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 immersion objective in a two-photon imaging microscope. Insert the filter cube for fret imaging that has a dichroic mirror at 505 nanometers and bandpass filters that span 460 nanometers to 500 nanometers for measuring ECFP and 520 nanometers to 560 nanometers for measuring Citron.

Then add ACSF to the well, containing the thinned skull window, and lower the 10x water immersion objective into the ACSF. Use the eyepiece, in conjunction with mercury lamp and GFP filter cube to locate the cNIFERs. Now, switch to the 40x water immersion objective.

Next, select the appropriate light path for two-photon imaging. Turn on the near-infrared femtosecond pulsed laser. Select the wavelength of 820 nanometers and a power setting of 5 to 15%. Set the PMT1 and PMT2 voltage to a sub-maximal value, typically 500 to 1000 volts, depending on the PMT.

Then set the gain to 1 for each channel and 0 the Z position for the objective. Lower the objective approximately 100 to 200 micrometers from the cortical surface, and start the x, y scan. Adjust the laser power gain and PMT voltage for each channel to optimize the signal to noise ratio of the cNIFERs fluorescence.

Next, use the software to restrict the imaging to a region that contains the cNIFERs cells as well as a background region. Select the Kalman line averaging for two for a suitable signal-to-noise ratio, and use a scan rate of 0.3 to 1 hertz at 4 microseconds per pixel. After that, draw an ROI around the cNIFER cells, surrounding about three to four cells per plane.

Set up real-time analysis of ROI average intensities. Then start acquisition to monitor the cNIFER fluorescence over time, and begin electrical stimulation or a behavioral experiment while monitoring fret.