6,124 Views
•
08:27 min
•
March 11, 2020
DOI:
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.
We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.
08:38
Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals
Related Videos
15495 Views
16:38
Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices
Related Videos
27244 Views
N/A
Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus
Related Videos
1031 Views
10:29
Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons
Related Videos
13989 Views
10:53
Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method
Related Videos
45959 Views
10:46
Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models
Related Videos
11235 Views
07:44
Evaluation of Synapse Density in Hippocampal Rodent Brain Slices
Related Videos
16900 Views
08:58
Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice
Related Videos
7418 Views
07:56
A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices
Related Videos
3197 Views
01:25
Microscopic Analysis of Synapses in Mouse Hippocampal Slices Using Immunofluorescence
Related Videos
31 Views
Read Article
Cite this Article
Dvorzhak, A., Grantyn, R. Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice. J. Vis. Exp. (157), e60113, doi:10.3791/60113 (2020).
Copy