Monocular visual deprivation is an excellent experimental protocol to induce primary visual cortical response plasticity. Normally, neurons in the binocular segment of the primary visual cortex in the mouse respond about twice as strongly to the stimulation of the contralateral eye as to the ipsilateral eye. However, suturing shut the contralateral eye during the critical period for ocular dominance plasticity result in the rapid loss of responsiveness by the V1 neurons to the contralateral eye stimulation and also followed by a subsequent increase in the response to the ipsilateral eye.
Here, we introduce the experimental protocol for monocular deprivation and to suggest a commonly used method to analyze the trend in ocular dominance during visual deprivation. Put the surgical tools in an aluminum box and autoclave them under 120 degrees Centigrade for half an hour. Prepare 2%agarose solution in water bath kettle and keep it at 75 degrees Centigrade.
Apply a thin layer of eye ointment to both eyes. Under the anatomical microscope with illumination, suture the eyelids. Make about four stitches.
Make two to three nodes of the thread. Apply three microliters of instant drying glue 502 on the head of the node to increase the stability of the node. Then cut the thread as short enough.
When the mouse are fully awake, place them into a separated holding cage. Before electrophysiological recording, use isoflurane to anesthetize the mouse. Remove the stitches and expose the eyeball.
Carefully trim the eyelid. Flush the eyes with contact lenses solutions and check the eyes for clarity. Only the mouse with good condition of the eyeballs can be used.
After anesthetizing the mouse, place the mouse on a stereotaxic frame. Use a heating pad to maintain the body temperature throughout the procedure. Apply the eye ointment on the surface of the eyes to keep it moist.
Remove the hair of the mouse to expose its skin. Incise an eight millimeters multiplied by eight millimeters area of skin between both ears to expose the skull and remove the scalp tissue. Then remove the overlying connective tissue with hydrogen peroxide.
Drill a hole in the skull above the cerebellum. Fix a small bone screw in the hole as the reference. Perform a small craniotomy of one millimeter in diameter in V1 binocular region.
Carefully remove the skull fragment without hurting the brain. Cover the exposed cortical surface with 2%agarose to prevent drying. Fix a tungsten electrode on the stereotaxic frame.
Place the tungsten electrode vertically on binocular V1 region to make sure that the cells that are recorded reacts to both eyes. The stimulus is separated by 30 degrees in a random block. A total of three to five blocks were presented in each measurement.
Mask one eye with nontransparent plastic plate. Present LCD1 to position in 23 centimeters from the mouse's eyes. Advance microelectrode slowly by the oil hydraulic micromanipulator.
Stop advancement high signal noise ratio signal when the electrode is advanced to layer four and start measurement. We amplify spike activities at a factor of 1, 000 and filter it from 300 to 10 kilohertz and then sample it at 40 kilohertz. Then mask the other eye and measure the contralateral eye's response.
Signals measured by single electrodes are population activities. Use software to separate spikes of different cells. First, filter the signal from 500 to 5, 000 hertz.
Set two cursors, one for positive deflection and one for negative deflection spikes. Set up spike templates. Set the templates area where it shows the greatest variation between different classes of spikes.
Use principal components analysis to separate them into clusters. Classify the spikes of a boundary by using the K means algorithm. Correlate the orientation with spike firing rate and plot the orientation tuning curves for the ipsilateral and contralateral eyes.
Then calculate the OD index for single cells which represents the contralateral and ipsilateral response Assign an OD score for each cell according to the OD index. Calculate the contralateral bias index according to the formula. This diagram show the orientation tuning curves of the ipsilateral and contralateral eye in monocular deprived and nondeprived mouse.
This protocol enables successful monocular visual deprivation and ocular dominance measurement from a deprived and a nondeprived mouse at critical period. We've just showed you how to accomplish this experiment and we had analyzed the change in ocular dominance during visual deprivation. During the experiment, it is important to avoid infection in the suturing process and interaction while electrophysiological recording.
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