Name: subcellular imaging of neuronal calcium handling in vivo
Uploaded: 2023-03-17T12:30:00
Description: Watch this Scientific Journal Video about Subcellular Imaging of Neuronal Calcium Handling In Vivo at JoVE.com

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.

1. Creating transgenic strains
<ol>
	<li>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&#39; UTR (see the discussion for more information)10. A list of plasmids and their sources can be found in Supplemental Table 1</stron...

<ol>
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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+,&#160;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...

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 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&#39;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 aging5,6. Specifically, abnormal Ca2+ uptake and release by the ER and mitochondria are linked to the onset of neuronal dysfunction in Alzheimer&#39;s disease, Parkinson&#39;s disease, and Huntington&#39;s disease6,7.
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&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100x/1.40 Oil objective&#160;&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td>&#160;UPlanSApo</td>
		</tr>
		<tr>
			<td>10x/0.40 Objective&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td>&#160;UPlanSApo</td>
		</tr>
		<tr>
			<td>22 mm x 22 mm Cover glass&#160;</td>
			<td>VWR&#160;</td>
			<td>48366-227&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose SFR&#160;</td>
			<td>VWR&#160;</td>
			<td>J234-100G&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Beam homogenizer</td>
			<td>Andor Technologies</td>
			<td></td>
			<td>Borealis upgrade to CSU-X1</td>
		</tr>
		<tr>
			<td>CleanBench laboratory table&#160;</td>
			<td>TMC&#160;</td>
			<td></td>
			<td>With vibration control</td>
		</tr>
		<tr>
			<td>Disposable culture tubes</td>
			<td>VWR&#160;</td>
			<td>47729-572</td>
			<td>&#160;13 mm x 100 mm</td>
		</tr>
		<tr>
			<td>Environmental chamber</td>
			<td>Thermo Scientific</td>
			<td>3940</td>
			<td>Set to 20 &#176;C</td>
		</tr>
		<tr>
			<td>Filter wheel or slider</td>
			<td>ASI</td>
			<td></td>
			<td>For 25 mm diameter filters</td>
		</tr>
		<tr>
			<td>FJH 185</td>
			<td>Caenorhabditis Genetics Center&#160;</td>
			<td>FJH 185</td>
			<td>Worm strain</td>
		</tr>
		<tr>
			<td>FJH 597</td>
			<td>Caenorhabditis Genetics Center</td>
			<td>FJH 597</td>
			<td>Worm strain</td>
		</tr>
		<tr>
			<td>GFP bandpass emission filter&#160;</td>
			<td>Chroma&#160;</td>
			<td></td>
			<td>525 &#177; 50 nm&#160;(25 mm diameter)</td>
		</tr>
		<tr>
			<td>ILE laser combiner&#160;</td>
			<td>Andor Technologies&#160;</td>
			<td></td>
			<td>4 laser lines&#160;</td>
		</tr>
		<tr>
			<td>ILE solid state 488 nm laser</td>
			<td>Andor Technologies&#160;</td>
			<td></td>
			<td>50 mW</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>Version 1.52a</td>
		</tr>
		<tr>
			<td>IX83 Spinning disk confocal microscope&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td>With Yokogawa CSU-X1 spinning disc</td>
		</tr>
		<tr>
			<td>iXon Ultra EMCCD camera&#160;</td>
			<td>Andor Technologies&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low auto-fluorescence immersion oil&#160;</td>
			<td>Olympus&#160;</td>
			<td>Z-81226</td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph&#160;</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td>Version 7.10.1&#160;</td>
		</tr>
		<tr>
			<td>Microscope control box</td>
			<td>Olympus</td>
			<td></td>
			<td>IX3-CBH</td>
		</tr>
		<tr>
			<td>Muscimol&#160;</td>
			<td>MP Biomedical&#160;/ Sigma</td>
			<td>02195336-CF&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>pAS1</td>
			<td>AddGene</td>
			<td>194970</td>
			<td>Plasmid</td>
		</tr>
		<tr>
			<td>pBSKS</td>
			<td>Stratagene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCT61</td>
			<td></td>
			<td></td>
			<td>Plasmid available from Hoerndli/Maricq lab upon request</td>
		</tr>
		<tr>
			<td>pJM23</td>
			<td></td>
			<td></td>
			<td>Plasmid available from Hoerndli/Maricq lab upon request</td>
		</tr>
		<tr>
			<td>pKK1&#160;</td>
			<td>AddGene&#160;</td>
			<td>194969</td>
			<td>Plasmid</td>
		</tr>
		<tr>
			<td>Polybead microspheres&#160;</td>
			<td>Polysciences Inc.&#160;</td>
			<td>00876-15</td>
			<td>&#160;0.094 &#181;m</td>
		</tr>
		<tr>
			<td>Stability chamber</td>
			<td>Norlake Scientific</td>
			<td>NSRI241WSW/8H</td>
			<td>Set to 15 &#176;C</td>
		</tr>
		<tr>
			<td>Stage controller</td>
			<td>ASI</td>
			<td></td>
			<td>With filter wheel control</td>
		</tr>
		<tr>
			<td>Standard microscope slide</td>
			<td>Premiere</td>
			<td>9108W-E</td>
			<td>75 mm x 25 mm x 1 mm</td>
		</tr>
		<tr>
			<td>Touch panel controller</td>
			<td>Olympus</td>
			<td></td>
			<td>I3-TPC</td>
		</tr>
		<tr>
			<td>Z-drift corrector&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td>IX3-ZDC2</td>
		</tr>
	</tbody>
</table>

subcellular imaging of neuronal calcium handling in vivo

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,&#160;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.

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...

Watch this Scientific Journal Video about Subcellular Imaging of Neuronal Calcium Handling In Vivo at JoVE.com

Subcellular Imaging of Neuronal Calcium Handling In Vivo

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.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

This technique can be used to advance our understanding of calcium handling in neurons, and how compartmentalized calcium can regulate different mechanisms important for neuronal function in vivo. The main advantage of this technique is that it allows for higher resolution rapid in vivo imaging under physiological conditions in C.elegans that may be combined with other fluorescent tools. This technique could provide insights into mechanism regulating calcium signaling in neurons in many different contexts.For example, during aging, neuronal injury, and neurodegeneration. Demonstrating this procedure will be Ennis Deihl, research technician. Kaz Knight, a PhD candidate, and Rachel Doser, a postdoctoral researcher from my lab.Begin by cloning expression vectors that contain a promoter followed by a calcium indicator, and three prime UTR of choice. Create transgenic C.elegans strains by microinjection of a DNA mix containing the rescue DNA Lin-15 plus in the calcium indicator transgene into the gonad of a one-day-old adult C.elegans. Maintain the injected parent worms at 20 degrees Celsius until the F1 progeny reach adulthood.Select transgenic adults by screening for the absence of the multi-vulva phenotype. That indicates expression of the extra chromosomal array containing the calcium indicator. Clonally passage adult F1 progeny that do not have the multi-vulva phenotype.Perform experiments on subsequent generations of the transgenic strains with an optimal expression of the calcium indicator. Use a microscope capable of long-term Time-lapse imaging. Locate the worm under a low magnification objective and then switch to a high magnification objective to localize the neurons.Acquire the images using an ultrasensitive camera capable of rapid image acquisition at more than 50 FPS. Next, use a standard emission filter for the calcium indicator. Add a Z-drift corrector for the acquisition of image streams longer than 10 seconds.In a 13-by-100-millimeter glass culture tube prepare three milliliters of 10%agar by dissolving molecular grade agar in M9 and microwave for several seconds. To make the agar pads, first, prepare two slides by adding two layers of laboratory tape. Then place a microscope slide between the two slides.Cut the tip of a 1000-microliter pipette tip and use it to pipette a small drop of agar onto the center of the cover slip. Flatten the agar by pressing another slide down on top of the agar. After cooling, cut the agar into a small disc using the opening of a 10-milliliter tube, and then remove the surrounding agar.Next, prepare the worm rolling solution by dissolving muscimol powder in M9 to create a 30-millimolar stock. Dilute the stock in a one-to-one ratio with polystyrene beads to make the rolling solution. To position the worm for imaging, first, place 1.6 microliters of the rolling solution onto the center of the agar pad.Then using a preferred worm pick, transfer a worm of the desired age into the rolling solution on the agar pad. Wait for about five minutes for the muscimol to reduce the worm movement, and then drop a 22-by-22-millimeter cover slip on top of the agar pad. For imaging neurites in the ventral nerve cord, roll the worm by slightly sliding the cover slip.To mount the worm on the microscope, place a drop of immersion oil onto the cover slip. Find the worm using a low magnification objective in brightfield. Then switch to 100x objective and locate the AVA neurite using the illumination of the GCaMP or mito GCaMP with the 488-nanometer imaging laser.For in vivo GCaMP imaging, adjust the imaging laser in acquisition settings until the basil GCaMP fluorescence is in the mid range of the dynamic range of the camera and set the exposure time to 20 milliseconds. After the AVA neurite is located using the GCaMP fluorescence at 100 times magnification, set the Z-drift corrector, and initiate continuous autofocusing. Manually correct the focal plane as needed before imaging.Acquire a stream of images at 100 times magnification. Using this protocol, cytoplasmic calcium was measured with high temporal and spatial resolution. The cell specific expression of GCaMP6F in the glutamatergic AVA command interneurons revealed a directional propagation of calcium influx.The subcellular quantification of GCaMP6F fluorescence showed that the onset of calcium influx was delayed in a portion of the neurite located only 10 micrometers away. Also, dendritic spine-like structures found along the AVA neurite showed flashes in GCaMP6F fluorescence that occurred independently of neurite activation and did not lead to a propagation of calcium influx. Further, the relative calcium levels were measured in the individual mitochondria in single neurites using a worm strain with a AVA specific expression of the mitochondrial matrix localized calcium indicator mito GCaMP.The results showed the synchronous uptake of a relatively large amount of calcium into a subset of mitochondria. Specifically, the calcium uptake into some mitochondria was synchronized, whereas neighboring mitochondria did not appear to take up calcium. Similarly, subsets of mitochondria showed rapid uptake and release of small amounts of calcium.This also appeared to be synchronous but only for a subset of mitochondria. Speed and fine manipulations are essential for positioning animals and retaining neuronal function. Animal health is also paramount.Even a little starvation is problematic. The hardest part to learn is the rapid orientation of the animals for confocal microscopy without damage. My advice is a lot of practice and good controls.This protocol could be applied to experiments where calcium is released from other subcellular locations in neurons, other neurons or cells other than neurons in C.elegans. Because this approach in C.elegans leaves animals intact, it is possible to address questions about regulation of subcellular calcium handling in vivo in single neurons of a complete nervous system.

Research

JoVE Journal

Neuroscience

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...