Supplementary Information Astrocyte signaling controls spike timing-dependent depression at neocortical synapses Rogier Min and Thomas Nevian Department of Physiology, University of Berne, Bern, Switzerland Supplementary information consists of: Supplementary Figure 1-5
a b Pyramidal Astrocyte Pyramidal Astrocyte Stim c Astrocyte 200 μm Pyramidal neuron 20 μm 40 mv -86 mv d -77 mv 200 ms Pyr Astro Stim Supplementary Figure 1 Identification of neocortical astrocytes and pyramidal neurons. (a) Low magnification infrared video microscopy image showing a thalamocortical brain slice. The stimulation electrode in L4 (Stim), as well as the recording pipettes for the pyramidal neuron and the astrocyte in L2/3 are highlighted. (b) Higher magnification infrared video microscopy image showing a recorded pair of a pyramidal neuron (left) and an astrocyte (right). (c) Voltage responses of the L2/3 astrocyte (left) and pyramidal neuron (right) shown in b in current-clamp mode. The voltage response to current injections from na up to 4 na ( na increments) into the astrocyte are shown. The astrocyte shows a passive response to these current injections. The voltage response to current injections from 40 pa up to 320 pa (40 pa increments) into the neuron are shown. The neuron shows regular action potential firing when depolarized above threshold, typical for cortical pyramidal neurons. (d) Response to extracellular stimulation in L4 for the same cell pair as shown in a c. Stimulation in L4 evokes an excitatory postsynaptic potential (EPSP) in the L2/3 pyramidal neuron.
a c e EPSP slope (norm) PPR EPSP slope (norm) t = 2 t = 25 ms 10 0 10 20 30 40 50 Baseline 10 0 t-ltd 10 20 30 THL DMSO 40 50 Normalized 1 mv 1 mv 2.0 2 mv b d f EPSC amp (norm) CV -2 After /CV-2 Control EPSP slope (norm) 10 t = 25 ms t = 2 2.0 EPSP After /EPSP Control 10 0 0 10 20 30 10 20 30 40 AM251 40 50 50 100 pa 20 ms Supplementary Figure 2 Properties of t-ltd at L4 to L2/3 synapses in barrel cortex. (a) Left, pooled and normalized EPSP slopes over time for experiments in which t-ltd was induced. The grey area indicates the AP-EPSP pairing period. Green symbols show experiments in which the timing interval between pre- and postsynaptic activity during pairing was Δt = 25 ms. This protocol induced robust t-ltd (1 ± 0.11; P = 07; n = 11). Blue symbols show experiments with Δt = 2. With this protocol no t-ltd was induced (0 ± 4; P = 0.99; n = 5; Δt = 25 ms vs Δt = 2: P = 18). Right, representative averaged EPSP during control (black) and after t-ltd induction with Δt = 25 ms (green) or Δt = 2 (blue). (b) Left, pooled and normalized EPSC amplitudes recorded in voltage clamp over time. AP-EPSP pairing at Δt = 25 ms (indicated by the shaded area) was performed in current clamp as for experiments in a. EPSC amplitudes significantly decreased after pairing, to a similar extent as EPSP slopes (5 ± 8; P = 2; n = 4). Right, representative averaged EPSCs during control (black) and after t-ltd induction (green). (c) Left, graph showing the paired pulse ratio (PPR; amplitude of EPSP 2 /EPSP 1 ; interval) during baseline and after t-ltd induction (Δt = 25 ms). Light symbols represent individual experiments; dark symbols show averaged PPR. t-ltd induction caused a significant increase in PPR (baseline: 0.90 ± 4; t-ltd: 2 ± 7; P = 43; n = 11) suggesting that t-ltd is expressed presynaptically. Right top, representative averaged EPSPs during control (black) and after t-ltd induction (green). Right bottom, same as top, but normalized to the amplitude of the first EPSP. (d) Analysis of the change in the coefficient of variation (CV -2 ) of EPSP slopes after the expression of t-ltd. Plotting the relative change in CV -2 (CV -2 After /CV-2 Control ) against the change in EPSP slope (EPSP After /EPSP Control ) revealed an almost linear relationship between the two for AP-EPSP pairing at t = 25 ms (circles), indicating a presynaptic locus of t-ltd. Pairing at t = 2 (squares) showed no change in CV -2. Light symbols represent individual experiments; solid symbols represents the average. (e) Same as in a, but for experiments in which tetrahydrolipstatin (THL; 5 µm), an inhibitor of the endocannabinoid synthesis enzyme diacylglycerol lipase, was added to the intracellular solution of the postsynaptic pyramidal neuron (turquoise) and vehicle controls (0.1% DMSO in pyramidal intracellular; green). THL prevented t-ltd (THL: 1.10 ± 0.11; P = 0; n = 6; DMSO: 1 ± 0.12; P = 48; n = 6; THL vs DMSO: P = 1), showing that postsynaptic diacylglycerol lipase activity is necessary for t-ltd. (f) Same as in a,e, but for experiments in which the CB1 receptor antagonist AM251 (5 µm) was added to the bath solution (magenta). AM251 significantly reduced the amount of t-ltd (0.91 ± 2; P = 04; n = 7; AM251 vs control: P = 47), showing that t-ltd requires cannabinoid receptor signaling. Data is presented as mean ± s.e.m.; indicates P < 5.
4 10 µm Roi 5 a b c 12 13 1 3 2 5 6 8 G/R 0.1 Roi 8 Number of roi 1 5 10 11 10 7 9 150 s 80 mv 150 s d f Normalized number of Ca 2+ transients Normalized amplitude of Ca 2+ transients 2.0 2.0 150 450 750 Time (s) 150 450 750 Time (s) e g Normalized width of Ca 2+ transients Fraction of processes 0 5 0 0.75 0.2 0 + 150 450 750 Time (s) Supplementary Figure 3 Astrocyte stimulation causes an increase in astrocyte Ca 2+ signaling. (a) Image of an astrocyte filled with Fluo-4 and Alexa-594, with regions of interest (rois) used for analysis. White rois indicate processes without significant increases in the number of Ca 2+ transients. Magenta rois indicate processes with increases in the number of Ca 2+ transients. (b) Fluorescence traces of two rois (Roi5 and Roi8) for the entire 15 min imaging session. Shaded area indicates time window of astrocyte depolarization (lower trace). (c) Raster plot of Ca 2+ transients over time for all rois indicated in a. Each Ca 2+ transient is represented by a tick. Magenta ticks indicate rois that showed significant increases in the number of Ca 2+ transients during stimulation. (d) Summary of the averaged normalized number of Ca 2+ transients over time. A significant increase in number was observed during astrocyte stimulation (n = 6). (e) Summary of the averaged normalized width of Ca 2+ transients over time. A significant increase in width was observed during astrocyte stimulation. (f) Summary of the averaged normalized amplitude of Ca 2+ transients over time. A significant change in amplitude was not observed. (g) Bar graph showing the fraction of astrocyte processes that showed increases (+), no changes (0) or decreases ( ) in the number of Ca 2+ transients during astrocyte stimulation. Data is presented as mean ± s.e.m.; indicates P < 5.
2.0 CV -2 After /CV-2 Control EPSP After /EPSP Control 2.0 Supplementary Figure 4 Astrocyte stimulation induced LTD (a-ltd) is expressed presynaptically. Analysis of the change in the coefficient of variation (CV -2 ) of EPSP slopes after the expression of a-ltd by astrocyte stimulation paired with afferent stimulation. Plotting the ratio CV -2 After /CV-2 Control (i.e. the relative change in CV -2 ) against the change in EPSP slope (EPSP After /EPSP Control ) revealed that the coefficient of variation decreased with the expression of a-ltd, indicating a presynaptic locus of a-ltd. Grey circles represent individual experiments; solid circle represents the average.
a +40 mv -70 mv Control CNQX D-AP5 Intracellular MK801 CNQX 100 pa b NMDA:AMPA ratio 0.2 Control D-AP5 MK801 Supplementary Figure 5 Intracellular inclusion of MK801 effectively blocks NMDA receptors. (a) Left, example traces from an experiment to determine the NMDA:AMPA ratio. A L2/3 pyramidal neuron was patched with a voltage-clamp intracellular solution (see methods). For determining the AMPA receptor component, EPSCs were evoked by extracellular stimulation in L4 while the neuron was held at 70 mv (bottom trace). For determining the NMDA receptor component, AMPA receptors were blocked with CNQX (10 µm) and EPSCs were evoked while the neuron was held at +40 mv (top trace). The NMDA receptor component was abolished by subsequent washin of D-AP5 (50 µm, purple trace). Right, example traces from an experiment to determine NMDA:AMPA ratio in a neuron loaded intracellularly with MK801 (1 mm). Under these conditions, no NMDA receptor component was observed at +40 mv (pink trace). (b) Summary bar graph showing NMDA:AMPA ratio under control conditions (7 ± 0.13; n = 4), after washin of D-AP5 (4 ± 1; control vs D-AP5: P = 11; paired t-test), and for neurons loaded with MK801 (5 ± 1; n = 4; control vs MK801: P = 16; unpaired t-test). Data is presented as mean ± s.e.m.; indicates P < 5.