Mutation of a NCKX eliminates glial microdomain calcium oscillations and enhances seizure susceptibility

Abstract

Glia exhibit spontaneous and activity-dependent fluctuations in intracellular Ca(2+), yet it is unclear whether glial Ca(2+) oscillations are required during neuronal signaling. Somatic glial Ca(2+) waves are primarily mediated by the release of intracellular Ca(2+) stores, and their relative importance in normal brain physiology has been disputed. Recently, near-membrane microdomain Ca(2+) transients were identified in fine astrocytic processes and found to arise via an intracellular store-independent process. Here, we describe the identification of rapid, near-membrane Ca(2+) oscillations in Drosophila cortex glia of the CNS. In a screen for temperature-sensitive conditional seizure mutants, we identified a glial-specific Na(+)/Ca(2+), K(+) exchanger (zydeco) that is required for microdomain Ca(2+) oscillatory activity. We found that zydeco mutant animals exhibit increased susceptibility to seizures in response to a variety of environmental stimuli, and that zydeco is required acutely in cortex glia to regulate seizure susceptibility. We also found that glial expression of calmodulin is required for stress-induced seizures in zydeco mutants, suggesting a Ca(2+)/calmodulin-dependent glial signaling pathway underlies glial-neuronal communication. These studies demonstrate that microdomain glial Ca(2+) oscillations require NCKX-mediated plasma membrane Ca(2+) flux, and that acute dysregulation of glial Ca(2+) signaling triggers seizures.

Figure 1.

Mutation of the NCKX zyd causes temperature- and bang-sensitive seizures. A, EMS-induced mutations isolated in CG2893/NCKX in three independently generated alleles of zyd. Alleles zyd² and zyd³ are missense mutations in a pore-lining domain of the exchanger (blue), known as the α1-repeat region. B, Alignment of mammalian and invertebrate NCKX α1-repeat regions, indicating zyd² and zyd³ mutations (blue = identical, green = similar residues). C, D, Representative voltage traces of spontaneous central pattern generator activity at larval third instar muscle 6 at 22°C (C) and 38°C (D) in wild-type and zyd¹ mutants (n > 8 preparations/genotype). E, Recovery time from seizures induced by mechanical stimulation (10 s of vortexing) in WT and zyd mutants at 22°C. n = 100 flies/genotype.

Figure 2.

zyd is required in cortex glia to regulate seizure susceptibility. A, Pan-glial repo-Gal4 driven expression of a wild-type UAS-zyd transgene rescues temperature-sensitive seizures in zyd¹ mutants, but pan-neuronal elav-Gal4 expression does not. B, Knockdown of zyd by pan-neuronal RNAi does not affect central pattern generator activity compared with control at 38°C, but knockdown of zyd by pan-glial- or cortex glial-driven (NP2222-Gal4) RNAi causes rapid, seizure-like discharges in the larval muscle. n > 5 preparations/genotype. C, Use of a collection of glial subpopulation specific Gal4 drivers to rescue zyd¹ TS seizures (with UAS-zyd), or to knockdown zyd (with UAS-zyd RNAi). Only Gal4 drivers with expression in cortex glia rescued adult zyd¹ seizures at 38°C and phenocopied zyd TS seizures by RNAi. NT, Not tested. n > 50 animals/genotype.

Figure 3.

ZYD is expressed in cortex glia. A, One hemisphere of the larval CNS in WT and zyd¹ stained with antisera to ZYD (purple) and ELAV (green), which labels neuronal nuclei. ZYD immunoreactivity outlines large chambers in the outer cortex where glial ensheathment of secondary neurons has not occurred. Scale bar, 40 μm. B, Optical section within the ventral cortex of the VNC in WT and zyd¹ third instar larvae. ZYD staining encapsulates individual neuronal soma. Scale bar, 20 μm. C, CNS stained with ZYD antisera in larvae expressing pan-glial GFP. Scale bar, 40 μm. D, Optical section of the CNS in larvae expressing pan-neuronal (elav-Gal4), pan-glial (repo-Gal4), or cortex glial (NP2222-Gal4) zyd RNAi. Scale bar, 10 μm.

Figure 4.

Adult-specific rescue of zyd mutants by conditional transgene expression rapidly reduces seizure susceptibility. Expression of UAS-zyd was controlled with the heat-inducible hsp70-Gal4 driver that is not expressed at room temperature and is activated above 30°C. zyd¹ mutants were reared at 22°C until adulthood (2 d posteclosion), then exposed to a brief 37°C heat pulse. TS seizure susceptibility was strongly reduced in zyd¹ mutants 6 h after transgene expression with hsp70-Gal4 (red bars). Genetically identical controls not receiving a heat pulse exhibited seizures at all time points (blue bars). n = 30 flies/time point.

Figure 5.

Microdomain Ca²⁺ oscillations in cortex glia require ZYD. A, Ca²⁺ transients occur in small regions of cortex glial membrane in the live larval ventral nerve cord. Scale bar, 10 μm. MyrGCaMP5 fluorescence (ΔF/F_avg) is shown for indicated regions in B. C, Frequency of microdomain Ca²⁺ transients in VNC cortex glia for wild-type and zyd¹ mutants at 25°C. n = 7 larvae/genotype. D, Time-lapse image series of a single cortex glial Ca²⁺ oscillation at 25°C and 38°C in wild-type larvae. Scale bar, 4 μm. E, MyrGCaMP5 fluorescence (ΔF/F_avg) for the regions indicated in the first panels of D. F, Representative images of myrGCamP5 fluorescence intensity in wild-type and zyd¹ at 25°C. Scale bar, 20 μm. G, Average myrGCaMP5 fluorescence in cortex glia of wild-type and zyd¹ at 25°C and 38°C. n = 10 larvae/condition. Error bars represent the SEM. t test: ***p < 0.001, **p < 0.01.

Figure 6.

Acute Ca²⁺ dysregulation in cortex glia causes seizures. A, The heat-activated dTRPA1 cation channel is closed at 22°C and open at temperatures ≥26°C, allowing Ca²⁺ influx. B, Ectopic expression of dTRPA1 in neurons (with elav-Gal4) and in cortex glia (with NP2222-Gal4) triggers seizure-like output from the central pattern generator. Voltage traces were recorded from the third instar larval muscle during application of a temperature ramp. Preparations were held at the indicated temperature for 2 min before recording. n > 6 larvae per genotype.

Figure 7.

Knockdown of calmodulin suppresses seizures in zyd mutants and paralysis due to ectopic glial TRPA1 activation. A, Central pattern generator activity recorded in larval muscle in control and following pan-glial expression of cam RNAi in zyd¹ mutants at 38°C. B, Western blot of adult heads expressing cam RNAi in the eye with gmr-Gal4. Tubulin was used as loading control. C, Quantification of seizure suppression in adult zyd¹ animals expressing pan-glial cam RNAi. n = 100 flies/genotype. D, Time course of paralysis upon exposure to 30°C in adult flies expressing pan-glial dTRPA1 (blue line) and flies expressing pan-glial dTRPA1 and cam RNAi (red line). n > 50 flies/genotype.