This study was supported by the National Natural Science Foundation of China (81571770, 81771925, 81861128001).

In this protocol, male C57Bl/6 mice were obtained from the Institute of Laboratory Animals of Sichuan Academy of Medical Sciences and Sichuan Provincial People&#39;s Hospital. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee, University of Electronic Science and Technology of China.
1. Monocular deprivation (MD) at postnatal day 28 in mice
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	<li>Put the surgical tools, the suture needle (0.25 mm diameter, string diameter 0.07 mm...

<ol>
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The authors declare that they have no competing financial interests.

We present a detailed protocol for MD and measuring OD plasticity by single unit recording. This protocol is widely used in visual neuroscience. Although the MD protocol is not complicated, there are some critical surgical procedures that must be followed carefully. First, there are two important details ensuring the quality of the stitching. The suture is sufficiently stable if the stitches are concentrated in the medial portion of the eyelid. Moreover, 3 &#956;L of glue is applied to the head of the knot to increase th...

Monocular visual deprivation is an excellent experimental paradigm to examine V1 plasticity. To study the importance of visual experience in neural development, David Hubel and Torsten Wiesel1,2 deprived kittens of normal vision in one eye at various time points and for varying periods of time. They then observed the changes in response intensity in V1 for the deprived and nondeprived eyes. Their results showed an abnormally low number of neurons reacting to the eye that had been sutured shut in the first three months. However, the responses from the neurons in the kittens remained identical in all respects to those of a normal adult cat&#39;s eye that was sutured shut for a year, and the kittens did not recover. MD in adult cats cannot induce OD plasticity. Therefore, the impact of visual experience on V1 wiring is strong during a brief, well-defined phase of development, before and after which the same stimuli have less influence. Such a phase of increased susceptibility to visual input is known as the critical period in visual cortex.
Although the mouse is a nocturnal animal, individual neurons in mouse V1 have similar properties to neurons found in cats3,4,5. In recent years, with the rapid development of transgenic technology, an increasing number of studies in visual neuroscience have used mice as an experimental model6,7,8. In mouse visual studies, neuroscientists use mutants and knockout mouse lines, which allow control over the genetic makeup of the mice. Although mice V1 lack OD columns, single neurons in the V1 binocular zone show significant OD properties. For example, most cells respond more strongly to contralateral stimulation than to ipsilateral stimulation. Temporary closure of one eye during the critical period induces a significant shift in the OD index distribution9,10,11. Therefore, MD can be used to establish an OD plasticity model to investigate how genes involved in neural developmental disorders influence cortical plasticity in vivo.
Here, we introduce an experimental method for MD and suggest a commonly used method (electrophysiological recording) to analyze the change in OD plasticity during monocular visual deprivation. The method has been widely used in many laboratories for more than 20 years12,13,14,15,16. There are other methods used in measuring the OD plasticity as well, such as chronic visual evoked potential (VEP) recording17, and intrinsic optical imaging (IOI)18. The significant advantage of this acute method is that it is easy to follow, and the results are remarkably reliable.

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monocular visual deprivation and ocular dominance plasticity measurement in the mouse primary visual cortex

Monocular visual deprivation is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the cortex to the contralateral eye to a stimulus is much stronger than the response of the ipsilateral eye in the binocular segment of the mouse primary visual cortex (V1). During the mammalian critical period, suturing the contralateral eye will result in a rapid loss of responsiveness of V1 cells to contralateral eye stimulation. With the continuing development of transgenic technologies, more and more studies are using transgenic mice as experimental models to examine the effects of specific genes on ocular dominance (OD) plasticity. In this study, we introduce detailed protocols for monocular visual deprivation and calculate the change in OD plasticity in mouse V1. After monocular deprivation (MD) for 4 days during the critical period, the orientation tuning curves of each neuron are measured, and the tuning curves of layer four neurons in V1 are compared between stimulation of the ipsilateral and contralateral eyes. The contralateral bias index (CBI) can be calculated using each cell&#39;s ocular OD score to indicate the degree of OD plasticity. This experimental technique is important for studying the neural mechanisms of OD plasticity during the critical period and for surveying the roles of specific genes in neural development. The major limitation is that the acute study cannot investigate the change in neural plasticity of the same mouse at a different time.

The experimental results described here enable successful MD and OD plasticity measurements from a deprived and nondeprived mouse during the critical period (P19&#8211;P32). Figure 1 shows how to perform single unit recordings in layer 4 from V1 the binocular zone for comparing responses in the ipsilateral and contralateral eye 4 days after MD. Figure 2 shows the spike sorting and orientation tuning measurements for stimulating the ipsilateral and contralateral ...

Watch this Scientific Journal Video about Monocular Visual Deprivation and Ocular Dominance Plasticity Measurement in the Mouse Primary Visual Cortex at JoVE.com

Monocular Visual Deprivation and Ocular Dominance Plasticity Measurement in the Mouse Primary Visual Cortex

Here, we present detailed protocols for monocular visual deprivation and ocular dominance plasticity analysis, which are important methods for studying the neural mechanisms of visual plasticity during the critical period and the effects of specific genes on visual development.

Monocular visual deprivation is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the ...

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.Thanks for watching our video.

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Developmental Biology

9.3K vistas.  University of Electronic Science and Technology of China. La privación visual monocular es un excelente protocolo experimental para inducir plasticidad de respuesta cortical visual primaria. Normalmente, las neuronas en el segmento binocular de la corteza visual primaria en el ratón responden aproximadamente dos veces más fuertemente a la estimulación del ojo contralateral que al ojo ipsilateral. Sin embargo, la sutura de cerrar el ojo contralateral durante el período crítico de plasticidad de dominancia ocular resulta en la rápida pérdida d...