genes,brain & behavior1

Genes, Brain and Behavior (2009) 8: 114–126 # 2009 The AuthorsJournal compilation # 2009 Blackwell Publishing Ltd/International Behavioural and Neural Genetics Society

Increased anxiety-like behavior in mice lacking theinhibitory synapse cell adhesion molecule neuroligin 2

J. Blundell†,‡, K. Tabuchi§, M. F. Bolliger§,

C. A. Blaiss†, N. Brose¶,**, X. Liu§,

T. C. Sudhof§,¶,†† and C. M. Powell*,†,‡‡

†Department of Neurology, The University of Texas Southwestern

Medical Center, Dallas, TX, USA ‡Present address: Department of

Psychology, Memorial University of Newfoundland, St John’s,

Newfoundland, Canada §Department of Neuroscience and¶Department of Molecular Genetics, The University of Texas

SouthwesternMedical Center, Dallas, TX, USA **Present address:

Department of Molecular Neurobiology, Max-Planck-Institute for

Experimental Medicine, Gottingen, Germany ††Howard Hughes

Medical Institute, and ‡‡Department of Psychiatry, The University

of Texas Southwestern Medical Center, Dallas, TX, USA

*Corresponding author: C. M. Powell, The University of Texas

Southwestern Medical Center, Dallas, TX, 75390-8813. E-mail: craig.

[email protected]

Neuroligins (NL) are postsynaptic cell adhesionmolecules

that are thought to specify synapse properties. Previous

studies showed that mutant mice carrying an autism-

associated pointmutation in NL3 exhibit social interaction

deficits, enhanced inhibitory synaptic function and

increased staining of inhibitory synaptic puncta without

changes inoverall inhibitory synapsenumbers. In contrast,

mutant mice lacking NL2 displayed decreased inhibitory

synaptic function. These studies raised two relevant ques-

tions. First, does NL2 deletion impair inhibitory synaptic

function by altering the number of inhibitory synapses, or

by changing their efficacy? Second, does this effect of NL2

deletion on inhibition produce behavioral changes? We

now show that although NL2-deficient mice exhibit an

apparentdecrease innumberof inhibitorysynapticpuncta,

the number of symmetric synapses as determined by

electron microscopy is unaltered, suggesting that NL2

deletion impairs the function of inhibitory synapses with-

out decreasing their numbers. This decrease in inhibitory

synaptic function in NL2-deficient mice correlates with

a discrete behavioral phenotype that includes a marked

increase in anxiety-like behavior, a decrease in pain sensi-

tivity and a slight decrease in motor co-ordination. This

work confirms that NL2 modulates inhibitory synaptic

function and is the first demonstration that global deletion

of NL2 can lead to a selective behavioral phenotype.

Keywords: Anxiety, autism, GABA, inhibition, neurexin, neu-roligin, nociception, pain, social interaction

Received 21 July 2008, revised 10 September, 15 October2008, accepted for publication 18 October 2008

Introduction

Neuroligins (NLs) are a family of ubiquitously expressedpostsynaptic cell adhesion molecules in the brain that interact

with neurexins (Ichtchenko et al. 1995, 1996) and aredifferentially localized to the postsynaptic specializations

of excitatory and inhibitory synapses (Graf et al. 2004;Ichtchenko et al. 1995, 1996; Song et al. 1999; Varoqueaux

et al. 2004). NL1 is enriched at postsynaptic densities ofexcitatory synapses in vivo (Song et al. 1999). NL2, however,

is preferentially localized to inhibitory synapses (Varoqueauxet al. 2004). Recent data suggest that NL3 is enriched in the

brain and appears to be localized to both excitatory andinhibitory synapses (Budreck & Scheiffele 2007). While the

results of early in vitro transfection experiments suggesteda role for NLs in synapse formation (Boucard et al. 2005; Chih

et al. 2005; Dean et al., 2003; Graf et al. 2004; Levinson et al.2005; Nam & Chen 2005; Prange et al. 2004), more recent

experiments in cultured neurons and in vivo suggest thatNLs are not required for synapse formation, but rather for

synapse specification and modulation (Chubykin et al. 2007;Varoqueaux et al. 2006).

Investigation of the role of NLs in vivo is critical for

understanding not only the molecular basis of synapsefunction and its role in complex behavior but also the

pathophysiology of autism spectrum disorder (ASD). In par-ticular, internal deletions in neurexin 1, a binding partner of

NL1, NL2 and NL3, have been observed in patients withautism (Feng et al. 2006; Szatmari et al. 2007). Understanding

how deletion of each of the neurexin 1’s postsynaptic binding

partners (i.e. NL1, NL2 and NL3) affects behavior is thus animportant step toward clarification of how neurexin 1 loss of

function may lead to autism. Furthermore, a point mutation inNL3 and multiple loss-of-function mutations in NL4 have been

discovered in individuals with X-linked autism (Chih et al.

2004; Comoletti et al. 2004; Jamain et al. 2003; Laumonnieret al. 2004; Yan et al. 2005), and three different nonsense

mutations in SHANK3 (Durand et al. 2007), a synaptic scaf-folding protein associated with NLs intracellularly (Meyer

et al. 2004), have been found in patients with ASDs. Whenwe introduced the autism-related R451C substitution in NL3

into mice, it caused an increase in inhibitory synaptic trans-

mission with no apparent effect on excitatory synapses(Tabuchi et al. 2007). Our NL2 knockout (KO) mice, which

exhibit reduced inhibitory synaptic transmission, now providea contrast to the NL3 R451C mutation mouse model that

shows enhanced inhibitory synaptic transmission.Consistent with its localization in vivo, NL2 appears to

function primarily at inhibitory synapses (Chubykin et al.

2007). Deletion of NL2 in mice leads to a decrease ininhibitory synaptic transmission (Chubykin et al. 2007).

Whether this effect of NL2 on inhibitory synapses is mediated

114 doi: 10.1111/j.1601-183X.2008.00455.x

through a change in inhibitory synapse numbers, and whetherthis effect of NL2 deletion leads to selective behavioral

deficits, remain critical questions. Analogous to findings inNL3 R451C mutant mice, we now demonstrate that the

decrease in inhibitory synaptic transmission in NL2 KOs isassociated with a decrease in the density of vesicular g-aminobutyric acid transporter (VGAT)-positive puncta abovea set threshold, but with no change in synapse number as

determined by electron microscopy (EM). Next, we deter-mined whether the decrease in inhibitory synaptic transmis-

sion induced by NL2 deletion produced a discrete behavioralphenotype consistent with a role for NL2 in a specific neural

circuit vs. non-specific, global alteration in brain function. Ourfindings suggest that global loss of NL2 leads to abnormalities

in specific behavioral domains referable to the decrease ininhibitory synaptic function. In particular, NL2 KO mice exhibit

increased anxiety-like behavior, decreased pain sensitivity anddecreased motor co-ordination, yet they show normal loco-

motor activity, social interaction and social learning.

Experimental procedures

Genetic manipulations

Neuroligin-2 KO mice were generated using SM1 embryonicstem cell clone derived from 129S6/SvEvTac mouse, and the

resulting chimeric mice were bred with C57BL/6J mice toobtain F1 heterozygous KO mice. Thus, the F1 mice were on

a 129S6/SvEvTac/C57BL/6J hybrid background. Knockoutmice were maintained by interbreeding mice heterozygous

for the NL2 allele for approximately 30 generations.Littermates were used for the breeding in some gener-

ations, although this was avoided as much as possible.Before the behavioral study, KO mice were backcrossed to

C57BL/6NCrl mice for two generations and subsequently to129S2/SvPasCrlf mice for another two generations. Resulting

NL2 heterozygote mice were interbred and resulting age- andsex-matched littermate pair offspring were used for behav-

ioral studies. The use of such a hybrid background minimizesthe possibility of deleterious recessivemutations that occur in

inbred strains being homozygous in the experimental mice.

Morphological analyses

Male NL2 KO and littermate control micewere anesthetized and

perfusion fixed with 4% paraformaldehyde in 100 mM phos-phate buffer (pH 7.4). Brainswere removed and immersion fixed

for 4 hours in 4% paraformaldehyde in 100 mM phosphatebuffer (pH 7.4) and cryoprotected with 30% sucrose in phos-

phate-buffered saline (PBS) for 2 days at 48C. Labels on glassvials storing brain samples were removed and coded for blind

experiment. A 30-mm serial parasagittal sections were cut ona cryomicrotome and blocked with 3% goat serum/0.3% Triton-

X-100 in PBS and incubatedwith anti-synaptophysin monoclonalantibody (Millipore, Billerica, MA, USA), anti-VGlut1 monoclonal

antibody (Synaptic System, Gottingen, Germany), and/or anti-VGAT polyclonal antibody (Millipore, Billerica, MA, USA) over-

night at 48C, followed by incubation with Alexa Fluor 488 or633 conjugated goat anti-mouse immunoglobulin G (Invitro-

gen, Eugene, OR, USA). Sections were transferred onto

SuperFrost slides and mounted under glass coverslips withVectashield with 4’,6-diamidino-2-phenylindole (Vector Labo-

ratories, Burlingame, CA, USA). For each brain section, areasincluding the center portion of the CA1 and CA3 subfields of

the hippocampus were imaged with a Leica TCS2 laserscanning confocal microscope (Leica Microsystems, Wetzlar,

Germany) at 63�, and the stratum radiatum layer of the CA1and CA3 regions (where the dendrites of pyramidal neurons

receive synaptic inputs) was magnified fivefold. For eachprotein of interest, images were acquired with identical

settings for laser power, photomultiplier gain and offset witha pinhole diameter. Images of identical regions (specified

above) were acquired from 15 sections from each of threeanimals/genotype. Images were imported into IMAGEJ 1.41

software for morphometric analysis. In the software, imageswere converted into binary data and thresholded to outline

immunopositive particles. Thresholds were determined tooutline as many immunopositive puncta as possible through-

out all images. Identical thresholds were used for the samesets of experiments (threshold ¼ 60 for synapsin and VGlut1

staining, threshold ¼ 20 for VGAT staining). The number andsize of puncta were detected using the ‘analyze particle’

module of the program. The average number and size ofimmunopositive puncta were normalized with data from

wildtype (WT) to determine synaptic density and size, respec-tively. Statistical significance was determined by Student’s

t-test. All of the data shown are mean � SEM.

Electron microscopy

Male NL2 KO littermates (four WT and four KO, 8 weeks of

age) were anesthetized and vascularly perfused through theheart with 2% paraformaldehyde and 1% glutaraldehyde in

100 mM phosphate buffer (pH 7.4) for the first 15 min. Brainswere removed and immersion fixed with 2% paraformalde-

hyde and 2% glutaraldehyde in the 100 mM cacodylate bufferovernight at 48C. The tissue was sectioned using a vibratome

at 200 mm thickness. The hippocampus of each section wasdissected out before the post-fixation (1 h) with 1% OsO4,

0.8% potassium ferricyanide and en bloc stained with 2%uranyl acetate for 15 min. After dehydration in a series of

ethanol up to 100%, slices were embedded in Poly/bed 812(Polysciences Inc., Warrington, PA, USA) for 24 h. Thin sec-

tions (65 nm) were made and post-stained with uranyl acetateand lead citrate, and viewed under a FEI Tecnai transmission

electron microscope at 120 kV accelerating voltage. All EMimages were captured by a 4k� 4k CCD camera at magnifica-

tions of 30 000, and quantitative analyses were conducted onthe digital EM micrographs of the same magnification. Images

were taken in the stratum radiatum layer of the CA1 hippo-campal region, and all images were within 20–30 mm of the

inner layer of pyramidal neuron cell bodies. A total of 258 EMmicrographs were analyzed. From the four KOmice, 30, 45, 25

and 25 images were randomly selected for analysis, and fromthe four WT mice, 42, 42, 26, and 36 images were randomly

selected for analysis. The measurement was performed with-out knowledge of the genotyping and was assisted by

METAMORPH 7.5 software (Molecular Devices, Union City,CA, USA). The final data were derived from the number of

synapses in the following sequence: Asymmetric/symmetric/

Genes, Brain and Behavior (2009) 8: 114–126 115

Increased anxiety-like behavior in mice

unidentifiable. The statistical significance was calculated withSIGMAPLOT 11 and MICROSOFT EXCEL 2003 SP3.

Western blot

Protein compositions were determined by immunoblotting onwhole brain tissues homogenized in PBS, 10 mM ethylenedia-

minetetraacetic acid, and proteinase inhibitors from four pairs ofP40 littermate mice per genotype. Proteins of 40 mg were

loaded per lane and blottedwith antibodies for synaptic proteinsand internal controls (b-actin or Rab GDP dissociation inhibitor).

Blots were reacted with 125I-labeled secondary antibodiesfollowed by STORM 820 PHOSPHOIMAGER (STORM 860 Amersham

Pharmacia Biotech) detection.

Behavioral overview

Mice were age-/sex-matched littermate progeny of heterozy-

gous/heterozygous matings tested behaviorally in two groups.Experimenters were blind to genotype. For all behavioral tests,

the number of NL2 KO littermate pairs was 22 (total of 44mice). No significant sex � genotype interactions were found

during the statistical analysis of any test (Table 1; NL2, n ¼ 10male pairs, 12 female pairs). For shock threshold, pain sensi-

tivity and the test of olfaction, however, only 15 littermate pairswere tested (30mice total) as some of themice were removed

for histological studies. All mice ranged from 2 to 4 months of

age during the behavioral testing, and within each group, micewere born within 4 weeks of each other. Less stressful

behaviors were tested first with more stressful procedures atthe end. The order of tests was as follows: locomotor, dark/

light box, open field, accelerating rotarod, social interactionwith a juvenile, social learning, social vs. inanimate preference

test, preference for social novelty test, social interaction withan adult caged conspecific, hot-plate sensitivity and shock

threshold. Mice were moved within the animal facility to thetesting room and allowed to habituate to the new location for at

least 1 h before behavioral testing. Significance was taken asP < 0.05 for all experiments and a complete description of

statistical results are given in Table 1.

Anxiety-like behavioral tests

The dark/light and open field tests were performed essen-

tially as described (Powell et al. 2004). In the dark/light test,one side of the apparatus was kept dark (room light entry

limited) while a light built into the top lit the other side(1700 lx, each chamber 25 � 26 cm). Mice were placed in

the dark side and allowed to freely explore the light and darksides for 10 min. Anxiety-like behavior was measured using

latency to enter the light side, time in the dark side andnumber of crosses into the light side. Locomotor activity

was also examined in both the light and dark sides of theapparatus. The open field test was performed for 10 min in

a brightly lit (�800 lx), 48 � 48 � 48 cm white plastic arenausing video tracking software from Noldus (ETHOVISION 2.3.19;

Wageningen, The Netherlands). Time spent in the center zone(15 � 15 cm) and frequency to enter the center was recorded.

Locomotor activity was also measured during the open fieldtest. Data were analyzed with a two-way ANOVA for genotype

and sex.

Accelerating rotarod

An accelerating rotarod designed for mice (IITC Life Sciences;Woodlands Hills, CA, USA) was used essentially as described

(Powell et al. 2004) except three sets of three trials wereperformed per day over 3 days. Briefly, the rotarod was

activated after placing a mouse on the motionless rod. The rodaccelerated from 0 to 45 r.p.m. in 60 s. Time to fall off the rod or

to turn one full revolution was measured. Data were analyzedwith a mixed ANOVA for genotype, sex and the repeated

measures of trial.

Hot-plate sensitivity

Mice were placed on a black, anodized, constant temperatureplate of 528C (IITC model 39 hot plate) covered with a Plex-

iglass enclosure. Latency to lick any pawwasmeasured.Micewere removed upon first paw lick or after 30 s if no response

was elicited, and the plate was cleaned with water betweenmice and allowed to return to room temperature. Data were

analyzed with a two-way ANOVA for genotype and sex.

Shock threshold

Footshock threshold analysis was performed by placing mice

in the fear conditioning apparatus (described in Powell et al.2004) for a 2-min habituation followed by a 2 s footshock with

an interstimulus interval of 20 s of gradually increasing

intensity from 0.05 mA at 0.05-mA intervals. The intensityrequired to elicit flinching, jumping and vocalizing was

recorded by an observer blind to genotype. Data wereanalyzed with a two-way ANOVA for genotype and sex.

Social interaction and social learning

Direct social interaction with a juvenile took place in a novel,empty, clear, plastic mouse cage under red light (Kwon et al.

2006). Following a 15-min habituation in the dark, the experi-mental and target mice were placed in the neutral cage for

2 min and allowed to directly interact. Time spent interactingwith the juvenile was scored by an observer blind to geno-

type. Social learning was assessed 3 days later by allowingmice to interact with the same juvenile for an additional

2 min. Again, time spent interacting with the juvenile wasscored. Data were analyzed with a three-way mixed ANOVA

with genotype and sex as between-subject factors and testsession as a within-subject factor.

Social vs. inanimate preference and preference for socialnovelty analyses were performed as described (Moy et al.

2004; Nadler et al. 2004) except room and door dimensionswere different (15 � 90 � 18.5 cm divided into three com-

partments of 15 � 29 cm separated by dividers with a central3.8 � 3.8 cm door), and video tracking software from Noldus

(ETHOVISION 2.3.19) was used to record mouse behavior (Kwonet al. 2006). In the test, mice were initially allowed to explore

the apparatus for 10 min. Then, mice were allowed to interactwith an empty cage in one compartment vs. a caged social

target in the far compartment for another 10 min. The test forsocial novelty involved a subsequent 10-min test in which mice

were allowed to interact with the familiar caged adult, or a novelcaged adult. Location of empty cages and target mouse as well

as novel vs. familiar mouse was counterbalanced. The test

116 Genes, Brain and Behavior (2009) 8: 114–126

Blundell et al.

Table 1: Detailed statistical analysis

n

Hippocampal

region Parameter Comparison Results

VGlut1/VGAT immunoreactivity (Figs 1 and 2)

Three pairs

(all male)

CA1 Density – VGlut1 puncta WT vs. NL2 KO Student’s t-test: t28 ¼ 0.07, P ¼ 0.95

Three pairs

(all male)

CA1 Density – VGAT puncta WT vs. NL2 KO Student’s t-test: t28 ¼ 2.63, P < 0.030

Three pairs

(all male)

CA1 Size – VGlut1 puncta WT vs. NL2 KO Student’s t-test: t28 ¼ 0.53, P ¼ 0.60

Three pairs

(all male)

CA1 Size – VGAT puncta WT vs. NL2 KO Student’s t-test: t28 ¼ 0.19, P ¼ 0.85

Three pairs

(all male)

CA3 Density – VGlut1 puncta WT vs. NL2 KO Student’s t-test: t28 ¼ 0.32, P ¼ 0.75

Three pairs

(all male)

CA3 Density – VGAT puncta WT vs. NL2 KO Student’s t-test: t28 ¼ 2.73, P < 0.011

Three pairs

(all male)

CA3 Size – VGlut1 puncta WT vs. NL2 KO Student’s t-test: t28 ¼ 0.35, P ¼ 0.73

Three pairs

(all male)

CA3 Size – VGAT puncta WT vs. NL2 KO Student’s t-test: t28 ¼ 0.69, P ¼ 0.50

Synapsin immunoreactivity (Fig. 3)

Three pairs

(all male)

CA1 Density WT vs. NL2 KO Student’s t-test: t28 ¼ 0.11, P ¼ 0.91

Three pairs

(all male)

CA1 Density WT vs. NL2 KO Student’s t-test: t28 ¼ 0.31, P ¼ 0.71

Three pairs

(all male)

CA3 Size WT vs. NL2 KO Student’s t-test: t28 ¼ 0.38, P ¼ 0.76

Three pairs

(all male)

CA3 Size WT vs. NL2 KO Student’s t-test: t28 ¼ 0.23, P ¼ 0.82

Electron microscopy (Fig. 4)

Four pairs

(all male)

CA1 Asymmetric synapses WT vs. NL2 KO Student’s t-test: t6 ¼ 1.75; P ¼ 0.13

Four pairs

(all male)

CA1 Symmetric synapses WT vs. NL2 KO Student’s t-test: t6 ¼ 1.02; P ¼ 0.35

n Test variant Parameter Comparison Results

Anxiety-like behavior (Figs 5 and 6)

22 pairs Open field Time in center/time

in periphery

WT vs. NL2 KO Two-way ANOVA: genotype: F1,40 ¼ 8.91,

P ¼ 0.0048

22 pairs Frequency in center WT vs. NL2 KO Two-way ANOVA: genotype: F1,40 ¼ 8.91,

P ¼ 0.0048

22 pairs Distance traveled WT vs. NL2 KO Two-way ANOVA: genotype: F1,40 ¼ 2.58,

P ¼ 0.12

22 pairs Velocity WT vs. NL2 KO Two-way ANOVA: genotype: F1,40 ¼ 2.57,

P ¼ 0.12

22 pairs Dark/light box Latency to enter

light side

WT vs. NL2 KO Two-way ANOVA: genotype: F1,40 ¼ 4.774,

P ¼ 0.0348.

22 pairs Time in dark side WT vs. NL2 KO Two-way ANOVA: genotype: F1,40 ¼ 36.23,

P ¼ 0.000001

22 pairs Crosses WT vs. NL2 KO Two-way ANOVA: genotype: F1,40 ¼ 16.17,

P ¼ 0.000025

22 pairs Activity in dark side WT vs. NL2 KO Two-way ANOVA: genotype: F1,40 ¼ 1.95,

P ¼ 0.17



was performed under red light and the box was wiped with

70% ethanol and air-dried between mice. Data were ana-lyzed with a three-way mixed ANOVA with genotype and sex

as between-subject factors and interaction target as

a within-subject factor.

Results

Effect of NL2 deletion on synapse density

One might expect that loss of the cell adhesion moleculeNL2 during development may lead to gross developmental

brain abnormalities. At the light microscopic level, however,

gross observation of brain sections by light microscopy didnot reveal any gross differences in anatomy or morphology

(not shown). Because NLs have been implicated in regula-

tion of the excitatory/inhibitory balance (Chubykin et al.2007; Varoqueaux et al. 2004) and NL2 causes a selective

decrease in inhibitory synaptic strength (Chubykin et al.2007), we examined the effects of NL2 loss on density of

excitatory and inhibitory synapses in vivo. We hypothesizedthat the functional effects of NL2 deletion might correlate

with changes in density of puncta labeled with inhibitorysynaptic markers.

Table 1: Continued

n

Hippocampal

region Parameter Comparison Results

22 pairs Activity in light side WT vs. NL2 KO Two-way ANOVA: genotype: F1,40 ¼ 26.52,

P ¼ 0.000007

Tests of pain sensitivity (Fig. 7)

15 pairs Hot plate Temperature WT vs. NL2 KO Two-way ANOVA: genotype: F1,26 ¼ 10.84,

P ¼ 0.0029

15 pairs Shock

threshold

Jump WT vs. NL2 KO Two-way ANOVA: genotype: F1,26 ¼ 11.66,

P ¼ 0.0021

15 pairs Vocalize WT vs. NL2 KO Two-way ANOVA: genotype: F1,26 ¼ 1.63,

P ¼ 0.21

15 pairs Flinch WT vs. NL2 KO Two-way ANOVA: genotype: F1,26 ¼ 0.81,

P ¼ 0.38

Three-chamber interaction test (Fig. 8)

22 pairs Social

preference

Time interaction WT vs. NL2 KO and

inanimate

target vs. social target

Three-way mixed ANOVA: genotype,

F1,40 ¼ 0.01, P ¼ 0.92, target,

F1,40 ¼ 25.30, P < 0.000011,

genotype � target

interaction, F1,40¼.72, P ¼ 0.40

22 pairs Social novelty Time interaction WT vs. NL2 KO

and familiar

target vs. novel target

Three-way mixed ANOVA: genotype,

F1,40 ¼ 0.95, P ¼ 0.33, target,

F1,40 ¼ 16.02, P < 0.00026,

genotype � target

interaction, F1,40¼1.94, P ¼ 0.17

Social interaction and social learning (Fig. 8)

22 pairs Social interaction

and learning

Time interaction WT vs. NL2 KO

and initial

session vs. recognition

session

Three-way mixed ANOVA; genotype,

F1,40 ¼ 1.50, P ¼ 0.23, session, F1,40 ¼ 97.69,

P < 0.000001, genotype � session

interaction, F1,40 ¼ 0.17, P ¼ 0.68

Motor co-ordination (Fig. 7)

22 pairs Rotarod Time to fall off WT vs. NL2 KO Three-way mixed ANOVA, genotype

F1,39 ¼ 7.70, P ¼ 0.0084,

sex F1,39 ¼ 10.13, P < 0.0029,

trial F26,1014 ¼ 10.88, P < 0.00001,

no genotype � trial

interaction F26,1014 ¼ 1.29, P ¼ 0.15

ANOVA, analysis of variance, Mixed ANOVA, ANOVA (two genotypes) with a repeated measures (day, time or trial); WT, wildtype. Fx,y, F ratio statistic is

used to determine whether the variances in two independent samples are equal, x,y are degrees of freedom (df). Degrees of freedom is

a measure of the number of independent pieces of information on which the precision of a parameter estimate is based. x ¼ number of groups

minus 1, y ¼ number of animals per group minus 1, multiplied by the number of groups. Information about main effects of sex and all interactions

involving sex has been included in this table only when the effect was significant (i.e. P < 0.05).


Blundell et al.

NL2 deletion resulted in a significant and selective decrease inthe density of inhibitory VGAT-positive (above a threshold level)

puncta in hippocampal fields CA1 and CA3 (Figs 1 and 2,P ¼ 0.030 and <0.011, respectively; see Table 1 for detailed

descriptions of all statistical results). No change was observed,however, in the density of VGlut1-positive (excitatory) puncta or

in the density of total puncta using synaptophysin as a globalsynaptic marker (Fig. 3; P > 0.05 for all comparisons). Further-

more, no change was observed in puncta size in NL2 KO mice(Figs 1–3; P > 0.05). The decrease in inhibitory synaptic density

in NL2 KO mice was not accompanied by compensatorychanges in NL1 or NL3 (Table 2) and was consistent with the

significant alterations in inhibitory synaptic function in vivo(Chubykin et al. 2007).

Decreased density of VGAT-positive puncta may bebecause of decreased inhibitory synapse density or because

of a decrease in the amount of VGAT within existingsynapses, such that fewer inhibitory synapses contain

sufficient VGAT to be detected. To distinguish betweenthese possibi l i t ies, we quantif ied symmetr ic and

Figure 1: VGAT-positive puncta above threshold are decreased in NL2-deficient hippocampal neurons in area CA1. (a)

Representative confocal images of WT and NL2 KO CA1 region of hippocampus double labeled with anti-VGLUT1 and VGAT antibodies.

(b) The density of VGAT-positive puncta is decreased in NL2 KO neurons, whereas VGLUT1-positive puncta is not changed. (c) There is no

significant change in size of VGLUT1 and VGAT-positive puncta in NL2 KO neurons. Density and size of VGLUT1 and VGAT-positive

puncta in mutant neurons are normalized to WT control. *P ¼ 0.029928. n ¼ 3 animals per genotype.



asymmetric synapse density using electron micrographsfrom NL2 KO mice and WT littermates. Consistent with

previous observations on NL1/2/3 triple KO and NL3 R451Cmutant knockin mice, no change was observed in the

number of symmetric or asymmetric synapses in NL2 KOmice (Fig. 4; P > 0.05).

In an effort to better understand the relationship betweenloss of NL2 and the decrease in inhibitory synaptic function,

we examined protein levels of 26 presynaptic and postsyn-aptic proteins in the brains of NL2 mice by Western blot

(Table 2). These experiments revealed only subtle and largelynonsignificant changes in the gross levels of these synaptic

markers as a result of NL2 deletion.

Anxiety-like behavior is increased in NL2 KO mice

Because NL2 is preferentially localized to inhibitory synapsesand deletion of NL2 leads to decreased inhibitory synaptic

function (Chubykin et al. 2007; Graf et al. 2004; Varoqueauxet al. 2004), we hypothesized that NL2 KOmice would exhibit

Figure 2: Density of VGAT-positive puncta above threshold are decreased in NL2-deficient CA3 region hippocampal neurons.

(a) Representative confocal images of WT and NL2 KO CA3 region of hippocampus double immunostained for VGLUT1 and VGAT. The

density (b) and size (c) of VGLUT1-positive puncta are normal, whereas density of VGAT-positive puncta is decreased in NL2 KO neurons.

Y axes depict punta density (b) and size (c) normalized to WT control. **P < 0.011. n ¼ 3 animals per genotype.


Blundell et al.

increased anxiety. As predicted, NL2 KO mice exhibited

increased anxiety-like behavior in two different behavioralassays. Specifically, NL2 KO mice spent less time in the

center compared with time in the periphery of an open fieldarena compared with WT littermates (Fig. 5a; P < 0.0048,

see Table 1 for detailed descriptions of all statistical results).In addition, the number of entries into the center was

significantly decreased in NL2 KO mice compared with WT(Fig. 5b; P < 0.0048). Importantly, distance traveled and

velocity of the NL2 KO mice were unaffected in the openfield (Fig. 5c,d; P ¼ 0.12 for both distance and velocity) in the

open field, indicating that the anxiety-like behavior in the NL2KO mice was not because of alterations in locomotor activity.

In a second test of anxiety, the dark/light box, NL2 KO mice

again exhibited increased anxiety-like behavior. NL2 KO micetook longer to enter the light side (Fig. 6a; P < 0.035), spent

more time in the dark side (Fig. 6b; 2P < 0.0000001) andentered the light side of the dark/light box less often than WT

(Fig. 6c; P < 0.000025). Of relevance is the fact that NL2 KO

mice showed normal activity in the dark (Fig. 6d; P ¼ 0.17)and decreased activity in the light (Fig. 6e; P < 0.000007),

consistent with an anxiety-like phenotype. See Table 1 for fullstatistical analysis of anxiety-like behavior and locomotor

activity in the open field and dark/light box.

NL2 KO mice exhibit decreased pain sensitivity and

motor co-ordination

Given the role of gamma-aminobutyric acid-mediated

(GABAergic) transmission in pain pathways (Enna & McCarson2006), we examined pain threshold in the NL2 KOmice. In a test

of hot-plate sensitivity, NL2 KO mice exhibited a significantly

longer latency to elicit a paw-lick reaction compared with WT(Fig. 7a; P < 0.0029). In a test for footshock sensitivity, despite

normal ‘flinch’ and ‘vocalization’ response thresholds (Fig. 7b;P > 0.05 for both flinch and vocalization), NL2 KOmice required

Figure 3: Synaptic density and size are normal in NL2-deficient hippocampal neurons. Representative confocal images of the CA1

(a, b, e, and f) and CA3 (c, d, g, and h) region of the hippocampus immunostained for synaptophysin. Scale bars ¼ 50 mm (a–d) and 10 mm(e–h). Density (i, P > 0.05) and size (j, P > 0.05) of synaptophysin positive puncta normalized toWT control are shown. n ¼ 3 animals per

genotype.



increased shock amplitude to obtain a ‘jump’ response com-

pared with WT (Fig. 7b; P < 0.0021).Interestingly, NL2 KO mice performed slightly worse than

controls on the accelerating rotarod. In particular, there was

a significant main effect of genotype (Fig. 7c; P < 0.0084)suggesting that the NL2 KO mice exhibited abnormal co-

ordination. However, there was no statistical interactionbetween genotype and trial (P ¼ 0.79) suggesting that motor

learning was normal in the NL2 KOmice. It is possible that theincreased footshock threshold required for a ‘jump’ response

in the NL2 KO mice might be in part because of this slightimpairment in motor co-ordination rather than decreased pain

sensitivity.

Social interaction is normal in NL2 KO mice

NL3 and NL4, neurexin 1, and Shank 3 mutations in humanshave been linked to ASD (Chih et al. 2004; Comoletti et al.

2004; Feng et al. 2006; Jamain et al. 2003; Laumonnier et al.2004; Szatmari et al. 2007); both NL3 human mutation

knockin mice (Tabuchi et al. 2007) and NL4 KO mice (Jamainet al. 2008) display deficits in social behavior and altered

excitatory to inhibitory balance, particularly decreased inhi-bition, has been hypothesized to be involved in human ASD

(Rubenstein & Merzenich 2003). Because of these links, wenext examined social behavior in NL2 KO mice. NL2 KO

mice exhibited normal social interaction in three differentsocial interaction measures. In a test for social vs. inanimate

preference, all mice showed a significant preference forinteracting with the social target (caged adult) compared

with interaction with the inanimate target (Fig. 8a;P < 0.000011). Furthermore, there was no difference

between NL2 KO and WT littermate controls in time spentinteracting with the targets (P ¼ 0.92). Immediately follow-

ing this task, mice were exposed simultaneously to both thealready familiar mouse and a novel mouse. Again, all mice

showed a significant preference for the novel mouse over

the familiar mouse (Fig. 8b; P < 0.00026), but there was nodifference between NL2 KO and WT mice in time spent

interacting with the targets (P ¼ 0.33). In a test of sociallearning and recognition, test mice were initially given

unrestricted exposure to a novel, conspecific juvenile for2 min, and then re-exposed to the same juvenile 3 days

Table 2: Synaptic protein composition in NL2 KO brain

% SEM P value

b-catenin 123.485 11.102807 0.1413438

CamKIIa 98.74953 1.118751 0.91972

Complexin1 102.1781485 3.403171328 0.640540056

CSP 96.53552409 3.48723068 0.471756001

GABAa-Ra 107.6538 3.301195 0.1289376

GluR1 99.34321914 4.32299936 0.944318611

Liprin 77.5336 6.254984 0.2005728

Munc-18 87.28692 7.170952 0.1297724

NL1 95.635178 14.794855 0.8056675

NL3 108.45325 2.260407 0.129107

NR1 100.5294293 12.40795845 0.972926516

NR2A 72.63036585 6.502714146 0.193094702

NR2B 117.2199784 9.569076908 0.319366302

NSF 101.7249404 4.298788187 0.791722811

PSD-95 107.0342778 5.683373597 0.385717671

Rab3A 94.52535 5.761818 0.5101761

SCAMP 93.1213572 18.07972692 0.725338527

SNAP-25 116.4453743 6.450997269 0.067852313

Synaptobrevin2 121.6617327 5.386123724 *0.010768002

Synapsin1a 97.96340397 4.370161128 0.667866658

Synapsin2b 98.87670922 2.242956983 0.834203731

Synaptophysin 97.31012036 3.94724896 0.612379609

Synaptotagmin1 91.72049 4.112077 0.366943

VAchT 94.72737 10.02643 0.6258743

VGAT 108.6743 8.773805 0.4047834

VGlut1 102.1125 1.953237 0.3805599

Protein levels (% of WT) in P40 NL2 KO brain homogenate, SEM and

P value (Student’s t-test) are listed (WT: n ¼ 4; KO: n ¼ 4). *P > 0.05.

Figure 4: Number of symmetric and asymmetric synapses are normal in NL2-deficient CA1 region of the hippocampus.

Representative EMmicrographs taken at themagnification of 30 000� fromWT (a) and NL2 KO (b). Both images show the ultrastructural

view of stratum radiatum of hippocampal CA1 region. The overall synaptic structure shows no obvious change in NL2 KO mice. The

arrows indicate symmetric synapses, arrowheads mark asymmetric synapses. Scale bar ¼ 800 nm for both (a) and (b). (c) Number of

asymmetric (P > 0.05) and symmetric (P > 0.05) synapses in CA1 did not differ across genotype. n ¼ 4 animals per genotype.


Blundell et al.

hence. While both WT and NL2 KO mice exhibited asignificant decrease in social interaction during the re-

exposure compared with the original exposure (Fig. 8c;P < 0.000001), there was no difference in social interaction

between the genotypes (Fig. 8c; P ¼ 0.23). See Table 1 forfull statistical analysis of social behavior.

Discussion

We previously demonstrated that deletion of NL2 in micedecreases inhibitory synaptic transmission (Chubykin et al.

2007), suggesting that the deletion decreases inhibitorysynapse numbers. Consistent with our results in NL3 mutant

mice and NL1/2/3 triple KO mice, we find that deletion of NL2does not alter the actual number of asymmetric or symmetric

synapses measured by EM, despite that fact that it inducesa significant decrease in the density of puncta stained above

threshold for markers of inhibitory synapses. Thus, NL2deletion may impair the function of inhibitory synapses

without decreasing their density. Decreased inhibition ledus to predict that the NL2 KO mice would display increased

anxiety-like behaviors. Indeed, NL2 KO mice displayedincreased anxiety on at least two independent tasks while

0.00

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WTNL2 KO

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)

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oci

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)

(a)

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600***

Tim

e in

dar

k si

de

(s)

(b)

Figure 6: NL2 KOmice exhibit increased anxiety-like behavior in the dark/light box. (a) NL2 KO mice exhibited increased anxiety-

like behavior compared with WT as measured by increased latency to enter the light side of the dark/light box. Legend in (a) applies

to panels (a)–(f). (b) NL2 KO mice spent more time in the dark side than WT. (c) NL2 KO mice entered the light side less often than WT.

(d) NL2 KO mice displayed normal activity in the dark side but were (e) less active in the light side compared with WT.

Figure 5: NL2 KO mice exhibit in-

creased anxiety-like behavior in the

open field arena. (a) NL2 KOmice display

decreased time spent in the center divided

by time spent in the periphery compared

with WT. Legend in (a) applies to panels

(a)–(d). (b) NL2 KOmice entered the center

less often that WT. (c) NL2 KO mice

displayed normal activity levels (for this

and all subsequent figures, **P < 0.01).



other complex behaviors, such as locomotor activity, social

interaction and social learning were normal.

NL2 and inhibitory synaptic function

NL2 KO mice exhibit decreased inhibitory synaptic stainingin the CA1 and CA3 regions of the hippocampus as meas-

ured by light microscopy. In particular, the density of VGAT-positive puncta (interpreted as inhibitory synapses) was

decreased in NL2 KO mice, whereas the density of VGlut-

positive puncta (interpreted as excitatory synapses) wasunchanged. A selective decrease in inhibitory synaptic

staining is consistent with data showing that NL2 over-expression in vitro increases inhibition, while deletion of

NL2 in vivo decreases inhibitory synaptic responses (Chu-bykin et al. 2007), with no effect on excitatory responses.

Furthermore, NL2 is localized exclusively to GABAergicsynapses (Varoqueaux et al. 2004), and NL1, NL2 and NL3

0 3 6 9 12 15 18 21 24 270

10

20

30 WTNL2 KO

Trial

Tim

e to

fal

l off

(s)

(c)

02468

1012

1416

WTNL2 KO *

Lat

ency

to

lick

(s)

(a)

Flinch Vocalize Jump0.0

0.1

0.2

0.3

0.4

0.5

0.6*

Sh

ock

(m

A)

(b)

Figure 7: NL2KOmice have decreased

pain sensitivity and motor co-ordina-

tion. (a) NL2 KO mice exhibit a longer

latency to paw-lick than WT as measured

with the hot-plate sensitivity test. Legend

in (a) applies to panels (a)–(c). (b) Com-

pared with WT, NL2 KO mice exhibit an

increased shock threshold for eliciting

jumping behavior but a comparable shock

threshold for eliciting flinching and vocal-

izing. (c) NL2 KO mice have deficits in

motor co-ordination compared with WT

as measured on the accelerating rotarod.

(P < 0.0084).

1st Interaction 2nd Interaction0

10

20

30 ********

Tim

e In

tera

ctio

n (

s)

(c)

no m

ouse

mou

se

no m

ouse

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se0

100

200

300 * *

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e In

tera

ctio

n (

s)

(a)

fam

ilar

nove

l

fam

ilar

nove

l0

100

200

300 * *

Tim

e In

tera

ctio

n (

s)

(b)

Figure 8: NL2 KO mice have normal

social behavior. (a) In a social vs. inani-

mate preference task, all mice showed

a significant preference for the social tar-

get, and the time spent interacting with

the social versus inanimate object was

unaffected by genotype. (b) In a preference

for social novelty task, all mice showed

a significant preference for the novel social

target, and the time spent with the novel

vs. familiar adult mouse did not differ

between the NL2 KO mice and WT. (c)

At day 1 (first interaction), NL2 KO mice

spent the same amount of time interacting

with a conspecific juvenile mouse as WT.

WT and NL2 KO mice spent significantly

less time interacting with the same juve-

nile 3 days hence (second interaction),

indicating significant social learning in WT

and NL2 KO mice.


Blundell et al.

triple KO mice compared with NL1, NL3 or NL1/3 KO miceas controls, exhibited a significant decrease in inhibitory

synaptic transmission using multiple electrophysiologicmeasures (Varoqueaux et al. 2006).

Despite a decrease in the density of VGAT-positive punctastained above threshold, we did not detect a decrease in

symmetric (presumed inhibitory) synapse density as meas-ured with EM in NL2 KO mice. Thus, our data indicate that

NL2 deletion likely causes a decrease in VGAT levels atindividual synapses. The lack of effect on synapse density

is consistent with results from NL3 R451C mutant mice(Tabuchi et al. 2007). In contrast to NL2 KO, NL3 R451C

mutant mice exhibit increased inhibitory synaptic staining andincreased inhibitory synaptic transmission; yet, this increase

in inhibition does not reflect an increase in inhibitory synapsedensity (Tabuchi et al. 2007). Tabuchi et al. (2007) concluded

that the R451C substitution in NL3 does not affect synapseformation, but appears to act downstream of synapse func-

tion to increase the average VGAT signal per synapse in NL3R451C mutant mice. Thus, alterations in NLs do not appear to

result in changes in synapse density but rather in changes insynapse function. Another possibility is that the NL2 deletion

results in a decrease in only a subset of inhibitory synapsesthat is too small to detect by EM in the larger population of

inhibitory synapses. Future studies examining the effect ofNL2 deletion (and NL3 R451C mutation) on specific subtypes

of inhibitory interneuron synapses will be necessary to

examine this possibility.

NL2 and behavior

Consistent with decreased inhibitory synaptic function, NL2KOmice demonstrate heightened anxiety-related behavior on

multiple measures. In particular, NL2 KOmice spent less timein the center and entered the center less often than controls

as measured in an open field. In addition, the NL2 KO miceexhibited increased latency to enter the light side, spent more

time in the dark side and crossed to the light side of the dark/light box less often than controls. Importantly, increased

anxiety-like behavior as seen in both tests was not becauseof alterations in locomotor activity. Generalized pharmaco-

logic manipulation of GABAA receptor function is known toalter anxiety levels (Dalvi & Rodgers 1996; Zarrindast et al.

2001), and GABAA-augmenting benzodiazepines are effectivein treating anxiety in humans. Thus, the increased anxiety in

NL2 KO mice is most likely referable to the decreasedinhibitory synaptic function.

In addition to increased anxiety, NL2 KO mice exhibitdifferences in nociception on two independent tasks. These

results are consistent with the role of GABAergic transmis-sion in pain pathways (Enna & McCarson 2006). In particular,

the GABA system, among others, plays a major role inmediating the analgesic action of morphine, a mu-opioid

agonist. Indeed, activation of mu-opioid receptors by mor-phine inhibits the release of GABA in many parts of the brain

(Stiller et al. 1996; Vaughan et al. 1997). Thus, it is notsurprising that deletion of NL2, which results in a decrease

in GABA function also causes decreased pain sensitivity.Given that rearrangements in regions that harbor NL2

genes (Konstantareas & Homatidis 1999; Zoghbi 2003),

mutations in NL3 and NL4 (Chih et al. 2004; Comoletti et al.2004; Jamain et al. 2003; Laumonnier et al. 2004; Yan et al.

2005) and mutations in the NL2-binding partner neurexin 1(Feng et al. 2006; Szatmari et al. 2007) have been implicated

in autism and decreased inhibition has been hypothesized tobe involved in human ASD (Rubenstein & Merzenich 2003),

we examined NL2 KO mice carefully for deficits in socialinteraction. NL2 KO mice show normal social behavior in

multiple measures. Thus, despite a link between the NL-binding partner neurexin-1 and autism, NL2 KO mice do not

display deficits in social behavior reminiscent of autism. Ofcourse, the absence of social interaction deficits in NL2 KO

mice with decreased inhibitory synaptic function does notmean that decreased inhibition cannot be associated with

autism or autism-relevant behavioral abnormalities. Indeed,increased anxiety is often an associated feature of ASDs.

Given the recent implication of neurexin-1 in ASD and theability of neurexins to bind to multiple NL isoforms including

NL2 (Boucard et al. 2005), NL2 KO behavioral abnormalitiesmay foreshadow a subset of neurexin-1 KO behavioral

abnormalities.Despite the dramatic increase in anxiety-like behavior, and

diminished nociception and motor co-ordination, NL2 KOmice exhibit normal locomotor activity, social interaction

and social learning. It is interesting that NL2 KO mice exhibitsuch selective abnormalities given the broad expression of

NL2 at inhibitory synapses throughout the brain. One might

predict that this is because of partial compensation by otherNL isoforms. However, as seen in Table 2, there was no

increase in NL1 or NL3 levels in NL2 KO mice. Indeed, therewere only very small overall changes of synaptic markers in

NL2 KOmice, suggesting that deletion of NL2 does not causea global change in the molecular composition of the brain.

Conclusions

Deletion of NL2 results in a decrease in inhibitory synapsefunction without a decrease in inhibitory synapse density.

This decrease in synaptic inhibition likely mediates theincreased anxiety-like behavior seen in NL2 KO mice and

may also contribute to decreased nociception and motorincoordination. Given that most published studies of NL

function have been carried out in culture, these studies ofthe role of NL in vivo represent a major advance in under-

standing the basic function of NLs

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Acknowledgements

The study was supported by grants from Autism Speaks (toC.M.P.) and the National Institute of Mental Health (MH065975-05 to C.M.P. and R37 MH52804-08 to T.C.S.), and gifts from theCrystal Charity Ball and the Hartwell Foundation (to C.M.P).


Blundell et al.

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