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Neuroscience JoVE (Open Access) Citable · DOI

In Situ Ca2+ Imaging of the Enteric Nervous System

DOI: 10.3791/52506-v
What you'll learn
  • ✓ Prepare live ENS tissue and load fluorescent Ca2+ indicators for imaging
  • ✓ Acquire and analyze Ca2+ imaging data from enteric neurons and glia
  • ✓ Interpret functional activity patterns in the enteric nervous system
Protocol

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

Difficulty
advanced
Total time
~2–3 hours per animal preparation and imaging session
Model organism
Mouse (strain unspecified)
Biosafety
BSL-1

Steps

1
Prepare and mount live enteric nervous tissue

Dissect and isolate intestinal preparations containing the ENS, then mount tissue in a suitable imaging chamber. This step preserves neuronal viability for subsequent Ca2+ imaging.

▶ 02:01
2
Load tissue with Fluo-4 calcium indicator dye

Apply Fluo-4 dye to the mounted ENS tissue to fluorescently label intracellular Ca2+ in neurons and glia, allowing optical detection of activity.

▶ 05:25
3
Acquire and analyze Ca2+ imaging data

Use widefield or confocal microscopy to record Ca2+ transients from enteric neurons and glia in real time, then process and analyze temporal dynamics of fluorescence signals.

▶ 06:42
4
Interpret functional activity and applications

Evaluate recorded Ca2+ signals to characterize neuronal and glial network activity, reflex circuits, and functional responses in the enteric nervous system.

▶ 09:10
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