Principles of miniSOG
As an engineered fluorescent protein, miniSOG will generate singlet oxygen by blue light illumination. Under blue light irradiation, miniSOG absorbs light energy and makes it transition from the ground state (S0) to the first excited state (S1). S1 is excited to the second excited state (S2) through internal transition energy. Based on S2, miniSOG is excited into a singlet state. The excited singlet state experiences internal energy transition and forms a more stable excited triplet state. The triplet state miniSOG can also decay back to the ground state by emitting a phosphorescent photon. This energy transfer step leads to the formation of singlet oxygen. This photochemical reaction process finally forms ROS, which destroys the amino acids, fats and unsaturated fatty acids in cells, and then causes cell death. The reaction is commonly known as a Type II photochemical process(Abrahamse and Hamblin 2016), which generate ROS more easily than Type I photosensitizer. Furthermore, miniSOG can kill neurons rapidly and effectively without detectable damages to surrounding tissues, and it is also reported ablating neurons at different developmental stages (Qi et al. 2012).
Constructs and molecular biology
The strain hpIs603, hpIs459, hpIs603; hpIs459 (Table 1) were used to detect neuronal activity in this paper. The construction method of miniSOG nematode strain was described in the previous research(Kawano et al., 2011). Briefly, as is shown in Fig. 1: firstly, the DNA fragment included target cell promoter, mito-miniSOG and marker protein, which was achieved by the coupled reaction. Then the DNA fragment was microinjected into the L4 stage nematode. Finally, the miniSOG nematode strain could be produced by passage culture. Three kinds of promoters used in this paper were from N2 C. elegans genomic DNA, including 5.1 kb Pnmr-1, 2.5 kb Punc-4, and 0.86 kb Plgc-55B genomic sequence with ATG start codon. The pnmr-1 used in this paper excludes the 2 kb internal fragment encoding cex-1, which interferes with the expression of the reporter gene. All C. elegans are cultured on standard nematode growth medium plates seeded with OP50 and are maintained at 22 °C incubators.
Fabrication of LED light
As is shown in the Fig. 2, the homemade LED light includes control box, lamp box, light-emitting part, power supply, switch and controller. Switch and controller are fastened in the control box. Culture dishes, shelf, little drawers, transparent heat insulation plate and LED lamp group are put in the lamp box. This homemade LED light has some advantages. First, a plurality of culture dish drawers can be inserted into the shelf of different heights, and the drawer handle is convenient to be arranged outside the culture dish drawer. Second, electric fans, radiators and heat shields are installed to ensure that the worms will not be heated to death. Third, there are six groups of lamps, and each group of lamps is composed of six 1W lamp beads, which can emit 470 nm blue light. More information can be found in Chinese patents (No. CN201910801243.8).
Neuron ablation
All members of motor neurons (MNs) and premotor interneurons (INs) were performed ablation on the plate using the homemade LED box. Worms were put on the standard NGM culture plates with a transparent lid, which could prevent dust in the air from entering into the medium. Then these whole plates with worms were illuminated under the homemade 470 nm blue LED light box for 30-45 minutes. Ablation was performed when the animals grow up to L1 or L2 stage. Later L4 stage animals were recorded for behavioral or calcium imaging analyses. Day 1 animals were recorded for behavioral analyses. Before the behavioral and calcium activity detection, the animals were examined for the RFP signals under the microscope.
Confocal Imaging
The later L4 stage hpIs603 nematode were anesthetized by 2.5 mM levamisole (Sigma-Aldrich) during M9 buffer. Fluorescence signals were captured when the worms alive by a Plan-Apochromatic 60×objective on a confocal microscope (FV3000, Olympus). Ten animals were recorded both in the control group (without the ablation of premotor INs and B-MNs) and ablated group (with the ablation of premotor INs and B-MNs) in the same conditions.
Behavioral analysis
The day 1 stage hpIs603 worms, maintained in standard culture conditions, was transferred to a 60 mm imaging plate seeded with a thin layer of OP50 with or without neurons ablation. Then these crawling worms were recorded for 3 minutes by a stereoscopic fluorescence microscope (Axio Zoom V16, Zeiss) equipped with a digital camera (acA2500−60um, Basler). All videos were captured with a 10X objective, at 10 frames every second. The worm images were skeletonized and divided into 33 segments (Fig. 4 & Fig. 5) by in-house written MATLAB scripts. In addition, we tracked the mid-point of the animals and calculated the velocity and direction of the animal’s movement between each frame. Finally, the curvatures of the whole worm, the angle between three joint points defined as the curvature of the middle point, were calculated and shown as color map(S. Gao et al. 2018).
Calcium imaging
DA9 MN activity recording was carried out as previous paper(S. Gao et al. 2018). In the ablated group, the worms were illuminated by blue light LED for 40 min in L2 stage and recorded after 20-24h. These worms (hpIs603;hpIs459) of an integrated strain expressing GCaMP6 in body-wall muscle cells were glued on a 2% agarose pad on a slide, suspended in the M9 buffer and imaged with a 60X objective CCD camera at 100 ms per frame. Data were collected by MicroManager and analyzed by ImageJ. The animals (hpIs603;hpIs459) of the control group weren’t illuminated by blue light LED and were carried out the same operation like the ablated group.
Electrophysiology
All electrophysiological experiments were performed with one-day-old adult C. elegans. The dissection and recording protocols were the same as in previous reports(S. Gao and Zhen 2011). Before the formal experiment, glass coverslips were coated by Sylgard 184 Silicone Elastomer (Dow Corning). The pipet solution and bath solution were prepared. The pipet solution includes (in mM): K-gluconate 115; KCl 25; CaCl2 0.1; MgCl2 5; BAPTA 1; HEPES 10; Na2ATP 5; Na2GTP 0.5; cAMP 0.5; cGMP 0.5, pH7.2 with KOH, ∼320 mOsm. cAMP and cGMP were included to maintain the activity and longevity of the preparation. The bath solution contains (in mM): NaCl 150; KCl 5; CaCl2 5; MgCl2 1; glucose 10; sucrose 5; HEPES 15, pH7.3 with NaOH, ∼330mOsm. Then worms were immobilized to the silicone surface by tissue adhesive glue (Histoacryl Blue, Braun) in M9 buffer. The cuticle on one side of the nematode was then cut to expose the internal organs. After the internal organs were sucked away with glass tube, the cuticle edge was gently glued down by WORMGLU (GluStitch Inc.) to expose the neuromuscular system. Body wall muscle cells were patched using 4−10 MΩ-resistant borosilicate pipettes (1B100F-4, World Precision Instruments). Pipettes were pulled by micropipette puller (P-1000, Sutter) and fire-polished by microforge MF-830 (Narishige). Membrane currents were recorded in the whole-cell configuration by EPC9 amplifier (HEKA, Germany), using the pulse and processed with Igor 6 (WaveMetrics) and Clampfit 10 software (Axon Instruments, Molecular Devices). Data were digitized at 10−20 kHz and filtered at 2.6 kHz. Chemicals were obtained from Sigma unless stated otherwise. Experiments were performed at room temperature.