Imaging Neurotransmitter Release Using Cell-Based Neurotransmitter Fluorescent Engineered Reporters

Take a mouse with a cranial window containing a thin skull layer to enable brain visualization.

The head movement is restricted using an attached head-bar.

The cortex is pre-injected with cell-based neurotransmitter fluorescent engineered reporters, or CNiFERs, which are reporter cells that allow real-time detection of neurotransmitter release.

The CNiFERs express a G-protein-coupled neurotransmitter receptor and a cytoplasmic calcium detector, which consists of a calcium-binding domain fused to donor and acceptor fluorophores.

Using a two-photon microscope, excite the detector to cause donor fluorescence emission, and enable CNiFER visualization.

Neuronal activity reaching the cortex causes neurotransmitter release.

These neurotransmitters bind to CNiFER receptors and activate signaling pathways that induce calcium release from the endoplasmic reticulum.

The increased cytoplasmic calcium binds to the detector, causing a conformational change that brings the fluorophores into proximity.

The donor transfers energy to the acceptor to stimulate acceptor fluorescence emission, indicating neurotransmitter release.

Place the imaging platform with the head restrained mouse under a 10x water immer