Glutamate indicators with increased sensitivity and tailored deactivation rates

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• Understanding neuronal input-output operations necessitates simultaneous measurement of synaptic transmission across numerous neuronal inputs in vivo.
• Existing tools face limitations in sensitivity, speed, and scale for comprehensive synaptic analysis.

Purpose of the Study:

• To engineer and characterize novel fluorescent protein-based glutamate indicators for high-resolution synaptic transmission monitoring.
• To develop variants optimized for either large-scale population recordings or rapid synaptic dynamics.

Main Methods:

• Screened 3365 variants of the glutamate indicator iGluSnFR3 in neuron culture.
• Selected and validated two high-performance variants (iGluSnFR4s and iGluSnFR4f) in mouse visual cortex.
• Utilized imaging of action-potential evoked signals on axons and visually-evoked signals on dendritic spines.

Main Results:

• Developed iGluSnFR4s (153 ms deactivation) for large-scale recordings and iGluSnFR4f (26 ms deactivation) for rapid dynamics.
• Demonstrated single-vesicle sensitivity in detecting local synaptic glutamate release.
• Successfully recorded naturalistic synaptic transmission in mouse vibrissal cortex and hippocampal CA1 dendrites.

Conclusions:

• iGluSnFR4s and iGluSnFR4f significantly enhance the sensitivity and scale or speed of tracking neural information flow in vivo.
• These indicators provide unprecedented capabilities for studying synaptic plasticity and neural circuit function.
• The developed indicators are valuable tools for advancing neuroscience research in intact brain preparations.