✓Image presynaptic dopamine release in real time using two-photon microscopy
✓Measure dopamine destaining kinetics to quantify neurotransmitter dynamics at individual terminals
Protocol
A new means to measure neurotransmission optically using fluorescent dopamine analogs.
Difficulty
advanced
Total time
~3–4 hours per animal (slice preparation, loading, imaging, and destaining)
Model organism
Mouse (striatum)
Biosafety
BSL-1
Steps
1
Understand optical dopamine imaging principles
Learn the basis for using fluorescent neurotransmitter analogs (FFNs) to visualize dopamine release at presynaptic terminals. Understand how FFN511 enables real-time optical measurement of neurotransmission.
▶ 01:15
2
Prepare acute striatal slices and load FFN511
Dissect striatal tissue, prepare acute brain slices, and incubate with fluorescent dopamine analog FFN511 to label dopaminergic terminals.
▶ 01:41
3
Image FFN511 fluorescence in brain slices
Use two-photon microscopy to visualize FFN511-labeled dopamine terminals and record baseline and stimulus-evoked fluorescence signals.
▶ 03:59
4
Monitor FFN511 destaining and dopamine clearance
Apply electrical or pharmacological stimulation and measure the rate of FFN511 fluorescence loss to quantify dopamine release and reuptake kinetics.
▶ 05:43
5
Analyze FFN511 loading and destaining results
Evaluate images and fluorescence time courses to confirm dopamine labeling, assess release dynamics, and validate signal specificity at individual presynaptic terminals.
▶ 07:03
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