Rapid and reversible optogenetic silencing of synaptic transmission by clustering of synaptic vesicles

Abstract

Acutely silencing specific neurons informs about their functional roles in circuits and behavior. Existing optogenetic silencers include ion pumps, channels, metabotropic receptors, and tools that damage the neurotransmitter release machinery. While the former hyperpolarize the cell, alter ionic gradients or cellular biochemistry, the latter allow only slow recovery, requiring de novo synthesis. Thus, tools combining fast activation and reversibility are needed. Here, we use light-evoked homo-oligomerization of cryptochrome CRY2 to silence synaptic transmission, by clustering synaptic vesicles (SVs). We benchmark this tool, optoSynC, in Caenorhabditis elegans, zebrafish, and murine hippocampal neurons. optoSynC clusters SVs, observable by electron microscopy. Locomotion silencing occurs with tau_on ~7.2 s and recovers with tau_off ~6.5 min after light-off. optoSynC can inhibit exocytosis for several hours, at very low light intensities, does not affect ion currents, biochemistry or synaptic proteins, and may further allow manipulating different SV pools and the transfer of SVs between them.

Fig. 1. OptoSynC inhibits behavior within seconds and recovers within minutes in the dark.

a Schematic illustrating SV clustering through homo-oligomerization of CRY2olig(535) upon blue light illumination. b Mean (±s.e.m.) swimming cycles of worms expressing optoSynC pan-neuronally. Illumination (470 nm, 0.1 mW/mm², 5 s / 25 s ISI) is indicated by blue rectangles; dotted line: one-phase decay fit (60 –120 s). c As in b, longer time course. Sustained inhibition of swimming by ongoing light pulses, and recovery in the dark. Dotted line: ‘plateau followed by one phase association’-fit. N2 – non-transgenic wild type. d Group data (speed of individual animals, median + inter-quartile range) before (0–1 min), during (1–2 min), and after (30–31; 35–36 min) blue illumination. Two-way ANOVA with Bonferroni correction between light and dark measurements of wild type and optoSynC expressing animals (sng-1(ok234) background); ***p < 0.001; ns – non-significant. Number of individual animals (n) from left to right: 44, 57, 49, 42, 91, 64, 33, 85, 65, 51, 82, 63; across N = 2 (wild type) and N = 3 (optoSynC) independent experiments with animals picked from independent populations; range of individual animals across each measured time point and over N independent experiments is indicated for b and c.

Fig. 2. Immediate inhibition of the LITE-1-dependent escape response by optoSynC.

a Mean ± s.e.m crawling speed analysis, blue light application (470 nm, 1 mW/mm², 5 s / 25 s ISI, indicated by blue rectangles), genotypes as indicated (optoSynC expressing animals are in sng-1(ok234) background). b Close-up of box indicated in a. c Group data, mean crawling speed of animals tested in N = 3 independent experiments (±s.e.m), analyzed as mean of time intervals before (295–320 s), and after (325–350 s) first light pulse; number of independent animals (n) across all independent experiments (N, i.e. animals picked from N independent populations) is indicated as range. d As in a, but using only 0.1 mW/mm² stimuli. e Close-up of box indicated in d. Dotted line represents one phase decay fit. f As in c, for data in d. In c, f, data are statistically analyzed with two-way ANOVA, Bonferroni correction, ns not significant.

Fig. 3. optoSynC activation reduces miniature post-synaptic current (mPSC) rate at the neuromuscular junction (NMJ) and can block cholinergic transmission for hours.

a Representative postsynaptic current traces recorded in body wall muscle cells of optoSynC-expressing animals; blue light pulses (470 nm, 8 mW/mm², 5 s / 5 s ISI) indicated by blue rectangles. b Close-up of regions indicated in a, before (0–10 s, dark blue) and after (95–105 s, light blue) blue light illumination. c Mean (±s.e.m.) mPSC frequency with blue light illumination of wild type and optoSynC expressing animals. d Group analysis of data (mean ± s.e.m.) in c, intervals before (0–30 s) and after (76–105 s) illumination. Number of independent animals is n = 8 (wild type) and n = 18 (optoSynC). e, f Analysis of mPSC amplitude (mean ± s.e.m.), as in c, d. g Paralysis of animals in response to 2 (or 0) mM aldicarb under continuous blue light illumination (470 nm, 0.05 mW/mm²), or in the dark, as indicated (mean ± s.e.m.). Two-way ANOVA with Bonferroni correction of the indicated number of animals (in b–f), or of N = 3 experiments averaged at the indicated time points with n independent animals as indicated (in g). ***p < 0.001, ns not significant.

Fig. 4. optoSynC activation causes clustering of SVs at the ultrastructural level.

a, b Transmission electron micrographs of representative cholinergic synapses from animals illuminated for 5 s (a, +light) with blue light (470 nm, 0.1 mW/mm²), or kept in darkness (b, −light) before high-pressure freezing. SVs (open black arrowheads), dense core vesicles (DCVs, white closed arrowheads), and dense projection (DP, closed black arrowheads) are indicated. c Distance analysis of nearest vesicles for each analyzed cholinergic micrograph; −light (n = 1473), +light (n = 819). d Relative frequency distribution of nearest vesicle distances shown in c. e Mean nearest distances per section; −light (n = 88), +light (n = 43). f Mean nearest distances per synapse; −light (n = 30), +light (n = 12). g, h Lengths of the PM and DP, respectively; −light (n = 88), +light (n = 43). Data in c, e–h are shown as median with 75–25% interquartile range (IQR). i–p, As for a–h, respectively, but for non-transgenic sng-1(ok234) mutant animals (n = 707, 41, 9 for −light, and 733, 40, 14 for +light; in k, n, l, o, m, p, respectively. Sections originated from two animals, and 9–30 synapses for each condition. Statistical test used: Mann–Whitney (two-tailed) in c, e, g–h, k, m–p; unpaired t-test (two-tailed) in f; Kolmogorov–Smirnov in d, l; ***p < 0.001, ns not significant.

Fig. 5. zf-optoSynC activation in zebrafish neurons blocks escape behavior.

a, b Pan-neuronal expression of eGFP in neurons of zebrafish larvae at the indicated developmental stages. Representative images show transient expression in Tg(elavl3.2:Gal4-VP16)mde4 animals injected with UAS:zf-optoSynC (n = 2 independent experiments). c Swimming behavior, triggered by continuous blue light (blue bar), in 4 dpf larvae expressing the respective transgene, as indicated. Mean ± s.e.m., numbers of individual animals are indicated. d Statistical analysis of data in c, as median and 75–25% IQR. Number of independent animals n = 7 (eGFP) and n = 6 (zf-optoSynC). e Touch response of 3 dpf larvae during blue light-induced zf-optoSynC/CRY2olig(535) clustering. Two experiments, number of animals is indicated. Unpaired Student’s t test (two-tailed) in d, and Fisher’s exact test (two-sided) in e.

Fig. 6. m-optoSynC activation in murine hippocampal neurons blocks synaptic transmission.

a Plot showing changes in normalized fluorescence intensity of mOrange2 by electrical field stimulation of neurons expressing mOrange2-SYP before (black line) and after 488 nm illumination for 30 s (green line). N = 2 independent cultures. n = 5 neurons and 116 synapses. b, c Normalized fluorescence intensity at 2 s or 25 s after electrical stimulation. Paired t-test. d–f Same as a-c, respectively, but in neurons expressing m-optoSynC (CRY2olig(535) inserted into SYP-mOrange2. Paired t-test. N = 3 independent cultures. n = 10 neurons, and 108 synapses. ns = not significant. *<0.05.

Fig. 7. Cell-specific optoSynC inhibition of cholinergic and GABAergic neurons.

a Swimming behavior in animals expressing optoSynC in cholinergic neurons. Blue light activation (470 nm, 0.1 mW/mm², 5 s/25 s ISI) indicated by blue rectangles. Dotted line: ‘plateau followed by one phase association’-fit. Error bars are s.e.m. Number of individual animals (n) across independent experiments (N, i.e. animals picked from N independent populations) is indicated as range. b Swimming cycles of individual animals, median and 25–75 % IQR of N = 2–3 experiments analyzed in time intervals before (0–1 min), during (1–2 min), and after (30–31 min) blue light illumination; number of individual animals (n) across all experiments from left to right: 58, 58, 57, 72, 53, 64. Two-way ANOVA with Bonferroni correction between light and dark measurements of wild type and cholinergic optoSynC expressing strains; ***p < 0.001. c, d as for a, b, but optoSynC was expressed in GABAergic neurons. Number of individual animals (n) from left to right: 77, 55, 86, 56, 98, 60.

Fig. 8. Expression of optoSynC in the single nociceptive neuron PVD attenuates PVD::Chrimson-evoked velocity increase.

a Mean ± s.e.m. normalized crawling speed of animals expressing Chrimson, or Chrimson and optoSynC in the PVD neuron. Red light stimulation (680 nm, 0.1 mW/mm², 1 s/5 s ISI) is indicated by red rectangles, activation of optoSynC with blue light (470 nm, 0.1 mW/mm², 5 s) by a blue rectangle and asterisk. Data acquisition started at 0 s, but animals are left to accommodate before starting the experiment. b Group data, mean crawling speed of N = 2–3 independent experiments (±s.e.m) analyzed during time intervals with (or without) blue pulse (320–325 s). Number of independent animals (n) across all independent experiments (N, i.e. animals picked from N independent populations) is indicated as range. c As in b, but during first red light pulse (360 s). d Light intensity titration of blue light responses of PVD::Chrimson animals (mean ± s.e.m.; 61–147 individual animals, across N = 2–3 independent experiments, were analyzed). Inset: Spectral overlap of CRY2 and Chrimson, derived from refs. ,. e As in a, but with only 25 µW/mm² optoSynC activation, avoiding optical crosstalk with Chrimson. f as in c, but during peaks of red pulses (380 s, 386 s, 392 s, 398 s, 404 s), for data in e. In b, c, f: one-way ANOVA with Bonferroni correction; ns non-significant. Number of independent experiments (N) and individual animals (n) is indicated.

Fig. 9. Comparison of tools for optogenetic silencing of synaptic transmission.

a Matrix showing time constants (activity on- and offset) of optogenetic tools for synaptic inhibition. Tools are grouped by their molecular nature/mechanism of action. Data was derived from refs. ,,,–,,,,,,b ACR2 expressed in C. elegans cholinergic motor neurons evokes rapid and complete inhibition of locomotion, associated with sustained after-effects. Mean (±s.e.m.) swimming cycles of animals expressing ACR2. Number of individual animals (n) across each measured time point and across N = 2 independent experiments (animals picked from N independent populations) is indicated as range.