Evaluation of mouse sleep behavior and glymphatic system function in response to manipulation of gut microbiota composition

1. 1. Proposal Narrative
Evaluation of mouse sleep behavior and glymphatic system function in response
to manipulations of gut­microbiota composition
University of Wisconsin­ La Crosse Department of Biology and Microbiology
Investigator: Jonathan Lendrum, Undergraduate SAH. Advisor: Bradley Seebach, PhD.
24 March 2015
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2. A. Abstract
The reason to why we sleep has been at the center of existential enigmas for as long as humans have inhabited
the earth. Sleep is a paradox. Evolutionarily, it is a highly conserved behavior occurring across all animal species
suggesting its vital importance. However, sleep concurrently places the participant in an extremely vulnerable position
among its external environment. Consequently, the basic evolutionary advantages of sleep must surmount the
conspicuous disadvantages produced by a behavior that dissociates the senses from its immediate surroundings, or it
would have been eliminated from our genome long ago. The recent discovery of the glymphatic system provides a
sufficient neurophysiological mechanism underlying perhaps, the most fundamental purpose of mammalian sleep and
the predominant reason why humans require ⅓ of our lives to be dedicated entirely to sleep.
B. Background/Statement of the Problem/Significance of the Project
Glymphatic System
In 2012, neuroscientists from the University of Rochester Medical Center discovered a previously unknown
waste clearance pathway for the mammalian central nervous system​1​
that provides a compelling neurophysiological
mechanism for us to consider as, perhaps the most prominent purpose of mammalian sleep. Analogous to conventional
lymphatic vasculature in the rest of the body, the glymphatic system (‘g’ for its dependence on CNS glial cells) has
comparable functions to a sink, however instead of water, this brain sink uses cerebrospinal fluid (CSF) to wash away
potentially harmful extracellular waste products, such as beta­amyloid associated with Alzheimer’s disease.​3,7
Metabolites, like beta­amyloid, progressively accumulate in the spaces between cells during daily activity and are
predominantly removed by the glymphatic brain sink through a plumbing matrix of fluid­filled channels. When you
fall asleep these channels open, washing away toxic byproducts from the sensitive neural tissues; the result of which is
likely responsible for the restorative properties of sleep.​8
Brain­Gut Microbiota Axis
In the last decade, mounting evidence suggests complex interactions between hosts and their microbial
communities, known as the microbiome, play a critical role in the development and maintenance of normal health.​2
These interactions include bidirectional communication between the microorganisms of the gut and the brain, referred
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3. to as the brain­gut microbiota axis.​3​
The brain­gut microbiota axis refers to the multitude of physiological processes in
which the microorganisms inhabiting the gastrointestinal tract can signal, “communicate” or influence host cognitive
function and vice­versa, the brain can also signal changes in gut diversity and composition. The gut is basically a giant
chemical factory and the types of bacteria that inhibit your gut, dictate many properties they confer to your health well
being by the certain chemicals they produce. There exist multiple anatomical pathways and physiological processes
allowing for these biochemical interactions between the gut­microbiota and the brain to occur, most of which are
regulated at neural, hormonal and immunological levels.​2,5
Approximately 100 trillion microorganisms reside in the gut of the average adult, outnumbering our human
cells by 10 to 1 and weighing nearly as much as our brain.​4​
The gut microbiome is a complex, dynamic community
influenced by subtle changes in diet, age, sleep, metabolic activity, genetics, geography, probiotic/antibiotic contact,
psychological stress and many more. The gut is dominated by an estimated 150,000 bacterial strains responsible for
harboring 9.9 million non­human genes, all of which are a collection of your life experiences. From everywhere you
have lived to the people you have had contact with and the food you have eaten, your gut microbiome is entirely
unique, a fingerprint of your individual history. These microorganisms are critical for digestion and drug
detoxification and influence a variety of important host functions ranging from metabolic activity and immune
response, to perhaps most remarkably, behavior and cognition.​2,5
C. Objectives
i. ​ ​It is the central objective of this two­part experiment to evaluate potential neurophysiological relationships
between the gut­microbiota compositions, the glymphatic system’s effectiveness in waste clearance and quantified
sleep­wake behavior in three randomized groups of BALB/C (m) genetically inbred mouse models.
​ii.​ ​Weeks 1­4 following potential acceptance of funding request shall be dedicated to part one of the
experimental design. The objective of part one is to measure changes in sleep duration in response to oral
administration of broad­spectrum antibiotics, probiotics, or a saline phosphate buffer solution to one of each three
randomized groups of mice. Researching how the consumption of antibiotic or probiotic supplements affect the
diversity of the gut microbiome should prove to be a successful opportunity to improve current theories regarding the
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4. magnitude of microbe­derived influence on the quantity and quality of host sleep behavior.
iii.​ ​Weeks 4­10 and beyond shall be reserved for part two of the experimental design. The objective of part
two is to quantify the removal of extracellular wastes by visually mapping the movement of a fluorescent tracer
infused in the cerebrospinal fluid of anesthetized mice. The movement of the fluorescent tracer through brain cavities
of anesthetized mice mimics the convective waste removal facilitated by the glymphatic system naturally during sleep;
allowing us to more accurately and appropriately evaluate the performance in response to the manipulations in gut
composition from part one of the experimental design.
D. Research Methods
A sample size of n=10­15 mice per parameter is commonly needed to detect moderate behavioral differences
using multivariate statistical analysis.​4​
Twice a day for three weeks, one group of randomly assorted mice is treated
with food supplement containing a broad­spectrum antibiotic mixture (Amoxicillin+Clarithromycin+
Metronidazole+Omeprazole), the second is treated with an established probiotic species (​Lactobacillus rhamnosus​ and
Bifidobacterium infantis) ​and finally the third group of mice is treated with a saline phosphate buffer as a control
group. Before the mice arrive at the HSC in the summer, the housing system Amy Cooper has made available for us to
use will be custom fit with pressure sensors lining the floor of the cages. In collaboration with Dr. Berns, replicating
research­established computational analysis of mouse sleep­wake dynamics using piezoelectric sensors (pressure
sensors) will allow me to quantify sleep­wake periods with 95% accuracy​4​
in the three groups of mice for the entirety
of the treatment cycle without the need for time­consuming and less accurate video analysis or other more invasive
methods. The pressure sensors use an established algorithm for pattern recognition of gross body movements in
rodents to automatically quantify sleep duration for small scale research purposes.​4 ​
This technology allows for us to
efficiently and inexpensively score sleep behavior in the antibiotic versus probiotic treated mice as their gut
microbiome composition undergoes drastic divergence from the control group in response to each subsequent
treatment. I expect all three treatments will result in statistically distinct groups of sleep dynamics in relation to their
altered gut microbiome compositions. Consequently, upon completion of part two of the experiment, the evaluation of
glymphatic waste clearance, I suspect we will observe impairment of the clearance pathways in mice treated with
broad­spectrum antibiotics and enhanced metabolite removal in mice treated with beneficial probiotic species. The
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5. results of part two will be compiled with the data from part one for comprehensive statistical analysis in order to
generate strong inferences based on our collective findings.
The second part of my experiment involves a sequential, histological assessment of each mouse’s brain tissues
to collectively quantify glymphatic activity for probiotic treated mice, antibiotic treated mice and control mice. The
effectiveness of the brain sink can be evaluated by mapping the movement of a fluorescent tracer throughout brain
cavities of anesthetized mice, mimicking the removal of extracellular wastes during sleep. Using clinically established
methods, ex vivo fluorescence imaging of brain tissues following infusion of a fluorescent tracer into the cerebrospinal
fluid of anesthetized mice is a technique that allows neuroscientists to create a map of the cerebral pathways directly
facilitating the clearance of our fluorescent tracer (i.e. mimicked waste product). These clinically­relevant techniques
were recently designed and standardized to carefully measure glymphatic activity for research and diagnostic imaging
purposes.​10​
An advantage of this evaluation method for glymphatic function is that once the animals have been
euthanized (upon completion of part­one behavioral phenotyping), the brains will be harvested from the animals and
the tissues frozen and can remain as such indefinitely without the risk of damage to our samples. This is helpful so we
are not restrained by a time frame dictated by the deterioration of quality tissue samples to acquire our data. Also, the
ability to freeze our tissue samples and return to them at a later time could protect the completion of the experiment
granted we run into unforeseen circumstances. Therefore, if necessary, the brain tissue analysis can extend into the
following year as agreed upon by Dr. Seebach and myself.
The histological equipment and fluorescent microscope necessary to replicate the methodology are available
in Dr. Seebach’s lab, other internal research labs and Gunderson Lutheran’s microbiology bench lab (Dr. Simon
Shelly) in HSC. The funds I am requesting will primarily be used for mice related expenditures (Table 1).
With this collection of data, I expect to implicate antibiotics with the impairment of waste clearance from the
brain. It is likely the antibiotic­induced dysbiotic state will quickly reduced diversity and abundance of the normal gut
flora typically uninhibited conferring their homeostatic properties, including sleep health. Additionally, I suspect the
probiotic­treated group will experience enhanced waste clearance during periods of sleep compared with the control
group for reasons contrary to that of antibiotic­induced dysbiosis. I plan for data acquisition and analysis from the
piezoelectric sensors to occur concurrently and comprehensively with the data from the evaluation of the brain sink’s
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6. waste clearance efficiency from the second part of the experiment. This deliberate extension of the experimental
design should greatly assist in narrowing the possible mechanisms­of­action responsible for the alteration of sleep
behavior, allowing us to not only interpret the quantity of sleep, but also the important ​quality ​component of sleep
(how thoroughly the glymphatic system removes waste per sleep cycle). If it were not for the physiological component
related to the quality of sleep, it would not be responsible to attempt any determination of ‘good’ sleep from ‘bad’
because sleep duration by itself is not a clearly defined or even well understood factor in health and disease. The
relationship could still be identified, but it would have less application, resulting in a weaker correlative study. This
would make it difficult to implicate the independent variables (antibiotic/probiotic/control treatments) as beneficial or
detrimental causative agents responsible for alterations in the brain sink’s aptitude for waste clearance, and the
hypothesized disparities in sleep behavior among the groups of treated mice. Additionally, part two operates in
complement with part one to improve the collective statistical significance of current and likely, future hypothesized
associations between host gut­microbiota interactions, sleep behavior and the glymphatic waste clearance pathway.
References
1) Benveniste, H., et al. 2012. A paravascular pathway facilitates CSF flow through the brain parenchyma and the
clearance of interstitial solutes, including amyloid­beta. ​Science Translational Medicine. ​4(147): 111­122.
2) Bonaz, B. 2013. Brain­gut interactions in inflammatory bowel disease. ​Gastroenterology.​ 144: 36­49.
3) Cryan, J. 2010. From bowel to behaviour: immune regulation of the brain–gut axis. ​Brain, Behavior, and Immunity.
24: S49.
4) Flores, A., et al. 2007. Pattern recognition of sleep in rodents using piezoelectric signals generated by gross body
movements. ​IEEE Transactions on Biomedical Engineering.​ 54(2): 225­233.
5) Foster, J., and Neufeld, K. 2013. Gut–brain Axis: How the microbiome influences anxiety and depression. ​Trends in
Neurosciences.​ 36: 305­12.
6) Nedergaard, M. 2013. Garbage truck of the brain. ​Science.​ 340: 1529­530.
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7. 7) Stilling, R. 2014. Microbial genes, brain & behavior – epigenetic regulation of the gut­brain axis. ​Genes, Brain and
Behavior​. 13: 69–86.
8) Underwood, E. 2013. Sleep: The brain's housekeeper? ​Science.​ 342: 301.
9) Xie, L., et al. 2013. Sleep dives metabolite clearance from the adult brain. ​Science.​ 342: 373­77.
10) Yang, L., et al. 2013. Evaluating glymphatic pathway function utilizing clinically relevant intrathecal infusion of
CSF tracer. ​Journal of Translational Medicine​. 11: 107­115.
Additional Fellowship Compliances
o​ ​American Association for Laboratory Animal Science (IACUC) application in process.
o​ ​Register for BIO­499 independent research with Dr. Seebach for summer semester, 2015.
o​ ​Registered and completed module 1 of the responsible conduct of research program (RCR).
Acknowledgements
Working collaborations and special thanks to: Dr. Barrett Klein (sleep and design consultant), Dr. Andrew
Berns (computer science and technology consultant), Dr. Simon Shelly (Gunderson Lutheran microbiology consultant),
Dr. David Reineke (statistical consultant) and Amy Cooper (animal care management).
E. Final Products and Dissemination
The content throughout my grant proposal is certainly dense and necessitates this section focus on
demonstrating the feasibility of the experiment. I first came across the discovery of the glymphatic system when
hunting for a topic to present at a sleep symposium held by sleep expert and experiment collaborator, Dr. Barrett
Klein’s Animal Behavior class in the spring of 2012. Since that time, not only has my interest in the glymphatic
system’s significance in health and disease grown, I have also found an academic passion for microbiology which lead
me to find the Human Microbiome Project (HMP), an NIH initiative launched in 2008 with the purpose of identifying
and characterizing the associations of microorganisms responsible for both healthy and diseased humans. Over the last
couple years I have become captivated by the research published in response to these discoveries and have remained
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8. educated on the techniques being developed for small­scale research applications.
In preparation for creating my first experimental design I have spent hundreds of hours studying primary
literature articles, initiating collaborative efforts with multiple organizations and working to acquire the proficiency
necessary to orchestrate a professional research experiment. Additionally, last semester I completed an online class
with Professor Robert Knight, Dr. Jessica Metcalf and Dr. Katherine Amato from the University of Colorado­ Boulder
called, “Gut Check: Exploring Your Microbiome”. This class was the first of its kind to be offered, dedicated entirely
to exploring current microbiome research. Along with my relevant academic endeavors in cellular and molecular
biology, microbiology and pre­veterinary sciences, the summation of these experiences have left me confident in my
ability to complete the objectives listed previous.
In conclusion, it is my hope that the Undergraduate Research and Creativity grant can provide an opportunity
for me to give back to UWL for all of the amazing experiences it has generously offered over the last few years. As
much as I plan to execute this experiment fastidiously, it is equally my intention to represent UWL and the many
people responsible for my success to the absolute best of my ability as a way of showing my appreciation and
gratitude for one of the most meaningful aspects of my life. I intend to validate the effort many people have exhausted
guiding me in my journey to be the best researcher that I can. Personally, I believe the utility of this experiment
extends beyond that of measuring independent variables, it is also a reflection of UWL’s aptitude for producing
students who have passion for creative and transformative sciences.
F. Budget justification
Research schedule and statement requesting $500 in supplies
Table 1 provides an itemized list of part­one lab consumables that are candidates to receive the requested
funds. Prices of lab consumables were generated from an assortment of suppliers, therefore, they are subject to slight
changes depending on the manufacturer of purchase.
The first thing you will notice is that the total expenditure is over $500; please note in addition to this request
for supplies I am seeking external funding. Also, the extension of our time frame has been considered as a viable
option if necessary, to build momentum prior to applying for larger extramural funding and grant opportunities.
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9. With that said, Dr. Seebach and I are working with collaborators within the university as well as the La Crosse
community (Gunderson Lutheran) with the goal of limiting our financial and temporal expenditures as much as
possible. Please refer back to Dr. Seebach’s letter of support for any additional concerns regarding resources, facilities,
supplies or financial management.
2. Letter of Support
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11. 3. Transcripts
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14. UWL Copyright Policy
“…If UW­L or UW System funding results in a copyrightable product, an agreement between the University (the
Chancellor or his/her designee) and the author for ownership should be negotiated before submission of a proposal, if
possible, and incorporated in the body of the proposal. The policies of the funding agency must be followed when
copyrightable material is developed under any extramural project.”
http://www.uwlax.edu/Grants/UW­L­Copyright­Policy/
After a discussion with the Dean of Science & Health, Dr. Bruce Riley and faculty advisor, Dr. Bradley
Seebach, the author(s) will reserve all rights if the experiment results in the development or creation of a copyrightable
product.​ ​All copyright, trademark, service marks, and other intellectual property are protected by and subject to
copyright and other intellectual property rights under United States law. Changes to the copyright policy may remain
available for negotiations up to any time, contingent on unanimous agreement between all parties involved.
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