Summary

Verwenden einen angepasste mikrofluidischen olfaktorische Chip für die Bildgebung neuronaler Aktivität als Reaktion auf Pheromone in männlichen C. Elegans Kopf Neuronen

Published: September 07, 2017
doi:
Please note that all translations are automatically generated. Click here for the English version.

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Disclosures

The authors have nothing to disclose.

Acknowledgements

Materials

Silicon WaferUniversity Wafer452
SU-8 2035MicroChemY111070-0500L1GL
DeveloperMicroChemY020100-4000L1PE
Wafer MaskCad/Art ServicesCustom order. Printed at 25,000 dpi.
Sylgard-184Ellsworth Adhesives184 SIL ELAST KIT 0.5KG
1.0 mm Dermal PunchesAcuderm Inc.P150
Soft TubingCole-PalmerEW-06419-01
Hard TubingIDEX Health & Science1622
PinsNew England Small TubeNE-1027-12
Blocking PinsNew England Small Tube0.415/0.425" OD x .500 LongBatch PB07027
3 mL syringesBD309657
30 mL syringesVitality Medical302832Used as buffer reservoirs.
Stainless Steel Blunt Needle 23 Gauge, Polyprolylene LuerComponent Supply CompanyNE-231PL-50
Stopcocks with Luer connections; 3-way; male lock; 5 flow pattern; non-sterileCole-PalmerEW-30600-07
Fisherfinest Premium Cover GlassFisher Scientific12-548-5M
Mercator Control System LF-5 Plasma SystemMercatorLF-5
Scotch TapeScotchBSN43575
Series 20 ChamberWarner InstrumentsP-2
Vacuum DesicatorBel-Art Scienceware42025000024 cm inner diameter.
Weigh BoatsCole-PalmerEW-01017-27
Classic Plus BalanceMettler ToledoPB1501-S/FACT
Glass Pasteur PipettesCole-PalmerEW-25554-06
Transfer pipettesGenesee Scientific30-202
OvenSheldon Manufacturing Inc9120993Model Number: 1500E.
60 mm, non-vented, sharp edge Petri dishesTriTech ResearchT3308
Zeiss Axio Observer.A1Zeiss
Hammamatsu Orca Flash 4.0 Digital CMOSHammamatsuC11440-22CU
Blue Fluorescent LightLumencorSOLA SM6-LCR-SA24-30V/7.9A DC.
Illumination AdaptorZeiss423302-0000
Series 1 and 2 Miniature Inert PTFE Isolation ValveParker001-0017-9003-way valve for controlling flow.
ValveLink8.2®AutoMate Scientific01-18Flow Switch Controller
Micro ManagerMicro-ManagerFree software, can be downloaded at: https://www.micro-manager.org/wiki/Download_Micro-Manager_Latest_Release
ImageJImageJFree software, can be downloaded at: https://imagej.nih.gov/ij/download.html
Agar, Bacteriological GradeApex9012-36-6
PeptoneApex20-260
CaCl<sub>2</sub>VWRBDH0224-1KG
MgSO<sub>4</sub>Sigma-Aldrich230391-1kg
CholesterolAlfa AesarA11470
EthanolSigma-Aldrich270741-4L
TetramisoleSigma-AldrichL9756-10(G)Store at 4 &deg;C.
FluoresceinSigma-AldrichFD2000S-250mgLight Sensitive. Store in photoprotective vials.
GlycerolSigma-AldrichG6279-1L
Ascaroside #3Synthesized in the Schroeder Lab (Cornell University).
NaClGenesee Scientific18-215
KH<sub>2</sub>PO<sub>4</sub>BDHBDH9268.25
K<sub>2</sub>HPO<sub>4</sub>J.T. Baker3252-025
ASH GCaMP3 lineCX10979 (KyEx2865 [p<em>sra</em>-6::GCAMP3 @ 100 ng/uL]). Developed in Bargmann lab. Provided from Albrecht Lab library.
CEM GCaMP6 lineJSR49 (FkEx98[p<em>pkd</em>-2::GCaMP::SL2::dsRED + pBX-1]; <em>pha</em>-1(e2123ts); <em>him</em>-5(e1490); <em>lite</em>-1(ce314)). Developed by Robyn Lints. Provided from Srinivasan Lab library.
<em>E. coli</em> (OP50)<em>Caenorhabditis</em> Genetics CenterOP50
"Reservoir"To create a Reservoir: A "30 mL syringe", is connected to a "Stopcock with Luer connections; 3-way; male lock; 5 flow pattern; non-sterile", which is connected to a "3 mL syringe" and a "Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer". The "Stainless Steel Blunt Needle 23 Gauge, Polyprolylene Luer" is then inserted into "Soft Tubing" approximately 1/3 of the way down the needle.

References

  1. Lagoy, R. C., Albrecht, D. R. Microfluidic Devices for Behavioral Analysis, Microscopy, and Neuronal Imaging in Caenorhabditis elegans. Methods Mol Biol. 1327, 159-179 (2015).
  2. Ben-Yakar, A., Chronis, N., Lu, H. Microfluidics for the analysis of behavior, nerve regeneration, and neural cell biology in C. elegans. Curr Opin Neurobiol. 19 (5), 561-567 (2009).
  3. Chronis, N. Worm chips: Microtools for C. elegans biology. Lab on a Chip. 10 (4), 432-437 (2010).
  4. Lee, H., Crane, M. M., Zhang, Y., Lu, H. Quantitative screening of genes regulating tryptophan hydroxylase transcription in Caenorhabditis elegans using microfluidics and an adaptive algorithm. Integr Biol (Camb). 5 (2), 372-380 (2013).
  5. Lockery, S. R., et al. A microfluidic device for whole-animal drug screening using electrophysiological measures in the nematode C. elegans. Lab Chip. 12 (12), 2211-2220 (2012).
  6. Mondal, S., et al. Large-scale microfluidics providing high-resolution and high-throughput screening of Caenorhabditis elegans poly-glutamine aggregation model. Nat Commun. 7, 13023 (2016).
  7. Larsch, J., Ventimiglia, D., Bargmann, C. I., Albrecht, D. R. High-throughput imaging of neuronal activity in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 110 (45), E4266-E4273 (2013).
  8. Akerboom, J., et al. Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics. Front Mol Neuro. 6, 2 (2013).
  9. Badura, A., Sun, X. R., Giovannucci, A., Lynch, L. A., Wang, S. S. H. Fast calcium sensor proteins for monitoring neural activity. Neurophotonics. 1 (2), 025008 (2014).
  10. Tatro, E. T. Brain-wide imaging of neurons in action. Front Neural Circuits. 8, 31 (2014).
  11. Tian, L., et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods. 6 (12), 875-881 (2009).
  12. Greene, J. S., et al. Balancing selection shapes density-dependent foraging behaviour. Nature. 539 (7628), 254-258 (2016).
  13. Greene, J. S., Dobosiewicz, M., Butcher, R. A., McGrath, P. T., Bargmann, C. I. Regulatory changes in two chemoreceptor genes contribute to a Caenorhabditis elegans QTL for foraging behavior. Elife. 5, (2016).
  14. Kim, K., et al. Two Chemoreceptors Mediate Developmental Effects of Dauer Pheromone in C. elegans. Science. 326 (5955), 994-998 (2009).
  15. McGrath, P. T., et al. Parallel evolution of domesticated Caenorhabditis species targets pheromone receptor genes. Nature. 477 (7364), 321-325 (2011).
  16. Schmitt, C., Schultheis, C., Husson, S. J., Liewald, J. F., Gottschalk, A. Specific Expression of Channelrhodopsin-2 in Single Neurons of Caenorhabditis elegans. PLoS ONE. 7 (8), e43164 (2012).
  17. White, J. G., Southgate, E., Thomson, J. N., Brenner, S. The Structure of the Nervous System of the Nematode Caenorhabditis elegans. Phil Trans of the Royal Soc of Lon. 314 (1165), 1 (1986).
  18. White, J. Q., et al. The sensory circuitry for sexual attraction in C. elegans males. Curr Biol. 17 (21), 1847-1857 (2007).
  19. Chronis, N., Zimmer, M., Bargmann, C. I. Microfluidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans. Nat Meth. 4 (9), 727-731 (2007).
  20. Chute, C. D., Srinivasan, J. Chemical mating cues in C. elegans. Semin Cell Dev Biol. 33, 18-24 (2014).
  21. Izrayelit, Y., et al. Targeted metabolomics reveals a male pheromone and sex-specific ascaroside biosynthesis in Caenorhabditis elegans. ACS Chem Biol. 7 (8), 1321-1325 (2012).
  22. Ludewig, A. H., Schroeder, F. C. Ascaroside signaling in C. elegans. WormBook. , 1-22 (2013).
  23. Narayan, A., et al. Contrasting responses within a single neuron class enable sex-specific attraction in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 113 (10), E1392-E1401 (2016).
  24. Srinivasan, J., et al. A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature. 454 (7208), 1115-1118 (2008).
  25. Sammut, M., et al. Glia-derived neurons are required for sex-specific learning in C. elegans. Nature. 526 (7573), 385-390 (2015).
  26. Sulston, J. E., Albertson, D. G., Thomson, J. N. The Caenorhabditis elegans male: postembryonic development of nongonadal structures. Dev Biol. 78 (2), 542-576 (1980).
  27. Hilliard, M. A., et al. In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents. The EMBO Journal. 24 (1), 63-72 (2005).
  28. Evans, T. C. Transformation and microinjection. WormBook. , (2006).
  29. Cáceres, I. d. C., Valmas, N., Hilliard, M. A., Lu, H. Laterally Orienting C. elegans Using Geometry at Microscale for High-Throughput Visual Screens in Neurodegeneration and Neuronal Development Studies. PLoS ONE. 7 (4), e35037 (2012).
  30. Schrodel, T., Prevedel, R., Aumayr, K., Zimmer, M., Vaziri, A. Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light. Nat Methods. 10 (10), 1013-1020 (2013).
  31. García, L. R., Portman, D. S. Neural circuits for sexually dimorphic and sexually divergent behaviors in Caenorhabditis elegans. Curr Opin Neurobiol. 38, 46-52 (2016).
  32. Clokey, G. V., Jacobson, L. A. The autofluorescent "lipofuscin granules" in the intestinal cells of Caenorhabditis elegans are secondary lysosomes. Mech Ageing Dev. 35 (1), 79-94 (1986).
  33. Coburn, C., et al. Anthranilate Fluorescence Marks a Calcium-Propagated Necrotic Wave That Promotes Organismal Death in C. elegans. PLoS Biology. 11 (7), e1001613 (2013).
  34. Macosko, E. Z., et al. A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans. Nature. 458 (7242), 1171-1175 (2009).
  35. Park, D., et al. Interaction of structure-specific and promiscuous G-protein-coupled receptors mediates small-molecule signaling in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 109 (25), 9917-9922 (2012).
Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

Play Video

Cite This Article
Reilly, D. K., Lawler, D. E., Albrecht, D. R., Srinivasan, J. Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons. J. Vis. Exp. (127), e56026, doi:10.3791/56026 (2017).

View Video