Calciumbilleddannelse i frit opførende caenorhabditis elegans med velkontrolleret, ikke-lokaliseret vibration
Summary
Rapporteret her er et system til calciumbilleddannelse i frit at opføre caenorhabditis elegans med velkontrolleret, ikke-lokaliseret vibration. Dette system gør det muligt for forskere at fremkalde ikke-lokaliserede vibrationer med velkontrollerede egenskaber ved nanoskala forskydning og kvantificere calciumstrømme under reaktioner fra C. elegans på vibrationerne.
Abstract
Ikke-lokaliserede mekaniske kræfter, såsom vibrationer og akustiske bølger, påvirker en lang række biologiske processer fra udvikling til homeostase. Dyr klare disse stimuli ved at ændre deres adfærd. Forståelse af de mekanismer, der ligger til grund for en sådan adfærdsændring, kræver kvantificering af neural aktivitet under adfærd af interesse. Her rapporterer vi en metode til calciumbilleddannelse i frit at opføre Caenorhabditis elegans med ikke-lokaliserede vibrationer af specifik frekvens, forskydning og varighed. Denne metode tillader produktion af velkontrollerede, ikke-lokaliserede vibrationer ved hjælp af en akustisk transducer og kvantificering af fremkaldte calciumresponser ved enkeltcelleopløsning. Som et bevis på princippet demonstreres calciumresponset fra en enkelt interneuron, AVA, under C. elegans flugtrespons på vibrationer. Dette system vil lette forståelsen af neurale mekanismer, der ligger til grund for adfærdsmæssige reaktioner på mekaniske stimuli.
Introduction
Dyr udsættes ofte for ikke-lokaliserede mekaniske stimuli såsom vibrationer eller akustiske bølger 1,2. Fordi disse stimuli påvirker homeostase, udvikling og reproduktion, skal dyr ændre deres adfærd for at klare dem 3,4,5. Imidlertid er de neurale kredsløb og mekanismer, der ligger til grund for en sådan adfærdsændring, dårligt forstået.
Mekanosensorisk adfærd i nematoden, Caenorhabditis elegans, er et simpelt adfærdsmæssigt paradigme, hvor orme normalt ændrer adfærd fra fremadgående bevægelse til et baglæns flugtrespons, når de støder på ikke-lokaliseret vibration6. Det neurale kredsløb, der ligger til grund for denne adfærd, består primært af fem sensoriske neuroner, fire par interneuroner og flere typer motorneuroner 7,8. Derudover er orme vant til sådanne mekaniske stimuli efter adskilt træning, der involverer gentagen stimulering 9,10,11. Derfor udgør denne enkle adfærdsmæssige reaktion et ideelt system til at undersøge neurale mekanismer, der ligger til grund for både ikke-lokaliseret vibrationsdrevet adfærd og hukommelse. En protokol for calciumbilleddannelse i frit opførende orme under påvirkning af ikke-lokaliserede vibrationer er illustreret. Sammenlignet med tidligere rapporterede systemer er dette system simpelt, fordi det ikke kræver et ekstra kamera til sporing; det giver os dog mulighed for at ændre frekvensen, forskydningen og varigheden af ikke-lokaliserede vibrationer. Fordi aktivering af AVA-interneuronerne inducerer det bagudrettede flugtrespons, blev orme, der samtidig udtrykker GCaMP, en calciumindikator, og TagRFP, et calcium-ufølsomt fluorescerende protein, under kontrol af en AVA-specifik promotor brugt som et eksempel (se Tabel over materialer for detaljer). Protokollen demonstrerer aktiveringen af AVA-neuroner, når en orm skifter fra fremadgående til baglæns bevægelse. Denne protokol letter forståelsen af den neurale kredsløbsmekanisme, der ligger til grund for mekanosensorisk adfærd.
Protocol
Representative Results
Discussion
Generelt kræver kvantificering af neural aktivitet indførelse af en sonde og / eller begrænsninger på dyrs kropsbevægelse. For undersøgelser af mekanosensorisk adfærd udgør den invasive introduktion af en sonde og begrænsninger i sig selv mekaniske stimuli. C. elegans giver et system til at omgå disse problemer, fordi dets funktioner er gennemsigtige, og fordi det har et simpelt, kompakt neuralt kredsløb, der kun består af 302 neuroner. Ved at kombinere disse fordele med den tidligere udviklede metod…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker Caenorhabditis Genetics Center for at levere de stammer, der anvendes i denne undersøgelse. Denne publikation blev støttet af JSPS KAKENHI Grant-in-Aid for Scientific Research (B) (Grant no. JP18H02483), om innovative områder “Science of Soft Robot” -projektet (Bevillingsnr. JP18H05474), PRIME fra Japan Agency for Medical Research and Development (bevillingsnummer 19gm6110022h001) og Shimadzu Foundation.
Materials
| Data anaylsis software | |||
| DualViewImaging.nb | author | For analysis of acquired data | |
| Mathematica12 | Wolfram | For running data anaysis software DualViewImaging | |
| Escherichia coli and C. elegans strains | |||
| E. coli OP50 | Caenorhabditis Genetics Center | OP50 | Food for C. elegans. Uracil auxotroph. E. coli B. |
| lite-1(ce314) strain | Caenorhabditis Genetics Center | KG1180 | Light-insensitive mutant |
| lite-1(ce314) strain expressing NLS-GCaMP-NLS and TagRFP under the control of the AVA-speciric promoter | author | ST12 | lite-1(ce314) mutant carrying the genes expressing NLS-GCaMP5G-NLS (NLS; nuclear localization signal) and TagRFP under the control of the flp-18 promoter as an extrachoromosomal arrays |
| Laser Doppler vibrometer | |||
| Lase Doppler vibrometer | Polytec Japan | IVS-500 | For quantifying frequency and displacement generated by the accoustic transducer |
| Mouse macro system | |||
| Assay.txt | Author | Script for temporally and specially controlling mouse cursol in Windows | |
| HiMacroEx | Vector | https://www.vector.co.jp/download/file/winnt/util/fh667310.html | Free download software for controling mouse cursor based on a script |
| Nematode growth media plate | |||
| Agar purified, powder | Nakarai tesque | 01162-15 | For preparation of NGM plates |
| Bacto pepton | Becton Dickinson | 211677 | For preparation of NGM plates |
| Calcium chloride | Wako | 036-00485 | For preparation of NGM plates |
| Cholesterol | Wako | 034-03002 | For preparation of NGM plates |
| di-Photassium hydrogenphosphate | Nakarai tesque | 28727-95 | For preparation of NGM plates |
| LB broth, Lennox | Nakarai tesque | 20066-95 | For culture of E. coli OP50 |
| Magnesium sulfate anhydrous | TGI | M1890 | For preparation of NGM plates |
| Potassium Dihydrogenphosphate | Nakarai tesque | 28720-65 | For preparation of NGM plates |
| Sodium Chloride | Nakarai tesque | 31320-05 | For preparation of NGM plates |
| Petri dishes (60 mm) | Nunc | 150270 | For preparation of NGM plates |
| Nonlocalized vibration device | |||
| Amplifier | LEPY | LP-A7USB | For stimulation with controllable vibration |
| Acoustic transducer | MinebeaMitsumi | LVC25 | For stimulation with controllable vibration |
| WaveGene Ver. 1.5 | Thrive | http://efu.jp.net/soft/wg/down_wg.html | Free download software for controling vibration property |
| Noninvasive calcium imaging | |||
| 2-Channel benchtop 3-phase brushless DC servo controller | Thorlabs | BBD202 | Compatible controller for MLS203-1 stages |
| 479/585 nm BrightLine dual-band bandpass filter | Semrock | FF01-479/585-25 | For acquisition of two channel images (GCaMP and TagRFP) |
| 505/606 nm BrightLine dual-edge standard epi-fluorescence dichroic beamsplitter | Semrock | FF505/606-Di01-25×36 | For acquisition of two channel images (GCaMP and TagRFP) |
| 512/25 nm BrightLine single-band bandpass filter | Semrock | FF01-512/25-25 | For acquisition of two channel images (GCaMP and TagRFP) |
| 630/92 nm BrightLine single-band bandpass filter | Semrock | FF01-630/92-25 | For acquisition of two channel images (GCaMP and TagRFP) |
| Computer | Dell | Precision T7600 | Windows7 with Intel Xeon CPU ES-2630 and 8 GB of RAM |
| High-speed x-y motorized stage | Thorlabs | MLS203-1 | Fast XY scannning stage |
| Image splitting optics | Hamamatsu photonics | A12801-01 | For acquisition of two channel images (GCaMP and TagRFP) generated by W-VIEW GEMINI Image spliting optics |
| LED light source | CoolLED | pE-4000 | For generating 470 nm and 560 nm excitation light |
| Microscope | Olympus | MVX10 | |
| sCMOS camera | Andor | Zyla | |
| x 2 Objective lens | Olympus | MVPLAPO2XC | Working distance 20 mm and numerical aperture 0.5 |
| Plasmid | |||
| pKDK66 plasmid | author | pKDK66 | Co-injection marker |
| pTAK83 plasmid | author | pTAK83 | Plasmid for expression of TagRFP under the control of the flp-18 promoter |
| pTAK144 plasmid | author | pTAK144 | Plasmid for expression of NLS-GCaMP5G-NLS under the control of the flp-18 promoter |
| Tracking software | |||
| homingback.vi | author | SubVi file for tracking a fluoresent spot of a worm through feedback control of sCMOS camera and x-y motorized stage | |
| LabVIEW | National instruments | For running tracking software | |
| Zyla Control ver.2.6CI.vi | author | For tracking a fluoresent spot of a worm through feedback control of sCMOS camera and x-y motorized stage |
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