Home › Immunology › Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala
Immunology JoVE (Open Access) Citable · DOI

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

DOI: 10.3791/53853-v
What you'll learn
  • ✓ Combine optogenetic viral transduction with freeze-fracture replica immunolabeling techniques
  • ✓ Quantify and map glutamate receptor expression at identified synapses
  • ✓ Analyze input-specific glutamate receptor distribution in amygdala circuits
  • ✓ Interpret electron microscopy data of synaptic receptor organization
Protocol

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Difficulty
advanced
Total time
~3–4 days per sample (including high-pressure freezing, replica preparation, immunolabeling, and EM imaging)
Model organism
Mouse (amygdala tissue, strain not specified)
Biosafety
BSL-1

Steps

1
Prepare and dissect mouse amygdala tissue

Extract fresh amygdala tissue from optogenetically transduced mice and prepare specimens for cryopreservation. Ensure rapid handling to preserve synaptic ultrastructure.

▶ 01:30
2
Apply high-pressure freezing to specimens

Use high-pressure freezing equipment to rapidly cryopreserve amygdala tissue, preventing ice crystal formation and preserving fine subcellular detail.

▶ 02:22
3
Prepare electron beam gun apparatus

Set up and calibrate the electron beam gun used in the freeze-fracture replication system prior to sample processing.

▶ 03:49
4
Perform freeze-fracture and metal replication

Fracture frozen tissue under vacuum and deposit platinum and carbon onto exposed membrane faces to create electron-dense replicas for imaging.

▶ 04:42
5
Conduct immunolabeling and image analysis

Apply primary and secondary antibodies against AMPA and NMDA receptors on replicas, then acquire and quantify electron microscopy images of labeled synapses.

▶ 06:26
6
Analyze receptor distribution at identified synapses

Quantify AMPA-R and NMDA-R immunolabeling patterns at functionally identified PIN/MGn-ITC synapses to determine input-specific glutamate receptor organization.

▶ 07:18
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