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Representative Results






Subcellular Imaging of Neuronal Calcium Handling In Vivo

Published: March 17th, 2023



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....

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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

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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.......

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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.......

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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.


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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 
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

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