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

Summary

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.

Abstract

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.

Introduction

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 Wiesel^1,2 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 cats^3,4,5. In recent years, with the rapid development of transgenic technology, an increasing number of studies in visual neuroscience have used mice as an experimental model^6,7,8. 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 distribution^9,10,11. 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 years^{12,13,14,15,16}. 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.

Protocol

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

1. 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.
2. Prepare a 2% agarose solution, put it in a water bath at 75 °C to avoid solidifying.
3. 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.
4. Apply a thin layer of petroleum-based eye ointment to both eyes.
5. 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.
6. 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.
7. Provide an intraperitoneal injection of buprenorphine (1 mg/kg) to the mouse.
8. 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.
9. When the mouse is fully awake place it into a separate holding cage.
10. Check the eyelids daily to ensure that they remain shut and uninfected. Exclude the mouse if an eyelid opening is found.
11. Before electrophysiological recording, use isoflurane mixed with oxygen to anesthetize the mouse (2% induction and 1.2–1.5% maintenance).
12. Remove the stitches with eye scissors to expose the eyeball. Carefully trim the eye lids.
13. 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

1. After anesthetizing the mouse, check for the depth of anesthesia by the lack of response to a toe pinch.
2. 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.
3. Use a heating pad to maintain the body temperature.
4. Apply a petroleum-based eye ointment on the surface of the eyes to keep them moist.
5. Remove the hair on the mouse's head to expose its skin. Rub the skin with alternating scrubs of iodine and 70% ethanol 3x.
6. 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.
7. Drill a 1 x 1 mm hole in the skull above the cerebellum. Affix a small bone screw in the hole as a reference.
8. 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.
9. Cover the exposed cortical surface with 75 μL of 2% agarose at 40 °C to prevent drying.
10. 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.
11. Use cotton swabs to remove the eye gel and apply silicon oil to the eye every 2 h.

3. Visual stimulation and electrophysiological recording

1. Mask the one eye with nontransparent plastic plate. Position an LCD monitor 23 cm from the mouse's eye.
2. Reduce the anesthesia to 0.5–0.8% when the mouse is fully anesthetized.
3. 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.
4. 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.
5. Measure the cell's response by stimulating the ipsilateral and contralateral eye separately. Present 3–5x total.
6. Measure the responses of five to eight cells in each penetration. Perform four to six penetrations in each mouse.
7. 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

1. 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.
2. 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.
3. Use principal component analysis to separate them into clusters. Clustering methods can vary between different laboratories.
4. Classify the spike of a boundary by using the K-means algorithm.
5. Correlate the orientation with the spike firing rate and plot the orientation tuning curves for the ipsilateral and contralateral eye.
6. Calculate the OD index for the single unit, which represents the contralateral/ipsilateral response strength ratio:
   
   where R_contra and R_ipsi are the cell's optimal response for the contralateral and ipsilateral eye, respectively, and R_spon is the cell's spontaneous activity.
7. 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.
8. Calculate the contralateral bias index (CBI):
   
   where N is the cell number, and n_x equals the cell number with OD scores equal to x.

Results

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. ...

Discussion

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 th...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
502 glue	M&G Chenguang Stationery Co., Ltd.	AWG97028
Acquizition card	National Instument	PCI-6250
Agarose	Biowest	G-10
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
Microscope	SOPTOP	SZMT1
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
Xylocaine	Huaqing