Microbial light-activatable proton pumps as neuronal inhibitors to functionally dissect neuronal networks in C. elegans

Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH

Optogenetic Long-Term Manipulation of Behavior and Animal Development

Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used “slow” ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the “dauer” larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development

High-throughput all-optical analysis of synaptic transmission and synaptic vesicle recycling in Caenorhabditis elegans

Synaptic vesicles (SVs) undergo a cycle of biogenesis and membrane fusion to release transmitter, followed by recycling. How exocytosis and endocytosis are coupled is intensively investigated. We describe an all-optical method for identification of neurotransmission genes that can directly distinguish SV recycling factors in C. elegans, by motoneuron photostimulation and muscular RCaMP Ca2+ imaging. We verified our approach on mutants affecting synaptic transmission. Mutation of genes affecting SV recycling (unc-26 synaptojanin, unc-41 stonin, unc-57 endophilin, itsn-1 intersectin, snt-1 synaptotagmin) showed a distinct ‘signature’ of muscle Ca2+ dynamics, induced by cholinergic motoneuron photostimulation, i.e. faster rise, and earlier decrease of the signal, reflecting increased synaptic fatigue during ongoing photostimulation. To facilitate high throughput, we measured (3–5 times) ~1000 nematodes for each gene. We explored if this method enables RNAi screening for SV recycling genes. Previous screens for synaptic function genes, based on behavioral or pharmacological assays, allowed no distinction of the stage of the SV cycle in which a protein might act. We generated a strain enabling RNAi specifically only in cholinergic neurons, thus resulting in healthier animals and avoiding lethal phenotypes resulting from knockdown elsewhere. RNAi of control genes resulted in Ca2+ measurements that were consistent with results obtained in the respective genomic mutants, albeit to a weaker extent in most cases, and could further be confirmed by opto-electrophysiological measurements for mutants of some of the genes, including synaptojanin. We screened 95 genes that were previously implicated in cholinergic transmission, and several controls. We identified genes that clustered together with known SV recycling genes, exhibiting a similar signature of their Ca2+ dynamics. Five of these genes (C27B7.7, erp-1, inx-8, inx-10, spp-10) were further assessed in respective genomic mutants; however, while all showed electrophysiological phenotypes indicative of reduced cholinergic transmission, no obvious SV recycling phenotypes could be uncovered for these genes

Endophilin A and B join forces with clathrin to mediate synaptic vesicle recycling in Caenorhabditis elegans

Synaptic vesicle (SV) recycling enables ongoing transmitter release, even during prolonged activity. SV membrane and proteins are retrieved by ultrafast endocytosis and new SVs are formed from synaptic endosomes (large vesicles—LVs). Many proteins contribute to SV recycling, e.g., endophilin, synaptojanin, dynamin and clathrin, while the site of action of these proteins (at the plasma membrane (PM) vs. at the endosomal membrane) is only partially understood. Here, we investigated the roles of endophilin A (UNC-57), endophilin-related protein (ERP-1, homologous to human endophilin B1) and of clathrin, in SV recycling at the cholinergic neuromuscular junction (NMJ) of C. elegans. erp-1 mutants exhibited reduced transmission and a progressive reduction in optogenetically evoked muscle contraction, indicative of impaired SV recycling. This was confirmed by electrophysiology, where particularly endophilin A (UNC-57), but also endophilin B (ERP-1) mutants exhibited reduced transmission. By optogenetic and electrophysiological analysis, phenotypes in the unc-57; erp-1 double mutant are largely dominated by the unc-57 mutation, arguing for partially redundant functions of endophilins A and B, but also hinting at a back-up mechanism for neuronal endocytosis. By electron microscopy (EM), we observed that unc-57 and erp-1; unc-57 double mutants showed increased numbers of synaptic endosomes of large size, assigning a role for both proteins at the endosome, because endosomal disintegration into new SVs, but not formation of endosomes were hampered. Accordingly, only low amounts of SVs were present. Also erp-1 mutants show reduced SV numbers (but no increase in LVs), thus ERP-1 contributes to SV formation. We analyzed temperature-sensitive mutants of clathrin heavy chain (chc-1), as well as erp-1; chc-1 and unc-57; chc-1 double mutants. SV recycling phenotypes were obvious from optogenetic stimulation experiments. By EM, chc-1 mutants showed formation of numerous and large endosomes, arguing that clathrin, as shown for mammalian synapses, acts at the endosome in formation of new SVs. Without endophilins, clathrin formed endosomes at the PM, while endophilins A and B compensated for the loss of clathrin at the PM, under conditions of high SV turnover

Endophilin A and B join forces with clathrin to mediate synaptic vesicle recycling in Caenorhabditis elegans

Synaptic vesicle (SV) recycling enables ongoing transmitter release, even during prolonged activity. SV membrane and proteins are retrieved by ultrafast endocytosis and new SVs are formed from synaptic endosomes (large vesicles—LVs). Many proteins contribute to SV recycling, e.g., endophilin, synaptojanin, dynamin and clathrin, while the site of action of these proteins (at the plasma membrane (PM) vs. at the endosomal membrane) is only partially understood. Here, we investigated the roles of endophilin A (UNC-57), endophilin-related protein (ERP-1, homologous to human endophilin B1) and of clathrin, in SV recycling at the cholinergic neuromuscular junction (NMJ) of C. elegans. erp-1 mutants exhibited reduced transmission and a progressive reduction in optogenetically evoked muscle contraction, indicative of impaired SV recycling. This was confirmed by electrophysiology, where particularly endophilin A (UNC-57), but also endophilin B (ERP-1) mutants exhibited reduced transmission. By optogenetic and electrophysiological analysis, phenotypes in the unc-57; erp-1 double mutant are largely dominated by the unc-57 mutation, arguing for partially redundant functions of endophilins A and B, but also hinting at a back-up mechanism for neuronal endocytosis. By electron microscopy (EM), we observed that unc-57 and erp-1; unc-57 double mutants showed increased numbers of synaptic endosomes of large size, assigning a role for both proteins at the endosome, because endosomal disintegration into new SVs, but not formation of endosomes were hampered. Accordingly, only low amounts of SVs were present. Also erp-1 mutants show reduced SV numbers (but no increase in LVs), thus ERP-1 contributes to SV formation. We analyzed temperature-sensitive mutants of clathrin heavy chain (chc-1), as well as erp-1; chc-1 and unc-57; chc-1 double mutants. SV recycling phenotypes were obvious from optogenetic stimulation experiments. By EM, chc-1 mutants showed formation of numerous and large endosomes, arguing that clathrin, as shown for mammalian synapses, acts at the endosome in formation of new SVs. Without endophilins, clathrin formed endosomes at the PM, while endophilins A and B compensated for the loss of clathrin at the PM, under conditions of high SV turnover

Electrophysiology verifies SV loading, release and recycling phenotypes.

<p>Photoevoked excitatory post-synaptic currents were measured in patch-clamped muscle, during repeated photostimulation of cholinergic neurons expressing ChR2, at 0.5 Hz. <b>A)</b> Representative original current traces of wild type, compared to mutants affecting SV loading (<i>unc-17(e113)</i> vAChT), SV recycling (<i>unc-26(s1710)</i> synaptojanin, or Ca²⁺ influx (<i>unc-2(ra612)</i> VGCC). <b>B)</b> Group data and statistical analysis, corresponding to the experiments in A. Wild type data is shown in black. Left panel: Mean inward currents (pA) ± SEM, in response to repeated stimulation with 10 ms blue light at 0.5 Hz. Right panel: Data was normalized to the first peak inward current [%]. Statistically significant differences were assessed by one-way ANOVA compared to wild type (* P < 0.05, ** P < 0.01, *** P < 0.001). For analysis of miniature PSC currents and frequencies of these strains, see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135584#pone.0135584.s005" target="_blank">S5 Fig</a>.</b></p

Establishing the R-OptIoN approach.

<p><b>A)</b> Schematic of the Ca²⁺ imaging approach showing single animal and bulk measurement conditions (lower inset, left and right, respectively, showing RCaMP fluorescence in BWMs). The 470 and 590 nm LEDs were used to stimulate ChR2 expressed in neurons, and for exciting RCaMP expressed in BWMs, respectively. The micro-wells (upper inset) were prepared in a thick 10% agar pad, pierced with a heated LED cooler element. <b>B)</b> Schematic of the epifluorescence microscope used for Ca²⁺ imaging. Two high power LEDs are coupled into the excitation light train with a dichroic mirror; light passes through a 470/593 nm double band-pass excitation filter and a 605 nm beam splitter onto the animals in the micro-wells. Emission light passes a 647 nm filter before reaching the camera. <b>C)</b> Comparison of changes in body length of animals expressing ChR2 in cholinergic motoneurons in wild type background (black trace), <i>unc-49(e407)</i> (GABA_AR) mutants (red trace), or <i>unc-26(s1710)</i> (synaptojanin) mutants (orange trace). In the absence of ATR, rendering ChR2 non-functional, no contraction is observed (grey trace). Shown is the mean normalized body length (± SEM; n = 8–21). Blue bar marks period of illumination. <b>D)</b> Light-induced activation of cholinergic neurons expressing ChR2 causes essentially identical changes in body length (contraction) with and without RCaMP expression in BWMs (red and black trace, respectively). In the absence of ATR, no contraction is observed (grey trace). Shown is the mean normalized body length (± SEM; n = 11–27 animals). <b>E)</b> Ca²⁺ response in BWMs of single animals fixed on polystyrene beads during photostimulation of ChR2 expressed in cholinergic motoneurons. Displayed are mean ΔF/F₀ values (± SEM) of wild type control (black trace), <i>unc-47(e307)</i> mutants (green trace), and <i>unc-49(e407)</i> mutants (red trace) of n = 5–15 animals. <b>F)</b> Ca²⁺ response in BWMs during photostimulation of ChR2 expressed in cholinergic motoneurons in bulk measurements. Displayed are mean ΔF/F₀ values (± SEM) of wild type control (black trace), <i>unc-47(e307)</i> mutants (green trace), and <i>unc-49(e407)</i> mutants (red trace). Shown is the mean of n = 3 measurements of ~ 1000 animals each. <b>G)</b> Ca²⁺ response in BWMs of single animals fixed on polystyrene beads during photostimulation of ChR2 expressed in GABAergic motoneurons. Displayed are mean ΔF/F₀ values (± SEM) of wild type control (black trace), <i>unc-47(e307)</i> mutants (green trace) and no-ATR control (grey trace), of n = 6–9 animals for each genotype/condition. <b>H)</b> Ca²⁺ response in BWMs during photostimulation of ChR2 expressed in GABAergic motoneurons in bulk measurements. Displayed are mean ΔF/F₀ values (± SEM) of wild type control (black trace), <i>snt-1(md290)</i> mutants (orange trace), <i>unc-47(e307)</i> mutants (green trace), and <i>unc-25(n2569)</i> mutants (magenta trace); means ± SEM of n = 3 measurements of ~1000 animals each.</p

Construction and characterization of a strain with specific RNAi sensitivity in cholinergic motoneurons.

<p><b>A)</b> The genetic background lacks <i>rde-1</i>, eliminating RNAi in all tissues. RDE-1 is selectively rescued in cholinergic neurons, by expression from the <i>unc-17</i> promoter. Furthermore, neurons are sensitized to RNAi by overexpression of the SID-1 dsRNA uptake facilitator (using the panneuronal <i>unc-119</i> promoter). <b>B)</b> Specific knockdown (feeding RNAi) of YFP in cholinergic neurons (using GFP-RNAi bacteria that also target YFP), in an animal expressing YFP panneuronally (punc-119::YFP). Left, mock-RNAi control; right, YFP knockdown, residual YFP fluorescence in ventral nerve cord mainly from GABAergic neurons. <b>C)</b> Group data and statistics of animals as shown in B. Mean ± SEM, background-corrected fluorescence values resulting from the whole nerve cord YFP fluorescence. Compared are animals treated with mock-RNAi vector (L4440) (n = 21) or with L4440::GFP RNAi (n = 15), showing statistically significant reduction of YFP fluorescence (t-test, *** P<0.001). <b>D)</b> Pan-neuronal YFP expression in the RNAi-sensitive strain ZX1800. Animals were treated with bacteria containing the mock-control (L4440) (upper images), or (lower images) the GFP RNAi vector. Reduced YFP expression in the ventral nerve cord can be recognized. In the right images, cholinergic (blue) and GABAergic (red) motoneurons are labeled, in maximum projections of confocal stacks; shown is an enlarged region of the ventral nerve cord surrounding the vulva. Anti-GFP RNAi renders most cholinergic neurons undetectable due to YFP mRNA knock-down.</p

Representation of Ca²⁺ imaging data for high throughput analyses and recapitulation of mutant phenotypes by cholinergic neuron RNAi.

<p><b>A)</b> Maximum-normalized ΔF/F₀ Ca²⁺ signals from bulk measurements, before and during 120 s cholinergic neuron ChR2 photostimulation, in wild type control (black trace), and <i>unc-26(s1710)</i> synaptojanin mutant (orange trace). The difference graph (wild type—mutant trace) is shown in green. Mean of n = 3 experiments with ~1000 animals each. The difference trace is further plotted in color-code below the graph, color scale is shown on the left. Blue bar marks the period of illumination. <b>B)</b> Comparing color-coded Ca²⁺ signal difference traces from bulk measurements in genomic mutants (left panel) and respective RNAi animals (right panel); genotypes or mRNAs targeted, as indicated. Data averaged from n = 3–18 experiments with ~1000 animals each.</p

Further analysis of selected genes from the RNAi screen cluster #1, by Ca²⁺ imaging (A, B) and by electrophysiology (C-G).

<p>The genomic mutants <i>C27B7</i>.<i>7(ok2978)</i>, <i>inx-10(ok2714)</i>, <i>inx-8(gk42)</i>, <i>spp-10(gk349)</i> and <i>erp-1(ok462)</i> were crossed either to the ChR2(C128S); RCaMP strain for Ca²⁺ imaging, or to the <i>zxIs6</i> ChR2(H134R) strain, for measuring photo-ePSCs. In A and B, normalized Ca²⁺ signal difference traces are shown as color coded, maximum normalized data, based on the mean ΔF/F₀ Ca²⁺ response (± SEM) from bulk measurements of RNAi or mutant and control (n = 3–5 experiments, ~1000 animals each). In E—G, the averaged photo-ePSC measurements of the respective wild type animals (black), <i>C27B7</i>.<i>7(ok2978)</i> (red), <i>inx-10(ok2714)</i> (yellow), <i>inx-8(gk42)</i> (green), <i>spp-10(gk349)</i> (blue), or <i>erp-1(ok462)</i> mutants (orange) are shown as inward currents in pA ± SEM (left panels). Statistically significant differences were calculated by one-way ANOVA for individual time points, always compared to wild type (* P < 0.05), or for the whole data set as two-way ANOVA (** P < 0.01, **P<0.001). Animals were stimulated for 10 ms with blue light at a frequency of 0.5 Hz (n = 7–12). On the right, the same data are represented, but normalized to the mean peak currents of the first stimulus. For representative original current records, as well as for mini ePSC current and frequency analysis, see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135584#pone.0135584.s005" target="_blank">S5 Fig</a>.</b></p