OptoDrum - FAQ
Many researchers contact us with their questions about measuring vision with the OptoDrum. What diseases is it good for? How do you properly perfom the experiments? How do you interpret the data? We have collected many of these questions here as a valuable resource. Please contact us if you have other questions or need further clarification. We also welcome your sharing additional tips and tricks.
Frequently asked questions (FAQ)
Is it possible to perform optomotor tests under scotopic conditions?
Yes, Striatech offers the “ScotopicKit” as an optional item for the OptoDrum. It contains a set of filter foils (neutral density filters) which can be placed in front of the OptoDrum monitors. A single filter has an optical density of 1.2, i.e. it reduces the brightness by 1.2 log units (about 16-fold). With 4 filters in front of each monitor (ND4.8, 63.000-fold reduction), the conditions inside the OptoDrum are well in the scotopic regime.
What is the typical mouse number per group?
Commonly, in research papers, 6 mice per group is an accepted number. In pharmacological studies (drug development) the group size is usually larger, around a dozen animals.
Is it better to use square wave gratings or sine wave gratings to trigger OMR or OKR?
The OptoDrum allows using both. We like square wave gratings, as the contrast edges between bars should be a good driver of retinal responses. Some researchers prefer sine wave gratings. As long as you do all your experiments the same way and compare your control and treatment groups under the same conditions, it should not matter.
Left/right eye: In my studies I treat one eye, the other eye is the control. Can the two eyes be measured independently with the OptoDrum?
Yes, this is possible. Some background: If you would stimulate only the right eye with a moving stimulus, you would observe that the optomotor reflex is only triggered for stimulus motion toward the left, but not toward the right. The same, but opposite, would happen if you only stimulated the left eye. Of, course, in normal experiments, both eyes of the animal see the stimulus at the same time. Still, optomotor movement to the left is triggered because the stimulus is seen by the right eye, and vice versa. The reason for this is that in rodents, the responsible brain circuits, which include the accessory optic system, or AOS, are wired in a mono-lateral fashion.
In the OptoDrum, you can independently analyze the reflex in the two directions. So, yes, if you only treat one eye, and the other eye serves as control, you can draw independent conclusions for each eye by analyzing the optomotor behavior separately for stimulus motion in different directions
How do you handle mouse overactivity/inattention during optomotor measurements?
Measurement should be done preferably in the morning to avoid inattention. In case the animal falls asleep inside the OptoDrum, you can wake it up by making some noise (e.g. scratch the screen frames by using a pen).
The best way to avoid overactivity, in general, are good handling practices. Animals used to being handled in a calm way will not mind as much being placed in the novel OptoDrum arena. Also, animals are much calmer if they are used to this new experimental environment. It helps to place the animal on the platform without showing a stimulus the day before the experiment, even if it just for 2 minutes. Further, as in every behavioral experiment, it is helpful to avoid further sources of distraction, such as many people entering the room, loud conversation, door banging, etc.
How do you do you prevent the animals from jumping off the stage during experiments?
Animals usually do stay on the platform. There are certain factors that support this. First, if an animal is well handled, it is much calmer. Second, it also helps that the OptoDrum opens at the front, not at the top. Placing the animal on the platform from the front reduces stress and the animal can get used to the new environment more quickly.
Also, animals are much calmer if they are used to the environment of the OptoDrum. It helps to place the animal on the platform without showing a stimulus the day before the experiment, even if it just for 2 minutes. Animals with motor dysfunction may slide off the platform; for such animals we used a wire around the stage as a "railing". It is best to attach the wire somewhat flexibly with 3-4 stripes of translucent tape (Scotch Tape, Tesa Film), so that the wire gives way if the animals tries to use it as support for climbing down.
Are different cell types assessed in acuity measures vs. contrast sensitivity measures?
One has to be careful when interpreting the optomotor reflex. As a behavioral output, it can be influenced by any alteration along the reflex pathway. The absence of an optomotor reflex can be caused by damage to the retina, the optic nerve, the involved midbrain regions, or muscular defects. Interpretation of the optomotor results therefore usually builds on existing knowledge about the disease ontology (photoreceptor death, optic nerve damage, etc). Ideally, the decline in optomotor abilities is directly correlated with disease progression; and in such cases, in-vivo and non-invasive optomotor measurements can fully substitute for invasive (and potentially terminal) other measurements, such as histological sections.
How can one interpret the contrast sensitivity curve (contrast sensitivity as a function of spatial frquency)?
The visual system - as all sensory systems - is mostly sensitive to change in the input (light), not so much to constant absolute values. The magnitude of this change is called contrast. The sensitivity of the system is the smallest contrast that can still be perceived. This contrast (= change of light intensity) can be a contrast over time (a step or flash of light) or a contrast across space (a brightness border). When smaller contrast can still be perceived, then we can say that the system is more sensitive.
While the sensitivity of a sensory system can be described in terms of “contrast”, it also depends on another property, namely on the frequency of the stimulus, in a fundamental way. All sensory systems have a frequency-response tuning - too low or too high frequencies cannot be perceived, and there is some optimum in the center of the sensitivity range. Similar to the contrast, also frequency can be a temporal property (“flicker”) or a spatial property (fine or coarse “pattern”).
In optomotor reflex measurements, we observe the reaction of an animal to a moving stripe pattern. This pattern has a certain spatial frequency (determined by the number of black-white bars surrounding the animal), and, given by the combination of this spatial frequency and the rotation speed, we can also assign a temporal frequency to the stimulus. Let’s say that the optomotor reflex is triggered by such a stimulus, i.e. this stimulus is within the sensitivity range for the optomotor reflex. We can then keep the frequency constant and determine the contrast sensitivity of the animal (for that frequency!) by reducing the contrast. When we repeat the same procedure for different frequencies of the stimulus, we will most likely get different values for the minimal contrast that can still trigger the optomotor reflex. As a result, we will obtain the so-called contrast-sensitivity function of the optomotor reflex.
Alternatively, when we start with a stimulus that triggers the optomotor reflex, we can leave the contrast constant and change the frequency of the stimulus. If we change the frequency enough (e.g. make the bars of the stripe pattern finer), we will eventually leave the range where the given contrast is sufficient to elicit the optomotor reflex. For the reflex to be triggered again, we would have to increase the contrast. However, what happens if the contrast is already maximal? The finest stripe pattern (highest spatial frequency) that still triggers the optomotor reflex at the maximal contrast, is called “visual acuity”.
Please note that measurements of the optomotor reflex do not measure the overall “visual abilities” of the animal. A fine stripe pattern that does not elicit the optomotor reflex may still be “seen” by the animal. Optomotor measurements only give the contrast-sensitivity function of THIS system, the optomotor reflex system.
In most research applications, the absolute value of the contrast sensitivity function (in particular: visual acuity) is not so important. Instead, one usually compares two conditions (healthy vs. sick, treated vs. untreated, young vs. old, etc.). In this way, the measurements are also “self-calibrating”. For example, depending on the hardware to display the stimulus (e.g. old monitors, new monitors, cardboard drum illuminated by a light source), the maximally achievable stimulus contrast will be different. When the hardware display can achieve higher contrast values, then the reported visual acuity will be higher as well. The comparative nature of the measurements (“treated is better than untreated”) take care of this problem.
Could visual abilities be quantified by counting "optomotor events" in a given time frame?
This is not how the OptoDrum quantifies optomotor behavior, but it could be a liable alternative. One caveat to reckon with, however, is that the underlying time interval (for example 2 min of observation) should be comparable between individual mice. The optomotor reflex is a relatively “weak” reflex in the sense that it can be overridden by many other behaviors, such as exploration, or cleaning and grooming. While the animal is doing those other things, it does not make sense to count the absence of optomotor events as an indication for “bad vision”. Effectively, you should only count times into your statistics where the animal could have performed optomotor behavior. In other words, you should take the animal's overall behavior into account, look only at those times where the animal does not do other things, and then count the number of events where the animal follows the rotating stimulus during that time. Maybe you even take into account how well or how strongly the animal follows the stimulus. All these considerations should improve the assessment of the animal’s optomotor behavior. But if you do all of this, then you are very close to how the OptoDrum analyzes the optomotor behavior of the animal.
Is the optomotoric reaction (OMR) comparable with the optokinetic reflex (OKR)?
Yes, under normal circumstances they are comparable. Just as a quick explanation: the optokinetic reflex, or OKR, underlies compensatory eye movements, while the optomotor reflex, or OMR, underlies compensatory head movements. Both reflexive movements combined stabilize the "moving world" on the retina. Here, the OKR is generally doing most of the work, and monitoring the eye movement (rather than head movement) would in principle be a more reliable and stable indicator of visual function. However, in order to monitor eye movement reliably, one has to restrain the animal. Monitoring head movement (triggered by the OMR) is therefore far less invasive, and can be done in a more high-throughput way. The automated detection and analysis of OMR by the OptoDrum software makes all of this very easy.
Can one measure color vision with the OptoDrum?
The OptoDrum can measure visual acuity and contrast sensitivity. Measuring color vision is a much more difficult problem. In principle, it is possible measure color vision (i.e. the ability to see two different colors as distinct) with the help of the optomotor reflex. For this, one would present bars of different color (example: blue/red) instead of different brightness (Example: black/white). The optomotor reflex will be triggered if the two colors are perceived as different (i.e. there is a color-contrast at the stripe boundary). While this is simple in theory, it is very difficult in practice. In order to properly test that the two colors are perceived as different, one has to make sure that the differently colored bars do not appear to have different brightness. Otherwise, there will be a brightness contrast in addition to a color contrast. In practice, it is very difficult to calibrate equal-luminance colors, in particular if one can only adjust three base colors, namely the “R”, “G”, and “B” of the computer monitor. Proper equal-luminance color calibration depends on the specifc spectral sensitivity of the photoreceptors in the species under investigation, on the spectral filter characteristics of the optical apperatus (cornea, lens, vitreous, retinal tissue), and the number of spectrally distinct photoreceptors. Further caveats are described in this publication: The Method of Silent Substitution for Examining Melanopsin Contributions to Pupil Control
Can the OptoDrum be used with rats?
The OptoDrum comes in two versions, the “normal” OptoDrum which is ideal for measuring the optomotor reflex in mice, and a larger version, OptoDrumPLUS, which can be used with both mice and rats.
Does the optomotor response work in albino rodents? Does the coat color affect the ability of tracking?
Technically, the coat color does not affect the tracking of the animal by the OptoDrum software. Biologically, it may affect the animal’s tracking of the stimulus. For albino (white) mice, for example, the data is inconsistent between studies. Some studies find that they do not have an optomotor reflex. Other studies find that they do have the reflex, but reversed: they reflexively move their heads in the wrong direction, opposite of the moving stripe pattern. This is due to a mistake in axonal crossing at the optic chiasm.
Can all mouse lines be assessed with optomotor measurements?
In principle yes. However, there are some exceptions.
For example, the mouse line DBA2/J is a model to study glaucoma. However, some studies find that these mice simply do not have an optomotor reflex, independent of their retinal phenotype (Barabas et al 2011). This finding is inconsistent between different labs, others find the optomotor reflex to nicely reflect the retinal phenotype (Rangarajan et al 2011, Burroughs et al 2011). Maybe there are different subtypes of DBA/2J mice with and without the ability to have an optomotor reflex. This highlights the importance to have a good control in each study, that shows that your phenotypic observation, for example lack of optomotor reflex, is really associated with the objective of your study.
The other example are albino mice. Here, the data is inconsistent between studies. Some studies find that they do not have an optomotor reflex. Other studies find that they do have the reflex, but reversed: they reflexively move their heads in the wrong direction, opposite of the moving stripe pattern. This is due to a mistake in axonal crossing at the optic chiasm. Again, this shows the importance to have a proper control.
In the OptoDrum, you can measure the optomotor reflex of all mice. As the examples above show, however, the absence of the optomotor reflex in a specific mouse line may reflect a biological deficit that may not be related to your primary research question.
Which diseases do first affect contrast sensitivity?
Patients with inherited retinal degenerations including rod-cone dystrophy (RCD), Stargardt disease (STGD) and Best disease have significant deficits in contrast sensitivity with relatively preserved visual acuity.
The anti-epileptic drug Vigabratin has ocular toxicity which manifests with visual field narrowing. Can this be measured with OMR?
The optomotor reflex (OMR) is very robust. For research purposes, this can be both an advantage and a disadvantage. In this case, it is likely a disadvantage. Regional damage to the retina, where the rest of the retina is basically unaffected, usually has a very minor influence on the optomotor behavior. The reason is that the healthy part of the retina is sufficient to drive the optomotor reflex in a normal way.
This being said, it may be worth to check the situation in every specific application. Maybe your drug has the devastating effect on part of the retina that you are aware of, but, in addition, a minor effect on the rest of the retina. And maybe the combination of both effects may corrupt the optomotor response enough to provide a meaningful and insightful behavioral readout. Such an in-vivo behavioral readout, if it exists, can be a great research benefit, as it allows to easily monitor your disease progression longitudinally, even daily if needed.
Striatech offers demos of the OptoDrum, to evaluate it in your specific research model. This will reveal whether or not optomotor measurements are a good readout.
Is the optomotor reflex useful to study corneal disease models?
If your corneal disease diminishes the image quality across the retina (e.g. blurring of the image), then this will impair the optomotor reflex outcome. If, on the other hand, you have a corneal injury that will result only in a local image degradation, this will likely not affect the reflex. This situation can be compared to local retinal damage, because the damaged region of the cornea will always be projected onto a fixed region on the retina.
Laser damage: Does retinal laser damage lead to neovascularization?
Not in all cases. The type of damage and the consequences depend on the settings of laser power and time. One can introduce just retinal scarring, or one can induce CNV.
What models are there for retinal or choroidal neovascularization (RNV, CNV)?
The following article in PubMed Central gives an introduction to animal models of RNV and CNV: Animal Models of Choroidal and Retinal Neovascularization
Glaucoma: Can glaucoma mouse models be tested for the optomotor reflex?
Diabetic Retinopathy: Can the optomotor reflex be used to study diabetic retinopathy?
NaIO3: With high doses of systemically injected NaIO3 (sodium iodite), most photoreceptors are dead, but one can sometimes see OMR visual acuity to drop by only half. Why?
The exact relationship between the physical and functional damage on the cellular and tissue level, and the corresponding effect on the optomotor reflex, needs to be established for each disease model individually. In case of NaIO3, the primary target is the retinal pigment epithelium with secondary photoreceptor death. This process does not only have a temporal component (when do I expect to see effects?) but also a spatial component. For systemic injection of NaIO3, the damage proceeds radially, it starts in the central retina and quickly also engulfs the peripheral retina.
The biology of the optomotor reflex is such that it is preserved quite robustly when there is sufficiently functional retina remaining in the periphery. The morphology of the retina can show severe damage in the center and mid-periphery, to an extent that absolutely no vision is possible anymore. In the far periphery, however, enough function can be preserved for some time to trigger the optomotor reflex when the stimulus is sufficiently easy (here: 50% easier than normal visual acuity). This phenomenon is similar to the observations with the model of local retinal laser damage where one needs a certain amount of damage to the retina before one can see a significant impact on the optomotor reflex.
NaIO3: Is the sodium iodite model a model for dry AMD?
No, but it mimics some characteristics of the disease including apoptosis of the photoreceptors after degenerating RPE cells and functional as well as morphological changes.