Supporting InformationHoon et al. 10.1073/pnas.1006946108SI Materials and MethodsAll experiments were performed on adult (8-to 10-wk-old) wild-type (WT) and NL4 or NL2 deletion-mutant (NL4-KO, NL2-KO;refs. 1 and 2) mice in compliance with the guidelines for thewelfare of experimental animals issued by the Federal Govern-ment of Germany, the National Institutes of Health, and theMax Planck Society.

Antibodies. For immunolabeling of NL4, a polyclonal rabbit an-tibody was used (1:1,000–1:2,000; ref. 2). To detect GABAA andglycine receptors (GlyRs), we used polyclonal guinea-pig anti-bodies specific for GABAA α1, α2, α3 or γ2 subunits (1:10,000,1:5,000, 1:4,000, and 1:2,500, respectively; ref. 3); pan-GlyR(mAb4a, 1:400; Synaptic Systems) or GlyRα1-specific (mAb2b,1:500; Synaptic Systems) monoclonal mouse antibodies; poly-clonal goat antibodies raised against GlyRα2 and α3 (both 1:400;Santa Cruz), and a polyclonal rabbit antibody specific forGlyRα4 (1:500; Chemicon). Specific retinal cell and synapsepopulations (Fig. S1) were detected with the following anti-bodies: Monoclonal mouse anti-PKCα (1:1,000; Biodesign), anti-GAD65 (1:1,000; Chemicon), anti-GAD67 (1:1,000; Chemicon),anti-PSD95 (1:1,000; Abcam), and anti-gephyrin (3B11, 1:1,000;Synaptic Systems); polyclonal goat anti-glycine transporter 1(1:1,000; Chemicon), guinea pig anti-VGLUT1 (1:2,000;Chemicon) or anti-VIAAT (1:1000; Synaptic Systems). Forbiochemical experiments, the antibodies used were as follows:Monoclonal mouse anti-collybistin (1:250; Transduction Labo-ratories) and anti-myc (9E10, 1:1,000; Sigma); polyclonal rabbitanti-NL4 (1:100; ref. 2), anti-NL2 (1:1,000; ref. 4), anti-gephyrin(3B11, 1:5,000; Synaptic Systems), anti-HA (1:2,000; Zymed),anti-human-IgG-Fc (HRP-coupled, 1:2,000; Pierce). Secondaryantibodies used for fluorescence labeling were anti-isotypicAlexa 488-, Alexa 555- or Cy5-conjugated antibodies (1:2,000;Molecular Probes).

Constructs. Myc-CB2SH3+ (5), GFP-Gephyrin (6), HA-NL2,and HA-NL3 (7) expression constructs have been described. AGST-CB2SH3+ fusion protein was generated by using thepGEX-4T-1 vector (Amersham Bioscience). Fc-tagged NL1,NL2, NL3, and NL4 constructs were generated by subcloningtheir respective cytoplasmic domain sequences into the pCMV-IgG-9 vector. HA-NL2ECD/NL1ICD and HA-NL2ECD/NL4ICDconstructs were created from rat and mouse NLs on the back-bone of a pcDNA3 HA-NL2 construct and include the extra-cellular domain of rNL2 (residues 1–670), and the transmem-brane and intracellular domains of mNL4 (residues 754–945)or rNL1 (residues 690–843). Sequencing of the full ORFs wasperformed to verify constructs in each case.

Immunohistochemistry in the Retina. The animals were deeplyanesthetized with Isofluran (DeltaSelect) and decapitated. Theeyes were quickly removed, the lens dissected out, and the eye-cups immersed in 2% paraformaldehyde in 0.1 M phosphatebuffer, pH 7.4 (PB) for 20 min. The fixation time was reduced to5 min for GABAA and GlyR labeling and increased to 3 h forglycine transporter 1 labeling. After fixation, the eyecups wererinsed in PB, and the retinae were isolated and cryoprotectedovernight in 30% sucrose in PB. Alternating pieces of WT andNL4-KO retinae were frozen on top of each other in tissue-freezing medium (Leica). These retina sandwiches were thensectioned vertically at 14 μm at the cryostat (8). For immuno-histochemistry, sections were preincubated in PB containing

0.2% gelatin and 0.1% Triton X-100 (PGT), incubated overnightwith primary antibodies in PGT, followed by a 1-h incubationwith fluorescent secondary antibodies in PGT.

Immunohistochemistry in the Brain and Spinal Cord. The animalswere deeply anesthetized with Isofluran (DeltaSelect) and killed.Brains were removed and frozen in −35 °C isopentane. Thelumbar part of the spinal cord was prepared by hydraulic ex-trusion (9) and washed in PB before being frozen in isopentane(−35 °C). Twenty-micrometer-thick sagittal and coronal sectionswere made at the cryostat, collected on slides, and immersion-fixed in methanol (−20 °C, 5 min). Sections were rinsed in PB,blocked for 1 h at room temperature in PB containing 5% nor-mal goat serum (NGS) and 0.1% Triton, then incubated over-night at 4 °C with primary antibodies in PB containing 3% NGSand 0.1% Triton (PNT). After several washes, the sections wereincubated for 1 h with fluorescent secondary antibodies in PNT.

Imaging and Quantification. Immunofluorescent sections and cellpreparations were viewed under an inverted Leica DMIRE2microscope connected to a TCS-SP2 AOBS confocal laser-scanning microscope (Leica Microsystems).All retina images used for quantification were acquired as

single optical sections with a 63× oil-immersion objective (N.A.1.4) and a digital zoom of 4. During acquisition, gain and offsetwere kept constant for a given labeling to allow for intensitycomparisons between images and genotypes. Images were fur-ther processed with AnalySIS. They were smoothed with an openfilter and objects (i.e., fluorescent clusters, corresponding tosynaptic puncta) were isolated by application of a separationfilter. In a selected region of interest (ROI) covering the entirewidth of the IPL, we measured the number (density) of all ob-jects above an intensity threshold designated as background (setto a gray value of 50 and maintained constant for all images). Forcolocalization studies, ROIs were drawn for each fluorescentpunctum in a given channel, superimposed on the complemen-tary channel, and the number of colocalized puncta were de-termined manually (10).For immunolabeling in the brain, overview images were taken

with a binocular (Leica MZ16); single-plane confocal imageswere taken with a 63× oil-immersion objective (N.A. 1.4) anda digital zoom factor of 8 for high magnification pictures.Immunolabeled COS cells were imaged as single-plane images

by using a 63× oil-immersion objective (N.A. 1.4) and a digitalzoom factor of 4. Quantification of specific microaggregate for-mation was performed as follows. In triple-transfected cells, thesize and shape of gephyrin aggregates was visually rated asforming “blobs” (large, rounded intracellular aggregates) or“microaggegates” (small submembranous aggregates), which aremutually exclusive. Blob-containing cells were scored as nega-tive, whereas microaggregate-containing cells were scored aspositive for activation.

Patch-Clamp Recordings from Retinal Ganglion Cells. Whole-cellpatch-clamp recordings from WT and NL4-KO retinal ganglioncells (RGCs) were performed on whole-mount retinal prepara-tions from 3-wk-old (P22–28) mice. All recordings were carriedout at room temperature and under dim-light conditions. Micewere not dark-adapted before experiments were carried out.Mice were deeply anesthetized with Isofluran (DeltaSelect) anddecapitated, and retinae were dissected in a low Ca2+ artificialcerebrospinal fluid (aCSF) containing 125 mM NaCl, 2.5 mM

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KCl, 1 mM MgCl2, 0.5 mM CaCl2, 25 mM glucose, 25 mMNaHCO3, 1.25 mM NaH2PO4, 0.4 mM ascorbic acid, 3 mMmyo-inositol, 2 mM Na-pyruvate at pH 7.3 when bubbled with carb-ogen (95% O2, 5% CO2). Retinae were mounted onto the ex-periment chamber with the ganglion cell layer facing up, and thecells were visualized by infrared differential interference contrastmicroscopy through a 60× water-immersion objective (Olympus;N.A. 1.00) of an upright microscope (Axioskop FS; Zeiss)equipped with a CCD camera (Till Photonics). During experi-ments, retinae were constantly perfused with low Ca2+ aCSF.Before recording from RGCs, the inner limiting membrane wasgently removed with a glass pipette (11). The intracellular pi-pette solution contained 55 mM Cs-Gluconate, 55 mM CsCl2,1 mM CaCl2, 10 mM EGTA, 10 mM Na-Hepes, 4 mM Mg-ATP,0.4 mM Na-GTP, 0.1 mM Alexa 488 (Molecular Probes) at pH7.3. The recordings were carried out with an EPC-10 patch-clamp amplifier (HEKA Elektronik) and the Pulse software.Patch pipettes were pulled on a PIP 5 vertical puller (HEKAElektronik) from borosilicate glass tubing (Hilgenberg). Whenfilled with internal solution, they had a resistance of 4–6 MΩ.Series resistance was <20 MΩ and routinely compensated (42%)during experiments. The signals were filtered by using the eight-pole Bessel filter, using a cutoff frequency of 5 kHz. The sam-pling rate during recordings was 20 kHz.To discriminate RGCs from displaced amacrine cells, also

localized in the ganglion cell layer, only cells with a diameter >15μm were recorded (11, 12). Further, only those cells generatingvoltage-activated sodium currents >2 nA were considered asRGCs (12, 13). Finally, the recorded cells were visualized posthoc to ascertain that they bore an axon projecting away fromthe ganglion cell layer, another distinguishing feature of RGCs.The visualization of the dendritic arbor of RGCs further allowedto distinguish ON-ganglion cells (with a superficial dendriticbranching) from OFF-ganglion cells (which branch deep into theIPL). The presence of an axon was first determined during re-cordings and further verified after recording when the retinaslice was fixed for 15 min in 2% paraformaldehyde (PFA) in PB,rinsed, mounted on a slide and viewed under an inverted TCS-SP2 confocal laser-scanning microscope (Leica Microsystems).Glycinergic mIPSCs were recorded at −70 mV, which is close

to the resting membrane potential of RGCs (estimated to beapproximately −65 mV; ref. 14), in the presence of NBQX(1 μM), AP5 (50 μM), and bicuculline (50 μM). After measuringsodium currents, TTX (1 μM) was applied to the bath to preventthe generation of action potentials. The frequency of glycinergicmIPSCs has been shown to be TTX-independent (15). GA-BAergic mIPSCs were also recorded at a −70 mV holding po-tential in aCSF containing NBQX (1 μM), AP5 (50 μM), andstrychnine (500 nM). In the absence of TTX, RGCs displayedvery robust GABAergic mIPSC activity, which frequently oc-curred in bursts. Because the high frequency of events hamperedthe analysis of individual mIPSCs, we generally bathed the reti-nae in TTX-supplemented external solution. TTX has beenshown to significantly reduce the frequency of GABAergicmIPSCs (15) in RGCs from rat retinal slices. It took severalminutes for the activity rate to decrease after TTX was washed in.Data analysis was carried out in IGOR Pro-6.1 (Wavemetrics).

mIPSCs were detected by using a sliding template algorithm (16).Peak amplitudes, decay time constants (from single exponentialfits) (11), and (20–80%) rise times were estimated from the av-erage mIPSC trace of each individual cell. In addition, the fre-quency distributions of decay time constants were obtained fromfitting exponentials to individual mIPSCs. Because small eventsproduced unreliable fits, only events with a peak amplitude ≥15pA were included in this analysis. Cumulative histogram of thedecay time constant (τ) values of individual glycinergic mIPSCswere computed for the entire population of WT and NL4-KORGCs. Note that the decay time course of glycinergic IPSCs is

voltage-dependent, with a slower decay at more positive mem-brane potentials (17, 18), which we confirmed here (τ ≈ 2.74 msat −50 mV, τ ≈ 3.01 ms at −30 mV). In previous studies, thedecay time constant from A-type ganglion cells was determinedat τ= 3.9 ± 2.5 ms (mean ± SD) at a holding potential of −5 mV(11). From our recordings performed at −70 mV, we would es-timate that the time constant would be ≈67% of the valuemeasured at −5 mV, which is close to the value we determinedfor the decay time constant of glycinergic mIPSCs from WTRGCs: τ = 2.42 ± 0.10 ms (mean ± SEM).

Multi-Electrode Array (MEA) Recordings. Recordings were per-formed by using 200/30 MEAs (60 electrodes, 30-μm diameter,200-μm spacing, 8 × 8 grid) and the MEA60BC amplifier (MultiChannel Systems). Mice were dark-adapted before experiments.Retinae were dissected under dim red light and kept in oxy-genated aCSF at 37 °C, containing 125 mM NaCl, 2.5 mM KCl, 2mM CaCl2, 1 mM MgCl2, 26 mM NaHCO2, 1.25 mM Na3PO4,25 mM glucose, equilibrated with 95% O2 and 5% CO2. Forrecordings, retina pieces were placed with the ganglion cell layerfacing the MEA. Potentials were digitized at 10 kHz and high-pass filtered at 100 Hz. To limit files size, 3-ms spike cutoutswere saved every time a threshold of −10 μV was crossed. Thesespike cutouts were sorted offline into individual units by usingOffline Sorter 2 (Plexon). Light stimuli were delivered by a greenlight emitting diode (LED) placed at the camera port of an in-verted microscope (BX-51; Olympus) triggered by a STG1004stimulus generator and MC-Stimulus 2.0 software (Multi Chan-nel Systems). Either an ON/OFF stimulus, consisting of a 1-slight pulse every 3 s, or a 10-s-long “white noise” stimulus wasdelivered 200 times. The latter pseudorandom white noisestimulus (19, 20) was generated by randomly assigning values of0 or 1- to 30-ms bins (SD/avg = 0.97). The trigger signal (light onor off) was recorded together with the action potentials and usedto calculate the spike-triggered average (STA) stimulus for eachrecorded cell. For the 1-s light stimulus, perievent histograms(10-ms bins) were calculated by Neuroexplorer 3 (NEX Tech-nologies) and expressed in spikes per second (Hertz). Sub-sequent analysis was carried out by using IGOR Pro-5.03(Wavemetrics). To determine amplitudes and latencies, perieventhistograms were smoothened by convolution with a Gaussian (σ =30 ms) as described (21). Cells were classified manually as “ON,”“OFF,” or “ON-OFF,” blinded for genotype. STAs were calcu-lated and analyzed in IGOR. Classification of response traces asmonophasic or biphasic and having a predominant positive ornegative peak was carried out manually and blinded for geno-type. The amplitude of monophasic STAs was measured froma 30-ms baseline to the peak. For biphasic STAs, the amplitudewas measured from peak to peak. The STA was calculated byaveraging for each cell the 500 ms of stimulus preceding anaction potential. Statistical analyses were carried out with JMP5 (SAS Institute) and Graph Pad Prism 4. Graphs representmean ± SEM.

Electroretinogram Recordings. Mice were dark-adapted overnight,anesthetized by i.p. injection of ketamine (125 μg/g) and xylazine(2.5 μg/g), and got one pupil dilated with 1% atropine sulfate.Surgery and subsequent handling were done under dim red light.A moistened ring-like AgCl wire electrode was placed on thecornea, a needle reference electrode was inserted s.c. above thenose, and a ground electrode into the tail. Full-field illumination(25 white LEDs) was used to produce light flashes of incrementalcalibrated intensities (0.0002–14 cds/m2; Mavolux IPL 10530).Scotopic (dark-adapted) responses were recorded (WT: n = 8;NL4-KO: n = 9 animals) for three different stimulus durations(0.1, 1, and 5 ms) with an interstimulus interval of 5 s (<1 cds/m2)or 17 s (>1 cds/m2) (22). Recorded potentials were amplified,filtered (band pass 400–4,000 Hz), and sampled at a rate of

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24 kHz. Ten responses were averaged for each light intensity. Foranalysis, amplitudes of the a-wave were measured relative to thebaseline, whereas the b-wave amplitudes were estimated relativeto the trough of the a-wave. Oscillatory potentials were sorted byusing a 30 Hz high-pass filter and the peak-to-peak amplitudeswere assessed. Latencies of the a- and b-waves were determinedas the temporal difference between light onset and time of peak.All data were analyzed by using MATLAB software.

Visual Acuity and Contrast Function Tests.Visual acuity was assessedby using the recently developed virtual reality optomotor system(23). Briefly, freely moving animals exposed to moving sine wavegratings of various spatial frequencies and contrasts reflexivelytrack the gratings by head movements as long as they can see thegratings. Spatial frequency at full contrast and contrast at sixdifferent spatial frequencies were varied by the experimenter untilthe threshold of tracking was determined. Because only motion inthe temporal-to-nasal direction evokes tracking, it was possible tomeasure thresholds and contrast functions for both eyes sepa-rately by reversing the direction of the moving gratings (24).

Yeast Two-Hybrid and in Vitro GST Pulldown Assays. Yeast-two-hybrid assays were performed as described (7). GST-pulldownassays were performed by using lysates from Escherichia coliBL21 DE3 cells expressing GST or GST-CB2SH3+. Glutathione-Sepharose beads were incubated with cleared lysates containingGST proteins for 2 h at 4 °C and subsequently with cleared ly-sates from 293T cells expressing NL1icd-Fc, NL2icd-Fc, NL3icd-Fc, or NL4icd-Fc overnight at 4 °C in TNE buffer (50 mMTris·HCl, 50 mM NaCl, 5 mM EDTA, 1 μM leupeptin, 1 μg/mLaprotinin, and 100 μM PMSF at pH 7.4). After washing, thebound proteins were analyzed by SDS/PAGE followed by im-munoblotting with a polyclonal, HRP-conjugated rabbit anti-human IgG-Fc antibody to detect Fc-tagged proteins.

Immunoprecipitation of Cross-Linked Material. For each sample(WT, NL2-KO, NL4-KO), cross-linking was performed onpostnuclear homogenates from brainstems and spinal cords oftwo adult mice. Homogenates were incubated on ice for 20 minwith 200 μM DSP (Pierce). The cross-linker was quenched in

Tris buffer. Each sample was centrifuged at 21,000 × g for15 min, and protein was extracted from pellets with 1% SDS inTNE buffer. Samples in 1% SDS were diluted with 7 volumes ofTNE buffer containing 1% Triton X-100. Either 10 μL of rabbit-anti-NL2 or rabbit-anti-NL4 antiserum was added to each sam-ple, and samples were incubated overnight at 4 °C. Fifty-micro-liter bed volume of Sepharose beads bearing immobilizedProtein A (Amersham) were added to the samples, and sampleswere incubated for 4 h at 4 °C. Beads were washed extensivelyand eluted in sample buffer containing 2% SDS and 7.5%β-mercaptoethanol, boiled for 5 min, and further incubated at60 °C for 30 min to ensure complete reductive cleavage of thecross-linker. Samples were subsequently probed for endogenousgephyrin and collybistin via SDS/PAGE and immunoblotting byusing standard procedures.

Transfection and Immunostaining of Cell Lines. COS7 cells wereplated directly onto glass coverslips, cultured in DMEMwith 10%FCS, and transfected by using FuGENE6 (Roche) with proce-dures based on standard protocols. Upon transfection in COS7cells, a pcDNA3 construct bearing the ORF of mNL4 was nottrafficked properly to the plasma membrane. Therefore, chimericconstructs expressing the NL2 extracellular domain fused to ei-ther NL1 or NL4 intracellular domains (including their trans-membrane domains) were generated and used instead. Cells werefixed 12 h after transfection in 4% paraformaldehyde, 5.5% su-crose in 100mMphosphate buffer at pH 7.4, 0.9%NaCl (PBS) for10 min at room temperature. Cells were blocked with 5% normalgoat serum and 0.1% gelatin in PB and incubated overnight at4 °C in polyclonal rabbit anti-HA for surface labeling of NLs.After permeabilization with 0.1% Triton X-100, the sampleswere further incubated with the monoclonal anti-myc antibody tovisualize collybistin. Secondary antibody staining was performedfor 1 h at room temperature by using anti-isotypic fluorophoreconjugated antibodies Alexa-555 and Cy5.

Statistics. Because all of the data passed the normality test, sta-tistical analysis between genotypes was carried out using the two-tailed unpaired (Welch corrected) t test.

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Fig. S1. Retinal architecture in NL4-deficient animals. Selected protein markers were used to assess the integrity of the retinal circuit in the absence of NL4.Immunolabeling for inhibitory (VIAAT) and excitatory (VGLUT1) presynapses, inhibitory (gephyrin) and excitatory (PSD-95) postsynapses, GABAergic amacrine(GAD65/67), glycinergic amacrine (GlyT1), rod bipolar (PKCα) cell populations, and GABAA receptor subtypes (α1, α2, α3, γ2 subunits) were compared in WT andNL4-KO retinae.

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Fig. S2. Expression of NL1, NL2, and NL3 in WT and NL4-KO retinae. Expression levels of NL1, NL2, and NL3 were comparable upon Western blot analysis ofWT and NL4-KO retinal homogenates (n = 5 × 4 retinae). Western blotting of actin was used as a loading control (A). The distribution (B) and number (C) ofNL2-immunopositive clusters were comparable in the IPL of WT and NL4-KO sections (n = 3 pairs). (Scale bar: 10 μm.)

Fig. S3. Spiking activity of NL4-KO RGCs in response to light flashes. RGC responses to a 1-s ON/2-s OFF stimulus paradigm (A). No change was observed in theoccurrence of different RGC types in the NL4-KO (B). NL4-KO RGCs were similar to WT cells with respect to their baseline activity (C) and maximum peakamplitudes for both ON (D) and OFF (E) responses.

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Fig. S4. NL4-specific labeling is observed throughout the brain. Moderate levels of NL4 immunoreactivity can be observed in the olfactory bulb, cortex,hippocampus, and striatum on sagittal sections of an adult mouse brain (A), whereas low levels were detected in the cerebellum. In the globus pallidus,thalamus, colliculus, substantia nigra, pons, and all brainstem nuclei, NL4 immunoreactivity is strong and forms synaptic clusters. Strikingly, the overall dis-tribution pattern of NL4 (A Upper) does not correlate with that of PSD-95 (A Lower), in line with an absence of NL4 from glutamatergic postsynapses. Of note,no unspecific labeling was observed on a similar section taken from a NL4-KO mouse brain and processed in parallel (B). (Scale bar, 1 mm.)

Fig. S5. NL4 localization in the CNS. In the barrel cortex (A, left panels), NL4-immunoreactivity is strongest albeit diffuse in layers IV and II/III. In the hip-pocampus (A, right panels), faint NL4 labeling can be observed in the stratum lacunosum moleculare, in the polymorphic layer of the dentate gyrus, and, toa lesser extent, in the stratum oriens. In contrast, NL4 is clustered specifically at inhibitory postsynapses in numerous brain areas, including the inferior colliculus(B) as illustrated by colocalization with the inhibitory postsynapse protein gephyrin (B). (Scale bars: A, 500 μm; B, 10 μm; detail, 1 μm.)

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Fig. S6. NL4 and NL2 are coclustered with gephyrin and collybistin in heterologous cells. Submembranous microaggregates containing NLs, gephyrin andcollybistin are generally observed in COS7 cells upon expression of HA-NL2 or HA-NL2ECD-NL4ICD, but not with HA-NL3 or a HA-NL2ECD-NL1ICD fusion protein.(Scale bar: 20 μm.)

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