This article describes a method for visualizing glutamate release and clearance in cortical neurons using electrophysiology and fluorescence imaging techniques. The protocol involves stimulating presynaptic neurons and recording fluorescence changes to assess synaptic activity.
Begin with an electrophysiology setup containing a mouse forebrain slice submerged in an oxygenated aCSF.
aCSF contains an ion-channel blocker that selectively inhibits the sodium-ion channels, preventing sodium-induced neuronal excitation.
The cortical neurons express fluorescent glutamate-detection sensor proteins.
Locate a single presynaptic neuron axonal bouton capable of releasing glutamate, an excitatory neurotransmitter, at the synapse, a junction where it connects with postsynaptic neurons.
Position a stimulation electrode near this bouton and apply depolarizing currents.
This excites the presynaptic neuron, causing a calcium ion influx and facilitating glutamate release.
Start fluorescence intensity recording around the selected bouton.
The released glutamate interacts with the sensor proteins and receptors on post-synaptic neurons.
Meanwhile, illuminate the selected area with laser light, causing the glutamate-bound sensor proteins to fluoresce; an increasing fluorescence intensity indicates glutamate release.
Over time, glutamate dissociates from the sensor proteins and enters the astrocyte, causing a decrease in fluorescence intensity, indicating glutamate clearance.
To visualize glutamate release and clearance using the microscope XY drives, place the tested glutamate sensor-positive bouton close to the view field center. After stopping the acquisition, click on the image with the left mouse button to determine the XY position of the resting bouton center. The XY coordinates of the set cursor will be displayed.
Using the calibration data, calculate the coordinates of the site where the laser beam should be sent for the excitation of the glutamate sensor fluorescence, using the formulas as indicated. To create a one-point sequence in the laser control software, select point in the Add to Sequence box on the Sequence page of the laser control software and set the runs and run delay to 0 and the sequence to run at TLL. Then, click Start Sequence.
In the camera control software, select the appropriate imaging parameters, and select external Start for the trigger mode. Click Take Signal in the camera control software. Then, initiate the experimental protocol laid down for the trigger device, and implement the experimental protocol trial with the appropriate timeline so that the camera will acquire 400 frames with a 2.48 kilohertz frequency during 1 trial with a 0.1 hertz or lower repetition frequency.
To identify pathological synapses, turn on the elevation routines and calculate the fluorescence intensity mean and standard deviation for the selected region of interest at rest. Determine and box the area occupied by pixels with a fluorescence intensity at rest greater than the mean plus 3 standard deviations, and determine a virtual diameter in microns, assuming a circular form of the super threshold area.
Plot the fluorescence intensity against time as the difference between the actual fluorescence intensity value and the fluorescence intensity value at rest divided by the fluorescence intensity value at rest. Determine the peak amplitude of the fluorescence response. Perform a monoexponential fitting for the decay from the peak of the fluorescence response, and determine the time constant of decay, tau d.
To estimate the maximal amplitude at a given synapse, select the pixel with the highest change in fluorescence intensity, which is the best indicator of the glutamate load presented to the clearance machinery of the synapse.
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