JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Published: March 17th, 2023

DOI:

10.3791/64928

1Department of Biomedical Sciences, Colorado State University College of Veterinary Medicine and Biomedical Sciences, 2Cellular and Molecular Biology Graduate Program, Colorado State University College of Veterinary Medicine and Biomedical Sciences
* These authors contributed equally

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo, where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Calcium ions (Ca2+) are highly versatile carriers of information in many cell types. In neurons, Ca2+ is responsible for the activity-dependent release of neurotransmitters, the regulation of cytoskeletal motility, the fine-tuning of metabolic processes, as well as many other mechanisms required for proper neuronal maintenance and function1,2. To ensure effective intracellular signaling, neurons must maintain low basal Ca2+ levels in their cytoplasm3. This is accomplished by cooperative Ca2+ handling mechanisms, including the uptake of Ca....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Creating transgenic strains

  1. Using a cloning method of choice8,9, clone expression vectors to contain the Pflp-18 or Prig-3 promoter (for AVA-specific signal in the ventral nerve cord), followed by the Ca2+ indicator of choice, and then a 3' UTR (see the discussion for more information)10. A list of plasmids and their sources can be found in Supplemental Table 1

Log in or to access full content. Learn more about your institution’s access to JoVE content here

These two protocols enable the rapid acquisition of differential Ca2+ levels within the subcellular regions and organelles of individual neurites in vivo with high spatial resolution. The first protocol allows for the measurement of cytoplasmic Ca2+ with high temporal and spatial resolution. This is demonstrated here using the cell-specific expression of GCaMP6f in the glutamatergic AVA command interneurons15, whose neurites run the entire length of the ventral nerve.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The first consideration when implementing the method described involves the selection of a Ca2+ indicator with ideal characteristics for the given research question. Cytoplasmic Ca2+ indicators typically have a high affinity for Ca2+, and the sensitivity of these indicators to Ca2+ is inversely related to the kinetics (on/off rate)16,17. This means that either Ca2+ sensitivity or kinetics will need to be.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was supported by the National Institutes of Health (NIH) (R01- NS115947 awarded to F. Hoerndli). We would also like to thank Dr. Attila Stetak for the pAS1 plasmid.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

NameCompanyCatalog NumberComments
100x/1.40 Oil objective  Olympus  UPlanSApo
10x/0.40 Objective Olympus  UPlanSApo
22 mm x 22 mm Cover glass VWR 48366-227 
Agarose SFR VWR J234-100G 
Beam homogenizerAndor TechnologiesBorealis upgrade to CSU-X1
CleanBench laboratory table TMC With vibration control
Disposable culture tubesVWR 47729-572 13 mm x 100 mm
Environmental chamberThermo Scientific3940Set to 20 °C
Filter wheel or sliderASIFor 25 mm diameter filters
FJH 185Caenorhabditis Genetics Center FJH 185Worm strain
FJH 597Caenorhabditis Genetics CenterFJH 597Worm strain
GFP bandpass emission filter Chroma 525 ± 50 nm (25 mm diameter)
ILE laser combiner Andor Technologies 4 laser lines 
ILE solid state 488 nm laserAndor Technologies 50 mW
ImageJNational Institutes of HealthVersion 1.52a
IX83 Spinning disk confocal microscope Olympus With Yokogawa CSU-X1 spinning disc
iXon Ultra EMCCD camera Andor Technologies 
Low auto-fluorescence immersion oil Olympus Z-81226
MetaMorph Molecular Devices Version 7.10.1 
Microscope control boxOlympusIX3-CBH
Muscimol MP Biomedical / Sigma02195336-CF 
pAS1AddGene194970Plasmid
pBSKSStratagene
pCT61Plasmid available from Hoerndli/Maricq lab upon request
pJM23Plasmid available from Hoerndli/Maricq lab upon request
pKK1 AddGene 194969Plasmid
Polybead microspheres Polysciences Inc. 00876-15 0.094 µm
Stability chamberNorlake ScientificNSRI241WSW/8HSet to 15 °C
Stage controllerASIWith filter wheel control
Standard microscope slidePremiere9108W-E75 mm x 25 mm x 1 mm
Touch panel controllerOlympusI3-TPC
Z-drift corrector Olympus IX3-ZDC2

  1. Berridge, M. J. Neuronal calcium signaling. Neuron. 21 (1), 13-26 (1998).
  2. Brini, M., Calì, T., Ottolini, D., Carafoli, E. Neuronal calcium signaling: Function and dysfunction. Cellular and Molecular Life Sciences. 71 (15), 2787-2814 (2014).
  3. Brini, M., Calì, T., Ottolini, D., Carafoli, E. Intracellular calcium homeostasis and signaling. Metal Ions in Life Sciences. 12, 119-168 (2013).
  4. Berridge, M. J. The inositol trisphosphate/calcium signaling pathway in health and disease. Physiological Reviews. 96 (4), 1261-1296 (2016).
  5. Schrank, S., Barrington, N., Stutzmann, G. E. Calcium-handling defects and neurodegenerative disease. Cold Spring Harbor Perspectives in Biology. 12 (7), 035212 (2020).
  6. Pchitskaya, E., Popugaeva, E., Bezprozvanny, I. Calcium signaling and molecular mechanisms underlying neurodegenerative diseases. Cell Calcium. 70, 87-94 (2018).
  7. Jadiya, P., et al. Impaired mitochondrial calcium efflux contributes to disease progression in models of Alzheimer's disease. Nature Communications. 10 (1), 3885 (2019).
  8. Zeiser, E., Frøkjær-Jensen, C., Jorgensen, E., Ahringer, J. MosSCI and gateway compatible plasmid toolkit for constitutive and inducible expression of transgenes in the C. elegans germline. PLoS One. 6 (5), 20082 (2011).
  9. Zhu, B., Cai, G., Hall, E. O., Freeman, G. J. In-FusionTM assembly: Seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques. 43 (3), 354-359 (2007).
  10. Evans, T. C. Transformation and Microinjection. WormBook. , (2006).
  11. Giordano-Santini, R., Dupuy, D. Selectable genetic markers for nematode transgenesis. Cellular and Molecular Life Sciences. 68 (11), 1917-1927 (2011).
  12. Stiernagle, T. Maintenance of C. elegans. WormBook. , (2006).
  13. Stiernagle, T. Maintenance of C. elegans: Transferring worms grown on NGM plates. WormBook. , (2006).
  14. Ogama, T. A beginner's guide to improving image acquisition in fluorescence microscopy. The Biochemist. 42 (6), 22-27 (2020).
  15. Mellem, J. E., Brockie, P. J., Madsen, D. M., Maricq, A. v. Action potentials contribute to neuronal signaling in C. elegans. Nature Neuroscience. 11 (8), 865-867 (2009).
  16. Zhang, Y., et al. Fast and sensitive GCaMP calcium indicators for imaging neural populations. bioRxiv. , (2021).
  17. Kerruth, S., Coates, C., Dürst, C. D., Oertner, T. G., Török, K. The kinetic mechanisms of fast-decay red-fluorescent genetically encoded calcium indicators. Journal of Biological Chemistry. 294 (11), 3934-3946 (2019).
  18. de Juan-Sanz, J., et al. Axonal endoplasmic reticulum Ca2+ content controls release probability in CNS nerve terminals. Neuron. 93 (4), 867-881 (2017).
  19. Fung, W., Wexler, L., Heiman, M. G. Cell-type-specific promoters for C. elegans glia. Journal of Neurogenetics. 34 (3-4), 335-346 (2020).
  20. Ali, F., Kwan, A. C. Interpreting in vivo calcium signals from neuronal cell bodies, axons, and dendrites: a review. Neurophotonics. 7 (1), 011402 (2019).
  21. Tian, L., et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nature Methods. 6 (12), 875-881 (2009).
  22. Mank, M., et al. A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nature Methods. 5 (9), 805-811 (2008).
  23. Birkner, A., Tischbirek, C. H., Konnerth, A. Improved deep two-photon calcium imaging in vivo. Cell Calcium. 64, 29-35 (2017).
  24. Ryan, K. C., Laboy, J. T., Norman, K. R. Deregulation of mitochondrial calcium handling due to presenilin loss disrupts redox homeostasis and promotes neuronal dysfunction. Antioxidants. 11 (9), 1642 (2022).
  25. Yang, H. H., et al. Subcellular imaging of voltage and calcium signals reveals neural processing in vivo. Cell. 166 (1), 245-257 (2016).
  26. Takahashi, N., Oertner, T. G., Hegemann, P., Larkum, M. E. Active cortical dendrites modulate perception. Science. 354 (6319), 1587-1590 (2016).
  27. Nicoletti, M., et al. Biophysical modeling of C. elegans neurons: Single ion currents and whole-cell dynamics of AWCon and RMD. PLoS One. 14 (7), 0218738 (2019).
  28. Church, P. J., Stanley, E. F. Single L-type calcium channel conductance with physiological levels of calcium in chick ciliary ganglion neurons. The Journal of Physiology. 496, 59-68 (1996).
  29. O'Hare, J. K., et al. Compartment-specific tuning of dendritic feature selectivity by intracellular Ca 2+ release. Science. 375 (6586), (2022).
  30. Mclntire, S. L., Jorgensen, E., Horvitz, H. R. Genes required for GABA function in Caenorhabditis elegans. Nature. 364 (6435), 334-337 (1993).
  31. Doser, R. L., Amberg, G. C., Hoerndli, F. J. Reactive oxygen species modulate activity-dependent AMPA receptor transport in C. elegans. The Journal of Neuroscience. 40 (39), 7405-7420 (2020).
  32. Wu, J., et al. A long Stokes shift red fluorescent Ca2+ indicator protein for two-photon and ratiometric imaging. Nature Communications. 5, 5262 (2014).
  33. Cho, J. -. H., et al. The GCaMP-R family of genetically encoded ratiometric calcium indicators. ACS Chemical Biology. 12 (4), 1066-1074 (2017).
  34. Smith, J. J., et al. A light sheet fluorescence microscopy protocol for Caenorhabditis elegans larvae and adults. Frontiers in Cell and Developmental Biology. 10, 1012820 (2022).
  35. Müller, M., et al. Mitochondria and calcium regulation as basis of neurodegeneration associated with aging. Frontiers in Neuroscience. 12, 470 (2018).
  36. Nikoletopoulou, V., Tavernarakis, N. Calcium homeostasis in aging neurons. Frontiers in Genetics. 3, 200 (2012).
  37. Chen, C. -. H., Chen, Y. -. C., Jiang, H. -. C., Chen, C. -. K., Pan, C. -. L. Neuronal aging: Learning from C. elegans. Journal of Molecular Signaling. 8 (1), 14 (2013).
  38. Saberi-Bosari, S., Huayta, J., San-Miguel, A. A microfluidic platform for lifelong high-resolution and high throughput imaging of subtle aging phenotypes in C. elegans. Lab on a Chip. 18 (20), 3090-3100 (2018).
  39. Sridhar, N., Fajrial, A. K., Doser, R. L., Hoerndli, F. J., Ding, X. Surface acoustic wave microfluidics for repetitive and reversible temporary immobilization of C. elegans. Lab on a Chip. 22 (24), 4882-4893 (2022).

Tags

Subcellular Imaging

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved