Neuron, Volume 78

Supplemental Information

Protocadherin 17 Regulates Presynaptic Assembly

in Topographic Corticobasal Ganglia CircuitsNaosuke Hoshina, Asami Tanimura, Miwako Yamasaki, Takeshi Inoue, Ryoji Fukabori, Teiko Kuroda,

Kazumasa Yokoyama, Tohru Tezuka, Hiroshi Sagara, Shinji Hirano, Hiroshi Kiyonari, Masahiko Takada,

Kazuto Kobayashi, Masahiko Watanabe, Masanobu Kano, Takanobu Nakazawa, and Tadashi Yamamoto

Inventory of Supplemental Information

Supplemental Data

Supplemental Figure 1 relates to main Figure 1

Supplemental Figure 2 relates to main Figure 2

Supplemental Figure 3 relates to main Figure 5

Supplemental Figure 4 relates to main Figure 5

Supplemental Figure 5 relates to main Figure 5

Supplemental Figure 6 relates to main Figure 6

Supplemental Figure 7 relates to main Figure 7

Supplemental Figure 8 relates to main Figure 8

Supplemental Movie 1 and 2 relate to Supplemental Figure 5

Supplemental Experimental Procedures

Supplemental References

Figure S1

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Figure S1, related to Figure 1. Expression of PCDH17 in mouse brain and anatomical connections

in PCDH17-expressing cortico-basal ganglia circuits

(A) Immunostaining for PCDH17 in serial coronal brain sections of a P10 mouse. (B) X-gal staining for

PCDH17 promoter-driven β-gal in coronal brain sections of a P10 PCDH17LacZ/+ mouse. (C)

Fluorescence of Alexa Fluor 488-conjugated cholera toxin subunit B (CTb-Alexa 488) in coronal brain

sections of an adult mouse, showing the injection site in the anterior striatum (left) and retrograde

labeling neurons in the mPFC, M1, and S1 (Right). (D) Double fluorescence histochemistry with CTb-

Alexa 488 and immunostained PCDH17 in a basal ganglia sagittal section of an adult mouse, showing

the injection site in the anterior striatum (left) and anterograde labeling axon terminals in the LGP,

MGP, and SNr (Right). (E) Images of basal ganglia sagittal sections of a P7 PCDH17-EGFP transgenic

mouse from the GENSAT database. Scale bars represent 1 mm (A, B, C, and E) and 0.5 mm (D). Amy,

amygdala; Au, auditory cortex; BST, bed nucleus of the stria terminalis; Cg, cingulate cortex; Ent,

entorhinal cortex; HTh, hypothalamus; LGP, lateral globus pallidus; M, motor cortex; M1, primary motor

cortex; M2, secondary motor cortex; MD, mediodorsal thalamus; MGP, medial globus pallidus; mPFC,

medial prefrontal cortex; NA, nucleus accumbens; OFC, orbitofrontal cortex; PAG, periaqueductal gray;

Rs, retrosplenial cortex; S, somatosensory cortex; S1, primary somatosensory cortex; SNr, substatia nigra

pars reticulata; Str, striatum; V, visual cortex.

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Figure S2

Figure S2, related to Figure 2. Complementary expression pattern between PCDH17 and PCDH10

mRNAs in basal ganglia

Double fluorescent in situ hybridization for PCDH17 and PCDH10 mRNAs in a basal ganglia sagittal

section of a P10 mouse. Scale bar represents 0.5 mm. LGP, lateral globus pallidus; MGP, medial globus

pallidus; SNr, substatia nigra pars reticulata.

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Figure S3

Figure S3, related to Figure 5. Generation and characterization of PCDH17−/− mice

(A) Schematic representation of the wild-type PCDH17 allele, the targeting vector, the targeted allele,

and the null allele deleted from the LacZ/Neo cassette. Mating with female CAG-Cre mice produced the

null allele. (B) PCR genotyping for PCDH17+/+, PCDH17+/−, and PCDH17−/− mice. (C) Immunoblotting

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for PCDH17 in P7 PCDH17+/+, PCDH17+/−, and PCDH17−/− mouse brain lysates. N-cadherin was used

as a loading control. (D) Immunostaining for PCDH17 in sagittal brain sections of P14 PCDH17+/+ and

PCDH17−/− mice. (E) Pre-embedding immunogold electron microscopy for PCDH17 at asymmetric

synapses in the anterior striatum of P14 PCDH17+/+ and PCDH17−/− mice. (F) Nissl staining in coronal

brain sections including the striatum of 3-week-old PCDH17+/+ and PCDH17−/− mice. (G)

Immunoblotting for various synaptic proteins in anterior and posterior striatal lysates of 3-week-old

PCDH17+/+ and PCDH17−/− mice. α-tubulin was used as a loading control. Scale bars represent 1 mm

(D) and 200 nm (E). Ctx, cerebral cortex; Sp, dendritic spine (blue); Str, striatum; t, presynaptic terminal

(red).

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Figure S4

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Figure S4, related to Figure 5. Normal axonal projections and topographic patterns in basal

ganglia of PCDH17−/− mice

(A) Immunostaining for Neurofilament (a general axon marker, eg. corticothalamic and thalamocortical

axons) in basal ganglia sagittal sections of P14 PCDH17+/+ and PCDH17−/− mice. (B) Double staining

for DARPP-32 (a marker of striatopallidal and striatonigral axons) and TH (a marker of nigrostriatal

axons) in basal ganglia sagittal sections of P14 PCDH17+/+ and PCDH17−/− mice. (C) Double

fluorescence of CTb-Alexa 488 and CTb-Alexa 555 in coronal brain sections of adult PCDH17+/+ and

PCDH17−/− mice, showing injection sites in anterior striatum and posterior striatum (left) and retrograde

labeling neurons in the prefrontal cortical area (Right). Higher-magnification images of each cortical

region are indicated on the right. (D) Fluorescence of CTb-Alexa 488 in basal ganglia sagittal sections of

adult PCDH17+/+ and PCDH17−/− mice, showing injection sites in anterior striatum (left) and

anterograde labeling axon terminals in the LGP, MGP, and SNr (Right). (E) Immunostaining for

PCDH10 in basal ganglia sagittal sections of P14 PCDH17+/+ and PCDH17−/− mice. (F) X-gal staining

for PCDH17 promoter-driven β-gal in basal ganglia sagittal sections of P14 PCDH17LacZ/+ and

PCDH17LacZ/− mice. Scale bars represent 1 mm. Ctx, cerebral cortex; LGP, lateral globus pallidus; MGP,

medial globus pallidus; mPFC, medial prefrontal cortex; M2, secondary motor cortex; SNc, substatia

nigra pars compacta; SNr, substatia nigra pars reticulata; Str, striatum; Th, thalamus.

Figure S5

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Figure S5, related to Figure 5. Overexpression of PCDH17 results in diffusion of synaptic vesicle

puncta and increases mobility of presynaptic clusters in cultured cortical neurons

(A) Localization of Synaptophysin-EGFP (Syn-EGFP) in 14 days in vitro (DIV) cortical neurons

transfected at DIV13 with Syn-EGFP plus mock plasmid or PCDH17-myc. (B) Diffusion of SVs was

quantified by measuring the average length of the major axis of Syn-EGFP puncta. n = 150 for each

condition from three independent cultures. (C) Live cell imaging of DIV14 cortical neurons that were

transfected at DIV13 with Syn-EGFP plus mock plasmid or PCDH17-mCherry. Images were captured

every 20 s. Frames at 2-min intervals from one representative mock-transfected and one PCDH17-

mCherry-overexpressing axon are presented. (D) Kymographs showing the movement of Syn-EGFP

along the axons. (E) A histogram displays the distribution of speeds of individual Syn-EGFP puncta. (F)

Average speed of Syn-EGFP puncta movement in axons of mock-transfected and PCDH17-mCherry-

overexpressing axons. n = 100 for each condition from three independent cultures. Scale bars represent 5

μm. Error bars indicate SEM. ***p < 0.001; Student's t-test. See also Movies S1 and S2.

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Figure S6

Figure S6, related to Figure 6. Other electrophysiological data for PCDH17−/− mice

(A) Summary of the 10-90% rise time and the decay time constant of evoked AMPA and NMDA EPSCs

at cortico-anterior striatal synapses and evoked IPSCs at striato-pallidal synapses (Left). Summary of the

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average amplitudes of AMPA EPSCs during prolonged repetitive stimulation of cortico-anterior striatal

afferents (Right). Data are expressed as mean ± SEM. (B) (Left) Representative traces of GABA

receptor-mediated IPSCs to paired stimuli of 50 ms interval from LGP neurons of 3-week-old

PCDH17+/+ and PCDH17−/− mice in response to stimulation of corresponding anterior striatum. (Right)

Summary graph showing the ratios of second to first IPSCs as a function of interstimulus interval (30,

50, 100, and 200 ms) (n = 7 for PCDH17+/+ and n = 8 for PCDH17−/− mice). There was a significant main

effect of genotype two-way ANOVA (p < 0.05; two-way ANOVA). Post-hoc tests revealed that there was a

significant difference between genotypes at 50 ms pulse interval (*p < 0.05). (C) Average normalized

response amplitudes of EPSCs from anterior MSNs of P16-18 PCDH17+/+ and PCDH17−/− mice during

the prolonged repetitive stimulation (10 Hz, 200 pulses) (n = 10 for PCDH17+/+ and n = 13 for

PCDH17−/− mice). Each point represents the average of 10 consecutive responses. Calibration bars = 0.2

nA and 20 ms (B). Error bars indicate SEM. There were no significant differences (p > 0.05; Mann-

Whitney U-test (A), two-way repeated measures ANOVA (C)). n.d., not determined.

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Figure S7

Figure S7, related to Figure 7. Other behavioral tests in PCDH17−/− mice

(A-C) Contextual and auditory fear conditioning tests. (A) Freezing responses on the conditioning day.

A tone was presented for 10 s (solid line), 170 s after placement of mice in the conditioning chamber; at

the end of the tone, mice were given a foot shock (arrow). (B) Freezing responses in the contextual fear

conditioning test. (C) Freezing responses in the auditory fear conditioning test. The same tone was

presented for 3 min (solid line), 3 min after the placement of mice into a testing chamber with novel

contexts. (n = 8 for PCDH17+/+ and n = 12 for PCDH17−/− mice) (D-E) Acoustic startle response and

prepulse inhibition (PPI) tests. (D) Mean amplitude of startle responses was measured. (E) Percentage

prepulse inhibition of the startle response was shown. Prepulse intensity was 80 dB. (n = 8 for

PCDH17+/+ and n = 12 for PCDH17−/− mice) (F) Tail flick test. The tail-flick latencies were measured.

(n = 8 for PCDH17+/+ and n = 12 for PCDH17−/− mice) Error bars indicate SEM. There were no

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significant differences (p > 0.05; Student's t-test).

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Figure S8

Figure S8, related to Figure 8. Expression of PCDH17 in basal ganglia of monkey brain

Immunostaining for PCDH17 in coronal sections of basal ganglia taken from a 2-month-old rhesus

monkey. Scale bar represents 5 mm. ac, anterior commissure; Cd, caudate nucleus; GPe, external

segment of the globus pallidus; GPi, internal segment of the globus pallidus; ic, internal capsule; LGN,

lateral geniculate nucleus; Put, putamen; SN, substantia nigra; Th, thalamus.

Movie S1, related Supplemental Figure 5. Live cell imaging of DIV14 cortical neurons that were

transfected at DIV13 with Syn-GFP and mock plasmid. Images were captured every 20 s.

Movie S2, related Supplemental Figure 5. Live cell imaging of DIV14 cortical neurons that were

transfected at DIV13 with Syn-GFP (green) and PCDH17-mCherry (magenta). Images were captured

every 20 s.

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Supplemental Experimental ProceduresDNA Constructs

The human PCDH17 full-length cDNA clone was purchased (Invitrogen). The mouse PCDH17 full-

length cDNA clone was raised from standard RT-PCR. The mouse PCDH10 cDNA clone was a kind

gift of the Kazusa DNA Research Institute. PCDH17 cDNAs were cloned into the expression vectors

pCMV-Tag5 (Agilent Technologies), pEGFP-N (Clontech), and pmCherry-N (Clontech) to express C-

terminal myc-, EGFP-, and mCherry-tagged proteins, respectively. PCDH10 cDNAs were cloned into

the expression vector pCMV-Tag5 to express C-terminal myc-tagged protein. For stable transfectants,

mouse PCDH17 and PCDH10 cDNAs were cloned into the expression vector pCX4-bsr (a gift from Dr.

T. Akagi; Akagi et al., 2003). To generate the PCDH17E-Fc fusion protein, the clone of human IgG1 Fc

region was raised from standard RT-PCR and its cDNA was cloned into the expression vector

pcDNA3.1 (Invitrogen) to construct pcDNA3.1-Fc. The extracellular domain of human PCDH17 cDNA

(PCDH17E) was cloned into the pcDNA3.1-Fc to express PCDH17E-Fc fusion protein. To generate

polyclonal antibodies, expression vectors for glutathione S-transferase (GST) were constructed by

inserting mouse cDNA fragments that encode portions of mouse PCDH17 corresponding to amino acid

residues 727-940 and 954-1157 and mouse PCDH10 corresponding to amino acid 738-932 into the

pGEX-6P or pGEX-4T (GE Healthcare). The mouse synaptohysin full-length cDNA clone was raised

from standard RT-PCR and cloned into the expression vector pEGFP-N (Clontech) to express C-

terminal EGFP tagged protein.

Antibodies

The following rabbit and guinea pig antibodies were generated against GST-fusion proteins for mouse

PCDH17 and PCDH10. Expression of GST-PCDH17 and GST-PCDH10 fusion proteins in E. coil

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BL21-strain was induced with isopropyl-β-D-thiogalactopyranoside, and the proteins were purified with

glutathione-Sepharose (GE Healthcare) and excised with PreScission Protease (GE Healthcare). New

Zealand White rabbits and Hartley guinea pigs were immunized with purified proteins. Antisera were

purified with HiTrap NHS-activated affinity columns (GE Healthcare). Rat monoclonal PCDH10

antibody was used (Aoki et al., 2003). Rabbit polyclonal GluN2A antibody was used (Taniguchi et al.,

2009). Rabbit polyclonal GluA1 and GluA2 antibodies were used (Fukaya et al., 2006). The following

antibodies were commercially available and used according to manufacturer’s protocols: N-cadherin

(mouse; BD Biosciences), α-tubulin (mouse; Sigma), DARPP-32 (mouse; BD Biosciences), β-

galactosidase (mouse; MBL), VGLUT1 (guinea pig; Millipore), PSD-95 (mouse; Affinity Bioreagents),

VGAT (guinea pig; Frontier Science), gephyrin (mouse; Synaptic Systems), myc (mouse; Santa Cruz

Biotechnology), human IgG Fc (goat; Jackson ImmunoResearch), Synaptophysin (mouse; Sigma),

GluN1 (mouse; Millipore), GluN2B (mouse; BD Biosciences), Neurofilament (mouse; Novus

Biologicals), Tyrosine hydroxylase (rabbit; Millipore), and GFP (rabbit; MBL).

Generation of PCDH17−/− Mice

Homologous recombination was used to replace the genomic region of PCDH17 that contains the first

exon, encoding a part of the extracellular cadherin domain, with the LacZ and neomycin-resistance (neo)

cassette. The DT-A fragment was ligated to the 3’ end of the targeting vector for negative selection. We

identified the PCDH17-targeted TT2 ES cell clones by Southern hybridization and then injected these

cells into blastocysts. Male chimeras were mated with C57BL/6 females to obtain heterozygous

(PCDH17+/−) F1 offspring with 75% pure C57BL/6 background. Heterozygous mice were crossed to

transgenic mice carrying the CAG-Cre transgene with C57BL/6 background (Sasaki and Miyazaki,

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1997) to remove the LacZ/neo cassette through Cre/loxP mediated excision. Heterozygous mice were

backcrossed successively to C57BL/6 genetic background. We intercrossed the progeny to produce wild-

type (PCDH17+/+) and homozygous (PCDH17−/−) mice with 96.88-99.61% pure C57BL/6 background.

Genotyping was performed by PCR with specific primers (P1: 5’-

GCGGGAGGAGCAAGTCTGGCAGTTAC-3’, P2: 5’-

CTAGTTCTAGAGCGGCCGATTTAAATTGG-3’, P3: 5’-

GACTTCTCCGTTGGGACCTTCATCAGC-3’) to amplify either the wild-type (P1/P3) or mutant

(P2/P3) allele. Detailed information regarding PCDH17 knockout mice (accession number CDB0608K)

is available at http://www.cdb.riken.jp/arg/mutant%20mice%20list.html. All experiments with mice

were carried out according to guidelines for animal use issued by the Committee of Animal

Experiments, University of Tokyo, Okinawa Institute of Science and Technology, Hokkaido University,

and Fukushima Medical University School of Medicine.

Intracranial Surgery

Adult mice were anesthetized with pentobarbital and subjected to unilateral or bilateral intrastriatal

injection of neuronal tracers. For single labeling of the specific pathway from the striatum, a solution

containing cholera toxin subunit B (CTb) conjugated to Alexa Fluor 488 (1.0 μg/μl) (Invitrogen) was

injected into one site (0.2 μl per site) of the anterior striatum through a glass micropipette, which was

stereotaxically introduced using the coordinates from an atlas of the mouse brain. For double labeling,

CTb conjugated to Alexa Fluor 488 (1.0 μg/μl) or to Alexa Fluor 555 (0.5μg/μl) (Invitrogen) was

injected into the anterior and posterior striatum, respectively (0.2 μl per site). The anteroposterior,

mediolateral and dorsoventral coordinates (mm) from bregma and dura were 1.6/1.3/2.5 (anterior

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striatum), 0.3/2.0/3.0 (posterior striatum). Injection was carried out at a constant flow rate of 0.1 μl/min

with a microinfusion pump (Eicom). For fluorescence detection, sections were prepared, followed by

staining with DAPI (Invitrogen) or immunohistochemistry about one week after the injections.

Cell Culture, DNA Transfection, Stable Transfectants, and Co-culture Experiments

HEK293T and CHO cells were cultured in DMEM + 10% FBS and transfected using FuGENE6 reagent

(Roche). For stable CHO transfectants, CHO cells were transfected with pCX4-bsr vectors and selected

with 10 μg/ml blasticidin S. Dissociated cortical neurons were prepared from E18 ICR mouse embryos.

Neurons were maintained in MEM (Invitrogen) supplemented with 2% B27 (Invitrogen),

penicillin/streptomycin (100 U/ml and 100 μg/ml, respectively), 2.5 mM glutamine, and 5% FBS and

plated on poly-L-lysine. For imaging analyses, neurons were transfected at DIV13 using the

Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s suggestions. For co-culture

between cortical neurons and CHO cells, neurons were transfected at DIV12 and CHO cells were

transfected at the same day. At DIV13, neurons were mixed with CHO cells at an 8:1 ratio. At DIV14,

these cells were subjected to immunocytochemistry using α-myc and α-GFP antibodies.

Immunoblotting

Mouse tissues and cultured cells were collected and homogenized in lysis buffer (50 mM Tris-HCl (pH

7.4), 120 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, and 50 U/ml

aprotinin). Protein concentrations were measured with a BCA protein assay kit (Pierce). Lysates were

resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore).

Membranes were blocked with 3% milk/0.1% Tween-20 in Tris-buffered saline for 1 hr and blotted with

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primary antibodies. Horseradish peroxidase-Renaissance Plus Reagent (Perkin-Elmer) was used to

visualize the immunoreactive proteins.

Fc Pull-down Assay

For purification of PCDH17E-Fc, HEK293T cells were transfected with pcDNA3.1-PCDH17E-Fc.

Twelve hr after transfection, the medium was replaced with serum-free DMEM/F12, supplemented with

penicillin/streptomycin and glutamine. Three days later the medium was collected and subjected to

purification with protein A-Sepharose (GE Healthcare). Purified proteins were separated on SDS–PAGE

gels and checked by CBB staining. For myc-tagged PCDH proteins, HEK293T transfected with

PCDH17-myc and PCDH10-myc, and lysed with lysis buffer C containing variable concentrations of

Ca2+ (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, and 100 U/ml aprotinin in addition

with 0-10 mM CaCl2 and 0-1 mM EDTA under the experimental conditions). PCDH-myc lysates were

incubated with PCDH17E-Fc for 2 hr at room temperature. Beads were washed with lysis buffer C and

bound proteins were resolved by SDS-PAGE and subjected to immunoblotting with α-myc antibody.

X-gal Staining

Brains were freshly obtained, immediately frozen and sectioned at 16 μm on a CM3050S cryostat (Leica

Microsystems). Sections were postfixed with 0.2% glutaraldehyde in phosphate-buffered saline (PBS)

for 5 min. Then they were washed with PBS and treated with X-gal staining buffer (1 mg/ml X-gal, 2

mM MgCl2, 5 mM potassium ferricyanide, and 5 mM potassium ferrocyanide in PBS). A blue

precipitate was generated by incubation for 2-3 hr at room temperature in the dark. After washing with

PBS, sections were photographed with a BZ-9000 Biorevo all-in-one fluorescence microscope

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(Keyence).

Fluorescent in Situ Hybridization

To generate cRNA probes, cDNA fragments of mouse PCDH17 cDNA (nucleotides 4634-5253;

GenBank accession number, NM_001013753.2) and mouse PCDH10 cDNA (3067-3651;

NM_001098171.1) were subcloned into the pBluescript II vector. For simultaneous detection of two

mRNAs by fluorescent in situ hybridization, PCDH17 cRNA and PCDH10 cRNA were labeled using

Digoxigenin (DIG)- and fluorescein (Flu)-labeling mixes (Roche), respectively. Under deep anesthesia

with diethylether, mouse brains were removed from the skull and immediately frozen in powdered dry

ice. Frozen sections 20 μm thick were obtained with a CM1900 cryostat (Leica Microsystems). Sections

were treated with the following incubation steps: fixation with 4% paraformaldehyde in phosphate buffer

(PB) for 10 min; washing in PBS for 10 min; acetylation with 0.25% acetic anhydride in 100 mM

triethanolamine-HCl (pH 8.0) for 10 min; and prehybridization for 1 hr in a hybridization buffer (50%

formamide, 50 mM Tris-HCl (pH 7.5), 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum

albumin, 600 mM NaCl, 200 μg/ml tRNA, 1 mM EDTA, and 10% dextran sulfate). Hybridization was

performed at 63.5°C for 12 hr in hybridization buffer supplemented with a mixture of two cRNA probes

each diluted 1:1,000. After stringent post-hybridization washing as described previously (Yamasaki et

al., 2010), sections were processed for immunohistochemical detection. Sections were blocked with

0.6% H2O2 in TNT buffer for 10 min, DIG blocking solution (1% blocking reagent (Roche)/10% normal

sheep serum in TNT buffer) for 30 min, and 0.5% TSA blocking reagent (PerkinElmer) in TNT buffer

for 30 min. DIG- and Flu-labeled probes were detected by a two-step method. The first detection

employed peroxidase-conjugated anti-Flu antibody (Invitrogen; 1:500 in DIG blocking solution for 1 hr)

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and the FITC-TSA plus amplification kit (PerkinElmer), and the second utilized peroxidase-conjugated

anti-DIG antibody (Roche; 1:500 in DIG blocking solution for 1 hr) and the Cy3-TSA plus amplification

kit (PerkinElmer). Residual activity of peroxidase introduced in the first detection was inactivated by

incubation with 1.0% H2O2 for 30 min. All washing and incubation solutions contained 0.0005% Tween

20. Sections were photographed with a FluoView FV1000 confocal microscope (Olympus).

Immunohistochemistry

Under deep pentobarbital anesthesia mice were transcardially perfused with 4% paraformaldehyde in

PBS. Brains were removed, frozen, and sectioned at 16 μm on a CM3050S cryostat. After wetting with

PBS, sections were blocked with 5% milk/0.2% Triton X-100 in PBS for 1 hr. They were then incubated

with primary antibodies in 0.2% Triton X-100 in PBS at room temperature overnight. After washing

with 0.2% Triton X-100 in PBS, sections were incubated with secondary antibodies (Molecular Probes)

at a 1:200 dilution for 2 hr. After washing with PBS, sections were photographed with a BZ-9000

Biorevo all-in-one fluorescence microscope (Keyence) for large-fields, a FluoView FV1000 confocal

microscope (Olympus), a TCS SP8 confocal microscope (Leica Microsystems) for small-fields, and a N-

SIM microscope (Nikon) for 3D super-resolution imaging.

STORM Imaging

For STORM experiments, sections were incubated with secondary antibodies dual-labeled with Alexa

Fluor 405 and Alexa Fluor 647 and dual-labeled with Cy3 and Alexa Fluor 647 (unlabeled secondary

antibody from Jackson ImmunoResearch) for 4-6 hr. After washing with PBS, sections were mounted in

imaging buffer made by mixing the following three solutions with a volume ratio of 620:70:7

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immediately before applying to the sections: Buffer B (50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 10%

glucose), 1 M mercaptoethylamine-HCl (pH 8.5), and GLOX solution (8 mM Tris-HCl (pH 8.0), 40 mM

NaCl, 56 mg/ml glucose oxidase, 3.4 mg/ml catalase). Clean coverglasses were placed on top of the

sections and excess imaging buffer was removed, after which the edges were sealed with nail polish.

STORM images were acquired using an N-STORM microscope (Nikon) with a perfect-focusing

system, but only two-dimensional imaging was performed. Two color STORM data were acquired by

imaging at 60 Hz with one frame of illumination using activation lasers (405 nm for Alexa Fluor 405–

647 or 561 nm for Cy3–Alexa Fluor 647) alternating with three frames of illumination with an imaging

laser (642 nm) (Dani et al., 2010). The focus on the slide was determined, and the out-of-focus signal

was reduced by illuminating with an imaging laser (642 nm) and by deactivating the fluorophores in the

samples above and below the focal plane. STORM analysis was performed using custom software (two-

color imaging, correction to the sample drift in the lateral direction during acquisition, and subtraction of

the crosstalk between the two imaging channels).

Immunoelectron Microscopy

Under deep pentobarbital anesthesia, mice were transcardially perfused with 4% paraformaldehyde in

PB (pH 7.2). Without post-fixation, 50-μm sections were prepared with a VT1000S slicer (Leica

Microsystems). Sections were incubated with 5% BSA/0.02% saponin in PBS for 30 min and then in a

primary antibody (1 μg/ml) diluted with 1% BSA/0.004% saponin in PBS overnight. After intensive

washing with PBS containing 0.004% saponin for 30 min, sections were incubated with a secondary

antibody linked to 1.4 nm gold particles (Nanoprobes) at 1:100 dilution for 4 hr, and immunogold

particles were intensified with a silver enhancement kit (Aurion). Sections were further treated with 1%

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osmium tetroxide for 15 min, stained with 2% uranyl acetate for 30 min, dehydrated and embedded in

Epon 812. Photographs were taken with an H-7100 electron microscope (Hitachi), and JEM-1200, 1400

electron microscopes (JEOL).

For quantitative analysis, plasma membrane-attached immunogold particles were counted on electron

micrographs and analyzed using ImageJ software. The mean number of membrane-attached gold

particles per 1 μm of plasma membrane was counted for each neuronal compartment (presynaptic

terminal, dendritic spine, and dendritic shaft). In each neuronal compartment, labeling density was

calculated on individual profiles, and compared with background immunogold labeling, which was

defined as labeling density in PCDH17 knockout mice. To carry out a semi-quantitative distribution

analysis of PCDH17 across synapses, synapse cross-section lengths were measured in the anterior

striatum and inner lateral globus pallidus. Immunogold particles in both pre- and postsynaptic

compartments were counted by taking the central 50% of the total synaptic cross-section length as the

'central' region, and the two ends, each 25% of the total synaptic cross-section length, as the 'peripheral'

region. An additional 25% length outside the synapse on each side was defined as the 'perisynaptic'

region, and 25~50% length outside the synapse as ‘extrasynaptic’ region. Lengths greater than 50%

outside the synapses were not counted.

Nissl Staining

Under deep pentobarbital anesthesia, mice were transcardially perfused with 4% paraformaldehyde in

PBS. Brains were removed, frozen, and sectioned at 16 μm on a CM3050S cryostat. After wetting with

PBS and DDW, sections were stained with 0.5% cresyl violet/0.01% acetic acid. Sections were then

rinsed with DDW and dehydrated in graded alcohols and Xylene. They were photographed with a BZ-

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9000 Biorevo all-in-one fluorescence microscope (Keyence).

Immunohistochemistry for Rhesus Monkey Brain

Two infant rhesus monkeys (Macaca mulatta; 1 and 2 months old) were deeply anesthetized with an

overdose of pentobarbital (50 mg/kg, i.v.) and killed by perfusion-fixation with 10% formalin in PB (pH

7.4). Brains were removed from the skull, post-fixed in the same fixative (fresh) overnight at 4 °C, and

saturated with 30% sucrose at 4 °C. Coronal sections were cut serially at 60 μm on a freezing

microtome. Every eighth section was processed for immunohistochemical staining by means of the

standard avidin-biotin-peroxidase complex (ABC) method. Following immersion with 1% skim milk,

sections were incubated for three days with anti-PCDH17 antibody at 1:200 dilution in PBS (pH 7.4)

containing 0.1% Triton X-100 and 2% normal donkey serum. Sections were then placed in the same

fresh incubation medium containing biotinylated secondary antibody (Jackson ImmunoResearch) at

1:1,000 dilution, followed by ABC Elite (Vector Laboratories). For visualization of the antigen, sections

were reacted in 50 mM Tris-HCl (pH 7.6) containing 0.04% diaminobenzidine, 0.04% nickel chloride,

and 0.002% hydrogen peroxide. Sections were photographed with a BZ-9000 Biorevo all-in-one

fluorescence microscope (Keyence). The experimental protocol was approved by the Institutional

Animal Care and Use Committees of Primate Research Institute, Kyoto University, and all experiments

were conducted in accordance with the Guidelines for Care and Use of Nonhuman Primates (Primate

Research Institute, Kyoto University).

Immunocytochemistry

CHO cells were fixed with methanol at −20°C for 5 min and neurons were fixed with 4%

27

paraformaldehyde/4% sucrose in PBS for 15 min at room temperature. Co-cultured neurons and CHO

cells were fixed with 4% paraformaldehyde/4% sucrose in PBS for 15 min at room temperature. After

washing with PBS, fixed cells were treated with 0.2% Triton X-100 in PBS for 5 min and blocked with

5% normal goat serum in PBS for 30 min. Cells were then incubated with primary antibodies in 1%

normal goat serum in PBS at 4°C for overnight. After washing with PBS, cells were incubated with

secondary antibodies (Invitrogen) at 1:500 dilution for 2 hr. Nuclei were stained with Hoechst 33342

(Invitrogen). For co-culture experiments between neurons and CHO cells, PCDH17-EGFP puncta in

neuons that were localized next to PCD17-myc or PCDH10-myc in CHO cells at contact points were

quantified. At least 100 puncta were counted for each condition from three separate cultures. For

overexpression studies in neurons, transfected neurons were stained using α-myc and α-GFP antibodies

to visualize overexpressed PCDH17 protein in Syn-GFP-labeled neurons. Cells were photographed with

a FluoView FV1000 confocal microscope (Olympus) and a TCS SP8 confocal microscope (Leica

Microsystems). To quantify the major axis of the Syn-EGFP puncta along axons, all images were

captured with the same exposure time. Images were imported into ImageJ software and puncta were

identified and analyzed at a mean intensity threshold of 10 and a minimum pixel size of 10. At least 150

puncta were measured per condition from three separate cultures. Data were statistically analyzed using

Student’s t-test. The difference was considered significant at p < 0.05.

Time-lapse Live Imaging

Neurons transfected with Syn-EGFP and PCDH17-mCherry were cultured on glass-bottomed dishes.

During observation, neurons were maintained on the IX-70 microscope stage (Olympus) in warm and

humid air supplemented with 5% CO2. Time-lapse images were captured at 20 s intervals for 10 min on

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an Olympus IX-70 inverted microscope (Olympus) controlled by Delta Vision Softworx (Applied

Precision) using a 100x objective lens. Deconvolution was performed and image stacks were quick

projected. Data analysis was carried out using ImageJ software. Large clusters with relatively low

mobility are localized at synaptic sites, constituting a population of SVs, while smaller and more mobile

clusters are believed to constitute a population of transport vesicles with directed, saltatory movements

(McAllister, 2007). To quantify the mobility of presynaptic clusters, the velocity of Syn-EGFP puncta

were obtained using the manual spot tracking plug-in of ImageJ. Kymographs were produced using the

kymograph plug-in. At least 100 puncta were measured per condition from three separate cultures. Data

were analyzed using Student’s t-test. The difference was considered significant at p < 0.05.

Electron Microscopy

Under deep pentobarbital anesthesia, male wild-type or knockout littermate mice at P21 were perfused

transcardially with 2% paraformaldehyde/1% glutaraldehyde in PB. The striatum and lateral globus

pallidus were then removed and cut into 1 mm thick sections. Sections were further immersed in 2%

paraformaldehyde/2.5% glutaraldehyde in PB for 3 hr, postfixed with 1% osmium tetroxide in PB for 1

hr, dehydrated in graded alcohols, and embedded in Epon 812. Ultrathin sections (70 nm) were prepared

on an ultramicrotome and stained with 2% uranyl acetate for 5 min and Reynold’s lead citrate solution

for 5 min. Electron micrographs were taken at a magnification of 5,000x using an H-7100 electron

microscope (Hitachi). Quantitative analysis of synapses in the striatum and lateral globus pallidus

employed 10 planar sections (each 1300 μm2 area) for three wild-type and knockout pairs in all regions.

The number of docked SVs located less than one SV diameter from the presynaptic membrane and the

total number of SVs per synapse were calculated. Synaptic cleft width in both asymmetric and

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symmetric synapses, spine area and PSD length in asymmetric synapses were also calculated with

ImageJ software. At least 240 synapses were counted per genotype from three wild-type and knockout

mouse pairs in all regions. Data were statistically analyzed with Student’s t-test. The difference was

considered significant at p < 0.05.

Electrophysiology

Sagittal brain slices containing the cortex and the striatum (300 μm thick) were prepared from male

wild-type or knockout littermate mice at P21-24 for the kinetics measurements, AMPA/NMDA ratio,

and paired-pulse ratio of evoked EPSCs, and the amplitude and frequency of mEPSCs, at P16-18 or P21-

23 for the evaluation of short-term synaptic depression, as described previously (Narushima et al., 2007).

In brief, mice were decapitated under anesthesia with 100% CO2, and brains were cooled in ice-cold,

modified external solution (120 mM Choline-Cl, 2 mM KCl, 8 mM MgCl 2, 28 mM NaHCO3, 1.25 mM

NaHPO4, and 20 mM glucose (bubbled with 95% O2 and 5% CO2)). Slices were cut using a Leica

VT1200 slicer (Leica Microsystems). For recovery, slices were incubated for at least 1 hr in normal

bathing solution (125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1.25 mM NaH2PO4, 26 mM

NaHCO3, and 20 mM glucose (pH 7.4) (bubbled with 95% O2 and 5% CO2)). The recording chamber

was perfused with the external solution supplemented with 100 μM picrotoxin for recording evoked

EPSCs. TTX (0.5 μM) and the NMDA receptor antagonist (R)-CPP (10 μM) were added to the

picrotoxin-containing external solution for recording mEPSCs.

Whole-cell recordings were made from MSNs in the anterior region of the striatum using an upright

microscope (BX50WI, Olympus) equipped with an infra-red CCD camera system (Hamamatsu

Photonics). MSNs were identified visually through their medium-sized, spherical somata, as well as

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from their electrophysiological properties (Narushima et al., 2007). Resistance of the patch pipette was

2–3 MΩ when filled with intracellular solution of the following composition: 140 mM CsCl, 10 mM

HEPES, 10 mM BAPTA-K4, 4.6 mM MgCl2, 4 mM Na2-ATP, 0.4 mM Na2-GTP (pH 7.3, adjusted with

CsOH). Membrane currents were recorded with an EPC9/2 amplifier (HEKA Electronik) and pipette

access resistance was compensated by 80%. PULSE software (HEKA Electronik) was used for

stimulation and data acquisition. Mini analysis program (Ver. 6.0.3, Synaptosoft Inc.) was used for

analysis of mEPSCs. Signals were filtered at 3 kHz and digitized at 20 kHz. To stimulate corticostriatal

axons, two glass micropipettes filled with normal saline were placed in the cerebral cortex or in the

underlying white matter. Stimulus pulses (duration: 0.1 ms; intensity: 0–80 V) were applied between the

pipettes to evoke EPSCs in MSNs. To measure the AMPA/NMDA ratio, EPSCs were first recorded at a

holding potential (Vh) of −70 mV and the peak amplitude of EPSCs was determined as the amplitude of

AMPA receptor-mediated EPSC. Then, Vh was set at +50 mV and the amplitude of EPSCs at 60 ms

from the stimulus was determined as the amplitude of NMDA receptor-mediated EPSCs. The paired-

pulse ratio was determined at Vh of −70 mV as the ratio of the second to the first peak amplitude of

AMPA receptor-mediated EPSCs at interstimulus intervals of 20, 30, 50, 70, 100, and 200 ms in normal

bathing solution (125 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1 mM MgSO4, 1.25 mM NaH2PO4, 26

mM NaHCO3, and 20 mM glucose (pH 7.4). To estimate short-term synaptic depression, EPSCs evoked

by a 10 Hz stimulus (200 times) were recorded in the external solution (150 mM NaCl, 2.5 mM KCl, 10

mM HEPES, 4 mM CaCl2 and 20 mM glucose (pH 7.4)) containing picrotoxin (100 μM), the CB1

receptor antagonist AM251 (2 μM), and (R)-CPP (10 μM).

To record from neurons in the LGP, sagittal brain slices containing the cortex, the striatum and the

LGP (300 μm thick) were prepared. Slices were incubated in the normal bathing solution (124 mM

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NaCl, 4.4 mM KCl, 2.5 mM CaCl2, 1.3 mM MgSO4, 1.0 mM NaH2PO4, 26 mM NaHCO3, and 11 mM

glucose (pH 7.4) (bubbled with 95% O2 and 5% CO2)). The recording chamber was perfused with the

external solution supplemented with NBQX (10 μM) and (R)-CPP (10 μM) for recording IPSCs. To

stimulate axons of MS neurons, bipolar electrodes were placed in the anterior dorsal striatum region near

the LGP. The paired-pulse ratio was determined at Vh of -70 mV as the ratio of the second to the first

peak amplitude of IPSCs at interstimulus intervals of 30, 50, 100, and 200 ms. Data were analyzed using

the Mann-Whitney U-test, two-way ANOVA with Fisher's LSD post-hoc analysis, or two-way repeated

measures ANOVA with Tukey post-hoc analysis. The difference was considered to be significant at p <

0.05.

Behavioral Tests

All behavioral experiments were performed as blind tests. Male mice, 7-9 weeks of age, were analyzed

for all experiments. Data were analyzed with Student’s t-test. The difference was considered significant

at p < 0.05.

Tail suspension test

Mice were suspended above the floor by fixing the ends of their tails to wire netting. Immobility was

quantified by observation during a 6-min test session.

Forced swim test

Mice were placed in a cylinder filled with water at 23-25°C. Immobility was quantified by observation

during a 10-min test session.

Open field test

The apparatus was a square arena (50 × 50 × 33.3 cm (W × D × H)) made of polyvinyl chloride. Mice

were placed in the perimeter and allowed to explore the apparatus for 15 min. Immobility time, total

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distance travelled in the area, rearing activity, and time spent in the center (30 × 30 cm) were analyzed

for 20 min with a Macintosh computer using Image OFCR 1.00 and Image OF circle 1.01 (O'Hara & Co.

Ltd.), modified software based on the public-domain NIH Image program.

Elevated plus maze test

The elevated plus maze (EP-3002; O′ Hara & Co. Ltd.) consisted of two open arms (25 × 5 cm) and two

closed arms of the same size extending from a central area (5 × 5 cm) and elevated 50 cm from the

ground. Mice were placed in the central square of the maze facing one of the open arms. Mouse behavior

was recorded during a 10 min test period by means of a Macintosh computer using Image OFCR 1.00

and Image OF circle 1.01 (O'Hara & Co. Ltd.). The amount of time spent in open or closed arms was

measured for 10 min.

Light - dark transition test

The light–dark box consisted of two compartments: a transparent polyvinyl chloride box (100 lux) and a

black polyvinylchloride box (both 19 × 19 × 19 cm). The two boxes were separated by a vertical sliding

door that remained open (5 × 5 cm). The amount of time spent in the transparent and black boxes was

measured for 10 min.

Contextual and auditory fear conditioning tests

Fear conditioning was conducted in a small conditioning chamber surrounded by a sound-attenuating

chest (CL-M3; O'Hara & Co., Ltd.). On Day 1, mice were placed in the conditioning chamber for 170 s

and then presented with a tone of 65 dB/10 kHz for 10 s through a speaker in the roof of the chest. At the

end of the tone presentation, a foot shock (2 s/0.35 mA) was paired with the tone. Freezing responses

were monitored for 1 min after the foot shock and the mice were then returned to their home cages. On

Day 2, the mice were placed in the conditioning chamber and freezing was scored for 6 min. On Day 3,

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the mice were placed in a novel chamber with contexts different from those of the conditioning chamber

to minimize freezing caused by contextual fear conditioning. Freezing was scored for 3 min before

presenting the tone and then for 3 min in the presence of the tone. Freezing responses were analyzed

with Image FZC 2.22sr2 software (O'Hara & Co., Ltd).

Acoustic startle response and prepulse inhibition tests

A startle reflex measurement system (SM-1001; Kinder Scientific) was used for assessing acoustic

startle responses and prepulse inhibition. A test session began by placing a mouse in a Plexiglas chamber

where it was left undisturbed for 2 min. The duration of white noise that was used as the startle stimulus

was 40 ms for all trial types. The startle response was recorded for 200 ms (measuring the response

every 1 ms) starting with the onset of the startle stimulus. The background noise level in the chamber

was 70 dB. The peak startle amplitude during the 200 ms sampling window was recorded as a measure

of the startle response. A test session consisted of six trial types (i.e., five types for startle stimulus trials

and one type for prepulse inhibition trial). The intensity of startle stimulus was 70, 90, 100, 110, and 120

dB. The prepulse sound was presented 100 ms before the startle stimulus and its intensity was 80 dB.

Eight blocks of the six trial types were presented in pseudorandom order such that each trial type was

presented once within a block. The average intertribal interval was 15 s (range, 10~20 s). The following

formula was used to calculate percentage prepulse inhibition of the startle response: 100 − [100 × (startle

response on prepulse trials/startle response on 120 dB startle trials)].

Tail flick test

Animals were gently held by hand so that the tail was placed in the groove of the tail-flick unit (Model

7360; Ugo Basile). The light was focused on the tail 1.5 cm from its tip by a foot-operated switch. The

tail-flick latencies were recorded and a cutoff time was set to 15 s to prevent thermal injury. Tail flick

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reactions were monitored in three consecutive trials at 10-sec intervals.

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Supplemental References

Akagi, T., Sasai, K., and Hanafusa, H. (2003). Refractory nature of normal human diploid fibroblasts

with respect to oncogene-mediated transformation. Proc. Natl. Acad. Sci. USA. 100, 13567–13572.

Fukaya, M., Tsujita, M., Yamazaki, M., Kushiya, E., Abe, M., Akashi, K., Natsume, R., Kano, M.,

Kamiya, H., Watanabe, M., and Sakimura, K. (2008). Abundant distribution of TARP gamma-8 in

synaptic and extrasynaptic surface of hippocampal neurons and its major role in AMPA receptor

expression on spines and dendrites. Eur. J. Neurosci. 24, 2177–2190.

McAllister, A.K. (2007). Dynamic aspects of CNS synapse formation. Annu. Rev. Neurosci. 30, 425–

450.

Narushima, M., Uchigashima, M., Fukaya, M., Matsui, M., Manabe, T., Hashimoto, K., Watanabe, M.,

and Kano, M. (2007). Tonic enhancement of endocannabinoid-mediated retrograde suppression of

inhibition by cholinergic interneuron activity in the striatum. J. Neurosci. 27, 496–506.

Sakai, K., and Miyazaki, J. (1997). A transgenic mouse line that retains Cre recombinase activity in

mature oocytes irrespective of the cre transgene transmission. Biochem. Biophys. Res. Commun. 237,

318–324.

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