Synaptic dysfunction and abnormal behaviors in mice lacking major

45 Pages·2011·0.85 MB·English

Human Molecular Genetics, 2011, Vol. 20, No. 15 3093–3108
doi:10.1093/hmg/ddr212
Advance Access published on May 10, 2011
Synaptic dysfunction and abnormal behaviors
in mice lacking major isoforms of Shank3
XiaomingWang1,{, Portia A.McCoy8, Ramona M.Rodriguiz4,7, YanzhenPan10, H. ShawnJe2,{,
Adam C.Roberts8, Caroline J.Kim7, JanetBerrios8, Jennifer S.Colvin4, DanielleBousquet-
Moore4, IsabelLorenzo10, GangyiWu11, Richard J.Weinberg9, Michael D.Ehlers2,§,
Benjamin D.Philpot8, Arthur L.Beaudet10, William C.Wetsel2,3,4,7
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and Yong-huiJiang1,2,5,6,∗ ow
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1Department of Pediatrics, 2Department of Neurobiology, 3Department of Cell Biology, 4Department of Psychiatry de
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and Behavioral Sciences, 5Duke Institute for Brain Science, 6Program in Genetic and Genomics and Cellular and fro
Molecular Biology and 7Mouse Behavioral and Neuroendocrine Analysis Core Facility, Duke University School of m h
Medicine, Durham, NC 27710, USA, 8Department of Cell and Molecular Physiology and 9Department of Cell and ttp
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Developmental Biology, University of North Carolina, Chapel Hill, NC 27599, USA and 10Department of Molecular ://a
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and Human Genetics and 11Department of Biophysics and Molecular Physiology, Baylor College of Medicine, ad
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Houston, TX 77030, USA m
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ReceivedJanuary24,2011;RevisedandAcceptedMay6,2011 .c
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SHANK3isasynapticscaffoldingproteinenrichedinthepostsynapticdensity(PSD)ofexcitatorysynapses. g
/a
SmallmicrodeletionsandpointmutationsinSHANK3havebeenidentifiedinasmallsubgroupofindividuals rtic
withautismspectrumdisorder(ASD)andintellectualdisability.SHANK3alsoplaysakeyroleinthechromo- le
-a
some 22q13.3 microdeletion syndrome (Phelan–McDermid syndrome), which includes ASD and cognitive b
s
dysfunction as major clinical features. To evaluate the role of Shank3 in vivo, we disrupted major isoforms tra
c
of the gene in mice by deleting exons 4–9. Isoform-specific Shank3e4–9 homozygous mutant mice display t/2
0
abnormal social behaviors, communication patterns, repetitive behaviors and learning and memory. /1
5
Shank3e4–9 male mice display more severe impairments than females in motor coordination. Shank3e4–9 /3
0
mice have reduced levels of Homer1b/c, GKAP and GluA1 at the PSD, and show attenuated activity-depen- 93
/2
dent redistribution of GluA1-containing AMPA receptors. Subtle morphological alterations in dendritic 5
2
spines are also observed. Although synaptic transmission is normal in CA1 hippocampus, long-term poten- 72
7
tiationisdeficientinShank3e4–9mice.WeconcludethatlossofmajorShank3speciesproducesbiochemical, 1
b
cellular and morphological changes, leading to behavioral abnormalities in mice that bear similarities to y g
human ASD patients with SHANK3 mutations. ue
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INTRODUCTION postsynaptic densities (PSDs) of excitatory synapses (1–3). ril 2
Shank3 is a large protein containing multiple conserved 0
1
Shank3[SH3andmultipleankyrin(ANK)repeatdomain3]is motifs, including an ANK repeat, a proline-rich cluster and 9
a member of the highly conserved Shank/ProSAP family of SH3, PDZ and SAM (sterile a-motif) domains (4–6).
synaptic scaffolding proteins, first identified as a component InvitrostudieshaveshownthattheANKdomaincaninteract
of the PSD-95/GKAP/Homer1 complex enriched in the with cytoskeletal proteins in the PSD (7), the proline-rich
∗To whom correspondence should be addressed at: Department of Pediatrics and Neurobiology, Duke University School of Medicine,
Durham,NC27710,USA.Tel: [phone];Fax: [phone];Email:[email protected]
†Presentaddress:DepartmentofBiochemistryandMolecularBiology,ShanghaiMedicalCollege,FudanUniversity,Shanghai,China.
‡Presentaddress:PrograminNeuroscienceandBehavioralDisorders,Duke-NUSGraduateMedicalSchool,Singapore169857,Singapore.
§Presentaddress:PfizerGlobalResearchandDevelopment,NeuroscienceResearchUnit,Groton,CT,USA.
# The Author 2011. Published by Oxford University Press. All rights reserved.
For Permissions, please email: [email protected]
3094 Human Molecular Genetics, 2011, Vol. 20, No. 15
domainofShank3caninteractwithHomer1b/cprotein(2)and backcrossed for more than seven generations onto a C57BL/
the PDZ domain can interact with the GluA1 subunit of the 6J background.
AMPA receptor (AMPAR) directly, and with the NMDA
receptor (NMDAR) through GKAP and the PSD-95 complex
Shank3 mRNA species and Shank3 isoform-specific
(1,2,8). In hippocampal cultures, expression of Shank3 in
deletion
aspiny neurons can recruit AMPARs for the formation of
new synapses, and siRNA-induced knockdown of Shank3 As predicted, a 940bp RT-PCR product was identified in
reduces spine densities and increases the lengths of spines Shank3 wild-type (Shank3+/+) mice, using primers anchoring
(9). It remains unclear whether these interactions occur exons 2 and 10 (Fig. 1B). In contrast, two smaller products
in vivo and whether they can influence synaptic function. (330 and 260bp) were detected in Shank3e4–9 brains (Sup-
SHANK3 maps to the critical region of chromosome plementary Material, Fig. S1A4). Sequencing confirmed that
22q13.3deletionsyndrome(alsoknownasPhelan–McDermid the 330bp product represented a fragment joining exons 2, 3 D
syndromeorPMS)(10–13).PMSpatientspresentwithpromi- and 10, whereas the 260bp product represented cryptic spli- ow
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nent autistic features, including severe language delay and cing from exons 2 to 10 (Supplementary Material, lo
a
moderate-to-severe intellectual disability (14,15). Heterozy- Fig. S1A5). Both transcripts resulted in truncated proteins, d
e
gmoiusssenmsiecraonddelfertaimones-sahnifdt mpouitnattimonustaotifoSnHs,AiNncKlu3d,ianrge rdeeponrotveod w26it0hbpprtermanastcurrieptstaopppecaorsdotnosbeinbreaxionn-sp1e1c.ifiIcn,tesrienscteinigtlyis, nthoet d fro
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in a small subgroup of individuals with autism spectrum dis- found in Shank3e4–9 kidney (Supplementary Material, h
order (ASD) with comorbidity for intellectual disability Fig. S1A4). ttp
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(16–21). These and other reports suggest that haploinsuffi- Westernblotwasperformedwithanantibodyraisedagainst ://a
ciency of SHANK3 may play a key role in PMS and ASD amino acids 1431–1590 of the human SHANK3 protein ca
d
(11,12,22,23). Interestingly, mutations in SHANK3 have (Q9BYB0) (Supplementary Material, Table S4). According e
m
been reported also in schizophrenia, suggesting that to GenBank (AB231013), the full-length mouse Shank3 ic
SHANK3 deficiency may contribute to both neurobehavioral protein (i.e. isoform 3a) should be (cid:3)190kDa. A band of .ou
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disorders (20,21). this size was found in Shank3+/+ but it was completely .c
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ASD is characterized by impairments in social behavior, absent in Shank3e4–9 brain (Fig. 1D). Although the Shank3b m
communication and language development, as well as by protein (AJ245904, (cid:3)77kDa) was predicted to be disrupted /hm
repetitive and stereotyped behaviors (24–26); comorbidities in Shank3e4–9 mice, we were unable to confirm this, since g/a
in intellectual disabilities and movement disorders are exon 21 is spliced out of this isoform and our antibody only rtic
common (27–29). A diagnosis of ASD in humans is purely binds this portion of Shank3. However, we observed bands le-a
based on behavioral assessment. Although the condition is at (cid:3)140 and (cid:3)170kDa in both genotypes with our antibody. bs
diagnosed more often in males than in females, the basis for These bands presumably represent different isoforms of tra
c
this sex difference is unknown (30,31). While a genetic com- Shank3, although we were not able to determine the corre- t/2
0
ponent is strongly implicated in the etiology of ASD, the sponding mRNA transcripts. /1
underlying molecular and neuropathophysiological basis of TheShank3genehas22exonsandspans(cid:3)58kbofgenomic 5/3
0
the condition is unknown in most ASD cases. Nevertheless, DNA. Alternative splicing of the SHANK3 gene has been 9
3
de novo missense mutations in exons 5–9 encoding the suggested(11,16,36)andwehaveidentifiedfiveintragenicpro- /2
5
ANK domain of SHANK3 and microdeletions of the entire motersaswellasextensivealternativesplicinginmouseShank3 27
2
SHANK3genehavebeenreportedinASD(16–19).Toinves- (Fig.1A).Thesepromotersandspliceformswereconfirmedfor 7
1
tigate the possible role of Shank3 in ASD, we generated bothhumansandmiceby5′RACE,RT-PCR,promoter–repor- b
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mutant mice lacking exons 4–9 which encode the ANK ter assays and in silico analyses (Fig. 1E and Supplementary g
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domain, disrupting expression of the major isoforms of Material,Fig.S1).Sequencesofthesenewtranscriptsaredepos- es
Shank3 (Shank3e4–9). These Shank3e4–9 homozygous ited in GenBank (HQ405757-HQ405766 and HO7636 t o
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mutants were subjected to biochemical, cellular, electro- 62-HO763663) (structures are found in Supplementary 0
5
physiological and behavioral analyses to elucidate the func- Material,Fig.S1B1–B8).Wehaveconfirmedatleast11differ- A
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tional significance of these major Shank3 proteins. entShank3mRNAspeciesinthemousebrain.Thetotalnumber ril 2
of mRNA species produced by combinations of alternatively 0
1
spliced exons and multiple promoters is expected to be 9
RESULTS sizable. Due to the limitations of our antibody, we were
unable to determine whether proteins are produced from all
Generation of Shank3 mutant mice
the mRNA transcripts we have identified. Nonetheless, by
WegeneratedShank3mutantmiceusingaconventionalgene- sequencing RT-PCR products, we have confirmed that tran-
targeting approach (32) where exons 4–9 of the Shank3 gene scripts(Shank3a–b)initiatedbypromoters1and2areabsent,
were deleted (Fig. 1A, B and C and Supplementary Material, whereas transcripts (Shank3c–e) from promoters 3–5 are
Fig. S1A1 and A2). The naturally occurring Disc1 (Disrupted present in the Shank3e4–9 brain (Fig. 1E). Transcript-specific
in Schizophrenia 1) mutation in the 129SvEv line of ES cells expression from promoter 6 (Shank3f) could not be verified,
was segregated from Shank3e4–9 at the beginning of back- because this promoter is embedded in exon 21, which is
crossing to avoid any possible confounding effects in our shared by other isoforms. Nevertheless, we conclude that
experiments (Supplementary Material, Fig. S1A3) (33–35). Shank3e4–9 mice possess isoform-specific deletions in which
All Shank3 mice used in the experiments had been the major Shank3a and Shank3b species, as well as other
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Figure1.GenerationandcharacterizationofShank3e4–9mice.(A)AdiagramofthestructureofthemurineShank3geneshowingpromotersandalternative t/2
0
splicingsites(theasteriskindicatesalternativelysplicedexons).Fiveintragenicpromoterswereidentified(seealsoSupplementaryMaterial,Fig.S1B):ANK, /1
ankyrinrepeats;SH3, Src homology3 domain;PDZ,postsynapticdensityprotein,Drosophila disklargetumor suppressor(DlgA) andZonula occludens-1 5/3
protein(Zo-1)domainproline-richdomain;SAM,sterilea-motifdomain.(B)GenomicmapandstructureoftheShank3genecoveringexons1–10andthe 0
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targetingconstructforthedeletionofShank3exons4–9.RT-F¼forwardprimersandRT-R¼reverseprimerforRT-PCRanalysisinSupplementaryMaterial, 3
Fig.S1A4.(C)GenotypingofShank3e4–9micebySouthernblotwithXbaIandthe3′ flankingprobe.The10.5kbfragmentisthewild-typeband;the6.4kb /25
fragmentisthemutantband.(D)WesternblotanalysiswithShank3e4–9(–/–)brainsamples;the190,170and140kDabandsareseenintheShank3+/+(+/+) 27
brainandonlythe140and170kDabandsareinShank3e4–9samples.(E)RT-PCRanalysisforspecificShank3transcriptsinShank3e4–9mice.Shank3aand 27
Shank3btranscriptswereabsentinShank3e4–9mice.Othertranscripts(Shank3c–e)frompromoters3–5werepresentinShank3e4–9mice. 1 b
y
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novelshorttranscripts(Shank3a1–2andShank3b1–2),aredis- of 50 Shank3e4–9 and 50 wild-type littermates from 21 u
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rupted(SupplementaryMaterial,Fig.S1B). litters of heterozygous breeding); detailed statistical analyses st o
for all behavioral results are provided in Supplementary n
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Material. Since social behavior is dependent upon olfaction, 5
A
Social behavior is abnormal in Shank3e4–9 mice this sense was evaluated in the Shank3 mice. No genotype p
Shank3e4–9micehadnormalMendelianratiosatweaning,had dfrioffmereensctreusswfeemreaolebssevrevresdusfosraloilnfeac(toSrhyanpkr3e+fe/r+e:nc0e.s50fo+r u0r.0in2e; ril 201
noapparentdevelopmentaldefectsandwereviableandfertile 9
withanormallifespan.ThebodyweightsofShank3e4–9mice Shank3e4–9: 0.41+0.2) or for urine from male breeders
versus saline (Shank3+/+: 0.51+0.04; Shank3e4–9:
at 8–12 months of age were slightly higher than those of
Shank3+/+ animals (Supplementary Material, Fig. S5B), con- 0.43+0.4) or for total interaction times with the paired
stimuli (Shank3+/+: 236+4.2 and 235+7.2s; Shank3e4–9:
sistent with the mild overgrowth seen in some human PMS
patients (14). However, the body weights of the mice at 3–4 242+4.3 and 228+6.7s, respectively). Hence, both geno-
months of age were not significantly different at the time of types are comparably able to discriminate urine from saline.
behavioral testing (see Supplementary Material, Behavioral In a sociability test, both social affiliation and social prefer-
studies). There were no differences for brain weights ence were examined. No stimulus preferences were observed
between adult Shank3e4–9 and Shank3+/+ mice at 2–4 for either genotype during the identical non-social 1–non-
months of age (Supplementary Material, Fig. S5C). social 1 (NS1–NS1) pairing (Fig. 2A and B). In the NS1–
We conducted extensive behavioral analyses on adult social 1 (NS1–Soc1) pairing, Shank3+/+ controls showed
Shank3e4–9 mice (3–6 months old and 4 cohorts of a total higher affiliation for the novel social stimulus than
3096 Human Molecular Genetics, 2011, Vol. 20, No. 15
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Ffoirgpurreefe2r.eSnocecsiabbeiltiwtyeeanndthientiedreancttiicoanlaNsSwoeblljeacstsu.ltIrnasthoeniNcSc1om–Smoucn1icpaatiiroinngs,aSrehaanbkn3o+rm/+a(l+in/S+h)amnka3lee4s–a9nmdifceem.(aAlesanpdreBfe)rNthoegneonvoetylpSeocorstsiemxudliufsf;eSrehnacneks3aer4e–e9v(i–d/e–n)t rticle-a
malesandfemalesshownoorreducedpreferencesforthisstimulus.WhenpresentedwithfamiliarandnovelSocstimuli,allmicepreferthenovelsocialpartner.(C) bs
Inthedyadictest,Shank3e4–9miceparticipateinbidirectionalsocialexchangeswithC3HpartnersforshorterperiodsoftimethanShank3+/+controlsandtheir tra
mpairctenearnsd.(thDe)irSrheasnpke3ctei4v–e9Cm3iHcepatarktneerlosnshgoerwtoredinuictieadtetimtheesirinfirssotcsiaolciinalteirnatcetriaocntsiothnatnhathneSrehsapnekc3t+iv/e+Smhaicnek.3(+E/+atnadrgFe)tsBaontdhthmeairleC(3EH)paanrdtnfeerms;anle¼(F1)0Smhiacnek/g3ee4n–o9- ct/20
type/sex.∗P,0.05,Shank3+/+versusShank3e4–9mice;#P,0.05,femalesversusmaleswithingenotype;‡P,0.05,comparedwiththeNS1–NS1testwithin /1
genotype;¤P,0.05,comparedwiththeNS1–Soc1testwithingenotype;&P,0.05,targetversuspartnerwithingenotype;vP,0.05,Shank3+/+socialpartner 5/3
versusShank3e4–9partner(seeSupplementaryMaterial,Fig.S2andTableS1).(G)Shank3e4–9malesemitmoreandShankee4–9femalesproducefewercallsrelative 09
toShank3+/+controls.(H)RepresentativespectrographsofultrasoniccallsformaleandfemaleShank3+/+andShank3e4–9mice.(IandJ)Thecallsemittedby 3/2
Shank3+/+miceareprimarilyoflongduration,whereasthosebyShank3e4–9micearemostlyofshortduration.(KandL)Shank3+/+malesproducemoremid-range 5
2
andhigh-frequencycallsoflongerdurationthanShank3e4–9males.DurationsdidnotvaryacrossfrequenciesforShank3e4–9mice;however,durationsofhigh- 7
2
frequencycallsbyShank3e4–9femaleswereshorterthanthosebyothergroups;n¼10mice/genotype/sex.∗P,0.05,Shank3+/+versusShank3e4–9mice;#P 7
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,0.05,femalesversusmaleswithingenotype;!P,0.05,comparedwithshortdurationorlow-frequencycalls;¼P,0.05,comparedwithmoderateduration b
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ormid-rangefrequencycalls. g
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Shank3e4–9 mice. Shank3e4–9 females interacted with the mice fail to show social affiliation. However, when forced to n 0
social stimulus more than Shank3e4–9 males. In the familiar choose between two social stimuli in the social preference 5 A
Sreogca1rd–lneossveolfsosecxia—l 2sh(oSwoecd1–siSmoicl2a)r psoaicriianlgp,rbeoftehregnecneostyfoprest—he treinstg, tShheannok3veml auntaimntasl.demonstrate social reference by prefer- pril 2
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novel animal. The reduced social affiliation in the NS1– Socialbehaviorswereexaminedalsoinadyadictest.Pre-test 19
Soc1 pairing in Shank3e4–9 mice was not due to decreased responseswerenotdifferentiatedbygenotype(Supplementary
interaction times, since both genotypes spent similar Material, Table S1). Shank3+/+ controls spent more time in
amountsoftime investigatingbothstimuli,regardless ofpair- bidirectional social interactions than Shank3e4–9 mice
ings (Supplementary Material, Table S1). Additionally, the (Fig. 2C). Latency to the first bidirectional social interaction
numbers of contacts between NS1 and NS1 stimuli did not was shorter in Shank3+/+ than in Shank3e4–9 mice, and was
differ between genotypes (Supplementary Material, Fig. S2A most protracted in mutant females (Fig. 2D). None of the
and B). Although contacts declined for Shank3+/+ mice Shank3e4–9 mice initiated the first social interaction; all
when social stimuli were present, habituation was not initiations were by C3H partners. By comparison, (cid:3)50% of
observed for Shank3e4–9 mice. Notably, Shank3e4–9 females thesocialexchangesbetweenShank3+/+targetsandC3Hpart-
made more NS1–Soc1 contacts than the Shank3+/+ controls. nerswereinitiatedbytargets,regardlessofsex.
Together, these results indicate that when given a choice of A dyadic test was used to analyze a broad range of social
interacting with a social or non-social stimulus, Shank3e4–9 responses. Shank3+/+ males spent less time investigating
Human Molecular Genetics, 2011, Vol. 20, No. 15 3097
their partners than Shank3+/+ females (Fig. 2E and F).None- was similar for Shank3e4–9 (160.6+19.3cm/5min) and
theless, investigation times by Shank3e4–9 mice were lower Shank3+/+ mice (171.1+21.6cm/5min), the ratio of foot-
than Shank3+/+ mice and their C3H partners. Possibly due misplacement errors to distance traveled was higher for
to this reduction, C3H partners of Shank3e4–9 mice engaged mutants (0.23+0.2) than for controls (0.13+0.01).
in fewer social investigations than pairings with Shank3+/+ Shank3e4–9 mice moved more slowly than Shank3+/+
controls. Although investigations were reduced, Shank3e4–9 animals, and the Shank3e4–9 males were slowest of all
mice spent more time self-grooming and sifting through (Fig. 3B). These data indicate that Shank3e4–9 mice have dif-
bedding materials than Shank3+/+ mice (Supplementary ficultiesinfinemotorcoordination,andmutantmalesaremore
Material, Fig. S2C and D); Shank3+/+ controls and their affected than mutant females.
C3H partners rarely engaged in these behaviors. Collectively, Miceweretestedinanopenfieldtodeterminewhethertheir
these data show that Shank3e4–9 mice engage in fewer social motor activities were aberrant. Locomotion was decreased in
interactions and are more likely to participate in non-social Shank3e4–9 males compared with all other groups (Fig. 3C). D
self-focused behaviors than Shank3+/+ mice. Additionally, Additional indices, including time spent in movement, ow
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social investigations are decreased in C3H partners to rearing activity and machine-scored stereotypical activities lo
Shank3e4–9 mice, indicating that the partners are less likely did not differ between genotypes (Supplementary Material, ad
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to sustain interactions with non-socially responsive mice. Table S2). Nevertheless, ethological scoring found that both d
male and female Shank3e4–9 mice participated in more self- fro
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Ultrasonic vocalizations are aberrant in Shank3e4–9 mice ignrdoiocmatiengththatanShSahnakn3k3e4+–/9+ mcoanletrsolse.ngTaoggeeththeer,irtheensveirfionndminegnst http
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UltrasoniccommunicationwasexaminedbyexposingShank3 less, and that grooming is more frequent in mutants than in ://a
mice to a non-familiar C3H mouse where no physical inter- Shank3+/+ controls. ca
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action could occur. Prior to mouse presentation, no ultrasonic Motor learning was evaluated with the accelerating rotorod e
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calls were recorded. At the presentation of the C3H mice, the (Fig.3DandE).Thelatencytofallwasincreasedacrosstrials ic
numbers of vocalizations by Shank3+/+ males and females for Shank3+/+ males, indicating motor learning. In contrast, .ou
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weresimilar(Fig.2G).However,Shank3e4–9malesmadesig- this latency was unchanged for Shank3e4–9 males and was .c
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nificantly more calls, and Shank3e4–9 females made signifi- abbreviated across test trials. No genotype differences were m
cantly fewer calls, than Shank3+/+ mice. The vocal observed for females. Hence, Shank3e4–9 males appear /hm
spectrograms revealed that Shank3+/+ mice produced more impaired on motor learning, whereas females are unaffected. g/a
complex patterns of calls than Shank3e4–9 mice (Fig. 2H). rtic
VwoitchamlizoarteiofnresqfuoerncSyhabnakn3d+s/a+ndmgarleeastewremreodluolnagtieorninofdfurerqatuieonn-, Shank3e4–9 mice display repetitive behaviors le-abs
cies within each call, than Shank3e4–9 males. Shank3+/+ Repetitive responses were examined in a novel environment tra
females made less complex calls than Shank3+/+ males, but with a hole-board test and in a familiar environment with a ct/2
they were longer in duration with more modulation of fre- novel object. With the hole-board, Shank3e4–9 mice engaged 0/1
quency bands than Shank3e4–9 females. Shank3e4–9 females in more head pokes (129.4+8.1) than Shank3+/+ controls 5/3
0
produced calls as single ‘chirps’ that were devoid of the mul- (101.5+6.6); no sex differences were seen. This enhance- 9
tiplefrequencybandsandcomplexitiesofShank3+/+controls. ment in head pokes was not due to reduced anxiety, because 3/2
5
When vocalizations were analyzed as percent durations, genotype responses were similar in the light–dark emergence 27
Shank3+/+ mice made more long than short duration calls and zero maze tests (Supplementary Material, Table S2). 27
1
(Fig. 2I and J); the converse was observed for Shank3e4–9 Moreover, increased head pokes by mutants cannot be b
y
mice—especiallyfemales.Whenthedurationofcallswasana- ascribed to novelty-seeking behaviors, because activity in g
lyzed as a function of frequencies, Shank3+/+ males and the center of the open field, and stretch-attend postures or ues
females emitted mid-range and high-frequency vocalizations head-dipping behaviors in the zero maze were similar t o
n
for longer periods of time than low-frequency calls (Fig. 2K between genotypes. 0
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and L). In contrast, the durations of calls by Shank3e4–9 Inahomecage,novelobjectexplorationwasscoredaccord- A
p
m9 afelemsadleids nemotitctehdanhgiegha-cfrroesqsuefrnecqyuecnacllisesf,owrhsehroeratserShdaunrakt3ioe4n–s iSnhgantok3i+nv/+estcigoanttirnoglsthceoonbtajcetcetdfrothme ionbsijdeectormoourtesidoeftethnefnreosmt. ril 20
1
than all other vocalizations and all other mice. Thus, outside than inside the nest (Fig. 3F). Conversely, 9
Shank3e4–9 mice emit aberrant ultrasonic vocalizations that Shank3e4–9 mice contacted the object more often from the
are altered in both duration and frequency. nest than outside it, and contacts from both locations were
higher than the Shank3+/+ mice. Maps were constructed to
analyze the patterns of object interactions. Regardless of
Motor behaviors are aberrant in Shank3e4–9 mice
sex, Shank3e4–9 mice made repeated contacts, but only on
In a neurophysiological screen, Shank3e4–9 mice displayed certain locations on the object (Fig. 3G and Supplementary
mild motor abnormalities as exemplified in tests of vertical Material, Fig. S3). By comparison, object contacts by
placement and delayed climbing down a vertical pole for Shank3+/+ controls were not repetitive. When Shank3e4–9
males and females and in contact righting for males (Sup- mice were not investigating the object, they were engaged in
plementary Material, Table S2). In the foot-misplacement self-grooming—a behavior that rarely occurred in
test, foot-faults were increased in Shank3e4–9 mice Shank3+/+ controls (Fig. 3H). Thus, upon encountering a
(Fig. 3A). Although the distance traveled in the apparatus novel object in their familiar environment, Shank3e4–9 mice
3098 Human Molecular Genetics, 2011, Vol. 20, No. 15
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Figure3.Shank3e4–9micedisplayabnormalitiesinmotorperformance.(A)Shank3e4–9(–/–)micemakemorefoot-misplacementsthanShank3+/+(+/+)con- t o
trols.(B)Shank3e4–9micemovemoreslowlythanShank3+/+mice;Shank3e4–9malesareslowerthanmutantfemales.(C)Spontaneouslocomotoractivityin n 0
twhiethopSehnanfike3ld+/i+slmowaleersinovSehraanlkl3fiev4e–9trmiaallseasntdhaintiisnpoethrteurrbgeroduopns.t(rDialasn4daEn)dM5octoomrlpeaarrendinwgiothntShheaanckc3e+l/e+raatinndgSrohtaonrko3de4is–d9effiemcieanletsi.n(FSh)aRnekp3ee4ti–ti9vmeafrleeqsuceonmcipeasreodf 5 Ap
oexbpjelcotreextphleornaotivoenlsofrbojemcti.nsSihdaenakn3de4o–u9tsmidaelethsesnpeesnttwseimreilhairghaemrofuonrtSshoafnkt3ime4e–9etxhpalnorSihnagntkh3e+/o+bjmecictse.f(rGom)Sihnasnidke3+a/n+dmoaultessidaendthfeemnaelsetstthyapnicSalhlaynlke3ft+t/h+enmeasltetso; ril 20
Shank3e4–9 females spent the most time exploring objects from the nest. (H) Shank3e4–9 spent more time self-grooming than Shank3+/+ controls; n¼10 19
mice/genotype/sex.∗P,0.05,Shank3+/+ versusShank3e4–9mice;#P,0.05,femalesversusmaleswithingenotype;+P,0.05,Shank3+/+ femalesversus
Shank3e4–9males;†P,0.05,innestcomparedwithoutofthenest.
engage in stereotyped investigations and participate in self- fornullactivity,startleresponsesorinhibitiontothethreepre-
stimulatory behavior. pulses(SupplementaryMaterial,TableS2).Inthewatermaze,
swimdistancedeclinedacrossacquisitiontestingforbothgen-
otypes; this reduction occurred less rapidly over the first
Learning and memory are deficient in Shank3e4–9 mice 3 days for Shank3e4–9 mice (Fig. 4A, left). When the hidden
platform was moved from the NE to the SW quadrant, swim
Cognitive performance wasexamined inShank3 mice bypre-
pulse inhibition, Morris water maze, novel object recognition distances initially increased for both genotypes on day 7
and social transmission of food preference (STFP) tests. For (Fig. 4A, right). Subsequently, it decreased for Shank3+/+
prepulse inhibition, no genotype differences were observed controls, but remained higher for Shank3e4–9 mice on days
Human Molecular Genetics, 2011, Vol. 20, No. 15 3099
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Figure4.Shank3e4–9micearedeficientinlearningandmemory.(A)Toreachthehiddenplatforminwatermaze,Shank3e4–9miceswamoverlongerdistancesonthe 1 b
Sfihrsatn3k3dea4y–s9omfaicceq.u(iBsitiaonndteCst)inVgisthibalneSphlaatnfko3rm+/+temstiicneg.Wwihthenntah¨ıveehi(dledfetn)panladtfowramtewr-amsamzeoveexdpetorieannceewd l(oEcxapt.i)on(,risgwhitm) Sdhiastnakn3c+e/+waasnidncSrheaasnekd3eo4n–9damysic9e.–1N1a¨ıfvoer y gu
Shank3e4–9males(B,left)andfemales(C,left)swamoverlongerdistancestoreachthevisibleplatformthanrespectiveShank3+/+controls.Swimdistances es
forexperiencedShank3e4–9males(B,right)andfemales(C,right)weresimilartothoseoftherespectiveShank3+/+controls.(DandE)Swimdistancesfor t o
Shank3+/+(D)andShank3e4–9(E)miceduringacquisition(days2,4and6)andreversalprobetests(days8,10and12).Duringacquisition,allmiceshoweda n 0
markedpreferencefortheNEquadrant.Atreversal,Shank3+/+ mice,butnotShank3e4–9mice,developedasignificantpreferencefortheSWquadrant.(F) 5 A
Shank3micewereevaluatedinthenovelobjectrecognitiontestconsistingoftraining(Train),andtestsforSTM,LTMandRM.Duringtraining,neithergenotype p
Sshhoawnke3dea4n–y9mpriecfee.r(eGnc)eTfootraelinthuemrboefrtshoefidoebnjeticctacloonbtjaeccttss.wSehraenskim3+il/a+rmbeitcweepernefgeerrneodtythpeesn.oOvbeljeocbtjceocnttoancttshedeScTliMne,dLfToMrSahnadnkR3M+/+tesmtsi;cperoevfeerreLncTeMswaenrdeRreMdutceesdtinfogr; ril 20
1
contactsremainedhighforShank3e4–9mice.(H)IntheSTFPtest,nogenotypedifferenceswerenotedforSTM.Shank3e4–9miceshowedadramaticallyreduced 9
preferenceforthedemonstratordietduringtheLTMandRMtests.Foralltests,n¼10mice/genotype/sex.∗P,0.05,Shank3+/+versusShank3e4–9mice;#P,
0.05,femalesversusmaleswithingenotype;^P,0.05,na¨ıveShank3e4–9miceversusexperiencedShank3e4–9mice;§P,0.05,versusNEquadrant;XP,0.05,
versusSWquadrant;†P,0.05,comparedwiththeSTMtest.
8–10.Theresultsforswimtimeweresimilar(Supplementary Probe tests for acquisition testing (days 2, 4, 6) failed to
Material, Fig. S4A). Representative tracings of swim paths detect significant genotype differences in swim distance
during acquisition supported the swim distance results (Sup- (Fig. 4D and E); all mice swam further in the NE than in
plementary Material, Fig. S4F, top left). Over reversal the other three quadrants. When the hidden platform was
testing, tracings revealed that Shank3e4–9 mice swam in moved to the SW quadrant, neither genotype displayed any
more circular search patterns (e.g. day 12) than the quadrant preference on day 8. However, by days 10 and 12,
Shank3+/+ mice (Supplementary Material, Fig. S4F, top Shank3+/+ miceswamfurtherintheSWthantheotherquad-
right). rants. In contrast, Shank3e4–9 mice failed to show any
3100 Human Molecular Genetics, 2011, Vol. 20, No. 15
quadrant preference. An examination of swim times revealed analyses with light and electron microscopy (Fig. 5E and F
similar results (Supplementary Material, Fig. S4D and E). andSupplementaryMaterial,Fig.S5A).Toexaminedendritic
Although tracings of the swim paths during acquisition spine morphology, hippocampal slice cultures were prepared
probe tests were similar between genotypes (Supplementary frompups6–7daysoldandneuronsweretransfectedbiolisti-
Material, Fig. S4F, bottom), the wide and circling patterns cally with a GFP plasmid (Supplementary Material,
of Shank3e4–9 mice became more prominent by the last day Fig.S5D).Althoughnogenotypedifferencesweredetectedin
of reversal. Together, these results suggest that spatial dendritic branching (Fig. 5A) or in spine densities and spine-
memory and plasticity may be deficient in Shank3e4–9 mice. headsizes(Fig.5BandD),dendriticspinesweresignificantly
The impaired performance in reversal training may also indi- longer in Shank3e4–9 (1.23+0.07mm, 678 spines from 7
cate inflexibility of behaviors in Shank3e4–9 mice. mice) than Shank3+/+ mice (0.76+0.06mm, 1183 spines
Over the first five trials of the visible platform test, na¨ıve from 8 mice; P,0.002) (Fig. 5C). Interestingly, overall,
Shank3e4–9 males swam further to reach the platform than Shank3e4–9micehadmorethinspinesthanShank3+/+controls; D
na¨ıve Shank3+/+ males; na¨ıve mutant females swam further however,thisdifferencedidnotachievestatisticalsignificance ow
on the first two trials than na¨ıve female controls (Fig. 4B (Shank3+/+ mice: stubby 151, mushroom 283 and thin spines nlo
and C, left). Notably, na¨ıve Shank3e4–9 females swam over 691; Shank3e4–9 mice: stubby 77, mushroom 172 and ad
e
shorter distances on the first six trials than na¨ıve Shank3e4–9 thinspine432,P.0.05).Theratioofstubby-andmushroom- d
males. To determine whether the protracted swim times of shapedspinestothinspineswasborderlinelowerinShank3e4–9 fro
m
na¨ıveShank3e4–9micecouldbeattributedtodelayedlearning mice [Shank3+/+ mice: (151+283)/691¼0.63+0.06; h
or to performance issues, na¨ıve and experienced mice were Shank3e4–9 mice: (77+172)/432¼0.58+0.07), P¼ ttp
s
compared.Althoughswimdistancesforna¨ıveandexperienced 0.052]. We used Golgi impregnation to examine dendritic ://a
Shank3+/+ controls did not differ (Fig. 4B and C, right), spine morphology in CA1 hippocampus of 4- and ca
d
experienced Shank3e4–9 males and females swam over 10-week-old Shank3e4–9 mice (Fig. 5G and H). Spine density e
m
shorter distances than respective na¨ıve mutants. Analyses of intheCA1hippocampuswasreducedandspinelengthwassig- ic
swim time revealed similar results (Supplementary Material, nificantlyincreasedin4-week-oldShank3e4–9micecompared .ou
Fig. S4B and C). Hence, deficiencies of na¨ıve Shank3e4–9 with Shank3+/+ mice (spine density: +/+, 1.49+0.09/mm; p.c
o
mice appear tobeduetotest familiarizationordelayed learn- –/–, 1.24+0.07/mm, P¼0.03; spine length: +/+, 0.94+ m
/h
ing, rather than specific sensory, motor or motivational 0.02mm; –/–, 1.05+0.03mm, P,0.003) (Fig. 5I and J). m
g
abnormalities. Cumulative frequencies for spine length are shown in Sup- /a
Episodic short-term (STM), long-term (LTM) and remote plementary Material, Fig. S5E and F. In contrast, spine rtic
memory(RM)wereanalyzedusinganovelobjectrecognition density was not significantly reduced but spine length was le-a
test.PreferencesforthenovelobjectbyShank3+/+ micewere increased in 10-week-old Shank3e4–9 compared with bs
high and unchanged across the STM, LTM and RM tests Shank3+/+ mice (spine density: +/+, 1.70+0.04/mm; –/–, tra
c
(Fig. 4F). In contrast, novel object preference was decreased 1.63+0.03/mm, P.0.05; spine length: +/+, 0.91+ t/2
for Shank3e4–9mice. Since poor performance could be attrib- 0.02mm; –/–, 0.85+0.01mm, P¼0.01) (Fig. 5K and L). 0/1
5
uted to object neophobia, the duration of object contacts and No gender difference was noted. The reduced density and /3
the numbers of object contacts were examined. The duration increasedspinelengthin4-week-oldShank3e4–9micearecon- 09
3
ofobjectcontactswassimilarforbothgenotypes(Supplemen- sistentwiththeobservationsofsiRNA-mediatedknockdownof /2
taryMaterial,TableS3).Shank3+/+andShank3e4–9micealso Shank3 in cultured neurons (9). The discrepancy for the spine 527
2
contactedtheobjectssimilarnumbersoftimesduringtraining density between cultured neurons of newborns and Golgi 7
and the STM test (Fig. 4G). However, Shank3+/+ mice made impregnation of 4-week-old mice and between 4- and 1 b
y
fewer object contacts than mutants during the LTM and RM 10-week-oldmice may beduetodifferences inthesensitivity g
u
tests, and these contact numbers were less than those during of the methods—although it could also reflect developmental es
training and the STM test. In contrast, object contacts were differences. t o
n
not reduced across tests for Shank3e4–9 animals. 0
In the STFP test, Shank3+/+ controls maintained a strong 5 A
Altered PSD proteins in Shank3e4–9 mice p
preference for the demonstrator diet during the STM, LTM ril 2
and RM tests, with little change across test-days (Fig. 4H). To examine whether the composition of PSD proteins was 0
1
However, this preference was lost by Shank3e4–9 mice altered in Shank3e4–9 animals, we performed quantitative 9
during the LTM and RM tests. Despite impairments in LTM immunoblotanalysesforcandidateShank3-interactingproteins
and RM in mutants, no genotype differences were discerned (Supplementary Material, Table S4). No genotype differences
for time spent interacting with the demonstrator mouse, the were detected in the levels of cytosolic proteins extracted
total amount of diets eaten or times investigating the diets frombrain(SupplementaryMaterial,Fig.S6AandB).Incon-
on each test-day (Supplementary Material, Table S3). Collec- trast,whenlevelsofsynapticproteinsfromthePSD-Ifraction
tively,theobjectrecognitionandSTFPresultsindicatethatat were examined, GKAP and Homer1b/c were reduced in
least LTM and RM are impaired in Shank3e4–9 mice. Shank3e4–9 compared with Shank3+/+ control PSD fractions
(85.2+6.1% of Shank3+/+ for GKAP, P¼0.03, n¼11
mice/genotype; 66.1+4.2%of Shank3+/+ for Homer1b/c, P
Dendritic spine morphology is altered in Shank3e4–9 mice
,0.001, n¼16 mice/genotype); no genotype distinctions
Brain anatomy of Shank3e4–9 mice at 2–4 months of age wereobserved forothersynapticproteinsatthePSD(Fig.6A
appeared normal by gross, histological and ultrastructural and C and Supplementary Material, Fig. S6C and D). No
Human Molecular Genetics, 2011, Vol. 20, No. 15 3101
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Figure5.DendriticspinemorphologywasalteredinShank3e4–9mice.(A)Low(left)andhigh(right)resolutionimagesofEGFP-expressingpyramidalneurons /2
fromShank3+/+(+/+)andShank3e4–9(–/–)mice.Quantificationofspinedensity(B),spinelength(C)andspine-headarea(D).Asignificantdifferencewas 52
7
revealedinspinelengthbutnotforspinedensityorspine-headarea(spinelength:1.23+0.07mm,678spinesfrom7Shank3e4–9mice;and0.76+0.06mm, 2
1183spinesfrom8Shank3+/+ mice;∗P,0.002,Shank3+/+ versusShank3e4–9mice).(EandF)RepresentativeelectronmicrographsofhippocampalCA1 71
striatum radiatum synapses from Shank3+/+and Shank3e4–9 mice. The PSD is visible as an electron-dense layer adjacent to the post-SPM by
(scalebar¼0.1mm).Atotalof71synapsesfrom3Shank3+/+miceand69synapsesfrom3Shank3e4–9miceweremeasured.Therewerenosignificantgeno- g
u
typechangesinPSDlengthorthickness.(GandH)RepresentativeimagesfromGolgistainingof4-week(G)and10-week-old(H)Shank3+/+andShank3e4–9 e
s
mice.Scalebar¼5mm.(IandJ)Spinedensity(I)andspinelength(J)in4-week-oldShank3e4–9mice(n¼15cellsfrom3+/+miceandn¼14cellsfrom3 t o
–le/n–gtmh)i.ce()K.AatnodtalLo)fS73p1inespidneenssfitryom(KS)haannkd3+s/p+inaendle6n4g9thsp(inLe)sifnrom10S-whaenekk3-oe4ld–9Smhaicnek3wee4r–e9mmeiacseure(nd¼(∗P40¼c0e.l0l3s fforromspin3e+de/n+sitymaicned;Pn,¼03.1003ceflolsrsfproinme n 05
3 –/– mice).Atotalof992spinesfromShank3+/+ and707spinesfromShank3e4–9miceweremeasured(∗P¼0.01forspinelength). A
p
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significant sex differences were noted. The AMPAR subunit Homer1b/c by co-immunoprecipitation (Supplementary 0
1
GluA1 and the NMDAR subunit NR2A were significantly Material,Fig.S6EandF). 9
reduced in membrane (SPM) fractions from Shank3e4–9 mice ThedistributionofsynapticPSD-Iproteinswasfurtherana-
(72.0+6.2% of Shank3+/+ for GluA1, P,0.002, n¼8 lyzed by immunostaining of hippocampal neuronal cultures
mice/genotype; 75.0+8.3% of Shank3+/+ for NR2A, P¼ fromShank34–9mice.Consistentwiththequantitativeimmuno-
0.02,n¼8mice/genotype).Levelsofotherreceptorsubunits, blots,immunostainingofHomer1b/candGluA1werereduced
includingtheAMPARGluA2andtheNMDARNR2Bsubunits, in Shank3e4–9 mice (Fig. 6E)—as confirmed by quantitative
werenotsignificantlyalteredinSPMfractionsfromShank3e4–9 analyses of Homer1b/c and GluA1 puncta (87.2+2.1 % of
mice (Fig. 6B and D). No sex differences were observed. Shank3+/+ for Homer1b/c, P,0.01, n¼17 cells/genotype;
Reduced levels of GluA1 and Homer1b/c were observed in 80.3+3.4% of Shank3+/+ for GluA1, P,0.001, n¼27
Shank3e4–9heterozygotescomparedwithShank3+/+ controls, cells/genotype)(Fig.6F).Thepunctasizesofimmunostaining
butthedifferencesweremoresubtlethanbetweenthehomozy- for Homer1b/c and GluA1 were also reduced in Shank3e4–9
gous mutant Shank3e4–9 and Shank3+/+ controls. In addition, mice (Supplementary Material, Fig. S5G). Although GKAP
we also detected an interaction between Shank3 and (n¼8/genotype)andNR2A(n¼8/genotype)immunostaining
3102 Human Molecular Genetics, 2011, Vol. 20, No. 15
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Figure6.AlteredproteincompositioninPSDandSPMfractionsofShank3e4–9mice.ImmunoblotanalysesofPSD(A)andSPM(B)fractionsfromindividual /25
Shank3+/+ and Shank3e4–9 mice for the indicated proteins. Pan-Shank (Shank1–3) antisera revealed an absence of the Shank3 band in Shank3e4–9 mice. 27
Homer1b/candGKAPinthePSDandGluA1andNR2AintheSPMarereducedinShank3e4–9mice.(CandD)QuantificationofproteinsinPSDfractions 27
bparosetedinosnirnesSuhlatsnskh3oe4w–n9icno(mAp)aarnedd(wBi)th,rSeshpaenckt3iv+e/l+y,snaomrpmleasli.zHedomuseinr1gba/cct(innf¼or1p6roetaecihn/qguenaonttyifipcea,t∗ioPn.,T0h.e0r0e1i)s,aGsKigAniPfic(nan¼tr1ed1uecatciohn/gienntohteypleev,∗ePls¼of0th.0e3i)n,dGicluatAe1d 1 by
(n¼8each/genotype,∗P,0.002)andNR2A(n¼8each/genotype,∗P¼0.02).Nosexdifferencewasobserved.Thereisnosignificantdifferencesforother gu
proteinsincludingHomer1a(n¼11each/genotype),GluA2(n¼8each/genotype)andNR2B(n¼8each/genotype).(E)Hippocampalneuronsindissociated es
culture(16–18DIV)fromShank3+/+orShank3e4–9mice(n¼8each/genotype)wereimmunostainedwithbassoonantibody(red)andwithGKAP,Homer1b/c, t o
n
GluA1(C-terminus)orNR2Aantibodies(green).Mergedimagesshownontheright.Scalebar¼5mm.(F)Normalizedintegrateddensityfortheindicated 0
(p∗rPote,in0s.0(1ShfoarnkH3o+m/+er¼1b1/c.0a0n).d∗SPig,nifi0c.0an0t1fdoirffGerleunAce1s).NwoeresigfnoiufincdantfodriffHeroemnceers1bw/cereanfodunGdlufoAr1GKprAotPeiannsdbNeRtw2eAe.n Shank3+/+ and Shank3e4–9 samples 5 Ap
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weredecreasedinShank3e4–9mice,thisdifferencewasnotstat- Shaffer-collateral synapses in 2–4-month-old Shank3e4–9 9
isticallysignificant,perhapsreflectingthedifferingsensitivities mice, using field post-synaptic potential (fPSP) recordings.
ofthesemethods,ordifferencesinsynapticversusnon-synaptic Shank3e4–9 mice showed reduced post-tetanic potentiation
poolsofthesemolecules.Together,thesefindingsindicatethat compared with Shank3+/+ animals (Shank3+/+ mice: 195+
specificsynapticscaffoldingproteinsandreceptorsubunitsare 22% baseline fPSP slope, n¼12; Shank3e4–9 mice:
alteredbythedisruptionofmajorShank3isoforms. 150+10% baseline fPSP slope, n¼8; P,0.001)
(Fig. 7A). Moreover, Shank3e4–9 mice exhibited reduced hip-
pocampal long-term potentiation (LTP) (Shank3+/+ mice:
Synaptic plasticity is impaired in Shank3e4–9 mice
165+9% baseline fPSP slope, n¼12; Shank3e4–9 mice:
The reductions in GluA1 subunits and other PSD proteins in 127+6% baseline fPSP slope, n¼8; P,0.001) (Fig. 7A).
Shank3e4–9 mice suggest a possible role for Shank3 in synap- No sex differences were observed. Despite the plasticity
tic function. To examine this possibility, we analyzed excit- defect, there were no apparent differences in basal synaptic
atory synaptic transmission and plasticity at the CA1 transmission between Shank3+/+ and Shank3e4–9 mice. The