An economical and highly adaptable optogenetics system for individual and population-level manipulation of Caenorhabditis elegans

Abstract

Background: Optogenetics allows the experimental manipulation of excitable cells by a light stimulus without the need for technically challenging and invasive procedures. The high degree of spatial, temporal, and intensity control that can be achieved with a light stimulus, combined with cell type-specific expression of light-sensitive ion channels, enables highly specific and precise stimulation of excitable cells. Optogenetic tools have therefore revolutionized the study of neuronal circuits in a number of models, including Caenorhabditis elegans. Despite the existence of several optogenetic systems that allow spatial and temporal photoactivation of light-sensitive actuators in C. elegans, their high costs and low flexibility have limited wide access to optogenetics. Here, we developed an inexpensive, easy-to-build, modular, and adjustable optogenetics device for use on different microscopes and worm trackers, which we called the OptoArm.

Results: The OptoArm allows for single- and multiple-worm illumination and is adaptable in terms of light intensity, lighting profiles, and light color. We demonstrate OptoArm's power in a population-based multi-parameter study on the contributions of motor circuit cells to age-related motility decline. We found that individual components of the neuromuscular system display different rates of age-dependent deterioration. The functional decline of cholinergic neurons mirrors motor decline, while GABAergic neurons and muscle cells are relatively age-resilient, suggesting that rate-limiting cells exist and determine neuronal circuit ageing.

Conclusion: We have assembled an economical, reliable, and highly adaptable optogenetics system which can be deployed to address diverse biological questions. We provide a detailed description of the construction as well as technical and biological validation of our set-up. Importantly, use of the OptoArm is not limited to C. elegans and may benefit studies in multiple model organisms, making optogenetics more accessible to the broader research community.

Keywords: Caenorhabditis elegans; Neuronal ageing; OptoArm; Optogenetics; Rhodopsin; Worm trackers.

Fig. 1

Channelrhodopsin-2 is a light-sensitive cation channel. A Channelrhodopsin-2 (ChR2) is rapidly opened by stimulation with blue (440–460 nm) light in an essentially nondesensitizing manner. ChR2 is permeable by multiple cations like Na⁺ and Ca²⁺ and enables strong and rapid membrane depolarization of the cell it is expressed in. B Multiple ChR2-expressing C. elegans strains exist, including but not limited to expressing in muscle cells (myo-3), cholinergic neurons (unc-17), and GABAergic neurons (unc-47). C The change in body length is often used as a readout of ChR2 activation (see B)

Fig. 2

Building the OptoArm only requires basic knowledge of electronics and thermal management. A The optimal electronic circuitry of the OptoArm. Inset: the variant of the circuitry used in this paper to test and validate the system. The color codes refer to the wires of the LED driver that is used in this paper. B The essential components required to build the electronic circuitry of the OptoArm: 1. Luxeon Rebel LED, 2. Thermal adhesives, 3. Heatsink, 4. Wire harness with potentiometer, 5. DC plug, 6. ON/OFF button, 7. LED driver, 8. lens with case, 9. Several lenses, 10. and 11. Lens holder. C The steps required to construct the OptoArm, detailed instructions per picture can be found in Table 3. STEP 1: connecting the solderless DC plug to the connecting wire harness. STEP 2: mounting the LED on a heatsink and soldering the connecting wire harness to the LED cathode and anode. STEP 3: Connecting the ON/OF switch to the LED driver and connecting the driver to the wire harness. Note, the pictures show a serial set-up, and not the recommend parallel wiring (see 2A, main). STEP 4: mounting the electronic circuitry to a standard flask clamp to finish the OptoArm. By placing the OptoArm in either a D fine-tuner or a E general lab standard, the system can be used for different applications. F The different thermal resistances (Rθ) in the heat flow between the LED junction, temperature test point, and bottom of the LED assembly

Fig. 3

The OptoArm provides the light intensity and stability required for optogenetic experiments. A The different lenses used for testing. B A schematic outlining the tested parameters with the three different lenses. C Raster showing the x,y coordinates used to measure the intensity of the LED. D The different integrated intensity profiles of the LED with and without lenses. The histograms show the zoomed-in distribution of intensity readings between a minimum and maximum intensity with a specified bin width. Red lines show the average intensity. E Smoothened histograms of D that show the increase in intensity when using different lenses with the OptoArm. F Schematic showing the method to assess the stability of intensity over time. G Distributions of intensity readings at the different timepoint without a lens (H) and with a 10° lens. All readings were performed at 448 nm

Fig. 4

The OptoArm fulfills all technical criteria in different experimental set-ups. A The OptoArm set-up used with a standard dissection microscope. B In experimental set-ups, the OptoArm is used with a specific angle (43°), thereby deviating from perpendicular illumination. C The different illumination profiles for the different lenses upon and illumination with the specified angle of incidence. D The different integrated intensity profiles for C. The histograms show the distribution of intensity readings between a minimum and maximum intensity with a specified bin width. Red lines show the average intensity. E Schematic showing the different visible microscopic areas (of the used microscope) when using different magnifications. The areas are on scale and can be directly compared to the intensity maps shown in D. 1.0× corresponds to a total magnification of 15×, and 4.0 to a total of 60×. The dotted red line represents the area in D with an intensity of > 1.6 mW/mm². F The OptoArm set-up with the WF-NTP. G The different integrated intensity profiles of the LED with the 21° lens, at different working distances with a fixed angle of incidence of 43°. Profiles were generated in the set-up with the WF-NTP. Circles represent the outline of a 3-cm NGM plate. H Histograms of intensity readings of the microscope-light (Gaussian) and WF-NTP backlight (flat-field). Red lines show the average intensity. All readings were performed at 448 nm. I Thrashing frequency of D1 worms, expressing ChR2 under the unc-17 (acetylcholine) or unc-47 (GABA) promotor, recorded with the WF-NTP, grown with or without ATR. There is no significant effect of the WF-NTP light on this behavior, n = 15, two-tailed unpaired Student’s t test (all: n.s.). J Body length of D1 worms recorded with the WF-NTP, grown with or without ATR. Body length was normalized by the mean of the paired ATR− condition. There is no significant effect of the WF-NTP light on this behavior, n = 15, two-tailed unpaired Student’s t test (all: n.s.). For I and J, there was no photo-stimulation with the OptoArm

Fig. 5

The OptoArm allows manual intensity adjustments, which modulate biological readouts. A To record worms in an inexpensive way, we used a Carson smartphone adapter to place and orient a cellphone camera to a microscope. A blue light filter was placed between the ocular and sample to avoid interference of the blue light with recording. B Schematic of the experimental outline: a priori determined intensities (0.2, 0.4, 0.8., 1.2., 1.6, and 1.8 mW/mm²) were set by adjusting the resistance of the potentiometer. At the different intensities, the ∆body length was measured. C The histograms show the distribution of intensity readings at a priori-specified intensities. Red lines show the average intensity. D Smoothened histograms of C that show the increase in intensity when adjusting the resistance of the potentiometer. E Pictures of a D1 worms (myo-3p::ChR2::YFP) just before and during illumination with 1.6 mW/mm² blue light. Scale bar: 200 μm. F The different body lengths (normalized to length before illumination) at different intensities of illumination with the OptoArm. Calculated via a midline approach, n = 10

Fig. 6

A semi-automatic perimeter approach is a reliable way of assessing changes in body length during and after optogenetic stimulation. A Schematic outline of image processing in Fiji to follow body length changes over time in a semi-automatic way. B Raw readings of body length (perimeter in pixels) of a worm expressing ChR2 in cholinergic neurons (unc-17p::ChR2::YFP) plotted against time in milliseconds, the graph represents n = 1. C The change in body lengths (normalized to length before illumination) of multiple worms, n = 10, when using a perimeter approach to estimate the worm length. ∆body length equals the change in length before illumination and during illumination. D Schematic of a worm with the midline and perimeter highlighted. E The relationship between the midline of the worm and the perimeter. n = 70 (35 still images of worms at light ON and 35 worms at light OFF). The spearman correlation was calculated (p < 0.001)

Fig. 7

Mutations in the cholinergic or GABAergic system affect ∆body length after optogenetic stimulation with the OptoArm. A A schematic showing the different mutant used to verify previously established results. B Schematic of the experimental outline: the change in body length was measured after blue light stimulation with an intensity of 1.6 mW/mm². C Kinetics of optogenetic stimulation in mean ± SEM body length of control worms and unc-26(s1710) and unc-47(e307) mutants expressing ChR2 in cholinergic neurons, n = 9–10. A perimeter approach was used. D Quantification of mean ± SEM normalized body length of control D1 worms and unc-26(s1710) and unc-47(e307) mutants expressing ChR2 in cholinergic neurons. Two-way ANOVA (Interaction, ATR, Genotype: p < 0.001) with post hoc Dunnett, n = 11–15 for ATR+ and n = 5–6 for ATR−. A midline approach was used. E Kinetics of optogenetic stimulation in mean ± SEM normalized body length of control worms and unc-26(s1710) and unc-47(e307) mutants expressing ChR2 in GABAergic neurons, n = 9-11. A perimeter approach was used. F Quantification of mean ± SEM normalized body length of control worms and unc-26(s1710) and unc-47(e307) mutants expressing ChR2 in GABAergic neurons. Two-way ANOVA (Interaction, ATR, Genotype: p < 0.001) with post hoc Dunnett, n = 10–15 for ATR+ and n = 5–6 for ATR−. A midline approach was used. G Long-term photo-stimulation of control worms, unc-26(s1710) and unc-47(e307) mutants expressing ChR2 in cholinergic neurons. Two-way repeated ANOVA with Geisser-Greenhouse correction (time, genotype, time × genotype and individual worms: p < 0.001) and post hoc Dunnett, n = 10. A midline approach was used. Blue bars represent “light ON.” Acetylcholine: zxIs6 (Punc-17::ChR2::YFP), zxIs3 (Punc-47::ChR2::YFP). *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001. Error bars represent S.E.M

Fig. 8

Population characteristics acquired with the WF-NTP are potential readouts of optogenetic stimulation with the OptoArm. A Schematic of experimental outline. D4 worms were assessed in liquid with the OptoArm being OFF and ON and population characteristics (bending frequency and eccentricity) were analyzed with the WF-NTP software. B Change in bends per 30 s when light is ON of worms grown with or without ATR. The percentual decline in thrashing capacity is annotated in the graph, n = 15. For acetylcholine: Mann-Whitney U test p < 0.001, GABA: two-tailed unpaired Student’s t test: p < 0.001. C Binned effect of blue light on swimming behavior of transgenic worms grown with or without ATR, n = 10. Two-way ANOVA (time, genotype, interaction: p < 0.001) with post hoc Sidak’s. D Change in bends per 10 s after optogenetic stimulation. Acetylcholine: only the last 10 s of 30-s illumination is used, GABA: only the first 10 s are used. n = 15. For acetylcholine: Mann-Whitney U test p < 0.001, GABA: two-tailed unpaired Student’s t test: p < 0.001. E Effect of blue light on eccentricity of worms grown with or F without ATR. n = 20–40, Mann-Whitney U tests acetylcholine + ATR: 0–10 s: not significant, 10–20 s: p = 0.0192, 20–30 s: p = 0.0033, for 0–30 s: p = 0.0427. Acetylcholine—ATR, and both GABA conditions: n.s. G The body length of worms expressing ChR2 under the unc-17 promoter after blue light stimulation. Bulk assayed worms (multiple) were compared to individual assayed worms (single). n = 15, two-tailed unpaired Student’s t test: n.s. Blue bars represent “light ON.” Acetylcholine: zxIs6 (Punc-17::ChR2::YFP), GABA: zxIs3 (Punc-47::ChR2::YFP). *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001

Fig. 9

Cholinergic neuronal ageing correlates best with the decline in motility. A Schematic showing the experimental outline. The ∆body length was determined for strains expressing ChR2 in muscle, cholinergic neurons and GABAergic neurons at several time intervals. At the same time, the movement capacity, represented as bends per 30 s in liquid, was also measured. B The decline in thrashing capacity in liquid of worms expressing ChR2 in cholinergic or GABAergic neurons. n = 15, two-way ANOVA (Age: p < 0.001, interaction and neuron type: n.s.). The experiment was triplicated, one representative experiment is shown (see D for the averages of the other two experiments). C The normalized ∆body length (elongation for GABA, contraction for muscle and cholinergic neurons) at different ages as measured by the midline approach. Every point represents the mean of an experiment with > 10 biological replicates each. Fitted lines represent a general linear model with 2nd-order polynomial fit. All points within one experiment were normalized by the intra-experimental average at D1 within each strain. D The average decline in thrashing ability over time. Every point represents the mean of an experiment with > 15 biological replicates each. All points were normalized by the inter-experimental average at D1 within each strain. This sets the average bends per 30 s to exactly 1.0 at D1. Fitted lines represent a general linear model with 2nd-order polynomial fit. E The correlation between the ∆body length and ∆movement capacity (normalized to the changes and capacity at D1 intra-experimentally). Fitted lines represent simple linear regression without restrictions (both slopes deviate significantly from zero: p < 0.001). The black line corresponds to a relation of x = y, in which the decline in movement is equal to the decline in ∆body length. F The ∆eccentricity (eccentricity before illumination minus eccentricity during illumination) at different ages after optogenetic stimulation. Each individual point represents the mean of n > 15 worms. Student’s t test: n.s. All experiments were replicated three times, all replicates are shown. The transparent zones around the fitted lines represent the confidence interval (95%). *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001

Fig. 10

Automation of the OptoArm allows fine adjustment of light pulse width and intensity. A The electronic circuitry of the OptoArm under the control of a microcontroller (an Arduino Uno in this paper). B By using a pulse width modulator output pin of Arduino the pulse width (I, II), time intervals (III), and intensity (IV) of the OptoArm can be easily regulated. C By using an LCD shield on top of an Arduino Uno, all parameters can be adjusted in live-modus without the intervention of a computer. There are 5 buttons that can be used to go UP and DOWN in the different programs to select parameters and to adjust them by pressing LEFT or RIGHT. All changes can be confirmed with the SELECT button (see Table 5). D The different programs of the automated OptoArm. Program 1 (I) allows testing the system and manually turning the LED ON and OFF (II). Here, the intensity of the system can be changed as well. Program 2 (III) can be used to give single-timed pulses of light (IV). Program 3 (V) can be used to give trains of single-time pulses. One can adjust both the pulse-time (IV), the waiting time, and the number of cycles (VI). E The software provides a clear overview of the steps that are performed. When a run is started and then finishes, the user can use the option “Rerun” to execute the same program without having to adjust the parameters again