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Distance Traveled Relativized to
Haibutaiton(%)
WT(WT) WT(H) KO(H)
Maternal Fmr1 Deficit Impairs Offspring Contextual Fear Memory Retrieval
Alex Koo ’18, Kevin Newhall ’17, Hadley Bergstrom, 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:
Fear Memory Acquisition Fear Memory Extinction
Subjects: Fmr1-KO mice (Consortium, 1994) on the FVB (FVB/NJ-Fmr1tm1Cgr)
background.
Group Sizes: KO(H) n=17; WT(H) n=21; WT(WT) n=21
All experiments and procedures were approved by Vassar College
Institutional Animal Care and Use Committee.
Discussion:
Phasic DA release in the VTA has been implicated in behavioral
flexibility and learning, while intra-VTA quinpirole and intra-BLA
sulpiride (a D2R antagonist) injections have been found to
reduce conditioned freezing response to contextual cues,
highlighting the role of the mesocorticolimbic dopamine
pathway in fear expression (Adamantidis et al. 2011; de Souza
Caetano, de Oliveira, & Brandão, 2013). Reduction in maternal
fmr1 expression leads to an impairment in contextual fear
retrieval in the progeny in a manner that reflects the DA
signaling dysregulation previously observed. However, the
mechanism governing the discrepancy in fear expression has
yet to be determined. While freezing has traditionally been
used in the literature as the standard measure of fear, it has
also been shown to vary with mouse strain (March et al.,
2014). Stiedl et al. (1999) argued that measuring behavioral
measures (e.g. activity and exploratory area) other than
autonomic responses (i.e. freezing) is necessary, showing a
correlation between the three variables. Furthermore, another
study found activity suppression to be a strong indicator of fear
comparable to freezing (Anagnostaras et al., 2000). In an
ongoing investigation to confirm the contextual retrieval deficit
in Het-derived mice, we are currently analyzing our existing
data. We are also investigating whether there is a deficit in
cued extinction learning, given the suppression of locomotor
activity relative to habituation in Het-derived mice at the end
of the extinction trial when compared to the responses of the
WT(WT) mice.
References:
Results:
Figure 3. Retrieval deficit is context, but not cue-specific
Figure 1. Fear memory acquisition unaffected by Fmr1 genotype
- 5x CS-US
- CS: 5kHz, 75 dB, 20s duration
- US: 1s, 0.6mA foot shock co-terminate
with CS
- ITI = 20-80s
- 70% ethanol odor
- 20x CS
- CS: 5kHz, 75 dB, 20s duration
- ITI = 20-80s
- 1% acetic acid odor
1. Adamantidis, A. R., Tsai, H. C., Boutrel, B., Zhang, F., Stuber, G. D., Budygin, E. A., Touriño, C., Bonci, A., Deisseroth, K., & de Lecea, L. (2011). Optogenetic interrogation of dopaminergic
modulation of the multiple phases of reward-seeking behavior. The Journal of Neuroscience, 31(30), 10829-10835.
2. Anagnostaras, S. G., Josselyn, S. A., Frankland, P. W., & Silva, A. J. (2000). Computer-assisted behavioral assessment of Pavlovian fear conditioning in mice. Learning & Memory, 7(1), 58-72.
Chu, D., Gale, J., Zupan, B. (2015) Maternal Fmr1 mutation reduces D2S expression in offspring VTA but not SN. Poster.
3. 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.
4. de Souza Caetano, K. A., de Oliveira, A. R., & Brandao, M. L. (2013). Dopamine D2 receptors modulate the expression of contextual conditioned fear: role of the ventral tegmental area and
the basolateral amygdala. Behavioural pharmacology, 24(4), 264-274.
5. Gunaydin, L. A., Grosenick, L., Finkelstein, J. C., Kauvar, I. V., Fenno, L. E., Adhikari, A., Lammel, S., Mirzabekov, JJ., Airan, R.D., Zalocusky, K.A., Tye, K.M., Anikeeva, P., Malenka, R.C., &
Deisseroth, K. (2014). Natural neural projection dynamics underlying social behavior. Cell, 157(7), 1535-1551.
6. 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.
7. Newhall, K., Zupan, B., (2014) Effect of educed maternal FMRP expression on reversal learning in male mice. Poster.
8. Stiedl, O., Radulovic, J., Lohmann, R., Birkenfeld, K., Palve, M., Kammermeier, J., SananBenesi, F. & Spiess, J. (1999). Strain and substrain differences in context-and tone-dependent fear
conditioning of inbred mice. Behavioural brain research, 104(1), 1-12.
9. 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.
10. 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.
From Stiedl, O., Radulovic, J., Lohmann, R., Birkenfeld, K., Palve, M., Kammermeier, J., Sananbenesi, F., & Spiess, J.
(1999). Strain and substrain differences in context-and tone-dependent fear conditioning of inbred mice. Behavioural
brain research, 104(1), 1-12.
Figure 2. Maternal Fmr1 deficit attenuates fear memory retrieval
24 hrs
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Mean Freezing (%)
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Distance Traveled (cm)
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Figure 4. Locomotor activity is a valid and FVB strain-
appropriate behavioral measurement of fear learning
Two-factor Mixed Measures ANOVA with a Greenhouse-Geisser correction found an effect of time
(F(11.354,635.831)=25.537, p<0.005) and genotype (F(2,56)=8.151, p=0.001) but no time*genotype interaction
(F(22.708,635.831)=1.132, p=0.304). Locomotor activity during all CS presentations except CS19 were lower than
that during habituation (p<0.05 to p<0.005). A Bonferroni post-hoc test revealed that WT(WT) mice showed lower
locomotor activity than WT(H) (p<0.01) or KO(H) (p<0.01) mice.
Two-factor Mixed Measures ANOVA found
an effect of time (F(2,56)=2.358, p=0.104)
but no effect of genotype ((F(5,
280)=82.032, p<0.001) or time*genotype
interaction (F(10,280)=1.18, p=0.304).
Fear memory acquisition is equal across
groups. Bonferroni post hoc showed that
locomotor activity was significantly
increased (CS1, p<0.01) and decreased
(CS2-5, p<.001 all) relative to habituation
period.
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Distance Traveled (cm)
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Acquisition Extincition
Distance Traveled (cm)
WT(WT) WT(H) KO(H)
ND
ND
**
***
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Fold change in locomotor activity
during habituation period (Ext/Acq)
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** ND
Two-factor Mixed Measures ANOVA found an effect
of time*genotype (F(2,55)=4.043, p=0.0023). All mice
show contextual fear learning as each group shows
reduced locomotor activity during habituation during
extinction relative to acquisition. A Bonferroni post-
hoc test revealed that WT(WT) mice showed reduced
locomotor activity during the extinction trial relative
to WT(H) (p<0.001) and KO(H) (p=0.004) mice. There
was a difference in fold change in locomotor activity
during the two habituation periods between groups
as determined by one-way ANOVA (F(2,57)=7.745,
p=0.001). A Bonferroni post-hoc test revealed that
WT(WT) mice showed reduced locomotor activity
relative to WT(H) (p=0.002) and KO(H) (p=0.015)
mice. Two-factor Mixed Measures ANOVA with a
Greenhouse-Geisser correction found an effect of
time (F(9.153, 585.787)=19.073, p<0.005) and
time*genotype (F(27.459,585.787)=1.586 p=0.030)​
but no effect of genotype (F(3,64)=1.840, p=0.149)
on distance traveled relativized to distance traveled
during habituation during extinction. **: p<0.01;
***: p<0.001; ND = no difference.
Acquisition Extinction
Highly variable and low levels of freezing in the FVB mouse strain
led to inconclusive acquisition and extinction trial results (above).
Strong correlation between freezing and attenuation of locomotor
activity as measures of fear in mice have previously been reported
by Stiedl et al. (1999).
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Mean Freezing (%)
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