An open-source, lockable mouse wheel for the accessible implementation of time- and distance-limited elective exercise.

Current methods of small animal exercise involve either voluntary (wheel running) or forced (treadmill running) protocols. Although commonly used, each have several drawbacks which cause hesitancy to adopt these methods. While mice will instinctively run on a wheel, the distance and time spent running can vary widely. Forced exercise, while controllable, puts animals in stressful environments in which they are confined and often shocked for "encouragement." Additionally, both methods require expensive equipment and software, which limit these experiments to well-funded laboratories. To counter these issues, we developed a non-invasive mouse running device aimed to reduce handler-induced stress, provide time- and distance-based stopping conditions, and enable investigators with limited resources to easily produce and use the device. The Lockable Open-Source Training-Wheel (LOST-Wheel) was designed to be 3D printed on any standard entry-level printer and assembled using a few common tools for around 20 USD. It features an on-board screen and is capable of tracking distances, running time, and velocities of mice. The LOST-Wheel overcomes the largest drawback to voluntary exercise, which is the inability to control when and how long mice run, using a servo driven mechanism that locks and unlocks the running surface according to the protocol of the investigator. While the LOST-Wheel can be used without a computer connection, we designed an accompanying application to provide scientists with additional analyses. The LOST-Wheel Logger, an R-based application, displays milestones and plots on a user-friendly dashboard. Using the LOST-Wheel, we implemented a timed running experiment that showed distance-dependent decreases in serum myostatin as well as IL-6 gene upregulation in muscle. To make this device accessible, we are releasing the designs, application, and manual in an open-source format. The implementation of the LOST-Wheel and future iterations will improve upon existing murine exercise equipment and research.

Introduction

While exercise is a safe and effective health strategy [1], the ability for laboratories to test hypotheses concerning the physiological adaptations brought about by movement in mouse models is hindered by the expense of commercial products [2]. To understand the systemic effects of untrained exercise and lower the barrier of entry for murine-exercise research, we have developed an open-source mouse running wheel that accurately tracks and displays the distance traveled and restricts wheel running at specified distances or times. The Lockable Open-Source Training Wheel (LOST-Wheel) has been designed for both standalone and computer connected scenarios. The only requirement for standalone mode is a USB power source. In this mode, the LOST-Wheel can log cumulative distance, which is visualized through the onboard screen. When a computer is connected to the wheel through the LOST-Wheel Logger application, more detailed data such as speed and time running can be collected, graphed, and exported. The LOST-Wheel was tested in both overnight (acute) and week-long (chronic) experiments. Samples from the acute, untrained, bout of exercise were subjected to analysis with real-time quantitative polymerase chain reaction and protein quantification through Luminex assays.

This design of the LOST-Wheel was inspired as an attempt to create an inexpensive, freely accessible, and human-relevant model of exercise. One drawback of voluntary exercise is the inability to limit running distances [3]. For studies that interrogate the dose dependent effect of exercise or to restrict running to certain times and distances, we implemented a microcontroller regulated locking mechanism to prevent wheel movement at the will of the investigator. Previous work using commercially available products has shown that wheel running produces dose-dependent effects on neuron proliferation and dendritogenesis [4]. Additionally, that the presence of an immobile wheel in a cage also elicits neurological effects in the absence of its use demands that such a device-exposure group should be included as a proper control in wheel-running experiments [4, 5]. Despite the average gait of a mouse being 5–6 cm [6], mice voluntarily run upwards of 7 hours and 20 km/night [3] when provided with a standard wheel [7, 8]. The use of a wheel for long periods cannot be explained by a single theory [9], and physiological effects differ substantially between strains of mice [10]. By limiting running distances, the locking capacity of the LOST-Wheel enables researchers to normalize voluntary exercise across animals. While forced exercise (treadmill running) is an alternative strategy that ensures a consistent distance across animals, the handling [11], confinement [12], and electrical shock [13] required for its implementation can induce stress and alter the biological responses being studied [14].

To verify the effectiveness of the LOST-Wheel and to evaluate the consequences of a single, untrained bout of exercise, we allowed a cohort of mice to perform voluntary exercise for a single night (their waking time) then performed RT-qPCR on gastrocnemius muscle and multiplex analysis of several myokines in serum. We confirmed exercise-induced physiological changes, including increases in gastrocnemius Il6 gene expression and a significant, negative, relationship between serum myostatin and the distance traveled by mice.

Methods

LOST-Wheel design and code

The LOST-Wheel was designed using Fusion360 (Autodesk, San Rafael, CA) and is composed of four pieces: main body, top face, servo pin, and wheel. Table 1 contains a component list for the electronics, bearings, axle, and hardware. Slicing the models for 3D printing was conducted using Cura (Ultimaker, Utrecht, Netherlands). All pieces were printed using fusion deposition modeling with polylactic acid (PLA) on an Ender3 V2 3D printer (Creality 3D, Shenzhen, China). The sketches used to program the LOST-Wheel were created in Arduino IDE (Arduino, New York City, NY) in Arduino/C++ language with the additional libraries, U8g2 and U8x8. A computer rendering, representative image, and wiring diagram for the LOST-Wheel are shown in Fig 1A–1C, respectively. In acute exercise experiments, the Timer Mode protocol was uploaded and set to begin when the wheel was powered on. For chronic exercise, the Distance Mode protocol was uploaded, and the threshold set to 106 m for unlimited running. The LOST-Wheel can be powered indefinitely; however, in the experiments of this manuscript, data was collected daily, at which time wheels were reset.

Table 1

Component list for the LOST-Wheel.

Component	Quantity
6 mm ID, 10 mm OD, x 3 mm bearing	3
6 mm axle cut to 65 mm	1
M3x5 self-tapping screw	1
M2x6 self-tapping screw	1
M2.3x8 self-tapping screw	8
M1.7x6 self-tapping screw	8
10 mm x 5 mm x 3 mm neodymium magnet	2
22-gauge, 2.54 mm breadboard jumper wires, 3 male, 7 female	10
Arduino Nano (or similar)	1
9g micro servo	1
KY-003 hall effect sensor	1
0.96 inch 128x64 OLED Screen I2C connection SSD1306 Driver	1

This list serves as a template for the electronics and hardware required for the device. Generic Arduino clones can be substituted as microcontrollers since they are often a fraction of the price. Magnet size and quantity can also be changed depending on availability and accuracy required.

Fig 1

Lost-Wheel assembly and testing.

A) Computer rendered design of the assembled LOST-Wheel. B) Completed assembly of the LOST-Wheel. C) Simplified wiring diagram of components. 22-gauge, 2.54 mm breadboard jumper wires are soldered to the microcontroller and connected to components using the attached plugs. Should a single component fail, this allows for easy replacement without resoldering. D) The LOST-Wheel Logger application can be used in conjunction with the LOST-Wheel to collect and plot additional data. E) Mice were allowed to run unrestricted for 7 days on the LOST-Wheel. Each morning, the distance was recorded, and wheels were reset (n = 5).

All files required to build and program the LOST-Wheel are available at https://github.com/jjbivona/LOSTwheel. The LOST-Wheel design files, manual, and LOST-Wheel Logger software are licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. To access a build video and setup tutorial, visit: https://www.youtube.com/channel/UCUp9zD0H99VcX0XXl2qGUmg.

LOST-Wheel logger

While the LOST-Wheel can accurately display distance using its onboard screen, more detailed information can be obtained using the LOST-Wheel Logger Application. The application was created in R and RStudio (version 4.1.1 and 1.4 respectively) using Shiny [15], ggplot2 [16], and Serial [17] packages. Users are instructed to enter an ID for the wheel, the associated communication port (COM port), and the duration of data collection. After information is entered and the start button is pressed, the logger restarts the wheel and collects data in one second intervals until the duration is met. The program then calculates the individual slopes between each data point to determine the maximum speed in meters/second. Using this information, the Logger can determine the amount of time the mouse has run. Previous literature indicates that untrained mice have an average speed of 1–2 km/h (0.28–0.56 m/s) [18]; therefore, a threshold of 0.2 m/s is applied to exclude non-running events. Finally, the LOST-Wheel Logger creates distance/time and velocity/time graphs and presents all information for the user. The number of wheels that can be simultaneously connected is limited by the number of universal serial bus (USB) ports available on the computer. In our laboratory, we use an inexpensive USB hub to expand the number of wheels connected. The current version of the LOST-Wheel Logger accommodates a single wheel. Therefore, to collect data from multiple wheels, the user must open a separate instance of RStudio for each wheel.

Mice

12–20 week old male C57BL/6J mice purchased from The Jackson Laboratory (Bar Harbor, ME) were housed in AAALAC-accredited animal facilities at the University of Vermont, and all experimental animal procedures were approved by the University of Vermont Institutional Animal Care and Use Committee, protocol #202100027. Mice were maintained on a 12 hour-light/dark cycle, beginning at 07:00 and 19:00, respectively, and provided chow and water ad libitum.

To examine the effect of a single, untrained bout of exercise, mice were brought from the vivarium and caged individually. A single LOST-Wheel, or an immobile “dummy” wheel, was introduced into each cage at 08:00. Wheels in the running group remained unlocked to acclimate the mice until 12:00, at which point the wheels locked. At 19:00 the wheels unlocked, and mice were allowed to run voluntarily for 12 hours, at which point they were euthanized by an intraperitoneal injection of pentobarbital (Euthasol, Midwest Veterinary Supply, Lakeville, MN), followed by exsanguination. Serum and gastrocnemius muscle were collected and snap frozen in liquid nitrogen.

For one week running experiments, wheels remained unlocked throughout the duration of the test. Distances were recorded at 09:00 each morning and the wheels were reset.

RNA isolation, quantitative real-time polymerase chain reaction (qRT-PCR), and protein quantitation

Total RNA was isolated from liquid nitrogen pulverized whole muscle using TRIzol reagent followed by a chloroform-isopropanol extraction (Thermo Fisher Scientific, Waltham, MA, USA). RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer. (Thermo Fisher Scientific). cDNA was synthesized from 100 ng of RNA using the qScript Supermix reagent kit per manufacturer’s instructions (Quantabio, Beverly, MA, USA). Quantitative real-time PCR was performed using iTaq Universal SYBR Green Supermix on a CFX96 Touch (Bio-Rad, Hercules, CA, USA), with the relative mRNA expression calculated using the threshold cycle (Ct; 2−ΔΔCt) method normalized to Gapdh expression. The following primer sequences were used (Integrated DNA Technologies, Coralville, IA, USA): Il6 forward 5’-CCCGGAGAGGAGACTTCACAG-3’, reverse 5’-GAGCATTGGAAATTGGGGTA-3’; Gapdh forward 5’-ACGACCCCTTCATTGACCTC-3’, reverse 5’-TTCACACCCATCACAAACAT-3’.

Serum samples were analyzed using a Milliplex Mouse Myokine Magnetic Bead Panel (Millipore Sigma, St. Louis, MO, USA) on a Luminex 100 xMAP Instrument (Bio-Rad) according to kit instructions.

Statistical analysis and figures

RT-qPCR and Milliplex data were analyzed and visualized using GraphPad Prism version 9.2.0 for Windows (GraphPad Software, San Diego, CA, USA) with unpaired t-tests and a linear regression, respectively. Significance is designated by p-values < 0.05. Fig 2A was created with BioRender.com.

Fig 2

Acute exercise of untrained mice.

A) Experimental setup. Mice were acclimated to the LOST-Wheel for four hours, at which point the wheel locked until the evening. Mice were allowed to run unrestricted for 12 hours, at which point the wheels relocked and mice were euthanized. B) RT-qPCR analysis of gastrocnemius muscle Il6 expression (n = 6-7/group, unpaired t test). C) Serum myostatin concentrations relative to the distance traveled of mice undergoing untrained acute exercise (n = 6, simple linear regression).

Results

Building and testing the LOST-Wheel

All components in Table 1 are readily available and the 3D printed models can be created using any entry-level 3D printer capable of printing in polylactic acid (PLA) filament. The wheel can be assembled using only a #1 Phillips head screwdriver, a soldering iron, and a wire cutter/stripper. Excluding a 3D printer, the entire apparatus can be created for less than 15 USD. The models can easily be modified to account for larger diameter bearings and drive shafts, additional wheel magnets, or changes in component mounting holes. A representative rendering and completed wheel are shown in Fig 1A and 1B, respectively. A simplified component wiring diagram is shown in Fig 1C. We have also created a series of videos that outline the assembly, programming, and cage setup, which can be accessed at https://www.youtube.com/channel/UCUp9zD0H99VcX0XXl2qGUmg.

The LOST-Wheel was tested for 7 days to evaluate durability and animal safety, during which mice steadily increased distance traveled per day (Fig 1D), averaging 2046.38 ± 2647.11 m on the first day and 8972.71 ± 2888.14 m at the end of the trial. One mouse did not use the wheel until the second day.

Locking criteria

To limit and control mouse exercise, we developed three separate modes for the LOST-Wheel. These are first edited by the user based on their experimental requirements and uploaded to the wheel through the Arduino IDE program. In Timer Mode, the user inputs the amount of time (in hours) for which they want the wheel to be sequentially locked and unlocked. This program was implemented for our acute voluntary exercise experiments. The second and third modes limit exercise by distance and time spent running, respectively, and allow for the normalization of exercise across animals within experimental groups.

Acute voluntary exercise by untrained mice

To examine the effects of a single bout of voluntary exercise on untrained mice, we subjected mice to a one-day acclimation and exercise protocol (Fig 2A). Mice were singly placed in cages containing either a LOST-Wheel or an immobile “dummy” wheel at 08:00. The wheel remained unlocked for four hours for acclimation, at which point the servo inserted a pin into the wheel to lock the device (12:00). At 19:00, the pin was withdrawn, and mice were allowed to voluntarily use the wheel for 12 hours. At the end of exercise, we collected serum and leg muscles, then measured expression and production of interleukin-6 (IL-6), a muscle-produced cytokine (myokine) reported to be induced in exercised human subjects [19] as well as in mouse models of acute exercise [20] and myostatin. We observed a significant increase in the expression of Il6 in the gastrocnemius muscle following acute exercise (Fig 2B). Interestingly, there was a significant correlation between distance traveled on the LOST-Wheel and serum myostatin measured by Luminex (Fig 2C). One mouse did not log any distance on the LOST-Wheel. This mouse was excluded from RT-qPCR measurements but included in measurements of serum myostatin as a sedentary control.

Discussion

This manuscript presents an open-source mouse wheel that can be affordably and efficiently assembled by investigators. The LOST-Wheel can be used in standalone mode to collect data on overall distances traveled. When used in combination with a computer, the wheel interfaces with the LOST-Wheel Logger application to calculate the amount of time spent running and top speed achieved. Finally, we verified the integrity of the device and observed physiological changes in serum and muscle gene expression from a single untrained bout of exercise.

Physiological alterations after running

While the primary objective was to assess the efficacy of the LOST-Wheel, there was merit in observing the effects of acute exercise. Muscle derived signaling molecules, termed myokines, are released during muscle contraction and indicate physiological adaptations following exercise. Most notably, IL-6 is highly upregulated and is believed to function differently from classic inflammatory signaling by instead increasing glucose sensitivity and uptake [21]. We observed increases in Il6 expression in the gastrocnemius muscle (Fig 2B); however, increased IL-6 protein concentrations were not detected in serum using a myokine multiplex panel, implicating its local effect in the muscle. Additionally, we observed a significant, negative, correlation between distance traveled and serum myostatin concentrations (Fig 2C). As a regulator of muscle growth and differentiation [22], this correlation implies that myostatin is dose dependently regulated by running distance. These results align with previous studies in humans and rats, in which myostatin was transiently decreased after bouts of acute exercise [23, 24].

Voluntary exercise versus forced exercise

While treadmill based forced exercise allows for controlled speed, duration, and incline of training, it increases corticosterone and norepinephrine levels, indicating a strong stress response that is not elicited by during voluntary exercise [25-28]. The shock, confinement, or handling of the mice can all contribute to the increased stress reported. Additionally, the presence of an immobile wheel can have the added benefit of environmental enrichment [29, 30]. Due to this effect, we advise using a locked LOST-Wheel or creating a dummy wheel (fully assembled without electronic components) for control groups [4, 5].

Durability of the LOST-Wheel

Several iterations of the LOST-Wheel were prototyped before using the Hall effect sensor and magnet combination. Originally, the wheel rotated on a rotary encoder, an electro-mechanical part that has a finite number of rotations (30,000–100,000) before wearing out. We also attempted using an infrared sensor, but cage bedding would often block the beam, rendering it useless. The magnet and Hall effect sensor bypass these problems and should remain operational indefinitely. The running surface and shaft can easily be removed and sprayed with ethanol to disinfect between uses. While mice have occasionally chewed the running surface, this has not hindered its balance or performance. We have also designed and provided a guard for the power cord that protects it from destructive animals.

Comparison to existing wheels

To verify data collection on the LOST-Wheel, we have compared our 7-day protocol to the well detailed and extensive work of de Bono and colleagues [18], who developed a method to track distances run by mice but that lacks the ability to limit exercise, requires an expensive data acquisition unit with software (Spike 2, https://ced.co.uk/prices/1401options, https://ced.co.uk/prices/softwareprices), and has limited customizability and programmability. We have developed the LOST-Wheel Logger App, which allows for detailed collection, analysis, and export of data similar to the Spike 2 program. Reassuringly, our device recapitulates the distances reported in the aforementioned work. Recently, an open-source wheel was introduced that tracks running distances using a similar Hall Effect sensor as the LOST-Wheel. However, this device does not allow for controlled exercise or additional data collection as it only collects cumulative distance measurements [31]. Forced exercise designs exist that can be used to exercise mice, but such devices require extensive machining and calibration [32] or the repurposing of an existing human-treadmill [33].

Cost

The LOST-Wheel provides an inexpensive alternative to commercial murine exercise devices. Similar distance tracking devices, without locking abilities, cost 300–400 USD per wheel, require an additional data acquisition unit (400–900 USD), and necessitate accompanying software (800–2500 USD) (price quotes are from correspondence with commercial retailers). The availability and shallow learning curve of Arduino-based microcontrollers allows for laboratories to create their own devices at a fraction of the price [34]. The 3D printed parts can be outsourced to university fabrication labs, commercial 3D print operations, or fabricated in-house as entry level fused deposition modeling printers have substantially decreased in price over the last decade, with entry level printers ranging from 200–300 USD.

Future designs

The LOST-Wheel was created out of necessity and to improve current research-based exercise protocols in experimental animals. We have shown that this inexpensive, open-source wheel can provide investigator-controllable exercise to small rodent research without modification of the cage. The value of an open-source project is that it allows researchers to easily implement changes that fit their research goals and budgets. Future iterations of the LOST-Wheel and Logger App can include wireless transmission of data to a smartphone through commercially available Bluetooth-Arduino adaptors. Other changes may include on-board data storage, operant conditioning modifications, or using the locking pin to provide resistance against the wheel to model weighted wheel hypertrophy-inducing exercise [35].

The code, manual, and 3D files in both .STL and .F3D format, can be found at: www.github.com/jjbivona/lostwheel.

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Click here for additional data file.

Videos for building and setting up the LOST-Wheel can be found at: https://www.youtube.com/channel/UCUp9zD0H99VcX0XXl2qGUmg.

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Click here for additional data file.

22 Nov 2021

PONE-D-21-33956An open source, lockable mouse wheel for the accessible implementation of time- and distance-limited elective exercisePLOS ONE

Dear Dr. Bivona III,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, all the reviewers were enthusiastic about the manuscript but there are a few minor changes hat will be necessary to finalize the manuscript.  These are fairly minor and can be re-reviewed editorially without need to resend to reviewers.  . Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ==============================

As you can see in the reviews, there are just a few changes required.  These mostly involve addition of references as well as the notation of two of the reviewers that you should eliminate or reword the use of the word hyperphsyiological regarding the amount of exercise performed by mice (they seen to run approximately 3-4km/night  under voluntary conditions (see Gerecke et al, 2010) and others referenced by reviewers.   Additionally, reviewer 3 asks for some more information on durability of the wheels as well as about how many can be run on a single computer (or can this be expanded by addition of a COM port hub? I look forward to your revised manuscript which should be of benefit to the scientific community.

==============================

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Reviewer #1: This communication provides an open source-based device as a means to study physical exercise, as modelled by wheel-running, in mice. The main issues which are successfully dealt with are both scientific (a device which permits to fix time and/or distance limits according to the experimenter will) and practical (a minor cost, compared to commercially available devices). I encourage the reader to long into the link providing the (detailed) video related to piece assembly.

My sole critics concern bibliographical issues, which however need to be taken into account:

(1) In the 2nd par. of the Introduction, it is indicated that "one drawback of voluntary exercise is the inability to limit running distances". In the authors' mind such a pitfall might probably extend to running times/periods. First, I encourage the authors to read two studies (Dostes et al., Hippocampus 2016; Dubreucq et al., Biol. Psychiatry 2013) wherein the time allowed to run was varied (showing e.g. that exercise-induced neurogenesis is running duration-dependent). Second, whether it is more meaningful to limit running distance or running duration is an open question. Indeed, rodents run for hours but these consist in successive, albeit extremely short, episodes with resting pauses aimed e.g. at drinking, resting, exploring or eating. Moreover, mice do run during the dark phase of the light/dark cycle but their maximal effort is observed during the first 3-4 hours (see the above mentioned study by Dubreucq as an illustration). Taken with the observation that each mouse bears its own active period and its own running speed, it is maybe more relevant to limit the time, rather than the distance, as the variable for wheel locking/unlocking.

(2) In the same paragraph, the authors use the word "hyperphysiological" for mouse running behaviour. I sincerely believe that this word should be removed because it might be prone to anthropomorphism. As humans, we consider these distances as enormous. However, mice (and rats) are able to do so without constraint. I encourage the authors to read the excellent review of Sherwin (Animal Behav 1998), which in addition, will allow the authors to change several of their (recent) references by older one which might be more relevant.

(3) Legends to the two figures should be separated from the main text and place at the end before the figures

(4) Discussion:

• The fact that circulating BDNF is not increased by acute exercise is somewhat surprising. How do the authors explain this observation?

• The paragraph comparing forced vs voluntary exercise is incorrect. Asz it stands, it seems that voluntary exercise does not increase corticosterone levels. Besides the observation that it is increased with wheel-running (see relevant figures in ref 22), the fact that corticosterone increases with exercise is logical and is also observed in humans (changing corticosterone to cortisol) because it provides a source of energy.

• The authors should mention the fact that their device is considered an environmental enrichment which, per se, can alter physiological and behavioural variables (see the abundant bibliography on that subject) hence indicating the need to host control mice with locked wheels (see the abovementioned study by Dostes et al. as an additional illustration).

• As mentioned by the authors, "free" wheel-running devices as the one reported in this study allow to easilly measure running performance under voluntary dimensions. However, these devices, by their "free access" dimension, do not permit to selectively measure running motivation. The latter requires operant conditioning paradigms (and hence expensive setups) wherein running access is rendered possible by prior lever pressing (see Iversen, J Exp Anal Behav 1993) or nose poking (see Muguruza et al., JCI Insight 2019) under fixed or variable ratio reinforcement schedules.

Reviewer #2: The article, regarding the building and validity of the LOSTwheel is very well done. The authors present clear information about materials, assembly, and implementation of the equipment. In addition, they do appropriate tests to verify their observational analysis of the running behavior of the mice. This is true both for not only whether they run or not, as well as for the distance that they ran. Thus, these results are in keeping with many other studies regarding wheel running behavior in mice, indicating that the design of the LOSTwheel is valid for running behavior. Importantly, access to a low cost strategy to make these wheels is invaluable to the many scientists who study this, but do not have large grants to buy the expensive monitoring cages available on the market. Thus, this research has broad implications for impacting the field, including to make this research more widely available. As someone with little experience with electrical builds, I appreciate the very comprehensive manual with clear, step by step pictures and instructions, as well as the online tutorials. The level of detail and clarity provided make this easily replicable.

Reviewer #3: This open source, lockable mouse wheel is a useful tool that may enable investigators to conduct mouse exercise trials without limitations due to equipment cost.

Some clarification in the manuscript statements is necessary:

1. Voluntary running wheels that allow restriction of running in time or distance increments are available, although the cost of the equipment may be prohibitive for some laboratories. This is not clear in all sections of the manuscript.

2. Previously published articles, referenced in the current manuscript (see De Bono et al., 2006) and (Gerecke et al., 2010) illustrating the amount of voluntary running that mice will perform in a given period of time (1 day to 90 days) refute the suggestion that mice will voluntarily run at ‘hyperphysiological” levels.

3. Some details about the potential protocols for the LOST-Wheel would be useful if clearly stated in the manuscript. For example, is one week (chronic), the maximum period of time for an exercise protocol (without restarting)?

How many wheels and COM ports may be run in a single experiment?

Approximately how long do the 3D printed wheel and magnet apparatus work efficiently with continued use and cleaning?

The Lost-Wheel manual is clear and appears easy to follow to build the wheel and controlling components. The video links provided are very helpful, however, parts of the Assembly video, especially the final assembly portion of the box housing the microcontroller, Nano, and Hall effect sensor, as well as the connection of the components would benefit from a closer view.

Minor comments:

The use of Gapdh for normalization of the expression of the gene of interest in qPCR is not a stable comparator in all cases (see(Desseille et al., 2017; Xu et al., 2018; Hildyard et al., 2019)). The housekeeping gene 18S rRNA has invariant expression and provides more stable comparison.

Reference #13 should be DeBono JPD (De Bono et al., 2006).

References:

De Bono JP, Adlam D, Paterson DJ, Channon KM (2006) Novel quantitative phenotypes of exercise training in mouse models. American journal of physiology Regulatory, integrative and comparative physiology 290:R926-934.

Desseille C, Deforges S, Biondi O, Houdebine L, D'Amico D, Lamazière A, Caradeuc C, Bertho G, Bruneteau G, Weill L, Bastin J, Djouadi F, Salachas F, Lopes P, Chanoine C, Massaad C, Charbonnier F (2017) Specific Physical Exercise Improves Energetic Metabolism in the Skeletal Muscle of Amyotrophic-Lateral- Sclerosis Mice. Front Mol Neurosci 10:332.

Gerecke KM, Jiao Y, Pani A, Pagala V, Smeyne RJ (2010) Exercise protects against MPTP-induced neurotoxicity in mice. Brain Res 1341:72-83.

Hildyard JCW, Finch AM, Wells DJ (2019) Identification of qPCR reference genes suitable for normalizing gene expression in the mdx mouse model of Duchenne muscular dystrophy. PLoS One 14:e0211384.

Xu H, Ren X, Lamb GD, Murphy RM (2018) Physiological and biochemical characteristics of skeletal muscles in sedentary and active rats. J Muscle Res Cell Motil 39:1-16.

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26 Nov 2021

Dear Dr. Smeyne,

We are excited to resubmit our manuscript, “An open-source, lockable mouse wheel for the accessible implementation of time- and distance-limited elective exercise” to PLOS One. The comments we received were helpful to complete the revision and are addressed below.

Best Regards,

Joseph J. Bivona III

Matthew E. Poynter

Reviewer #1:

This communication provides an open source-based device as a means to study physical exercise, as modelled by wheel-running, in mice. The main issues which are successfully dealt with are both scientific (a device which permits to fix time and/or distance limits according to the experimenter will) and practical (a minor cost, compared to commercially available devices). I encourage the reader to long into the link providing the (detailed) video related to piece assembly.

My sole critics concern bibliographical issues, which however need to be taken into account:

(1) In the 2nd par. of the Introduction, it is indicated that "one drawback of voluntary exercise is the inability to limit running distances". In the authors' mind such a pitfall might probably extend to running times/periods. First, I encourage the authors to read two studies (Dostes et al., Hippocampus 2016; Dubreucq et al., Biol. Psychiatry 2013) wherein the time allowed to run was varied (showing e.g. that exercise-induced neurogenesis is running duration-dependent). Second, whether it is more meaningful to limit running distance or running duration is an open question. Indeed, rodents run for hours but these consist in successive, albeit extremely short, episodes with resting pauses aimed e.g. at drinking, resting, exploring or eating. Moreover, mice do run during the dark phase of the light/dark cycle but their maximal effort is observed during the first 3-4 hours (see the above mentioned study by Dubreucq as an illustration). Taken with the observation that each mouse bears its own active period and its own running speed, it is maybe more relevant to limit the time, rather than the distance, as the variable for wheel locking/unlocking.

We would like to thank Reviewer #1 for their kind words and constructive response. Our revised submission has included the following references in the second paragraph of the introduction and within the discussion as they further provide the case for appropriate controls and dose dependent effects of exercise:

Dostes S, Dubreucq S, Ladevèze E, Marsicano G, Abrous DN, Chaouloff F, et al. Running per se stimulates the dendritic arbor of newborn dentate granule cells in mouse hippocampus in a duration-dependent manner. Hippocampus. 2016;26(3):282-8. doi: https://doi.org/10.1002/hipo.22551.

Dubreucq S, Marsicano G, Chaouloff F. Emotional consequences of wheel running in mice: Which is the appropriate control? Hippocampus. 2011;21(3):239-42. doi: https://doi.org/10.1002/hipo.20778.

(2) In the same paragraph, the authors use the word "hyperphysiological" for mouse running behaviour. I sincerely believe that this word should be removed because it might be prone to anthropomorphism. As humans, we consider these distances as enormous. However, mice (and rats) are able to do so without constraint. I encourage the authors to read the excellent review of Sherwin (Animal Behav 1998), which in addition, will allow the authors to change several of their (recent) references by older one which might be more relevant.

Both Reviewer #1 and #3 have raised concerns about the use of hyperphysiological and upon examination, we agree that this term should be removed from the manuscript. Consequently, we have done so. We have also added the recommended paper (Sherwin, 1998) to the introduction as we enjoyed this perspective on the reasons why animals utilize running wheels. With our background in immunology, we often do not appreciate the behavioral aspects of murine research that the review highlights.

(3) Legends to the two figures should be separated from the main text and place at the end before the figures

We have reviewed the PLOS One guidelines and believe the format is correct.

(4) Discussion:

• The fact that circulating BDNF is not increased by acute exercise is somewhat surprising. How do the authors explain this observation?

In our experiment, BDNF was not detectable in the serum of both the control and exercise groups. We have removed this sentence from the results and discussion since BDNF and other myokines are not included as data and do not contribute to the manuscript. In humans, BDNF appears to be transiently found after exercise and is returned to baseline after about 10 minutes (Schmidt-Kassow et al 2012). We speculate this rapid utilization may also be the case in mice. It would appear other groups have also had trouble detecting serum BDNF (Delezie et al., 2019, Klein et al., 2010).

Schmidt-Kassow M, Schädle S, Otterbein S, Thiel C, Doehring A, Lötsch J, Kaiser J. Kinetics of serum brain-derived neurotrophic factor following low-intensity versus high-intensity exercise in men and women. Neuroreport. 2012 Oct 24;23(15):889-93. doi: 10.1097/WNR.0b013e32835946ca. PMID: 22964969.

BDNF is a mediator of glycolytic fiber-type specification in mouse skeletal muscle

Julien Delezie, Martin Weihrauch, Geraldine Maier, Rocío Tejero, Daniel J. Ham, Jonathan F. Gill, Bettina Karrer-Cardel, Markus A. Rüegg, Lucía Tabares, Christoph Handschin. Proceedings of the National Academy of Sciences Aug 2019, 116 (32) 16111-16120; DOI: 10.1073/pnas.1900544116

Klein AB, Williamson R, Santini MA, Clemmensen C, Ettrup A, Rios M, Knudsen GM, Aznar S. Blood BDNF concentrations reflect brain-tissue BDNF levels across species. Int J Neuropsychopharmacol. 2011 Apr;14(3):347-53. doi: 10.1017/S1461145710000738. Epub 2010 Jul 7. PMID: 20604989.

• The paragraph comparing forced vs voluntary exercise is incorrect. Asz it stands, it seems that voluntary exercise does not increase corticosterone levels. Besides the observation that it is increased with wheel-running (see relevant figures in ref 22), the fact that corticosterone increases with exercise is logical and is also observed in humans (changing corticosterone to cortisol) because it provides a source of energy.

Upon reviewing literature, we have included several sources that support the statement that voluntary exercise stimulates reduced levels of corticosterone compared to forced exercise. At Reviewer #1’s suggestion, we have removed the statement that voluntary exercise increases corticosterone levels and replaced it with mention that the wheel itself is an enrichment device.

• The authors should mention the fact that their device is considered an environmental enrichment which, per se, can alter physiological and behavioural variables (see the abundant bibliography on that subject) hence indicating the need to host control mice with locked wheels (see the abovementioned study by Dostes et al. as an additional illustration).

We appreciate this suggestion and have added it into the discussion.

• As mentioned by the authors, "free" wheel-running devices as the one reported in this study allow to easilly measure running performance under voluntary dimensions. However, these devices, by their "free access" dimension, do not permit to selectively measure running motivation. The latter requires operant conditioning paradigms (and hence expensive setups) wherein running access is rendered possible by prior lever pressing (see Iversen, J Exp Anal Behav 1993) or nose poking (see Muguruza et al., JCI Insight 2019) under fixed or variable ratio reinforcement schedules.

The value of an open-source design is that all files can be modified (and we encourage it). We are in the early stages of creating a resisted LOST-Wheel for hypertrophic exercise. Based on the review by Sherwin and the references suggested by the reviewer, there is certainly a need to incorporate an operant condition feature. We would be willing to collaborate and help design such a device.

Reviewer #2:

The article, regarding the building and validity of the LOSTwheel is very well done. The authors present clear information about materials, assembly, and implementation of the equipment. In addition, they do appropriate tests to verify their observational analysis of the running behavior of the mice. This is true both for not only whether they run or not, as well as for the distance that they ran. Thus, these results are in keeping with many other studies regarding wheel running behavior in mice, indicating that the design of the LOSTwheel is valid for running behavior. Importantly, access to a low cost strategy to make these wheels is invaluable to the many scientists who study this, but do not have large grants to buy the expensive monitoring cages available on the market. Thus, this research has broad implications for impacting the field, including to make this research more widely available. As someone with little experience with electrical builds, I appreciate the very comprehensive manual with clear, step by step pictures and instructions, as well as the online tutorials. The level of detail and clarity provided make this easily replicable.

We thank Reviewer 2 for their time to review our manuscript and their encouraging comments. One of our goals was to make the assembly and coding of the LOST-Wheel as approachable as possible so that others can implement it in their labs. We hope that this is the case.

Reviewer #3:

This open source, lockable mouse wheel is a useful tool that may enable investigators to conduct mouse exercise trials without limitations due to equipment cost.

Some clarification in the manuscript statements is necessary:

1. Voluntary running wheels that allow restriction of running in time or distance increments are available, although the cost of the equipment may be prohibitive for some laboratories. This is not clear in all sections of the manuscript.

We have edited a sentence in the introduction to address this point and maintain this perspective through the remainder of the manuscript.

2. Previously published articles, referenced in the current manuscript (see De Bono et al., 2006) and (Gerecke et al., 2010) illustrating the amount of voluntary running that mice will perform in a given period of time (1 day to 90 days) refute the suggestion that mice will voluntarily run at ‘hyperphysiological” levels.

We have been convinced through the comments of Reviewer #3 and #1 that the use of hyperphysiological is incorrect and anthropomorphizes the typical distances that mice run. Thus, we have removed these sentences from the manuscript.

3. Some details about the potential protocols for the LOST-Wheel would be useful if clearly stated in the manuscript. For example, is one week (chronic), the maximum period of time for an exercise protocol (without restarting)?

We have included additional information into the methods section that indicate the modes that were uploaded.

How many wheels and COM ports may be run in a single experiment?

We have added a sentence in the methods to address this question. The number of wheels that can be run is limited by the number of USB ports available on the computer. In our laboratory, we use an inexpensive USB hub splitter to expand the number of wheels connected. The current version of the LOST-Wheel Logger can only accommodate a single wheel. Therefore, to collect data from multiple wheels, the user must open a separate instance of RStudio for each wheel. This is a minor inconvenience but works effectively.

Approximately how long do the 3D printed wheel and magnet apparatus work efficiently with continued use and cleaning?

We have added the following paragraph to the discussion to address this comment:

Several iterations of the LOST-Wheel were prototyped before using the Hall effect sensor and magnet combination. Originally, the wheel rotated on a rotary encoder, an electro-mechanical part that has a finite number of rotations (30,000-100,000) before wearing out. We also attempted using an infrared sensor, but cage bedding would often block the beam, rendering it useless. The magnet and Hall effect sensor bypass these problems and should remain operational indefinitely. The running surface and shaft can easily be removed and sprayed with ethanol to disinfect between uses. While mice have occasionally chewed the running surface, this has not hindered its balance or performance. We have also designed and provided a guard for the power cord that protects it from destructive animals.

The Lost-Wheel manual is clear and appears easy to follow to build the wheel and controlling components. The video links provided are very helpful, however, parts of the Assembly video, especially the final assembly portion of the box housing the microcontroller, Nano, and Hall effect sensor, as well as the connection of the components would benefit from a closer view.

We appreciate the reviewer’s comments on the assembly video and plan to increase the size of the images projected in the sections pertaining to the final assembly during future updates.

Minor comments:

The use of Gapdh for normalization of the expression of the gene of interest in qPCR is not a stable comparator in all cases (see(Desseille et al., 2017; Xu et al., 2018; Hildyard et al., 2019)). The housekeeping gene 18S rRNA has invariant expression and provides more stable comparison.

We acknowledge that housekeeping genes are often the topic of debate. Yet, determining the best one may be situation specific. It appears through the references that for dystrophic, atrophic, and recovering muscle, 18S is a proven candidate. For exercise, it may be worth investigating 18S further, since ribosomal biogenesis can be linked to exercise induced hypertrophy.

Wang X, Zhao H, Ni J, Pan J, Hua H, Wang Y. Identification of suitable reference genes for gene expression studies in rat skeletal muscle following sciatic nerve crush injury. Mol Med Rep. 2019 May;19(5):4377-4387. doi: 10.3892/mmr.2019.10102. Epub 2019 Mar 28. PMID: 30942461; PMCID: PMC6472138.

Regulation of Ribosome Biogenesis in Skeletal Muscle Hypertrophy. Vandré Casagrande Figueiredo and John J. McCarthy. Physiology 2019 34:1, 30-42

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7 Dec 2021

An open-source, lockable mouse wheel for the accessible implementation of time- and distance-limited elective exercise

PONE-D-21-33956R1

Dear Dr. Bivona III,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Richard Jay Smeyne

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I am excited for this paper to be published in PLoS One and feel that this may have a significant impact on the field; due to its ease of use and low cost. On a personal level, having worked in exercise, I look forward to its publication, at which point I will access the information and give this a try. Thank you for an nice piece of work.

Reviewers' comments:

10 Dec 2021

PONE-D-21-33956R1

An open-source, lockable mouse wheel for the accessible implementation of time- and distance-limited elective exercise

Dear Dr. Bivona III:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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