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Neuroscience JoVE (Open Access) Citable · DOI

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

DOI: 10.3791/50915-v
What you'll learn
  • ✓ Perform laser-scanning photostimulation to map forebrain circuit connectivity
  • ✓ Prepare acute brain slices and perform whole-cell recordings with optogenetic stimulation
  • ✓ Characterize functional topography and synaptic properties of neuronal populations
  • ✓ Visualize and verify channelrhodopsin expression in targeted projections
Protocol

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations in vitro.

Difficulty
advanced
Total time
~4–6 hours per animal (surgery, slice preparation, recording, and imaging)
Model organism
Mouse
Biosafety
BSL-1

Steps

1
Perform stereotaxic surgery and viral injection

Conduct stereotaxic surgery on mice to target forebrain regions and inject channelrhodopsin-expressing viral vectors into specific projection pathways for optogenetic manipulation.

▶ 01:29
2
Prepare acute brain slices for electrophysiology

Extract and slice mouse brain tissue containing transduced neurons to generate acute slices suitable for whole-cell patch-clamp recording.

▶ 01:52
3
Perform laser-scanning photostimulation and recording

Use focused laser scanning to photostimulate channelrhodopsin-expressing neurons while performing simultaneous whole-cell electrophysiological recordings to map circuit connectivity and measure synaptic responses.

▶ 02:37
4
Image fixed slices to verify expression

Fix recorded brain slices and perform fluorescence imaging to anatomically confirm channelrhodopsin expression in recorded neurons and targeted projections.

▶ 04:22
5
Analyze connectivity in V1-V2 and S1-VP circuits

Examine channelrhodopsin expression patterns and map functional connectivity between primary visual and somatosensory cortical regions and higher-order targets.

▶ 05:16
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