitory signalling in the mammalian brain is mediated by gamma-aminobutyric acid type A receptors (GABAARs), which are targets for anti-epileptic therapy such as benzodiazepines. GABAARs undergo tightly regulated trafficking processes that are essential for maintenance and physiological modulation of inhibitory strength. The trafficking of GABAARs to and from the membrane is altered during prolonged seizures such as in Status Epilepticus (SE) and has been suggested to contribute to benzodiazepine pharmacoresistance in patients with SE. However, the intracellular signalling mechanisms that cause this modification in GABAAR trafficking remain poorly understood. In this study, we investigate the surface stability of GABAARs during SE utilising the low Mg(2+) model in hippocampal rat neurons. Live-cell imaging of super ecliptic pHluorin (SEP)-tagged α2 subunit containing GABAARs during low Mg(2+) conditions reveals that the somatic surface receptor pool undergoes down-regulation dependent on N-methyl-d-aspartate receptor (NMDAR) activity. Analysis of the intracellular Ca(2+) signal during low Mg(2+) using the Ca(2+)-indicator Fluo4 shows that this reduction of surface GABAARs correlates well with the timeline of intracellular Ca(2+) changes. Furthermore, we show that the activation of the phosphatase calcineurin was required for the decrease in surface GABAARs in neurons undergoing epileptiform activity. These results indicate that somatic modulation of GABAAR trafficking during epileptiform activity in vitro is mediated by calcineurin activation which is linked to changes in intracellular Ca(2+) concentrations. These mechanisms could account for benzodiazepine pharmacoresistance and the maintenance of recurrent seizure activity, and reveal potential novel targets for the treatment of SE.

Keywords: Calcium signaling; Epilepsy; GABA(A) receptor; Surface stability; Trafficking.

Fig. 1

Somatic surface α_2SEP-GABA_ARs decrease upon low Mg²⁺ treatment. (A) Representative average intensity projection of α_2SEP expression in control and low Mg²⁺ treated neurons over time (0–3 min and 10–20 min). Scale bar, 10 μm. (B) Kymograph (a line scan vertically projected over time) showing somatic (left; scale bar, 5 μm) and clustered (right; arrow heads indicate clusters; scale bar, 2 μm) α_2SEP fluorescence intensity over the movie in control (aCSF) conditions and in the presence of low Mg²⁺ (grey bar). Red bar on the right indicates a decrease in somatic fluorescence intensity upon low Mg²⁺ treatment. (C) Average fluorescence intensity of somatic α_2SEP GABA_AR F/F₀: control (green, n = 9 cells) and low Mg²⁺ (blue, n = 7). (D) Time course of diffuse α_2SEP GABA_AR F/F₀: control (green, n = 9 cells); low Mg²⁺ (purple, n = 7). (E) Time course of α_2SEP GABA_AR clusters F/F₀: control (green, n = 9 cells) and low Mg²⁺ (light blue, n = 7). (F) Bar graph of ROI's F/F₀: soma (left), diffuse (middle) clusters (right). Significant loss of fluorescence in the soma compared to control at 20 min following low Mg²⁺ treatment (p = 0.02). Diffuse fluorescence is not altered at 20 min (p = 0.36) after low Mg²⁺ treatment. Fluorescence intensity of α_2SEP GABA_AR clusters is unaltered following low Mg²⁺ treatment (t = 20 min; p = 0.28) compared to control. (F') At 60 min after low Mg²⁺ treatment somatic (p = 0.09), diffuse (p = 0.85) and clustered (p = 0.42) fluorescence intensity are not significantly altered.*p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

NMDARs mediate low Mg²⁺ induced somatic α_2SEP GABA_AR surface decrease. (A) Representative images of α_2SEP GABA_AR fluorescence in control, low Mg²⁺ and low Mg²⁺ with dAPV (low Mg²⁺/dAPV) treated neurons as an average intensity projection over time (0–3 min and 10–20 min). Somatic α_2SEP GABA_AR loss highlighted in red; scale bar, 10 μm. (B) Kymograph showing somatic (left; scale bar, 5 μm) fluorescence intensity over the movie (duration: 60 min) in control (aCSF) conditions and in the presence of low Mg²⁺ and low Mg²⁺/dAPV (grey bar). Red bar on the right indicates decrease in somatic fluorescence intensity upon low Mg²⁺ treatment and blocking of low Mg²⁺ induced effect by dAPV (B). (C) Time course of somatic α_2SEP GABA_AR F/F₀: control (green, n = 7 cells), low Mg²⁺ (blue, n = 8 cells) and low Mg²⁺/dAPV (dark blue, n = 6 cells). (C') Summary of somatic F/F₀ at 20 min after low Mg²⁺ treatment. Low Mg²⁺ induces a significant decrease (p < 0.001) in somatic fluorescence intensity, which is inhibited by application of NMDAR blocker dAPV (p < 0.001). ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Low Mg²⁺ treatment induces intracellular Ca²⁺ accumulation. (A) Representative average intensity projection of fluo-4 loaded neurons (coloured squares indicate individual cell bodies). (B) Raw fluorescence intensity of spontaneous Ca²⁺ transients in a neuron (bottom) and correlating kymograph (top; segmented line through somatic ROI) showing fluorescence changes over time under control (aCSF) conditions. (B') Raw fluorescence intensity reporting low Mg²⁺ (grey bar) induced Ca²⁺ accumulation (bottom) and kymograph (top) indicating increase in fluorescence intensity. (C) Quantification of increase in intracellular Ca²⁺ (n = 21 cells). Between 10 and 20 min (averaged data points from min 10 to min 20) after low Mg²⁺ induction, intracellular Ca²⁺ is significantly elevated (p < 0.001) compared to baseline (averaged data points from min 0 to min 3). At 50–60 min intracellular Ca²⁺ has decreased (p < 0.001) compared to 10–20 min and significantly increased (p = 0.02) compared to the baseline. *p < 0.05, ***p < 0.001. Scalebar, 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Clustered α_2SEP GABA_ARs decrease upon low Mg²⁺/NMDA treatment. (A) Representative average intensity projection of α_2SEP GABA_AR fluorescence in control and low Mg²⁺/NMDA treated neurons over time (0–3 min and 10–20 min). Somatic α_2SEP GABA_AR fluorescence highlighted in red; scale bar, 10 μm. (B) Kymograph showing somatic (left; scale bar, 5 μm) and clustered (right; scale bar, 2 μm) α_2SEP GABA_AR fluorescence intensity over the movie in control (aCSF) conditions and in the presence of low Mg²⁺/NMDA (grey bar). Red bar on the right indicates decrease in somatic α_2SEP GABA_AR fluorescence intensity upon low Mg²⁺/NMDA treatment. (C) Average fluorescence intensity time course of clustered α_2SEP GABA_AR F/F₀: control (green, n = 7 cells) and low Mg²⁺/NMDA, (light blue, n = 9). (D) Time course of diffuse α_2SEP GABA_AR F/F₀: control (green, n = 7 cells); low Mg²⁺, (purple, n = 9). (E) Time course of somatic α_2SEP GABA_AR F/F₀: control (green, n = 7 cells) and low Mg²⁺ (blue, n = 9). (F) Bar graph of ROI's F/F₀: clusters (left), diffuse (middle) soma (right). Significant loss of fluorescence in the clusters compared to control at 20 min following after low Mg²⁺ treatment (p = 0.0008). Diffuse fluorescence is not altered upon low Mg²⁺ treatment at 20 min (p = 0.36) after low Mg²⁺ treatment. Diffuse fluorescence is unaltered following low Mg²⁺ treatment (t = 20 min; p = 0.58) compared to control. (F') At 60 after low Mg²⁺ treatment clustered fluorescence intensity is still significantly reduced (p < 0.001), diffuse (p = 0.99) and somatic (p = 0.63) fluorescence are not significantly altered. *p < 0.05, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Calcineurin mediates the decrease of somatic surface GABA_ARs during low Mg²⁺ treatment. (A) Representative images of α_2SEP GABA_AR fluorescence in control, control with CAIP (control/CAIP), low Mg²⁺ and low Mg²⁺ with CAIP (low Mg²⁺/CAIP) treated neurons as an average intensity projection over time (0–3 min and 10–20 min). Scale bar, 10 μm. (B) Kymographs showing somatic (scale bar: 5 μm) fluorescence intensity over the movie (duration: 30 min) in control conditions, control/CAIP and in the presence of low Mg²⁺ and low Mg²⁺/CAIP (grey bar). Red bar on the right indicates decrease in somatic fluorescence intensity upon low Mg²⁺ treatment. (C) Average fluorescence intensity of α_2SEP GABA_AR F/F0: control (light green, n = 6 cells); low Mg²⁺ (dark blue, n = 7 cells); control/CAIP (dark green, n = 6); low Mg²⁺/CAIP (purple, n = 6). (C') Bar graph showing quantification of α_2SEP GABA_AR F/F₀ at t = 20 min. Somatic fluorescence intensity of low Mg²⁺ treated cells is significantly decreased compared to control at t = 20 min (dark blue bar, p < 0.05). Treatment of low Mg²⁺ perfused cells with a calcineurin autoinhibitory peptide prevents the change in fluorescence intensity (dark green bar, p < 0.05). Treatment with calcineurin autoinhibitory peptide alone does not significantly alter the fluorescence intensity of GABA_AR α_2SEP (magenta bar, p > 0.05). *p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)