Carlisle2011p16505J_Neurosci.pdf

Cellular/Molecular

Deletion of Densin-180 Results in Abnormal Behaviors

Associated with Mental Illness and Reduces mGluR5 and

DISC1 in the Postsynaptic Density Fraction

Holly J. Carlisle,

* Tinh N. Luong,

* Andrew Medina-Marino,

* Leslie Schenker,

Eugenia Khorosheva,

Tim Indersmitten,

Keith M. Gunapala,

Andrew D. Steele,

Thomas J. O’Dell,

Paul H. Patterson,

and Mary B. Kennedy

Division of Biology, California Institute of Technology, Pasadena, California 91105, and

David Geffen School of Medicine, University of California, Los

Angeles, Los Angeles, California 90095

Densin is an abundant scaffold protein in the postsynaptic density (PSD) that forms a high-affinity complex with

CaMKII and

-actinin.

To assess the function of densin, we created a mouse line with a null mutation in the gene encoding it (

LRRC7

). Homozygous knock-out

mice display a wide variety of abnormal behaviors that are often considered endophenotypes of schizophrenia and autism spectrum

disorders. At the cellular level, loss of densin results in reduced levels of

-actinin in the brain and selective reduction in the localization

of mGluR5 and DISC1 in the PSD fraction, whereas the amounts of ionotropic glutamate receptors and other prominent PSD proteins are

unchanged. In addition, deletion of densin results in impairment of mGluR- and NMDA receptor-dependent forms of long-term depres-

sion, alters the early dynamics of regulation of CaMKII by NMDA-type glutamate receptors, and produces a change in spine morphology.

These results indicate that densin influences the function of mGluRs and CaMKII at synapses and contributes to localization of mGluR5

and DISC1 in the PSD fraction. They are consistent with the hypothesis that mutations that disrupt the organization and/or dynamics of

postsynaptic signaling complexes in excitatory synapses can cause behavioral endophenotypes of mental illness.

Introduction

In excitatory synapses of the CNS, activity-dependent regulation

of synaptic strength is mediated by a large protein complex called

the postsynaptic density (PSD) (Kennedy, 2000). Genetic vari-

ants of PSD proteins have been associated with cognitive defects

or mental illness in humans (Durand et al., 2007; Pinto et al.,

2010). For example, copy number variants (CNVs) of synGAP, a

prominent component of the PSD, are associated with non-

syndromic mental retardation and autism spectrum disorders

(ASDs) (Hamdan et al., 2009; Pinto et al., 2010); CNVs of the

PSD scaffold protein shank3 have been linked to ASDs (Durand

et al., 2007; Bourgeron, 2009; Pinto et al., 2010); and the 3q29

microdeletion syndrome, which involves mild mental retarda-

tion and sometimes autism, includes deletion of the gene for the

PSD scaffold protein sap97 (Willatt et al., 2005). Although men-

tal illnesses are most precisely defined in humans, a number of

abnormal behaviors in rodents that are similar to those found in

humans have been defined as “behavioral endophenotypes” (iso-

lated behavioral features) associated with mental illness. These

include endophenotypes associated with schizophrenia (Powell

and Miyakawa, 2006) and with ASDs (Silverman et al., 2010).

Mutations in PSD proteins, including synGAP and calcium/

calmodulin-dependent protein kinase II (CaMKII), can produce

some of these endophenotypes (Yamasaki et al., 2008; Brandon et

al., 2009; Guo et al., 2009). Here we show that mice with a dele-

tion of the PSD scaffold protein densin-180 (densin) display

several behavioral endophenotypes often associated with schizo-

phrenia and with ASDs.

Densin is sufficiently abundant in the PSD that it was among

the first proteins identified by sequencing and cloning from the

PSD fraction (Apperson et al., 1996). Its domain structure sug-

gests that it functions as a scaffold protein, and it has been re-

ported to bind directly to CaMKII,

-actinin,

-catenin, and

shank (Strack et al., 2000; Walikonis et al., 2001; Izawa et al., 2002;

Quitsch et al., 2005). However, the normal physiological roles of

its various associations are not known. Here we show that dele-

tion of densin causes a reduction in the level of

-actinin globally

in brain homogenate and in the PSD fraction and selectively re-

duces the amounts of the metabotropic glutamate receptor

Received Nov. 8, 2010; revised July 29, 2011; accepted Aug. 15, 2011.

Authorcontributions:H.J.C.,T.N.L.,A.M.-M.,E.K.,T.I.,K.M.G.,A.D.S.,T.J.O.,P.H.P.,andM.B.K.designedresearch;

H.J.C., T.N.L., A.M.-M., L.S., E.K., T.I., K.M.G., and T.J.O. performed research; H.J.C., T.L., A.M.-M., L.S., E.K., T.I.,

K.M.G.,A.D.S.,T.J.O.,P.H.P.,andM.B.K.analyzeddata;H.J.C.,T.N.L.,A.M.-M.,A.D.S.,T.J.O.,P.H.P.,andM.B.K.wrote

the paper.

*H.J.C., T.N.L., and A.M.-.M. contributed equally to this work.

This research was supported by National Institutes of Health Grants NS17660 and NS028710 (M.B.K.) and

MH609197 (T.J.O.), the Gordon and Betty Moore Foundation Center for Integrative Study of Cell Biology (M.B.K. and

H.J.C.), the Howard Hughes Medical Institute (A.M.-M.), National Science Foundation Grant 0543651 (T.J.O.), the

McGrath Foundation (P.H.P.), and the Broad Fellows in Brain Circuitry program (K.M.G. and A.D.S.). We thank J.

Sanes(HarvardUniversity,Boston,MA)andP.Seeburg(MaxPlankInstituteforMedicalResearch,Munich,Germany)

for mutant mouse strains and Sarah Cantor for help with experiments.

Correspondence should be addressed to Mary B. Kennedy, Division of Biology 216-76, California Institute of

Technology, Pasadena, CA 91105. E-mail: kennedym@its.caltech.edu.

H. J. Carlisle’s present address: Department of Neuroscience, Amgen, Thousand Oaks, CA 91360

Andrew Medina-Marino’s present address: Epidemic Intelligence Service, Center for Disease Control, Chicago, IL

60657.

DOI:10.1523/JNEUROSCI.5877-10.2011

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31(45):16194 –16207

mGluR5 and the scaffold protein DISC1 in the PSD fraction. In

contrast, the amount of CaMKII in the PSD is not reduced. At the

cellular level, we show that activation of CaMKII by NMDA

receptors (NMDARs) is subtly altered in neurons from the

knock-out animals. Furthermore, both low-frequency- and

(

)-3,5-dihydroxyphenylglycine (DHPG)-induced long-term

depression (LTD) are abnormal, and spine morphology is altered

in knock-out animals. Our results support the general hypothesis

that mutations that alter the organization and/or dynamics of

synaptic signal transduction can result in behavioral endopheno-

types associated with mental illness.

Materials and Methods

Generation of the densin knock-outs

The densin knock-out mouse line was generated by deleting exon 3 of the

LRRC7

gene, which contains the transcriptional start site of the densin

message. The deletion was generated by homologous recombination to

introduce loxP sites, followed by introduction of Cre recombinase

utero

. Clone 456C10 was selected from a CITB mouse bacterial artificial

chromosome (BAC) library (Research Genetics) encoding genomic se-

quences of strain 129S1Sv (recently renamed 129S3Sv/ImJ) because it

hybridized with a cDNA probe encoding exon 3 of the

LRRC7

gene. The

presence of exon 3 in the BAC clone was confirmed by PCR. The BAC

DNA insertion junctions were sequenced and aligned with the known

genomic sequence of the

LRRC7

gene. A 9.9 kb region of clone 456C10

that included exon 3 and its surrounding introns was cloned into the

pKO Scrambler 907 vector (Stratagene). The long arm of the densin

targeting construct contained a 7.8 kb fragment, including intron 2, exon

3, and part of intron 3; the short arm contained a 2.1 kb fragment of

intron 3. The first loxP site was inserted 4.8 kb upstream of exon 3. A

hygromycin selection cassette, flanked on both sides by loxP sites, was

cloned into the short arm of the targeting sequence 1.1 kb downstream

from exon 3. The linearized targeting construct (25

g) was electropo-

rated into 1

cells per cuvette of mouse ES (CJ7) cells. Transfected

ES cells were grown in the presence of hygromycin (200

g/ml) for 7– 8 d

to select for homologous recombinants. Two recombinant clones with

integration of both arms were identified and confirmed by PCR. One

clone (2G8) exhibited a normal karyotype and was expanded for the

generation of densin knock-out ES cells. Ninety-eight 129B6 blastocysts

injected with ES cells from the 2G8 clone were implanted into seven

pseudo-pregnant mothers. Chimeric pups exhibiting a

90% agouti

coat color were used for subsequent breeding.

Eight adult male chimeras were mated to C57BL/6 EIIaCre

expressing female mice (The Jackson Laboratory) developed in the lab-

oratory of H. Westphal (NIH, Bethesda, MD; Lakso et al., 1996). F1

generation offspring were screened for mosaic Cre-recombination pat-

terns by PCR. F1 animals exhibiting genomic mosaicism were subse-

quently mated to wild-type C57BL/6 mice. Segregation of the EIIaCre

transgene and Cre-recombined alleles was monitored by PCR in the F2

generation. F2 generation

EIIaCre

densin

(total excision loxP 1/3

recombination pattern) males were liberally mated to wild-type C57BL/6

females to generate a large F3 population of animals heterozygous for the

densin deletion. Deletion was verified by PCR. Densin knock-out mice

were maintained as heterozygotes. Animals with the loxP 1 plus loxP 2/3

pattern (floxed densin, not used in this study) were bred as homozygotes.

Behavioral cohorts were bred from a line outcrossed into C57BL/6 seven

times. All other experiments were performed with mice that had been

outcrossed twice and maintained as F2.

Genotyping of densin knock-out mice

Tissue from tail clippings or ear punches was lysed in 100

l of Viagen

direct PCR tail mix supplemented with Proteinase K overnight at 55–

60°C. To genotype embryos, we prepared DNA from either the tail or leg

of the embryo harvested at the time that the brain was dissected to make

neuronal cultures. Samples were heated at 85°C for 40 min to inactivate

Proteinase K and then centrifuged for 10 min at 16,000

. Template

DNA was sedimented by centrifugation for 10 min at 16,000

and

amplified by PCR in 1

Coraload (Qiagen) PCR buffer, 0.625 m

MgCl

, 0.3 m

dNTP, 1.25 U/2.5

l of Taq polymerase (Qiagen), and 1

pm/

l of the following primers: Lox Pray Up (5

-GAGATGCTCTCA

AGATAGACATG-3), Lox Pray low (5

-CTCCAATTCTGAAGCCAG

TAG-3

), and Posthygro2 (5

-ACAGAACTGGCTTCTGTCCAC- 3

The temperature was cycled as follows: 11 cycles of 95°C for 30 s, 58°C for

30 s, 72°C for 2 min; 21 cycles of 94°C for 40 s, 56°C for 30 s, 72°C for 2

min; 1 cycle of 72°C for 5 min. PCR products were fractionated on a 1.6%

TBS agarose gel with ethidium bromide. A DNA band at 187 bp identifies

a wild type mouse, a 257 bp identifies a knock-out mouse, and 187 and

257 bp bands indentify heterozygous mice.

Verification of densin deletion

Aliquots containing equal amounts of total protein (10 or 50

g) mea-

sured by the bicinchoninic acid method (Pierce) with BSA as standard

were fractionated by SDS-PAGE, transferred to nitrocellulose, and

blocked as described below. Membranes were incubated with rabbit anti-

densin antisera CT245 (1:2500) against the PDZ domain, mouse anti-

densin polyclonal antibody M2 (1:2500) against the mucin homology

domain, or mouse anti-densin polyclonal antibody LRR (1:1000) against

the LRR domain, diluted into 20 m

Tris, 150 m

NaCl, and 0.1% Tween

20 (Apperson et al., 1996). Bound antibodies were detected with fluores-

cent secondary antibodies and visualized with the Odyssey Infrared Im-

aging System (Li-Cor Bioscience) as described below (see SDS-PAGE

immunoblot analysis).

Other mouse strains

GFP line M transgenic mice (a gift from Joshua Sanes, Harvard Univer-

sity, Boston, MA) were maintained as homozygotes in a C57BL/6 back-

ground (Feng et al., 2000).

GFP

transgenics were crossed with F2

generation

densin

mice.

GFP

densin

offspring were crossed

to produce

GFP

/densin

and

GFP

/densin

mice that were

used for spine analysis. GluN1 knock-out mice (a gift from Peter See-

burg, Max Planck Institute, Heidelberg, Germany) were maintained as

heterozygotes.

GluN1

mutants were crossed with F2 generation

densin

mice to produce GluN1/densin heterozygotes.

GluN1

densin

mice (F3) were subsequently crossed to produce GluN1/den

sin double knock-out embryos (

⁄

of embryos, 6.3%) and littermate

controls for immunocytochemistry of neuronal cultures. Individual em-

bryos were genotyped after removal of neurons to be cultured.

Fixation and Nissl staining

Densin knock-out and wild-type littermate pairs, aged 8 –12 weeks, were

perfused transcardially with 4% formaldehyde, 15% saturated picric acid

in 0.1

PBS for 20 min. Forebrains were dissected and postfixed over-

night at 4°C, and then 50

m coronal sections were cut with a vibratome

and stored at

20°C in 50 m

phosphate buffer, 15% glucose, 30%

ethylene glycol. Sections were washed in PBS (10 m

NaHPO

, 120 m

NaCl, pH 7.4), mounted on Supermount Plus slides, and dried over-

night. Slides were delipidated in a series of decreasing ethanol solutions:

95% (15 min), 70% (1 min), and 50% (1 min). Afte

r a 5 min water rinse,

slides were submerged in 0.5% cresyl violet, 0.125% glacial acetic acid in

distilled water for 5 min, rinsed for 30 s in distilled water, and destained

in 96% ethanol/0.5% acetic acid for 5 min. Slices were cleared in isopro-

panol (5 min), followed by 5 min in isopropanol/xylene (1:2 parts) and

four rinses in xylene for 2 min each. Slides were sealed with Permount

and dried at room temperature overnight. Slides were imaged at 2.5

(Plan-Neofluar 2.5

/NA 0.075) and 5

(Plan-Neofluar 5

/NA 0.15)

magnification.

GFAP immunohistochemistry

Fifty-micrometer coronal sections were incubated for 10 min in a solu-

tion containing PBS, 0.5% Triton X-100, and 0.5% H

to permeabilize

the tissue and eliminate endogenous peroxidase activity and then incu-

bated in blocking solution containing 0.1

PBS and 5% normal goat

serum (Invitrogen) for 10 min, followed by overnight incubation at 4°C

in blocking solution plus anti-glial fibrillary acidic protein (GFAP) anti-

body (1:500; Millipore). Sections were washed in PBS (three times for 5

min) and incubated with biotinylated goat anti-mouse IgG (1:200; Vec-

tor Laboratories) in blocking buffer fo

r2hat

room temperature and then

washed (three times for 5 min) in 0.1

PBS and incubated in avidin–

Carlisle et al.

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Deletion of Densin Produces Abnormal Behavior

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31(45):16194 –16207

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biotin–peroxidase complex (1:100; Vector Laboratories) in 0.1

PBS for

30 min. Sections were developed in 20% diaminobenzidine (Vector Lab-

oratories), mounted, dried, and coverslipped with Entillin (Thermo

Fisher Scientific).

Behavioral studies

Mice were acclimated to cages in the behavioral facility for at least 2 weeks

before behavioral testing. The male cohort consisted of 15 densin knock-

out and 23 wild-type mice, and the female cohort consisted of nine

densin knock-outs and 14 wild-type mice. Mice from both cohorts had

been backcrossed seven times into a C57BL/6 background. Females were

housed in groups of four. Initially, most of the males were group housed

with littermates, with an equal number of wild-type and knock-out males

singly housed. As the experiments progressed, most of the males were

separated because they were fighting. The cohort was tested during their

light cycle, except when indicated.

Open field.

Mice were placed in the corner of a plastic 50

50 cm

square box and allowed to explore the box for 10 min. Aerial video

footage was captured using Picolo (frame grabber) with Media Cruise

software. The path the mouse traveled and the duration of time spent in

the center quadrant (25

25 cm) were analyzed using Ethovision 3.0

software.

Object-place recognition.

Each mouse was acclimated (10 min) to an

open box (50

50 cm) and then returned to its home cage (5 min). It was

then allowed to explore (5 min) the open box in which two objects had

been placed in opposite corners. Mice were returned to their home cages

(10 min) and then reintroduced to the same box (5 min) in which one of

the objects had been moved to the opposite corner while the other object

remained stationary. The preference for the moved object was measured

by calculating the number of investigations of each object. Investigations

were defined as head movements in the direction of the object in which

the tip of the nose was within 2 cm of the object.

Novel object recognition.

At 24 h after the place preference test, mice

were reacclimated to the testing box (10 min) and then returned to the

home cage (5 min). The mice were then exposed to the same objects used

in the place preference test (5 min). Afte

r a 5 min intertrial period, one of

the objects was replaced with a novel object in the same location, and the

number of investigations of each object was counted.

Prepulse inhibition.

Prepulse inhibition (PPI) was assessed on a cohort

of female wild-type (

12) and densin knock-out (

13) littermates

(11–15 weeks old) during the dark cycle. The male cohort was unsuitable

for this test because fighting has been shown to alter the PPI response

in mice. Mice were restrained in a cylindrical Plexiglas tube on a

platform situated in a chamber (SR Labs) in which the involuntary

startle response is measured by an accelerometer underneath the plat-

form on which the mice are resting. The equipment was operated with

“Startle” software to expose the mice to the following trials: no startle

noise, just the startle noise (120 db), or a prepulse (of 3 or 6 db over

background) before the startle noise. Background noise was 65 db.

Prepulse inhibition was calculated as follows: 100%

((startle-

.pulse)

(startle.prepulse))/(startle.pulse).

Nest building.

2 inch square piece of cotton nesting material was

placed in the wire food racks of singly caged densin knock-out and wild-

type mice, at a position low enough to be easily reached (Deacon, 2006).

The unshredded nesting material remaining on the rack or on the cage

floor was weighed at 12 h intervals for 72 h.

Home-cage activity.

A home-cage activity monitoring system with au-

tomated behavioral detection was used to continuously track the activi-

ties of the mice over a 24 h period at 8, 10, and 12 weeks of age after

acclimating to the facility for 2 weeks (Steele et al., 2007).

Clasping.

Twenty-four knock-out (15 males, 9 females) and 30

wild-type (19 males, 11 females) mice were suspended by their tails

for 1 min

30 cm above a table top and observed for hindlimb and

forelimb clasping.

Rotarod.

Mice were placed on an accelerating rotarod (

6to

50 rpm

in 240 s), and the duration of time the mice remained on the rod was

measured. Mice were trained on the rotarod for 2 consecutive days, two

times each day separated by a 10 min rest period. Mice were tested on the

third day, and the average of two trials was recorded. The maximum

duration of a trial was 300 s.

Beam crossing.

Mice were placed at one end of a beam, and the time

required to cross to the escape box at the other end (80 cm away) was

measured by motion detectors. Mice were trained on two beams (12 and

6 mm wide) on 2 consecutive days. The mice rested for 10 min in their

home cages between training sessions on the two beams. Each mouse

crossed each beam three times on training days, starting with the 12 mm

beam and ending with the 6 mm beam. On test day, the times for two

trials in which the mice did not stop while crossing the beam were

averaged.

Assessment of aggressive behavior.

During the course of the behavioral

experiments, male mice housed with their littermates were monitored for

aggressive behavior. Aggression was defined as chasing, biting, or initiat-

ing a fight. Often the victim was confirmed by bite marks on the back and

rear.

Slice preparation and extracellular recording

Hippocampal slices were obtained from 4- to 10-month-old mice for

long-term potentiation (LTP) experiments and from 3- to 4-week-old

mice for LTD experiments. Mice were anesthetized with halothane and

killed by cervical dislocation. Hippocampal slices (400

m thick) were

prepared by standard techniques and maintained in an interface-slice-

type recording chamber (at 30°C), perfused with oxygenated (95%

–5% CO

) artificial CSF (ACSF) containing the following (in m

124 NaCl, 4.4 KCl, 25 NaHCO

, 1 NaH

, 2 CaCl

, 1.2 MgSO

, and 10

glucose. After slices had recovered for at least 1 h, a bipolar, nichrome

wire stimulating electrode was placed in stratum radiatum of the CA1

region to activate Schaffer collateral– commissural fiber synapses, and an

extracellular glass microelectrode filled with ACSF (resistance, 5–10

) was used to record evoked field EPSPs (fEPSPs). The presynaptic

fiber stimulation was adjusted to evoke fEPSPs with amplitude of

50%

of the maximal fEPSP amplitude for each slice. fEPSPs were then elicited

at 0.02 Hz during the baseline period of the experiment and after LTP or

LTD induction.

Whole-cell recordings

Slices were maintained in a submerged-slice recording chamber, and

inhibitory synaptic transmission was blocked with picrotoxin (100

)

that was added to a modified ACSF (4.0 m

CaCl

, 4.0 m

MgSO

, and

2.4 m

KCl). The CA3 region of the slices was removed to prevent burst-

ing in the absence of GABAergic inhibition. Low resistance (2– 6 M

)

patch electrodes were filled with a solution containing the following (in

): 102 cesium gluconate, 17.5 CsCl, 10 tetraethylammonium-Cl, 5

QX314, 4.0 Mg-ATP, 0.3 Tris-GTP, and 20 HEPES, pH 7.2. EPSCs

evoked by presynaptic Schaffer collateral fiber stimulation (0.2 Hz) were

recorded at membrane potentials of

80 or

40 mV, and the AMPA

receptor and NMDAR-mediated components of the synaptic currents

were estimated by measuring EPSC amplitude 5 and 50 ms after EPSC

onset, respectively. In these experiments, the intensity of presynaptic

fiber stimulation was adjusted to elicit EPSCs (at

80 mV) with peak

amplitudes of

200 pA. Statistical comparisons in these experiments

were performed using ANOVAs followed by Student–Newman–Keuls

tests for multiple pairwise comparisons. Miniature EPSCs (mEPSCs)

were recorded at

80 mV in cells bathed in a modified ACSF containing

4.0 m

CaCl

, 2.4 m

MgSO

, 2.4 m

KCl, 100

picrotoxin, and

0.5– 0.75

tetrodotoxin. mEPSCs were analyzed using a template-

based event detection routine in pClamp 10 (Molecular Devices) and a

threshold of 6 pA. Statistical comparisons of mEPSC amplitude and

interevent interval distributions were performed using the Kolmogorov–

Smirnov test.

Analysis of spines

Three

GFP

/densin

and

GFP

/densin

littermate pairs, 3– 6

months in age, were perfused transcardially with fixative (as described

above), and 50

m coronal sections were cut with a vibratome and

mounted with Prolong Gold antifade reagent. Images were acquired with

a Carl Zeiss 510 Meta confocal microscope with a 63

/NA 1.4 lens and

optical zoom. Images of dendrites were reconstructed from 40

0.2-

m optical sections and preprocessed with blind iterative deconvo-

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31(45):16194 –16207

Carlisle et al.

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Deletion of Densin Produces Abnormal Behavior

lution software (Autodeblur) from Autoquant. Spine morphology was

analyzed using 3DMA spine analysis software developed in the labora-

tory of Brent Lindquist (Stony Brook University, Stony Brook, NY) (Koh

et al., 2002). The investigator was blind to genotype during image acqui-

sition and analysis of spine morphology.

PSD fractions

PSD fractions were prepared from seven to eight pooled wild-type or

densin knock-out mice matched by age (8 –16 weeks) and sex. Forebrains

(excluding olfactory bulbs) were dissected from mice killed by cervical

dislocation, rinsed in buffer A (0.32

sucrose, 1 m

NaHCO3, 1 m

MgCl2, 0.5 m

CaCl2, 0.1 m

PMSF, and 1 mg/L leupeptin), and ho-

mogenized with 12 up and down strokes at 900 rpm in 14 ml of buffer A.

Homogenates were diluted to 35 ml in buffer A and centrifuged at 1400

for 10 min. The pellet was resuspended in 35 ml of buffer A, homoge-

nized (three strokes), and centrifuged at 710

for 10 min. Superna-

tants were combined and centrifuged at 13,800

for 10 min. The pellet

was resuspended in 8 ml of buffer B (0.32

sucrose, 1 m

NaHCO3),

homogenized with six strokes, and layered onto a sucrose gradient (10 ml

each of 0.85, 1.0, and 1.2

sucrose in 1 m

NaH

buffer). The

gradient was centrifuged fo

r2hat

82,500

in a swinging bucket rotor.

The synaptosome-enriched layer at the interface of 1.0 and 1.2

sucrose

was collected, diluted to 15 ml with solution B, and added to an equal

volume of buffer B containing 1% Triton X-100. The mixture was stirred

for 15 min at 4°C and centrifuged for 45 min at 36,800

. The pellet

containing the PSD-enriched, Triton X-100-insoluble fraction was re-

suspended in 300

lof40m

Tris, pH 8, with a 25 gauge needle and 1 ml

syringe. Samples were aliquoted, frozen in liquid nitrogen, and stored at

80°C.

SDS-PAGE and immunoblot analysis

Equal amounts of protein from each sample (15–25

g) was dissolved in

SDS-PAGE sample buffer, heated at 90°C for 5 min, fractionated on

12.5% acrylamide gels, and electrically transferred to nitrocellulose

membranes (Schleicher & Schuell) in 25 m

Tris, 200 m

glycine, and

20% methanol. Membranes were blocked with Odyssey blocking buffer

(Li-Cor Biosciences) and then incubated in primary antibody solution:

phospho-thr

286

CaMKII [Affinity BioReagents (ABR)], CaMKII (ABR),

-actin (Sigma), PSD-95 (ABR),

-actinin (Sigma), DISC1 (C-terminal;

Sigma), mGluR5 (Millipore),

-catenin (Transduction Labs),

-catenin

(Transduction Labs), Densin (Kennedy Lab),

-CaMKII (Kennedy Lab),

PSD-93 (ABR), synGAP (ABR), GluN1 (ABR), GluN2A (Kennedy Lab),

GluN2B (Kennedy Lab), GluA1 (Millipore), and GluA2 (Millipore).

Bound antibodies were detected with IRdye700- or IRdye800-conjugated

secondary antibody (Rockland) and visualized with the Odyssey Infrared

Imaging System (Li-Cor Biosciences).

Cell culture and treatment

Hippocampi dissected from mice at embryonic day 15 or 16 were tritu-

rated and plated into wells of 24-well plates coated with 50 ng/ml poly-

-lysine (Sigma) and 2 ng/ml laminin (BD Biosciences) at a density of

50,000 neurons per well in Neurobasal medium (Invitrogen) supple-

mented with B-27 (Invitrogen) and glutamax-I (Invitrogen) as described

previously (Brewer et al., 1993). At 16 –18 DIV, cultures were pretreated

for 30 min with 5

TTX, gently washed twice in HEPES-control salt

solution (HCSS) (in m

: 120 NaCl, 5.4 KCl, 0.8 MgCl

, 1.8 CaCl

,10

NaOH, 20 HEPES, and 5.5 glucose, pH 7.4) and then exposed to 10

bicuculline methiodide (Tocris Bioscience) and 10

glycine (Sigma)

dissolved in HCSS. After treatment, the HCSS was removed, and cultures

were solubilized in lysis buffer [3% SDS, 20 m

Tris-Cl, pH 7.5, 10 m

EGTA, 40 m

-glycerophosphate, 2.5 m

MgCl

, and Protease Inhibi

tor Complete (Roche)]. Lysates were heated at 90°C for 5 min, and pro-

tein concentrations were determined by the bicinchoninic acid method

(Pierce) using bovine serum albumin as standard.

Cell culture immunocytochemistry

Hippocampal neurons were dissociated with trypsin and mechanical trit-

uration, plated on glass coverslips coated with poly-

-lysine (Sigma),

and maintained as described above. After 18 –21 DIV, coverslips contain-

ing neurons were rinsed in PBS and placed briefly in ice-cold methanol.

The methanol was replaced with

20°C methanol, and coverslips were

incubated for 10 –15 min. Cells were rinsed and incubated in h-PBS (450

NaCl and 20 m

phosphate buffer, pH 7.4) for 15 min, blocked with

5% normal goat serum and 0.05% Triton X-100 in h-PBS fo

r1hat

4°C,

and then incubated overnight with primary antibodies: rabbit anti-

PSD-95 (Cell Signaling Technology) and mouse anti-

CaMKII (ABR).

Coverslips were washed three times (15 min/wash) in blocking buffer

followed by incubation with goat anti-mouse conjugated to Alexa Fluor

568 and goat anti-rabbit conjugated to Alexa Fluor 488 secondary anti-

bodies (Invitrogen) at room temperature for 1 h. Coverslips were washed

once in blocking buffer for 15 min, twice in PBS for 15 min, and then

postfixed in 2% paraformaldehyde in PBS for 10 min, followed by two

washes in PBS for 10 min each. Finally, coverslips were mounted on

microscope slides with a drop of Prolong antifade reagent (Invitrogen)

and allowed to dry overnight. Images were acquired on a Carl Zeiss

Axiovert 200M fluorescent microscope equipped with a 63

/1.4 oil ob-

jective and a high-resolution CCD camera (Axiocam MRm) controlled

by Carl Zeiss AxioVision 3.1 imaging software. A fixed exposure time was

set for all images in an experiment to a time that gave submaximal pixel

brightness for wild-type images in that experiment. Images were ana-

lyzed with the NIH ImageJ software program. PSD-95 was used as a

marker for the PSD region. Threshold for PSD-95 immunostaining was

set to allow all recognizable PSD-95 puncta to be included in the creation

of a mask. The PSD-95 mask was then overlaid onto the CaMKII image.

Intensities of CaMKII puncta that colocalized with the PSD-95 mask

were recorded for 15–20 neurons per embryo. Each genotype was ana-

lyzed at least three different times from litters dissected from three dif-

ferent pregnant females. Mutant animals were always compared with

wild-type littermates.

Statistics

Data are presented as mean

SEM, with

indicating the number of

experiments, as stated. Statistical analyses of two groups were measured

using Student’s

tests (two-tailed). One-sample -tests (two-tailed) were

used to determine whether datasets that were normalized to matched

control values were significantly different from 100%. Statistical analyses

on data containing more than two groups were performed using the

one-way ANOVA test, followed by Tukey–Kramer analysis, to account

for multiple comparisons. The Kolmogorov–Smirnov method was used

to assess whether datasets had Gaussian distributions, as required for

tests and ANOVA analyses. In cases in which the data were not Gaussian,

nonparametric tests were used as stated.

Results

Generation of densin knock-out mice

The

LRRC7

gene, which encodes densin, was disrupted by loxP/

Cre-mediated excision of exon 3, which contains the transcrip-

tion start site. ES cells were transfected with a targeting construct

that contained a LoxP site inserted into intron 2 and a hygromy-

cin selection cassette flanked by LoxP sites inserted into intron 3.

After the altered gene was incorporated into the germ line, Cre

recombinase was expressed

in utero

to produce a mixture of three

additional genotypes: a deletion of the hygromycin selection cas-

sette, a floxed exon 3 conditional knock-out, or deletion of both

exon 3 and the hygromycin cassette resulting in a full knock-out

(Fig. 1

; Materials and Methods). All the studies described here

were performed on the full knock-out. Deletion of exon 3 in

densin knock-out mice was verified by performing PCR on

genomic DNA (Fig. 1

). Immunoblots stained with antibodies

specific for the N-terminal LRR domain, the mucin homology

domain, or the C-terminal PDZ domain confirmed that no den-

sin protein was expressed in the knock-out mice (Fig. 1

The densin mutants are viable and born at the expected Men-

delian ratio. During the first 2– 6 weeks after birth, they have

significantly lower body weights compared with their wild-type

littermates (Fig. 2

) and have an

20% mortality rate. The

disparity in body weight becomes much less dramatic by 11 weeks

Carlisle et al.

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Deletion of Densin Produces Abnormal Behavior

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31(45):16194 –16207

• 16197

of age, and the mutants that survive the

first 6 weeks of life appear to have a nor-

mal lifespan. In addition to their smaller

body size, the knock-out mice clasp when

suspended by the tail, a behavior often ob-

served in mouse models of neurodegen-

eration (data not shown). Nissl-stained

sections of mutant forebrains did not re-

veal any gross abnormalities in brain

structure (Fig. 2

), and immunostaining

for GFAP did not show abnormal levels of

astrogliosis (Fig. 2

Densin knock-out mice have behavioral

endophenotypes associated with

mental illness

Although schizophrenia, ASDs, and other

mental illnesses are distinctively human

diseases, researchers working with various

partial animal models of these diseases

have defined the term endophenotype to

mean a “heritable phenotypic indicator

that reflects discrete components of

pathophysiological processes more proxi-

mal to particular sets of predisposing

genes than the actual clinical diagnosis”

(Gottesman and Gould, 2003; Arguello

and Gogos, 2006). As part of our initial

characterization of the functional effects

of densin deletion, we examined the per-

formance of densin knock-out mice in be-

havioral tests and found that they display

many behaviors that have been suggested

as endophenotypes associated with hu-

man mental illness (Arguello and Gogos,

2006).

One such endophenotype is impaired

short-term memory. We chose tests of

short-term memory that use novelty-

preference paradigms to take advantage of

a rodent’s natural curiosity about novel

situations. Hippocampus-dependent short-

term memory was assessed with a one-

trial object-place recognition task in

which the mice were allowed to investi-

gate two objects for 5 min during a train-

ing trial. After a 10 min delay

, a 5 min test

trial was administered in which the mice

were allowed to investigate the same two

objects, but with one of the objects moved to a novel location. As

expected, the wild-type mice investigated the moved object sig-

nificantly more frequently than the stationary object (Fig. 3

). In

contrast, the knock-out mice did not show a preference for the

moved object, suggesting they have impaired spatial memory of

the training configuration.

We assessed hippocampus-independent short-term memory

with the one-trial object-recognition test. This test is identical to

the object-place recognition test, except that a novel object is

substituted in the same location for one of the training objects

during the test trial. Again, wild-type mice showed a significant

preference for the novel object, whereas the knock-out mice did

not (Fig. 3

). Thus, the densin knock-out mice appear to have

deficits in both hippocampus-dependent and

-independent forms

of short-term memory. Interestingly, the knock-out mice displayed

hyperlocomotive behavior when interacting with the objects during

the short-term memory tasks, investigating the objects twice as fre-

quently as wild-type mice (Fig. 3

). In addition to short-term

memory deficits, hyperactivity in response to stress or novelty has

been suggested as an animal model of psychomotor agitation that

often occurs in schizophrenia (Arguello and Gogos, 2006). It is un-

likely that the failure of the densin knock-out mice to show a prefer-

ence for novel objects resulted from a ceiling effect related to their

hyperlocomotion when interacting with the objects. The highest

number of average interactions recorded for the mutants, 17 in 5

min during the first exposure to objects (Fig. 3

), represents one

interaction every 17.6 s, a rate that is far below the maximum possi-

ble interactions in 5 min.

Figure 1.

Disruption of the

LRRC7

gene encoding densin.

, Targeting vector and recombined floxed alleles. The targeting

construct for exon 3 included a LoxP site inserted into intron 2 and a hygromycin selection cassette flanked by LoxP sites inserted

into intron 3 (LoxP sites indicated by black arrowheads). Expression of Cre recombinase

in utero

resulted in the deletion of the

hygromycin selection cassette, producing a floxed exon 3 conditional knock-out, or in the deletion of both exon 3 and the hygro-

mycin cassette, producing a full knock-out.

, Southern blot analysis of PCR products from DNA of wild-type (Wt), heterozygous

(Het), and knock-out (KO) littermates.

, Immunoblot analysis of expression of densin protein in forebrain homogenates of

6-week-old mice. Aliquots containing equal amounts of total protein were fractionated and blotted as described in Materials and

Methods with antibodies raised against three distinct regions of densin: N-terminal LRR domain, mucin homology domain, and

C-terminal PDZ domain.

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Carlisle et al.

•

Deletion of Densin Produces Abnormal Behavior

PPI is a common measure of sensorimotor gating related to

preattentive processing that is often deficient in patients with

schizophrenia as well as other psychiatric diseases (Powell et al.,

2009). In healthy subjects, a low-intensity pulse of sound preced-

ing an acoustic startle stimulus inhibits the startle reflex. We

found tha

ta3or6dB

prepulse of sound induced

50% greater

inhibition of the startle reflex in wild-type mice than in knock-

out mice (Fig. 3

). These data mean that densin deletion results

in a deficit in PPI.

Deficits in nesting behavior have been observed in mouse

models with schizophrenia-like features and can be induced with

psychotomimetic agents (Powell and Miyakawa, 2006). These

deficits are hypothesized to be related to social withdrawal, a

common negative symptom in schizophrenia and autism (Powell

and Miyakawa, 2006). We observed that male densin mutants

had a profound deficit in nest-making behavior. We measured

this behavior by providing sets of mice with identical squares of

cotton. The wild-type mice shredded the cotton and made fully

formed nests within 24 h. In contrast, only 1 of 16 knock-out

mice shredded any of the cotton within a 72 h period (Fig. 3

To confirm that the knock-out mice do not have motor defi-

cits that might confound the results of other behavioral tests, we

measured their balance and coordination in the rotarod and

beam-crossing tests. Densin knock-out and wild-type mice re-

mained on an accelerating rotarod for similar lengths of time

(Fig. 4

). On the beam-crossing test, the

knock-out mice crossed the beam signifi-

cantly more quickly than wild-type mice,

consistent with the observation that the

knock-out mice become hyperactive in

novel or stressful situations (Fig. 4

Overall, the densin mutants have similar

levels of coordination, endurance, and

balance to their wild-type counterparts,

despite their differences in size and clasp-

ing behavior.

Abnormal aggression is rare in hu-

mans with schizophrenia, and it is

not considered a feature of the disease.

However, some mouse models with

schizophrenia-like features do display ab-

normal aggression (Pletnikov et al., 2008).

We observed that male densin knock-outs

were aggressive when group-housed even

with littermates. Males in nearly all cages

containing densin heterozygotes or full

knock-outs eventually had to be separated

because of excessive fighting. Over a sev-

eral hour period during behavioral exper-

iments, we observed fighting in

82% of

11 cages containing at least one knock-out

mouse and in two of three cages contain-

ing one heterozygous knock-out and one

wild-type mouse. In contrast, no fighting

was observed in four cages that contained

only wild-type mice. We did not quantify

this behavior further because the fighting

occasionally resulted in injury to the mice.

Although it is not a core symptom of

schizophrenia, anxiety is often comorbid

with schizophrenia (Achim et al., 2011)

and autism. Anxiety in mice can be mea-

sured by the open-field test. Mice have an

innate fear of open space and tend to avoid the center of the

open-field apparatus. Typically, they explore the outer edges of

the box first and then gradually explore the center.

Their level of

anxiety is considered to be inversely correlated with the

per-

centage of time spent in the center quadrant of the open field.

Densin mutants spent a significantly smaller fraction of time in

the center quadrant compared with wild-type mice, indicating

that they have significantly higher levels of anxiety (Fig. 5

Using automated behavior analysis, we tracked typical behav-

iors of the mice in their home cages during day and night cycles

over a period of weeks (Steele et al., 2007). We found that, al-

though the knock-out mice become hyperactive in novel situa-

tions, they are significantly more sedentary than wild-type mice

in the context of a familiar environment. In fact, the densin mu-

tants showed a threefold reduction in the amount of time en-

gaged in high-energy behaviors, such as hanging vertically from

the wire food rack and jumping (Fig. 5

). Knock-out mice spent

the same amount of time sleeping as wild-type mice but showed a

more than twofold increase in twitching during resting periods,

suggesting that their sleep may be interrupted frequently.

In summary, densin knock-out mice have significant deficits

in hippocampus-dependent and -independent short-term mem-

ory, prepulse inhibition, and nesting behavior. In addition, they

display abnormal aggression and anxiety, and they become phys-

ically agitated in response to novelty. Many of these behavioral

Figure 2.

Densin knock-out mice have normal gross neuroanatomy and low body weight.

, At 3 weeks of age, densin knock-

out mice had significantly lower body weights (6.8

0.4 g,

15 mice) than wild-type mice (12.3

0.5 g;

17 mice;

****

0.0001). By 11 weeks of age, the disparity in their weights was much less dramatic (wild-type, 27.1

1.1 g,

21;

densinknock-out,23.9

1.3g;

19;

0.062).Significancewasdeterminedwithtwo-tailed

test.ErrorbarsrepresentSEM.

, Representative pictures of wild-type (left) and densin knock-out littermates (right) at 3 weeks of age.

, Representative

Nissl-stainedcoronalsectionsfromwild-type(left)anddensinknock-out(right)mice(bregma

1.7mm).Bottomrowshows5

images of the dentate gyrus.

, Immunohistochemical staining for GFAP is qualitatively similar in wild-type (left column) and

knock-out (right column) sections. Representative images are shown of sensory cortex (top row) and the CA1 region of hippocam-

pus (bottom row). Heterozygous, Het; Wt, wild type.

Carlisle et al.

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Deletion of Densin Produces Abnormal Behavior

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31(45):16194 –16207

• 16199

defects have been reported in other mouse models with schizo-

phrenia and autism-like features and are considered endopheno-

types related to human mental illness (Powell and Miyakawa,

2006; Silverman et al., 2010).

Loss of densin reduces the amount of

-actinin in brain

homogenates and selectively reduces the levels of DISC1 and

mGluR5 in the PSD fraction

Because densin interacts with a specific set of other PSD proteins,

we hypothesized that its loss might alter the composition of the

PSD in a way that disrupts synaptic function and thus produces

behavioral abnormalities. As an initial test of this hypothesis, we

isolated PSD fractions from homogenates of several sets of wild-

type and densin knock-out forebrains and compared the

amounts of core PSD proteins. We measured the known densin-

binding proteins

-CaMKII,

-actinin,

-catenin, and

-catenin.

Only the amount of

-actinin was significantly reduced in knock-

out PSD fractions (32%); it was also reduced in total brain ho-

mogenates (33%), suggesting that expression or stability of

-actinin is impaired in the absence of densin (Fig. 6).

-Actinin

has been reported to bind two proteins previously implicated in

schizophrenia, DISC1, and mGluR5 (Millar et al., 2003; Cabello

et al., 2007). We measured their amounts and found that both

DISC1 and mGluR5 are normal in brain homogenates of densin

knock-out mice but significantly reduced (

30%) in PSD frac-

tions compared with wild type (Fig. 6). We detected no signifi-

cant differences in the amounts of other core PSD proteins (

CaMKII, PSD-95, PSD-93, and synGAP; data not shown) or of

ionotropic glutamate receptor subunits (GluN1, GluN2A,

GluN2B, GluA1, and GluA2; data not shown) in PSD fractions

from knock-out mice. Thus, the absence of densin selectively

reduces the steady-state level of

-actinin in brain and in the PSD

fraction, whereas the amounts of DISC1 and mGluR5 are re-

duced just in the PSD fraction. The latter result was not expected

Figure 3.

Short-term memory, sensorimotor gating, and nest building are impaired in den-

sin knock-out mice.

, Knock-out (KO) mice did not show increased preference for the moved

object(B*)inaplacepreferencetestdesignedtomeasurehippocampus-dependentshort-term

spatial memory (stationary object, 15.1

1.7 investigations; moved object, 13.5

1.2 inves-

tigations;

15 mice), whereas wild-type (Wt) mice did prefer the moved object as measured

by a significant increase in the number of investigations of the moved object (stationary object,

6.2

0.7 investigations; moved object, 10.3

1.0 investigations;

20 mice; *

0.05).

Despite not showing a preference for the moved object during the testing session, densin

knock-out mice investigated objects A and B more frequently on average than the wild-type

mice during the training session (wild type, 8.4

0.5 investigations,

40 trials; knock-out,

16.1

0.8 investigations,

30 trials; ****

0.0001).

, Densin knock-out mice were

similarly unable to discriminate between a novel object (C) and a previously viewed object in a

hippocampus-independentnovelobjectrecognitiontask(previouslyviewed,10.6

1.8inves-

tigations; novel, 11.3

2.0 investigations), whereas the wild-type mice showed an increase in

the number of investigations of the novel object (previously viewed, 5.3

0.7 investigations;

novel, 10.2

1.1 investigations;

15 mice). Knock-out mice again showed an increased

average number of investigations of objects A and B during the training session compared with

wild-type mice (wild type, 6.2

0.5 investigations,

40 trials; knock-out, 12.2

1.0

investigations,

30 trials;

0.0001).

, Knock-out mice have abnormal sensorimotor

gating. Startle induced by a 120 dB pulse of sound was significantly less inhibited by a prepulse

(3 or 6 dB) in the knock-out (3 dB, 15

5% PPI; 6 dB, 33

5% PPI;

13 knock-out mice)

compared with wild-type mice (3 dB, 42

5% PPI; 6 dB, 60

5% PPI;

12 wild types;

0.01, ANOVA).

, Knock-out mice show impaired nest building. Wild-type mice shred-

ded

60% of the supplied cotton within 12 h and all had fully formed nests within 48 h (black

dots,

13 wild types). Only one of the knock-out mice removed and shredded the cotton; all

others left the cotton unshredded (gray dots,

16 knock-out mice). Wild-type mice had

significantly less unshredded cotton in their cages at all measured time points after

0.001, Mann–Whitney

test). Error bars represent SEM.

Figure 4.

Densin knock-out mice have normal motor skills.

, Male and female knock-out

(KO) mice performed as well as the wild types (Wt) on the rotarod test. Male wild-type mice

stayed on the accelerating rotarod for 191

9s(

20 mice), whereas male knock-out mice

stayed on for 187

11s(

15 mice). Female wild-type mice stayed on the accelerating

rotarodfor219

10s(

11mice),whereasfemaleknock-outmicestayedonfor198

14s

(

9 mice).

, Male knock-out mice crossed the 12 mm beam as fast as wild types (male wild

types, 4.6

0.4 s; male knock-outs, 3.9

0.4 s) and crossed the 6 mm beam significantly

faster than wild types (male wild types, 6.8

0.7 s; male knock-outs, 4.9

0.5 s; *

0.05,

two-tailed

test).

, Similarly, female knock-out mice crossed the 12 mm beam as fast as wild

types (female wild types, 3.3

0.3 s; female knock-outs, 3.5

0.3 s) and crossed the 6 mm

beam significantly faster than wild types (female wild types, 5.9

0.6 s; female knock-outs,

4.1

0.4 s; *

0.05, two-tailed

test).

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Carlisle et al.

•

Deletion of Densin Produces Abnormal Behavior

and may be an indirect effect of loss of

-actinin or may be caused

by disruption of other, as yet unknown, protein associations.

Synaptic transmission is normal in densin knock-out neurons

We also assessed whether the synapses of densin knock-out mice

display normal glutamate receptor activity. Whole-cell record-

ings revealed no difference in the relative contributions of AMPA

receptors and NMDARs to EPSCs in CA1 pyramidal cells from

wild-type and densin knock-out mice (Fig. 7

). To rule out the

possibility that a proportional shift in both types of glutamate

receptors had occurred in the densin mutants, we also compared

the amplitude and frequency of mEPSCs in CA1 pyramidal cells

and found no difference in their distribution, amplitude, or

fre-

quency between wild-type and

knock-out neurons (Fig. 7

Thus, densin is not necessary for recruitment and normal func-

tion of NMDARs at synapses, and hypofunction of NMDARs is

not the underlying cause of the abnormal behavior of the densin

knock-outs (Belforte et al., 2010).

Loss of densin alters the steady-state

activation of CaMKII and its activation

in response to synaptic activity

CaMKII is highly concentrated in the PSD

fraction and in spines of cultured excit-

atory neurons, suggesting that one or

more docking sites within the PSD medi-

ate its postsynaptic accumulation (Ken-

nedy et al., 1983; Kennedy, 2000). Densin

and the GluN2B subunit of the NMDAR

both have high-affinity binding sites for

CaMKII that may play significant roles in

targeting CaMKII to the PSD (Omkumar

et al., 1996; Strack and Colbran, 1998;

Leonard et al., 1999; Walikonis et al.,

2001; Barria and Malinow, 2005; Zhou et

al., 2007). To formally test the hypothesis

that densin acts as a docking site for CaM-

KII in the PSD and examine the relative

importance of the two potential docking

sites, we compared the localization of

CaMKII in spines of neurons lacking den-

sin, lacking synaptic NMDARs, and those

lacking both.

We generated neurons lacking synap-

tic NMDARs by culturing hippocampal

neurons from GluN1 knock-out embryos

(Fukaya et al., 2003). Engineered mice

that are missing the cytosolic tails of

GluN2A and GluN2B have been created

(Sprengel et al., 1998); however, homozy-

gous deletion of the tail of GluN2B results

in failure of movement of all subunits of

NMDARs to synapses (Sprengel et al.,

1998). Deletion of the GluN1 subunit

similarly causes GluN2 subunits to be re-

tained in the endoplasmic reticulum and

results

failure

movement

NMDARs to synapses (Fukaya et al.,

2003). Nonetheless, excitatory synapses of

neurons cultured from GluN1 knock-out

embryos still form PSDs containing PSD-

95. We cultured neurons lacking both

densin and synaptic NMDARs from em-

bryos bred to contain both deletions as

described in Materials and Methods.

We estimated the amount of CaMKII docked at PSDs in each

phenotype by measuring the integrated intensity of immuno-

staining for CaMKII that colocalized with PSD-95 (Fig. 8

). By

this measure, PSDs on densin knock-out neurons did not show a

significant reduction in colocalization of CaMKII with PSD-95,

consistent with our finding that PSD fractions from densin

knock-out mice contain normal amounts of CaMKII (Fig. 5).

This result means that densin is not required for accumulation of

CaMKII in PSDs. The GluN1 knock-out neurons showed only a

15% reduction in CaMKII staining colocalizing with PSD-95,

indicating that synaptic NMDARs also are not required for accu-

mulation of CaMKII in PSDs. However, in neurons lacking both

densin and synaptic NMDARs (Densin/GluN1 double knock-

out), the amount of CaMKII colocalizing with PSD-95 was re-

duced by

50%. Staining for CaMKII appeared lighter in

dendritic shafts of double knock-out neurons as well as in spines

(Fig. 8

, bottom right); however, the integrated intensity of all

Figure 5.

Densin knock-out mice have increased levels of anxiety and reduced home-cage activity and are aggressive with

littermates.

,Knock-out(KO)micedisplayedanxiety-likebehaviorsinanopenfield.Thepercentageoftimeknock-outmicespent

in the center quadrant of the open field was significantly less than wild types (Wt) during the first 2 min (wild type, 10.0

1.4%;

knock-out, 3.4

1.3%;

20 wild-type mice, 15 knock-out mice; **

0.01), 4 min (wild type, 14.0

1.8%; knock-out,

3.6

1.0%;

17 wild-type mice, 10 knock-out mice; ***

0.001), and 8 min (wild type, 15.3

4.0%; knock-out, 6.3

1.8%;

8 wild-type mice, 7 knock-out mice; *

0.05, two-tailed

test; error bars represent SEM) of exploration in the open

field. Right shows representative paths traveled by wild-type (left) and knock-out (right) mice during 10 min of exploration in an

open field. The inner gray box marks the boundaries of the center quadrant.

, Densin knock-out mice are hypoactive in a

home-cage setting. The heat plot shows behaviors detected by an automated home-cage monitoring system over a 24 h period at

8, 10, and 12 weeks of age (weeks 1, 2, and 3, respectively). Behaviors are expressed as the fraction of frames that the behavior was

detectedandnormalizedtowild-typevalues.Lightbluerepresentsbehaviorsforwhichtherewasa

2.5-folddecreasecompared

with wild-type. Bright yellow represents behaviors for which there was a

2.5-fold increase compared with wild-type. Knock-out

mice spent significantly less time engaged in high-activity behaviors, such as hanging, jumping, and rearing.

Carlisle et al.

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Deletion of Densin Produces Abnormal Behavior

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•

31(45):16194 –16207

• 16201

staining for CaMKII in each image divided by the total number of

pixels with staining above background was not significantly re-

duced in the images of double knock-out neurons compared with

wild-type controls. Thus, it is not clear whether the total amount

of CaMKII was reduced in the double knock-out neurons or just

dispersed more broadly throughout the neurons. The simplest

explanation for these data is that densin and the tails of NMDAR

subunits both can act as docking sites for CaMKII in the PSD and

each of them can partially or completely compensate for the ab-

sence of the other in performing this function. However, when

both proteins are missing, considerably less CaMKII is localized

to spines, and it may be reduced in concentration in dendrites as

well.

We wondered whether the absence of densin might alter the

micro-localization of CaMKII within the PSD. For example, if

more CaMKII is bound to NMDARs in the absence of densin, one

might predict that the rate or magnitude of activation of CaMKII

would be increased in response to synaptic activity in densin

knock-out neurons. To test whether this is the case, we treated

hippocampal cultures with bicuculline, a GABA

receptor antag

onist that causes enhanced glutamate release from excitatory syn-

apses through disinhibition of inhibitory synapses (Ivanov et al.,

2006). We found that the resulting activation of CaMKII, mea-

sured by an increase in autophosphorylation at threonine-286,

was approximately twofold higher in densin knock-out neurons

compared with wild type (Fig. 8

). Conversely, the basal level of

autophosphorylation of threonine-286 in untreated densin

knock-out neurons was reduced to

75% that of wild-type neu-

rons (Fig. 8

). Together, these data indicate that densin is not

required for localization of CaMKII in the PSD. However, it may

be important for precise positioning of CaMKII in relation to

other regulatory molecules, such that absence of densin alters

both the steady-state activation of CaMKII and the dynamics of

its activation in response to neuronal activity.

Long-term depression is impaired in densin

knock-out neurons

We reasoned that mislocalization of mGluRs and/or of CaMKII

might alter induction or stability of synaptic plasticity. We mea-

sured two commonly studied forms of LTP and found them un-

affected by the densin deletion. Brief 100 Hz stimulation of

Schaffer collateral fibers induced an approximate twofold in-

crease in fEPSP amplitudes in wild-type and knock-out hip-

pocampal slices 60 min after LTP induction (Fig. 7

). A 30 s train

of 5 Hz stimulation (theta stimulation) also produced similar

levels of fEPSP potentiation (

50% increase over baseline) in

wild-type and knock-out slices (Fig. 7

). Thus, LTP appears nor-

mal in the densin mutants during the first hour after induction.

We observed a trend toward larger potentiation in the first 5 min

after either of the induction stimuli, but the trend did not reach

statistical significance. We conclude that the aberrant activation

of CaMKII we observed during the first 5 min after activation of

NMDARs on cultured neurons (Fig. 8

) does not significantly

affect induction of LTP under conditions that produce near-

maximal potentiation in slices.

In contrast, both low-frequency stimulation and DHPG-

induced forms of LTD are impaired in the densin knock-out

mice. A 15 min train of 1 Hz stimulation caused a 25% reduction

of fEPSP amplitudes in wild-type slices but no reduction in the

densin knock-out slices (Fig. 7

). This form of LTD has been

shown to be dependent on activation of NMDARs (Ho et al.,

2004). Bath application of DHPG, a group 1 mGluR agonist that

activates mGluR1 and mGluR5, induces a non-NMDAR-

dependent form of LTD (Lu

̈ scher and Huber, 2010). A 10 min

application of DHPG (100

) induced a

50% transient de-

pression in fEPSP amplitude and a stable

25% depression after

washout of the drug in wild-type slices but only a

10% depres-

sion in the knock-out slices (Fig. 7

). Thus, loss of densin impairs

both NMDAR-dependent and mGluR-dependent LTD, suggest-

ing that the defect lies at a biochemical step downstream of re-

ceptor activation.

Loss of densin alters the morphology of spines on

hippocampal neurons

When spine synapses are remodeled to increase or decrease syn-

aptic strength, the number of AMPA-type receptors is increased

or decreased and the actin cytoskeleton is remodeled to increase

or decrease the size of the spine head. The biochemical regulatory

pathways that alter the spine actin cytoskeleton are tightly linked

to those that regulate surface AMPA receptors (Carlisle and Ken-

nedy, 2005). CaMKII, along with other PSD enzymes, partici-

pates in this regulation of the actin cytoskeleton (Carlisle et al.,

2008). Abnormalities in spine shape were reported in mouse

models in which DISC1 isoforms were genetically deleted or

knocked down with siRNA (Kvajo et al., 2008). To investigate

whether loss of densin deranges the regulatory machinery that

shapes spine morphology in adult mice, we first crossed densin

knock-out mice with a transgenic line that sparsely expresses GFP

in the CA1 region of the hippocampus (Feng et al., 2000). Spine

morphology was examined in three-dimensional reconstructions

Figure 6.

Levels of

-actinin, DISC1, and mGluR5 are reduced in PSD fractions from densin

knock-out mice. Protein levels in brain homogenates and PSD fractions from several sets of

mice, each containing seven to eight pooled wild-type (WT) brains and seven to eight pooled

densin knock-out (KO) brains, were determined by immunoblot. Average levels in fractions

from knock-outs were normalized to those of corresponding wild types. The densin-binding

protein

-actinin was reduced by 33% in brain homogenates and 32% in PSD fractions from

knock-out mice compared with wild type (

4 subcellular fractionations). The

-actinin-

binding partners DISC1 and mGluR5 were reduced by

30% in PSD fractions from knock-out

mice but were not reduced in brain homogenates (

3 subcellular fractionations for DISC1;

5 brain homogenates and 3 PSD preparations for mGluR5). Loss of densin did not alter the

levels of other known densin-interacting proteins:

-catenin,

-catenin, or

-CaMKII. Statis-

tical significance was determined with one-sample

tests (null hypothetical mean

100).

Error bars represent SEM. The amounts of actin or of PSD-95 (as appropriate) were quantified

and used as loading controls. Bottom panels show representative bands from immunoblots of

homogenates and PSD fractions. *

0.05.

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31(45):16194 –16207

Carlisle et al.

•

Deletion of Densin Produces Abnormal Behavior

of confocal images of labeled pyramidal

cell dendrites from densin knock-out and

wild-type mice containing the GFP label.

The necks of spines were significantly

more elongated in densin knock-out

mice. Furthermore, when spines were cat-

egorized by morphological type, we found

that the proportion of mushroom spines

was increased by 31% and the number of

stubby spines decreased by 22% in the

knock-out mice, whereas the total num-

ber of spines was not significantly changed

(Fig. 9

). The necks of densin knock-

out mushroom spines were significantly

longer and thinner than wild-type mush-

room spines (Fig. 9

), but the aver-

age head diameter was unchanged (Fig.

). These data indicate that the defect

or defects in biochemical signaling in the

spine induced by loss of densin occur at

positions in the regulatory network that

alter steady-state spine morphology. They

provide additional evidence that densin is

important for proper kinetic tuning of

regulatory pathways in the spine.

Discussion

Molecular and cellular effects of

deletion of densin

The domain structure of densin, which in-

cludes binding sites for several proteins,

suggests that it functions as a scaffold

molecule (Apperson et al., 1996; Strack et

al., 2000; Walikonis et al., 2001; Izawa et

al., 2002; Ohtakara et al., 2002). As such, it

would be expected to influence the orga-

nization and dynamics of signaling path-

ways in the PSD in which it is highly

concentrated. Mutation or deletion of

large scaffold proteins can lead to com-

plex behavioral or pathological pheno-

types, apparently because such proteins

influence more than one cellular function.

A prominent example is huntingtin, the

mutation of which interferes with neuro-

nal transcription, intracellular transport,

and synaptic transmission (Imarisio et al.,

2008; Marcora and Kennedy, 2010) and

causes the complex phenotypes associated

with Huntington’s disease. Another ex-

ample is DISC1, which has a wide range of

postulated binding partners and is located

in mitochondria, the centrosome, and ex-

citatory synapses (James et al., 2004; Bran-

don et al., 2009; Hayashi-Takagi et al.,

2010). Mutation of DISC1 in humans

produces increased risk of schizophrenia

and bipolar disorder (Millar et al., 2000).

We show that loss of densin leads to

reduction in the amounts of at least three

other proteins in the PSD fraction. The

steady-state level of the direct binding

partner of densin, the cytoskeletal protein

Figure 7.

Evoked NMDAR currents, mEPSCs, and LTP amplitude are normal in knock-out hippocampal slices, but LTD is im-

paired.

, Loss of densin does not alter the ratio of NMDA/AMPA receptor currents. Top traces show representative whole-cell

voltage-clamp recordings of EPSCs from wild-type (Wt) and knock-out (KO) CA1 pyramidal neurons at two different postsynaptic

membranepotentials(

80and

40mV).TheleftgraphshowstheNMDAR-mediatedEPSCs(estimatedfromtheEPSCamplitude

50 ms after EPSC onset) normalized to the AMPA receptor-mediated component of the EPSC (estimated from the EPSC amplitude

5 ms after EPSC onset). There is no difference in EPSCs between wild-type cells (black bars;

3 wild-type mice, 13 cells) and

knock-out cells (gray bars;

3 knock-out mice, 14 cells) at either membrane potential. The right graph shows the comparison

of weighted mean decay time constants of NMDAR-mediated EPSCs calculated from double-exponential fits to the decay of the

synaptic currents recorded at

40 mV. No difference in the decay characteristics was observed between wild-type and knock-out

mice.

, Loss of densin does not affect the amplitude or frequency of mEPSCs. Top traces show representative whole-cell voltage-

clamp recordings from CA1 pyramidal neurons. Cumulative amplitude (left graph) and interevent interval (right graph) distribu-

tions were similar for both genotypes (

3 wild-type mice, 13 cells;

3 knock-out mice, 13 cells). Inset graphs show averages

of mEPSC amplitudes and interevent intervals.

, High-frequency and theta-induced forms of LTP in the CA1 region of the

hippocampus are normal in knock-out mice.

, Similar levels of fEPSP potentiation were recorded 60 min after 100 Hz stimulation

(2 trains, each o

f 1 s duration, delivered at time

0) for both genotypes (wild-type fEPSPs were 182

8% of baseline,

densin knock-out fEPSPs were 193

6% of baseline,

4).

, The 5 Hz stimulation for 30 s (delivered at

0) potentiated

fEPSPs to similar levels in both genotypes (wild-type fEPSPs were 159

13% of baseline,

4 wild-type mice;

knock-out

fEPSPs were 161

4% of baseline 45 min after 5 Hz stimulation,

5 knock-out mice). The trend in

and

toward

smaller fEPSPs in the wild type at 5–10 min after stimuli was not statistically significant.

, Low frequency-induced LTD is

impaired in knock-out hippocampal slices. Wild-type fEPSPs were depressed to 74

7% of baseline (

7 wild-type

mice) 60 min after the start of a 15 min train of 1 Hz stimulation, whereas knock-out fEPSPs returned to baseline levels

(98

6% of baseline,

8 knock-out mice). There was a significant difference in the amplitudes of the fEPSPs between

the two genotypes at

60 (

0.05, two-tailed

test).

, The group 1 mGluR agonist DHPG induced a lower level of

fEPSP depression in knock-out compared with wild-type slices. Bath application of DHPG depressed wild-type fEPSPs to

12% of baseline, whereas knock-out fEPSPs were only reduced to 87

5.2% of baseline during the first 5 min of drug

application (

0.016, two-tailed

test;

6 wild-type, 6 knock-out mice). At 35 min after washout of DHPG (

45),

wild-type fEPSPs were reduced to 78

6.4% of baseline and knock-out fEPSPs were reduced to 92

2.2% of baseline

(

0.031, one-tailed

test). Error bars represent SEM.

Carlisle et al.

•

Deletion of Densin Produces Abnormal Behavior

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31(45):16194 –16207

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