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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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 system1 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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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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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% accuracy4 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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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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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) 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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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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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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3. Transcripts

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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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