Supplementary Figure 1 SybII and Ceb are sorted to distinct vesicle populations in astrocytes.
(a) Exemplary images for cultured astrocytes co-immunolabeled with SybII and Ceb antibodies. SybII accumulates at the cell s periphery, whereas Ceb is scattered throughout the cytoplasm. Note the lack of colocalization between the two v-snare proteins as depicted in the higher magnification image. (b) Ceb colocalizes with the large dense core (LDCV) marker protein NPY. (c) Coimmunolabeling of the vesicular glutamate transporter (vglut) and SybII shows a high degree of colocalization between the two proteins. (d) Cytofluorograms (pixel plotted in grey values) for the indicated groups underline the lack of colocalization between SybII and Ceb (r 2 =0,34) and a good colocalization between Ceb and NPY (r 2 =0,84) and SybII and vglut (r 2 =0,94). (e) Line scan analyses for the indicated antigens (dashed lines in a-c). (f) Syb and Ceb colocalize with their respective cargo significantly better than with the other v-snare variant (Data were collected from 3 preparations; SybII vs Ceb, n=12; Manders coeff.: Ceb vs NPY, n=11, p<0.0001; sybii vs vglut, n=12, p<0.0001, one-way ANOVA versus SybII vs Ceb). All data are represented as mean ± s.e.m.
Supplementary Figure 2 Antibodies are specific for their respective antigens. (a) Confocal images of sybiiko astrocytes co-immunolabelled with affinity purified antibodies for sybii and ceb. Note that sybii staining is abolished in ko astrocytes. (b) Exemplary images of co-staining with ceb and sybii in cebko astrocytes. Ceb staining is abrogated in cebko cells. (c) Coimmunolabeling of NPY and ceb in NPYko astrocytes. NPY immunoreactivity is abolished in NPYko astrocytes. (d) Line scan analyses for the indicated antigens (dashed lines in a-c).
Supplementary Figure 3 Ca 2+ signaling is unaffected in v-snare-deficient astrocytes. (a,b) Confocal high magnification images of a dko astrocyte transfected with either cebmrfp (a) or sybiimrfp (b) and subsequently immunolabeled with NPY. Lentiviral expressed ceb-mrfp and endogenous NPY display a high degree of colocalization (Pearson s coefficient: 0.98±0.02; n=12), whereas sybii-mrfp and NPY show little colocalization (Pearson s coefficient: 0.54±0.001; n=11). (c) Exemplary confocal images of astrocytes loaded with Fluo4AM (10 µm) before (upper image) and
after stimulation (lower image) with 1 mm glutamate. (d) Time-course of stimulus dependent increase in Fluo4AM fluorescence in WT, sybiiko, cebko, dko, dko+ceb and dko+sybii. No increase could be observed in WT cells treated with mglur5 antagonist MPEP (20 µm) and unstimulated cells. (e) Quantification of Fluo4AM fluorescence at the signal maximum for all groups in (d). No difference in the maximum fluorescence could be observed for any of the conditions. Note that in the absence of a stimulus or pre-incubation of cells with MPEP a metabotropic glutamate receptor antagonist abolished increase in Fluo4AM (Data were collected from 3 preparations; WT, n=17; sybiiko, n=18, p=0.48; cebko, n=21, p=0.38; dko, n=22, p=0.34; dko+sybii, n=15 p=0.41; dko+ceb, n=19, p=0.42; +MPEP, n=21, p<0.0001; not stim., n=22, p<0.0001, one-way ANOVA vs WT). ***p<0.001. All data are represented as mean ± s.e.m.
Supplementary Figure 4 iglusnfr monitors glutamate release in astrocytes and neurons. (a) Exemplary image of an astrocyte transfected with iglusnfr during stimulation with 20 µm DHPG. Arrows point to quantal-like glutamate release. Dashed lines indicate the cellular borders of the
astrocyte, where they could be visualized. (b) Exemplary traces of single regions of interest in WT, sybiiko and cebko astrocytes. WT and cebko cells respond to DHPG with discrete, quantal-like fluorescent signals monitoring glutamate release. The slowly developing fluorescence offset is likely due to increase in ambient glutamate. No fluorescence increase could be detected in sybiiko astrocytes. (c) Glutamate secretion is abolished in sybiiko, dko astrocytes and in the presence of 50 µm MPEP, but remained unchanged in cebko cells (vs WT, cebko, p=0.996; sybiiko, p<0.001; dko, p<0.001; WT+MPEP, p<0.001; one-way ANOVA). (d) iglusnfr expression is unchanged between the groups (versus WT: cebko, p=0.133; sybiiko, p=0.130; dko, p=0.141; WT+MPEP, p=0.198; one-way ANOVA). (e) The amplitude of the transient glutamate flashes is unaltered between WT and cebko cells (events: WT, 648; cebko, 668; threshold: 5xSD of noise, WT, 0.0156±1.3x10-3 ; cebko, 0.0181±3.2x10-3 ). Inset displays the average of 10 exemplary events. Bar, 0.1 F/F 0, 320 ms. (f) WT and cebko cells continuously secrete glutamate during DHPG application. Data was collected from 3 preparations; WT, n=13; cebko, n=12; sybiiko, n=13, dko, n=11, WT+MPEP, n=9. (g) Exemplary images of autaptic hippocampal neurons transfected with iglusnfr before (upper panel: F 0 ) and during electrical stimulation (lower panel: F). (h) Representative traces of iglusnfr fluorescence in WT and sybiiko neurons. Top trace indicates the stimulation frequency. Note the lack of fluorescence increase in the sybiiko neurons where glutamatergic signaling is abolished. (i) Mean fluorescence increase upon single action potentials (p=0.000096). (j) Expression level is unchanged in sybiiko neurons. Data was collected from WT, n=9; sybiiko, n=7, p=48 from 2 preparations, student s t-test. ***p<0.001. All data are represented as mean ± s.e.m.
Supplementary Figure 5 Loss of gliotransmitter secretion differentially affects the pool of readily releasable vesicles and paired pulse ratio. (a) Representative traces of the average postsynaptic response to 5s application of hypertonic solution (500 mm sucrose) from neurons cultured on WT, sybiiko and cebko astrocytes. (b,c) v- SNARE dependent secretion affects the readily releasable pool charge and release probability (RRP, sucrose: sybiiko, p<0.001; cebko, p<0.001; P R : sybiiko, p<0.001; cebko, p=0.77, one-way ANOVA vs WT.). Data was collected from recordings with WT (n=20), sybiiko (n=19) and cebko (n=19) 3 preparations. (d) Normalized EPSCs measured with an inter-stimulation-interval of 50ms. (e) Paired pulse ratio is only increased for neurons grown on sybiiko astrocytes. Data was extracted from the first two EPSCs of the 20 Hz train (amplitude ratio: EPSC2/EPSC1) and collected from 3
preparations, WT, n=62; sybiiko, n=28; cebko, n=35; dko, n=29 (ANOVA vs wt: sybiiko, p=0.009; cebko,p=0.43; dko, p=0.5). All data are represented as mean ± s.e.m.
Supplementary Figure 6 Spontaneous release is unaffected by extracellular application of NPY and ATP.
(a) Representative recordings of quantal signaling from neurons grown on WT or cebko astrocytes treated daily with either the vehicle or 10 nm NPY. (b) mepsc frequency, amplitude and charge are unchanged for the indicated groups (Data were collected from 3 preparations; WT+vehicle, n=24; cebko+vehicle, n=25; cebko+npy, n=26; for mepsc frequency, amplitude and charge all p-values ranged between 0.101 and 0.935, one-way ANOVA vs WT+vehicle). (c) Exemplary traces of spontaneous mepscs recorded in neurons plated on WT and cebko astrocytes treated daily either with 10 nm NPY/ATP in the medium or a vehicle. (d) mepsc frequency, amplitude and charge are unaffected by NPY/ATP co-application (Data were collected from 3 preparations; WT+vehicle, n=19; cebko+vehicle, n=20; cebko+npy/atp, n=21; for mepsc frequency, amplitude and charge all p- Values ranged between 0.233 and 0.969, one-way ANOVA vs WT+vehicle). (e) Representative recordings of mepscs from WT neurons grown on WT and cebko astrocytes, treated with 150 nm DPCPX/BIIE0246 (Data were collected from 3 preparations; adenosine A1 and NPY Y2 receptor antagonists; WT+vehicle, n=24; cebko+vehicle, n=19; WT+150 nm, DPCPX/BIIE0246, n=18; cebko+150 nm DPCPX/BIIE0246, n=20). (f) mepsc frequency, amplitude and charge for the conditions described in (e) were unaffected by the treatment (for mepsc frequency, amplitude and charge all p-values ranged between 0.21 and 0.982, one-way ANOVA vs WT+vehicle). All data are represented as mean ± s.e.m.
Supplementary Figure 7 Tonic ATP/NPY and glutamate release from astrocytes regulates synaptic transmission.
(a) Consecutive trains of APs (20Hz; interval time 1 min) recorded either in the presence of Ringer s solution or ATP/NPY (10 µm). (b,c) Amplitude of the first evoked response and RRP size were significantly reduced in the presence of ATP/NPY in neurons grown on WT and cebko astrocytes. Data was collected from 2 preparations. (Ringer: WT, n=9; amp: p=0,641, RRP, p=1,0; cebko, n=9 amp: p=0,706; RRP, p=0,691; ATP/NPY: WT, n=9; amp: p<0.001; RRP, p<0.001; cebko, n=9; amp: p<0.001, RRP: p<0.001, t-test) (d) Acute application of the A1 antagonist DPCPX and NPY-receptor antagonist BIIE0246 (indicated by the bar) strongly and reversibly augmented the evoked EPSC amplitude (recorded at 0.2 Hz) of neurons grown on WT astrocytes. No effect was seen for neurons plated on cebko astrocytes. Scale bar, 2nA, 20ms (e,f) The EPSC amplitude is specifically increased for WT, but not for cebko astrocytes (WT,DPCPX/BIIW0246: p<0.001; cebko, DPCPX/BIIE0246 p=0,37; t-test). Data were collected from 3 preparations, WT, n=16; cebko, n=14 cells; ***p<0.001. (g) Acute application of the kainate receptor antagonist NS-102 (indicated by the bar) strongly suppressed the EPSC amplitude (recorded at 0.2 Hz) in neurons grown on WT astrocytes to levels of cells plated with sybiiko cells. No changes were observed in sybiiko cells; bar, 2nA; 15ms. (h,i) EPSC amplitude is only decreased during NS-102 application in neurons grown on WT astrocytes. NS-102 had no effect on neurons grown on sybiiko cells (WT, NS-102: p<0.001, recovery, p=0,272; sybiiko, NS-102 p=0.419; recovery, p=0.482, t-test). Data was collected from 3 preparations from WT, n=17 and sybiiko, n=19 cells. ***p<0.001, t-test. All data are represented as mean ± s.e.m.
Supplementary Figure 8 Loss of distinct secretion pathways in astrocytes modulates the number of readily releasable vesicles. (a) Schematic drawing of the experimental protocol. Neurons were electrically stimulated with the
patch pipette (20Hz/2s) while fluorescence changes were monitored at 10Hz simultaneously. At the end of the recording cells were perfused with NH 4 Cl to unquench syn-ph fluorescence. (b) Exemplary recordings of quantal signaling from neurons transfected with Syn-pH grown without glia, on sybiiko or cebko astrocytes. (c) mepsc frequency is strongly reduced in cells grown without or on sybiiko astrocytes (mepsc freq: no glia, p<0.001; sybiiko, p<0.001; cebko, p=0.53; mepsc ampl.: no glia, p=0.79; sybiiko, p=0.82; cebko, p=0.83; mepsc charge: no glia, p=0.73; sybiiko, p=0.76; cebko, p=0.68, one-way ANOVA vs WT) (d) Time course for the decline of the evoked amplitudes. (e,f) v- SNARE mediated astrocytic secretion similarly affects the evoked amplitude (1 st EPSC) and RRP (first EPSC ampl.: no glia, p<0.001; sybiiko, p=0.0031; cebko, p<0.001; RRP charge: no glia, p<0.001; sybiiko, p<0.001; cebko, p<0.001, one-way ANOVA vs WT). Data was collected from 4 preparations; WT, n=20; sybiiko, n=17; cebko, n=16; non-glia, n=9; not stimulated, n=5; **p<0.01, ***p<0.001. All data are represented as mean ± s.e.m.
Supplementary Figure 9 v-snare-dependent gliotransmitters release does not influence the number of synapses. (a) Exemplary confocal images of autaptic neuronal cultures grown without glia cells or on WT, sybiiko, cebko or dko astrocytes immunolabelled with the presynaptic marker protein anti-bassoon.
(b) The number of synapses per microisland is reduced in the absence of astrocytes but not affected by their v-snare deficiency (no glia, p<0.001; sybiiko, p=0.994; cebko, p=0.994; dko, p=0.985; oneway ANOVA vs WT). Data was collected from 3 preparations, WT, n=17; no-glia, n=16; sybiiko, n=19; cebko, n=19; dko, n=19; ***p<0.001 All data are represented as mean ± s.e.m.
Supplementary Figure 10 Proposed model for v-snare-dependent gliotransmitter secretion pathways modulating synaptic efficacy. (a) Astrocytes release NPY and ATP through a ceb-dependent secretion pathway. NPY acts on presynaptic NPY Y2 receptors and ATP (rapidly converted into adenosine (ado)) may act on presynaptic adenosine A1 receptors to decrease the RRP. (b) SybII mediated glutamate release from astrocytic SLMVs exerts its potentiating effect of synaptic signaling (increasing the RRP size and P r )
through activation of presynaptic kainate receptors (KAR) or NMDA(NR2B) 2 and mglur receptors 6. (a)+(b) In the full protein context of WT cells the antagonistic effects of the v-snare dependent secretion pathways compensate each other (as evidenced by the dko phenotype) to increase the dynamic range of synaptic signaling.