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 Ca^....

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

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