*For correspondence:
tmastro@caltech.edu (TLM);
kennedym@its.caltech.edu (MBK)
Competing interests:
The
authors declare that no
competing interests exist.
Funding:
See page 15
Received:
11 October 2019
Accepted:
14 January 2020
Published:
15 January 2020
Reviewing editor:
Leslie C
Griffith, Brandeis University,
United States
Copyright Mastro et al. This
article is distributed under the
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Attribution License,
which
permits unrestricted use and
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credited.
A sex difference in the response of the
rodent postsynaptic density to synGAP
haploinsufficiency
Tara L Mastro
1
*, Anthony Preza
1
, Shinjini Basu
2
, Sumantra Chattarji
2,3
,
Sally M Till
2
, Peter C Kind
2,3
, Mary B Kennedy
1
*
1
Division of Biology and Biological Engineering, Caltech, Pasadena, United States;
2
Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences,
University of Edinburgh, Edinburgh, United Kingdom;
3
Centre for Brain
Development and Repair, Bangalore, India
Abstract
SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the
scaffold protein PSD-95. We previously reported that heterozygous deletion of
Syngap1
in mice is
correlated with increased steady-state levels of other key PSD proteins that bind PSD-95, although
the level of PSD-95 remains constant (Walkup et al., 2016). For example, the ratio to PSD-95 of
Transmembrane AMPA-Receptor-associated Proteins (TARPs), which mediate binding of AMPA-
type glutamate receptors to PSD-95, was increased in young
Syngap1
+/-
mice. Here we show that
only females and not males show a highly significant correlation between an increase in TARP and a
decrease in synGAP in the PSDs of
Syngap1
+/-
rodents. The data reveal a sex difference in the
adaptation of the PSD scaffold to synGAP haploinsufficiency.
Introduction
SynGAP is a Ras/Rap GTPase Activating Protein that is specifically expressed in neurons and is highly
concentrated in the postsynaptic density (PSD) of glutamatergic synapses in the brain (
Chen et al.,
1998
;
Kim et al., 1998
). Mutations that cause heterozygous deletion or dysfunction of the human
gene
Syngap1
cause a severe form of intellectual disability (synGAP haploinsufficiency, also called
Mental Retardation type 5 [MRD5]) often accompanied by autism and/or seizures (
Berryer et al.,
2013
;
Hamdan et al., 2011
;
Hamdan et al., 2009
). In mice, heterozygous deletion of the gene
Syn-
gap1
causes similar neurological deficits; homozygous deletion causes death a few days after birth
(
Komiyama et al., 2002
;
Vazquez et al., 2004
).
One function of synGAP is to regulate the balance of active Ras and Rap at the postsynaptic
membrane (
Walkup et al., 2015
), thereby controlling the balance of exocytosis and endocytosis of
AMPA-type glutamate receptors (
Zhu et al., 2002
) and contributing to regulation of the actin cyto-
skeleton (
Tolias et al., 2005
). In a recent paper in eLife (
Walkup et al., 2016
), we postulated that
synGAP also helps to regulate anchoring of AMPA-type glutamate receptors (AMPARs) in the PSD.
AMPARs are tethered to the scaffold protein PSD-95 by auxiliary subunits called TARPs (Transmem-
brane AMPA Receptor-associated Proteins,
Tomita et al., 2003
). TARPs contain a PDZ ligand that
binds to PDZ domains in PSD-95. An early event in induction of long-term potentiation (LTP) is
increased trapping of AMPARs that is mediated by enhanced binding of TARPs to PDZ domains
(
Opazo and Choquet, 2011
;
Tomita et al., 2005
). SynGAP is also anchored in the PSD by binding
of its
a
1 splice variant to the PDZ domains of PSD-95 (
Kim et al., 1998
;
McMahon et al., 2012
;
Walkup et al., 2016
). SynGAP is nearly as abundant in the PSD fraction as PSD-95, which suggests
that it occupies a large fraction of the PDZ domains and can compete with TARPs for binding to
PSD-95 (
Chen et al., 1998
;
Dosemeci et al., 2007
). During induction of LTP, calcium/calmodulin-
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dependent protein kinase II (CaMKII) phosphorylates synGAP, increasing the rate of inactivation of
Rap relative to Ras, and, at the same time, causing a decrease in the affinity of synGAP-
a
1 for the
PDZ domains of PSD-95 (
Walkup et al., 2015
;
Walkup et al., 2016
). We postulated that the
decreased affinity of synGAP for PSD-95 might contribute to induction of LTP by allowing TARPs
and their associated AMPARs to compete more effectively for binding to the PDZ domains and thus
increase their anchoring in the PSD. If this hypothesis is correct, one consequence could be that
induction of LTP would be disrupted in synGAP heterozygotes because the transient shift in compe-
tition for PDZ binding by synGAP would be less potent because of loss of a copy of S
yngap1
. A sec-
ond possible consequence could be that the steady state level of TARPs bound to PSD-95 in PSDs
would be increased in synGAP heterozygotes because the steady state level of synGAP is reduced.
In the study that prompted the present work (
Walkup et al., 2016
), we measured the ratios to
PSD-95 of TARPs, LRRTM2, neuroligin-1 and neuroligin-2 in PSD fractions prepared from six pooled
forebrains of
wild type
(WT) mice and six of S
yngap1
+/-
(HET) mice. The WT animals comprised three
9.5 and two 7.9 week old males and one 12.5 week old female. The HETs comprised three 12.5
week old males, one 7.9 week old male, and two 9.5 week old females. The mean ratio of synGAP
to PSD-95 was 25% less in PSDs from the HET mice compared to WT. As we had predicted, the
mean ratio of TARPs to PSD-95 showed a small (12%) but significant increase in PSDs from the HET
animals compared to WT. We also found a small but significant increase in the mean ratio of
LRRTM2 (14%) and neuroligin-2 (9%) to PSD-95. The mean ratio of neuroligin-1 to PSD-95 was
unchanged.
Because the number of pooled brains in this previous study was small and WT and HET pools
were not perfectly balanced by developmental age or sex, we set out to expand these findings with
a larger data set gathered from PSDs isolated from individuals rather than from pooled animals.
Data from individuals allowed us to use a more rigorous statistical measure of correlation, the well-
established Spearman’s rank correlation coefficient r. Comparison of mean levels of two proteins in
pooled samples is not a perfect measure of the correlation between the two levels in individuals. It is
possible to have a correlation between protein levels in individuals that is not reflected as a differ-
ence between mean levels. Spearman’s r tests whether a monotonic correlation exists between the
rank order of magnitudes of two variables in a data set. We used it to examine the correlation of lev-
els of synGAP with levels of four other proteins in individual PSD fractions. If the rank orders of two
variables correlate perfectly, Spearman’s r is 1; if there is no correlation, it is zero; and if the ranks
are perfectly anti-correlated, it is
1.
When the data were averaged for WT and HET animals in this large data set, we were surprised
to find that the mean TARP/PSD-95 ratio in PSDs was not different between WT and HET animals, in
contrast to our earlier finding. However, when we calculated Spearman’s r for individual data sets,
we made the unexpected discovery that a strong inverse correlation between the levels of TARP and
synGAP is present only in females and not in males. The large and highly significant inverse correla-
tion in HET females drives a significant inverse correlation data from all HET animals and from all
female animals. The inverse correlation is not found in any subset of animals that contains only
males. We also established that a similar sex difference is present in rats, as well as mice. We
repeated the finding of the earlier study that there is no difference in the level of neuroligin-1
between WT and HET rodents; but, there is a small increase in the amount of neuroligin-2 in HETs.
Finally, we made the additional discovery that the level of synGAP correlates positively with the level
of the NMDA-type glutamate receptor GluN2B. In other words, the level of NR2B is reduced in the
PSDs of HET rodents.
Results
Creation of rat synGAP KO by the CRISPR-Cas9 method
CRISPR/Cas9 technology was used to establish a
Syngap1
KO rat line that harbors a frameshift muta-
tion in exon8 of
Syngap1
(
Figure 1A
), which prevents expression of the protein. SynGAP protein
expression level is reduced by 50% in HET knockout rats compared to wild-type (WT) and is absent
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in homozygous knockouts (
Figure 1B
). While SynGAP KO rats die perinatally, SynGAP HET rats
appear healthy and fertile.
Average synGAP/PSD-95 and TARP/PSD-95 ratios in WT and HET
rodents
We devised a method to isolate PSD fractions from individual mice and rats as described under
Materials and Materials and methods. PSD fractions were prepared from the forebrains of 165 indi-
vidual rodents, comprising 82 WT and 83 HETs. The total sample included 81 females (39 WT, 42
HET), and 84 males (43 WT, 41 HET). In each category, approximately half of the animals were rats
and half were mice; approximately half were 7.5 weeks old and half were 12.5 weeks old. The ratio
of synGAP/PSD-95 and TARP/PSD-95, averaged over all of the rodents, are summarized in the two
bars labeled ‘All’ (
Figure 2A and B
, left). As expected, the synGAP/PSD-95 ratio (
Figure 2A
, left) is
reduced by 22% in HET rodents compared to WT (the WT level is indicated by a dotted line). How-
ever, the ratio of TARP to PSD-95 (
Figure 2B
, left) is not significantly different, even when the results
were averaged for animals grouped by sex, species, and age (
Figure 2A and B
), except for seven
wk old female mice in which the ratio of TARPs to PSD-95 was significantly reduced compared to
WT. This value may have been influenced by a developmental effect causing lower overall expression
of TARPs in 7 week old mice. We also noted more variability in the averaged ratios of TARP to PSD-
95 for females (
Figure 2B
, right) compared to males (
Figure 2B
, middle). Taken as a whole, the aver-
aged results do not reproduce our original finding in
Walkup et al. (2016)
.
Spearman’s correlation coefficient reveals that the synGAP/PSD-95 and
TARP/PSD-95 ratios are inversely correlated only in females
We examined the correlation between levels of TARP and synGAP among individual rodents in each
sample using the more sensitive measure, Spearman’s r. We used Spearman’s r rather than Pearson’s
r for these measurements because many of the data sets showed a non-normal distribution. Pear-
son’s r is valid only for normally distributed data.
Figure 1.
Generation of synGAP null rats. (
A
) SynGAP targeting strategy introduces a frame shift mutation into
exon 8. (
B
) Quantification of synGAP immunoblots (inset) of cortical homogenates was performed as described in
Materials and methods.
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Figure 2.
Averaged ratios of synGAP and TARPs to PSD-95 in PSDs from WT and HET Rats and Mice. PSDs were
purified from the brains of individual animals as described under Materials and methods. The ratios of synGAP to
PSD-95 (
A
) and TARPs to PSD-95 (
B
) were determined as described under Materials and methods and in
Figure 2—figure supplement 1
. Ratios from HET animals (bars) are normalized to the ratios from WT animals
(dotted lines). Antibodies against synGAP, TARPS, and PSD-95 are the same as those used in
Walkup et al.
(2016)
. The antibody against synGAP (AB_2287112) recognizes all isoforms of synGAP. The antibody against
TARPs (AB_877307) recognizes TARP-
g
2,
g
3,
g
4, and
g
8. The sample sizes for each group and the significance tests
are as follows. A) all animals WT = 79 and HET = 78, one-tailed Wilcoxon matched-pairs signed rank test; male
mouse 7.5 weeks WT = 11 and HET = 9, one-tailed Student T-test; male mouse 12.5 weeks WT = 11 and HET = 8,
one-tailed Student T-test with Welch’s correction; male rat 7.5 weeks WT = 11 and HET = 10, one-tailed Student
T-test; male rat 12.5 weeks WT = 10 and HET = 11, one-tailed Student T-test; female mouse 7.5 weeks WT = 10
and HET = 12, one-tailed Student T-test with Welch’s correction; female mouse 12.5 WT = 9 and HET = 9, one-
tailed Student T-test; female rat 7.5 weeks WT = 10 and HET = 10, one-tailed Student T-test; female rat 12.5
weeks WT = 9 and HET = 10, one-tailed Student T-test. B) all animals WT = 77 and HET = 80, two-tailed Wilcoxon
matched-pairs signed rank test; male mouse 7.5 weeks WT = 10 and HET = 9, two-tailed Student T-test; male
mouse 12.5 weeks WT = 10 and HET = 10, two-tailed Mann Whitney test; male rat 7.5 weeks WT = 10 and
HET = 10, two-tailed Student T-test; male rat 12.5 weeks WT = 10 and HET = 1, two-tailed Student T-test; female
mouse 7.5 weeks WT = 9 and HET = 10, two-tailed Student T-test; female mouse 12.5 WT = 9 and HET = 10, two-
tailed Mann Whitney test; female rat 7.5 weeks WT = 10 and HET = 10, two-tailed Student T-test; female rat 12.5
Figure 2 continued on next page
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The average intensities of staining for proteins differed significantly between the cohorts, presum-
ably because of developmental changes in protein expression. We therefore normalized the ratios
for all cohorts to account for these average differences, as described under Materials and methods.
The normalization enabled us to look for correlations between ratios among individuals across
cohorts.
Figure 3B and C
contain plots for all WT and all HET animals, respectively. These data show that,
at steady state in vivo, lower amounts of synGAP in PSDs from the HET animals (
Figure 3C
) correlate
with higher amounts of TARP; whereas there is no correlation in WT animals (
Figure 3B
). This finding
supports our original report (
Walkup et al., 2016
).
Figure 2 continued
weeks WT = 9 and HET = 10, two-tailed Student T-test with Welch’s correction. Significance: * for p
0.05, ** for
p
0.01, *** for p
0.001, and **** for p
0.0001.
The online version of this article includes the following figure supplement(s) for figure 2:
Figure supplement 1.
Measurement of densities and calculation of ratios.
Figure 3.
Correlation of the ratios TARPs/PSD-95 and synGAP/PSD-95 among individual animals. Each point represents mean ratios for a single animal.
Corrected ratios and Spearman’s rank correlation coefficients were determined as described under Materials and methods. (
A
) All animals, including all
genotypes, ages, species, and sexes; n = 152. (
B
) All WT animals, including all ages, species, and sexes; n = 76. (
C
) All HET animals, including all ages,
species, and sexes; n = 76. (
D
) All female animals, including all genotypes, ages, and species; n = 75. (
E
) All WT females including all ages and species;
n = 36. (
F
) All HET females, including all ages and species; n = 39. (
G
) All male animals, including all genotypes, ages, and species; n = 77. (
H
) All WT
males, including all ages and species; n = 40. (
I
) All HET males, including all ages and species; n = 37. Black symbols, WT; Orange symbols, HET.
P-values for Spearman’s rank correlation coefficient are one-tailed. Significant p-values are shown in red.
The online version of this article includes the following figure supplement(s) for figure 3:
Figure supplement 1.
Intensity of PSD-95 bands on immunoblots.
Figure supplement 2.
Correlation analysis between synGAP/PSD-95 and TARP/PSD-95 for data from 7 and 12 week old female mice and rats.
Figure supplement 3.
Correlation analysis between synGAP/PSD-95 and TARP/PSD-95 for data from 7 and 12 week old male mice and rats.
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The data from all females (
Figure 3D
) show an inverse correlation between the two ratios that
just reaches statistical significance. In contrast, the data from all males (
Figure 3G
) shows no correla-
tion. Similarly, WT females and WT males (
Figure 3E and H
) show no correlation. Strikingly, the data
from HET females (
Figure 3F
) show the largest inverse correlation of all the data sets, with a Spear-
man’s r =
0.498 and a p-value=0.0006. HET males (
Figure 3I
) show no significant correlation. The
strong inverse correlation between the amount of synGAP and the amount of TARP in PSDs from
HET females (
Figure 3F
) likely drives the inverse correlation observed for pooled HET animals
(
Figure 3C
) and pooled females (
Figure 3D
).
We established that the amounts of PSD-95 per PSD protein are not statistically different
between HETs and WT or between male and female subgroups; they are also not correlated with
synGAP levels among individuals (
Figure 3—figure supplement 1
). Thus, differences among individ-
uals in the target protein/PSD-95 ratio can be interpreted as actual differences in the concentrations
of the target protein in the PSD fractions.
These results mean that, between 7.5 and 12.5 weeks of age, synGAP haploinsufficiency has a
much greater effect on the content of TARPs in the PSDs of female animals than in those of males.
The simplest explanation is that in HET females, the structure of the PSD, which is determined by
multiple equilibria among several proteins, is such that TARP and synGAP compete directly for bind-
ing to PSD-95; whereas in HET males, this particular competition is not significant. Possible underly-
ing mechanisms are outlined in the Discussion.
We also compared data sets from mice and rats at 7.5 weeks and 12.5 weeks (
Figure 3—figure
supplements 2
and
3
). These data sets were small (9 or 10 animals). Nevertheless, they show a sta-
tistically significant inverse correlation between TARP/PSD-95 and synGAP/PSD-95 in HET female
mice at both 7.5 and 12.5 weeks. In data from HET rats at 7.5 weeks, the inverse correlation is very
close to significance; at 12.5 weeks, it is less significant, but still shows a trend. In the corresponding
males, none of the data sets shows a statistically significant inverse correlation. More data would be
required to make a definitive conclusion, but the results suggest that competition between synGAP
and TARP for binding to PSD-95 in females is more prominent at 7 weeks than at 12 weeks, and
more prominent in mice than in rats.
Effect of synGAP haploinsufficiency on the relative levels of other PSD
proteins
In our previous paper, we examined the levels of neuroligins 1 and 2 (NLG-1,–2), and of the surface
protein LRRTM2. In this study, we re-examined the effect of reduction of synGAP on the levels of
NLG-1 and 2 in the PSD and looked at the effect on levels of GluN2B, a subunit of the NMDA-type
glutamate receptor that binds most avidly to PDZ2 of PSD-95. We predicted that the level of
GluN2B would be less affected than TARPs or NLGs by reduction of synGAP because synGAP has
lower affinity for PDZ2 than for PDZ1 and PDZ3 (
Walkup et al., 2016
).
The ratios of the three proteins to PSD-95 in HET and WT rodents, averaged over the same PSD
fractions shown in
Figure 2A and B
, are shown in the bars labeled ‘All’ in
Figure 4A,B and C
(left).
GluN2B exhibits a highly significant reduction of about 10% in HETs compared to WT; NLG-1 shows
no change; and NLG-2 increases significantly by about 7%. There is no significant difference in these
ratios between rat and mouse, males and females, or between 7.5 week and 12.5 week old animals.
The absence of any change in NLG-1 and the slight increase in NLG-2 recapitulate our findings in
Walkup et al. (2016)
.
Spearman’s correlation coefficient reveals that the levels of GluN2B
and levels of synGAP in PSDs are positively correlated
Figure 5
contains ratios of GluN2B to PSD-95 plotted against ratios of synGAP to PSD-95 measured
in the same set of individual PSDs shown in
Figure 3A,D and G
. Data for all animals (
Figure 5A
),
female animals (
Figure 5B
), and male animals (
Figure 5C
) show positive Spearman’s r values of
~
0.4
with p-values indicating a highly significant difference from zero. The positive correlation is present
in both WT (black) and HET (orange) animals. These results support our hypothesis that synGAP
does not compete with GluN2B for binding to PSD-95. To the contrary, the data suggest that a
higher level of synGAP leads to higher localization of GluN2B; and, therefore, NMDA-type gluta-
mate receptors, to the PSD (see Discussion).
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Figure 4.
Averaged ratios of GluN2B, NLG1, and NLG-2 to PSD-95 in mice and rat syngap1 HETs. PSDs were
purified as in
Figure 2
. Ratios were determined as described under Materials and methods. Ratios from HET
animals (bars) are normalized to the ratios from WT animals (dotted lines). (
A
) GluN2B/PSD-95. Sample sizes and
significance tests are as follows: all animals WT = 81 and HET = 82, two-tailed Wilcoxon matched-pairs signed
rank test; male mouse 7.5 weeks WT = 11 and HET = 10, two-tailed Student T-test; male mouse 12.5 weeks
WT = 11 and HET = 10, two-tailed Student T-test; male rat 7.5 weeks WT = 11 and HET = 10, two-tailed Student
T-test; male rat 12.5 weeks WT = 10 and HET = 11, two-tailed Student T-test; female mouse 7.5 weeks WT = 10
and HET = 12, two-tailed Student T-test; female mouse 12.5 WT = 9 and HET = 9, one-tailed Student T-test;
female rat 7.5 weeks WT = 9 and HET = 10, two-tailed Student T-test with Welch’s correction; female rat 12.5
weeks WT = 9 and HET = 9, two-tailed Student T-test with Welch’s correction. (
B
) NLG-1/PSD-95. Sample sizes
and significance tests are as follows: all animals WT = 81 and HET = 83, two-tailed Wilcoxon matched-pairs signed
rank test; male mouse 7.5 weeks WT = 10 and HET = 10, two-tailed Student T-test; male mouse 12.5 weeks
Figure 4 continued on next page
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Spearman’s correlation coefficient shows no significant correlation
between levels of NLG-1 and 2 and levels of synGAP in PSDs
Our previous results showed only a small effect of synGAP haploinsufficiency on the level of NLG-2
in PSDs. The pooled data in
Figure 4C
reproduces those findings. However, the significance of
Spearman’s r between levels of NLG-2 and synGAP in PSDs shows only a strong trend toward an
inverse correlation (
Figure 6A
). As in our previous paper, both
Figure 4B
and
Figure 6B
show no
effect of synGAP haploinsufficiency on the amount of NLG-1 in PSDs.
Discussion
The most striking new result of this research advance is the discovery of a sex difference in the adap-
tation of the PSD scaffold to synGAP haploinsufficiency. Specifically, we show that a decrease in the
steady-state concentration of synGAP in rodent PSDs correlates with a higher concentration of
TARPs in PSDs only in females and not in males. In female HETs, the rank correlation coefficient
between the concentrations of TARP and synGAP in PSDs is
0.5, which suggests a relatively high
competition between the two proteins for binding to PDZ domains of PSD-95 in vivo. This competi-
tion does not affect the composition of PSDs in male HETs. SynGAP haploinsufficiency causes about
4% of cases of sporadic intellectual disability (ID) in humans, often accompanied by seizures and
autistic behaviors (
Berryer et al., 2013
). If the sex difference in the interaction of synGAP with
TARPs in the rodent PSD is also present in humans, it might result in differences between girls and
boys in the prevalence of some of the associated symptoms.
The PSD is formed by multiple interactions among the major scaffold proteins and ‘client’ signal-
ing proteins which are concentrated in the PSD by their association with the scaffold proteins (
Ken-
nedy, 2016
;
Kennedy et al., 2005
;
Sheng and Kim, 2011
). At any one time, the composition of a
PSD is a dynamic equilibrium among all the possible protein associations, driven by the relative con-
centrations of each protein in a spine, and the relative affinities of their mutual binding domains (e.g.
Gray et al., 2006
). The simplest interpretation of our present result isthat there is a difference
between males and females in the composition or regulation of proteins in PSDs which causes the
concentration of TARPs in PSDs to be sensitive to the steady-state concentration of synGAP in
females, but not males. This occurs despite the fact that there is no difference between males and
females in the amount of synGAP in PSDs of either genotype.
TARPs are subunits of AMPARs, and have been shown to immobilize AMPARs at the synaptic site
by binding to PDZ domains of PSD-95 (
Opazo and Choquet, 2011
;
Tomita et al., 2005
). In our
recent eLife paper, we postulated that synGAP-
a
1, which contains a PDZ ligand, competes with
TARPs for binding to PSD-95 and therefore helps to limit the number of AMPARs immobilized at the
synapse (
Walkup et al., 2016
). This hypothesis has two possible corollaries. One is that transient
phosphorylation of synGAP by CaMKII during induction of LTP, which reduces the affinity of its PDZ
ligand for PSD-95, will allow more binding of TARP to the PDZ domains; and thus contribute to
increased trapping of AMPARs. The second corollary, which is addressed in this study, concerns the
steady-state composition of PSDs in
Syngap
+/-
rodents. If, at steady-state, the concentration of
Figure 4 continued
WT = 11 and HET = 10, two-tailed Student T-test; male rat 7.5 weeks WT = 11 and HET = 10, two-tailed Student
T-test; male rat 12.5 weeks WT = 10 and HET = 11, two-tailed Student T-test; female mouse 7.5 weeks WT = 10
and HET = 12, two-tailed Student T-test; female mouse 12.5 WT = 10 and HET = 10, two-tailed Student T-test;
female rat 7.5 weeks WT = 10 and HET = 11, two-tailed Mann-Whitney test; female rat 12.5 weeks WT = 9 and
HET = 10, two-tailed Student T-test with Welch’s correction. (
C
) NLG-2/PSD-95. Sample sizes and significance tests
are as follows: all animals WT = 79 and HET = 79, one-tailed Wilcoxon matched-pairs signed rank test; male
mouse 7.5 weeks WT = 10 and HET = 10, one-tailed Student T-test; male mouse 12.5 weeks WT = 11 and
HET = 10, one-tailed Student T-test; male rat 7.5 weeks WT = 11 and HET = 10, one-tailed Student T-test; male rat
12.5 weeks WT = 10 and HET = 11, one-tailed Student T-test; female mouse 7.5 weeks WT = 9 and HET = 12, one-
tailed Student T-test with Welch’s correction; female mouse 12.5 WT = 10 and HET = 9, one-tailed Student T-test;
female rat 7.5 weeks WT = 9 and HET = 10, one-tailed Student T-test; female rat 12.5 weeks WT = 9 and HET = 8,
one-tailed Student T-test with Welch’s correction. Significance: * for p
0.05, ** for p
0.01, *** for p
0.001, and
**** for p
0.0001.
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Neuroscience
Figure 5.
Correlation of the ratios GluN2B/PSD-95 and synGAP/PSD-95 in individual animals. Each point
represents a single animal. Black, WT; Orange, HET. (
A
) All animals including all genotypes, ages, species, and
sexes. n = 158. (
B
) All female animals, including all genotypes, ages, and species. n = 77. C) All male animals,
Figure 5 continued on next page
Mastro
et al
. eLife 2020;9:e52656.
DOI: https://doi.org/10.7554/eLife.52656
9 of 17
Research advance
Biochemistry and Chemical Biology
Neuroscience