implantation of a head bar on top of the mouse skull

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

In response to visual stimuli in the environment, our eyes move to shift our gaze, stabilize retinal images, track moving targets, or align the foveae of two eyes with targets located at different distances from the observer, which are vital to proper vision1,2. Oculomotor behaviors have been widely used as attractive models of sensorimotor integration to understand the neural circuits in health and disease, at least partly because of the simplicity of the oculomotor system3. Controlled by three pairs of extraocular muscles, the eye rotates in the socket primarily around three corresponding axes: elevation and depression along the transverse axis, adduction and abduction along the vertical axis, and intorsion and extorsion along the anteroposterior axis1,2. Such a simple system allows researchers to evaluate the oculomotor behaviors of mice easily and accurately in a lab environment.
One prime oculomotor behavior is the optokinetic reflex (OKR). This involuntary eye movement is triggered by slow drifts or slips of images on the retina and serves to stabilize retinal images as an animal&#39;s head or its surroundings move2,4. The OKR, as a behavioral paradigm, is interesting to researchers for several reasons. First, it can be stimulated reliably and quantified accurately5,6. Second, the procedures of quantifying this behavior are relatively simple and standardized and can be applied to evaluate the visual functions of a large cohort of animals7. Third, this innate behavior is highly plastic5,8,9. Its amplitude can be potentiated when repetitive retinal slips occur for a long time5,8,9, or when its working partner vestibular ocular reflex (VOR), another mechanism of stabilizing retinal images triggered by vestibular input2, is impaired5. These experimental paradigms of OKR potentiation empower researchers to unveil the circuit basis underlying oculomotor learning.
Two non-invasive methods have primarily been used to evaluate the OKR in previous studies: (1) video-oculography combined with a physical drum7,10,11,12,13 or (2) arbitrary determination of head turns combined with a virtual drum6,14,15,16. Although their applications have made fruitful discoveries in understanding the molecular and circuit mechanisms of oculomotor plasticity, these two methods each have some drawbacks which limit their powers in quantitatively examining the properties of the OKR. First, physical drums, with printed patterns of black and white stripes or dots, do not allow easy and quick changes of visual patterns, which largely restricts the measurement of the dependence of the OKR on certain visual features, such as spatial frequency, direction, and contrast of moving gratings8,17. Instead, tests of the selectivity of the OKR to these visual features can benefit from computerized visual stimulation, in which visual features can be conveniently modified from trial to trial. In this way, researchers can systematically examine the OKR behavior in the multi-dimensional visual parameter space. Moreover, the second method of the OKR assay reports only the thresholds of visual parameters that trigger discernible OKRs, but not the amplitudes of eye or head movements6, 14,15,16. The lack of quantitative power thus prevents analyzing the shape of tuning curves and the preferred visual features, or detecting subtle differences between individual mice in normal and pathological conditions. To overcome the above limitations, video-oculography and computerized virtual visual stimulation had been combined to assay the OKR behavior in recent studies5,17,18,19,20. However, these previously published studies did not provide enough technical details or step-by-step instructions, and consequently it is still challenging for researchers to establish such an OKR test for their own research.
Here, we present a protocol to precisely quantify the visual feature selectivity of OKR behavior under photopic or scotopic conditions with the combination of video-oculography and computerized virtual visual stimulation. Mice are head-fixed to avoid the eye movement evoked by vestibular stimulation. A high-speed camera is used to record the ocular movements from mice viewing moving gratings with changing visual parameters. The physical size of the eyeballs of individual mice is calibrated to ensure the accuracy of deriving the angle of eye movements21. This quantitative method allows comparing OKR behavior between animals of different ages or genetic backgrounds, or monitoring its change caused by pharmacological treatments or visual-motor learning.

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

Quantification of Visual Feature Selectivity of the Optokinetic Reflex in Mice

We're interested in the capacity of retinal system in health and disease mouse model. Currently, we are using optokinetic reflex or OKR insured, which is our involuntary eye movement that serves to stabilize retinal images to understand how the retinal system contributes to the adaptive plasticity in retinal innate behaviors. OKR model studies have advanced our understanding of innate behavior plasticity by demonstrating that prolonged retinal stimulation or impaired by similar ocular reflex can increase the behavior amplitude.And by investigating the underlying molecular synaptic and secretory mechanisms. We combined video oculargraphy and computerized virtual visual stimulation to precisely quantify the OKR behavior invokes by truncating with freely changing parameters. This procedure is relatively simple and can be standardized to accommodate large scale studies.Our protocol accurately analyzes to include shapes and prefer visual features, detects subtle differences in normal and pathological conditions, and also can monitor changes in OKR due to pharmacological treatments or visual motor learning. The high precision and the quantitative power make it possible to compare repetitive OKR measurements of the same eyes in longitudinal studies and their different pharmacological treatments or under neurosurpreterations. It also provides opportunities to study the neurosurpres and the circuit mechanisms of OKR plasticity.

Watch this Scientific Journal Video about Implantation of a Head Bar on Top of the Mouse Skull at JoVE.com

Implantation of a Head Bar on Top of the Mouse Skull  

After anesthetizing the mouse, place it on a heating pad to maintain its body temperature. Apply a layer of lubricant eye ointment to both eyes and cover them with aluminum foil to protect them from light illumination. Then inject carprofen subcutaneously at a dose of 20 milligrams per kilogram to reduce the pain.Wet the fur on top of the skull with 4%chlorhexidine gluconate and shave the area. Disinfect the exposed scalp twice using 70%isopropyl alcohol and chlorhexidine alcohol. Next, inject bupivacaine subcutaneously at the incision site.Then remove the scalp with scissors to expose the dorsal surface of the skull, including the posterior frontal bone, parietal bone and interparietal bone. Apply several drops of 1%lidocaine and one to 100, 000 epinephrine on the exposed skull to reduce local pain and bleeding. Then scrape the skull with a Meyhoefer curette to remove the fascia, and clean it with PBS.Gently blow compressed air onto the skull surface until the moisture is gone and the bone turns whitish. Then apply a thin layer of super glue to the exposed surface of the skull, including the edge of the cut scalp. Followed by a layer of acrylic resin.Now place a stainless-steel head bar along the midline on top of the skull. And apply more acrylic resin, starting from the edge of the head bar, until its base is completely embedded in the acrylic resin. Wait for about 15 minutes until the acrylic resin hardens.Subcutaneously inject one milliliter of lactated Ringer's solution. Then return the mouse to a cage placed on a heating pad until the animal is fully mobile.

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Research

JoVE Journal

Neuroscience

257 visualizações.  University of Toronto Mississauga. Depois de anestesiar o rato, coloque-o numa almofada de aquecimento para manter a temperatura corporal. Aplique uma camada de pomada lubrificante para os olhos em ambos os olhos e cubra-os com papel alumínio para protegê-los da iluminação da luz. Em seguida, injete carprofeno por via subcutânea em uma dose de 20 miligramas por quilograma para reduzir a dor.Molhe o pelo na parte superior do crânio com gluconato de clorexidina a 4% e raspe a área. Desinfetar o couro cabeludo expos...