Fluorescence Imaging of Synaptic Calcium Dynamics in an In Vitro Model of Amyotrophic Lateral Sclerosis

Abstract

Source: Krishnamurthy, K. et al. Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis. J. Vis. Exp. (2021)

This video demonstrates real-time calcium imaging to monitor synaptic activity in ALS-mutant neurons expressing a genetically encoded calcium biosensor. It outlines the steps for preparing the neurons for imaging, stimulating calcium dynamics, and recording fluorescence to measure real-time changes in intracellular calcium levels, which reflect neuronal synaptic activity.

Protocol

1. Fluorescence imaging of Gcamp6m calcium transients

1. Transfect primary rodent cortical neurons cultured on 35 mm glass-bottom dishes with 500 ng of Gcamp6m.
   NOTE: If desired, co-transfect neurons with a plasmid of interest containing a fluorescent tag in the red or far-red range.
2. Incubate neurons with low potassium chloride (KCl) artificial cerebrospinal fluid (aCSF) (Table 1) for 15 min, 48 h post-transfection, and then mount the dish on the imaging platform.
3. Visualize GCaMP6m fluorescence using a fluorescein isothiocyanate (FITC) filter (488 nm) and a 20x or 40x objective.
4. Select the imaging field and engage perfect focus. Next, take a single still image with brightfield, FITC, and fluorescence marker channels to mark neuronal boundaries.
5. Initiate Run Now in the acquisition software. Carry out the baseline recording for 5 min and then perfuse with aCSF containing 50 mM KCL (Table 1).
   NOTE: The goal of this imaging is to measure evoked calcium transients. Should a neuron have basal firing activity and calcium fluxes during the pre-stimulation period, it is not used in data analysis. Instead, only cells with stable background fluorescence are used. Post-stimulation periods can also be extended to 60 min for calcium transients, with or without additional continuous high KCl perfusion.
6. Save the experiment and analyze data using confocal software. The experiment may be stopped at this point, and the analysis can be performed later.​
   NOTE: If the cells do not require transfection for the expression of proteins of interest, this protocol may be performed at any time point in vitro when seeking to examine calcium transients. Following a test of control cells to ensure the measurement of calcium transients, the experimenter should be blinded to the genetic or pharmacologic conditions of each dish tested to minimize bias.

2. Image analysis

1. Open time-lapse images with confocal software.
2. Align images in time-lapse series by the command series: Image | Processing | Align Current Document. Select Align to the First Frame.
3. Select regions of interest (ROIs) along neurites using the ROI selection tool, a bean-shaped icon on the right of the image frame. Also, mark an ROI representing background fluorescence intensity.
   NOTE: ROIs are selected by choosing areas of distinctly separated puncta along neuronal tracks indicated by the brightfield still image. At least five ROIs per neuron are chosen for analysis. The background ROI is chosen in a region of the field of view that does not contain neurites.
4. Measure raw fluorescence over time for selected ROIs using the following command series: Measure | Time Measurement.
   1. Initiate the Measure function at the upper portion of the Time Measurement panel. A graphical representation of raw fluorescence over time and quantitative data are both generated.
      NOTE: Each data point represents the raw fluorescence for that ROI for the frame associated with that specific measured time point.
5. Export raw fluorescence intensities to the spreadsheet software.

Table 1: Composition of artificial cerebrospinal fluid (aCSF) buffers.

Low KCL aCSF buffer (pH 7.40 to 7.45)
Reagent	Concentration
HEPES	10 mM
NaCl	140 mM
KCl	5 mM
Glucose	10 mM
CaCl2 2H2O	2 mM
MgCl2 4H2O	1 mM
High KCL aCSF buffer (pH 7.40 to 7.45)
Reagent	Concentration
HEPES	10 mM
NaCl	95 mM
KCl	50 mM
Glucose	10 mM
CaCl2 2H2O	2 mM
MgCl2 4H2O	1 mM

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
20x air objective	Nikon		For imaging
40x oil immersion objective	Nikon		For imaging
B27 supplement	Thermo Scientific	17504044	Neuronal growth supplement
BD Syringes without Needle, 50 mL	Thermo Scientific	13-689-8	Part of gravity perfusion assembly
Biosafety cell culture hood	Baker	SterilGARD III SG403A	Asceptic cell culturing, transfection, and dye loading
Beta-mercaptoethanol	Millipore Sigma	M3148	For culturing and maintenance of neuronal cultures
Bovine Serum Albumin	Millipore Sigma	A9418	For preparing neuronal cultures
Calcium chloride dihydrate	Millipore Sigma	223506	Component of aCSF solutions
Cell culture carbon dioxide incubator	Thermo Scientific	13-998-123	For culturing and maintenance of neurons
Confocal microscope	Nikon	Eclipse Ti +A1R core	For fluorescence imaging
CoolSNAP ES2 CCD camera	Photometrics		For image acquisition
D-Glucose	Millipore Sigma	G8270	Component of aCSF solutions
FITC Filter cube	Nikon	77032509	For imaging Gcamp calcium transients
Glass bottom petri dishes (Thickness #1.5)	CellVis	D35-10-1.5-N	For growth of neurons on imaging-compatible culture dish
HEPES	Millipore Sigma	H3375	Component of aCSF solutions
High KCl artifical cerebrospinal fluid (aCSF)			For imaging.
Horse serum	Millipore Sigma	H1138	For culturing and maintenance of neurons
Laminin	Thermo Scientific	23017015	For preparing neuronal cultures
Leibovitz's L-15 Medium	Thermo Scientific	11415064	For preparing neuronal cultures
Leibovitz's L-15 Medium, no phenol red	Thermo Scientific	21083027	For preparing neuronal cultures
L-Glutamine (200 mM)	Thermo Scientific	25030149	Neuronal culture supplement
Lipofectamine 2000 Transfection Reagent	Thermo Scientific	11668019	For neuronal transfections
Low KCl artifical cerebrospinal fluid (aCSF)			For imaging.
Magnesium chloride	Millipore Sigma	208337	Component of aCSF solutions
Microsoft Excel	Microsoft		Software for data analysis/normalization
Nalgene Filter Units, 0.2 µm PES	Thermo Scientific	565-0020	Filter unit for aCSF solution
Neurobasal medium	Thermo Scientific	21103049	For culturing and maintenance of neuronal cultures
NIS-Elements Advanced Research	Nikon		Software for image capture and analysis
Nunc 15 mL Conical tubes	Thermo Scientific	339650	For preparing neuronal culture and buffer solutions
Nunc 50 mL conical tubes	Thermo Scientific	339652	For preparing neuronal culture and buffer solutions
Optiprep	Millipore Sigma	D1556	For preparing neuronal cultures
Papain	Millipore Sigma	P4762	For preparing neuronal cultures
Penicillin-Streptomycin (10,000 U/mL)	Thermo Scientific	15140122	To prevent bacterial contamination of neuronal cultures
Perfusion system	Warner Instruments	SF-77B	For exchange of aCSF
Perfusion tubing	Cole-Parmer	UX-30526-14	Part of gravity perfusion assembly
pGP-CMV-Gcamp6m plasmid	Addgene	40754	For imaging calcium transients
Poly-D-lysine hydrobromide	Millipore Sigma	P7886	Coating agent for glass bottom petri dishes
Potassium chloride	Millipore Sigma	P3911	Component of aCSF solutions
Sodium bicarbonate	Millipore Sigma	S5761	Component of aCSF solutions
Sodium Chloride	Millipore Sigma	S9888	Component of aCSF solutions
Stage Top Incubator	Tokai Hit		For incubation of live neurons during imaging period
TRITC Filter cube	Nikon	77032809	For imaging FM4-64
Vibration Isolation table	New Port	VIP320X2430-135520	Table/stand for microscope