Home › Cell Biology › Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction
Cell Biology JoVE (Open Access) Citable · DOI

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

DOI: 10.3791/55122-v
What you'll learn
  • ✓ Load calcium-sensitive dyes into frog nerve endings via nerve stump retrograde transport.
  • ✓ Record and quantify fast calcium transients at the neuromuscular junction using fluorescence microscopy.
  • ✓ Analyze calcium imaging data to extract kinetic parameters from peripheral nerve terminals.
Protocol

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

Difficulty
advanced
Total time
~4–6 hours per preparation (dye loading 2–3 hrs, imaging and analysis 2–3 hrs)
Model organism
Frog (Rana pipiens or related species)
Biosafety
BSL-1

Steps

1
Prepare calcium-sensitive dye loading solution

Mix and prepare the calcium dye solution according to protocol specifications, ensuring proper concentration and solubility for retrograde nerve loading.

▶ 01:03
2
Load dye through isolated frog nerve stump

Apply the dye solution to the severed nerve stump to allow retrograde transport of the calcium indicator into the nerve endings at the neuromuscular junction.

▶ 01:53
3
Prepare stained tissue for microscopy examination

Wash, mount, and position the labeled neuromuscular preparation on the microscope stage under appropriate environmental conditions for imaging.

▶ 05:40
4
Capture fluorescence video with CCD camera system

Record high-speed digital video of calcium transients in response to nerve stimulation using a sensitive CCD camera and appropriate optical filters.

▶ 08:22
5
Analyze calcium transient kinetics and parameters

Extract, quantify, and statistically evaluate calcium transient amplitudes, rise times, decay kinetics, and other functional metrics from the recorded video sequences.

▶ 10:06
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