Abstract
Astrocytic uptake of GABA through GABA transporters (GATs) is an important mechanism regulating excitatory/inhibitory balance in the nervous system; however, mechanisms by which astrocytes regulate GAT levels are undefined. We found that at mid-pupal stages the Drosophila melanogaster CNS neuropil was devoid of astrocyte membranes and synapses. Astrocyte membranes subsequently infiltrated the neuropil coordinately with synaptogenesis, and astrocyte ablation reduced synapse numbers by half, indicating that Drosophila astrocytes are pro-synaptogenic. Shortly after synapses formed in earnest, GAT was upregulated in astrocytes. Ablation or silencing of GABAergic neurons or disruption of metabotropic GABA receptor 1 and 2 (GABABR1/2) signaling in astrocytes led to a decrease in astrocytic GAT. Notably, developmental depletion of astrocytic GABABR1/2 signaling suppressed mechanosensory-induced seizure activity in mutants with hyperexcitable neurons. These data reveal that astrocytes actively modulate GAT expression via metabotropic GABA receptor signaling and highlight the importance of precise regulation of astrocytic GAT in modulation of seizure activity.
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Acknowledgements
We are grateful to S. Waddell (University of Oxford), G. Miesenböck (University of Oxford), J. Carlson (Yale University), V. Budnik (University of Massachusetts Medical School), B. Ganetsky (University of Wisconsin-Madison) and M. Tanouye (University of California, Berkley) as well as the Vienna Drosophila RNAi Center and the Bloomington Stock Center for generously providing fly stocks. We thank the Transgenic RNAi Project (TRiP) at Harvard Medical School (US National Institutes of Health NIGMS R01-GM084947) for providing transgenic RNAi fly stocks and/or plasmid vectors. The antibodies nc82, anti-PDF, anti-Fasciclin II and anti-Elav, developed respectively by E. Buchner, J. Blau, C. Goodman and G.M. Rubin, were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. We thank the University of Massachusetts Medical School electron microscopy facility, in particular L. Strittmatter, for expert technical assistance with TEM studies. We thank C. Merlin for expert advice on real-time PCR experiments. We thank N. Fox for comments on the manuscript. We thank D. Bergles and all members of the Freeman laboratory for discussion on the manuscript. The project described was supported by Award Number S10RR027897 from the National Center For Research Resources. T.S. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (DFG). This work was supported by NINDS grant R01NS053538 (to M.R.F.). M.R.F. is an Investigator with the Howard Hughes Medical Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center For Research Resources or the National Institutes of Health.
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A.K.M. performed the experiments; T.S. generated GAT antibodies; M.R.F. supervised the project; A.K.M. and M.R.F. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Astrocytes infiltrate the neuropil throughout the central brain during late metamorphosis
(a) Confocal section through central brain showing astrocyte infiltration during late metamorphosis. Astrocyte membranes are labeled by UAS-mCD8::GFP expression using the alrm-GAL4 driver (green), and neuropil is labeled by nc82 antibody staining (red). Scale bar = 50μm. (b) Confocal section through the AL region showing astrocyte morphology at adult stages. Astrocyte membranes are labeled by UAS-mCD8::GFP expression using the alrm-GAL4 driver (green), and neuropil is labeled by nc82 antibody staining (red). Cell bodies, marked by arrow heads, reside along the periphery of neuropil regions while astrocyte processes are present within the neuropil. Scale bar = 10μm.
Supplementary Figure 2 Initial phases of astrocyte infiltration
Confocal section through the central brain, zoomed in on only a few cells, in order to highlight the change in astrocyte morphology during the initial phases of infiltration. Astrocyte membranes are labeled by UAS-mCD8::GFP expression using the alrm-GAL4 driver (green). Scale bar = 10μm.
Supplementary Figure 3 Identification of astrocytes after ablation procedures
(a) Confocal section through AL of adult animals where astrocytes are labeled by anti-GAT antibody staining (green), neuropil is labeled by anti-HRP antibody staining (red), and glial nuclei are labeled with anti-Repo staining (blue). Repo+ nuclei belonging to GAT+ cells were identified as individual astrocytes within neuropil regions of interest. Scale bar = 10μm. (b) High magnification images of Repo+ nuclei belonging to GAT+ cells in our various regions of interest and ablation conditions. Arrows point to examples of cells that are both Repo+ and GAT+ (astrocytes). Arrow heads point to Repo+ nuclei that do not belong to GAT+ cells. Scale bar = 5μm.
Supplementary Figure 4 Ultrastructure of neuropil after astrocyte ablation
Low magnification image of AL neuropil tissue ultrastructure following the 30°C astrocyte ablation procedure. Asterisks mark mitochondrial structures. Many mitochondrial structures looked unhealthy and ruptured in adult tissue following astrocyte ablations. However, this trend was not observed at earlier stages. Arrows point to structures that we suspect are astrocyte membranes. These structures become extremely difficult to identify following astrocyte ablations. Scale bar = 1μm.
Supplementary Figure 5 Constitutive ablation of astrocytes during late metamorphosis
Confocal section through AL of animals ∼60 and ∼84 h APF that were undergoing the 30°C astrocyte ablation procedure. Astrocytes are successfully ablated during development. Astrocytes are labeled by anti-GAT staining (green) and neuropil is labeled by anti-HRP staining (red). Scale bar = 10μm.
Supplementary Figure 6 Astrocyte ablations result in higher frequency of immature synaptic structures at 84 h APF
(a) Quantification of the number of immature synaptic structures in the AL of 84 h APF animals (n = 19 sections, Control; n = 20 sections, Hid) (b) Stacked representation of the number of synapses with the number of immature synaptic structures in the AL of 84 h APF animals (n = 19 sections, Control; n = 20 sections, Hid). (c) Sum of the number of synapses and immature synaptic structures in the AL of 84 h APF animals and adult animals (n = 19 sections, Control 84 h APF; n = 20 sections, Astro> Hid 84 h APF; n = 23 sections, Control Adult; n = 27 sections, Astro>Hid Adult. ***P≤0.001, unpaired Student’s t-test. Error bars, s.e.m.
Supplementary Figure 7 Morphology of cortex glia and ensheathing glia are grossly unaltered following astrocyte ablations
Confocal section through region surrounding AL, showing cortex and ensheathing glia morphology by anti-Draper stain, and location of glial nuclei by anti-Repo stain. Scale bar = 10μm
Supplementary Figure 8 GAT is not expressed in neurons
The pan-neuronal driver, elav-GAL4, was used to express UAS-gat RNAi. Adult brains were stained with anti-GAT and anti-Elav antibodies. Confocal section through region surrounding AL shows that GAT+(red) cells are not Elav+ (green), and that gat knockdown in neurons has no effect on GAT expression. Scale bar = 10μm
Supplementary Figure 9 Temperature shift scheme for GABA neuron ablations
The gad-GAL4 driver and tub-GAL80ts were used to conditionally express UAS-hid in GABA neurons specifically during metamorphosis.
Supplementary Figure 10 Distribution of GABA release sites and astrocyte morphology are grossly unaltered following inhibition of GABA neuron activity during synaptogenesis
The gad-GAL4 driver was used to either co-express UAS-syt::eGFP and UAS-shits or express UAS-syt::eGFP alone. Dominant negative Shits was conditionally expressed during synaptogenesis, and 84 h APF animals were dissected and stained with anti-GAT antibody. Confocal section through AL shows astrocyte morphology (red) and distribution of GABAergic pre-synaptic sites (GABA neuron>Syt::eGFP). Scale bar = 10μm
Supplementary Figure 11 Temperature shift scheme for adult specific inhibition of GABABR1/2 signaling in astrocytes
Tub-GAL80ts with UAS-GABABR1 RNAi or UAS-GABABR2 RNAi were expressed under the control of the alrm-GAL4 driver. RNAi expression was induced in 1 day old adult animals for 7-10 days.
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Supplementary Text and Figures
Supplementary Figures 1–12 (PDF 9108 kb)
Typical behavior of animals following severe astrocyte ablations: control
One day old control animals after undergoing severe ablation temperature shifts (30°C). Animals do not display any overt behavioral defects. (MOV 3049 kb)
Typical behavior of animals following severe astrocyte ablations: fully eclosed
Example of behavior displayed by 1 day old animals following severe (30°C) astrocyte ablation. Flies are fallen over with non-inflated wings. Movement is mostly in the legs and sometimes in the proboscis. This is in striking contrast to control animals (Supplementary Video 1). (MOV 3768 kb)
Typical behavior of animals following severe astrocyte ablations: partially eclosed
Example of an animal that partially eclosed after severe astrocyte ablation. This animal is still alive, demonstrated by its ability to extend its proboscis. (MOV 3287 kb)
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Muthukumar, A., Stork, T. & Freeman, M. Activity-dependent regulation of astrocyte GAT levels during synaptogenesis. Nat Neurosci 17, 1340–1350 (2014). https://doi.org/10.1038/nn.3791
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