Home › Cell Biology › An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System
Cell Biology JoVE (Open Access) Citable · DOI

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

DOI: 10.3791/52408-v
What you'll learn
  • ✓ Generate co-cultures combining iPSC-derived neurons, primary cortical neurons, and astrocytes
  • ✓ Apply optogenetics to detect synaptic connectivity between neuronal populations
  • ✓ Perform electrophysiological recordings to assess neuronal circuit formation
Protocol

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Difficulty
advanced
Total time
~5–7 days (including differentiation and maturation phases)
Model organism
Human iPSC-derived neurons, mouse primary cortical neurons, mouse astrocytes
Biosafety
BSL-1

Steps

1
Dissociate cortical tissue into single-cell suspension

Extract and enzymatically dissociate mouse cortical tissue to obtain primary neurons for culture. This step yields viable single cells ready for downstream processing.

▶ 02:12
2
Electroporate primary neurons with channelrhodopsin-2

Introduce channelrhodopsin-2 expression into primary cortical neurons via electroporation and plate cells on prepared culture substrates.

▶ 03:52
3
Differentiate human neural progenitor cells in co-culture

Culture iPSC-derived neural progenitor cells alongside primary cortical neurons and astrocytes to allow synaptic integration and circuit formation.

▶ 05:14
4
Record electrophysiology from iPSC-derived neurons

Perform patch-clamp or whole-cell recordings from iPSC-derived neurons to measure light-evoked responses and assess connectivity with channelrhodopsin-2-expressing primary neurons.

▶ 06:55
5
Analyze co-culture synaptic connectivity results

Evaluate electrophysiological data to confirm formation of functional synaptic contacts between iPSC-derived and primary cortical neuronal populations.

▶ 09:05
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