Supplemental Information. Differential Regulation. of Evoked and Spontaneous Release. by Presynaptic NMDA Receptors

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1 Neuron, Volume 96 Supplemental Information Differential Regulation of Evoked and Spontaneous Release by Presynaptic NMDA Receptors Therése Abrahamsson, hristina You hien hou, Si Ying Li, Adamo Mancino, Rui Ponte osta, Jennifer Anne rock, Erin Nuro, Katherine Anne uchanan, Dale Elgar, Arne Vladimir lackman, Adam Tudor-Jones, Julia Oyrer, William Todd Farmer, Keith Kazuo Murai, and Per Jesper Sjöström

2 meps amp (pa) meps freq (Hz) GluN-specific NMDAR blockade pa 5 ms meps amplitude meps frequency pa -3 s 4 Ro Ro baseline Ro difference NMDA:AMPA (%) meps frequency 9 control Ro NMDA:AMPA ratio 9 control Ro NMDA:AMPA ratio NMDA:AMPA ratio.5 Ro time (min) 4 6 Figure S. At P3-P5, Postsynaptic NMDARs ontain the GluN Subunit, Related to Figure (A) Sample experiment showing that, in. mm Mg ++, wash-in of the GluN-specific NMDAR antagonist Ro reduced mini frequency (.58 ±.4 Hz to.45 ±., p <.5) and NMDA:AMPA ratio (.6 ±. to.9 ±.5, p <.) but not amplitude (- ±.5 pa to - ±. pa, p =.36) as expected from combined pre- and postsynaptic effects. Top: The difference current (black) is consistent with the relatively slow kinetics of GluN-containing NMDAR-mediated current (Paoletti et al., 3) that affects the peak mini current relatively little. Inset figures: umulative histograms. () Ro robustly reduced mini frequency as compared to controls, consistent with the existence of prenmdars at in L5 Ps from P3-P5 animals. aseline mini frequency was.57 ±.6 Hz, n = 9, considerably lower than at P-P6 (e.g. p <. compared to Figure S). () At P3-P5 as opposed to at P-P6 (see Figure S), Ro reduced the NMDA:AMPA ratio, demonstrating that postsynaptic NMDARs in L5 Ps of mouse visual cortex are sensitive to GluN-specific blockade, as previously demonstrated in the rat (Stocca and Vicini, 998). aseline NMDA:AMPA ratio was.8 ±., n = 9, somewhat higher than at P-P6 (p <.5), presumably because currents of NMDAR containing the GluN subunit decay more slowly than those with GluNA (Paoletti et al., 3).

3 Non-specific NMDAR blockade GluN-specific NMDAR blockade 5 pa 5 ms baseline difference 5 pa 5 ms baseline Ro difference meps amplitude meps amplitude meps amp (pa) pa -4 meps amp (pa) pa -4 Ro meps freq (Hz) meps frequency s meps freq (Hz) 3 meps frequency s Ro NMDA:AMPA ratio NMDA:AMPA ratio NMDA:AMPA NMDA:AMPA Ro 3 time (min) time (min) meps frequency 8 control Ro D NMDA:AMPA (%) 5 5 NMDA:AMPA ratio 8 control Ro Figure S. At P-P6, prenmdars ontain the GluN Subunit whereas Postsynaptic NMDARs do not, Related to Figure (A) Sample experiment in. mm Mg ++ to relieve NMDARs of Mg + block showing that wash-in of the non-specific NMDAR antagonist reduced mini amplitude (-6 ±.3 pa to -4 ±. pa, p <.), frequency (.9 ±.5 Hz to.4 ±.5 Hz, p <.), and NMDA:AMPA ratio (. ±.5 to.7 ±.3, p <.) as expected from combined pre- and postsynaptic effects. Top: The difference current (black) is consistent with an NMDAR-mediated current. Inset figures: umulative histograms. () Sample recording showing a reduction of mini frequency (.3 ±.8 Hz to. ±.5 Hz, p <.) but not amplitude (-6 ±. pa to -6 ±. pa, p =.79) or NMDA:AMPA ratio (. ±.7 to.3 ±.5, p =.9) due to wash-in of Ro in. mm Mg ++, showing that pre- but not postsynaptic NMDARs are sensitive GluN-specific blockade. Top and insets as in (A). () oth and Ro robustly reduced mini frequency as compared to controls, in agreement with the view that prenmdars are sensitive to GluN-specific blockade (rasier and Feldman, 8; Sjöström et al., 3; Woodhall et al., ). The additional reduction of mini frequency due to results from the mini detection software missing events that drop below the detection threshold (STAR Methods) and does not imply that blocks prenmdars better than Ro does. aseline mini frequency was.8 ±. Hz, n = 9.

4 (D) but not Ro reduced the NMDA:AMPA ratio, showing that post- but not presynaptic NMDARs have undergone the GluN-to-A developmental switch in L5 Ps of P-6 mice, as previously demonstrated in the rat (Stocca and Vicini, 998) (also see Sjöström et al., 3). aseline NMDA:AMPA ratio was.4 ±., n = 9. 3

5 -8-4 Mock wash, n = baseline, data mock wash, data baseline, model mock wash, model EPS amp (pa) -8-4 Reduced calcium, n = 4 baseline, data a wash, data baseline, model a wash, model -8-4 Ro wash-in, n = baseline, data Ro wash-in, data baseline, model Ro wash-in, model 5 EPS number vesicle U SE (%) -5 Depletion 4 recovery (%) 5 Recovery * * ctrl a Ro ctrl a Ro Figure S3. omputer Modelling Supports the Finding that PreNMDARs Regulate RRP Replenishment Rate during Evoked Release, Related to Figure 3 (A) The TM short-term depression model (Tsodyks and Markram, 997) was fitted (open symbols) using ayesian inference (see STAR Methods and osta et al., 3) to data (closed symbols) from the three conditions: mock wash-in, reduced a +, and Ro wash-in. Note that Ro wash-in (but not lowered a + ) reduced the steady-state response amplitude, indicating a decreased RRP replenishment rate. () The vesicle usage parameter U SE was decreased by both lowered a + ( a ) and by Ro washin ( Ro ) relative to controls ( ctrl ), implying a reduced p r in both conditions. () Ro wash-in but not lowered a + increased the recovery time constant t recovery, demonstrating that Ro wash-in had effects beyond those resulting from reduced a + influx. 4

6 RIM expression Synaptic protein levels RIM RIM fl/+ RIM fl/fl PSD95 RIM +/+ RIM -/- 5 vglut 5 % RIM +/+ % 5 GluN 5 RIM fl/+ RIM fl/fl GluN PSD95 vglut GluN GluN IP: GluN o-immunoprecipitation IP: GluN RbIg Input Input RIM fl/fl RbIg Input 5 RIM GluN Figure S4. An Association of GluN-RIM is Evidenced by haracterization of WT and RIMab Knockout Mice, Related to Figure 4 (A) RIM expression levels were progressively reduced in cortex of heterozygous and homozygous KO mice (n = 5 animals per condition). Expression was not, however, abolished in cortex from homozygous RIM deletion animals, in keeping with the specificity of the Emx promoter for Ps (Gorski et al., ), which leaves RIM intact at inhibitory synapses. As numerical values depend on exposure time, this quantification does not imply that overall % of RIM is left in homozygous mice, only that gradually less RIM is present in homo- and heterozygous deletion mice compared to controls. This graded reduction is consistent with RIM haploinsufficiency (see main text). () The expression levels of the key synaptic proteins PSD95, vglut, GluN, and GluN were not detectably affected by homozygous RIM KO (n = 4 animals per condition, except for GluN: n = 3). () Left: GluN IP with blotting for RIM demonstrates a band of the expected size, consistent with GluN and RIM being associated in the same complex. Right: GluN immunoprecipitation (IP) followed by blotting for GluN reveals an appropriately sized band compared to in total lysate (Input). RbIg: Rabbit immunoglobulin negative control. Note that the RIM band is stronger than in A, where only.5% of input was loaded. 5

7 Figure S5. Emx-re Drives Expression in a Majority of Neurons, Related to Figure 4 (A) Sample PLSM red-channel image of acute slice from ;Ai9 tdtom/+ mice (STAR Methods) showing only a handful of Ps that were not labelled by tdtomato (black pyramid-shaped regions). () Laser-scanning Dodt contrast (STAR Methods) of the same slice illustrates the straight-forward identification of Ps. () Overlay of images in A and illustrate how labeled versus unlabeled Ps were counted. (D) Thirty-six of 4 Ps (9%) in the sample image in panels A- were labelled. (E) Across 3 mice, 87% ± % of cells were labelled. Although the incidence of tdtomato-expressing Ps in ;Ai9 tdtom/+ mice need not correspond perfectly to the rates of Ps with RIM KO in ;RIMab fl/+ or ;RIMab fl/fl mice, this verifies previous results showing that Emx mice drive re expression in a majority of neocortical excitatory neurons (Gorski et al., ). 6

8 EPSP amp (mv) PPR * RIM fl/+ RIM fl/+ RIM fl/fl RIM fl/fl Figure S6. Evoked Release is Reduced in RIMab KO Mice, Related to Figures 4 and 5 Across 385 paired L5 P recordings, RIMab KO led to smaller EPSP amplitude (A, ) and increased PPR ratio (, D) in a gene-dosage-dependent manner, in keeping with the previously reported role for RIMs in short-term depression and vesicle priming (Südhof, ). denotes WT, RIMab fl/fl ;no- re, and ;RIMab +/+ pooled, as they were indistinguishable (amplitude p ANOVA =.7 and PPR p ANOVA =.45; STAR Methods). % D % 5 5 EPSP amp (mv) PPR ;RIM fl/+ ;RIM fl/fl 3 ;RIM fl/+ ;RIM fl/fl 3 7

9 µm µm 5%dG/R 5ms 5mV * dg/r (%) E dg/r (%) RIM fl/+ RIM fl/+ Dendritic calcium signals D RIM fl/fl Axonal calcium signals F RIM fl/fl % % dg/r (%) dg/r (%) ;RIM fl/+ ;RIM fl/fl ;RIM fl/+ ;RIM fl/fl 5 Figure S7. Axonal but not Dendritic Spike-Triggered a + Transients are Reduced in RIMab KO Mice, Related to Figure 6 (A) Sample PLSM stack showing an axon collateral (arrow heads) branching off the main axon (asterisk), with position of linescan indicated in inset image. () The spike-evoked a + signal was measured in a 5-ms-long window (grey box) positioned 5 ms after the onset of the current injection (five 5-ms-long current pulses at 5 Hz, see STAR Methods). The a + signal (blue; average of six linescans) was box-filtered at 7 ms for clarity, but signal was measured on unfiltered data. (, D) RIMab KO had no effect on spike-mediated a + transients in dendritic compartments, indicating no appreciable role for RIM on the postsynaptic side. (E, F) a + signals were reduced in axonal boutons, in keeping with the previously reported role for RIMs in scaffolding VDs (Südhof, ). These findings are also consistent with the reduced evoked responses (Figure S6). denotes WT and ;RIMab +/+ pooled, as no difference was found (p =.3; STAR Methods). 8

10 5 ; control, n=, n= control meps amp (%) 5 control 5 time (min) ;RIM fl/+ control, n=6, n=9 3 5 control meps amp (%) 5 control 5 time (min) ;RIM fl/fl 3 control, n=7, n= 4 5 control meps amp (%) 5 control time (min) Figure S8. PreNMDAR-Dependent Regulation of Spontaneous Release is Unaffected by RIMab Knockout, Related to Figure 7 wash-in reversibly reduced mini frequency but not amplitude in ;RIMab +/+ L5 Ps (A, red), as expected from prior findings (Figure ) (erretta and Jones, 996; orlew et al., 7; Sjöström et al., 3). Results in heterozygous ;RIMab +/fl (, red) or homozygous ;RIMab fl/fl KO Ps (, red) were the same, indicating that prenmdar-mediated regulation of spontaneous release did not directly need RIM. Mock wash-in controls are shown in blue (A-). 9

11 amp (pa) freq (Hz) RIM fl/+ RIM fl/+ RIM fl/fl RIM fl/fl Figure S9. Spontaneous Release is Increased in Homozygous RIMab KO Mice, Related to Figure 7 Homozygous but not heterozygous RIMab KO led to larger mini amplitude (A, ) and frequency (, D). denotes RIMab fl/fl ;no-re and Emx re/re pooled, as they were not different (amplitude p ANOVA =.94 and frequency p ANOVA =.89; STAR Methods). % D % amp (pa) 4 freq (Hz) -5 ;RIM fl/+ ;RIM fl/fl 6 - ;RIM fl/+ ;RIM fl/fl 8

12 Table S. P-P onnectivity Rates were Unaffected by RIMab KO, Related to Figure 7 ategory onnections found Pairs tested onnectivity rate (%) Number of cells RIM +/ % 473 ;RIM fl/ % 63 ;RIM fl/fl % 386 Totals % 349 No differences in connectivity were found (p =., three-category chi-squared test; p =.9, ochran-armitage test for decreasing trend). denotes WT, RIMab fl/fl ;no-re, and ;RIMab +/+ pooled, as they were not significantly different (38/33, 53/474, 34/69 respectively, p =.3, three-category chi-squared test; STAR Methods). For simplicity, and to avoid possible experimenter bias (e.g. slice cutting angle, depth of patching in the slice), only paired recording data from one experimenter (T.A.) was used for this analysis. Table S. RIMab KO did not Affect L5 P asal Dendrite Spine Densities, Related to Figure 7 ategory Spine density per µm Number of dendritic segments Number of cells Postnatal age RIM +/+.9 ± ±.3 ;RIM fl/+.4 ±. 3 3 ± ;RIM fl/fl.7 ± ±. Spine densities were not different (p =.39, Kruskal-Wallis). Animal ages of this data set were not biased (p =.4, Kruskal-Wallis). denotes WT and ;RIMab +/+ pooled, as they were not different (.8 ±.3 per µm, n =, 3. ±., n =, p =.67, Wilcoxon-Mann-Whitney two-sample rank test; STAR Methods).