1. Researchers are investigating relationships between intestinal microbiota compositions and sleep function in mice. They administered antibiotics to deplete gut bacteria in 5 mice and a high-fat diet to alter gut bacteria in another 5 mice, with 5 mice on a low-fat diet as controls. 2. The researchers recorded the sleep behaviors of the mice and collected fecal and tissue samples. They now seek funding to analyze the microbial compositions in the fecal samples using next-generation DNA sequencing to understand how altered sleep behaviors relate to bacterial imbalances in the gut. 3. The objective is to investigate relationships between gut microbiota diversity, colonic permeability, and sleep-wake patterns in mice to better understand potential connections between gut health and

1. 1. Proposal Narrative
Project Title: Induction of intestinal microbial imbalances through antibiotic gavage, high­fat
diet: effects on the microbiota­gut­brain axis and sleep behavior in mice
Investigators: Jonathan Lendrum​1​
, Sumei Liu​1​
, Bradley Seebach​1​
, Barrett Klein​1​
and Andrew
Berns​2
1​
Department of Biology, University of Wisconsin­ La Crosse
2​
Department of Computer Science, University of Wisconsin­ La Crosse
A. Abstract
Myself, along with fellow UWL collaborators, are currently investigating possible
relationships between the compositions of intestinal microbiota and sleep function in a pilot
study using 15, C57BL/6 mice. At the end of summer 2015, we used a broad­spectrum antibiotic
cocktail to deplete intestinal bacteria in five of the mice. In addition, we used a high­fat diet to
alter the compositions of intestinal microbiota to another five mice. The last five were fed a
low­fat diet and served as the control group. The sleep­wake behaviors of all the mice were
recorded throughout the duration of the experiment by a custom­fitted video surveillance system.
With the support from a previous undergraduate research and creativity grant, we have finished
animal treatment and collection of all tissue and fecal samples. Now, I would like to ask for a
continuation of support to analyze the microbial compositions of the mouse fecal samples we
collected over the past summer. This will allow us to draw relationships between altered sleep
behaviors and bacterial imbalances in the gut.
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2. B. Background/Statement of the Problem/Significance of the Project
Throughout much of human history, the purpose of sleep has been at the center of
existential enigmas. Newborns sleep an astounding 14 to 17 hours a day; as adults, we spend
nearly 1/3 of our lives asleep. Sleep is a paradox. Evolutionarily, it is a highly conserved
behavior occurring across all animal species, suggesting its vital importance. Alternatively, it
places the participant in a vulnerable position among its external environment. Consequently,
the evolutionary advantages of sleep must surmount the immediate disadvantages caused by a
behavioral state that dissociates the senses from its surroundings, or the requirement for sleep
would have been lost from the genome long ago. The glymphatic system, a recently­discovered,
water­based drainage system for neural tissues that is active during sleep phases—but inactive
during wakefulness—offers just such a strong advantage for animals that sleep: to provide the
nearly one­hundred billion (10​11​
) neurons within the brain an energy­efficient pathway for the
removal of toxic, metabolic waste products that increasingly accumulate in compact spaces
between highly sensitive neural tissue during periods of extended wakefulness (6).
Cerebrospinal Fluid (CSF), described by Hippocrates in the 4​th​
Century B.C. as the “vital
spirit”, is a fluid composed of 99% water, and circulates throughout the central nervous system.
Water possesses an array of truly unique chemical properties, hence its title as ‘the universal
solvent’, that are often overlooked and important to appreciate in this context. Its hydrogen
atoms are pulled tightly to the oxygen atom, causing an uneven charge distribution. This
polarity causes neighboring water molecules to align in a transient, flickering orientation,
promoting equal distributions of any heat added to the system. Additionally, water exhibits
cooperative behavior such as surface tension, or capillary action that allow for water to “creep”
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3. and stick to small spaces. And finally, water has a large dielectric constant, which is the measure
of a solvent’s ability to diminish the electrostatic attractions between dissolved ions, thus,
serving as a qualified effector to restore a homeostatic environment to the brain.
We are critically interested in the regulation of energy states, sleep, and waste clearance
from commensal bacterial population that exists in the human (mammalian) gut. The discovery
of the glymphatic system and improving knowledge of the peripheral lymphatic system of the
brain provides a possible, explanatory link for ​how​ and ​why​ bacterial populations may regulate
sleep phases, and why changes in the types of bacteria in the gut (as a result of poor nutrition)
may be associated with poor sleep, bowel dysfunction and a broad range of nervous system
disorders.
Glymphatic System Implications
Development of multi­photon microscopes allowed researchers to visualize the brain in
ways previously unavailable. Using fluorescent tracers carefully injected into the CSF of mice,
researchers from Rochester Medical Center in New York were able to visualize, in real time, the
movement of those molecules as the CSF
circulation highway carried them along for the ride
(6). They found that CSF circulates around the
outside of the brain while awake, but shortly after
falling asleep special water channels located on the
brain’s surface that are normally closed to prevent
CSF access, quickly open. And like the opening of
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4. a dam, the watery CSF (and fluorescent tracers) rushes through the aquaporin­4 water channels
and directly into waste­laden brain tissue (9). The CSF is following an osmotic gradient created
by the solute­poor CSF rapidly gaining access to mass accumulations of unwanted neuronal
waste products that became trapped in the brain since waking from the last episode of sleep.
Therefore, normal sleep behavior may represent an ultimately involuntary, but semiflexible
threshold that will soon become quantifiable through mathematical modeling of metabolite
‘saturation points’ within regions of the brain. Additionally, insufficient sleep duration and/or
chronic impairment in the waste clearance pathway have been shown to greatly contribute to
advancing neurodegeneration (1). Furthermore, the CSF continues to percolate through
microscopic fluid channels that resemble many small, winding rivers. The CSF rivers flow
along anatomical glymphatic pathways exchanging with the pollutants it contacts, such as
beta­amyloid associated with Alzheimer’s disease (1). The CSF, increasing in solute
concentration, begins to slow as it acquires an increasing amount of bulk waste along the
glymphatic pathway, eventually fusing with peripheral lymphatic vessels responsible for
surveying and directing the disposal of wastes back into the blood circulation (10). Finally, once
in the blood, the brain’s garbage travels to the liver and kidneys where it is appropriately
detoxified and excreted from the body.
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5. Microbiota­Gut­Brain­(Sleep?) Axis
o​ ​The ​gut microbiota provide a pathway for optimal energy intake (maximizing absorption),
sleep provides the pathway for optimal energy conservation (waste removal).
The brain is the body’s major energy­consuming organ, and it controls aspects of the
function of every other organ. The brain is particularly fastidious about maintaining healthy gut
function because of its critical dependence on water and energy, a service that only the intestinal
portal provides. Thus the gut and brain have coevolved intimately; hence one has ‘gut feelings’.
However, only recently has there been an appreciation
for the role of commensal gut microbiota in health and
disease. This has led to an emerging concept suggesting
the microorganisms inhabiting the large intestine
possess a unique ability to communicate with the brain,
and thus modulate behavior in what is now referred to
as the microbiota­gut­brain axis (Figure 2)(4). The
complex diversity of the gut microbiome consists of
approximately 40,000 bacterial strains in 1800 genera,
harboring up to 10 million non­human genes (500 times
that of human cells) that are needless to say, not well understood (2,7). It has been demonstrated
that each person is home to a truly unique enteric bacterial fingerprint; however, there appears
to exist a certain homeostatic balance that confers cognitive health benefits, such as reduced
anxiety or depression (2). Curiously, sleep patterns between individuals also exhibit a similar
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6. fragile homeostatic balance. The restorative properties fall between insufficient sleep (less than
5 hours a night) and excessive sleep (greater than 9 hours a night), but the parameters are widely
distributed between individuals. The objective of our study is to examine relationships between
intestinal microbial compositions and sleep behaviors. We hope our study will make
contributions towards the unraveling of the mystery between these intriguing and mostly
unexamined relationships. Understanding potential connections between gut microbiota and
sleep behavior may assist with the development of novel therapeutics to deter brain
degeneration and other conditions associated with congruent brain, gut and sleep dysfunction.
C. Objectives
The broad objective of our continuing project is to investigate the relationships between the
diversity of gut microbiota, colonic epithelial permeability, and sleep wake patterns in C57BL/6
male mice. The objective of this grant is to acquire additional funding to analyze fecal bacteria
compositions using next­generation DNA sequencing. Next­generation sequencing analyzes the
entire genome allowing for precise determination of microbial diversity and composition down
to the genus and species level.
D. Research Methods
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7.
We manipulated the compositions of gut microbiota through two methods in order to
evaluate the effects on the microbiota­gut­brain axis and sleep behaviors. We gavaged a
broad­spectrum antibiotic cocktail consisting of ampicillin, neomycin, metronidazole and
vancomycin to deplete gut­microbiota in one group of five mice. To a different group of five
mice we used high­fat (60% kcal) diet feeding, which has been shown to alter bacterial
compositions in the gut. From July 16th until September 5th we completed treatments with
antibiotics and high­fat diet, ran multiple studies and collected the necessary gut, brain and fecal
tissues and have since been begun analyzing data and interpreting preliminary results. However,
we are in need of additional support to analyze the microbial compositions of previously
collected mouse fecal samples in order to continue.
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8. We plan to use next­generation sequencing technique for the analysis. Next­generation
sequencing precisely identifies nearly all of the DNA sequences found within a sample; and
using those sequences we can compare and extrapolate the microbial diversity of the fecal pellet
down to the genus and species level with great accuracy. We need this sensitivity in order to
understand how our intervention of antibiotics and high­fat feeding changed the organization of
intestinal microorganisms. This is a crucial step that must be taken because nearly all of the other
components of the study are inherently dependent on the results of fecal analysis, and without it
the relationship to our sleep hypothesis means very little. Additionally, next­generation
sequencing is the preferred method for experiments involving the microbiota­gut­brain axis. In
order for our results from this preliminary study to be productive, it is important that we do not
sacrifice the quality of our design to spend slightly less money. For example, there are alternative
methods of analysis that could be applied within our university for a lesser cost. However, the
antiquated technique we would be required to use here at UWL is extremely time consuming,
significantly less sensitive, reliable, and seldom seen in publications within the last decade.
E. Final Products and Dissemination
I plan to present our research at the following regional and national conferences.
Presentation Opportunities
● ​i.​ ​Seven Rivers Undergraduate Research Symposium, November 6​th​
2015.
​ii.​ ​National Conference for Undergraduate Research, April 9th 2016.
● ​iii.​ ​Experimental Biology, April 2​nd​
­6​th​
, http://experimentalbiology.org/2016
● ​iv.​ ​Annual UWL celebration of Student Research and Creativity
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9. F. Budget justification
Table 1. Itemized list of expenditures.
Item Expenditure
Genomic DNA Sequencing (UWBC)
1 Initial Sample $575 $575
14 Each Additional Sample $75 $1,050
Total expenditure $1,625.00
This project has multiple sources of funding due to its collaborative nature, including a
previous undergraduate research and creativity grant that was used to purchase our
pharmaceutical­grade antibiotics and the low fat control, high­fat mouse diets used over the
summer. I am applying for financial assistance from the URCG to cover most of the costs
associated with sending our experimental fecal samples to the University of Wisconsin­Madison
Biotechnology Center (UWBC) for comprehensive analysis of fecal microbial content using
Illumina Next­Generation Genomic Sequencing.
“​The UWBC DNA Sequencing Facility was established to provide cost­effective, cutting
edge sequencing resources, available to the University of Wisconsin campus as well as
colleagues at public and private institutions.”​ ​http://www.biotech.wisc.edu/
2. Letter of Support
To: Undergraduate Research and Creativity Grant Review Committee
I am writing to you in support of Jonathan Lendrum’s application for the undergraduate
research and creativity grant. Jonathan is currently a major in Biology­Biomedical concentration
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10. at the University of Wisconsin ­ La Crosse. I have known Jonathan since fall 2014 as his course
instructor for Comparative Animal Physiology. He received an A in the class, with an
impressive 95% on the cumulative final. He had a great attitude, was highly motivated, and
genuinely interested in the subject.
Most of the undergraduate research students take a research project from their faculty
advisor and work on the project under the faculty advisor’s direct supervision. Jonathan took a
very different route. He developed his own original ideas and actively reached out to faculty
advisors who might be able to help him to test his ideas. He is very interested in the links
between gut microbiota and brain function. Specifically, he would like to test a hypothesis that
perturbation of gut microbiota by antibiotic treatment or high­fat diet activates gut­brain axis
and alters sleep behavior. Since this is a relatively broad topic, Jonathan sought help from
several UWL faculty members from different disciplines, including Dr. Andrew Berns from
Computer Science Department, Drs. Barrett Klein, Sumei Liu, and Brad Seebach from Biology
Department. I (Sumei Liu) am one of his faculty advisors to help him test how perturbation of
gut microbiota changes gut permeability. Increasing in gut permeability may open up a route for
gut microbiota or their metabolites to enter the body, active the gut­brain axis, and change brain
function. Jonathan received an undergraduate research and creativity grant in spring 2015 to
start the project. During this phase of the project, he treated the mice with broad­spectrum
antibiotics and high­fat diet for 2 weeks. Broad­spectrum antibiotics would deplete the gut
microbiota, while high­fat diet would change the composition of gut microbiota. He videotaped
the sleep behaviors of the animals continuously before and during the treatment period. He
collected all samples and is currently doing data analysis.
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11. One of the critical components of the project is to determine the changes in gut bacteria
population after antibiotic or high­fat diet treatment. By knowing the gut bacteria composition,
we can relate the changes in gut permeability and sleep behavior to certain population of
bacteria in the gut. Jonathan plans to send the fecal samples from our experiment to the
University of Wisconsin­Madison Biotechnology Center for next­generation DNA sequencing,
a technique that can determine the microbial composition down to genus and species level. An
additional support by the undergraduate research and creativity grant will help him achieve this
goal.
Jonathan is a very independent undergraduate researcher. This is his project. He designed
the experiments, carried out the experiments, and even monitored the progress of the project
among several collaborators, including faculty members and undergraduate research students. I
view myself as his collaborator rather than a supervisor. I fully support his application and will
help him as much as I can. Aside from the experiments, I will be available for advice on data
analysis and preparation and submission of abstracts and manuscripts. Due to the nature of the
experiments, four months will be required to complete the proposed experiments and an extra
2­3 months will be needed to finish data analysis and preparation of abstracts and manuscripts.
If I can be of any further assistance, please feel to contact me.
Sincerely,
Sumei Liu
Associate Professor, Department of Biology
3. Transcripts
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15. 4. Previously Funded Research Information (if applicable)
If you do have not received previous funding, you may delete this section. If you have received previous funding more than
once, duplicate this table for each project.
Title of Proposal:
Induction of intestinal microbial imbalances through antibiotic gavage, high­fat diet:
effects on the microbiota­gut­brain axis and sleep behavior in mice
When did you receive funding for undergraduate research/creativity? How much funding did you
receive? Include semester and year.
$1,778.00 on April 13, 2015
Who funded your work (Undergraduate Research and Creativity Committee, College Deans, a faculty
research grant, or other source)?
URCC, faculty grants and collaborators.
Describe the results/outcomes of your research/creativity project?
In the process of data analysis.
Did /will you present your findings at the UW-La Crosse annual Celebration of Research and Creativity?
If so, what year?
I plan to present at the upcoming UWL CRC.
Did/will you present your findings at any other conferences? If so, explain.
I also plan to present at the conferences listed previously in the proposal as well as seek
out other opportunities.
Did/will you submit a final report to UW-L’s ​Journal of Undergraduate Research​? If so, what year?
I plan to submit our final report in 2016.
Did/will you publish your findings in any other journals? If so, explain.
I plan to submit our final report to a few professional journals in 2016 such as
Experimental Biology, or PLOS ONE.
References
1. Alzheimer Disease: Whole­brain imaging of the glymphatic system–a new strategy to
investigate amyloid clearance in AD. ​Nature Reviews Neurology.​ 9: 184.
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,
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16. 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 Engineerin.​ 54(2):
225­233.
5. Foster, Jane A., and Karen­Anne Mcvey Neufeld. 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.
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 drives 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.
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