Name: monocular visual deprivation and ocular dominance plasticity measurement in the mouse primary visual cortex
Uploaded: 2020-02-08T15:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Monocular Visual Deprivation and Ocular Dominance Plasticity Measurement in the Mouse Primary Visual Cortex at JoVE.com

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
<ol>
	<li>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 &#176;C for 0.5 h. Sterilize the hood with 75% ethanol. Dry the surgical tools in a drying oven.</li>
	<li>Prepare a 2% agarose solution, put it in a water bath at 75 &#176;C to avoid solidifying.</li>
	<li>Use isoflurane mixed with oxygen to anesthetize the mouse (2% induction and 1.2&#8211;1.5% maintenance). Fix the mouse on the stereotaxic apparatus and use a heat regulating device to maintain the mouse body temperature at 37 &#176;C and prevent hypothermia.</li>
	<li>Apply a thin layer of petroleum-based eye ointment to both eyes.</li>
	<li>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.</li>
	<li>Knot the thread 2&#8211;3x and then trim the thread. Apply 3 &#956;L of instant drying glue on the knot to increase its stability. Then cut the extra suturing thread.</li>
	<li>Provide an intraperitoneal injection of buprenorphine (1 mg/kg) to the mouse.</li>
	<li>Transfer the mouse onto a heating pad to maintain its body temperature at 37 &#176;C and prevent hypothermia and monitor it until it regains consciousness.</li>
	<li>When the mouse is fully awake place it into a separate holding cage.</li>
	<li>Check the eyelids daily to ensure that they remain shut and uninfected. Exclude the mouse if an eyelid opening is found.</li>
	<li>Before electrophysiological recording, use isoflurane mixed with oxygen to anesthetize the mouse (2% induction and 1.2&#8211;1.5% maintenance).</li>
	<li>Remove the stitches with eye scissors to expose the eyeball. Carefully trim the eye lids.</li>
	<li>Flush the eye with lens solution and check the eye under a microscope for clarity. Exclude mice with corneal opacities or signs of infection.</li>
</ol>
2. Craniotomy in the mouse V1 binocular region after monocular deprivation on the 4th day
<ol>
	<li>After anesthetizing the mouse, check for the depth of anesthesia by the lack of response to a toe pinch.</li>
	<li>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.</li>
	<li>Use a heating pad to maintain the body temperature.</li>
	<li>Apply a petroleum-based eye ointment on the surface of the eyes to keep them moist.</li>
	<li>Remove the hair on the mouse&#39;s head to expose its skin. Rub the skin with alternating scrubs of iodine and 70% ethanol 3x.</li>
	<li>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.</li>
	<li>Drill a 1 x 1 mm hole in the skull above the cerebellum. Affix a small bone screw in the hole as a reference.</li>
	<li>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&#8211;lambda +1.67 mm; M-L: -2.6&#8211; -3.0 mm; D-V: 0&#8211;1 mm). Carefully remove the skull fragment without hurting the brain.</li>
	<li>Cover the exposed cortical surface with 75 &#956;L of 2% agarose at 40 &#176;C to prevent drying.</li>
	<li>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.</li>
	<li>Use cotton swabs to remove the eye gel and apply silicon oil to the eye every 2 h.</li>
</ol>
3. Visual stimulation and electrophysiological recording
<ol>
	<li>Mask the one eye with nontransparent plastic plate. Position an LCD monitor 23 cm from the mouse&#39;s eye.</li>
	<li>Reduce the anesthesia to 0.5&#8211;0.8% when the mouse is fully anesthetized.</li>
	<li>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&#8211;450 &#956;m in depth). Ensure that the amplification factor is set at 1,000, the filter at 300&#8211;100 Hz, and the sample rate at 40 Hz.</li>
	<li>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.</li>
	<li>Measure the cell&#39;s response by stimulating the ipsilateral and contralateral eye separately. Present 3&#8211;5x total.</li>
	<li>Measure the responses of five to eight cells in each penetration. Perform four to six penetrations in each mouse.</li>
	<li>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 &#956;m apart in the V1 binocular zone.</li>
</ol>
4. Off-line spike sorting and data analysis
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Use principal component analysis to separate them into clusters. Clustering methods can vary between different laboratories.</li>
	<li>Classify the spike of a boundary by using the K-means algorithm.</li>
	<li>Correlate the orientation with the spike firing rate and plot the orientation tuning curves for the ipsilateral and contralateral eye.</li>
	<li>Calculate the OD index for the single unit, which represents the contralateral/ipsilateral response strength ratio: 
	<img alt="Equation 1" src="/files/ftp_upload/60600/60600eq1a.jpg" /> 
	where Rcontra and Ripsi are the cell&#39;s optimal response for the contralateral and ipsilateral eye, respectively, and Rspon is the cell&#39;s spontaneous activity.</li>
	<li>Assign OD scores to 1&#8211;7 as follows: &#8722; 1 to &#8722;0.75 = 1; &#8722;0.75 to &#8722;0.45 = 2; &#8722;0.45 to &#8722;0.15 = 3; &#8722;0.15 to 0.15 = 4; 0.15 to 0.45 = 5; 0.45 to 0.75 = 6; and 0.75 to 1 = 7.</li>
	<li>Calculate the contralateral bias index (CBI): 
	<img alt="Equation 2" src="/files/ftp_upload/60600/60600eq2a.jpg" /> 
	where N is the cell number, and nx equals the cell number with OD scores equal to x.</li>
</ol>

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E., Gordon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+period+for+the+monocular+deprivation+effect+in+rats:+assessment+with+sweep+visually+evoked+potentials.">Critical period for the monocular deprivation effect in rats: assessment with sweep visually evoked potentials.</a> Journal of Neurophysiology. 81 (1), 121-128 (1999).</li><li>Wang, L., Sarnaik, R., Rangarajan, K. V., Liu, X., Cang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+receptive+field+properties+of+neurons+in+the+superficial+superior+colliculus+of+the+mouse.">Visual receptive field properties of neurons in the superficial superior colliculus of the mouse.</a> Journal of Neuroscience. 30 (49), 16573-16584 (2010).</li><li>Niell, C. 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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 ...

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Developmental Biology

9.8K Views.  University of Electronic Science and Technology of China. Monoculaire visuele ontbering is een uitstekend experimenteel protocol om primaire visuele corticale respons plasticiteit induceren. Normaal gesproken reageren neuronen in het verrekijkersegment van de primaire visuele cortex in de muis ongeveer twee keer zo sterk op de stimulatie van het contralaterale oog als op het ipsilaterale oog. Echter, hechten sloot het contralaterale oog tijdens de kritieke periode voor oculaire dominantie plasticiteit resulteren in het snelle verlies van responsivit...

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

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

Neuroscience

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Medicine

Using Looming Visual Stimuli to Evaluate Mouse Vision

The visual system in the central nervous system processes diverse visual signals. Although the overall structure has been characterized from the retina through the lateral geniculate nucleus to the visual cortex, the system is complex. Cellular and molecular studies have been conducted to elucidate the mechanisms underpinning visual processing and, by extension, disease mechanisms. These studies may contribute to the development of artificial visual systems. To validate the results of these studies, behavioral vision testing is necessary. Here, we show that the looming stimulation experiment is a reliable mouse vision test that requires a relatively simple setup. The looming experiment was conducted in a large enclosure with a shelter in one corner and a computer monitor located on the ceiling. A CCD camera positioned next to the computer monitor served to observe mouse behavior. A mouse was placed in the enclosure for 10 minutes and allowed to acclimate to and explore the surroundings. Then, the monitor projected a program-derived looming stimulus 10 times. The mouse responded to the stimuli either by freezing or by fleeing to the hiding place. The mouse&#39;s behavior before and after the looming stimuli was recorded, and the video was analyzed using motion tracking software. The velocity of the mouse movement significantly changed after the looming stimuli. In contrast, no reaction was observed in blind mice. Our results demonstrate that the simple looming experiment is a reliable test of mouse vision.

To examine mouse vision, we conducted a looming test. Mice were placed in a large square arena with a monitor on its ceiling. The looming visual stimulus consistently evoked freezing or flight reactions in the mice.

The visual system in the central nervous system processes diverse visual signals. Although the overall structure has been characterized from the retina ...

Behavior

Assessing Binocular Central Visual Field and Binocular Eye Movements in a Dichoptic Viewing Condition

Macular degeneration typically results in heterogeneous binocular central visual defects. Currently available approaches to assess central visual field, like the microperimetry, can test only one eye at a time. Therefore, they cannot explain how the defects in each eye affect the binocular interaction and real-world function. Dichoptic stimulus presentation with a gaze-controlled system could provide a reliable measure of monocular/binocular visual fields. However, dichoptic stimulus presentation and simultaneous eye-tracking are challenging because optical devices of instruments that present stimulus dichoptically (e.g., haploscope) always interfere with eye-trackers (e.g., infrared video-based eye-trackers). Therefore, the goals were 1) to develop a method for dichoptic stimulus presentation with simultaneous eye-tracking, using 3D-shutter glasses and 3D-ready monitors, that is not affected by interference and 2) to use this method to develop a protocol for assessing central visual field in subjects with central vision loss. The results showed that this setup provides a practical solution for reliably measuring eye-movements in dichoptic viewing condition. In addition, it was also demonstrated that this method can assess gaze-controlled binocular central visual field in subjects with central vision loss.

Presented here is a protocol for assessing binocular eye movements and gaze-controlled central visual field screening in participants with central vision loss.

Macular degeneration typically results in heterogeneous binocular central visual defects. Currently available approaches to assess central visual field, ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the different aspects of visual, cross-modal and developmental plasticity along the mammalian visual system1-4.
Here, we demonstrate an elegant and straightforward technique for the removal of one or both eyes in the mouse, which is validated in mice of 20 days old up to adults. Briefly, a disinfected curved forceps is used to clamp the optic nerve behind the eye. Subsequently, circular movements are performed to constrict the optic nerve and remove the eyeball. The advantages of this technique are high reproducibility, minimal to no bleeding, rapid post-operative recovery and a very low learning threshold for the experimenter. Hence, a large amount of animals can be manipulated and processed with minimal amount of effort. The nature of the technique may induce slight damage to the retina during the procedure. This side effect makes this method less suitable as compared to Mahajan et al. (2011)5 if the goal is to collect and analyze retinal tissue. Also, our method is limited to post-eye opening ages (mouse: P10 - 13 onwards) since the eyeball needs to be displaced from the socket without removing the eyelids. The in vivo enucleation technique described in this manuscript has recently been successfully applied with minor modifications in rats and appears useful to study the afferent visual pathway of rodents in general.

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

The removal of eyes, also called enucleation, provides a useful strategy to study aspects of visual, cross-modal, and developmental plasticity along the mammalian visual system since it induces irreversible partial (monocular) or complete (binocular) vision loss. Here we describe a highly reproducible and straightforward approach to perform in vivo enucleation.

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the ...

Biology

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...

Evaluating Optokinetic Reflex Responses in Mice: A New High-Precision Protocol

Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize retinal images. Due to its importance and robustness, the OKR has been used to study visual-motor learning and to evaluate the visual functions of mice with different genetic backgrounds, ages, and drug treatments. Here, we introduce a procedure for evaluating OKR responses of head-fixed mice with high accuracy. Head fixation can rule out the contribution of vestibular stimulation on eye movements, making it possible to measure eye movements triggered only by visual motion. The OKR is elicited by a virtual drum system, in which a vertical grating presented on three computer monitors drifts horizontally in an oscillatory manner or unidirectionally at a constant velocity. With this virtual reality system, we can systematically change visual parameters like spatial frequency, temporal/oscillation frequency, contrast, luminance, and the direction of gratings, and quantify tuning curves of visual feature selectivity. High-speed infrared video-oculography ensures accurate measurement of the trajectory of eye movements. The eyes of individual mice are calibrated to provide opportunities to compare the OKRs between animals of different ages, genders, and genetic backgrounds. The quantitative power of this technique allows it to detect changes in the OKR when this behavior plastically adapts due to aging, sensory experience, or motor learning; thus, it makes this technique a valuable addition to the repertoire of tools used to investigate the plasticity of ocular behaviors.

Author Spotlight: An Accurate and Quantitative Approach to Study Visual Feature Selectivity of the Optokinetic Reflex in Mice 

Here, we describe a standard protocol for quantifying the optokinetic reflex. It combines virtual drum stimulation and video-oculography, and thus allows precise evaluation of the feature selectivity of the behavior and its adaptive plasticity.

The optokinetic reflex (OKR) is an essential innate eye movement that is triggered by the global motion of the visual environment and serves to stabilize ...

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

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