Fluorogenic Targeting of Voltage-Sensitive Dyes to Neurons.

Area of Science:

• Neuroscience
• Biochemistry
• Synthetic Chemistry

Background:

• Voltage-sensitive fluorescent dyes are crucial for imaging neuronal activity.
• Existing genetically encoded voltage reporters have limitations in sensitivity and speed.
• Targeting fluorescent probes to specific cell types remains a challenge.

Purpose of the Study:

• To develop a novel method for targeted voltage imaging in specific neurons.
• To enhance the sensitivity and speed of voltage-sensitive fluorescent dyes.
• To enable precise visualization of neuronal electrical activity.

Main Methods:

• Utilized enzyme-catalyzed reactions to activate voltage-sensitive fluorescent dyes (VoltageFluor) on cell surfaces.
• Chemically modified VoltageFluor with a cleavable ether group, requiring porcine liver esterase (PLE) for activation.
• Targeted PLE expression to specific cells, including mammalian neurons, using neuron-specific promoters.

Main Results:

• Demonstrated successful in vitro and in cell-based substrate validation of modified VoltageFluor with PLE.
• Achieved targeted staining of mammalian neurons, enabling clear detection of neuronal action potentials in single trials.
• Developed VoltageFluors targeted by esterase expression (VF-EXs) with VF-EX2 showing a nearly twofold higher signal-to-noise ratio than genetically encoded reporters.
• Successfully used VF-EX2 to investigate neuromodulatory effects of serotonin in cultured hippocampal neurons.

Conclusions:

• A combined approach of synthetic chemistry and biochemistry enables bright and fast voltage imaging in genetically defined neurons.
• This method overcomes limitations of existing voltage imaging techniques, offering improved sensitivity and specificity.
• The VF-EX system provides a powerful tool for interrogating neuronal function and neuromodulation.