Webinar Q&A Report - InsideScientific

Webinar Q&A Report: Automated Quantitative Measures of Forelimb Function in Rats and Mice

Q: what are examples of liquid rewards for mice? A: Our mouse users so far have used water (with water restriction) and peanut oil (as a supplement with no food or water restriction). Although not yet tested with the MotoTrak system, sweetened condensed milk is perhaps the most popular liquid reward for mice reported in the literature, and it is compatible with Vulintus’ liquid dispensers.

Q: I may have missed this (my apologies!) but how long was each animal left in the apparatus during a session? A: The session duration can, of course, vary depending on your particular model, but most labs run animals for two 30 minute sessions per day, one in the morning and one in the afternoon.

Q: Do you limit diet outside of reaching activity? Do you have to food deprive animals to do these tasks? A: Yes, animals training with food pellet rewards are typically food deprived. Once behavioral shaping has formed the initial association between food rewards and the task manipulandum, animals typically won’t need supplemental food on top of food rewards, except on days in which they don’t perform the task (such as weekends, perhaps). It’s important in this case to use a food reward pellet with a complete nutritional profile, as opposed to a basic sucrose or sugar pellet. Complete nutritional profile pellets are available from Bio-Serv or Purina.

Q: What percentage of the mouse body weight should be lost in order for them to do the task? A: In our experience, there’s no “magic” body weight loss that translates into a “eureka” moment. Generally speaking, you’ll want to follow guidelines set by your local IACUC. In our experience, we typically start training after a single night of food restriction (12-24 hours), and animals often make the association between the manipulandum and reward quickly enough that they might not experience more than a 5% reduction in weight relative to age-matched controls. 1

For food deprivation, if the animal isn’t progressing in shaping enough to receive significant food/water by the end of the second day, we’ll supplement with vivarium stock food to maintain body weight above 85% of agematched controls.

Q: How long does the mouse need to learn this task? A: For the isometric pull task, mice require ~10-20 days of training to become proficient at the task and then another ~5-10 days to establish a baseline (see Becker et al., 2016), which is ~1 week longer than what’s seen with rats. We’re still refining the mouse versions of the supination and lever press tasks, so we don’t yet have a reliable estimate of the training time for mice on those tasks.

Q: Does the training for isometric pull task take longer than traditional methods? A: With rats that were concurrently trained on isometric pull, pasta matrix, and skilled reaching, it took an average of ~17 days to learn the isometric pull task to criterion, an average of ~11 days to learn pasta matrix to criterion, and an average of ~10 days to learn skilled reaching to criterion. So, yes, training does seem to take about 1 week longer than traditional methods. However, once trained, there was significantly less variation in performance between animals on the isometric pull task than on the traditional tasks.

Q: You mentioned someone is using the lever press task for bradykinesia. Do you have information on the output of motor impairment/how impaired are they to controls/how do you show this? Or videos? A: Yes, the use of the lever press task for measuring bradykinesia was published in a methods paper in 2013 (Hays et al., J Neurosci Methods, 2013). In a rat model of ischemic stroke, the time in which it took animals to double-press a lever increased from 408 ms to 946 ms, on average, pre- to post-lesion, respectively. Videos of the task are included in the supplementary data for the paper.

Q: Are rat models better than Mice to assess motor deficits? Since I use mice and they recover pretty quickly (few days’ post-stroke) without any treatment. A: There may be some contention over this, but generally I (Drew Sloan) believe rats have an advantage of size when it comes to training, testing, and surgical procedures, but that mice and rats will show roughly similar impairments from similar models if the methods can be appropriately scaled. The one MotoTrak mouse behavior publication to-date (Becker et al., 2016) does show a lasting deficit after a photothrombotic stroke model that was significant out to ~4 months, so detection of chronic deficits in mice is certainly possible. The biggest driver of differences between the mouse and rat behaviors, in the experience of our beta testers, is the relative size of reward to body weight. The smallest food pellet reward that can be reliably used with our pellet dispensers is 20 mg, which is considerably larger relative to a mouse’s typical body weight than the 45 mg food pellet used with rats relative to a rat’s typical body weight. Liquid rewards can be aliquoted more proportionally, but water deprivation and food deprivation are not necessarily motivationally equivalent. 2

As we gather more information and experience from mouse system beta testers, we hope to come up with a suggested reward paradigm for mice that can produce results on par with those shown from the rats.

Q: Can you train animals on all tasks so you can then measure improvements in all tasks? A: So far, no lab has trained animals on all of the tasks, but some have trained animals on multiple tasks. Dr. Hays’ lab has studied rehabilitation generalization with rats trained successfully on both the supination and isometric pull tasks (publication in preparation). Although the study used only one MotoTrak system task, my (Drew Sloan) 2015 paper in PLoS One used rats trained concurrently to perform three tasks: the isometric pull task, pasta matrix, and skilled pellet reaching.

Q: Have any groups used MotoTrak with subtler models of motor dysfunction (dystonia models for instance)? A: We’ve not used it in any dystonia models, so I (Seth Hays) can’t give you a direct answer. However, we have used the systems across a pretty wide range of injury models from relatively minor distal median nerve injuries to severe SCI. Using the adaptive thresholding scheme, I suspect the systems would be useful even for pretty subtle, minor changes in forelimb performance.

Q: How many mice could you train/test in a day? A: How many animals can be run in a day is really a function of the desired length of the training session and the diligence with which experimenters switch out animals. Ideally, one mouse needs two 30-minute sessions per day for training, and an experimenter can remove one rat from the system and start the next in less than 2 minutes. With one system then, theoretically, one experiment could run 8 mice in one 8-hour day. In practice, however, small delays tend to creep into the schedule and an estimate of 6 animals per system per day is more realistic.

Q: Is food pellet retrieval automatically scored? How can the researcher ensure the food/reward has actually been taken? A: No, the food pellet retrieval isn’t currently automatically confirmed, although a capacitive sensor at the pellet receiver can at least confirm that the animal approached the receiver. Currently, if reward delivery needs to be confirmed, you would have to do that manually. In practice, however, particularly with food deprived animals, rewards are rarely ignored.

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Q: In your experience are these forelimb tasks learned more easily with one paw or the other - are rodents left or right pawed? What causes the rat or mouse to grab the handle? A: In our experience, yes, rodents show handedness, but won’t exclusively use one paw unless forced to. With no partition to force left- or right-paw usage, handedness is shown mostly in which paw animals reach with first, with the other paw reaching out shortly thereafter if the first reach attempt missed. Both the rats and mice learn to grasp the handle through a simple shaping paradigm in which any small bump of the manipulandum causes a reward to be dispensed, so in those stages the manipulandum almost acts more like a button to be pressed. As training progress, the manipulandum is retracted farther outside the cage, requiring animals to reach and grab.

Q: Has any work been done to use this equipment as a physical therapy device for animals (ie. treating an animal following an orthopedic injury)? A: Since those involved in MotoTrak’s development primarily came from a neuroscience background, most studies with the system so far involving rehabilitation or physical therapy have focused on neuropathologies, but the system would perfect for repetitive physical therapy for almost any forelimb model.

Q: For Dr. Hays. Have you ever tested animals in other less specific tests, more generic as walking tests and observed any improvements? Could it be a clue of further generalization? A: We have evaluated animals using other more standard motor tests, including grip strength, cylinder, and automated walking assessment. These are indeed great ways to assess generalization. Although we haven’t yet published any of the data (the manuscripts are being prepared now), we appear to see generalization in some contexts.

Q: Can the MotoTrak System be used during awake-behaving electrophysiological recordings? A: Yes. The MotoTrak system has standard BNC trigger outputs for synchronizing behavior with electrophysiology equipment, and the cage has access for a commutator on the top panel. Several labs are currently using the MotoTrak system in conjunction with EMG recordings, cortical recording, peripheral stimulation, and optogenetic stimulation.

Q: For Dr. Hays – How exactly did you stimulate the VNS in rat subjects? Also, how is this done with patients in clinical trials? A: In rats, we implant a bipolar stimulating cuff around the left cervical vagus nerve. The leads are tunneled subcutaneously to a two-channel connector affixed to the skull. The connector is then encapsulated in acrylic. During stimulation, a wire is plugged into the connector and allows delivery of stimulation from an isolated pulse stimulator. Much more detailed information can be found in our publications.

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Here are a few: DOI:10.1016/j.brs.2015.12.007; DOI: 10.1177/1545968315616494; DOI:10.1016/j.nbd.2013.08.002; DOI: 10.1161/STROKEAHA.114.006654; DOI:10.1038/nature09656. Stimulation pairing in patients was performed similar to that in rats, but the implantation procedures differed. A helical stimulating cuff was placed around the left cervical vagus nerve, and electrical stimulation pulses were delivered via an implantable pulse generator located under the clavicle (similar to placement for a pacemaker). More details regarding the human studies can be found here: DOI: 10.1161/STROKEAHA.115.010477.

Q: Have you ever used treat (sucrose) pellets rather than food as reward, or is it not necessary if diet is limited? A: Yes, we’ve used sucrose pellets effectively as a reward with these tasks, but in that case you’ll need to supplement their diet with a complete nutritional profile food, which can reduce animals’ appetite for treats and therefore reduce your daily trial count. Using complete-nutrition food pellet rewards, many of which are available in treat-like flavors, maximizes daily trial count and very often provides sufficient daily food to maintain or increase body weight with no supplementation.

Q: Do you use an altered light/dark cycle and only train during their dark cycle? A: Most, but not all, of the labs currently using MotoTrak systems use a 12:12 reversed light/dark cycle, with the behavior run during the dark part of the cycle. A few labs use a non-reversed cycle and run during the light part of the cycle. We haven’t yet done any analysis to determine what effects the light cycle has on these particular tasks, but, anecdotally, it doesn’t appear that light-cycle-testing labs get substantially fewer trials than darkcycle-testing labs, and vice versa.

Q: Dr. Hays -- why didn't the groups of rats who received VNS do better 1 week after stroke then the rats who did not receive VNS? If after 1 week none of the groups had received VNS shouldn't their performance at week 1 be the same? A: I apologize for not explaining the timeline better. Week 1 is an aggregation of all the sessions during the first week of receiving VNS. The preceding time point, labelled “POST”, shows performance one week after lesion before allocation to treatment group. As you correctly note, performance at this time point is the same between groups, allowing an accurate comparison of the effects of the therapy.

Q: For Dr. Hays – in your research how did you induce stroke and why do you do it that particular way? A: The work that I presented used two different (but rather similar) methods to induce ischemic stroke. In the first, we used injections of endothelin-1, a vasoconstricting peptide, directly into 8 locations in the motor cortex (a selection of references detailing the model: DOI:10.1016/j.nbd.2013.08.002; DOI:10.1016/j.neurobiolaging.2016.03.030; DOI: 10.1177/1545968314521006).

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In the second, we performed the same injections, but also added a ninth injection into the dorsolateral striatum (details here: DOI: 10.1177/1545968315616494). We chose these models because they result in substantial, reliable deficits in forelimb function and similar lesion sizes. Many other methods are available to model ischemic stroke. A nice description and comparison of endothelin models can be found here: DOI:10.1016/j.expneurol.2006.04.012. Other models use middle cerebral artery occlusion. There are clear limitations to all animal models of stroke. That’s one reason that my lab uses a variety of mechanistically distinct models of neurological injury, including ischemic stroke, hemorrhagic stroke, traumatic brain injury, spinal cord injury, and peripheral nerve injury. By testing our findings in a variety of models, we can determine how different aspects of each model influence our effects.

Q: Sometimes stroke causes language and speech problems, how can we use mice and rats to help humans overcome these problems? A: Great question. Aphasia is certainly an issue in many stroke patients. While there is really solid literature on speech discrimination in rats, rodents don’t produce speech (at least not the way we think about it) and thus don’t provide a great model for testing speech-specific therapies. However, understanding how to maximize the benefits of rehabilitation using new therapeutic strategies may be useful, as these could potentially be used to improve speech therapy after stroke.

Q: How fully automated is the training/testing process? What specifically does a researcher need to do to run a test? A: From start to finish, the experimenter is only required to transfer an animal into the cage, enter in the animal’s name and desired training/test stage into the computer, press “start” to start a session, press “stop” at the end of a session, and to remove the animal from the cage. All other aspects of the behavior are automated and monitored by the computer. In total, per session, the system requires ~2 minutes of experimenter time for setup.

Q: How many rats/mice can be trained simultaneously? A: Similar to the answer to the previous questions about how many mice one system can be run in a day, the number of animals that can be trained simultaneously depends on the desired length of the training session, the diligence with which experimenters switch out animals, and the number of available MotoTrak systems. One system can, ideally, run 8 animals in one 8-hour day, but perhaps more realistically run 6 animals given the small delays that tend to creep into schedules. With multiple systems then, the total number of animals that can be trained simultaneous is 6 times however many systems are available.

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Q: What sort of system training does Vulintus provide? A: We have written setup and training documentation that is available and downloaded from our website 24 hours a day. In addition to that, we generally like to do video or phone chats to talk about system setup and individual lab needs, and we’re always available for further email, video, or phone chats to discuss any issues you might have.

If you have additional questions for Vulintus regarding content from their webinar or wish to receive additional information about their solutions for animal behavior and forelimb function tests, please contact them by phone or email:

Vulintus, Inc. 17217 Waterview Pkwy, Ste 1.202BB, Dallas, TX 75252 USA Phone: +1 (844) 668 6872 Email: [email protected]

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