Emily Winokur is a recent graduate who majored in Neuroscience and Behavioral Biology and minored in Sociology. She was awarded a Fall 2015 Independent Grant which she used to conduct research on sensory perception under Dr. Gary Miller.
The protein vesicular monoamine transporter 2 (VMAT2) is critical for dopaminergic neuronal survival because of its role in packaging monoamines into synaptic vesicles. Neurodegenerative diseases, such as Parkinson’s disease (PD), are thought to be caused by disruption of the usual functioning of vesicular release and uptake of monoamines including dopamine in the nigrostriatal system. This leads to dopaminergic cell death, impaired movement and increased anxiety- and depressive-like symptoms (Pifl, 2014).
Our laboratory developed transgenic mice which express 5% of normal levels of VMAT2 (VMAT2-Lo) and mice with a threefold increase VMAT2 protein level (VMAT2-Hi). VMAT2-Lo mice recapitulate many aspects of human PD including olfactory deficiencies, but show no deficits in the perception of touch or visual abilities compared to wild-type mice. While prior research explores the non-motor symptoms of PD in VMAT2-Lo mice, little research focuses on the effect of over-expression of VMAT2. My goal of performing this project was to use sensory assays to characterize the sensory acuity of VMAT2-Hi mice in order to appropriately interpret behavior in additional affective behavioral analyses.
I used a visual cliff assay to test the visual acuity of the two genotypic groups. The general idea, adapted from Carter et al, 2010 (Guide to Research Techniques in Neuroscience), is to construct a “cliff” using a cardboard box with plexi-glass overhanging from the box on one side. I placed a checkerboard tablecloth that reached the floor under the plexi-glass to enhance the appearance or a drop-off. As described by the protocol, the “cliff” side was deemed as the overhanging portion while the “safe” side was considered to be where the glass overlaps with the cardboard. Mice with lower visual acuity will show no preference for the “safe” portion of the plexi-glass and cross to the overhanging portion more frequently and with less initial hesitation.
The
apparatus shown below was used for the entirety of the sensory test. I drew a red dot in the middle of the “safe”
side and always placed the mouse in that location for consistency. Each trial
was five minutes, and the top portion of the apparatus was cleaned prior to
each one. I used the video tracking software, Topscan 2.0, to record latency to
cross to the “cliff” side, number of crosses, and the proportion of the testing
period spent on each side. For each measure, there was no difference between
the VMAT2-HI and wild-type mice. These data might be compromised by the fact
that mice retained untrimmed whiskers, which they may use to explore the visual
cliff. I’m considering rerunning the test with trimmed whiskers in order to
isolate the genotypic differences in the visual system in this assay.
A buried food assay was used to test the non-social
olfactory abilities of the VMAT2-HI and wild-type mice. Approximately 16 hours
before testing, I removed all food from the cage. I gave each mouse one Fruit
LoopÒ in the bottom left corner on top of the cage
bedding. This ensured that the smell was familiar, and the following morning,
all the mice had eaten the food. For the experiment, each mouse was placed in a
clean cage without a nestlet, the material used for the mouse to make bedding,
and allowed 5 minutes to habituate to the new environment. I then placed the
mouse back in its original cage while I buried a new Fruit LoopÒ at the
bottom right corner of the new cage. For each subject, the food was placed such
that it was not visible from the surface. With each trial, I placed the mouse
at the top of the cage relative to the food and recorded the amount of time for
the mouse to find the food. The timer was stopped when the mouse deliberately
dug at the Fruit LoopÒ and not just when it sniffed
around the area.
When analyzing the results, my graduate mentor would not
allow me to know the genotypes of the mice. Instead, she gave me a list of
“group A” and “group B” which allowed me to analyze the differences between the
groups without causing bias on further behavioral analyses. As indicated below,
group A found the food reward much more quickly than B. An unpaired t-test
yielded a p-value of 0.0196, thereby providing enough evidence to reject the
null hypothesis that there is no difference between the groups. Further sensory
analyses will be performed to determine differences in sensitivity to touch and
taste as well as characterize genotypic differences in anxiety- and
depressive-like symptoms.
This semester has been my first experience conducting
research independently and asking my own research questions. It has been the
first time that I am in charge of all the aspects of designing and executing
the behavioral tests. It has been challenging to use and modify existing behavioral
assays to answer my initial research questions. I have had issues with
scheduling times to use the behavioral rooms, mice trying to jump off of the
visual cliff apparatus, waiting for IACUC approval, and learning to work with
mice. With the challenges, I am learning to trust myself as a researcher. I am
excited to expand on my current research and further develop my research skills
in my remaining semesters at Emory.
Visit the Undergraduate Research Programs website to learn more about applying for Independent Research Grants.
Visit the Undergraduate Research Programs website to learn more about applying for Independent Research Grants.
References:
Carter, Matt, and Jennifer C. Shieh. Guide to
Research Techniques in Neuroscience. Amsterdam: Elsevier/Academic Press, 2010.
Print. 39-71
Caudle W.M., Colebrooke R.E., Emson P.C.,
Miller G.W. Altered vesicular dopamine storage in Parkinson’s disease: a
premature demise. Trends in Neuroscience,
2008; 31(6): 303-307
Lohr K.M., Bernstein I.A., Stout K.A., Dunn
A.R., Lazo C.R., Alter S.P., Wang M, Li Y, Fan Y, Hess E.J., Yi H, Vecchio
L.M., Goldstein D.S., Guillot T.S., Salahpour A, Miller G.W. Increased
vesicular monoamine transporter enhances dopamine release and opposes Parkinson
disease-related neurodegeneration in vivo. PNAS,
2014; 111(27): 9977-9982
Pifl C., Rajput A., Reither H., Blesa J.,
Cavada C., Obeso J.A., Rajput A.H., Hornykiewicz O. Is Parkinson’s Disease a
Vesicular Dopamine Storage Disorder? Evidence from a Study in Isolated Synaptic
Vesicles of Human and Nonhuman Primate Striatum. J. Neuroscience, 2014; 34(24): 1210-1218
Taylor T.N., Caudle W.M., Shepherd K.R.,
Noorian A, Jackson C.R., Iuvone P.M., Weinshenker D, Greene J.G., Miller G.W.
Nonmotor Symptoms of Parkinson’s Disease
Revealed in an Animal Model with Reduced Monoamine Storage Capacity. J. Neuroscience, 2009; 29(25): 8103-8108
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