This study examined auditory fear conditioning in a mouse model of Fragile X syndrome. Mice from three genotypes - Fmr1 knockout (KO) mice with a heterozygous mother (KO(H)), wild-type (WT) mice with a heterozygous mother (WT(H)), and WT mice with a WT mother (WT(WT)) - underwent fear conditioning and extinction training. The results showed no significant differences between genotypes in context fear, fear conditioning acquisition, or fear extinction. This suggests that the Fmr1 mutation does not impact auditory fear conditioning or extinction in this mouse line on an FVB background.
Grin1 knockdown mice (Grin1KD) have a loss-of-function mutation that is caused by the insertion of foreign DNA into an intron of the Grin1 gene. Although this mouse does not model a
specific patient variant, it is a useful model to understand how brain functions are altered by a global reduction of NMDA receptors. Grin1KD mice display many phenotypes that are similar to patient symptoms, including increased motor activity, decreased muscle tone, increased stereotypic movements,
and impairments in multiple domains of cognition. We asked whether phenotypes of Grin1KD mice could be improved by genetic rescue of the knockdown mutation in adult mice. To do this, we used a tamoxifen inducible Cre recombinase to excise the mutation and restore the Grin1 gene to a wildtype configuration. We discovered that several forms of cognition were improved or even normalized when genetic rescue was performed in adult mice. Our results suggest that the adult brain has sufficient plasticity to overcome developmental insults to NMDA receptor function, which has important implications for patients with GRIN disorders.
While gene therapy may be possible in the future, there are a number of pharmacological interventions that can be tested in this model and quickly translated to patients. As an example, we
tested the ability of a ketogenic diet or dietary ketone ester to improve behavioural phenotypes of Grin1KD mice. We discovered that either the ketogenic diet or beta-hydroxybutyrate improved the Grin1KD phenotypes of hyperactivity, sociability, sensory processing, and spatial memory. Similar studies could be used to test novel pharmacological agents that increase NMDA receptor activity.
Mating behaviour of the small hive beetle, Aethina tumida. Matshidiso Pitswane
Behavioural Ecology: The objectives of the study was to check if there is a relationship between size and copulation and mounting time, (2) to test if there is correlation between antennating and copulation/mounting frequency or time, and (3) compare the general behaviour performance of each male when they are alone with the female (experiment one) vs. when the other male is present (experiment two).
cloning. Second, it is sensitive. Activities canbe detected WilheminaRossi174
cloning. Second, it is sensitive. Activities can
be detected in the purified GST-ORF pools
that simply cannot be detected in extracts or
cells, the starting point of both conventional
purification and expression cloning. Because
the GST-ORFs are individually expressed at
high levels and are largely free of extract
proteins after purification, activities can be
measured for hours without competing activ-
ities that destroy the substrate, the product, or
the enzymes.
In addition to the conventional use demon-
strated here, this array could be used in two
other ways: (i) to determine the range of poten-
tial substrate proteins for any protein-modifying
enzyme (such as a protein kinase) before genet-
ic or biochemical tests to establish authentic
substrates and (ii) to identify genes encoding
proteins that bind any particular macromole-
cule, ligand, or drug. Thus, one could rapidly
ascribe function to many presently unclassified
yeast proteins, complementing other genomic
approaches to deduce gene function from ex-
pression patterns, mutant phenotypes, localiza-
tion of gene products, and identification of in-
teracting partners.
References and Notes
1. H. Simonsen and H. F. Lodish, Trends Pharmacol. Sci.
15, 437 (1994).
2. Plasmid pYEX 4T-1 (Clontech, Palo Alto, CA) was
modified by the addition of a 140-nucleotide recom-
bination domain, 39 of its Eco RI site, linearized within
the recombination domain by restriction digestion,
and cotransformed with a genomic set of reamplified
ORFs that had the same ends as the linearized plas-
mid [ J. R. Hudson Jr. et al., Genome Res. 7, 1169
(1997)] into strain EJ 758 [MATa his3-D200, leu2-
3,112, ura3-52, pep4::URA3], a derivative of JHRY-
20-2Ca (5). Transformants obtained on synthetic
minimal (SD) 2 Ura drop-out plates [F. Sherman,
Methods Enzymol. 194, 3 (1991)] (.100 in all cases,
and more than five times the cut vector in 97% of the
cases) were eluted in batch and saved in 96-well
microtiter plates. The library contains 6080 ORF-
containing strains and 64 strains with vector only.
3. Cell patches were inoculated in SD 2 Ura liquid
medium, grown overnight, reinoculated, and grown
overnight in SD 2 Ura 2 Leu medium, and then
inoculated into 250 ml of SD 2 Ura 2 Leu medium,
grown to absorbance at 600 nm of 0.8, and induced
with 0.5 mM copper sulfate for 2 hours before har-
vest [I. G. Macreadie, O. Horaitis, A. J. Verkuylen,
K. W. Savin, Gene 104, 107 (1991)]. Cells were re-
suspended in 1 ml of buffer [50 mM tris-HCl (pH 7.5),
1 mM EDTA, 4 mM MgCl2, 5 mM dithiothreitol (DT T),
10% glycerol, and 1 M NaCl] containing leupeptin (2
mg/ml) and pepstatin (1 mg/ml), and extracts were
made with glass beads [S. M. McCraith and E. M.
Phizicky, Mol. Cell. Biol. 10, 1049 (1990)], followed
by supplementation with 1 mM phenylmethylsulfo-
nyl fluoride and centrifugation. GST-ORF fusion pro-
teins were purified by glutathione agarose chroma-
tography in buffer containing 0.5 M NaCl, essentially
as described [ J. ...
Grin1 knockdown mice (Grin1KD) have a loss-of-function mutation that is caused by the insertion of foreign DNA into an intron of the Grin1 gene. Although this mouse does not model a
specific patient variant, it is a useful model to understand how brain functions are altered by a global reduction of NMDA receptors. Grin1KD mice display many phenotypes that are similar to patient symptoms, including increased motor activity, decreased muscle tone, increased stereotypic movements,
and impairments in multiple domains of cognition. We asked whether phenotypes of Grin1KD mice could be improved by genetic rescue of the knockdown mutation in adult mice. To do this, we used a tamoxifen inducible Cre recombinase to excise the mutation and restore the Grin1 gene to a wildtype configuration. We discovered that several forms of cognition were improved or even normalized when genetic rescue was performed in adult mice. Our results suggest that the adult brain has sufficient plasticity to overcome developmental insults to NMDA receptor function, which has important implications for patients with GRIN disorders.
While gene therapy may be possible in the future, there are a number of pharmacological interventions that can be tested in this model and quickly translated to patients. As an example, we
tested the ability of a ketogenic diet or dietary ketone ester to improve behavioural phenotypes of Grin1KD mice. We discovered that either the ketogenic diet or beta-hydroxybutyrate improved the Grin1KD phenotypes of hyperactivity, sociability, sensory processing, and spatial memory. Similar studies could be used to test novel pharmacological agents that increase NMDA receptor activity.
Mating behaviour of the small hive beetle, Aethina tumida. Matshidiso Pitswane
Behavioural Ecology: The objectives of the study was to check if there is a relationship between size and copulation and mounting time, (2) to test if there is correlation between antennating and copulation/mounting frequency or time, and (3) compare the general behaviour performance of each male when they are alone with the female (experiment one) vs. when the other male is present (experiment two).
cloning. Second, it is sensitive. Activities canbe detected WilheminaRossi174
cloning. Second, it is sensitive. Activities can
be detected in the purified GST-ORF pools
that simply cannot be detected in extracts or
cells, the starting point of both conventional
purification and expression cloning. Because
the GST-ORFs are individually expressed at
high levels and are largely free of extract
proteins after purification, activities can be
measured for hours without competing activ-
ities that destroy the substrate, the product, or
the enzymes.
In addition to the conventional use demon-
strated here, this array could be used in two
other ways: (i) to determine the range of poten-
tial substrate proteins for any protein-modifying
enzyme (such as a protein kinase) before genet-
ic or biochemical tests to establish authentic
substrates and (ii) to identify genes encoding
proteins that bind any particular macromole-
cule, ligand, or drug. Thus, one could rapidly
ascribe function to many presently unclassified
yeast proteins, complementing other genomic
approaches to deduce gene function from ex-
pression patterns, mutant phenotypes, localiza-
tion of gene products, and identification of in-
teracting partners.
References and Notes
1. H. Simonsen and H. F. Lodish, Trends Pharmacol. Sci.
15, 437 (1994).
2. Plasmid pYEX 4T-1 (Clontech, Palo Alto, CA) was
modified by the addition of a 140-nucleotide recom-
bination domain, 39 of its Eco RI site, linearized within
the recombination domain by restriction digestion,
and cotransformed with a genomic set of reamplified
ORFs that had the same ends as the linearized plas-
mid [ J. R. Hudson Jr. et al., Genome Res. 7, 1169
(1997)] into strain EJ 758 [MATa his3-D200, leu2-
3,112, ura3-52, pep4::URA3], a derivative of JHRY-
20-2Ca (5). Transformants obtained on synthetic
minimal (SD) 2 Ura drop-out plates [F. Sherman,
Methods Enzymol. 194, 3 (1991)] (.100 in all cases,
and more than five times the cut vector in 97% of the
cases) were eluted in batch and saved in 96-well
microtiter plates. The library contains 6080 ORF-
containing strains and 64 strains with vector only.
3. Cell patches were inoculated in SD 2 Ura liquid
medium, grown overnight, reinoculated, and grown
overnight in SD 2 Ura 2 Leu medium, and then
inoculated into 250 ml of SD 2 Ura 2 Leu medium,
grown to absorbance at 600 nm of 0.8, and induced
with 0.5 mM copper sulfate for 2 hours before har-
vest [I. G. Macreadie, O. Horaitis, A. J. Verkuylen,
K. W. Savin, Gene 104, 107 (1991)]. Cells were re-
suspended in 1 ml of buffer [50 mM tris-HCl (pH 7.5),
1 mM EDTA, 4 mM MgCl2, 5 mM dithiothreitol (DT T),
10% glycerol, and 1 M NaCl] containing leupeptin (2
mg/ml) and pepstatin (1 mg/ml), and extracts were
made with glass beads [S. M. McCraith and E. M.
Phizicky, Mol. Cell. Biol. 10, 1049 (1990)], followed
by supplementation with 1 mM phenylmethylsulfo-
nyl fluoride and centrifugation. GST-ORF fusion pro-
teins were purified by glutathione agarose chroma-
tography in buffer containing 0.5 M NaCl, essentially
as described [ J. ...
Se han hecho públicos los resultados de un prometedor trabajo encabezado por investigadores del Hospital Infantil de Boston y la Facultad de Medicina de Harvard, que ha conseguido recuperar, utilizando terapia génica, parte de la audición de ratones sordos. El artículo, que ha merecido la portada de la prestigiosa revista Science Translational Medicine, promete abrir un abanico terapéutico para el tratamiento de la sordera genética en los seres humanos.
Translating from Animal Models to Human Schizophrenia - Insights into Pathoph...wef
Presentation made by Dr. Tony Grace at the Schizophrenia Research Forum's live webinar of May 4, 2017 - Dopamine in Schizophrenia—Cortical and Subcortical Pathophysiology - review recording of session at http://www.schizophreniaforum.org/forums/dopamine-schizophrenia%E2%80%94cortical-and-subcortical-pathophysiology
Se han hecho públicos los resultados de un prometedor trabajo encabezado por investigadores del Hospital Infantil de Boston y la Facultad de Medicina de Harvard, que ha conseguido recuperar, utilizando terapia génica, parte de la audición de ratones sordos. El artículo, que ha merecido la portada de la prestigiosa revista Science Translational Medicine, promete abrir un abanico terapéutico para el tratamiento de la sordera genética en los seres humanos.
Translating from Animal Models to Human Schizophrenia - Insights into Pathoph...wef
Presentation made by Dr. Tony Grace at the Schizophrenia Research Forum's live webinar of May 4, 2017 - Dopamine in Schizophrenia—Cortical and Subcortical Pathophysiology - review recording of session at http://www.schizophreniaforum.org/forums/dopamine-schizophrenia%E2%80%94cortical-and-subcortical-pathophysiology
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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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30.00
40.00
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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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
MeanPercentFreezing
Conditioned Stimulus
KO(H)
WT(H)
WT(WT)
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20.00
30.00
40.00
50.00
1
MeanPercentFreezingDifference
KO(H) WT(H) WT(WT)