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Neural engineering with photons as synaptic transmitters

Nature Methods volume 20, pages 761–769 (2023)Cite this article

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Abstract

Neuronal computation is achieved through connections of individual neurons into a larger network. To expand the repertoire of endogenous cellular communication, we developed a synthetic, photon-assisted synaptic transmission (PhAST) system. PhAST is based on luciferases and channelrhodopsins that enable the transmission of a neuronal state across space, using photons as neurotransmitters. PhAST overcomes synaptic barriers and rescues the behavioral deficit of a glutamate mutant with conditional, calcium-triggered photon emission between two neurons of the Caenorhabditis elegans nociceptive avoidance circuit. To demonstrate versatility and flexibility, we generated de novo synaptic transmission between two unconnected cells in a sexually dimorphic neuronal circuit, suppressed endogenous nocifensive response through activation of an anion channelrhodopsin and switched attractive to aversive behavior in an olfactory circuit. Finally, we applied PhAST to dissect the calcium dynamics of the temporal pattern generator in a motor circuit for ovipositioning. In summary, we established photon-based synaptic transmission that facilitates the modification of animal behavior.

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Fig. 1: A nose-touch defect derived from mutations in synaptic transmission.
Fig. 2: Equipping animals with calcium-dependent light emitters and postsynaptic light sensors.
Fig. 3: PhAST rescues nociceptive avoidance behavior and postsynaptic neuronal activity.
Fig. 4: Desensitization through ACR1 activation in interneurons.
Fig. 5: Rewiring attraction to avoidance behavior.
Fig. 6: PhAST uncovers a disinhibitory role of VC neurons.

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Data availability

The AutoCad files of the microfluidic Trap‘N’Slap devices have been deposited on Zenodo.org at https://doi.org/10.5281/zenodo.7551719. Some strains will be deposited to the Caenorhabditis Genetics Center. Plasmids listed in Supplementary Table 4 have been deposited to Addgene under the following IDs: 199312 (pNMSB139), 199311 (pNMSB131), 199321 (pNMSB88), 199322 (pNMSB109), 199319 (pNMSB91), 199317 (pNMSB97), 199316 (pNMSB96), 199314 (pNMSB104), 199310 (pNMSB132), 199309 (pNMSB84) and 199308 (pNMSB99). Source data are provided with this paper.

Code availability

The Monte Carlo simulation code and software to analyze chemotaxis behavior and other code developed to support the analysis can be accessed under Gitlab::NMSB, which can be used under an open-source license permitting not-for-profit research applications (see file license.txt).

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Acknowledgements

We thank the members of the Neurophotonics and Mechanical Systems Biology (NMSB) and Superresolution & LightNanoscopy (SLN) laboratories for discussions and suggestion throughout the work and for use of their microscopes. We thank the Institute of Photonic Sciences Biology Lab and NanoFabrication Lab (NFL) for support with animal maintenance and SU8 lithography, respectively. We thank C. Bargmann (Rockefeller University), S. Xu (University of Michigan), S. van der Heuvel (Utrecht University), A. Gottschalk (Goethe University), P. Askjaer (CABD Sevilla), T. Kirkland (Promega), J. Cerón (Idibell) and the Caenorhabditis Genetics Center (National Institutes of Health, Office of Research Infrastructure Programs (P40 OD010440)) for providing reagents and animals; Y. Janin, P. Gorostiza, R. Kittel, T. Durduran and A. Kim for discussions and suggestions on bioluminescence, optogenetics, photon propagation and microfabrication; and I. Rabinowitch, P. Gorostiza and P. Askjaer for critical comments on the manuscript. M.K. acknowledges financial support from the European Research Council (ERC MechanoSystems, 715243), Human Frontiers Science Program (HFSP CDA00023/2018), PID2021-123812OB-I00 project funded by Ministerio de Cienca e Innovacion/Agenica Estatal de Investigacion (MCIN/AEI /10.13039/501100011033/FEDER, Union Europea; Severo Ochoa program for Centers of Excellence in R&D, CEX2019-000910-S; RYC-2016-21062), from Fundació Privada Cellex, Fundació Mir-Puig and from Generalitat de Catalunya, in addition to funding through H2020 Marie Skłodowska-Curie Actions (754510 to A.G.) and Mineco (FPIPRE2019-088840 to N.S.).

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Authors and Affiliations

  1. Institut de Ciències Fotòniques, Castelldefels, Spain

    Montserrat Porta-de-la-Riva, Adriana Carolina Gonzalez, Neus Sanfeliu-Cerdán, Shadi Karimi, Nawaphat Malaiwong, Aleksandra Pidde, Luis-Felipe Morales-Curiel, Pablo Fernandez, Sara González-Bolívar, Cedric Hurth & Michael Krieg

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  1. Montserrat Porta-de-la-Riva
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Contributions

M.P.R. was responsible for animal husbandry, molecular biology, CRISPR, CRE recombination, optogenetic and nociceptive experiments, egg-laying and olfactory behavior, microscopy, data analysis and manuscript writing. A.C.G. was responsible for microfluidics, calcium imaging, bioluminescence imaging and the first manuscript draft. N.S.C. was responsible for behavioral assays and molecular biology. C.H., S.G. and S.K. were responsible for microfabrication, microfluidic design and simulations. L.F.M.C. was responsible for bioluminescence imaging, microscopy and photon propagation. N.M. was responsible for calcium imaging, behavior and genetics. A.P. and P.F. were responsible for data analysis and programming. M.K. was responsible for the study concept, acquisition of funding, data analysis, software programming and manuscript writing.

Corresponding author

Correspondence to Michael Krieg.

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Extended data

Extended Data Fig. 1 Nose touch avoidance behavior in wild-type and eat-4 vesicular glutamate transporter mutant.

(a) Schematic of the behavioral assay. The yes/no response of a single animal is recorded as it navigates into a user-controlled obstacle (eyebrow hair). (b, c) Wiring of the nociceptive avoidance circuit with the representative result of an experiment of 10 trials conducted on 10 animals in (b) wild-type and (c) eat-4(ky5) mutants. Circuit nodes colored according to their neurotransmitter (yellow=glutamate; red=acteylcholine; gray=silent). Dashed arrow indicates broken connection in the eat-4(ky5) mutation. Grid plots show the yes/no response, color-coded according to its outcome, of individual animals as they navigated into the eyebrow hair. (d) Summary of touch tests of both genotypes. Each blue dot corresponds to the average from ten touch tests per animal, N = number of tested animals in total. A jitter was applied to visually separate individual datapoints only for plotting purposes. Only positive datapoints are shown. Floating axis on the right shows the bootstrapped distribution of their paired median difference (right axis). Filled circle indicates median, vertical bar indicates 95%CI on the median. Two-sided p-value derived from a Wald test.

Source data

Extended Data Fig. 2 Mechanosensitivity of ASH does not depend on eat-4.

(a,b) Schematic of the neuronal circuit with ASH highlighted in blue and glutamatergic edges in yellow and nodes colored corresponding their neurotransmitter usage (red, acteylcholine; yellow, glutamate). Stacked kymographs and average ASH:jRGECO1a fluorescence after nose touch in the Trap`N’Slap device after 10s in (a) control and (b) eat-4(ky5) animals. Shaded area indicates SD around the mean (black trace). Broken connections in the eat-4(ky5) mutant are indicated as gray dotted lines.

Source data

Extended Data Fig. 3 Efficiency of ChR2-HRDC to induce reversal behavior through AIB and AVA in the eat-4 neurotransmitter mutants.

(a-c) Reversal response of animals expressing ChR2-HRDC in (a) AVA, in (b) AIB and (c) in both AIB and AVA in wild-type and eat-4 mutant background. Each dot is the yes or no response of an animal to a 1 s blue light stimulus. Solid line corresponds to a binary logistic regression of the measured response as a function of the light intensity with the 95% confidence bands. N=30 animals, each tested 10 times.

Source data

Extended Data Fig. 4 PhAST rescues calcium signaling but not behavior in the eat-4(ky5) mutation.

(a,b) Schematic of the experimental circuit manipulation. Nodes are colored according to the LUT in (b), gray dotted edges indicate genetically perturbed synaptic connections in the eat-4(ky5) mutation. (b) Image plot and average jRGECO1a fluorescence after nose touch in the microfluidic device for AVA (khaki, blue) and AIB (olive, violet) in eat-4(ky5) animals expressing the light pathway in absence of the cofactors. Shaded area indicates SD around the mean (black trace). The vertical bar indicates the duration of the mechanical stress. (c,d) Schematic of the circuit with TeNL expression in ASH (blue) and light-restored edges shown with blue arrows. (d) Image plot and average jRGECO1a fluorescence for AVA (khaki, blue) and AIB (olive, violet) for the same animals as in (b) but in presence of both cofactors. Shaded area indicates SD around the mean (black trace). (e) Behavioral response of eat-4(ky5) animals expressing the light pathway in presence and absence of all cofactors. (f) Summary of nose touch in eat-4(ky5) animals. Each blue dot corresponds to the average from ten touch tests per animal, N=30 animals in total. Black dot and vertical bars indicate median ± 95% confidence interval (CI). Numbers on gray brackets indicate the two-sided p-value derived from a Wald test. The floating axis indicates the bootstrapped distribution of the paired median difference (PMD) between HIKarazine single treated animals and double-treated animals. Red point indicates median, vertical bar 95%CI. Overlap of the CI with zero indicates low effect size and likely statistically insignificant distributions. Inset shows the log-odds ratio of finding a treated animal responding compared to the untreated mutant control.

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Extended Data Fig. 5 ASH-specific loss in glutamatergic signaling causes nose touch deficiency.

(a,b) Schematic of the genetic strategy for ASH-specific ablation of eat-4 and the location of the two loxP sites flanking exons 1 and 2, (a) before and (b) after CRE recombination. The circuit indicates glutamatergic (yellow) and cholinergic (red) edges, with numbers of synaptic contacts above the arrow. (c) Stacked kymographs of individual jRGECO1a videos and average AIB:jRGECO1a fluorescence after nose touch in the microfluidic device in control and eat-4(loxP) animals in absence of CRE recombinase. Shaded area indicates SD around the mean (black trace). N=12 recordings. (d) Representative animal showing the split-CRE recombination pattern using a recombination reporter with a BFP-mCherry switch for successful recombinations coexpressing an sra-6p::GCaMP7 construct to highlight ASH (see also Supplementary Fig. 11). Scale bar = 50μm. (e-g) Raster plots show representative datasets for (e) eat-4(loxP) control, (f) gpa-14p::CRE single transgenics and (g) ASH(eat-4) loss-of-function animals after gpa-14p::CRE expression, color-coded according to its outcome. See also Supplementary Text 2. (h) Summary of the nose touch response for the control and conditional eat-4 knockout in ASH. Blue dots correspond to the average of ten touch tests per animal, N=30 animals in total. Only for display purposes, a scatter of 10% was applied to each datapoint to avoid overlap. Black dot indicates median, vertical bar indicates 95% confidence interval on the median. Numbers on gray brackets indicate the two-sided p-value derived from a Wald test. Floating axis indicates the paired median difference between the floxed mutant and the control animals, derived from bootstrapping 100 independent distributions from the experimental data set. Black circle indicates median, vertical bar indicates 95%CI on the median.

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Extended Data Fig. 6 Nose touch response of soluble TeNL.

(a) i) Fluorescence micrograph of the individual transgenes used to express TeNL in ASH and ChR2-HRDC in AIB and AVA. ii) Luminescence of the TeNL in ASH. Results are representative of eight (fluorescence) and six (luminescence) worms imaged. Scale bars = 50μm. (b) Nose touch avoidance response of the conditional eat-4(loxP) mutant allele in ASH, coexpressing TeNL in ASH and ChR2-HRDC in AIB and AVA supplemented with the cofactors indicated. (c) Summary of the scores for the nose touch experiment on 30 animals in all conditions tested. Each blue dot corresponds to the average from ten touch tests per animal. Only for display purposes, a scatter of 10% was applied to each datapoint to avoid overlap. Numbers on gray brackets indicate the two-sided p-value derived from a Wald test. Floating axis indicates the paired median difference, derived from bootstrapping 100 independent distributions from the experimental data set. As indicated, filled circles indicates median + / − 95%CI. Overlap of the CI with zero indicates low effect size and likely statistically insignificant distributions.

Source data

Extended Data Fig. 7 Nose touch response of synaptically localized TeNL.

(a) Fluorescence micrograph i) of the synaptically localized Nanolantern, fused to SNG-1 Synaptogyrin and the other transgenes. Cyan arrowheads point towards the presumptive synapses indicated by high SNG-1::TeNL intensities. Scale bars = 40 μm. ii) Calcium saturated bioluminescence micrograph of the SNG-1::TeNL. Image is representative of a set of 4 derived from 2 independent lines. (b) Nose touch avoidance response of an animal carrying the ASH-specific eat-4(loxP) mutant allele in absence and presence of indicated cofactors, coexpressing synaptic SNG-1::TeNL in ASH and ChR2-HRDC in AIB and AVA, displayed as a grid plot, color-coded according to its outcome (blue=positive response, gray=negative response to nose touch). (c) Summary of the scores for the nose touch experiment on 30 animals in all conditions tested. Each blue dot corresponds to the average from ten touch tests per animal. Only for display purposes, a scatter of 10% was applied to each datapoint to avoid overlap. Horizontal bar indicates median, vertical bar indicates 95% confidence interval on the median. Floating axis indicates the paired median difference, derived from bootstrapping 100 independent distributions from the experimental data set. Red point indicates median, red vertical bar indicates 95%CI. Overlap of the CI with zero indicates low effect size and likely statistically insignificant distributions. Numbers on gray brackets indicate the two-sided p-value derived from a Wald test.

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Extended Data Fig. 8 Red-shifted Luciferase/ChRmine pair is not sufficient to replace endogenous neurotransmitters.

(a) i) Representative fluorescence micrograph (N=8) of the individual transgenes used to express CaMBI in ASH and ChRmine:SL2:jRGeCO1a in AIB and AVA. ii) Bioluminescence of the CaMBI in ASH. Scale bars = 40 μm. (b) Quantification of the nose touch response of animals expressing ChRmine in AVA and AIB in presence an absence of all cofactors. Each red dot corresponds to the average from ten touch tests per animal, One representative experiment displayed of a total of three (N=30 animals). Only for display purposes, a scatter of 10% was applied to each datapoint to avoid overlap. Black circle indicates median, vertical bar indicates 95% confidence interval on the median. Floating axis indicates the paired median difference (PMD), derived from bootstrapping 100 independent distributions from the experimental data set. Red point indicates median, red vertical bar indicates 95%CI. Overlap of the CI with zero indicates low effect size and likely statistically insignificant distributions. Numbers on gray brackets indicate the two-sided p-value derived from a Wald test.

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Extended Data Fig. 9 Sensitizing a sexually dimorphic behavior in males with PhAST.

(a) Schematic of the ASH nociceptive avoidance circuit with nodes indicating presynaptic (ASH) and postsynaptic (AVA, AIB, RIM) neurons; edges are color-coded according to neurotransmitter (yellow, glutamate). Gray dashed edges correspond to inactive neurotransmission in the males due to absence of synaptic connections. Numbers on the edges indicate numbers of connections found in EM reconstruction in males and (hermaphrodites). (b) Stacked kymographs of individual Ca2+ recordings after nose touch in the microfluidic device from (top) AVA and (bottom) AIB in male animals expressing the red-shifted Ca2+ indicator jRGECO1a. Black line indicates a 2-s stimulus delivered at t=10 s. Plots below the kymographs depict mean + / − SD (N=recordings per condition). (c) Individual responses of male animals expressing the PhAST system in presence and absence of the ATR and Hikarazine cofactors, compared to untreated hermaphrodites of the same genotype (N=30 animals). (d) Top: Representative confocal microscopy picture of ASH expressing ChRmine::wrmScarlet. Scale bar = 20 μm. Bottom: Individual responses of 30 animals expressing ChRmine in ASH, hermaphrodites and male animals responding to 5μW/mm2 of 595nm light in presence and absence of ATR. (e) Male avoidance response to orange light, upon cell-specific ChRmine expression in ASH after illuminating individual animals with 5μW/mm2 of 595nm light. Animals have been selectively treated with ATR, HIK or the two cofactors before illumination. For all graphs, a scatter of 10% was applied to each datapoint to avoid overlap, only for display purposes. Each blue dot corresponds to the average from ten touch tests per animal. Black circle indicates median, vertical bar indicates 95% confidence interval on the median. Numbers on gray brackets indicate the two-sided p-value derived from a Wald test; Floating axis indicates the paired median difference, derived from bootstrapping 100 independent distributions from the experimental data set. Red point indicates median, red vertical bar indicates 95%CI. Overlap of the CI with zero indicates low effect size and likely statistically insignificant distributions.

Source data

Extended Data Fig. 10 vm2 calcium recordings in animals expressing TeTx in VC or VC+HSN .

(a,b) Spontaneous calcium recordings of vm2 in animals expressing TeTx in VC neurons in presence of ATR (a) and both ATR+FFz (b). Histogram on top indicates spike time interval (STI) and violin plot to the right shows distribution of peak intensities. (c) p-values for the pairwise combinations derived from a two-sided K–S-test for the ‘spike’ time interval and the calcium peak amplitude. All conditions treated with ATR, except control. ‘Light’ conditions were treated with ATR and HIK. Control condition is untreated wild-type population of worms. (d,e) Spontaneous calcium recordings of vm2 in animals expressing TeTx in VC and HSN neurons (a) in absence and (b) in presence of ATR+FFz. Histogram on top indicates spike time interval (STI) and violin plot to the right shows distribution of peak intensities. Spike times (p-value = 0.4747, two-sided, pairwise K–S-test) and peak amplitudes (p-value = 0.5653, two-sided, pairwise K–S-test) are not significantly different between ATR and ATR-FFz treated animals. N=number of recordings.

Source data

Supplementary information

Supplementary Information

Supplementary Text 1–5, Supplementary Figures 1–12 and supplementary references.

Reporting Summary

Peer Review File.

Supplementary Video 1

Nociceptive avoidance behavior. Representative video of an animal navigating into an obstacle in (a) wild-type and (b) eat-4(ky5) background.

Supplementary Video 2

Pneumatic stimulation of a trapped animal inside the Trap‘N’Slap device. Representative video of a wild-type animal subjected to a 2.5-bar stimulus, recorded using brightfield microscopy.

Supplementary Video 3

Calcium imaging under mechanical stimulation in ASH. Representative video of the fluorescence intensity of (a) control and (b) eat-4(ky5) mutant animals expressing jRGECO1a in ASH. Animals were immobilized in the microfluidic device during the presentation of a 2-s mechanical stimulus after 10 s. Scale bar, 30 m, frame rate of 10 Hz. Anterior to the left. Same color LUT as in Fig. 1.

Supplementary Video 4

Calcium imaging under mechanical stimulation in AVA. Representative video of the fluorescence intensity of (a) control and (b) eat-4(ky5) mutant animals expressing jRGECO1a in AVA. Animals were immobilized in the microfluidic device during the presentation of a 2-s mechanical stimulus after 10 s. Framerate of 10 Hz. Anterior to the left.

Supplementary Video 5

Calcium imaging under mechanical stimulation in AIB. Representative video of the fluorescence intensity of (a) control and (b) eat-4(ky5) mutant animals expressing jRGECO1a in AIB. Animals were immobilized in the microfluidic device during the presentation of a 2-s mechanical stimulus after 10 s. Scale bar, 40 m, frame rate of 10 Hz. Anterior to the left.

Supplementary Video 6

Optogenetic stimulation of AVA. Representative video of a reversal response to optogenetic stimulation of an animal expressing ChR2-HRDC in AVA in presence (right) and absence (left) of the photosensitizer ATR.

Supplementary Video 7

Optogenetic stimulation of AIB. Representative video of a reversal response to optogenetic stimulation of an animal expressing ChR2-HRDC in AIB in presence (right) and absence (left) of the photosensitizer ATR.

Supplementary Video 8

Crawling animal with calcium-sensitive Nanolantern reporting body wall muscle activity. Representative video of a freely crawling animal expressing a calcium-sensitive Nanolantern in body wall muscles. Increases in intensity on the concave side of the body bend indicates that the calcium influx increases quantum yield of the Nanolantern probe.

Supplementary Video 9

Calcium activity in vm2. Three representative videos for vm2 calcium activity in untreated control animals expressing vm2::ChR2-HRDC::jRGECO1a (left), in animals expressing TeTx in HSN and treated with ATR (middle) and animals expressing TeTx in HSN and treated with ATR+FFz (right). Scale bar, 20 μm.

Supplementary Tables

Supplementary Tables 1–4. Supplementary Table 1. Summary and characteristics of strains used in this study. Supplementary Table 2. Plasmids and their DNA sequences. Supplementary Table 3. CRISPR and mutagenesis sequences. Compilation of the crRNAs, homology repair templates used in CRISPR/Cas9 edits and primers used for directed mutagenesis. Supplementary Table 4. C. elegans-specific PhAST tools developed during this study.

Supplementary Data

Source Data for Supplementary Figs. 1–11

Source data

Source Data for Figs. 1–6 and Extended Data Figs. 1–10

Statistical source data for figures and Extended Data figures.

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Porta-de-la-Riva, M., Gonzalez, A.C., Sanfeliu-Cerdán, N. et al. Neural engineering with photons as synaptic transmitters. Nat Methods 20, 761–769 (2023). https://doi.org/10.1038/s41592-023-01836-9

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