Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2.

Abstract

Autism spectrum disorders comprise a range of neurodevelopmental disorders characterized by deficits in social interaction and communication, and by repetitive behaviour. Mutations in synaptic proteins such as neuroligins, neurexins, GKAPs/SAPAPs and ProSAPs/Shanks were identified in patients with autism spectrum disorder, but the causative mechanisms remain largely unknown. ProSAPs/Shanks build large homo- and heteromeric protein complexes at excitatory synapses and organize the complex protein machinery of the postsynaptic density in a laminar fashion. Here we demonstrate that genetic deletion of ProSAP1/Shank2 results in an early, brain-region-specific upregulation of ionotropic glutamate receptors at the synapse and increased levels of ProSAP2/Shank3. Moreover, ProSAP1/Shank2(-/-) mutants exhibit fewer dendritic spines and show reduced basal synaptic transmission, a reduced frequency of miniature excitatory postsynaptic currents and enhanced N-methyl-d-aspartate receptor-mediated excitatory currents at the physiological level. Mutants are extremely hyperactive and display profound autistic-like behavioural alterations including repetitive grooming as well as abnormalities in vocal and social behaviours. By comparing the data on ProSAP1/Shank2(-/-) mutants with ProSAP2/Shank3αβ(-/-) mice, we show that different abnormalities in synaptic glutamate receptor expression can cause alterations in social interactions and communication. Accordingly, we propose that appropriate therapies for autism spectrum disorders are to be carefully matched to the underlying synaptopathic phenotype.

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LETTER
doi:10.1038/nature11015
Autistic-like behaviours and hyperactivity in mice
lacking ProSAP1/Shank2
Michael J. Schmeisser1*, Elodie Ey2,3,4*, Stephanie Wegener5*, Juergen Bockmann1, A. Vanessa Stempel5, Angelika Kuebler1,
Anna-Lena Janssen1, Patrick T. Udvardi1, Ehab Shiban1{, Christina Spilker6, Detlef Balschun7, Boris V. Skryabin8,9,
Susanne tom Dieck10, Karl-Heinz Smalla11, Dirk Montag12, Claire S. Leblond2,3,4, Philippe Faure13, Nicolas Torquet2,3,4,
Anne-Marie Le Sourd2,3,4, Roberto Toro2,3,4, Andreas M. Grabrucker1, Sarah A. Shoichet5, Dietmar Schmitz5, Michael R. Kreutz6,
Thomas Bourgeron2,3,4, Eckart D. Gundelfinger11& Tobias M. Boeckers1
Autismspectrumdisorderscomprisearangeofneurodevelopmental
disorderscharacterizedbydeficitsinsocialinteractionandcommun-
ication,andbyrepetitivebehaviour1.Mutationsinsynapticproteins
such as neuroligins2,3, neurexins4, GKAPs/SAPAPs5and ProSAPs/
Shanks6–10wereidentifiedinpatientswithautismspectrumdisorder,
but the causative mechanisms remain largely unknown. ProSAPs/
Shanks build large homo- and heteromeric protein complexes at
excitatory synapses and organize the complex protein machinery of
the postsynaptic density in a laminar fashion11,12. Here we dem-
onstratethatgeneticdeletionofProSAP1/Shank2resultsinanearly,
brain-region-specificupregulationofionotropicglutamatereceptors
at the synapse and increased levels of ProSAP2/Shank3. Moreover,
ProSAP1/Shank22/2mutants exhibit fewer dendritic spines and
show reduced basal synaptic transmission, a reduced frequency of
miniature excitatorypostsynapticcurrents and enhancedN-methyl-
D-aspartate receptor-mediated excitatory currents at the physio-
logicallevel.Mutantsareextremelyhyperactiveanddisplayprofound
autistic-like behavioural alterations including repetitive grooming
as well as abnormalities in vocal and social behaviours. By com-
paring the data on ProSAP1/Shank22/2mutants with ProSAP2/
Shank3ab2/2mice, we show that different abnormalities in synaptic
glutamate receptor expression can cause alterations in social inter-
actions and communication. Accordingly, we propose that appro-
priate therapies for autism spectrum disorders are to be carefully
matched to the underlying synaptopathic phenotype.
Many of the recently identified autism spectrum disorders (ASD)
candidate genes code for proteins of excitatory synapses13–15, suggesting
that these disorders may arise from molecular imbalances of synaptic
connections. Inthis context,targeted disruptionof theProSAP2/Shank3
gene in mice resulted in molecular perturbations of glutamatergic
synapses and profound autistic-like behaviour16–19. Here we generated
mice lacking all isoforms of ProSAP1/Shank2 (Fig. 1A and Supplemen-
tary Fig. 1a–g) to decipher the interrelation between ProSAP1/Shank2
protein levels, synaptic architecture, neurophysiology and behaviour in
mice.
Heterozygous ProSAP1/Shank21/2(expressing approx. 50% of
ProSAP1/Shank2 protein, Fig. 1A) and homozygous ProSAP1/
Shank22/2mutantswereviable,but theirsurvival ratewaslowercom-
pared with wild-type littermates (Supplementary Fig. 2a). Although
body weight was reduced (Supplementary Fig. 2b–e), adult mutants
displayed normal appearance and overall brain morphology (data not
shown). However, hindlimb clasping was observed (Supplementary
Fig. 2f), similarly to some other mouse models of ASD20,21.
Owing to high expression of ProSAP1/Shank2 in the hippocampus
during spinogenesis22and as patient-based mutations in ProSAP1/
Shank2wererecentlyshowntoalterdendriticspinesinthehippocam-
pus23,weassessedspinedensityandsynapticultrastructureintheCA1
region. We found a small reduction of spine numbers in ProSAP1/
Shank22/2mutants (Fig. 1B, a) whereas postsynaptic density (PSD)
length or thickness was not significantly altered (Fig. 1B, b).
Biochemical analysis revealed higher levels of the N-methyl-D-
aspartate receptor (NMDAR) subunit GluN1 and ProSAP2/Shank3
in whole brain PSDs of ProSAP1/Shank22/2mice (Supplementary
Fig. 3a). Interestingly, ProSAP2/Shank3 upregulation specifically
occurred at synapses, as protein and messenger RNA (mRNA) levels
were not changed significantly in whole brain of mutant versus wild-
type animals (Supplementary Fig. 3b, c). Further evidence for local
compensation was apparent by subfractionation experiments and
transient knockdown of ProSAP1/Shank2 in primary hippocampal
cultures, resulting in a rapid increase of GluN1 and ProSAP2/
Shank3 at synaptic sites (Supplementary Fig. 3b, d).
Based on the ProSAP1/Shank2 expression profile in wild-type
mouse brain (Supplementary Fig. 4), we biochemically isolated crude
synaptosomal fractions from cortex, hippocampus and striatum of
wild-type, ProSAP1/Shank21/2and ProSAP1/Shank22/2mice at
postnatal day (P)25 and P70 to examine molecular alterations with
respect to brain regions and development. The major change com-
paredwithwildtypeswasanearlyincreaseofNMDARsubunitsinthe
hippocampus and striatum of ProSAP1/Shank22/2mutants. Notably,
this increase was sensitive to ProSAP1/Shank2 gene dosage as it was
also observed in ProSAP1/Shank21/2mice, but to a lesser extent. At
P70 the upregulation of ProSAP2/Shank3 was observed in all brain
regionsinvestigated(Fig.1C,DandSupplementaryFigs5and7;cortex
data not shown). We compared these observations with molecular
synaptic changes when major isoforms of ProSAP2/Shank3 are not
presentandanalysedProSAP2/Shank3ab2/2mice(similartorecently
published Shank3 mutants17, Supplementary Fig. 6). Especially in the
striatum, we observed a clear difference between ProSAP1/Shank22/2
andProSAP2/Shank3ab2/2micewithrespecttotheirsynapticcontent
of ionotropic glutamate receptors. The levels of most subunits were
higher in ProSAP1/Shank22/2and lower in ProSAP2/Shank3ab2/2
mice. Interestingly, apart from the increase of ProSAP2/Shank3 in
*These authors contributed equally to this work.
1Institute for Anatomy and Cell Biology, Ulm University, 89081 Ulm, Germany.2Human Genetics and Cognitive Functions, Institut Pasteur, 75724 Paris CEDEX 15, France.3CNRS, URA 2182 ‘Genes,
Synapses and Cognition’, Institut Pasteur, 75724 Paris CEDEX 15, France.4University Paris Diderot, Sorbonne Paris Cite ´, Human Genetics and Cognitive Functions, 75013 Paris, France.5Neuroscience
Research Center, Cluster of Excellence NeuroCure, Charite ´, 10117 Berlin, Germany.6PG Neuroplasticity, Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany.7Laboratory of Biological
Psychology, Department of Psychology, Catholic University of Leuven, 3000 Leuven, Belgium.8Institute of Experimental Pathology (ZMBE), University of Muenster, 48149 Muenster, Germany.
9Interdisciplinary Center for Clinical Research (IZKF), University of Muenster, 48149 Muenster, Germany.10Max Planck Institute for Brain Research, Department of Synaptic Plasticity, 60528 Frankfurt,
Germany.11Department of Neurochemistry, Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany.12Neurogenetics Special Laboratory, Leibniz Institute for Neurobiology, 39118 Magdeburg,
Germany.13UniversityParis06,CNRS,UMR7102,75005Paris,France.{Presentaddress:KlinikumrechtsderIsar,TechnischeUniversita ¨tMu ¨nchen,NeurosurgeryDepartment,IsmaningerStr.22,81675
Munich, Germany.
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ProSAP1/Shank22/2mice, we detected a vice versa upregulation of
ProSAP1/Shank2 in ProSAP2/Shank3ab2/2mice (Fig. 1C, D and
Supplementary Figs 5–7). This phenomenon was not due to an
increase of transcript levels and was observed in whole brain PSDs
from both animal models (Supplementary Fig. 8a–c).
To analyse how the altered molecular composition of ProSAP1/
Shank22/2synapses influences synaptic transmission, we performed
extracellular field and whole-cell patch clamp recordings from CA1
pyramidal cells in acute hippocampal slices. Field excitatory
postsynaptic potentials (fEPSPs) were decreased by approximately
40% in ProSAP1/Shank22/2(Fig. 2A) as well as ProSAP1/Shank21/2
animals (Supplementary Fig. 9a). The reduced synaptic transmission
wasnotonlyfoundinyoungmice(P21–P28),butalsoinolderanimals
(3monthsofage,Fig.2A,c,investigatedinProSAP1/Shank22/2only).
There was no evidence for genotypic differences in the excitability of
presynapticfibres,theintrinsicfiringthresholdandthewhole-cellinput
resistance of CA1 pyramidal cells (Supplementary Fig. 9b–d). When
analysing miniature excitatory postsynaptic currents (mEPSCs) from
CA1 pyramidal cells (Fig. 2B), ProSAP1/Shank22/2mice showed a
significant reduction in mEPSC frequency. There was no evidence for
differences in mEPSC amplitudes and a-amino-3-hydroxy-5-methyl-
4-isoxazole propionic acid (AMPA)-mediated whole-cell currents
(reflecting the total number of synaptic plus extrasynaptic AMPA
receptors) (Fig. 2C). To probe for possible changes in NMDAR-
mediated excitatory synaptic transmission, we compared the relative
contribution of NMDA versus AMPA receptors to evoked EPSCs. In
agreement with the upregulation of NMDAR subunits in hippocampi
of ProSAP1/Shank22/2mice (see Fig. 1C, D), we found an approxi-
mately30%increasedNMDA/AMPAratioinmutantsversuswildtypes
(Fig. 2D). We further analysed synaptic plasticity. NMDAR-dependent
long-term potentiation induced by high-frequency stimulation of the
Schaffer collaterals was slightly enhanced in ProSAP1/Shank22/2mice
(Fig.2E).Wefoundnoevidenceforalterationsinlong-termdepression
between genotypes (Supplementary Fig. 9e). As imbalanced excitation/
Hippocampus
Whole brain
B
Cerebellum
Forebrain
A
+/+ –/–
ProSAP1E/Shank2E
ProSAP1A/Shank2A
ProSAP1/Shank2
170
130
100
70
55
β-Actin
+/+ +/– –/–
+/+ +/+ –/–
0
20
40
60
80
100
120
ProSAP1/Shank2 mRNA (%)
Hippocampus
P = 0.07
+/+
***
0
2
4
6
8
10
12
14
16
18
20
Spine density (spines per 10 μm)
**
b
CA1
+/+
*
+/+
*
–/–
0
100
0.2
0.4
0.6
0.8
1
200300
PSD length (nm)
400500600
700
Cumulative frequency
+/+
–/–
0
100
200
300
400
nm
0
0.2
0.4
0.6
0.8
1
305580105130
Cumulative frequency
0
20
40
60
80
+/+
–/–
nm
PSD thickness (nm)
C
Cortex P70
ProSAP1/Shank2–/–
ProSAP2/Shank3αβ–/–
Hippocampus P70
Striatum P70
GluN1
GluN2A
GluN2B
GluA1
GluA2
GluA3
Shank3
Shank2
GluN1
GluN2A
GluN2B
GluA1
GluA2
GluA3
Shank3
GluN1
GluN2A
GluN2B
GluA1
GluA2
GluA3
Shank3
Shank2
Shank2
AMPAR
NMDAR
AMPAR
NMDAR
AMPAR
NMDAR
01–101–1
*
*
*
*
**
+ +
+
+
***
**
*
*
*
Relative to wild typeRelative to wild type
D
ProSAP2
Shank3
ProSAP1
Shank2
NMDAR
AMPAR
P25P70P70
ProSAP1/Shank2–/–
ProSAP2/Shank3αβ–/–
Hippocampus
Cortex
Striatum
NMDAR
AMPAR
–/–
+/– –/–
Increase
a
+/+ –/–
Decrease
Figure 1 | Cyto-architechtural and molecular changes in ProSAP1/
Shank22/2mousebrain. A,Westernblotofpooled(n510)wholebrains(left
panel) and cerebella (upper right panel) from wild-type (1/1) and ProSAP1/
Shank22/2(2/2)miceasindicated.ProSAP1/Shank2isoformsaremarkedby
arrowheads: ProSAP1/Shank2 (black), ProSAP1A/Shank2A (white),
ProSAP1E/Shank2E (grey). Forebrain wild-type homogenate was used as
control to differentiate cerebellar isoforms. Total ProSAP1/Shank2 mRNA
(middlepanel)andproteinlevels(rightlowerpanel)fromwild-type,ProSAP1/
Shank1/2(1/2) and ProSAP1/Shank22/2hippocampi. B, a, Representative
images of secondary dendrites from CA1 hippocampal neurons of adult wild-
type and ProSAP1/Shank22/2mice (Golgi–Cox staining, scale bar: 1 mM) and
quantificationof spine density from n56 wild-type(white bar)andProSAP1/
Shank22/2(black bar) littermate pairs. B, b, Representative electron
microscopy images of CA1 synapses from wild-type and ProSAP1/Shank22/2
animals. Synaptic vesicles (arrowheads), PSDs (arrows) and dendritic spines
(asterisks).Scalebar: 100nm.AnalysisofPSDlengthandthicknessfrom wild-
type and ProSAP1/Shank22/2animals (right panel). Data are presented as
cumulative frequency plots, small insets depict median values compared
between wild-type (white bars) and ProSAP1/Shank22/2(black bars) animals.
n5220PSDs forsixwild types andn5215PSDs forsixProSAP1/Shank22/2
mice.C,Semi-quantitativeanalysisofproteinsincrudesynaptosomalfractions
from different brain regions of wild-type, ProSAP1/Shank22/2and ProSAP2/
Shank3ab2/2mice as indicated. Mutant protein was normalized to wild-type
levels and is plotted as relative change of expression levels. D, Colour-coded
visualization of protein levels (ProSAP1/Shank2, ProSAP2/Shank3, NMDAR,
AMPAR) in ProSAP1/Shank22/2or ProSAP2/Shank3ab2/2brains (cortex,
hippocampus,striatum)attheindicatedtimepoints(P25,P70).C,D,Redbars/
colourindicateelevated,bluebars/colourdecreased,proteinlevels.A–D,1/1,
wildtypes;1/2,ProSAP1/Shank21/2;2/2,ProSAP1/Shank22/2.Alldataare
presented as mean6s.e.m.; all P values are derived from unpaired, two-tailed
Student’s t-tests (*P,0.05, **P,0.01, ***P,0.001).
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Page 3

inhibition ratios have been repeatedly implicated in models of autism24,
wealsoanalysedGABAergic(c-aminobutyricacid-mediated)synaptic
transmission. Frequency and amplitude of inhibitory postsynaptic
currents (both miniature and spontaneous) were largely unchanged
inProSAP1/Shank22/2mice(SupplementaryFig.10a,b).Basedonthe
electrophysiological analyses, we conclude that merely glutamatergic
transmission is impaired in ProSAP1/Shank2 mutants.
Despite these synaptic abnormalities, ProSAP1/Shank22/2mice
displayed functional working memory, motor coordination, olfaction
and object recognition (Supplementary Fig. 11a–g). The most
remarkablebehaviouralphenotypewashyperactivity.Whencompared
with wild-type littermates, male and female ProSAP1/Shank22/2mice
displayedtwicetheleveloflocomotoractivityintheopenfield(Fig.3a,
b)andinothertests(SupplementaryFig.12a–e).ProSAP1/Shank22/2
malesdisplayedanincreasedlevelofanxietyduringthelight–darkbox
test (Supplementary Fig. 12f, g). Compared with wild types, digging
boutsofProSAP1/Shank22/2miceweresignificantlyshorter(Fig.3c),
and self-grooming in ProSAP1/Shank22/2females was significantly
extended (Fig. 3d). These stereotyped behaviours, however, were less
severe compared with other mouse models of ASD such as ProSAP2/
Shank3ab2/2mutants17,20or BTBR T1tf/J mice25.
We next examined social behaviour. During free same-sex social
interactions (resident–intruder), the latency for the first contact did
not differ significantly (Fig. 4a), but both male and female ProSAP1/
Shank22/2mice had difficulties maintaining social contacts or were
less interested in them (Fig. 4b). During free interactions of a tested
malemousewithanoestrusC57BL/6femalemouse,thelatencyforthe
first contact was significantly longer for ProSAP1/Shank22/2males
than wild-type males (Fig. 4a), but no impairment in contact main-
tenance was detected (Fig. 4b). During the three-chamber test, both
male and female mutants displayed a reduction in conspecific recog-
nition or in their interest for social novelty compared with wild types
(Supplementary Fig. 13a–d).
Cumulative fraction of mEPSCs
–0.3 –0.2–0.1
Fibre volley (mV)
1.2
0.8
1.4
0.0
–1.0
+/+
A
Bb
D a
+/+
E ac
C
bc
+/–
–/–
–/–
–/–
+/+
–/–
+/+
+/+
+/+
+/+
–/–
–/–
–/–
–/–
+/+
–/–
+/+
–/–
+/+
–/–
+/+
–0.5
0.0
*
*
*
*
fEPSP slope (mV/ms)
0
mEPSC frequency (Hz)
–15
–10
–5
0
–40–200
0.0
0.5
1.0
Amplitude (pA)
05 10
–600
–400
–200
0
20 nM AMPA
Time (min)
Whole-cell current (pA)
0.0
0.5
1.0
NMDA/AMPA ratio
010 203040
0.5
1.0
1.5
2.0
2.5
3.0
**
***
Time (min)
fEPSP slope (norm.)
0
+/+ –/–
20
40
60
80
100
Potentiation (%)
0.5 mV
5 ms
0.2 mV
5 ms
20 pA
1 s
5 pA
20 ms
100 pA
50 ms
100 pA
50 ms
–0.4
–0.3
–0.2–0.1
–0.8
–0.4
0.0
Fibre volley (mV)
fEPSP slope (mV/ms)
0
a
a
b
c
+/+ –/–
b
Cummulative fraction
of mEPSCs
Figure 2 | Imbalancedhippocampalglutamatergicsynaptictransmissionin
ProSAP1/Shank22/2mice. A, Input–output curves for basal synaptic
transmission. As illustrated in the sample traces (A, a, averages of six fEPSPs)
and in the quantification (A, b), ProSAP1/Shank22/2(2/2) mice suffer from
reduced synaptictransmissioncompared with wild-typecontrols (1/1) at the
age of P21–P28 (two-way analysis of variance (ANOVA): P,0.05; 1/1:
number of experiments (n) and number of animals (N)58 (3); 2/2: n511
(4)). This defect is also found in mice that are 3 months of age (A, c) (two-way
ANOVA: P,0.05; 1/1: n (N)57 (3); 2/2: n (N)57 (3)). B, mEPSCs in
CA1 pyramidal cells. B, a, Sample traces of individual recordings (left) and an
average ofallmEPSCevents (right). B, b, The frequency ofmEPSCsisreduced
inProSAP1/Shank22/2(Student’st-test:P,0.05;1/1:n(N)512(4);2/2:n
(N)516 (5)). B, c, Cumulative fraction distribution of mEPSC amplitudes
(two-sampleKolmogorov–Smirnovtest:P50.96;1/1:504events,n(N)512
(4); 2/2: 630 events, n515 (5)). Inset: mean mEPSC amplitudes (Student’s
t-test: P50.37; sample sizes as above). C, Whole-cell currents evoked by bath
application of 20nM AMPA (Student’s t-test: P50.9; 1/1: n (N)58 (3);
2/2: n (N)59 (3)). D, NMDA/AMPA ratios estimated from compound
EPSCs evoked at 140 and 260mV, respectively. As illustrated in the sample
traces (D, a) and the quantification (D, b), the ratio of synaptic NMDA versus
AMPA receptors is significantly increased in ProSAP1/Shank22/2animals
(Student’st-test:P,0.05;1/1:n(N)518(8);2/2:n(N)519(6)).E,Long-
termpotentiationisincreasedinProSAP1/Shank22/2mice,asevidentfromthe
sample traces (E, a), the average time plot (E, b) (two-way ANOVA: P,0.01;
1/1: n (N)530(5); 2/2: n534 (6)) and the ratio of fEPSP slopes 30min
after versus before induction of long-term potentiation (E, c) (Student’s t-test:
P,0.001; sample sizes as above). All data are presented as mean6s.e.m.
*P,0.05, **P,0.01, ***P,0.001.
0
100
200
300
400
500
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0
2
4
6
8
10
12
14
a
******
**
bcd
+/+
–/–
******
**
********
******
–/–
+/–
+/+
Distance travelled (m)
Digging bout duration (s)
Grooming bout duration (s)
Figure 3 | Increased locomotor activity and stereotypical behaviours in
ProSAP1/Shank22/2mice. a, Examples of trajectories of a wild-type mouse
and a ProSAP1/Shank22/2mouse in 30min exploration of the open field.
b,Distancetravelledbymaleandfemalemiceduring30minfreeexplorationof
a circular maze. c, Mean digging bout duration in male and female mice.
d, Mean self-grooming bout duration in male and female mice. Data are
presented as mean6s.e.m. (Mann–Whitney U-tests: *P,0.05; **P,0.01;
***P,0.001). Unlessotherwisespecified,(n1/1516,n1/2516,n2/2516)
males and (n1/1516, n1/2516, n2/2513) females were tested.
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During free social interactions or when isolated as pups, mice emit
ultrasonic vocalizations. In pups, ProSAP1/Shank22/2females, but
not males, called at a significantly higher rate than wild-type females
at P4 and P10 (Supplementary Fig. 14a, b). In adults, during male–
male social interactions, few calls were recorded and there was no
significantdifferencebetweengenotypes(Fig.4c,d).Incontrast,during
female–female interactions, we observed a significantly longer latency
to emit the first vocalization (Fig. 4c) and significantly fewer vocaliza-
tions (Fig. 4d) for pairs involving a ProSAP1/Shank22/2mouse com-
pared with pairs involving wild-type females. Notably, in pairs
involving ProSAP1/Shank22/2mutants, mice uttered more short
and unstructuredcalls and fewer mixedcalls than pairs involving wild
types(SupplementaryFig.14c,e).Inthesocio-sexualcontextofamale
in the presence of an oestrus female, the latency forthe first ultrasonic
vocalization was significantly longer in pairs involving ProSAP1/
Shank22/2males than pairs involving wild types (Fig. 4c). Similarly
to females, more short and unstructured calls were emitted (Sup-
plementary Fig. 14d, e).
In conclusion, based on this study and on previous reports, mice
lacking any member of the ProSAP/Shank family display recurrent
featuresobservedinanimalmodelsforASD:thatis,preservedworking
memory, but increasedanxiety and abnormalities in both social inter-
actions and vocalizations (Supplementary Fig. 15)16–19,26–29.
In summary, here we demonstrate that altered glutamatergic
neurotransmissioncanleadtothecoresymptomsofASD.Inaddition,
this study shows that ProSAP1/Shank2 and ProSAP2/Shank3 seem to
serve different interrelated functions at excitatory synapses, especially
inglutamatereceptortargeting/assembly.However,theexactmolecular
mechanisms are still to be deciphered. In any case, our comparative
analysis of mice lacking either ProSAP1/Shank2 or major isoforms of
ProSAP2/Shank3revealsthatmutationsofverysimilarproteinswithin
the same synaptic pathway can have different molecular consequences
(for example, excess or deficit of glutamate receptors), but both lead to
abnormal social and vocal behaviours. Future studies should tell
whether gene- or pathway-specific therapies are necessary to modulate
or even reverse the pathophysiology of ASD.
METHODS SUMMARY
Biochemistry,Golgistainingandelectronmicroscopy.Asubfractionationpro-
tocol30was performed to obtain subcellular fractions from brain tissue of juvenile
and/oradultwild-type,ProSAP1/Shank2andProSAP2/Shank3mutantmicefrom
both sexes. After immersion in Golgi–Cox solution for 21 days, adult brains were
cutin 200mm sagittalsectionsto develop Golgi–Cox stainingforanalysis of spine
density. Further, adult mice were perfused, brains were dissected out, stained and
cut in ultrathin sections to be examined by electron microscopy.
Electrophysiology. Extracellular field and whole-cell patch-clamp recordings
were performed in horizontal hippocampal slices from mice of both sexes.
Evoked postsynaptic responses were induced by electrical stimulation of
Schaffer collaterals in CA1 stratum radiatum. fEPSPs were recorded in stratum
radiatum.Long-termpotentiationwasinducedbyasingletetanusof100pulsesat
100Hz. Long-term depression was induced by 15min paired pulse stimulation at
1Hz with 50ms between single pulses. mEPSCs, whole-cell AMPA currents and
inhibitorypostsynapticcurrents(IPSCs)wererecordedinwhole-cellpatch-clamp
configurationfromCA1pyramidalcellsvoltage-clampedat260mV. Forestima-
tion of NMDA/AMPA ratios, compound EPSCs were evoked at 260 and
140mV.
Behavioural analysis. Three cohorts of mice (C57BL/6 background) were tested.
Cohort 1 included pups for the developmental study to examine pup vocal beha-
viour, motor coordination, olfaction and developmental milestones. Adult beha-
viour was tested on cohort 2 in the following order: light–dark anxiety test, open
field, Y-maze, three-chamber test, self-directed and digging behaviours, resident–
intrudertest,malebehaviourinpresenceofanoestrusfemale,buried-foodfinding
test and object recognition. Cohort 3 was used for the general neurological exam-
ination, juvenile body weight and to analyse motor coordination.
All animal procedures were in accordance with institutional, state and govern-
ment regulations (Tu ¨bingen: O.103; Berlin: LAGeSo, T0100/03; Paris: CEEA Ile-
de-France Comite ´ 1).
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 17 November 2011; accepted 8 March 2012.
Published online 29 April; corrected 13 June 2012 (see full-text HTML version for
details).
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2.
3.
4.
5.
6.
7.
8.
9.
Latency first contact (s)
0
20
40
60
80
100
Time in contact (s)
0
50
100
150
200
Latency first call (s)
0
20
40
60
80
100
Call rate (calls min–1)
0
50
100
150
200
–/– +/–+/+
B6
B6 B6
B6
B6
B6
B6B6B6
B6B6 B6
*
* **
*
**
**
*
**
*
*
ab
cd
Figure 4 | Abnormalities in social and vocal behaviour of ProSAP1/
Shank22/2miceintheresident–intrudertestandduringtheinteractionofa
male with an oestrus female. a, Latency for the first contact in same-sex free
interactions (C57BL/6 resident–intruder) when the resident mouse was
isolatedfromweaningon(males)orfor3daysbeforetheexperiment(females),
andintheinteractionofamalewithaC57BL/6oestrusfemale.b,Timespentin
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n2/2516). c, Latency for the first ultrasonic vocalization in same-sex free
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anoestrusfemale.d,Rateofcallingduringsame-sexfreeinteractionswhenthe
resident mouse was isolated from weaning on (males) or for 3 days before the
experiment (females), and in the interaction of a male with an oestrus female.
Data are presented as mean6s.e.m. (Mann–Whitney U-tests: *P,0.05;
**P,0.01;***P,0.001).Unlessotherwisespecified,(n1/1516,n1/2516,
n2/2516)malesand(n1/1516,n1/2516,n2/2513)femaleswere tested.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank M. Manz, R. Zienecker, S. Gerlach-Arbeiter, N. Damm,
H. Riederer, C. Jean,S. Rieckmann, S. Hochmuth and K. Sowa for technicalassistance.
M.J.S.,A.-L.J.andP.T.U.aremembersoftheInternationalGraduateSchoolinMolecular
Medicine at Ulm University. M.J.S. is further supported by Baustein 3.2 (L.SBN.0081),
E.E. by the Fondation de France and the Agence Nationale de la Recherche (ANR)
FLEXNEURIM (ANR09BLAN034003), S.W. and A.V.S. by the Deutsche
Forschungsgemeinschaft (DFG) (GRK 1123), A.M.G. by Baustein 3.2 (L.SBN.0083),
S.A.S by the DFG (EXC 257), D.S. by the DFG (SFB 618, SFB 665, EXC 257), the
Bundesministerium fu ¨r Bildung und Forschung (BMBF) (BCCN, BFNL) and the
EinsteinFoundation,M.R.K.bytheDFG(SFB779),C.S.L.,R.T.,N.T.,A.LS.andT.B.bythe
ANR (ANR-08-MNPS-037-01 - SynGen), Neuron-ERANET (EUHF-AUTISM), Fondation
OrangeandtheFondationFondaMentale,P.F.bytheBettencourt-SchuellerFondation,
R.T.,T.B.,P.F.bytheCNRSNeuroinformatic,E.D.G.bytheDFG(SFB779)andtheBMBF
(EraNET Neuron), and T.M.B. by the DFG (Bo 1718/3-1 and 1718/4-1; SFB 497/B8).
Author Contributions M.J.S., E.E., J.B., C.S., D.B., S.t.D., K.H.S., D.M., D.S., M.R.K., T.B.,
E.D.G. and T.M.B. designed the outline of this study. J.B. and B.V.S. generated, and
J.B., C.S. and S.A.S. supervised breeding of, the ProSAP1/Shank2-mutant mice. J.B.
supervised breeding of the ProSAP2/Shank3-mutant mice. M.J.S., A.K., A-L.J., P.T.U.
and A.M.G. performed all the biochemistry, real-time PCR, Golgi stainings, electron
microscopy, transfection of primary neurons and immunohistochemistry, E.E., C.S.,
D.M.,C.S.L.,P.F.,N.T.andA.LS.thebehaviouralexperiments,andS.W.,A.V.S.andD.B.
the electrophysiological experiments. E.S. conducted the survival analysis. M.J.S.,
E.E., S.W., A.V.S., C.S., D.B., D.M., R.T. and A.M.G. performed all data analyses and
jointly drafted the manuscript with S.A.S., D.S., M.R.K., T.B., E.D.G. and T.M.B. All
authorsreadandapprovedthefinalversion.M.J.S.,E.E.andS.W.contributedequally
to this study. We thank H.-J. Kreienkamp, Hamburg, for providing the pan-Shank
antibody ‘189.3’.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to T.M.B. (tobias.boeckers@uni-ulm.de).
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METHODS
Animals.TwomouseC57BL/6genomicXbaIDNAfragmentscodingforexonVI
and VII of the ProSAP1/Shank2 PDZ domain were subcloned from a bacterial
artificial chromosome (pBelo-BAC II 21259) yielding a 16.3-kilobase (kb)
chromosomalDNAfragment.Fortargetvectorconstruction,thelongarm,coding
forexonVI,wasclonedasanEcoRI–XbaIfragment(7.7kb)intopBluescriptIISK
vector(ThermoScientific)followedbyanXbaI–XhoIfragment(1.1kb)codingfor
exonVIIinwhichaloxPsequencewasintegratedintotheuniqueSphIsite.Thefrt
PGKneo frt loxP cassettewas cloned into the XhoI site followed by the short arm
(2.8 kb) as an XhoI–SphI fragment. The linearized targeting construct was elec-
troporatedintorecombinantinbredembryonicstemcells(RI-EScells)(passage15
(129/SV3129/SV-CP)) at 25mF and 400V (Gene Pulser; Bio-Rad). After elec-
troporation,cellswereplatedontoculturedishes(100mmdiameter)containinga
gamma-irradiated monolayer of G418-resistant PMEF cells. Thirty-two hours
later, 350mg of G418 (Invitrogen) per millilitre and 0.2mM 29-deoxy-29-fluoro-
b-D-arabinofuranosyl-5-iodouracil (Moravek Biochemicals and Radiochemicals)
were added to the culture medium. The medium was replaced every day, and
colonies were picked and analysed 8 days after plating. Cells were expanded in
HEPES-buffered Dulbecco’s modified Eagle’s medium supplemented with 15%
fetal bovine serum (Thermo Scientific), 1,000U of recombinant leukaemia inhib-
itory factor (Millipore) per millilitre, non-essential amino acids, L-glutamine,
b-mercaptoethanol and antibiotics (penicillin 100Uml21, and streptomycin
100mgml21). For electroporation, 23107cells were re-suspended in 20mM
HEPES (pH 7.4), 173mM NaCl, 5mM KCl, 0.7mM Na2HPO4, 6mM dextrose
and 0.1mM b-mercaptoethanol. Homologous recombination was tested by
Southern blot analysis. Embryonic stem cells were transiently transfected with a
FRT-recombinase plasmid to delete the selection cassette, leaving a single frt site.
Correctly targeted embryonic stem cells were microinjected into 3.5-day-old
B6D2F1 blastocysts and transferred to the uteri of 2.5 day pseudopregnant CD1
females. Pregnant mice carried pups to term and born chimaeras were identified
by agouti coat colour contribution. For the germ-line transmission, male
chimaeras were crossed to C57BL/6 female mice. Heterozygous offsprings
(ProSAP1/Shank21/frt) were confirmed by Southern blot analysis and further
tested by PCR for the presence of the targeted allele. Cre-mediated excision was
performedinvivobycross-breedingmiceharbouringaCMVpromoterdrivenCre
transgene resulting in heterozygous ProSAP1/Shank2-deficient mice (ProSAP1/
Shank1/2).ProSAP2/Shank3mutantsweregeneratedbyGenoway(Lyon,France)
and raised on a C57BL/6 background. The targeting strategy is shown in
SupplementaryFig.6.Allmicewerekeptinspecificpathogen-freeanimalfacilities
and all mouse procedures were performed in compliance with the guidelines for
the welfare of experimental animals issued by the Federal Government of
Germany and further approved by the ethical committee of Ile-de-France
(CEEA Ile-de-France Comite ´ 1).
Primary antibodies. Two polyclonal ProSAP1/Shank2 antibodies were used for
thisstudy.Thefirstone(PRCpabSA6045)wasdirectedagainstaminoacids355–
509, the second one (ppI-SAM pab SA5193) against amino acids 826–1259 of rat
ProSAP1/Shank2. Both antisera were produced in rabbits and guinea pigs and
have previously been characterized31. Furthermore, a novel polyclonal ProSAP2/
Shank3 antibody was produced (PRC pab). This antibody was directed against
aminoacids781–1009and1260–1392withintheproline-richclusters(PRC)ofrat
ProSAP2/Shank3. For antibody production, partial complementary DNAs
(cDNAs) of rat ProSAP2/Shank3 (base pairs 2116–2800 and 3550–3948, respect-
ively) were each cloned into the bacterial expression vector pGEX-4T (GE
Healthcare). The corresponding glutathione S-transferase (GST)–ProSAP2/
Shank3 fusion proteins were expressed in Escherichia coli BL21, purified and
injected at the same time in rabbits to generate antiserum. For all experiments
conductedinthisstudy,bothProSAP1/Shank2antiseraandtheProSAP2/Shank3
antiserum were each purified against the other familymembers. For this purpose,
COS-7 cells were transfected with pEGFP–Shank1, pEGFP–ProSAP1/Shank2
and/or pEGFP–ProSAP2/Shank3 constructs (all constructs have been described
previously32). The corresponding fusion proteins were collected, coupled to mag-
netic beads and loaded onto a column-based system (Miltenyi Biotec). The anti-
serawerethenpurifiedbyhavingthemflowsubsequentlythroughtheappropriate
columns (the green fluorescent protein (GFP)–Shank1 and GFP–ProSAP2/
Shank3 columns in case of the ProSAP1/Shank2 antisera, the GFP–Shank1 and
GFP–ProSAP1/Shank2columns in case of the ProSAP2/Shank3antiserum). Two
different pan-Shank antibodies were further used in this study. The first, pan-
Shank ‘189.3’ was directed against the PDZ domain of human Shank1 and is
known to detect Shank1 as well as ProSAP1/Shank2 (ref. 33). The second, pan-
Shank ‘Clone N23B/49’, was purchased from Millipore and directed against the
SH3/PDZ domains of rat ProSAP1/Shank2 (amino acids 84–309). It is known to
recognize all three ProSAP/Shank proteins, though. (The antigen sequence is
approximately 75% identical to the corresponding sequence in ProSAP2/
Shank3andapproximately70%tothatinShank1.)Thefollowingantibodieswere
purchased from commercial suppliers: PSD95 (Abcam), b3-tubulin (Covance),
GluN2A (NR2A), GluN2B (NR2B) (both Millipore), GKAP/SAPAP, Shank1 (IF)
(Novus Biologicals), b-actin, GluN1 (NR1), Shank1 (WB) (all from Sigma-
Aldrich), pan-GluA, GluA1, GluA2, GluA3 (all from Synaptic Systems).
Biochemistry and quantitative immunoblotanalyses.Toobtainthesubcellular
fractionsfrommousebrainanalysedinthisstudy(homogenates,solublefractions,
crude synaptosomal fractions, synaptic plasma membranes, one-triton extracted
PSD fractions), a subcellular fractionation procedure was performed as described
previously30with minor modifications. In brief, tissue from mouse brain of both
sexes (either brain regions and/or whole brain) was homogenized in HEPES-
buffered sucrose (320mM sucrose, 5mM HEPES, pH 7.4) containing protease
inhibitormixture(Roche).The homogenate wastaken for analysis and/or further
centrifuged at 1,000g for 10min at 4uC to remove cell debris and nuclei. The
supernatant was spun for 20min at 12,000g to obtain the soluble and the crude
synaptosomalfraction (pellet P2). This fraction wasused for the broad analysis of
scaffold and receptor protein levels throughout various brain regions at two
developmentalstages(P25,P70).Forsomeexperiments,apooledP2fractionfrom
10 whole mouse brains was further fractionated by sucrose density gradient cent-
rifugation(0.8/1.0/1.2sucrose)at200,000gfor2hat4uC.Purifiedsynaptosomes/
synaptic plasma membranes were collected at the 1.0–1.2M interface. To obtain
the one-triton extracted PSD fraction, synaptosomes were re-suspended in five
volumes of 1mM Tris pH 8.1, stirred for 15min on ice in buffer containing 0.5%
Triton X-100 and centrifuged at 33,000g for 30min. Equal amounts of 10–20mg
protein per lane were separated by SDS–polyacrylamide gel electrophoresis,
stained with Coomassie gel staining solution (Thermo Scientific) or blotted onto
polyvinylidene fluoride or nitrocellulose membranes using standard protocols.
After incubation with specific primary antibodies, immunoreactivity was visua-
lized on X-ray film (GE Healthcare) using HRP-conjugated secondary antibodies
(Dako) and a SuperSignal detection system (Thermo Scientific). For quantifica-
tion, the films were scanned and the grey value of each band was analysed by
ImageJ (National Institutes of Health, http://rsb.info.nih.gov/ij/) and normalized
to the grey value of b-actin and/or b3-tubulin.
Immunohistochemistry. Immunohistochemical stainings were performed on
frozen brain sections (5mm) of adult mice from both sexes. Ice-cold methanol
(220uC) was used for fixation. Cell membranes were permeabilized with Triton
X-100 (0.5% Triton X-100 in 10mM PBS) followed by blocking with hydrogen
peroxide solution (to inactivate endogenous peroxidase) and 2% BSA (to block
unspecific binding sites). The sections were subsequently incubated with the first
antibodyin0.5%BSAovernightat4uCinthewetchamber.Fordiaminobenzidene
staining, the sections were incubated with a biotinylated secondary antibody and
detectionwasascertainedusingtheABCVectastainKit(VectorLaboratories)and
DAB solution (0.6% DAB, 0.1% hydrogen peroxide in 5mM Tris/HCl, pH 7.4).
For immunofluorescence staining, the sections were incubated with an Alexa
Fluor 568 (red) fluorescence labelled secondary antibody (Invitrogen) and cell
nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI).
Golgi staining. Dissected adult mouse brainsfrom both sexes were immersed for
21 days in Golgi–Cox solution (1% potassium dichromate, 1% mercuric chloride,
8% potassium chromate). The brains were subsequently dehydrated and sagittal
sections (200mm) were cut using a vibratome. Golgi–Cox staining was developed
byincubationin16%ammoniafor30minbeforeembedding.Z-stackimageswere
taken using an upright Axioscope (Zeiss). For quantitative analysis of spine
density, we analysed at least three neurons (pyramidal neurons from CA1
hippocampus) from at least three independent wild-type and ProSAP1/
Shank22/2or wild-type and ProSAP2/Shank3ab2/2littermate pairs.
Electron microscopy. Adult mice from both sexes were transcardially perfused
withfixative(2%paraformaldehyde,2.5%glutaraldehyde,1%saccharosein0.1M
cacodylate buffer, pH 7.3) and brains were dissected out and postfixed overnight
(2%glutaraldehyde, 1%saccharose in 0.1M cacodylatebuffer).Afterdehydration
and staining with 2% uranyl acetate, the material was embedded in epoxy resin.
Ultrathin sections were cut using an ultramicrotome. After lead citrate staining,
the sections (CA1 hippocampus) were examined using an EM 10 electron micro-
scope. For quantitative analysis of PSD length and thickness, we analysed at least
threeindependentwild-typeandProSAP1/Shank22/2orwild-typeandProSAP2/
Shank3ab2/2littermate pairs.
Real-timequantitativePCR.IsolationoftotalRNAfrommousebraintissuewas
performed using the RNeasy kit as described by the manufacturer (Qiagen).
Isolated RNA was eluted in 20ml of RNase-free water and stored at 280uC. For
the reverse transcriptase (RT)-mediated PCR studies, first strand synthesis and
real-time quantitative RT–PCR amplification were performed in a one-step,
single-tube format using the QuantiFast SYBR Green RT–PCR kit (Qiagen).
Thermal cycling and fluorescent detection were performed using the Rotor-
Gene-Q real-time PCR machine (model 2-Plex HRM) (Qiagen). The qRT–PCR
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was assayed in 0.1ml strip tubes in a total volume of 20ml reaction mixture
containing1ml of undiluted total RNA, 2ml of QuantiTect Primer Assay oligonu-
cleotides,10mlof23QuantiFastSYBRGreenRT–PCRMasterMixsupplemented
with ROX (5-carboxy-X-rhodamine) dye, 6.8ml of RNase-free water (supplied
with the kit) and 0.2ml of QuantiFast RT Mix. RT. Amplification conditions were
asfollows:10minat50uCand5minat95uC,followedby40cyclesofPCRfor10s
at 95uC for denaturation, 30s at 60uC for annealing and elongation (one-step).
During the extension, real-time fluorescence measurements were recorded by the
PCR machine, thus monitoring real-time PCR amplification by quantitative ana-
lysis of the fluorescence emission. The SYBR Green I reporter dye signal was
measured against the internal passive reference dye (ROX) to normalize non-
PCR-related fluctuations in fluorescence that occur from reaction tube to reaction
tube.Resultingdatawereanalysedusingthehydroxymethylbilanesynthasegeneas
aninternalstandardtonormalizetranscriptlevels.Cyclethreshold(ct)valueswere
calculated by Rotor-Gene-Q Software (version 2.0.2). Cycle threshold values indi-
catethePCRcyclenumberatwhichthemeasuredfluorescenceoftheindicatordye
(SYBRGreenI),accordanttothequantityofamplifiedPCRproducts,isincreasing
in a linear fashion above background. All qRT–PCR reactions were run in dupli-
catesinthreeindependentexperiments,andmeanctvaluesforeachreactionwere
takenintoaccountforcalculationsofdataanalysis.Toascertainprimerspecificity,
ameltingcurvewasobtainedfortheampliconproductstodeterminetheirmelting
temperatures. Melting curve was driven from 60uC to 95uC rising in 1uC steps
whereas fluorescence was recorded continuously. For negative controls and to
check for reagent contamination, a complete reaction mixture was used in which
the RNA sample was replaced by RNase-free water. Real-time quantitative PCR
wasperformedusingoligonucleotidestoinvestigateexpressionofProSAP1/Shank2
andProSAP2/Shank3(validatedprimerpairs,QuantiTectprimerassay,Qiagen)in
tissue from wild-type littermates, ProSAP1/Shank21/2, ProSAP1/Shank22/2and
ProSAP2/Shank3ab2/2mutants. All consumables used for the extraction of total
RNA and real-time PCR analysis were purchased from Qiagen.
Primaryhippocampalculturesfromrat.Primarycellcultureofrathippocampal
neurons (embryonic day 18 (E18)) was performed as described previously32.
Hippocampal neurons were seeded on poly-L-lysine (0.1mgml21; Sigma-
Aldrich) coated coverslips at a density of 33104cells per well. Cells were grown
in Neurobasal medium, complemented with B27 supplement, 0.5mM
L-glutamine, and 100 Uml21penicillin/streptomycin (all Invitrogen) and main-
tainedat37uCin5%CO2.NeuronsweretransfectedwithProSAP1/Shank2short
hairpin RNA (shRNA) (sequence: TG CCT TCA CCA AGA AGG AA) and/or
scrambledcontrolconstructs32at12daysinvitro(DIV12).AfterfixationatDIV14
andimmunostainingwiththeappropriateantibodies,picturesweretakenwithan
uprightAxioscopemicroscopeequippedwithaZeissCCDcamera.Quantification
offluorescencesignalswasperformedusingImageJ(NationalInstitutesofHealth,
http://rsb.info.nih.gov/ij/). For evaluation, fluorescent puncta (ProSAP2/Shank3,
Shank1, GluN1, GluN2A, GluN2B, GluA1, GluA2 and GluA3) along dendrites
within the field of view were analysed and the signal intensity values of GFP co-
localizing (shRNA-ProSAP1/Shank2 or scrambled RNAi expressing cells) and
non-GFP co-localizing puncta compared.
Statistical analysis for the methods mentioned above was performed using
Microsoft Excel for Macintosh and data were tested for significance using two-
tailed, Student’s t-test and ANOVA. P values,0.05 were stated as significant
(*P,0.05, **P,0.01, ***P,0.001).
Electrophysiology. ProSAP1/Shank2 mutants were raised on a C57BL/6 back-
ground and wild-type littermates were used as a control in all experiments. The
experimenters were blind to the genotype of the tested animals for data collection
and analyses. Hippocampal slices were prepared from animals of both sexes aged
P21–P28 (unless indicatedotherwise), andas previously described34. Briefly, mice
were anaesthetized with isoflurane and decapitated. Brains were rapidly removed
and transferred to ice-cold artificial cerebrospinal fluid (ACSF) slicing solution
containing (in mM): 87 NaCl, 26 NaHCO3, 50 sucrose, 25 glucose, 2.5 KCl, 1.25
NaH2PO4,3MgCl2,0.5CaCl2,pH7.4.Tissueblockscontainingthehippocampus
were mounted on a Vibratome (Leica VT1200) and cut into horizontal slices of
300mm. Slices were incubated in slicing solution at 35uC for 30min, cooled to
room temperature and transferred to ACSF containing (in mM): 119 NaCl, 26
NaHCO3, 10 glucose; 2.5 KCl, 2.5 CaCl2, 1.3 MgCl2, 1 NaH2PO4. All ACSF was
equilibrated with carbogen (95% O2, 5% CO2). Slices were stored under sub-
merged conditions for 30min to 6h before being transferred to a submerged
recording chamber (Luigs and Neumann) where they were perfused with ACSF
maintained at room temperature at a rate of 3–4mlmin21.
Extracellular field and whole-cell patch-clamp recordings were performed.
Stimulation and recording pipettes were pulled from borosilicate glass capillaries
(Harvard Apparatus; 1.5mm outside diameter, with a micropipette electrode
puller (DMG Universal Puller)). Evoked postsynaptic responses were induced
bystimulatingSchaffercollaterals(0.1Hz)inCA1stratumradiatumwithpipettes
of 20mm tip diameter (filled with ACSF), using a stimulus isolater (ISO-flex,
A.M.P.I.). fEPSPs were recorded with the same kind of pipettes that were placed
in stratum radiatum. fEPSP slopes were determined as dV/dt of the 20–80%
amplitude from averages of five individual traces. Long-term potentiation was
induced by a single tetanus of 100 pulses at 100Hz. For long-term depression
experiments, CA1 was isolated by a microcut set at the border of CA2/CA3
immediately before the experiment, and recordings were made in the presence
of 1mM gabazine. Long-term depression was induced by 15min paired-pulse
stimulation at 1Hz with 50ms between singlepulses. For whole-cell patch-clamp
recordings, pipettes had resistances of 2–3 MV. CA1 pyramidal cells were held at
260mV in voltage-clamp mode (not corrected for liquid junction potential).
Series resistance (not compensated) was constantly monitored and was not
allowed to increase beyond 22MV or change more than 20% during the experi-
ment. mEPSCs and whole-cell AMPA currents were recorded in the presence of
1mM tetrodotoxin, 0.1mM cyclothiazide and 1mM gabazine with a potassium-
based intracellular recording solution containing (in mM): 135 K-gluconate, 10
HEPES, 0.5 EGTA, 20 KCl, 2 MgATP and 5 phosphocreatine. Osmolarity was
300mOsm;pHwasadjustedto7.2withKOH.mEPSCeventsweredetectedwitha
threshold-based algorithm and their amplitudes calculated from a 1ms time win-
dowaroundthepeak.Eventswerealignedbytheirrisetimebeforeaveraging.The
mEPSC frequency was determined from a 3min time window. The analysis of
mEPSCamplitudesincludedthefirst42eventsofeachrecordedcell(onecellwith
fewereventswas excludedfrom amplitude analysis). Cellularinputresistance was
calculatedfromthesteady-statecurrentmeasuredinresponsetoahyperpolarizing
test pulse of 50ms duration at a holding potential of 260 mV. NMDA/AMPA
ratios were recorded in ACSF containing 4mM CaCl2, 4mM MgCl2and 1mM
gabazine with a caesium-based intracellular recording solution containing (in
mM): 145 CsCl, 10 HEPES, 0.2 EGTA, 2 MgCl2, 2 NaATP, 0.5 NaGTP and 5
phosphocreatine. Osmolarity was 305mOsm;pHwasadjustedto7.2with CsOH.
For estimation of NMDA/AMPA ratios, compound EPSCs were evoked at 260
and 140 mV. Stimulation was adjusted to produce a single-peaked short-latency
response. Ten consecutive EPSCs for each holding potential were averaged. The
AMPA receptor-mediated component of the EPSC was estimated by measuring
the peak amplitude of the averaged EPSC at 260 mV. The NMDAR-mediated
componentwasestimatedat140mVbymeasuringtheamplitudeoftheaveraged
EPSC 75ms after stimulation. IPSCs were recorded in the presence of 10mM
NBQX and 10mM APV with a potassium-chloride-based intracellular recording
solutioncontaining(inmM):145KCl,10HEPES,0.1EGTA,2MgCl2,2Na2ATP.
Osmolaritywas305mOsm;pHwasadjustedto7.3withKOH.FormIPSCrecord-
ings, 1mM tetrodotoxin was added to the bath. The IPSC frequency was deter-
mined from a 2min time window. The analysis of mIPSC (sIPSC) amplitudes
include the first 120 (260) events of each recorded cell. All drugs were purchased
from Tocris Bioscience.
Recordings were performed with an Axopatch 700A Amplifier (Axon
Instruments). Data were acquired using a BNC-2090 adaptor chassis, digitized
at 5kHz (PCI 6035E A/D Board, National Instruments), filtered at 1kHz and
recorded in IGOR Pro 4.0 using custom made plug-ins (WaveMetrics).
Analyses were performed using custom-written procedures in IGOR Pro
(WaveMetrics) and MATLAB (The Mathworks). mEPSCs were analysed with
NeuroMatic (http://www.neuromatic.thinkrandom.com). Data in graphs and the
text are presented as mean6s.e.m. Statistical comparisons between groups were
performed in GraphPad Prism (GraphPad Software) with unpaired two-tailed
Student’s t-tests and two-way repeated-measures ANOVA. A Kolmogorov–
Smirnov test was performed in MATLAB. Results were considered significant at
P,0.05.Stimulusartefactswereblankedinsampletraces.Samplesizesaregivenas
number of experiments (n) and number of animals (N). Data in bar graphs are
presented as mean with standard error; grey circles show individual data points.
Behavioural studies. The behavioural studies included several cohorts of mice.
Cohort 1 (backcrossed for 10 generations on C57BL/6) was used for the develop-
mental study of pups. Cohort 2 included 3- to 6-month-old adult mice (back-
crossed for 11 generations on C57BL/6). The general neurological examination
and the rotarod experiments were conducted with cohort 3 of 6- to 8-month-old
adult mice (backcrossed for 11 generations on C57BL/6). We tested the offspring
(that is, wild-type, ProSAP1/Shank21/2and ProSAP1/Shank22/2littermates) of
ProSAP1/Shank21/2mice crossings. The experimenters were blind of the geno-
type of the tested animals for data collection and analyses.
General parameters indicative of the health and neurological state were
addressed following the neurobehavioural examination described by Whishaw
et al.35and the tests of the primary screen of the SHIRPA protocol except startle
response36. For the developmental study, males were separated from pregnant
females 1 or 2 days before birth. Births were checked each morning and evening.
Pups of both sexes (cohort 1) were individually identified with long-lasting sub-
cutaneous tattoos (green tattoo paste, Ketchum Manufacturing) on the paws on
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P1.Eachpupwasisolatedfromdamandlittermatesandplacedinasmallenclosure
with soft plastic surface in a soundproof chamber (temperature T52461uC).
Audiorecordingslasted5minandwereconductedevery2days(P2,P4,P6,P8,P10
and P12). Recording hardware (UltraSoundGate 416-200, condenser ultrasound
microphone Polaroid/CMPA) and software (Avisoft SASLab Pro Recorder) were
from Avisoft Bioacoustics (sampling frequency 300kHz; fast Fourier transform
length 1,024 points; 16-bit format). After each audio recording, developmental
milestones(weight,opening of the eyes, extroversion ofthe ears and incisiveerup-
tion) were noted. Tests for general sensory perception and motor coordination
(righting reflex (P2–P12), home cage odour preference (P7), reaction to a click
soundfromapen2cmbehindtheears(P14))wereconductedat least30minafter
audio recordings.
Rightingreflex.Thepupwasturnedonitsbackonaflatsurface.Thelatencyuntil
it reached a normal position (face down) was measured. When the pup did not
succeed in righting up, latency was set at the maximum time allowed (120s).
Preference for home cage odour. A plastic cage (20cm330cm36 cm) was
divided in three zones. One side of the cage was covered with bedding from the
nest cage; the other side was covered with fresh bedding. A neutral zone (width
2.5cm)separatedthese twozones.The 7-day-oldpup wasplacedin the middleof
the neutral zone. Time spent by the nose of the pup in each zone was measured
within 60s. This test was repeated for each pup and the mean of the two tests was
used in the analyses.
To examine adult behaviour, mice of both sexes aged between 3 and 6 months
(cohort 2) were tested in the following order: light–dark anxiety test, open field,
Y-maze, three-chamber test, self-directed and digging behaviour, resident–intruder
test, male behaviour in presence of an oestrus female, buried-food finding test and
objectrecognition.Allanimalswereweighedat4monthsofage,andtheirweightwas
comparedwithage-andsex-matchedcommerciallyavailableC57BL/6mice.Atthe
sameage,micewereheldbythetail20cmaboveatableforapprox.1mintocheckfor
hindlimb clasping. Males were housed individually from weaning onwards, given
theirhighaggressivenesstowardseachother.Femaleswerehousedingroups(except
for the resident–intruder test; see hereafter). At least 2days elapsed between two
consecutive tests. Unless otherwise specified, we tested and analysed 16 males per
genotype and 16 females per genotype (but only 13 ProSAP1/-Shank22/2females).
Settingswerecleanedwithsoapandwater,anddriedwithpapertowelsbetweeneach
mouse.Whenbeddingwasused,newfreshbeddingwasusedforeachmouse.Tests
using audio-recordings were conducted in a soundproof chamber.
Light–dark anxiety test. The setting consisted of a white, brightly illuminated
compartment (1300lx) connected to a black, very dark one (3lx) through a small
door. The tested mouse was introduced into the white compartment and allowed
to explore the whole apparatus for 5min. The latency to enter the dark compart-
ment,thetimespentineachcompartmentandthenumberoftransitionsbetween
compartments were measured.
Open field. The tested mouse was allowed to explore freely for 30min a round,
open-field arena of 1m diameter (100lx in the centre of the arena). Automatic
detection of the mouse (custom software of P. Faure) recorded the total distance
travelled,andthetimespentinthecentralzoneversustimespentattheperipheryof
the maze.
Y-maze. The tested mouse was allowed to explore freely a Y-shaped labyrinth for
3min.Thenumberofvisitsineacharmofthelabyrinthandsequencesofentrance
in each arm were recorded.
Three-chamber test. A Plexiglas cage was divided in three compartments as
previously described37. Both side compartments contained an empty perforated
cup (side compartments: 150lx; central compartment: 140lx). First, the tested
mouse was allowed to explore freely the whole setting, with all doors open for
10min (phase 1). After this habituation period, the mouse was restricted in the
central compartment, while an unfamiliar C57BL/6 mouse of the same sex
(stranger 1) was placed under one of the cups (sides alternated between each
mouse). The tested mouse was then allowed to explore the whole apparatus for
10min (phase 2). After that, it was restricted to the central compartment while
another unfamiliar C57BL/6mouse ofthesame sex(stranger2) was placedunder
the other cup. The tested mouse could then again freely explore the whole appar-
atusfor10min(phase3).Inallthreephases,timespentineachcompartmentand
numberoftransitionsbetweencompartmentswereautomaticallyrecorded.Times
spentincontactwiththecupcontainingthemouse(stranger1)andtheemptycup
were manuallymeasuredinphase2.Timesspentincontact with thecupcontain-
ing the unfamiliar mouse (stranger 2) and in contact with the cup containing the
familiar mouse (stranger 1) were manually measured in phase 3.
Self-directedanddiggingbehaviours.Thetestedmousewasplacedinanewtest
cage (Plexiglas, 50cm325cm330cm; 100lx; clean sawdust bedding) in a
soundproof chamber. After 10min habituation, itsbehaviourwas video-recorded
for10min.Werecordedthetimespentself-groominganddigginginthebedding,
as well as the number of self-grooming and digging bouts.
Resident–intruder test. Female mice were housed individually 3days before the
testing day to increase their social motivation. Males were reared in social isolation
fromweaningonwards(seeabove).Thetestedmousewasleft30minforhabituation
in the test cage (Plexiglas, 50cm325cm330cm; 100lx; clean sawdust bedding;
soundproof chamber38). After this time, an unfamiliar C57BL/6 mouse of the same
sexwasintroduced.Thetwoanimalswereallowedtointeractfreelyfor4min.Social
interactions were videotaped continuously (high-resolution Sony XCD-SX90CR
video camera). At the same time, ultrasonic vocalizations were recorded with a
condenser ultrasound microphone Polaroid/CMPA, the interface UltraSoundGate
416-200 and the software Avisoft SASLab Pro Recorder from Avisoft Bioacoustics
(sampling frequency: 300kHz; fast Fourier transform length: 1024points; 16-bit
format). Behaviours were encoded manually. We recorded the latency for the first
contact, the time spent in contact, as well as the latency for the first ultrasonic
vocalization, the call rate and the distribution of the different call types.
Male–female interactions. All males had a previous contact of 3days with a
femaleatleast3daysbeforetheexperiment.The testedmalewasleft inatest cage
(Plexiglas, 50cm325cm330cm; 100lx; clean sawdust bedding; soundproof
chamber)tohabituatefor10min.AnunfamiliarC57BL/6femaleinoestrus(tested
though vaginal smears) was then introduced. Ultrasonic vocalizations and social
interactions were recorded for 3min with the same setting as cited above. The
latency forthe first contact,the timespentincontact,aswell asthelatency for the
first ultrasonic vocalization, the call rate and the distribution of the different call
types, were recorded.
Buried-food finding test. Four days before testing, female mice were housed
individually, like males. Each day, all mice were given four pieces of cocoa-
flavouredcrispedricecereals(CocoPops,Kellogg’s)withrestrictedaccesstotheir
usual food (SAFE) but water ad libitum. Mice that did not eat their cocoa-
flavoured cereals were excluded from the test. Twelve hours before testing, mice
were food-deprived, with water ad libitum. On the day of the test, six pieces of
Coco Pops were buried 1.5cm under sawdust bedding in a clean cage. The tested
mousewasplacedintheoppositecorner.Thelatencyforthemousetoretrievethe
food was measured (maximum time: 3min). For each animal tested, a new clean
cage with fresh bedding was used.
Object recognition. To test ProSAP1/Shank2 mutants’ ability to differentiate
objects, we used the set up of the three-chamber test. In the habituation phase, the
mouse was allowed to explore the whole setting freely, without any object inside.
After that, the mouse was restricted in the central compartment and one identical
electricsocketwasplacedineachsidecompartment.Themousewasagainallowedto
explore the setting freely with thetwo identical objects for 10min. After this second
period,whilethemousewasrestrictedinthecentralcompartment,oneofthesockets
wasreplacedbyacopperjoint.Again,themousewasallowedtoexplorethecomplete
setting for 10min. In all three phases, the time spent in each compartment and the
number of transitions between compartments was automatically recorded. Time
spent in contact with each object was manually measured in each phase.
Rotarodtest.Animalsofcohort3receivedtwotrainingsessions(3hinterval)ona
rotarod apparatus (TSE Systems) with increasing speed from 4 to 40r.p.m. for 5
min. After4 days,mice were testedat 16,24,32 and 40r.p.m.constantspeed. The
latency to fall off the rod was measured39.
Analyses of audio recordings. In the pup developmental study, given that there
were no significant differences between the number of ultrasonic vocalizations
automatically detected (pulse train detection analyses from Avisoft SASLab Pro;
hold time 7ms) and the number of calls manually detected (data not shown), we
used the automatic detection of calls to compare the global call rate between
genotypes for each age. For vocalizations recorded in adult animals, we manually
detected the calls in the software Avisoft SASLab Pro (Avisoft; 75% overlap; time
resolution 0.853ms; frequency resolution 293Hz; Hamming window).
For pups and adults, we labelled the calls with the labelling function of Avisoft
SASLabPro.Eachcallwasclassifiedinonecallcategoryof11,adaptedfromref.40as
follows.(1)Short:durationshorterthan5ms;frequencyrange#6.25kHz.(2)Flat:
durationlongerthan5msandfrequencyrange#6.25kHz.(3)Upward:increasein
frequency; frequency range greater than 6.25kHz with only one direction of fre-
quency modulation. (4)Downward: decreaseinfrequency; frequencyrangegreater
than 6.25kHz with only one direction of frequency modulation. (5) Modulated:
frequency modulations in more than one direction; frequency range greater than
6.25kHz. (6) Complex: addition of one or more frequency component (not
harmonic). (7) One frequency jump: inclusion of one jump in frequency without
timegapbetweenthetwofrequencycomponents.(8)Frequencyjumps:inclusionof
more than one jump in frequency without time gaps between the two consecutive
frequencycomponents.(9)Mixed:inclusionofanoisy(‘unstructured’)partwithina
puretonecall.(10)Unstructured:nopuretonecomponentidentifiable;‘noisy’calls.
(11)Others:includeallthecallsthatdidnotfitinanyoftheprecedingcategories(for
example, calls combining features of several of the previous call types).
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Unless otherwise specified, we used Mann–Whitney U-tests to compare the
behaviouraltraitsmeasuredbetweengenotypesorwithingenotypesbecauseofthe
non-normaldistributionofthedataandthelimitedsamplesizeinmanycases.We
compared the distribution of the different call types between genotypes using x2
tests.AllanalyseswereconductedwiththecomputingandstatisticalsoftwareR(R
Developmental Core Team 2009) and data are presented as mean6s.e.m. For
exact values of statistical analyses, see Supplementary Tables.
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synaptogenesis and synapse maturation. EMBO J. 30, 569–581 (2011).
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receptor interacting protein defines a novel family of multidomain proteins
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35. Wishaw, I. Q., Haun, F. & Kolb, B. in Modern Techniques in Neuroscience (eds
Windhorst, U. & Johansson, H.) 1243–1275 (Springer, 1999).
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37. Nadler, J. J. et al. Automated apparatus for quantitation of social approach
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