Subcellular Imaging of Neuronal Calcium Handling In Vivo

Summary

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.

Abstract

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.

Introduction

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 function^1,2. 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 Ca²⁺ into organelles such as the endoplasmic reticulum (ER) and mitochondria. These processes, in addition to the arrangement of Ca²⁺-permeable ion channels in the plasma membrane, result in heterogeneous levels of cytoplasmic Ca²⁺ throughout the neuron.

Ca²⁺ heterogeneity during rest and neuronal activation allows for the diverse, location-specific regulation of Ca²⁺-dependent mechanisms¹. One example of the concentration-specific effects of Ca²⁺ is the release of Ca²⁺ from the ER through inositol 1,4,5-trisphosphate (InsP₃) receptors. Low Ca²⁺ levels in combination with InsP₃ are required for the opening of the receptor's calcium-permeable pore. Alternatively, high Ca²⁺ levels both directly and indirectly inhibit the receptor⁴. The importance of Ca²⁺ homeostasis for proper neuronal function is supported by evidence suggesting that disrupted intracellular Ca²⁺ handling and signaling is an early step in the pathogenesis of neurodegenerative disorders and natural aging^5,6. Specifically, abnormal Ca²⁺ uptake and release by the ER and mitochondria are linked to the onset of neuronal dysfunction in Alzheimer's disease, Parkinson's disease, and Huntington's disease^6,7.

The study of Ca²⁺ dyshomeostasis during natural aging or neurodegeneration requires the longitudinal observation of Ca²⁺ levels with subcellular resolution in a living, intact organism in which the native cellular architecture (i.e., the arrangement of synapses and distribution of ion channels) is maintained. To this end, this protocol provides guidance on the use of two readily available, genetically encoded Ca²⁺ sensors for recording Ca²⁺ dynamics in vivo with high spatial and temporal resolution. The representative results acquired using the described method in C. elegans demonstrate how the expression of Ca²⁺ indicators in the cytoplasm or mitochondrial matrix of single neurons can allow for the rapid acquisition of fluorescent images (up to 50 Hz) that illustrate the Ca²⁺ dynamics with the additional capability of discerning the Ca²⁺ levels within single spine-like structures and individual mitochondria.

Protocol

1. Creating transgenic strains

1. Using a cloning method of choice^8,9, 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.
2. Follow the established protocol for the creation of transgenic strains via the microinjection of a DNA mix (<100 ng/μL; see Supplemental Table 1 for the concentrations of each plasmid) containing the Ca²⁺ indicator transgene (~20 ng/μL) and the rescue DNA lin-15⁺ (selection marker) into the gonad of a 1 day old adult C. elegans¹⁰ (genotype: lin-15(n765ts); phenotype: multi-vulva)^10,11.
   NOTE. Maintain lin-15(n765ts) parents at 15 °C to suppress temperature-sensitive mutation.
3. Maintain the injected parents at 20 °C until the F1 progeny reach adulthood to allow for transgenic selection using the absence of the multi-vulva phenotype.
4. Clonally passage¹³ adult F1 progeny that do not have the multi-vulva phenotype as this indicates the expression of the extrachromosomal array containing the Ca²⁺ indicator¹¹.
5. Assess the expression of the Ca²⁺ indicator in the F2 or F3 progeny that do not have the multi-vulva phenotype by mounting and imaging 1 day old adults as described later in section 3.
   NOTE. A relatively high expression of the indicator is required for the rapid acquisition of images as described here (see the discussion for more detail).
6. Perform experiments on subsequent generations of the transgenic strains with an optimal expression of the Ca²⁺ indicator (see the discussion for more detail), maintaining the strains under standard conditions (20 °C on NGM plates)¹².

2. Optical setup

1. Use a microscope capable of long-term time-lapse imaging.
   NOTE. The representative data were acquired using an inverted spinning disk confocal microscope equipped with 488 nm and 561 nm excitation lasers.
2. Use a low-magnification objective (i.e., 10x/0.40) for the crude localization of the worm.
3. To achieve subcellular resolution, switch to and localize the neurons using a high-magnification objective.
   NOTE. A 100x/1.40 NA oil objective was used to acquire the representative data in this study.
4. Acquire the images using an ultra-sensitive camera capable of rapid image acquisition (>50 fps).
5. Use a standard emission filter for the Ca²⁺ indicator of choice (i.e., a 525 nm ± 50 nm emission filter for GCaMP6f and mitoGCaMP).
6. Add a Z-drift corrector (ZDC) for the acquisition of image streams longer than 10 s, as the desiccation of the agar pad during the imaging session causes the neurite to drift out of focus within several seconds.
   NOTE. If a microscopy set-up does not allow for ZDC, or if long (>20 min) imaging periods are desired, then apply a border of silicon grease or melted petroleum jelly around the agar pad to slow the desiccation.

3. Preparation of worms for imaging

1. Preparing the agar pads
   1. Make 3 mL of 10% agar by dissolving molecular grade agar in M9¹² in a 13 mm x 100 mm glass culture tube and microwaving for several seconds (see Table of Materials).
      NOTE. Molten agar can be kept on a heat block for up to 1 h or can be made fresh each time agar pads are needed.
   2. Place a microscope slide between two additional slides that each have two layers of laboratory tape (see Figure 1A-i).
   3. Cut the tip of a 1,000 μL pipette tip (without a filter), and use it to pipette a small drop of agar onto the center coverslip (Figure 1A-i).
   4. Flatten the agar by pressing another slide down on top of the agar (Figure 1A-ii).
   5. After cooling, cut the agar into a small disc using the opening of a 13 mm x 100 mm glass culture tube (Figure 1A-iii), and then remove the surrounding agar.
2. Preparing the worm-rolling solution
   1. Dissolve muscimol powder in M9 to create a 30 mM stock. Separate into 50 μL aliquots, and store at 4 °C.
   2. Thaw a new aliquot of 30 mM muscimol every 3-5 days (and store at 20 °C while in use).
   3. Dilute a 30 mM muscimol stock in a 1:1 ratio with polystyrene beads to make the rolling solution.
3. Positioning a worm for imaging
   1. Place 1.6 μL of the rolling solution onto the center of the agar pad.
      NOTE: The amount of liquid should be adjusted for imaging younger (less) or older animals (more).
   2. Using the preferred worm pick (i.e., a glass or platinum wire pick)¹³, transfer a worm of the desired age without the multi-vulva phenotype¹¹ into the rolling solution on the agar pad (Figure 1A-iv).
      NOTE. The representative data are from 1 day old hermaphrodites (GCaMP6f strain = FJH185; mitoGCaMP strain; FJH597; see Supplemental Table 1), which can be identified by the presence of only one row of eggs. See the discussion for more detail on working with worms of different ages.
   3. Wait for ~5 min for the muscimol to reduce the worm movement, and then drop a 22 mm x 22 mm coverslip on top of the agar pad, physically restricting the worm movement.
   4. For imaging neurites in the ventral nerve cord, roll the worm into the orientation shown in Figure 1B,C by lightly sliding the coverslip.
      NOTE. To visualize the ventral nerve cord, position the worm with the head upward, and roll the worm until the intestine is on the right side of the proximal portion and the left for the distal portion of the worm (from the viewer's perspective). Invert this orientation (Figure 1C) for imaging neurites in the dorsal nerve cord.
4. Mounting the worm on a microscope
   1. Place a drop of immersion oil onto the coverslip before mounting it onto the microscope stage.
   2. Find the worm using a low-magnification objective (10x) objective in brightfield.
   3. Switch to the 100x objective, and adjust the focus.
   4. Locate the AVA neurite using the illumination of the GCaMP or mitoGCaMP with the 488 nm imaging laser.
      NOTE: Use small adjustments to prevent squishing the worm or pulling off the coverslip.

4. Acquisition of high-resolution image streams

1. In vivo GCaMP imaging
   1. Set the exposure time to 20 ms.
   2. Adjust the imaging laser and acquisition settings until the basal GCaMP fluorescence is in the mid-range of the camera's dynamic range¹⁴. Refer to the discussion for suggestions on optimizing the imaging parameters using other microscopy setups.
      ​NOTE. For the optical setup used in this study, set the 488 nm imaging laser to the following output settings: laser power = 50% and attenuation = 1. Modify the additional acquisition settings as follows: EM gain = 200 and pre-amplifier gain = 1.
   3. After the AVA neurite is located using the GCaMP fluorescence at 100x magnification, set the ZDC (see Table of Materials) to a range of ±30 μm, and initiate continuous autofocusing. Manually correct the focal plane as needed before imaging.
   4. Acquire a stream of images at 100x magnification. Durations as long as 10 min can be achieved with the setup used in this study.
2. In vivo mitoGCaMP imaging
   1. Follow steps 4.1.1-4.1.2 as needed to acquire the basal mitoGCaMP fluorescence in the mid-range for the camera's dynamic range¹⁴.
      NOTE. For the optical set-up used in this study, set the 488 nm imaging laser to the following output settings: laser power = 20% and attenuation = 10. Modify the acquisition settings to the following: EM gain = 100 and pre-amplifier gain = 2.
   2. After the mitochondria in the AVA neurite are located using the mitoGCaMP fluorescence, set the ZDC as described above in step 3.3.
   3. Acquire a stream of images at 100x magnification.

5. Image stream analysis

1. Open the image stream in an appropriate image software for analyzing the pixel values in an image series (see Table of Materials).
2. Using the Polygon Selection tool, define the subcellular region of interest (ROI) containing GCaMP or mitoGCaMP.
3. Click on Analyze > Tools > ROI Manager. In the ROI Manager, click Add and then More > Multi Measure. In the Multi Measure window, click OK to automatically collect the average, minimum, and maximum pixel intensities of the ROI defined above for each frame of the image stream for further analysis.
   NOTE. It is recommended that the average pixel intensity (F_avg) is normalized to the minimum intensity (F_min) for each ROI using the ratio F_avg/F_min to account for fluorescence differences due to positioning in the z-plane. Discard the image streams with worm movement during imaging since this would also affect the fluorescence measurements.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
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 homogenizer	Andor Technologies		Borealis upgrade to CSU-X1
CleanBench laboratory table	TMC		With vibration control
Disposable culture tubes	VWR	47729-572	13 mm x 100 mm
Environmental chamber	Thermo Scientific	3940	Set to 20 °C
Filter wheel or slider	ASI		For 25 mm diameter filters
FJH 185	Caenorhabditis Genetics Center	FJH 185	Worm strain
FJH 597	Caenorhabditis Genetics Center	FJH 597	Worm 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 laser	Andor Technologies		50 mW
ImageJ	National Institutes of Health		Version 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 box	Olympus		IX3-CBH
Muscimol	MP Biomedical / Sigma	02195336-CF
pAS1	AddGene	194970	Plasmid
pBSKS	Stratagene
pCT61			Plasmid available from Hoerndli/Maricq lab upon request
pJM23			Plasmid available from Hoerndli/Maricq lab upon request
pKK1	AddGene	194969	Plasmid
Polybead microspheres	Polysciences Inc.	00876-15	0.094 µm
Stability chamber	Norlake Scientific	NSRI241WSW/8H	Set to 15 °C
Stage controller	ASI		With filter wheel control
Standard microscope slide	Premiere	9108W-E	75 mm x 25 mm x 1 mm
Touch panel controller	Olympus		I3-TPC
Z-drift corrector	Olympus		IX3-ZDC2