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Auditory Fear Conditioning in a Mouse Model of Fragile X Syndrome
Alex Koo ’18, Kevin Newhall ’17, Dr. Hadley Bergstrom, Dr. Bojana Zupan
Psychology Department, Neuroscience and Behavior Program, Vassar College, Poughkeepsie NY
Background:
Dysfunction of the Fmr1 gene leads to an absence of
Fragile X Mental Retardation Protein (FMRP), causing
Fragile X Syndrome (FXS), the most common heritable
cause of intellectual disability and monogenic form of
autism (Schaefer & Mendelsohn, 2008). Mouse fmr1 is
homologous to human Fmr1, and the fmr1 knock out (KO)
mouse models FXS (Consortium, 1994). Previous studies in
our lab have shown that maternal genotype is a marker for
dopamine (DA) dependent behavioral changes in the
offspring, including hyperactivity, abnormal sociability, and
altered appetitive learning strategy (Zupan & Toth, 2008;
Newhall & Zupan, 2014). Additionally, a maternal genotype
dependent reduction in expression of the dopamine D2
autoreceptor (D2aR) is associated with this behavioral
phenotype and possibly contributes to it by increased tonic
and attenuated phasic levels of dopamine (Chu, Gale, &
Zupan, 2015). The mesocorticolimbic dopamine pathway
projects to the amygdala where dopamine signaling has
been shown to be necessary for fear conditioning
(Steinberg et al., 2013). Suppression of phasic DA release
following administration of a D2aR agonist quinpirole has
been shown to impair fear conditioning (Nader & LeDoux,
1999). If reduction in maternal fmr1 expression
dysregulates offspring DA signaling, then we asked
whether fear conditioning may also be disrupted in a
maternal genotype-specific manner in our mouse model.
Methods:
Fear Conditioning Schedule:
Subjects: Fmr1-KO mice (Consortium, 1994) on the FVB (FVB/NJ-Fmr1tm1Cgr)
background.
Group Sizes: KO(H) n=9; WT(H) n=10; WT(WT) n=9
All experiments and procedures were approved by Vassar College
Institutional Animal Care and Use Committee.
Discussion:
• Freezing behavior has been shown to vary with mouse strain,
with FVB line freezing about 25% and 129 line freezing 45% of
cued recall CS presentation (March et al., 2014).
• Although we have high variability in our groups, across genotypes
our fmr1 KO mice on an FVB background freeze at similar rates to
other FVB mice in the published literature (March et al., 2014).
• One study using fmr1 KO mice on a hybrid FVB/129 strain showed
WT mice freeze significantly more during presentation of the CS
compared to fmr1 KO (Paradee et al., 1999), though other studies
using fmr1 KO mice on just an FVB strain were unable to replicate
these results (Van Dam et al., 2000).
• Lesions of the orbital prefrontal cortex have been shown to block
extinction of a conditioned fear response (Zelinksi et al., 2010).
Previous work in our lab has shown orbital prefrontal cortical
mediated reversal learning deficits in fmr1 KO mice (Stoff, Erazo,
Newhall, Mendoza, Chan & Zupan, 2016). This deficit, however,
was observed in appetitive reversal learning and not extinction of
a conditioned fear response.
• Our results confirm other studies that also show no effect on fear
conditioning in fmr1 KO mouse line on FVB background (Van Dam
et al., 2000).
Further Research:
In order to better elucidate the role of the Fmr1 KO mutation in fear
conditioning, this study should be replicated in a non-FVB fmr1 KO
mouse line that has higher basal response rates to fear conditioning.
Additionally, we hope to examine the effect of the fmr1 mutation on
acquisition and established extinction of the fear memory. As
dysregulation of dopamine signaling has been associated with other
maternal fmr1 genotype-dependent behaviors, it may be of interest
to assess the efficacy of DAergic agonists/antagonists on modulating
fear conditioning.
References:
Results:
Figure 2. CS+ Extinction.
Figure 1. Context Fear.
- 3x CS-US
- CS: 5kHz, 75 dB,
20s duration
- US: 1s, 0.6mA
foot shock
co-terminating
with CS
- ITI = 20-80s
- Ethanol odor
- 5 minute
re-exposure
- Ethanol
odor
- 20x CS
- CS: 5kHz, 75 dB
20s duration
- ITI = 20-80s
- Acetic acid odor
KO(H) WT(H) WT(WT)
1. Chu, D., Gale, J., Zupan, B. (2015) Maternal Fmr1 mutation reduces D2S expression in offspring
VTA but not SN. Poster.
2. Consortium, Helm, R. Van Der, Oerlemans, F., Hoogeveen, T., & Oostra, B. A. (1994). Fmrl
Knockout Mice : A Model to Study Fragile X Mental Retardation, 78, 23–33.
3. Nader, K., & LeDoux, J. (1999). The dopaminergic modulation of fear: quinpirole impairs the
recall of emotional memories in rats. Behavioral Neuroscience, 113(1), 152–165.
4. Newhall, K., Zupan, B., (2014) Effect of educed maternal FMRP expression on reversal learning in
male mice. Poster.
5. Paradee, W., Melikian, H. E., Rasmussen, D. L., Kenneson, A., Conn, P. J., & Warren, S. T. (1999).
Fragile X mouse: Strain effects of knockout phenotype and evidence suggesting deficient
amygdala function. Neuroscience, 94(1), 185–192.
6. Steinberg, E. E., Keiflin, R., Boivin, J. R., Witten, I. B., Deisseroth, K., & Janak, P. H. (2013). A
causal link between prediction errors, dopamine neurons and learning. Nature Neuroscience,
16(7), 1–10.
7. Stoff, E., Erazo, J., Newhall, K., Mendoza, M., Chan, C., & Zupan, B. (2016). Deficits in PFC-Dependent
Reversal Learning in fmr1 Knockout Mice. Poster.
8. Van Dam, D., D’Hooge, R., Hauben, E., Reyniers, E., Gantois, I., Bakker, C. E., … De Deyn, P. P.
(2000). Spatial learning, contextual fear conditioning and conditioned emotional response in
Fmr1 knockout mice. Behavioural Brain Research, 117(1-2), 127–136.
9. Zupan, B., & Toth, M. (2008). Wild-type male offspring of fmr-1+/- mothers exhibit
characteristics of the fragile X phenotype. Neuropsychopharmacology: 33(11), 2667–75.
10. Zelinski, E.L., Hong, N.S., Tyndall, A.V., Halsall, B., McDonald, R.J. (2010). Prefrontal cortical
contributions during discriminative fear conditioning, extinction, and spontaneous recovery in rats.
Exp. Brain Res. 203: 285-297
MeanPercentFreezing
**
Context A Context A Context B
No difference between groups in
context fear test (1-Way ANOVA,
F(2,25)=1.130, p=0.399).
No marked change in percent
difference between first 3 CS+ and
last 3 CS+ (1-Way ANOVA,
F=(2,25)=0.800, p=0.460).
- 3x CS
- CS: 5kHz, 75 dB
20s duration
- ITI = 20-80s
- Acetic acid odor
Context A Context A Context B Context B
Figure 3. Percent Difference Between Recall & Extinction.
No significant difference between
groups in extinction of CS+
(Repeated Measures ANOVA,
F(2,25)=0.726, p=0.494), but
significant effect of time
(F(162.821)=6.744, p<0.001).
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CS 1 CS 2 CS 3
MeanPercentFreezing
KO(H)
WT(H)
WT(WT)
No significant difference between
groups in extinction recall test
(Genotype: F(2,25)=.815,p=.454,
Genotype*Time F(4,50)=.349,
p=.844), but there is a trend in time
(F(2,50)=2.900, p=.064).
Figure 4. Extinction Recall Test.
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MeanPercentFreezing
Conditioned Stimulus
KO(H)
WT(H)
WT(WT)
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MeanPercentFreezingDifference
KO(H) WT(H) WT(WT)