This study investigated differences in microRNA expression between two rat strains with different responses to drugs. Previous research found differences in miR-26a and miR-484 expression in the nucleus accumbens of high-responder and low-responder rats after cocaine exposure, using qPCR. The current study used in situ hybridization to validate these findings and identify the neuroanatomical locations of miRNA expression. Results showed downregulation of miR-26a in the nucleus accumbens core of low-responder rats exposed to cocaine compared to saline, consistent with prior qPCR results. However, results for miR-484 were not statistically significant. This study provides neuroanatomical information on miRNA expression differences between rat strains in response

1. MicroRNA differences in two rat strains related to drug abuse
S.S. Kitchin1, N.J. Ethen1, D.E. Hamilton2, B.S. Carter2,R.C. Thompson2
1Undergraduate Research Opportunity Program, University of Michigan, Ann Arbor, MI
2Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI
Abstract
Drug addiction is a widespread phenomenon that can cause
significant changes to neurobiology and behavior. Additionally,
individuals differ in their responses to drugs and susceptibility to
drug addiction. However, the underlying molecular mechanisms of
vulnerability to drug addiction are not well characterized. To
investigate this phenomenon with an animal model, we have used
two strains of rats that have been selectively bred based upon their
novelty seeking traits, high-responders (HR) and low-responders
(LR). Previously, our laboratory performed Taqman qPCR arrays on
RNA samples derived from cocaine and saline injected HR and LR
animals. We discovered differences in basal miRNA expression
patterns between the strains as well as differential miRNA
regulation by strain in response to drug treatment. In order to
verify these findings, I ran miRNA in situ hybridizations (ISH) in
tissue sections from these rats to determine the level of the
targeted miRNA expression in the core and shell of the nucleus
accumbens, measured by optical density. I gathered
neuroanatomical information of where different miRNAs are
expressed in two different strains of rats in response to acute
cocaine or saline injections. We have validated that miR-484 and
miR-26a are expressed in the nucleus accumbens, which is an
important brain region in the reward pathway. These results allow
us to begin to understand how the drug experience is different
between two different strains of rats. Extrapolating beyond the
animal model, it could eventually lead to understanding why drugs
affect different people in unique ways.
*p= 0.041
Conclusion
• LR tissue exposed to cocaine showed down-
regulation of miR-26a.
• MiR-26a results do not correlate with previous
qPCR studies.
• Discrepancy between ISH and qPCR results are
possibly derived from differences in the
biological make-up of the animal, the
sensitivity of the experiments, or a possible
error in the protocol.
• Results from miR-484 not statistically
significant, and as such cannot be used to draw
conclusions from.
References
1] Blanchard, Mathieu M., Daniel Mendelsohn, and Jennifer A.
Stamp. "The HR/LR Model: Further Evidence as an Animal
Model of Sensation Seeking." Neuroscience & Biobehavioral
Reviews 33.7 (2009): 1145-154. Print.
2] Carlezon, WA, and RA Wise. "Rewarding Actions of
Phencyclidine and Related Drugs in Nucleus Accumbens Shell
and Frontal Cortex." Journal of Neuroscience 16.9 (1996): 3112-
122. Web of Science. Web. 2 Apr. 2013.
3]Paxinos, George, and Charles Watson. The Rat Brain in
Stereotaxic Coordinates. United States: ACADEMIC PR-ELSEVIER
SCIENCE (MO), 2004. Print.
4]Greco, S.J. and P. Rameshwar. “MicroRNAs regulate synthesis
of the neurotransmitter substance P in human mesenchymal
stem cell-derived neuronal cells.” Proceedings of the National
Academy of Sciences, 2007. 104(39): p. 15484-15489.
5] "National Institute on Drug Abuse." National Institute on
Drug Abuse. NIH, n.d. Web. 16 Apr. 2013.
Results
Figure 3: Expression of miR-484 and miR-26a cocaine vs. saline
Figure 3: A) Expression of miR-484 cocaine vs. saline. (B) Expression of miR-
484 per animal (even, saline; odd, cocaine). (C) Expression of miR-26a cocaine
vs. saline. (D) Expression of miR-484 per animal (even, saline; odd, cocaine).
A. B.
D.C.
Future
Future studies will characterize changes in
miR-26a and miR-484 in HR animals as well as
continuing to characterize changes in miRNA
expression as a result of exposure to cocaine.
Further investigation of a variety of miRNA will
enable a more comprehensive understanding of
the changes in neurobiology and behavior that
are caused by cocaine exposure.
Methods
• Rat Treatment: After 1 hour in test cages, rats will be injected with either saline or cocaine. On the first test day, cocaine
treated animals will receive injections of escalating doses of cocaine (5, 10 and 20 mg ⁄ kg), with 1 hour between each injection
(saline treated animals will be treated with equal doses/injections of saline). Over12 days, the rats will be injected once a day
with 30 mg ⁄ kg of cocaine (or saline).
• In-Situ Hybridization: Used radioactive in-situ hybridizations (ISH). ISH protocol used 33P radiation to tag an miRNA probe at the
5’ end. We confirmed specificity of probe using mismatch miRNA (miRNA strand with two nucleotides switched). The ISH was
run with cocaine and saline treated animals to compare relative miRNA expression.
• Scanning and Analyzing Film: Scanned ISH film with ScanMaker 1000XL Pro Flatbed Scanner using SilverFast Ai Imaging
Software. Scanned images were then analyzed for optical density using the Image J program. Each animal has two slides, and
an individual slide contains two tissue samples. Each slide was measured five times, once for each nucleus accumbens (miR
26a, core; miR 484, shell) of the two tissue samples, and then once for background (optical density that is present in non-
radioactive areas). We then determined the mean of all optical density measurements per slide. Once all the means were
calculated for both cocaine and saline treated animals, a comparison of the saline/cocaine treated animals was done using a
two-tailed t-test (considered significant if p < 0.05).
B.
C.
A.
Core. Region of miR- 26a
data sampling.
Shell. Region of miR-
484 data sampling.
Figure 2:
(A) Rat brain atlas marking nucleus
accumbens core and shell region.3
Marked regions are areas of data
sampling.
(B) Representative tissue sample
from miR-484 ISH. C.
(C) Representative tissue sample
from miR-26a ISH.
Introduction
• Cocaine blocks dopamine receptors, thereby increasing dopamine levels in synapses.5
• miRNA, which act as post-transcriptional regulators, have a role in the changes in response to drug
exposure. 4
• HR animals are much more likely to self-administer drugs compared to LR animals.1
• Previous qPCR findings have shown that LR animals exposed to cocaine have higher expression of
miR-26a and miR-484 in nucleus accumbens, a region associated with reward processing2,5.
• ISH enables validation of qPCR data and additional neuroanatomical data regarding the location of
expression of miR-26a and miR-484.
Figure 2:miR-26a and miR-484 are expressed in the nucleus accumbens
NS
Hypothesis
Expression levels of miR-484 and miR-26a in the nucleus accumbens of LR animals
exposed to cocaine will be different than those exposed to saline.
A. B.
D. miRNA Comparison P-Value FC
mmu-miR-26a Core LR S v C 0.048 2.654
mmu-miR-484 Shell LR S v C 0.028 3.322
Figure 1: Introduction and background images and data
(A) Actions of cocaine at dopamine receptors5. (B)
Post-transcriptional regulation role of miRNA4. (C) Reward
pathways in response to drugs and other natural
rewards5. (D) Previous qPCR findings
C.
p=0.660
Acknowledgements
To the Thompson Lab, thank you for your gracious mentorship.
To UROP, thank you for the chance to broaden my horizons.
To David and Brad, thank you for your endless patience.
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ISH qPCR
ISHmicroRNA-26afoldchangevs.qPCR
Nucleus Accumbens core
MicroRNA-26a Expression: ISH vs.
qPCR