Supplemental Information Super-resolution Imaging of Chemical Synapses in the Brain Adish Dani, Bo Huang, Joseph Bergan, Catherine Dulac, Xiaowei Zhuang This supporting information file includes Figure S1 Figure S2 Figure S3 Figure S4 Figure S5 Table S1 Movie S1
Supplemental Figures: A Cy3 Cy3 Cy3 Photoactivation of dye pairs Cy2 A405 Cy2 A405 Cy2 A405 532 nm 460 nm 405 nm B Activation / Imaging Laser 532 nm 460 nm 405 nm repeat many cycles Activation events in a local region summed over all imaging cycles N Cy3 N Cy2 I 10I 460 532 n 0 n 0 n 0 n 0 n 0 n 0 N Cy3 N A405 n 0 n 0 n 0 n Cy3 n Cy2 n A405 CCD frame Figure S1 (related to Figure 1). Schemes for multicolor STORM and cross-talk correction. (A) -based activator-reporter probes for multicolor STORM. The three activator-reporter pairs, Cy3-, Cy2- and A405- can be distinguished by the wavelength of the activation laser that they each respond to. Cy3- is specifically activated by the 532 nm light, Cy2- is specifically activated by the 460 nm light, and A405- is specifically activated by the 405 nm light. (B) Color cross-talk subtraction scheme. The upper panel shows the activation (532 nm, 460 nm and 405 nm) and imaging laser () pulse sequence, in which each activation laser pulse is followed by three imaging frames. This sequence is then repeated over many imaging cycles. The lower panel shows the composition of activation events detected in the imaging frames. N Cy3, N Cy2 and N A405 refer to the specific activation contributions caused by the 532 nm, 460 nm and 405 nm activation pulses, respectively. They are present only in the imaging frames immediately following the activation pulses. n 0 refers to the nonspecific activation contribution caused by the imaging laser, which is present in all imaging frames. n Cy3, n Cy2 and n A405 denote the total numbers of activation events in the first imaging frames following the activation pulses. A small cross-activation contribution due to activation of Cy3- by the 460 nm laser is also shown (green bar in the n Cy2 column). In the cross-talk subtraction procedure, the above numbers are calculated by summing over all imaging cycles for better statistics.
Figure S2 (related to Figure 3). Low-magnification image of Bassoon and Homer1 in the olfactory bulbs and nearby orbital cortex. Bassoon (red) and Homer1 (green) are immunostained with antibodies against the N-terminal domain of Bassoon and C terminal domain of Homer1, respectively. The AOB, MOB and Orbital Cortex regions are marked.
Figure S3 (related to Figure 4). The axial positions of synaptic proteins. Various synaptic proteins (blue) were imaged together with Bassoon[N] (red) and Homer1[C] (green). The axial position distributions of PSD95, CaMKII, NR2B, GABA B R1, Piccolo[N], Bassoon[C] and RIM1 are shown here and those of Homer[N], Shank1, GluR1 and Piccolo[C] are shown in Figure 4A-D. For PSD-95, three-color imaging of PSD-95, Bassoon[N], and Homer1[C] was difficult due to a conflict in antibody host and subtypes. We therefore used twocolor imaging to position PSD-95 along with the Bassoon[C]. The position of Bassoon[C] has already been determined in our synaptic coordinate system, enabling us to unambiguously place the average axial position of PSD-95 with the knowledge that PSD-95 is a postsynaptic protein.
Figure S4 (related to Figure 6). Correlation plots of the number of localization points (N) of NR2B, GluR1, Bassoon, and Shank1 versus the number of localization points of Homer1 for synapses in the AOB and MOB regions. (A) N NR2B vs. N Homer1. The correlation coefficient is 0.27 in the AOB and 0.67 in the MOB. (B) N GluR1 vs. N Homer1. The correlation coefficient is 0.29 in the AOB and 0.34 in the MOB. (C) N Bassoon vs. N Homer1. The correlation coefficient is 0.35 in the AOB and 0.51 in the MOB. (D) N Shank vs. N Homer1. The correlation coefficient is 0.25 in AOB and 0.51 in MOB.
Figure S5 (related to Figure 7). Electrical activity recording in the AOB region of a ChR2- YFP expressing mouse upon the VNO stimulation by light. (Left panel) Sagital cross-section of the AOB from an ORC-V mouse. YFP fluorescence (green) marks the glomerular layer and the tract from a DiI coated electrode (red) shows the recording sites penetrating through the AOB. (Right panel) Electrical activity recorded from eight electrode sites with 100 µm spacing along the dorsal-ventral axis. Blue dashes on the x-axes indicate the time of light stimulation (50 ms duration, 1 second inter-stimulus interval). The top five voltage traces recorded in the AOB region show electrical activities in sync with the light activation. As an internal control, the bottom three voltage traces, recorded from electrode sites that were outside the AOB show no significant modulation of electrical activity in response to light stimulation.
Supplemental Tables: Table S1 (related to all figures): Primary antibodies used in this work Target protein Species Clone Subtype Catalogue number Supplier Bassoon Mouse SAP7F407 IgG2a 13249 Abcam (N-terminal) Bassoon (C-terminal) Homer1 (1b/c) Rabbit Polyclonal 141003 Synaptic Systems Rat Polyclonal 5877 Millipore (C-terminal) Homer1(1a/b/c) (N-terminal) Piccolo (C-terminal) Piccolo (N-terminal) Rabbit Polyclonal 160003 Synaptic Systems Rabbit Polyclonal 142003 Synaptic Systems Rabbit Polyclonal 20664 Abcam RIM1 Rabbit Polyclonal 140003 Synaptic Systems PSD95 Mouse 6G6-1C9 IgG2a 2723 Abcam Shank1 Mouse N22/21 IgG1 75-064 NeuroMab CaMKII Mouse 6G9 IgG1 22609 Abcam GluR1 Rabbit Polyclonal 31232 Abcam GABA B R1 Guinea Pig Polyclonal 2256 Millipore NMDAR2B Mouse 13/NMDAR2B IgG2b 610416 BD Biosciences c-fos Rabbit Polyclonal Sc-52 Santa-Cruz
Supplemental Movie: Movie S1 (related to Figure 2). Three-dimensional view of Bassoon and Homer1 clusters in a synapse. In this movie, the Bassoon (red) and Homer1 (green) pair is rotated, allowing its 3D morphology to be clearly visualized. The side and face views of this synapse is shown in Figure 2A-C.