Activity-dependent regulation of astrocyte GAT levels during synaptogenesis

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 (GABA(B)R1/2) signaling in astrocytes led to a decrease in astrocytic GAT. Notably, developmental depletion of astrocytic GABA(B)R1/2 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.

Figure 1. Astrocyte infiltration and synaptogenesis are temporally correlated during late metamorphosis

(a) Confocal section through the AL region showing astrocyte infiltration at several timepoints during 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 = 10µm. (b) Ultrastructure of AL neuropil at several timepoints during metamorphosis, highlighting the progression in synapse development. Arrows point to pre-synaptic sites and asterisks mark post-synaptic structures. Synaptic structures are prominent starting at 72 h APF. Scale bar = 0.5µm. (c) Quantification of the number of synaptic structures in the AL (n ≥ 20 sections for each timepoint), MB (n ≥ 6 sections for each timepoint), and SPSL (n ≥ 9 sections for each timepoint) during late metamorphosis. Error bars, s.e.m.

Figure 2. Genetic ablation of astrocytes during synaptogenesis

(a) Temperature shift scheme for astrocyte ablations. The alrm-GAL4 driver and tub-GAL80^ts were used to conditionally express UAS-hid in astrocytes specifically during late metamorphosis. Varying degrees of GAL80 activity and UAS-hid expression were achieved by varying the incubation temperature during late metamorphosis. Low-level Hid expression was achieved at 25°C and maximal Hid expression was achieved at 30°C. (b) Confocal slice of central brain and ventral nerve cord of adult animals immunostained with anti-GAT antibody to visualize astrocytes. Astrocyte staining is moderately reduced following Hid expression in astrocytes at 25°C, and robustly reduced after Hid expression in astrocytes at 30°C, indicating moderate and severe ablation conditions, respectively. Scale bar = 10µm. (c) Confocal sections through AL of adult animals where astrocytes are labeled by anti-GAT antibody staining (green) and neuropil is labeled by anti-HRP antibody staining (red). Astrocyte processes can fully cover neuropil space when ablations are performed at 25°C. Large regions of neuropil are left unoccupied by astrocyte processes when ablations are performed at 30°C. Scale bar = 10µm. (d) Number of astrocytes remaining in the AL, MB, SOG, and TGab of the adult CNS following moderate (25°C) astrocyte ablations (n = 5 brains for control and >Astro>Hid AL, MB, SOG, and TGab). (e) Number of astrocytes remaining in the AL, MB, SOG, and TGab of the adult CNS following severe (30°C) astrocyte ablations (n = 7 brains, control AL; n = 8 brains, >Astro>Hid AL; n = 5 brains, control and >Astro>Hid MB; n = 5 brains, control and >Astro>Hid SOG; n = 5 brains, control TGab; n = 7 brains, >Astro>Hid TGab). Fewer astrocytes remain when ablations are performed at 30°C, compared to 25°C; demonstrating the varying degrees of astrocyte ablation achieved by the two temperature conditions. (f) Fates of animals undergoing ablation procedure (n ≥ 30 flies for each condition). The majority of animals struggle to eclose when undergoing severe astrocyte ablations (30°C). *P≤0.05, **P≤0.01, ***P≤0.001, unpaired Student’s t-test. Error bars, s.e.m.

Figure 3. Synapse number, but not gross neural architecture, is altered when astrocytes are ablated during late metamorphosis

(a) Ultrastructure of synapses in the AL at adult and 84 h APF animals following 30°C astrocyte ablation. Morphology of mature synaptic structures is unaltered by ablation. Arrows point to pre-synaptic sites and asterisks mark post-synaptic structures. Scale bar = 0.5µm. (b) Quantification of post-synaptic density (PSD) length in AL, MB, and SPSL regions of the adult brain after 30°C astrocyte ablation (n = 80 PSDs from 12 sections, AL control; n = 81 PSDs from 12 sections, AL Astro>Hid; n = 47 PSDs from 7 sections, MB control; n = 50 PSDs from 7 sections, MB Astro>Hid; n = 58 PSDs from 7 sections, SPSL control; n = 63 PSDs from 7 sections, SPSL Astro>Hid. (c) Quantification of the percentage of synapses with T-bar morphology in the AL, MB, and SPSL regions of the adult brain after 30°C astrocyte ablation (n = 23 sections, AL control; n = 24 sections, AL Astro>Hid; n = 7 sections, MB control; n = 11 sections, MB Astro>Hid; n = 8 sections, SPSL control; n = 8 sections, SPSL Astro>Hid. (d) Quantification of the number of synaptic structures in the AL, MB, and SPSL regions of adult and 84 h APF animals following 30°C astrocyte ablation (n = 17 sections, AL adult control; n = 22 sections, AL adult Astro>>Hid; n = 19 sections, AL 84 h APF control; n = 20 sections, AL 84 h APF >Astro>Hid; n = 17 sections, MB adult control; n = 22 sections, MB adult Astro>>Hid; n = 19 sections, MB 84 h APF control; n = 20 sections, MB 84 h APF >Astro>Hid; n = 17 sections, SPSL adult control; n = 22 sections, SPSL adult Astro>>Hid; n = 19 sections, SPSL 84 h APF control; n = 20 sections, SPSL 84 h APF Astro>>Hid. (e) Brain architecture is grossly unaltered following astrocyte ablations performed at 30°C. Projected confocal z-stacks showing (i) glomeruli structure in brains stained with nc82 antibody (red), (ii) morphology of PNs marked by GH146-QF/QUAS-mCD8::GFP, (iii) morphology of axonal projections and arborizations of PNs, (iv) morphology of PDF neuron axonal projections in the central brain, (v) morphology of PDF neuron dendritic arborizations in the optic lobe. Scale bar = 10µm for (i) and (iii); 50µm for (ii); 25µm for (iv) and (v). (f) Quantification of the number of PNs per hemisphere (n = 6 brains, control and Astro>>Hid) and (g) PDF⁺ neurons per hemisphere (n = 6 brains, control; n = 5 brains, Astro>>Hid) following 30°C astrocyte ablation. ***P≤0.001, unpaired Student’s t-test for (b–d), and (f–g). Error bars, s.e.m.

Figure 4. GAT is exclusively expressed in astrocytes and activated during synaptogenesis

(a) UAS-gat RNAi was expressed in astrocytes using the alrm-GAL4 driver. Western blots performed on larval CNS and adult brain lysates were probed with anti-GAT antibody to confirm specific knockdown of GAT in astrocytes. GAT runs at approximately 50kDa. (b) Quantification of GAT levels from Western blot analysis shown in (a) (n = 3 experiments, 3rd instar larva; n = 3 experiments, adult). (c) The alrm-GAL4 driver was used to co-express UAS-mCD8::GFP and UAS-gat RNAi, or express UAS-mCD8::GFP alone. Adult brains were stained with anti-GAT antibody. Confocal section through AL shows that GAT (red) localizes specifically to astrocyte membranes (green). Scale bar = 10µm. (d) Western blot performed on WT brain lysates from several stages of metamorphosis was probed with anti-GAT antibody. (e) Quantification of GAT levels from Western blot analysis shown in (d) (n = 3 experiments). (f) Adult brains expressing UAS-syt::eGFP using the gad-GAL4 driver were stained with anti-GAT antibody. GABAergic pre-synaptic sites (GABA neuron>Syt::eGFP) and GAT protein are present through out the central brain. High magnification images of the AL and MB regions show that GABAergic pre-synaptic sites and GAT proteins are in close association. Scale bar = 20µm. *P≤0.05, ***P≤0.001, unpaired Student’s t-test for (b) and 1-way ANOVA with Tukey’s post hoc test for (e). Error bars, s.e.m. Full-length blots are presented in Supplementary Fig. 12.

Figure 5. GAT expression is sensitive to GABA neurons

(a) Confocal sections showing anti-GAT immunostaining (green) in the central brain and AL region after ablation of GABA neurons. GABA neurons are labeled by UAS-mCD8::mcherry expression using the gad-GAL4 driver (red). Significant reduction in GAT levels is seen throughout the central brain in correspondence with a reduction in GABA neurons. Localization of GAT appears unaltered by GABA neuron ablations, as highlighted in images from the AL. Animals were 84 h APF upon preparation. Scale bar = 20µm. (b) Quantification of the number of GABA neurons in the ventral medial region of the central brain (n = 5 brains for control and GABA neuron>>Hid). (c) Quantification of the mean GAT intensity in the AL and SOG regions (n = 8 brains for control and GABA neuron>>Hid for AL and SOG). (d) Quantification of the number of astrocytes in the AL and SOG regions (n = 4 brains, control AL; n = 5 brains, GABA neuron>>Hid AL, n = 4 brains, control SOG; n = 5 brains, GABA neuron>>Hid SOG). ***P≤0.001, unpaired Student’s t-test for (b) and (d), paired Student’s t-test for (c). Error bars, s.e.m.

Figure 6. GAT expression is fine tuned in response to GABA release specifically during synaptogenesis

(a) Temperature shift scheme for conditional manipulation of GABA neuron activity. The temperature sensitive constructs, UAS-shi^ts and UAS-trpA1, as well as UAS-Kir2.1 with tub-GAL80^ts, were activated in a conditional manner using the gad-GAL4 driver. Dominant negative shi^ts expression, Kir2.1 expression, or TrpA1 activation was induced at 30°C. (b) Western blots performed on brains 84 h APF following various neuronal manipulations using the gad-GAL4 driver. Blots were probed with anti-GAT antibody. GABA neurons were “inactivated” with expression of UAS-hid UAS-shi^ts UAS-TNT, and UAS-Kir2.1. Alternatively, GABA neurons were “activated” using UAS-trpA1(c) Quantification of GAT levels from Western blot analysis shown in (b) (n = 4 experiments, Hid; n = 3 experiments, Shi^ts; n = 5 experiments, TNT; n = 3 experiments, Kir2.1; n = 3 experiments, TrpA1). GAT levels are significantly reduced when GABA neurons are “inactivated.” This is in contrast to unaltered GAT levels when GABA neurons are “activated.” (d) Relative gat mRNA levels are unaltered when GABA neuronal activity is silenced by expression of UAS-hid (n = 3) or UAS-TNT (n = 3) using the gad-GAL4 driver. (e) Western blots performed on adult brains following adult specific silencing of GABA neuronal activity by expression of UAS-shi^ts or UAS-kir2.1 using the gad-GAL4 driver. (f) Quantification of GAT levels from Western blot analysis shown in (e) (n = 3 experiments, Shi^ts; n = 3 experiments, Kir2.1). **P≤0.01, ***P≤0.001, paired Student’s t-test for (c) and (f), unpaired Student’s t-test for (d). Error bars, s.e.m. Full-length blots are presented in Supplementary Fig. 12.

Figure 7. GAT expression is modulated through astrocytic metabotropic GABA receptors

(a) Western blots performed on brains 84 h APF following inhibition of GABA_BR1/2 signaling in astrocytes using the alrm-GAL4 driver. Blots were probed with anti-GAT antibody. GABA_BR1/2 signaling was impaired by expressing UAS-GABA_BR1 RNAi UAS-GABA_BR2 RNAi, or UAS-ptx. (b) Quantification of GAT levels from Western blot analysis shown in (a) (n = 5 experiments, GABA_BR1 RNAi; n = 3 experiments, GABA_BR2 RNAi; n = 3 experiments, ptx). (c) Relative gat mRNA levels are unaltered when astrocytic GABA_BR1/2 signaling is perturbed by expressing UAS-GABA_BR2 RNAi (n = 3 experiments) or UAS-ptx (n = 3 experiments) using the alrm-GAL4 driver. (d) Western blots performed on adult brains following adult specific inhibition of GABA_BR1/2 signaling in astrocytes. Blots were probed with anti-GAT antibody. (e) Quantification of GAT levels from Western blot analysis shown in (d) (n = 3 experiments). (f) Western blots performed on adult brains following inhibition of GABA_BR1/2 signaling throughout development in astrocytes using the alrm-GAL4 driver. Blots were probed with anti-GAT antibody. GABA_BR1/2 signaling was impaired by expressing UAS-GABA_BR1 RNAi or UAS-GABA_BR2 RNAi though out development. (g) Quantification of GAT levels from Western blot analysis shown in (f) (n = 3 experiments, GABA_BR1 RNAi; n = 4 experiments, GABA_BR2 RNAi). *P≤0.05, **P≤0.01, paired Student’s t-test for (b), and (g), unpaired Student’s t-test for (c), and 1-way ANOVA with Tukey’s post hoc test for (e). Error bars, s.e.m. Full-length blots are presented in Supplementary Fig. 12.

Figure 8. Regulation of GAT through astrocytic metabotropic GABA receptors can modulate neurotransmission

(a) Flies were vortexed for 10 seconds to provide mechanical stimulation (“bang”) and induce paralysis in bang sensitive mutants. The percent of flies recovering from paralysis is shown as a function of time. Flies without the eas^PC80 mutation do not display a bang sensitive phenotype, and therefore 100% of these flies display normal behavior within the first 10 s. eas ^PC80 flies and eas ^PC80 flies carrying only the alrm-GAL4 driver or UAS constructs display similar recovery response profiles to each other. GABA_BR2 signaling was inhibited in eas ^PC80 flies by expressing UAS-ptx or UAS-GABA_BR2 RNAi using the alrm-GAL4 driver. Recovery profiles are shifted toward shorter recovery times when GAT levels are reduced via inhibition of astrocytic GABA_BR1/2 signaling in eas ^PC80 flies (n > 100 flies for each genotype). (b) Mean recovery time calculated from data shown in (a). (c) Western blot performed on adult brain lysates was probed with anti-GAT antibody. In comparison to eas ^PC80 flies or eas ^PC80 flies carrying the alrm-GAL4 driver, GAT expression was reduced in eas ^PC80 flies expressing UAS-ptx or UAS-GABA_BR2 RNAi using the alrm-GAL4 driver. (d) Quantification of GAT levels from western blot analysis shown in (c) (n = 3 experiments). ***P≤0.001, 1-way ANOVA with Tukey’s post hoc test for (b) and repeated measures ANOVA with Tukey’s post hoc test for (c). Error bars, s.e.m. Full-length blots are presented in Supplementary Fig. 12.