Jana F Liewald, Martin Brauner, Gregory J Stephens, Magali Bouhours, Christian Schultheis, Mei Zhen & Alexander Gottschalk

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1 nature methods Optogenetic analysis of synaptic function Jana F Liewald, Martin Brauner, Gregory J Stephens, Magali Bouhours, Christian Schultheis, Mei Zhen & Alexander Gottschalk Supplementary figures and text: Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 Supplementary Figure 7 Supplementary Figure 8 Supplementary Table 1 Supplementary Table 2 Characterization of photo-evoked changes in body length Expression patterns of ChR2::YFP in ACh and GABA neurons Length chronograms in wild type and mutant animals after lightevoked transmitter release Synaptic physiology is normal in ChR2::YFP expressing animals Origin of the enhanced ACh mediated contraction phenotype in presynaptic mutants Photostimulation trains can be applied for long time periods ACh and GABA photo-epscs during 2Hz photostimulation trains at different intertrain intervals (ITI) Single ACh and GABA photo-epscs with 20 or 60s interstimulus intervals (ISI) Summary of photo-evoked ACh- and GABA-mediated contraction and relaxation after 520 ms of illumination of all mutants tested Summary of ACh and GABA photo-epscs in the various mutants analyzed Supplementary Results Supplementary Methods Note: Supplementary Videos 1 6 are available on the Nature Methods website.

2 Supplementary Figure 1 Characterization of photo-evoked changes in body length (a) Neuronal connectivity controlling ventral muscles, exemplified for A- and D-type motor neurons. Analogous circuits control dorsal muscles. (b) Time course of changes in relative body length of zxis6 transgenic animals during light-evoked ACh release, triggered by continuous blue-light illumination (open grey boxes, indicated by grey bar), or 10ms illumination (closed black boxes, indicated by black bar) (n = 10). (c) Light induced changes in mean relative body length in animals expressing ChR2 in different sets of cholinergic neurons from different promoters (n = 10). (d) Light-evoked changes in body elongation or contraction were compared in adult animals and L4 larvae (n = 10). (e) Weak positive correlation between absolute body length in pixels and the extent of contractions effected by light-induced ACh release in adults and larvae. In each panel, the number of animals analyzed (n) is indicated. Error bars represent the standard error of the mean, s are given after Student s t-test (* P < 0.05; ** P < 0.01; *** P < 0.001), as reported throughout. 1

3 Supplementary Figure 2 Expression patterns of ChR2::YFP in ACh and GABA neurons (a) ChR2::YFP in cholinergic neurons shows a normal wiring pattern of the neurons. Arrows point to motoneuron cell bodies, commissures connecting ventral neurons to dorsal muscles are indicated by arrowheads. (b) Analogous analysis as in (a), for ChR2::YFP expressed in GABAergic neurons. (c) Confocal stacks of transgenic animals expressing ChR2::YFP in cholinergic or GABAergic neurons. Mutations do not affect expression pattern of ChR2 or neuronal wiring. Genotype of mutants as indicated. All animals were young adults, body length is ~1mm for the wild type, and diameter of neuronal cell bodies is 2-3µm. 2

4 Supplementary Figure 3 Length chronograms in wild type and mutant animals after lightevoked transmitter release (a) ACh release (transgene zxis6) was photo-evoked for 10s (indicated by the blue bar), in wild type animals (n = 59), as well as in unc-49(e407) GABA A R single mutants (n = 20) and unc-49(e407); snb-1(md247) synaptobrevin double mutants (n = 20). The length of the animals was automatically analyzed in single movie frames at 15fps, normalized, and averaged (b) GABA release (transgene zxis3) was triggered in wild type animals (n = 39) and snb-1(md247) mutants (n = 19), and analyzed as in (a). 3

5 Supplementary Figure 4 Synaptic physiology is normal in ChR2::YFP expressing animals (a) Miniature postsynaptic currents (mpscs) were analyzed in zxis6 animals and compared to the wild type. Frequency and amplitude of events showed no statistically significant differences. (b) Exogenous neurotransmitter application evoked typical currents in zxis3 and zxis6 animals: Averaged currents evoked by pressure-application of ACh (5x10-4 M; left) or GABA (10-4 M; right) were no different between transgenic zxis3, zxis6 and wild type animals. (c) Photo-evoked ACh, but not GABA epscs could be blocked by tubocurare. Top: Representative traces for ACh photo-epscs evoked by 10ms light stimuli (indicated by black bar) in Ringer`s (left), in Ringer`s containing tubocurare (0.5mM; middle) and after washout with Ringer`s (right). Bottom left: Corresponding mean ACh photo-epscs. Bottom right: Mean GABA photo-epscs after 10ms light stimuli were not significantly different in Ringer`s (left) compared to Ringer`s containing tubocurare (0.5mM; right). Number of experiments and mean ± s.e.m. is indicated. 4

6 Supplementary Figure 5 Origin of the enhanced ACh mediated contraction phenotype in presynaptic mutants (a) ACh mediated contractions triggered by two different light intensities (1.6 mw/mm² and mw/mm²) in unc-49(e407) single mutants and snb-1(md247); unc-49(e407) as well as unc-13(n2813); unc-49(e407) double mutants. Double mutants contracted more strongly under low-light conditions. (b) Direct triggering of muscle contractions via ChR2 expressed in muscle cells, as previously described 1, in wild type, unc-13(n2813) or snb-1(md247) mutant animals. Results indicate higher sensitivity of muscle contractions in response to depolarization in mutants, particularly at low-light conditions. (c) Light-induced contractions after treatment with phorbol 12-myristate 13-acetate (PMA). ChR2 was expressed in cholinergic neurons, GABAergic neurons, or muscle cells. Controls were treated with equal amounts of DMSO, used to dissolve PMA. Number of animals analyzed as indicated. 5

7 Supplementary Figure 6 Photostimulation trains can be applied for long time periods (a) Representative 60s traces evoked by 10ms photostimuli at 2Hz for GABA and (b) ACh photo-epscs. (c) Representative traces evoked by 10ms photostimuli at 0.5Hz for GABA and (d) ACh photo-epscs. Displayed are 60s (left) and 240s traces (right). (e) Comparison of normalized ACh photo-epscs (relative to the first evoked current) in wild type (black, n = 7) and unc-13(n2813) (red, n = 6) animals evoked by 10ms photostimuli at 2Hz. (f) Normalized currents evoked by 10ms photostimuli at 0.5Hz are nearly the same for GABA (n = 5) and ACh (n = 5) photo-epscs, exhibiting only weak decay. 6

8 Supplementary Figure 7 ACh and GABA photo-epscs during 2Hz photostimulation trains at different intertrain intervals (ITI) Representative traces (left) and mean values (right) for photo-epscs evoked by 5 consecutive 10ms light pulses at 2Hz. Four consecutive ACh photo-epscs trains with 20s ITI (Top; n = 3) or 60s ITI (Middle; n = 3). Bottom: Four consecutive GABA photo-epscs trains with 20s ITI (n = 3-4). 7

9 Supplementary Figure 8 Single ACh and GABA photo-epscs with 20 or 60s interstimulus intervals (ISI) Representative traces (left) and mean values (right) for ACh photo-epscs evoked by single 10ms light pulses at 20s (Top; n = 4) or 60s ISI (Middle; n = 4-5). Bottom: Analogous GABA photo-epscs at 20s ISI (n = 6). 8

10 Supplementary Table 1 Summary of photo-evoked ACh- and GABA-mediated contraction and relaxation after 520 ms of illumination of all mutants tested ACh-mediated contraction after 520 ms illumination Body length (% initial length) s.e.m. n = (to WT or control) Mutant Genotype Wild type Wild type no ATR *** vgat unc-47(e307) *** Synaptotagmin snt-1(md290) ** Synaptobrevin snb-1(md247) ** UNC-13 null unc-13(e1091) * UNC-13 hypomorph unc-13(n2813) *** Synaptojanin unc-26(s1710) *** AP180 Clathrin Adaptor unc-11(e47) * GABAAR unc-49(e407) *** GABAAR unc-49(e407) mw/mm² Double mutants snb-1(md247); unc-49(e407) ns unc-13(n2813); unc-49(e407) ns snb-1(md247); unc-49(e407) mw/mm² *** unc-13(n2813); unc-49(e407) mw/mm² ** Contraction directly evoked (ChR2 expression in muscle) after 520 ms illumination Body length (% initial length) s.e.m. n = (to WT) Mutant Genotype Wild type Synaptobrevin snb-1(md247) ns UNC-13 hypomorph unc-13(n2813) * Wild type mw/mm² Synaptobrevin snb-1(md247) mw/mm² ** UNC-13 hypomorph unc-13(n2813) mw/mm² * GABA-mediated elongation after 520 ms illumination Body length (% initial length) s.e.m. n = (to WT) Mutant Genotype Wild type Wild type no ATR *** vgat unc-47(e307) *** Synaptobrevin snb-1(md247) ** Syntaxin unc-64(js115); rescue in ACh neurons *** Synaptotagmin snt-1(md290) ns Synaptojanin unc-26(s1710) ns * p<0.05 ** p<0.01 *** p<0.001 The experimental accuracy of our measurements, given by the pixel size of the camera, is ca. 0.3% of the whole length of an adult animal. If s.e.m. values are below this limit, this is due to averaging of large numbers of animals. 9

11 Supplementary Table 2 Summary of ACh and GABA photo-epscs in the various mutants analyzed cholinergic photo-epscs 10 ms pulse 1000 ms peak 1000 ms steady state (to WT) (pa ± s.e.m.) n = (to WT) (pa ± s.e.m.) n = Mutant Genotype (pa ± s.e.m.) n = Wild type 1,401 ± ,299 ± ± 8 11 GABAAR unc-49(e407) 1,440 ± ns 1,383 ± ns 87 ± 18 9 ns UNC-13 hypomorph unc-13(n2813) 635 ± *** 737 ± * 21 ± 5 6 *** UNC-13 null unc-13(e1091) 255 ± 33 8 *** Synaptojanin unc-26(s1710) 938 ± * Synaptotagmin snt-1(md290) 620 ± *** (to WT) GABAergic photo-epscs 10 ms pulse 1000 ms peak 1000 ms steady state (to WT) (pa ± s.e.m.) n = (to WT) (pa ± s.e.m.) n = Mutant Genotype (pa ± s.e.m.) n = Wild type 803 ± ± ± vgat unc-47(e307) 8 ± 6 10 *** 23 ± *** GABAAR unc-49(e407) 6 ± 4 10 *** 6 ± 4 10 *** Syntaxin unc-64(js115); rescue in ACh neurons 191 ± 62 7 *** 107 ± 33 6 *** 0 ± 0 6 ** UNC-13 null unc-13(e1091) 255 ± 33 8 *** Synaptojanin unc-26(s1710) 785 ± ns Synaptotagmin snt-1(md290) 403 ± 84 4 * (to WT) Displayed are means ± s.e.m. for 10 and 1000 ms light pulses, n - values, and P values (* P < 0.05; ** P < 0.01; *** P < 0.001). All statistically significant differences refer to wild type values. 10

12 SUPPLEMENTARY RESULTS Origin of the coiling phenotype evoked by photo-triggered ACh release Upon illumination of zxis6 animals, we observed dorsal coiling within a few seconds (Fig. 1b main manuscript and Supplementary Video 4). What may cause the coiling phenotype? Cholinergic neurons trigger contraction on one side of the body, while they also innervate GABAergic motoneurons to inhibit muscle activity contra laterally 2 (Supplementary Fig. 1a), thus generating body bends and sinusoidal locomotion. In unc- 49(e407) GABA A R mutants, we found more pronounced shortening after 520ms of illumination (Fig. 1b,c,e main manuscript), but no coiling (Fig. 1b main manuscript and Supplementary Video 6). Thus concurrent activation of GABAergic neurons by cholinergic neurons may be the cause for the coiling. However, the promotor Punc-17, used for expression of ChR2 in zxis6 animals, is expressed in all cholinergic neurons, including 13 interneurons outside of the neuromuscular system 3. The observed coiling phenotype in AChreleasing animals could thus be a consequence of interneuron activity. To investigate this, we expressed ChR2 from other promoters expressed in cholinergic neurons, i.e. Punc-4 and Pacr- 5. Expression of ChR2::YFP could be observed in these cells, however, at lower expression levels, particularly for Pacr-5. Consequently, we could not evoke any effects in Pacr- 5::ChR2::YFP expressing animals. Yet, we could evoke weak contractions in Punc- 4::ChR2::YFP expressing animals, as well as mild coiling, especially of the posterior half of the animal. Thus, even though Punc-4 was reported to be expressed also in two interneurons (AVFL and AVFR, in which Punc-17 is not active 3, 4 ), the coiling phenotype appears not to be evoked by interneuron activity. Taken together, these observations indicate that in the wild type, photostimulation of cholinergic neurons causes indirect stimulation of GABAergic neurons, which reduces AChmediated contractions (apparently more pronounced ventrally) and effects coiling. Unexpectedly enhanced ACh-mediated contractions in exo- and endocytosis mutants Unexpectedly, in all presynaptic mutants tested, ACh-mediated photo-evoked contractions were larger than in the wild type (Fig. 4b main manuscript), even though photoepscs were smaller, at least as measured for unc-13(n2813 and e1091), unc-26(s1710) and snt-1(md290) mutants (Fig. 3a,b main manuscript). How could this be explained? One possibility is that in synaptic mutants the indirectly evoked GABA release is even more affected than the directly photostimulated release of ACh, since both types of synapses are impaired, and GABA neurons need to be stimulated by ACh neurons (Supplementary Fig. 11

13 1a). Consistent with this idea, light-induced coiling, which depends on GABA transmission and was absent in unc-49 GABA A R mutants, appeared less pronounced in most synaptic mutants tested (data not shown), and unc-49(e407) animals showed stronger contractions than the wild type (both after 520 ms and in long-term behavioral experiments; Fig. 1c,e main manuscript and Supplementary Fig. 5a). Therefore we tested if snb-1; unc-49 and unc-13; unc-49 double mutants would contract less than unc-49 single mutants. Yet, contractions in the double mutants were still equal or even more pronounced (Supplementary Figs. 3a and 8a). One possibility could be that, due to the high light power, large amounts of ACh were released that could compensate for presynaptic defects. Thus, we reduced the light to about 4% compared to previous experiments (0.065 mw/mm²). This triggered less contractions in unc-49 mutants (92.2 ± 1.2 % vs ± 0.5 % for high light power; n = 10; P = 0.027; Supplementary Fig. 8a), whereas the double mutants contracted as strong as before (and not less): snb-1; unc-49: 85.9 ± 1.0 %; n = 10; P < 0.001, unc-13; unc-49: 87.3 ± 0.6 %; n = 10; P = Therefore, the lack in GABA signaling causes even stronger contractions in these double mutants. Yet, the presynaptic mutants do not contract less than the unc-49 single mutants, thus an indirect GABA effect appears not to contribute to the hypercontraction phenotype of presynaptic mutants. The experiment at high light power, where double mutants contracted no more than unc-49 single mutants, may be misleading, since it is possible that animals can not contract to more than 85 %, otherwise also here the double mutants may have contracted more (this is actually seen during a 10s stimulation, i.e. the snb-1; unc-49 double mutants initially contract more strongly than unc-49 single mutants; Supplementary Fig. 3a). Another possibility is that muscle cells may compensate for a deprivation in presynaptic neurotransmitter release by up-regulating neurotransmitter receptors or their sensitivity. However, as previously shown, unc-13(n2813) animals have normal postsynaptic responses to pressure-applied ACh 5, thus enhanced contractions after photo-evoked ACh release in these mutants can not be explained by alterations in nachr sensitivity. However, it may be that muscles in these presynaptic mutants alter their physiology, i.e. downstream of nachrs, to better respond to the small depolarizing inputs they receive. Thus, the small amount of ACh released in response to the photostimulus could trigger stronger contractions in these mutants, as compared to the wild type. If this were the case, direct depolarization of muscles should have stronger effects in mutants that are defective for presynaptic neurotransmitter release. To this end, we induced contractions by direct photo-depolarization of muscles (ChR2 expressed from the myo-3 promoter 1 ) in the wild type, snb-1(md247), and unc-13(n2813) mutants. Since different sensitivity to depolarization may become apparent especially when 12

14 the depolarization is small, we compared contractions induced by normal amounts of blue light to those induced by low light power (1.6 vs mw/mm²). Using normal stimulus strength, contractions did not differ significantly between snb-1(md247) mutants and wild type, while unc-13(n2813) contracted more strongly (contraction after 520 ms: wild type: 92.9 ± 0.3 %, n = 20; snb-1: 91.7 ± 0.5 %, n = 20, P = 0.055; unc-13: ± 0.73 %, n = 13; P = 0.018; Supplementary Fig. 8b). Interestingly, at low light power, both snb-1 and unc-13 mutants contracted significantly more than the wild type (wild type: ± 0.30 %, n = 35; snb-1: ± 0.52 %, n = 10, P = ; unc-13: ± 0.52, n = 20; P = 0.031). Thus, presynaptic defects in ACh release may indeed affect the sensitivity of the postsynaptic contractile apparatus to depolarization such that smaller activating signals have larger behavioral effects, at least in snb-1 and unc-13 mutants. This could explain why the presynaptic mutants we tested showed more pronounced contractions than the wild type in response to photo-triggered ACh release, even though the inward currents measured were significantly smaller than in the wild type. However, we can not rule out other possibilities or additional effects accounting for the enhanced contractions in the mutants tested. For example, it is conceivable that a lack in GABA signaling may also influence the outcome of direct ChR2-mediated muscle depolarization, e.g. the extent of contractions may be stronger if there is no (tonic) GABAergic input to muscles. Phorbol esters, that stimulate the priming factor UNC-13, increase ACh-mediated contractions To investigate how conditions of constitutively increased ACh release would affect photoevoked contraction behavior, we cultivated animals in the presence of phorbol esters (phorbol-12-myristate-13-acetate - PMA), which stimulate the priming factor UNC We observed significantly enhanced contractions in zxis6 animals grown in the presence of PMA (P < 0.001; Supplementary Fig. 8c). Thus, potentially, also mutants that have increased ACh release may show enhanced contractions. Therefore in case of an unknown mutant, additional experiments should demonstrate the exact nature of the mutation (i.e. increasing or reducing release). In analogous experiments with PMA-treated zxis3 animals, no obvious enhancement of GABA-mediated elongation was apparent, and also animals expressing ChR2 in muscle did not respond differently, whether or not they were cultivated in the presence of PMA. 13

15 SUPPLEMENTARY METHODS Genetics C. elegans strains were cultivated on nematode growth medium (NGM). The following genotypes were used: N2 (wild type), lin-15(n765ts), unc-49(e407), unc-13(n2813), unc- 13(e1091), unc-47(e307), snb-1(md247), snt-1(md290), unc-26(s1710), unc-11(e47) and the GABA(-) syntaxin mosaic strain EG3817 (unc-64(js115); oxex705[punc-17:syx; Pglr- 1:SYX; Pacr-2:SYX]) 7. The following transgenic strains were prepared: ZX280: N2; zxex17[pmyo-3::chop-2(h134r)::yfp] 1, ZX426: N2; zxis3, ZX448: lin-15(n765ts); zxex70[punc-17::chop-2(h134r)::yfp;lin-15+], ZX460: N2; zxis6, ZX462: snt-1(md290); zxis3, ZX463: unc-47(e307); zxis3, ZX465: unc-26(s1710); zxis3, ZX466: snb-1(md247); zxis3, ZX497: unc-49(e407); zxis6, ZX498: snb-1(md247); zxis6, ZX503: unc-11(e47); zxis6, ZX511: unc-26(s1710); zxis6, ZX512: GABA(-) syntaxin mosaic strain EG3817 (unc- 64(js115); oxex705[punc-17:syx; Pglr-1:SYX; Pacr-2:SYX]); zxis3, ZX518: unc-13(n2813); zxis6, ZX521: snb-1(md247); unc-49(e407); zxis6, ZX529: unc-13(n2813); unc-49(e407); zxis6, ZX531: unc-47(e307); zxis6, ZX532: snt-1(md290); zxis6, ZX533: snb-1(md247); zxex17, ZX571: lin-15(n765ts);zxex71[punc-4::chop-2(h134r)::yfp;lin-15+], ZX582: lin- 15(n765ts);zxEx72[pacr-5::chop-2(H134R)::yfp;lin-15+], ZX583: unc-13(n2813); zxex17, ZX584: unc-13(e1091); zxis6 Molecular Biology As previously described, a truncated ChR2 cdna encoding amino acids 1 to 315 with a H134R mutation and a YFP-coding region at the C-terminus was used 1. The punc-47::chop- 2(H134R)::yfp construct was generated by amplifying a 1.44 kb genomic fragment upstream of the unc-47 start codon via PCR, using primers 5 - CCCCGCAAGCTTGTTGTCATCACTTCAAACTTTTCAATG-3 and 5 - CCCCGCTGATCACTGTAATGAAATAAATGTGACGCTGT-3. After HindIII/BclI cleavage, the fragment was ligated with a pmec-4::chop-2(h134r)::yfp vector 1, where pmec- 4 was removed using HindIII and BamHI. To generate the punc-17::chop-2(h134r)::yfp construct we excised the chop-2(h134r)::yfp coding region and the subsequent unc-54 3 UTR of the pmec-4::chop-2(h134r)::yfp plasmid using a BamHI site (blunt ended with Klenow fragment after cleavage) and an ApaI site. The resulting fragment was subcloned into the HincII/ApaI sites of vector RM#348p (a gift from James Rand). 14

16 Generation of transgenic animals Transgenic C. elegans were obtained by microinjection of DNA into the gonads of lin- 15(n765ts) nematodes, by standard procedures, together with a lin-15 genomic DNA rescue construct. Extrachromosomal arrays were chromosomally integrated via UV irradiation to generate the integrated transgenes zxis3[punc-47::chop-2(h134r)::yfp; lin-15 + ] and zxis6[punc-17::chop-2(h134r)::yfp;lin-15 + ]. The resulting strains were backcrossed to the wild type at least 5 times. Electrophysiology Recordings from dissected C. elegans body muscle were essentially performed as described previously 1. We used an EPC10 amplifier with head stage and Pulse software (HEKA, Germany) to clamp the cells to a holding potential of -60mV. Alternatively, data were recorded through a digidata 1440A and an axopatch 1D amplifier using Clampex10 software and processed with Clampfit10 (all Axon Instruments, Molecular Devices, USA). An 80db lowpass Bessel filter was used to filter signals at 5kHz. Light activation was performed using an LED lamp (KSL-70, Rapp OptoElectronic, Germany) at a wavelength of 470nm (8mW/mm 2 ), and controlled by the HEKA or Axon amplifier software. Electric stimuli were applied with a DS8000 Digital Stimulator (W.P.I., USA) as described 8 Fluorescence Microscopy Expression of ChR2::YFP in cholinergic or GABAergic neurons of C. elegans was analyzed on an Axiovert 200 (Zeiss) equipped with an YFP-specific excitation/emission filter set. Images were obtained with a CoolSNAP HQ2 camera (Roper Scientific) and MetaVue software. Furthermore, an LSM confocal laser scanning microscope (Zeiss) was used to analyze nervous system structure in detail. 15

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