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 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'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.
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'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.
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'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
- Put the surgical tools, the suture needle (0.25 mm diameter, string diameter 0.07 mm) and cotton swabs in an aluminum box and autoclave them at 120 °C for 0.5 h. Sterilize the hood with 75% ethanol. Dry the surgical tools in a drying oven.
- Prepare a 2% agarose solution, put it in a water bath at 75 °C to avoid solidifying.
- Use isoflurane mixed with oxygen to anesthetize the mouse (2% induction and 1.2–1.5% maintenance). Fix the mouse on the stereotaxic apparatus and use a heat regulating device to maintain the mouse body temperature at 37 °C and prevent hypothermia.
- Apply a thin layer of petroleum-based eye ointment to both eyes.
- Under the anatomical microscope with illumination, suture the eyelid on one eye. Make the needle pass though both sides of the eyelid 2x (Figure 1A) and make about four stitches.
- Knot the thread 2–3x and then trim the thread. Apply 3 μL of instant drying glue on the knot to increase its stability. Then cut the extra suturing thread.
- Provide an intraperitoneal injection of buprenorphine (1 mg/kg) to the mouse.
- Transfer the mouse onto a heating pad to maintain its body temperature at 37 °C and prevent hypothermia and monitor it until it regains consciousness.
- When the mouse is fully awake place it into a separate holding cage.
- Check the eyelids daily to ensure that they remain shut and uninfected. Exclude the mouse if an eyelid opening is found.
- Before electrophysiological recording, use isoflurane mixed with oxygen to anesthetize the mouse (2% induction and 1.2–1.5% maintenance).
- Remove the stitches with eye scissors to expose the eyeball. Carefully trim the eye lids.
- Flush the eye with lens solution and check the eye under a microscope for clarity. Exclude mice with corneal opacities or signs of infection.
2. Craniotomy in the mouse V1 binocular region after monocular deprivation on the 4th day
- After anesthetizing the mouse, check for the depth of anesthesia by the lack of response to a toe pinch.
- Place and fix the mouse on the stereotaxic apparatus. Adjust the height of the ear bar and tooth rod to keep the brain flat and stable.
- Use a heating pad to maintain the body temperature.
- Apply a petroleum-based eye ointment on the surface of the eyes to keep them moist.
- Remove the hair on the mouse's head to expose its skin. Rub the skin with alternating scrubs of iodine and 70% ethanol 3x.
- Incise an 8 x 8 mm area of the skin between the ears to expose the skull and remove the scalp tissue. Then remove the overlying connective tissue with 30% hydrogen peroxide.
- Drill a 1 x 1 mm hole in the skull above the cerebellum. Affix a small bone screw in the hole as a reference.
- Perform a small craniotomy of 1 mm in diameter in the V1 binocular region from the contralateral hemisphere to the deprived eye (Figure 1B, A-P: lambda -0.51–lambda +1.67 mm; M-L: -2.6– -3.0 mm; D-V: 0–1 mm). Carefully remove the skull fragment without hurting the brain.
- Cover the exposed cortical surface with 75 μL of 2% agarose at 40 °C to prevent drying.
- Fix a tungsten electrode on the stereotaxic frame. Place the tungsten electrode vertically on the surface of the exposed cortex, the binocular region of V1, to make sure that the cells that are recorded react to both eyes.
- Use cotton swabs to remove the eye gel and apply silicon oil to the eye every 2 h.
3. Visual stimulation and electrophysiological recording
- Mask the one eye with nontransparent plastic plate. Position an LCD monitor 23 cm from the mouse's eye.
- Reduce the anesthesia to 0.5–0.8% when the mouse is fully anesthetized.
- Advance the microelectrode electrode slowly with an oil hydraulic micromanipulator. Stop it when a high signal-to-noise ratio is observed and the electrode is advanced to layer 4 (Figure 1C, approximately 250–450 μm in depth). Ensure that the amplification factor is set at 1,000, the filter at 300–100 Hz, and the sample rate at 40 Hz.
- Present a full-field moving sinusoidal grating (Figure 1D, 12 directions, 100% contrast, 2 Hz of temporary frequency, 0.04 cycles per degree of spatial frequency) on the LED monitor.
- Measure the cell's response by stimulating the ipsilateral and contralateral eye separately. Present 3–5x total.
- Measure the responses of five to eight cells in each penetration. Perform four to six penetrations in each mouse.
- After the recording, adjust the isoflurane flow rate to 5% or greater, continue isoflurane exposure for 1 min, and then perform the cervical dislocation.
NOTE: Separate penetrations were spaced at least 200 μm apart in the V1 binocular zone.
4. Off-line spike sorting and data analysis
- Detect spikes when the raw signal crosses a threshold level. Align captured spikes on the first positive or negative peak. Use software to detect spikes from different cells.
- Set two cursors: one for positive and the other for negative deflection. Set the spike template (Figure 2A). Set the template area to that with the most significant variation between different classes of spikes.
- Use principal component analysis to separate them into clusters. Clustering methods can vary between different laboratories.
- Classify the spike of a boundary by using the K-means algorithm.
- Correlate the orientation with the spike firing rate and plot the orientation tuning curves for the ipsilateral and contralateral eye.
- Calculate the OD index for the single unit, which represents the contralateral/ipsilateral response strength ratio:
where Rcontra and Ripsi are the cell's optimal response for the contralateral and ipsilateral eye, respectively, and Rspon is the cell's spontaneous activity.
- Assign OD scores to 1–7 as follows: − 1 to −0.75 = 1; −0.75 to −0.45 = 2; −0.45 to −0.15 = 3; −0.15 to 0.15 = 4; 0.15 to 0.45 = 5; 0.45 to 0.75 = 6; and 0.75 to 1 = 7.
- Calculate the contralateral bias index (CBI):
where N is the cell number, and nx equals the cell number with OD scores equal to x.
The experimental results described here enable successful MD and OD plasticity measurements from a deprived and nondeprived mouse during the critical period (P19–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 eyes. For spike sorting, the spike template was established by clustering the principal component weights of the spikes. As an example in (Figure 2A,B), cell 01 and cell 02 were classified by spike sorting. The orientation tuning curves of single units were measured by stimulation of the ipsilateral and contralateral eyes. Figure 2C,D shows the orientation tuning curves of four sample cells, in which two are from the mouse that underwent MD and the others from the mouse that did not. Our results show that the firing rate was relatively close by stimulating the contralateral and ipsilateral eye in the mouse 4 days after MD was performed (Figure 2C). However, the firing rate obtained by stimulating the contralateral eye was much stronger than that obtained by stimulating the ipsilateral eye in the nondeprived mouse (Figure 2D). Figure 3A shows the distribution of OD scores for all units and the CBI index for a mouse that underwent MD (P28, 4 days after MD). Figure 3B shows the OD scores for all units and the CBI index from a nondeprived C57/BL6 mouse (P26, no MD). The CBI index is 0.38 for an MD mouse and 0.67 for another one without MD.
The results show that the response of the V1 neurons to the contralateral eye to a stimulus was much stronger than the response of the ipsilateral eye in the binocular segment of the nondeprived mouse. However, 4 days after MD in the critical period, the response of most neurons to stimulation to the contralateral eye was relatively close or weaker than the response to the ipsilateral eye. Therefore, the V1 neurons in critical periods have significant OD plasticity. MD alters the relative strength of the cell's response by stimulating the contralateral and ipsilateral eye.
Figure 1: Schematic of the visual deprivation experiment. (A) A schematic for suturing the eyelid. The needle passes through the eyelid 2x and then 2–3 knots are made. Images 1–4 show the position where the needle passes through the eyelid. (B) Recording schematic in an anesthetized, head-fixed mouse. An enlarged view of V1 is displayed in the gray circle, and the binocular zone is indicated with dark gray. The recording sites within the binocular zone are shown with small circles. (C) The coronal plane of the mouse brain and the recording sites are shown in the layer 4 of V1. (D) Illustrations of the visual stimulus of different orientations. Twelve different orientations were totally used in each measurement. Please click here to view a larger version of this figure.
Figure 2: Illustrations of data analysis procedure. (A) Spikes were sorted using a commercial software (see Table of Materials). The waveform in green shows the filtered signal (0.3–10 kHz). Two cells were sorted from the filtered data. (B) Example of the separation of spiking activity on a single microelectrode by spike sorting. The spike sorting method is a principal component of the analysis. (C) Orientation tunings from two cells to respond to the contralateral (red solid line) and ipsilateral (red dotted line) stimuli in a monocular-deprived mouse (cell 01 and cell 02). (D) Orientation tuning from two cells to respond to contralateral (blue solid line) and ipsilateral (blue dotted line) eye stimuli in a nondeprived mouse (cell 03 and cell 04). The error bar indicates the standard error of the mean (SEM) in each measurement. The black line indicates spontaneous activity. Please click here to view a larger version of this figure.
Figure 3: Shift in the OD index by MD. We recorded the cell's response from the binocular zone in the contralateral brain by stimulating the ipsilateral and contralateral eyes individually. We calculated and added the OD index for the single units. (A) Distribution of OD scores for 38 neurons recorded from a C57/BL6 mouse that underwent MD from P28–P32. (B) Distribution of OD scores for 38 neurons from a nondeprived C57/BL6 mouse. Please click here to view a larger version of this figure.
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 μL of glue is applied to the head of the knot to increase the stability of the knot to prevent eye reopening. Second, some key steps should be taken to improve wound healing and reduce discomfort. The suture method is very important for the protocol. Previous studies have proven that a simple continuous suturing pattern has the benefits of better wound healing and shorter suturing time19,20. The thread should be thin and stable to avoid causing a large wound and reduce discomfort. A suture needle with a diameter of approximately 0.25 mm is suitable for suturing and two to three knots are necessary.
There are also some key points that need to be paid attention to in the recording. Control of anesthetic concentration is an important factor in electrophysiological recordings. In anesthetized animals, the experiment is very easy to control, and the results are highly stable and reliable. Many previous studies used urethane as an anesthetic. However, it is difficult to use urethane to control the depth of anesthesia in mice. At lower levels, the mice are not fully anesthetized, and at higher levels, the mice are prone to death. Isoflurane is more appropriate as an anesthetic in a mouse study. While it is almost impossible to obtain good neural activity in the neocortex of mice that are receiving over 1% isofurane21, most V1 neurons have good visual evoked activity at lower levels of isoflurane. Thus, start with a higher concentration of isoflurane (1%) to anesthetize the mouse, and then reduce the isoflurane (0.5–0.8%) when the mice are completely anesthetized. Besides, alternating measurements from the deprived eye and the nondeprived eye can ensure the accuracy of the experimental results. It is not appropriate to measure one eye's response many times and then measure the other eye, because the electrode may move, and the intensity of the cell's response may change during long-term recordings. Furthermore, this protocol targets neurons in layer 4, which is the primary thalamo-recipient layer in V1. But in older mice, which do exhibit OD plasticity primarily mediated by open eye potentiation, it is better to record in layers 2 or 3, which retain plasticity beyond the critical period. Therefore, it is important to determine the cortical laminar in recording.
There are still some limitations in the methods. Calculating a relatively accurate CBI index requires 4–6 penetrations and more than 30 units because recording too few samples will lead to inaccurate results. But it is not easy to obtain more than 30 high-quality units from a single mouse. A better method is to use multiunit recording, which can provide enough units in a single measurement. In addition, VEP recording and IOI can also be used to measure OD plasticity17,18. Single unit recording involves the activity of individual neurons, while VEP involves recording the activity of the sum of neurons near the electrode. But single unit recording data yield no information on synchronization among neurons, while VEP amplitudes depend on temporal synchronization among neurons22. A reversal grating is often used for VEP measurement. The most commonly used reversal frequency is 3–4 Hz. However, the exact value is determined by the computer refresh rate when presenting the grating. OD plasticity is measured by comparing the average of VEP amplitudes evoked by the deprived and nondeprived eye. The IOI technique can effectively detect the blood oxygen level-dependent signal evoked by contralateral and ipsilateral stimulation. It could show the OD plasticity of a large area in V1.
In summary, single unit recording and IOI are suitable for acute anesthesia experiments. In the future, MD and OD plasticity measurements in conjunction will be widely used in the study of neural plasticity as a experimental method.
The authors declare that they have no competing financial interests.
This study was supported by the National Natural Science Foundation of China (81571770, 81771925, 81861128001).
|502 glue||M&G Chenguang Stationery Co., Ltd.||AWG97028|
|Acquizition card||National Instument||PCI-6250|
|Amplifier||A-M system||Model 1800|
|Atropine||Aladdin Bio-Chem Technology Co., Ltd||A135946-5|
|Brain Stereotaxic Apparatus||RWD Life Science Co.,Ltd||68001|
|Cohan-Vannas spring scissors||Fine Science Tools||15000-02|
|Contact Lenses Solutions||Beijing Dr. Lun Eye Care Products Co., Ltd.||GM17064|
|Cotton swabs||Henan Guangderun Medical Instruments Co.,Ltd|
|Fine needle holder||SuZhou Stronger Medical Instruments Co.,Ltd||CZQ1370|
|Forcep||66 Vision Tech Co., Ltd.||53320A|
|Forcep||66 Vision Tech Co., Ltd.||53072|
|Forcep||66 Vision Tech Co., Ltd.||#5|
|Heating pad||Stryker||TP 700 T|
|Illuminator||Motic China Group Co., Ltd.||MLC-150C|
|Isoflurane||RWD Life Science Co.,Ltd||R510-22|
|LCD monitor||Philips (China) Investment Co., Ltd.||39PHF3251/T3|
|Noninvasive Vital Signs Monitor||Mouseox|
|Oil hydraulic micromanipulator||NARISHIGE International Ltd.||PC-5N06022|
|Petrolatum Eye Gel||Dezhou Yile Disinfection Technology Co., Ltd.||17C801|
|Spike2||Cambridge Electronic Design, Cambridge, UK||Spike2 Version 9|
|Surgical scissors||66 Vision Tech Co., Ltd.||54010|
|Surgical scissors||66 Vision Tech Co., Ltd.||54002|
|Suture Needle||Ningbo Medical Co.,Ltd||3/8 arc 2.5*8|
|Tungsten Electrode||FHC, Inc||L504-01B|
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