High resolution imaging of single synapses expressing a fast glutamate sensor allows a detection of local mismatch between transmitter release and uptake. In the case of disease, this method can be used to identify dysfunctional synapses. For autofluorescence correction, first, place a brain slice from the mouse of interest into the recording chamber of a one-photon microscope.
Submerge the slice in oxygenized, artificial cerebrospinal fluid and use the 20x water-immersion objective to locate the dorsal striatum. Fix the slices with a nylon grid on a platinum harp to minimize the tissue movement, and switch to the 63x water-immersion objective. Using a high pass filter at 510 nanometers, acquire an image of the autofluorescent and glutamate sensor positive structures together.
Using a high pass filter at 600 nanometers, acquire a second image of the autofluorescent structures alone. To define the range, use the mean intensities of the 10 brightest and 10 darkest pixels to scale the red and yellow images. Then, perform a subtraction of the yellow minus red image and rescale the subtracted image to generate a standard 8-bit TIFF file for convenient visualization of the bouton of interest.
To perform a search for responsive boutons, you need a suitable glass micropipette for electrical stimulation. Use a micropipette puller to produce stimulation pipettes from borosilicate glass capillaries with internal tip diameters of about one micrometer. To probe for action potential dependent release of glutamate from a set of candidate boutons, select a 63x magnification and a 510 nanometer emission filter.
Load the subtracted image to allow the placement of a glass stimulation electrode next to a fluorescent varicosity, avoiding the proximity of additional axons. In what varicosities associated with a maximum bifurcation or allocation within deeper part of the slice. Place the stimulation electrode near the bouton of interest and turn off the light, as the recordings must be performed in complete darkness.
Then, turn on the multi-channel bath application system, with one channel delivering the standard bath solution and the other channels delivering the necessary blockers of the ion channels, transporters, or membrane receptors, including tetrototoxin to block action potential generation. Control the flow at the site of recording and turn on the stimulator to deliver depolarizing current pulses of two to 10 microamps to the stimulation pipette. The release is now activated by direct calcium influx through voltage gated channels.
To visualize glutamate release in clearance, using the microscope X, Y drives, place the tested glutamate sensor positive bouton close to the viewfield center. After stopping the acquisition, click on the image with the left mouse button to determine the X, Y position of the resting bouton center. The X, Y 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 zero, 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 one 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 three standard deviations, and determine a virtual diameter in microns assuming a circular form of the supra 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, TauD. 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.
Single synapse imaging can be used to identify two classes of corticostriatal synapses using the size and paired pulse ratio criteria. At stimulus intervals of 20 to 50 milliseconds, the smaller interenterochephalic terminals are prone to paired pulse depression, while the larger pyramidal tract terminals showed paired pulse facilitation. Tests on motor behavior performed on wild-type mice and mice expressing a Huntington phenotype, reveal a significant positive coorelation between the results obtained for the total path run in the open field, and the step over latency.
Further, single synapse glutamate imaging shows that symptomatic Huntington mice exhibited a deficit in the speed of juxtasynpatic glutamate decay as reflected in the TauD values of the glutamate responses to single synapse stimulation. In wild-type animals, such prolongation was only observed after the application of a selective, non-transportable inhibitor of glutamate uptake. Evaluation of the probability of occurrence of a given TauD value in slices from wild-type mice, and mice expressing the symptoms of Huntington's disease, revealed that in wild-type animals, TauD never exceeds 15 milliseconds.
In symptomatic Huntington's disease however, 40%of the synapses exhibit TauD values between 16 and 58 milliseconds, despite a tendency for a reduction in the amount of released glutamate. Therefore, TauD might be regarded as a biomarker for dysfunctional synapses in Huntington's disease, and may further be used to verify functional recovery in experiments targeting astrocytic glutamate transport. This protocol for glutamate monitoring at individual corticostriatal synapses may help to clarify the role of glutamate uptake deficiency in the pathogenesis of neurodegenerative disease.
Single synapse imaging is particularly useful for exploring pre-synaptical sites of excitatory synapses.
