RESEARCH ARTICLE
An open-source, lockable mouse wheel for
the accessible implementation of time- and
distance-limited elective exercise
Joseph J. Bivona, IIIID1,2*, Matthew E. Poynter1*
1 Department of Medicine and Vermont Lung Center, University of Vermont Larner College of Medicine,
Burlington, Vermont, United States of America, 2 Cellular, Molecular, and Biomedical Sciences Doctoral
Program, University of Vermont, Burlington, Vermont, United States of America
* JJ.Bivona@uvm.edu (JJB); matthew.poynter@med.uvm.edu (MEP)
Abstract
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 scien-
tists 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 imple-
mented 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 imple-
mentation of the LOST-Wheel and future iterations will improve upon existing murine exer-
cise equipment and research.
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OPEN ACCESS
Citation: Bivona JJ, III, Poynter ME (2021) An
open-source, lockable mouse wheel for the
accessible implementation of time- and distance-
limited elective exercise. PLoS ONE 16(12):
e0261618. https://doi.org/10.1371/journal.
pone.0261618
Editor: Richard Jay Smeyne, Thomas Jefferson
University, UNITED STATES
Received: October 23, 2021
Accepted: December 6, 2021
Published: December 21, 2021
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0261618
Copyright: © 2021 Bivona, Poynter. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All model, code, and
manual files are available for download at: https://
github.com/jjbivona/LOSTwheel.
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 com-
puter 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 microcon-
troller 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 phys-
iological 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 sin-
gle night (their waking time) then performed RT-qPCR on gastrocnemius muscle and multi-
plex 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
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Funding: This study was made possible by support
from the Larner College of Medicine and the
Vermont Lung Center T32HL076122. The funders
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript.
Competing interests: The authors have declared
that no competing interests exist.
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 (Ardu-
ino, 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 Dis-
tance 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.
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 com-
munication 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 Log-
ger 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
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.
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universal serial bus (USB) ports available on the computer. In our laboratory, we use an inex-
pensive 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 accli-
mate 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.
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).
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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 spectrophotom-
eter. (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). Quanti-
tative 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’-TTCACACCCATCA
CAAACAT-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 lin-
ear regression, respectively. Significance is designated by p-values < 0.05. Fig 2A was created
with BioRender.com.
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 compo-
nent 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
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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
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).
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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 measure-
ments 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 exer-
cise. Most notably, IL-6 is highly upregulated and is believed to function differently from clas-
sic 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, cor-
relation between distance traveled and serum myostatin concentrations (Fig 2C). As a regula-
tor 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 pres-
ence 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 opera-
tional indefinitely. The running surface and shaft can easily be removed and sprayed with etha-
nol to disinfect between uses. While mice have occasionally chewed the running surface, this
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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 dis-
tances 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 exer-
cise mice, but such devices require extensive machining and calibration [32] or the repurpos-
ing 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 labo-
ratories 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 imple-
ment 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 commer-
cially available Bluetooth-Arduino adaptors. Other changes may include on-board data stor-
age, operant conditioning modifications, or using the locking pin to provide resistance against
the wheel to model weighted wheel hypertrophy-inducing exercise [35].
Supporting information
S1 Data. The code, manual, and 3D files in both .STL and .F3D format, can be found at:
www.github.com/jjbivona/lostwheel.
(TXT)
S1 Video. Videos for building and setting up the LOST-Wheel can be found at: https://
www.youtube.com/channel/UCUp9zD0H99VcX0XXl2qGUmg.
(TXT)
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Acknowledgments
The authors thank Jeffery L. Brabec and Colleen E. Yancey for their help debugging code.
Author Contributions
Conceptualization: Joseph J. Bivona, III.
Data curation: Joseph J. Bivona, III.
Formal analysis: Joseph J. Bivona, III.
Funding acquisition: Joseph J. Bivona, III.
Investigation: Joseph J. Bivona, III.
Methodology: Joseph J. Bivona, III.
Project administration: Joseph J. Bivona, III, Matthew E. Poynter.
Software: Joseph J. Bivona, III.
Supervision: Matthew E. Poynter.
Visualization: Joseph J. Bivona, III.
Writing – original draft: Joseph J. Bivona, III.
Writing – review & editing: Joseph J. Bivona, III, Matthew E. Poynter.
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An open-source, lockable mouse wheel for the accessible implementation of time- and distance-limited elective exercise.
Bivona, Joseph J,Poynter, Matthew E