Neuron
Article Protocadherin 17 Regulates Presynaptic Assembly in Topographic Corticobasal Ganglia Circuits Naosuke Hoshina,1,9 Asami Tanimura,3 Miwako Yamasaki,4 Takeshi Inoue,1 Ryoji Fukabori,5 Teiko Kuroda,6 Kazumasa Yokoyama,1 Tohru Tezuka,1 Hiroshi Sagara,2 Shinji Hirano,7 Hiroshi Kiyonari,8 Masahiko Takada,6 Kazuto Kobayashi,5 Masahiko Watanabe,4 Masanobu Kano,3 Takanobu Nakazawa,1,3 and Tadashi Yamamoto1,9,* 1Division
of Oncology Proteomics Laboratory The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan 3Department of Neurophysiology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan 4Department of Anatomy, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan 5Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan 6Systems Neuroscience Section, Primate Research Institute, Kyoto University, Inuyama 484-8506, Japan 7Department of Neurobiology and Anatomy, Kochi Medical School, Nankoku-City 783-8505, Japan 8Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan 9Cell Signal Unit, Okinawa Institute of Science and Technology, Onna-son 904-0495, Japan *Correspondence:
[email protected] http://dx.doi.org/10.1016/j.neuron.2013.03.031 2Medical
SUMMARY
Highly topographic organization of neural circuits exists for the regulation of various brain functions in corticobasal ganglia circuits. Although neural circuit-specific refinement during synapse development is essential for the execution of particular neural functions, the molecular and cellular mechanisms for synapse refinement are largely unknown. Here, we show that protocadherin 17 (PCDH17), one of the nonclustered d2-protocadherin family members, is enriched along corticobasal ganglia synapses in a zone-specific manner during synaptogenesis and regulates presynaptic assembly in these synapses. PCDH17 deficiency in mice causes facilitated presynaptic vesicle accumulation and enhanced synaptic transmission efficacy in corticobasal ganglia circuits. Furthermore, PCDH17 / mice exhibit antidepressant-like phenotypes that are known to be regulated by corticobasal ganglia circuits. Our findings demonstrate a critical role for PCDH17 in the synaptic development of specific corticobasal ganglia circuits and suggest the involvement of PCDH17 in such circuits in depressive behaviors.
INTRODUCTION The basal ganglia comprise a group of subcortical nuclei that includes the striatum, the globus pallidus, and the substantia nigra. These nuclei receive input from the cerebral cortex and send output to the thalamus, constituting corticobasal gangliathalamocortical loops that govern various brain functions associated with complex motor action, reward-based learning, cogni-
tion, emotion, and motivation (Redgrave et al., 2010; Utter and Basso, 2008). To perform these different functions, individual cortical areas project to discrete regions of the basal ganglia in a highly topographic manner (Alexander and Crutcher, 1990; Redgrave et al., 2010). Thus, prefrontal cortical areas provide input to anterior regions of the striatum; sensorimotor cortical areas project to central dorsolateral regions; and the parietal cortex provides input to more posterior regions (Draganski et al., 2008; Takada et al., 2001; Wiesendanger et al., 2004). Dysfunctions along the corticobasal ganglia circuit lead to neurological and neuropsychiatric diseases, including Parkinson’s disease, obsessive-compulsive disorder, schizophrenia, and depressive disorder (Krishnan and Nestler, 2008; Simpson et al., 2010; Utter and Basso, 2008). Therefore, clarification of the precise topography and pathway-specific synapse development in corticobasal ganglia circuits is crucial for understanding the mechanisms that regulate respective brain functions. The anatomical topography of neural circuits generally emphasizes distinct functional units. Functional establishment of this topography requires circuit-specific differentiation and refinement of synapses. Recent studies suggest that synapse development in individual circuits is organized by various types of synaptic interaction molecules (Williams et al., 2010). Proteins of the cadherin superfamily, including the protocadherin family, are thought to participate in synapse-specific interactions (Shapiro and Colman, 1999; Williams et al., 2011; Zipursky and Sanes, 2010). This family of proteins is expressed in synaptic junctions between different types of neurons in neural circuits (Kim et al., 2007; Redies, 2000). Owing to their highly selective adhesive interactions, cadherin-catenin complexes are required for both pre- and postsynaptic development (Arikkath and Reichardt, 2008; Togashi et al., 2002). Although some cadherin members are expressed in specific zones of the basal ganglia (Hertel et al., 2008), their roles in circuitspecific synaptic development and their physiological significance remain unclear. Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc. 839
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
After enormous numbers of synapses are formed, subsequent synaptic refinement is an essential step for the completion of functional neural circuits. Recently, molecular mechanisms that control recruitment and localization of synaptic vesicles (SVs) to presynaptic locations have attracted much attention (Goda and Davis, 2003; Ziv and Garner 2004). Furthermore, dynamic regulation of presynaptic SV by neural activity is thought to be a fundamental process involved in presynaptic plasticity (Hopf et al., 2002; Regehr, 2012). Several lines of evidence suggest that trans-synaptic cell adhesion molecules, such as cadherin superfamily proteins, SynCAM family proteins, and the neurexin-neuroligin complex, help trigger presynaptic assembly (Ziv and Garner, 2004). Ablation of N-cadherin or b-catenin in neurons results in reduced SV assembly in presynaptic terminals (Bamji et al., 2003; Stan et al., 2010), suggesting that cadherincatenin adhesive complexes play a pivotal role in localizing SVs to presynaptic compartments (Arikkath and Reichardt, 2008). Although some protocadherins are thought to participate in presynaptic assembly, roles of individual protocadherin members in vivo in synapse refinement and function are not well understood. In the present study, we addressed the biological significance of PCDH17, a nonclustered d2-protocadehrin family member. Our results indicate that PCDH17 plays a crucial role in the regulation of presynaptic vesicle assembly in corticobasal ganglia circuits. Furthermore, PCDH17 deficiency leads to altered presynaptic function in the corticostriatal pathway. We also observed antidepressant-like phenotypes in PCDH17 / mice. These results provide new insights into the mechanisms underlying the synaptic development of specific corticobasal ganglia circuits and the physiological role of depression-related behaviors. RESULTS PCDH17 Is Expressed along Corticobasal Ganglia Circuits in a Topographic Manner PCDH17, a member of the nonclustered d2-protocadherin family, is a transmembrane protein that displays the six extracellular cadherin domains and two cytoplasmic motifs, CM1 and CM2, that are conserved in this family (Redies et al., 2005; Figure 1A). Immunoblot analysis showed that mouse PCDH17 is specifically expressed in the brain (Figure 1B) and that its expression level is high during early synaptogenesis (postnatal weeks 1–2) (Figure 1C). At postnatal day 10, immunohistochemistry revealed that PCDH17 is distributed in the striatum, lateral globus pallidus (LGP), medial globus pallidus (MGP), and substantia nigra pars reticulata (SNr) of the basal ganglia in a highly zone-specific manner (Figure 1D). To precisely evaluate the expression pattern of PCDH17 in basal ganglia, we co-stained PCDH17 and DARPP-32, which are expressed in striatal medium spiny neurons (MSNs) and which are distributed in almost all basal ganglia nuclei. It was found that PCDH17 is distributed in anterior regions of the striatum, including the anterior dorsal striatum and the anterior nucleus accumbens, inner regions of the LGP and MGP, and posterior regions of the SNr (Figure 1E and see Figure S1A available online). To characterize PCDH17expressing cells along the corticobasal ganglia circuits, we performed X-gal staining of brain slices from PCDH17 heterozy840 Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc.
gous mice expressing LacZ under control of the PCDH17 promoter (Figure S3A). b-gal-positive neurons were localized in the anterior striatum, inner LGP, inner MGP, and posterior SNr. This staining pattern is virtually identical to that observed with PCDH17 antibody (Figures 1F and S1B). Costaining of b-gal and DARPP-32 revealed that anterior striatal MSNs express PCDH17 (data not shown). b-gal-positive neurons were also identified in the cerebral cortex; The b-gal signal was strongest in the medial prefrontal cortex, high in the cingulate cortex and motor cortex, and moderate to low in the somatosensory cortex and posterior part of the cortex (Figures 1G and S1B). In addition, the b-gal signal was strong in layer V neurons, including those that project to MSNs (Figures 1G and S1B), although it was also strongly expressed in layer II/III neurons of the medial prefrontal cortex. We next investigated whether PCDH17-expressing regions were anatomically connected in the corticobasal ganglia pathway using the fluorescent neuronal tracer, cholera toxin subunit B (CTb) conjugated to Alexa Fluor 488. When CTb was injected into the anterior striatum for retrograde tracing, medial prefrontal cortical neurons were mostly labeled (Figure S1C). CTb can also be used for anterograde tracing (Angelucci et al., 1996). Accordingly, double fluorescence histochemistry with CTb and immunostained PCDH17 showed that CTb anterograde-labeled anterior striatal axon terminals accurately identify PCDH17-positive zones in basal ganglia (Figure S1D). In addition, the Gene Expression Nervous System Atlas (GENSAT) database (http://www.gensat.org/ index.html) contains PCDH17 promoter-driven EGFP-expressing transgenic mice. Their EGFP expression patterns are similar to PCDH17 expression patterns in basal ganglia, reflecting PCDH17-expressing pathways (Figure S1E). Overall, PCDH17 was expressed at the high level in both pre- and postsynaptic neurons in the medial prefrontal cortex and anterior striatum, as well as the inner LGP, inner MGP, and posterior SNr. In other words, PCDH17 is expressed along the anatomically connected corticobasal ganglia pathways in a highly topographic manner. Complementary Expression Patterns of PCDH17 and PCDH10 in Corticobasal Ganglia Circuits Because protocadherin 10 (PCDH10), another d2-protocadherin family member, is highly expressed in the striatum (Aoki et al., 2003), we next compared expression patterns of both of these proteins in basal ganglia. Double immunostaining of PCDH17 and PCDH10 showed that while PCDH17 is distributed in the anterior striatum, PCDH10 is distributed in the posterior striatum (Figure 2A). Therefore, expression of the two protocadherins was complementary along the anteroposterior axis. Their distributions are also complementary in the LGP and MGP; PCDH17 displays an inner distribution, but PCDH10 displays an outer distribution within these regions (Figure 2A). Furthermore, in contrast to the distribution of PCDH17 in the posterior SNr, PCDH10 is distributed in the anterior SNr (Figure 2A). Doublefluorescent in situ hybridization demonstrated that both PCDH17 and PCDH10 mRNAs also exhibit complementary expression patterns in basal ganglia (Figure S2). Thus, these findings indicate that PCDH17 and PCDH10 delineate topographic features of this pathway.
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
Figure 1. Expression of PCDH17 along Corticobasal Ganglia Circuits in a Topographic Manner (A) A schematic representation of PCDH17 domain structure. ss, signal sequence; CA, cadherin-like domain; TM, transmembrane domain; CM1/2, conserved motifs among the d-protocadherin family; aa, amino acid. (B) Immunoblotting for PCDH17 in P10 mouse tissue lysates (30 mg). (C) Immunoblotting for PCDH17, PCDH10 and N-cadherin in mouse brain lysates at the indicated ages. a-tubulin was used as a loading control. (D) Immunostaining for PCDH17 in a sagittal brain section of a P10 mouse. (E) Double staining for PCDH17 and DARPP-32 in a basal ganglia sagittal section of a P10 mouse. (F) X-gal staining for PCDH17 promoter-driven b-gal in a sagittal brain section of a P10 PCDH17LacZ/+ mouse. (G) X-gal staining for PCDH17 promoter-driven b-gal in coronal brain sections of a P10 PCDH17LacZ/+ mouse. Higher-magnification images of each cortical area are indicated on the right. Scale bars represent 1 mm (D, F, and G) and 0.5 mm (E). LGP, lateral globus pallidus; MGP, medial globus pallidus; mPFC, medial prefrontal cortex; M1, primary motor cortex; M2, secondary motor cortex; NA, nucleus accumbens; Ob, olfactory bulb; SNr, substantia nigra pars reticulata; Str, striatum; S1, primary somatosensory cortex. See also Figure S1.
We next compared the protein expression patterns of PCDH17 and PCDH10 in the cerebral cortex and thalamus, particularly in the prefrontal cortex and the mediodorsal thalamus, as these regions are anatomically and functionally incorporated into the corticobasal ganglia-thalamocortical loops (McCracken and Grace, 2009; McFarland and Haber, 2002). In
the prefrontal cortex, while PCDH17 is distributed in the medial prefrontal cortex, PCDH10 expression is higher in the orbitofrontal cortex, indicating partially complementary expression patterns (Figure 2B). In subregions of the mediodorsal thalamus, PCDH17 and PCDH10 expression are also expressed in a somewhat complementary manner (Figure 2C). Thus, expression of Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc. 841
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
Figure 2. Complementary Expression Pattern between PCDH17 and PCDH10 in Corticobasal Ganglia Circuits (A–C) Double staining for PCDH17 and PCDH10 in P10 mice in a basal ganglia sagittal section (A), prefrontal cortex coronal section (B), and mediodorsal thalamus coronal section (C). Scale bars represent 0.5 mm. CL, centrolateral thalamic nucleus; CM, central medial thalamic nucleus; CMD, central mediodorsal thalamus; IMD, intermediodorsal thalamus; LGP, lateral globus pallidus; LHb, lateral habenula; LMD, lateral mediodorsal thalamus; MGP, medial globus pallidus; MHb, medial habenula; MMD, medial mediodorsal thalamus; mPFC, medial prefrontal cortex; M2, secondary motor cortex; OFC, orbitofrontal cortex; PC, paracentral thalamic nucleus; PV, paraventricular thalamic nucleus; SNr, substantia nigra pars reticulata. See also Figure S2.
PCDH17 and PCDH10 are largely complementary throughout the corticobasal ganglia-thalamocortical loop circuits in a highly topographic manner. We note that expression of PCDH17 and PCDH10 partially overlaps in some cortical and thalamic areas, which could explain the presence of integrative and converging trans-circuits in these areas (Draganski et al., 2008). PCDH17 Is Localized at Perisynaptic Sites in Basal Ganglia We examined the subcellular localization of PCDH17 in basal ganglia using high-resolution structured illumination microscopy (SIM) to acquire 3D images with resolution approaching 100 nm (Schermelleh et al., 2008). We performed immunostaining of PCDH17 in addition to VGLUT1 and PSD-95, markers of the pre- and postsynaptic compartments of corticostriatal excitatory synapses, respectively. SIM images of the anterior striatum revealed that PCDH17 proteins are present in punctate structures, a subset of which is associated with VGLUT1 and PSD-95 (Figure 3A). 3D reconstructed images showed that PCDH17 is localized next to juxtaposed pairs of VGLUT1 and PSD-95 (Figure 3A). Using stochastic optical reconstruction microscopy (STORM), 842 Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc.
another new, super-resolution imaging technique, distributions of synaptic proteins can be measured with nanometer precision (Dani et al., 2010). Two-color 2D STORM images clearly resolved the PCDH17/VGLUT1 protein distribution and the PCDH17/ PSD-95 protein distribution as adjacent, but discrete molecular structures (Figure 3B). We next used pre-embedding immunogold electron microscopy to analyze PCDH17 ultrastructural localization in asymmetric synapses of MSNs, most of which are thought to be corticostriatal excitatory synapses (Surmeier, et al., 2007). In MSNs of the anterior striatum, immunogold particles indicating PCDH17 localization were observed in presynaptic boutons, dendritic shafts, and dendritic spines, where the majority of particles were attached to the plasma membrane (Figures 3C and 3D). To quantify the distribution of PCDH17 at synapses in detail, we divided each synapse cross-section into central, peripheral, perisynaptic, and extrasynaptic regions, and scored each region for immunogold particles (Nakazawa et al., 2006). Many of the membrane-associated particles were perisynaptically localized at both pre- and postsynaptic sites in an apposed manner (Figures 3C and 3D). Furthermore, in the inner regions of the LGP, 3D-SIM imaging revealed that PCDH17
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
puncta are associated with VGAT and gephyrin, markers of the pre- and postsynaptic compartments of striatopallidal inhibitory synapses, respectively (Figure 3E). STORM imaging also clearly resolved the PCDH17/VGAT protein distribution and the PCDH17/gephyrin protein distribution as neighboring molecular structures (Figure 3F). At the ultrastructural level, PCDH17 particles were mostly located at perisynaptic sites in inhibitory symmetric synapses (Figures 3G and 3H). These findings indicate that PCDH17 is localized in both excitatory and inhibitory perisynaptic sites in basal ganglia nuclei. PCDH17-Mediated Intercellular Homophilic Interaction Given that most cadherin family members exhibit calciumdependent homophilic interactions, we then investigated PCDH17-mediated homophilic interactions using biochemical assays. We prepared a soluble form of the Fc-fused extracellular domain of PCDH17 (PCDH17E-Fc) with independently prepared myc-tagged, full-length PCDH17 (PCDH17-myc) at various Ca2+ concentrations (Figure 4A). Immunoblotting revealed that PCDH17E-Fc interacted with PCDH17-myc in solutions at Ca2+ concentration >1 mM (Figure 4B). This interaction was abolished in the presence of the calcium chelator, EDTA, further supporting the conclusion that the observed homophilic binding is calcium-dependent (Figure 4B). Furthermore, PCDH17 did not exhibit heterophilic interactions with PCDH10 (Figure 4C). The specificity of intercellular interactions of PCDH17 was also examined using CHO cells stably expressing PCDH17 or PCDH10. In a coculture system with these stable cell lines, PCDH17 was localized at contact sites between PCDH17-expressing cells, but not at contact sites with cells expressing PCDH10 (Figure 4D). In addition, we also performed coculture experiments between cortical neurons expressing PCDH17-EGFP and CHO cells expressing either PCDH17myc or PCDH10-myc. A significant portion of PCDH17-EGFP in neurons was localized next to PCDH17-myc in CHO cells at contact points, but not PCDH10-myc in CHO cells (Figure 4E). Taking these results together with the finding that PCDH17 is mainly localized at both excitatory and inhibitory perisynaptic sites (Figure 3), we conclude that PCDH17 mediates homophilic intercellular interactions at synapses in basal ganglia (Figure 4F). PCDH17–/– Mice Show Increased Numbers of Synaptic Vesicles at Presynaptic Terminals in Zone-Specific Basal Ganglia Pathways To examine the physiological role of PCDH17, we generated PCDH17 / mice (Figure S3A). The success of the procedure was confirmed by Southern blot (data not shown) and PCR analysis (Figure S3B). We confirmed the absence of the PCDH17 protein in PCDH17 / mice by immunoblotting and immunostaining (Figures S3C and S3D). The loss of PCDH17 in PCDH17 / mice was also confirmed by immunoelectron microscopy (Figure S3E). Quantitative analysis in the anterior striatum verified a 96% reduction in numbers of immunogold particles in comparison with wild-type mice. The numbers of PCDH17 / mice produced followed a Mendelian segregation pattern and these mice attained normal body size and appeared healthy (data not shown). Histological analysis using Nissl-stained coro-
nal sections from the central nervous system from PCDH17 / mice did not show any gross abnormalities in cytoarchitecture (Figure S3F). In addition, the absence of PCDH17 did not affect the expression of synapse-specific markers, including N-cadherin, Synaptophysin, VGLUT1, PSD-95, NMDA receptor subunits, and AMPA receptor subunits in the anterior and posterior striatum (Figure S3G). We examined whether axonal projections were abrogated in the absence of PCDH17. Immunostaining analyses showed that PCDH17 deficiency did not affect overall axonal projections, including corticothalamic/thalamocortical projections, striatopallidal/striatonigral projections, and nigrostriatal projections (Figures S4A and S4B). Therefore, in contrast to the abnormal axonal projection phenotypes observed in PCDH10 / mice (Uemura et al., 2007), the overall circuitry in basal ganglia appeared to be intact in PCDH17 / mice. We next evaluated whether ablation of PCDH17 affected the topographic connections within the corticobasal ganglia circuits. In retrograde tracing, local injections of CTb-Alexa Fluor 488 into the anterior striatum and CTb-Alexa Fluor 555 into the posterior striatum resulted in the labeled signals in medial prefronatal cortex and motor cortex, respectively, in both wild-type and PCDH17 / mice (Figure S4C). Thus, PCDH17 deficiency did not affect projection topography in corticostriatal pathways. Anterograde tracing using CTb-Alexa Fluor 488 revealed no defects of projection topography of PCDH17 / striatal axons onto output nuclei (Figure S4D). We also found that the PCDH10 expression pattern and PCDH17 promoter-driven b-gal pattern were unaffected in basal ganglia (Figures S4E and S4F). These findings strongly suggested that PCDH17 is not involved in topographic map formation along corticobasal ganglia circuits. We then performed electron microscopic analysis of synaptic morphology in 3-week-old wild-type and PCDH17 / mice. In excitatory asymmetric synapses of the anterior striatum, where PCDH17 expression was evident, PCDH17 deficiency caused significant increases in the number of docked SVs and the total number of SVs per presynaptic terminal, whereas in the posterior striatum where PCDH17 expression was weak, these parameters did not exhibit significant changes (Figures 5A and 5B). Other parameters, such as average postsynaptic spine area, PSD length, synaptic cleft width, and the density of asymmetric synapses, were similar in wild-type and PCDH17 / sections (Figures 5B and 5C). We also investigated inhibitory symmetric synapses of the LGP as output nuclei from the striatum. Increased numbers of docked SVs and total SVs per presynaptic terminal were observed in the inner LGP, but not in the outer LGP after PCDH17 ablation (Figures 5D and 5E). Synaptic cleft width and density of symmetric synapses were similar in both zones (Figures 5E and 5F). There were no changes in synapse densities (Figures 5C and 5F) or the expression of presynaptic proteins in PCDH17 / mice (Figure S3E). Only an increased number of docked SVs and total SVs per presynaptic terminal were observed in PCDH17 / neurons. Taken together, PCDH17 appears to regulate presynaptic SV assembly at both excitatory synapses on MSNs and inhibitory synapses on LGP neurons in each zone-specific region. Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc. 843
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
Figure 3. PCDH17 Is Localized at Perisynaptic Sites in Basal Ganglia (A) 3D SIM imaging for PCDH17, VGLUT1, and PSD-95 in the anterior striatum of P14 mice. (B) STORM imaging for PCDH17/VGLUT1 and PCDH17/PSD-95 in the anterior striatum of P14 mice. (C) Pre-embedding immunogold electron microscopy for PCDH17 at asymmetric synapses in the anterior striatum of P14 mice.
(legend continued on next page)
844 Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc.
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
Overexpression of PCDH17 Results in Diffusion of Synaptic Vesicle Puncta and Increases Mobility of Presynaptic Clusters in Cultured Cortical Neurons To evaluate the role of PCDH17 in SV assembly in vitro, we overexpressed PCDH17 in cultured cortical neurons in concert with synaptophysin-EGFP (Syn-EGFP), which is an SV marker used to monitor SV assembly (Bamji et al., 2003). In the axons of control cells, Syn-EGFP exhibited rounded, punctate clusters. In contrast, Syn-EGFP puncta that were originally associated with PCDH17 puncta became more diffuse in PCDH17-overexpressing neurons (Figures S5A and S5B), suggesting that expression of PCDH17 inhibits the accumulation of SVs. Because inhibition of SV assembly to the presynaptic terminal promotes SV cluster movements (Bamji et al., 2003), we examined whether overexpression of PCDH17 affected the mobility of SV clusters. While Syn-EGFP large puncta were stable or slow-moving in control axons, Syn-EGFP large puncta were relatively mobile and exhibited coordinated movement with PCDH17-mCherry puncta in PCDH17-overexpressing axons (Figures S5C–S5E; Movies S1 and S2). These findings demonstrate that PCDH17 clusters are mobile elements and that PCDH17 overexpression promotes the mobility of SV clusters along axons. PCDH17–/– Mice Exhibit Altered Synaptic Transmission Efficacy at Corticoanterior Striatal Synapses To further characterize the role of PCDH17 in synaptic function, we performed electrophysiological analysis of corticostriatal synapses in acute slices. Since MSNs in the anterior portion of the striatum strongly express PCDH17 (Figures 1D and 1E), we made whole-cell recordings from MSNs in the anterior striatum in wild-type and PCDH17 / mice of about three weeks of age. To assess spontaneous synaptic transmission, we measured miniature excitatory postsynaptic current (mEPSC). Both the frequency and amplitude of mEPSCs in PCDH17 / MSNs were comparable to those in wild-type MSNs (Figure 6A), suggesting that the number of functional synapses is not altered in the absence of PCDH17. We next analyzed the AMPA and NMDA receptor-mediated components of evoked EPSCs. No significant differences were observed in the 10%–90% rise time and the decay time constant of either the AMPA or NMDA receptor-mediated EPSCs between wild-type and PCDH17 / mice (Figure S6A). Furthermore, the AMPA/NMDA ratio was not altered in PCDH17 / mice, compared to wild-type mice (Figure 6B). These results indicate that basic properties of AMPA and NMDA receptors at corticostriatal synapses and their relative contributions to corticostriatal synaptic transmission are not altered in PCDH17 / mice.
To examine possible presynaptic changes in PCDH17 / mice, we next analyzed the paired-pulse ratio of evoked AMPA receptor-mediated EPSCs at a range of interstimulus intervals. We observed that the paired-pulse ratio exhibited a tendency to increase in PCDH17 / mice (Figure 6C). These results would suggest that PCDH17 deficiency may affect presynaptic function at corticostriatal synapses. However, post-hoc tests did not reveal significant difference between genotypes at any pulse interval. To test whether presynaptic function of GABAergic inhibitory synapses was altered in PCDH17 / mice, we analyzed the paired-pulse ratio of evoked inhibitory postsynaptic currents (IPSCs) at anterior striatal-LGP synapses. We made whole-cell recordings from neurons in the inner portion of the LGP where PCDH17 was strongly expressed (Figures 1D and 1E) and stimulated the corresponding portion of the anterior striatum. We found that the paired-pulse ratio of IPSCs was significantly increased in PCDH17 / mice at inter-pulse interval of 50 ms (Figure S6B), although basic properties of GABA receptors were not changed (Figure S6A). Taken together, these results suggest that PCDH17 would be important for the presynaptic function in both excitatory and inhibitory synapses in the basal ganglia. We then assessed the recycling process of SVs in presynaptic terminals by measuring synaptic depression induced by prolonged repetitive stimulation. Synaptic depression is reported to reflect a presynaptic cycling process in which depleted docked vesicles are replenished by reserve pool vesicles (Bamji et al., 2003; Cabin et al., 2002). We applied repetitive stimulation (10 Hz, 200 pulses) to corticostriatal fibers and measured the amplitude of AMPA receptor-mediated EPSCs relative to that of the first response (Figure 6D). Since repetitive stimulation can release endocannabinoid 2-arachidonoyl glycerol from MSNs (Kano et al., 2009; Maejima et al., 2005), we applied a cannabinoid CB1 receptor antagonist to block the endocannabinoid-mediated retrograde suppression of corticostriatal synapses and to evaluate unadulterated presynaptic function. In wild-type and PCDH17 / mice at P21–P23, the normalized EPSC amplitude decreased gradually during prolonged repetitive stimulation, but the depression of the normalized EPSC amplitude was significantly weaker in PCDH17 / mice (Figure 6D). There was no significant difference in average amplitudes of the first ten EPSCs between wild-type and PCDH17 / mice (Figure S6A). Since the numbers of total SVs was increased in PCDH17 / synapses at three weeks of age (Figures 5A and 5B), the weaker synaptic depression following repetitive stimulation is thought to result from the increased number of vesicles available for release during enhanced activity. In contrast, the extent of synaptic depression was
(D) (Left) The mean number of immunoparticles for PCDH17 per 1 mm of plasma membrane of presynaptic terminals, spines, and dendrites in the anterior striatum. (Right) Histogram for the tangential distribution of PCDH17 at asymmetric synapses. (E) 3D SIM imaging for PCDH17, VGAT, and gephyrin in the inner LGP of P14 mice. (F) STORM imaging for PCDH17/VGAT and PCDH17/gephyrin in the inner LGP of P14 mice. (G) Pre-embedding immunogold electron microscopy for PCDH17 at symmetric synapses in the inner LGP of P14 mice. (H) (Left) The mean number of immunoparticles for PCDH17 per 1 mm of the plasma membrane of presynaptic terminals and dendrites. (Right) Histogram for the tangential distribution of PCDH17 at symmetric synapses. Boxed regions in (A) and (E) are enlarged on the right; orthogonal views of them are shown to the right and bottom. (D and H) The length of analyzed plasma membrane is indicated in parentheses. The red bar indicates presynaptic distribution and the blue bar indicates postsynaptic distribution of PCDH17. The gray shadow indicates the interior of synapses. Scale bars represent 2 mm (A and E) and 200 nm (B, C, F, and G). Dn, dendrite (blue); Sp, dendritic spine (blue); t, presynaptic terminal (red).
Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc. 845
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
Figure 4. PCDH17-Mediated Intercellular Homophilic Interaction (A–C) Fc pull-down assays. (A) Diagram of PCDH17E-Fc, PCDH17-myc and PCDH10-myc. (B) Lysates of HEK293T cells expressing PCDH17-myc were subjected to pull-down assays using PCDH17E-Fc in buffer containing the indicated concentrations of CaCl2 and EDTA (1 mM). (C) Lysates of HEK293T cells expressing mock plasmids, PCDH10-myc and PCDH17-myc, were subjected to pull-down assays with PCDH17E-Fc in buffer containing 1 mM CaCl2. Amounts of bound PCDH-myc, total PCDH-myc and loading PCDH17E-Fc were analyzed by immunoblotting. (D) Immunofluorescence for PCDH17-myc and PCDH10-myc in confluent CHO cells stably expressing each protocadherin (left) and coculture of two stable CHO cell lines (right). (E) (Upper) Immunofluorescence for PCDH17-EGFP in 14 days in vitro (DIV) cortical neurons and PCDH17-myc (left) or PCDH10-myc (right) in CHO cells after co-culture. Dashed white lines indicate the border of CHO cells. Higher-magnification images of PCDH17-EGFP signals are indicated on the bottom. (Lower) Quantification of PCDH17-EGFP puncta in neurons that indicate close localization to PCDH17-myc or PCDH10-myc in CHO cells. At least 100 puncta were counted for each condition from three separate cultures. (F) Schematic representation of PCDH17 mediation of homophilic binding at an excitatory synapse and an inhibitory synapse. Scale bars represent 20 mm (D) and 10 mm (E). Error bars indicate SEM. **p < 0.01; Student’s t test.
similar in wild-type and PCDH17 / mice at P16–P18 (Figure S6C), suggesting that PCDH17’s regulation of synaptic depression is developmental stage-dependent. Taken together, these electrophysiological data suggest that overall synaptic transmission efficacy is enhanced at anterior corticostriatal excitatory synapses in PCDH17 / mice. PCDH17–/– Mice Exhibit Antidepressant-like Behaviors To examine behavioral abnormalities in PCDH17 / mice, we performed a battery of behavioral tests for evaluating sensory 846 Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc.
and motor functions, cognition, anxiety, and depression. We employed the tail suspension test and the forced swim test, which are widely used for assessing antidepressant-like activity in mice. In both tests, PCDH17 / mice were less immobile than wild-type mice (Figures 7A and 7B), suggesting that PCDH17 / mice were less susceptible to depression than wild-type mice. Alternatively, the reduced immobility of PCDH17 / mice in these tests might have been caused by an increase in spontaneous activity (Cryan and Holmes, 2005). To check this possibility, we performed the open field test to measure
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
spontaneous locomotor activity of PCDH17 / mice. The results showed that there were no significant differences between wildtype and PCDH17 / mice in terms of immobility time, total distance traveled, or the amount of rearing activity observed during the test (Figure 7C), suggesting that spontaneous activity was normal in PCDH17 / mice. As alteration in fearfulness can also affect performance in tests for depression (Cryan and Holmes, 2005), we also analyzed anxiety-related behavior. We did not detect any significant abnormalities in PCDH17 / mice in terms of ‘‘time spent in center areas’’ in the open field test, ‘‘time spent in open arms’’ in the elevated plus-maze test, or ‘‘time spent in the light box’’ in the light-dark transition test (Figures 7C–7E), suggesting that the anxiety level of PCDH17 / mice is normal. Performance of PCDH17 / mice also appeared normal in the contextual fear conditioning test, auditory fear conditioning test, acoustic startle response test, prepulse inhibition test, and tail-flick test (Figure S7). Collectively, PCDH17 / mice showed reduced susceptibility to depression, but other general behaviors, such as locomotor activity, anxiety behavior, fear learning, startle response, and pain behavior were normal. PCDH17 Is Topographically Expressed in Corticobasal Ganglia Circuits of Primates To better understand the possible functional role of PCDH17 in human depressive disorders, it is important to analyze its expression in primate brain. Here, we examined PCDH17 protein expression in corticobasal ganglia circuits of infant rhesus monkeys by immunostaining. Intense PCDH17 immunoreactivity was generally observed in the frontal lobe and the striatum, although the regional density differed among cortical areas or striatal sectors (Figure 8A). In the frontal lobe, PCDH17 signals were strong in the medial prefrontal cortex (area 32), the rostral part of the anterior cingulate cortex (area 24), and the medial part of the dorsolateral prefrontal cortex (area 9). PCDH17 signals were of intermediate strength in other prefrontal areas (such as areas 46 and 11) and the motor-related areas (such as areas 6 and 4). By contrast, PCDH17 immunoreactivity was weak in the other (parietal and temporal) cortical areas that include the somatosensory areas (areas 3 and 40) (Figures 8A and 8B). Throughout the cortex, PCDH17 signals were apparent in layers V and VI (Figure 8B). Thus, cortical PCDH17 expression was rather specific to the frontal lobe, with a rostrocaudal gradient. Likewise, PCDH17 immunoreactivity in the striatum was found in both the caudate nucleus and the putamen with a clear rostrocaudal gradient (Figure 8A). In addition, PCDH17 expression occurred in the external and internal segments of the globus pallidus and the substantia nigra in a topographic manner (Figure S8). These results indicate that the overall expression pattern of PCDH17 in primates is largely consistent with that in mice. DISCUSSION Topographically parallel organization is essential for information processing along corticobasal ganglia circuits. In this study, we showed that PCDH17 and PCDH10 display spatially complementary expression patterns along corticobasal ganglia circuits,
suggesting that the expression of these protocadherins reflects the topographic organization of the pathway. Then, using PCDH17 / mice, we demonstrated that PCDH17 regulates presynaptic vesicle assembly and synaptic transmission efficacy in corticostriatal pathways. Finally, we found that PCDH17 / mice display less depression-like behavior, and that they manifested normal sensorimotor and cognitive functions and anxiety level, suggesting that PCDH17 is specifically involved in depressionrelated behavior. Based on neuroanatomical and neuroimaging studies in primates, an anteroposterior gradient of corticostriatal connections has been proposed (Draganski et al., 2008). Specifically, neurons in prefrontal cortical areas, including the medial prefrontal cortex, project to anterior regions of the striatum, sensory, and motor cortical neurons to central dorsolateral regions, and parietal cortical neurons to more caudal regions, forming the limbic and associative loops, the sensorimotor loop, and the perceptual loop, respectively. Because PCDH17 was mostly expressed along the medial prefrontal cortex-anterior striatal pathways in a topographic manner, in both rodents and primates, it is likely that PCDH17 is anatomically and functionally conserved in the corticobasal ganglia circuits of higher primates as well. Therefore, the PCDH17-expressing neuronal pathway could correspond to the prefrontal cortical loops in primates used for processing some aspects of motivational and executive functions. As the complementary expression patterns of PCDH17 and PCDH10 appear at E14.5 and gradually develop in the embryonic mouse striatum until birth (our unpublished data), we assumed that the expression of these protocadherins was reciprocally regulated by positional information in the embryonic striatum. Nevertheless, PCDH17 is dispensable in this topographic map formation (Figure S4), although it has crucial roles in the synaptic development of this pathway. In addition to these protocadherins, some axon guidance molecules that are involved in topographic map formation in the visual and olfactory systems (Luo and Flanagan, 2007; Sakano, 2010), are also expressed in the embryonic striatum in a zone-specific manner. Like PCDH17, Netrin-1 exhibits an expression pattern with a high-anterior to low-posterior gradient in embryonic striatal regions (Powell et al., 2008). In contrast, similar to PCDH10, Ephrin-A5 and Semaphorin-3A exhibit expression patterns with low-anterior to high-posterior gradients (Dufour et al., 2003; Wright et al., 2007). Therefore, these axon guidance molecules might organize the topographic map delineated by PCDH17 and PCDH10 expression in the embryonic basal ganglia. PCDH10 / mice exhibit axonal growth defects in the striatum and die within the first several weeks after birth (Uemura et al., 2007), whereas PCDH17 / mice do not die prematurely and are not characterized by abnormal striatal axonal growth. These phenotypic differences may be at least partially attributable to different protein distributions in embryonic striatal fibers; PCDH10 (Uemura et al., 2007), but not PCDH17 (our unpublished data), is distributed around striatal fibers at E14.5. It should be noted that similar to that of PCDH17, the expression of PCDH10 peaks during early synaptogenesis. A recent paper showed that PCDH10 is required for activity-dependent Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc. 847
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
(legend on next page)
848 Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc.
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
synapse elimination in cultured neurons (Tsai et al., 2012). Therefore, PCDH10 may function not only in axonal growth, but also in synaptic development of corticobasal ganglia circuits. Conditional depletion of PCDH10 would be informative in elucidating its possible roles in synaptic development in that circuit. What are the molecular mechanisms by which PCDH17 regulates SV assembly in developmental synapses? One report showed that homophilic interactions of PCDH8, in cis or trans, decrease dendritic spine density (Yasuda et al., 2007). Evidence that PCDH17 mediates intercellular homophilic interactions and is localized at perisynaptic sites may imply that homophilic interactions of PCDH17 regulate SV assembly in presynaptic terminals, although the cellular and molecular mechanisms need to be clarified. Several lines of evidence demonstrate that the N-cadherin-b-catenin adhesion complex plays a central role in recruiting SVs to presynaptic terminals (Arikkath and Reichardt, 2008). SV clusters are surrounded by actin filaments, suggesting that localization of SVs is dependent upon F-actin (Bamji 2005). Thus, the N-cadherin-b-catenin complex and its associated F-actin regulation are thought to play a part in presynaptic SV assembly. Given that some d-protocadherin members, such as PCDH8 and PCDH10, act as negative regulators of N-cadherin (Nakao et al., 2008; Yasuda et al., 2007), it is likely that PCDH17 also perturbs the function of the N-cadherin-b-catenin complex and inhibits SV assembly forces. Furthermore, as the cytoplasmic domain of PCDH17, like that of PCDH10 and PCDH19, interacts with the WAVE complex (our unpublished data; Nakao et al., 2008; Tai et al., 2010), PCDH17-WAVE complex machinery might affect the N-cadherin-b-catenin complex and its associated actin cytoskeleton, resulting in delocalization of SVs in presynaptic terminals. Further detailed analyses are required for clarification of the role of PCDH17 in SV assembly. The abundance and localization of presynaptic SVs are critical for regulation of synaptic physiology. In N-cadherin and b-catenin knockout synapses, the response with respect to the EPSC amplitude during repetitive stimulation is significantly smaller, suggesting that the cadherin-catenin complex positively regulates the availability of SVs for release during high activity (Bamji et al., 2003; Ju¨ngling, et al., 2006). Considering the possible negative regulation of N-cadherin by d-protocadherins (Nakao et al., 2008; Yasuda et al., 2007), increased numbers of SVs in PCDH17 knockout synapses could contribute to the ready availability of SVs for neurotransmitter release. This idea is supported by our electrophysiological data that PCDH17 knockout synapses exhibited less
synaptic depression following repetitive stimulation of input fibers. It is assumed that paired-pulse depression is affected by SV transitions in the pools as well as by neurotransmitter release probability (Regehr, 2012). It might be possible that PCDH17 deficiency decreases paired-pulse depression as a result of higher vesicle replenishment into release sites. Although it is also possible that the presynaptic release probability is changed at PCDH17 knockout synapses, the precise roles of PCDH17 in presynapses need to be analyzed in future studies. In addition to its regulatory role in presynaptic function, PCDH17 may have additional roles in postsynaptic function considering the both pre- and postsynaptic localization of PCDH17. Our observation that loss of PCDH17 affects depressionrelated behaviors might suggest that altered synaptic function in the aforementioned PCDH17-expressing corticobasal ganglia circuits could play an important role in depressive behaviors. Accordingly, dysregulated functional activity within an extended network, including medial prefrontal cortex and striatum, is a key symptom of depression in humans (Krishnan and Nestler, 2008; Price and Drevets, 2012). Optogenetic stimulation of the medial prefrontal cortex-mediated pathways in rodents is reported to control depression-related behaviors (Covington et al., 2010; Warden et al., 2012). Furthermore, our hypothesis may be supported by evidence that PCDH17 is strongly expressed in the primate prefrontal cortical area and associated regions that are most crucial for depression. Although PCDH17 was also expressed in amygdala, hypothalamus, and other mesolimbic areas, future studies with neural pathway-specific PCDH17 conditional knockout mice could clarify the possible relationship between topographic corticobasal ganglia circuits and depression-related behaviors. Moreover, it will be of considerable importance to search for mutations in PCDH17 in human mood disorders. EXPERIMENTAL PROCEDURES Detailed experimental procedures are provided in the Supplemental Information. Experiments were conducted according to the institutional ethical guidelines for animal experiments. Generation of PCDH17–/– Mice Details can be found in Supplemental Experimental Procedures.
Intracranial Surgery Intracranial surgery was performed as previously described (Fukabori et al., 2012).
Figure 5. Increased Synaptic Vesicle Number at Presynaptic Terminals in the Topographic Basal Ganglia Pathways of PCDH17–/– Mice (A) Electron microscopic images of the anterior and posterior striatum of 3-week-old PCDH17+/+ and PCDH17 / mice. (B) Quantification of asymmetric synapses (docked SV number, total SV number, synaptic cleft, spine area, and PSD length) in the striatum. At least 240 synapses were counted per genotype from three mouse pairs. (C) Quantification of synapse number in 1,300 mm2 of the striatum per genotype from three mouse pairs. (D) Electron microscopic images of the inner and outer LGP of 3-week-old PCDH17+/+ and PCDH17 / mice. (E) Quantification of symmetric synapses (docked SV number, total SV number, synaptic cleft). At least 240 synapses were counted per genotype from three mouse pairs. (F) Quantification of synapse number in 1,300 mm2 of the LGP for each genotype from three mouse pairs. Scale bars represent 200 nm. Error bars indicate SEM. **p < 0.01; ***p < 0.001; Student’s t test; SV, synaptic vesicle. See also Figures S3, S4, and S5.
Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc. 849
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
Figure 6. Altered Short-Term Synaptic Plasticity at Corticoanterior Striatal Synapses in PCDH17–/– Mice (A) (Left) Representative traces of mEPSCs from anterior MSNs of 3-week-old PCDH17+/+ and PCDH17 / mice. (Right) Summary bar graphs showing average mEPSC amplitude and frequency (n = 13 for PCDH17+/+ and n = 14 for PCDH17 / mice). (B) (Left) Representative traces of AMPA and NMDA receptor-mediated EPSCs from anterior MSNs elicited by stimulation of corticostriatal afferents in 3-week-old PCDH17+/+ and PCDH17 / mice. AMPA and NMDA EPSCs were recorded at a holding potential of 70 mV and +50 mV, respectively. (Right) Summary bar graph showing ratios of NMDA to AMPA EPSC amplitudes (n = 25 for PCDH17+/+ and n = 23 for PCDH17 / mice). Amplitudes of AMPA EPSCs were measured at their peaks (leftward arrowheads), whereas those of NMDA EPSCs were measured at 60 ms from the stimuli (downward arrowheads). (C) (Left) Representative traces of AMPA receptormediated EPSCs to paired stimuli of 70 ms interval from anterior MSNs of 3-week-old PCDH17+/+ and PCDH17 / mice. (Right) Summary graph showing ratios of second to first EPSC amplitudes as a function of interstimulus interval (20, 30, 50 70, 100, and 200 ms) (n = 25 for PCDH17+/+ and n = 29 for PCDH17 / mice). There was a significant main effect of genotype (p < 0.01; two-way ANOVA), although post-hoc tests did not reveal significant difference between genotypes at any pulse interval. (D) (Upper) Representative traces of EPSCs from anterior MSNs of 3-week-old PCDH17+/+ and PCDH17 / mice during prolonged repetitive stimulation (10 Hz, 200 pulses). Only responses 1–3 (black) and 51–53 (red) are shown. (Lower) Summary graph showing average normalized response amplitudes of EPSCs (n = 12 for PCDH17+/+ and n = 9 for PCDH17 / mice). Each point represents the average of 10 consecutive responses. Calibration bars = 10 pA and 200 ms (A), 0.4 nA and 20 ms (B), 0.1 nA and 20 ms (C), 0.1 nA and 50 ms (D). Error bars indicate SEM. *p < 0.05; Mann-Whitney U-test (A and B), two-way repeated-measures ANOVA (D). See also Figure S6.
850 Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc.
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
Figure 7. Antidepression-like Behaviors in PCDH17–/– Mice (A) Tail suspension test. (Left) Immobility time was measured for 6 min. (Right) Cumulative immobility time during the last 4 min of the 6 min test session (n = 8 for PCDH17+/+ and n = 12 for PCDH17 / mice). (B) Forced swim test. (Left) Immobility time was measured for 10 min. (Right) Cumulative immobility time during the last 6 min of the 10 min test session (n = 19 for PCDH17+/+ and n = 21 for PCDH17 / mice). (C) Open field test. Immobility time, total distance traveled, rearing number and time spent in center areas were measured (n = 8 for PCDH17+/+ and n = 12 for PCDH17 / mice). (D) Elevated plus maze test. Time spent per compartment (open arms [O] or closed arms [C]) was measured (n = 8 for PCDH17+/+ and n = 12 for PCDH17 / mice). (E) Light-dark emergence test. Time spent per compartment (lighted areas [L] or dark areas [D]) was measured (n = 11 for PCDH17+/+ and n = 9 for PCDH17 / mice). Error bars indicate SEM. *p < 0.05; Student’s t test. See also Figure S7. Cell Culture, DNA Transfection, Stable Transfectants, Coculture Experiments, and Immunocytochemistry Neuronal culture was performed as previously described (Nakazawa et al., 2008). Details can be found in Supplemental Experimental Procedures.
Nissl staining, and immunohistochemistry in rhesus monkey brain were basically performed as described (Dani et al., 2010; Lu et al., 2012; Takeuchi et al., 2010; Taniguchi et al., 2009; Yamasaki et al., 2010).
Fc Pull-Down Assay The Fc pull-down assay was performed as previously described (Kazmierczak et al., 2007).
Time-Lapse Live Imaging Time-lapse imaging analysis was performed as previously described (Oshimori et al., 2009).
Histology X-gal staining, fluorescent in situ hybridization, immunohistochemistry, STORM imaging, pre-embedding immunogold electron microscopy,
Electron Microscopy Transmission electron microscopy analysis was performed as previously described (Goto et al., 2008).
Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc. 851
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
Figure 8. Expression Pattern of PCDH17 in Monkey Brain Immunostaining for PCDH17 in coronal sections taken from a 2-month-old rhesus monkey. Each number indicates Brodmann’s area. (A) Overview of PCDH17 expression along the rostrocaudal axis. (B) Higher-magnification images of cortical areas specified in (A). Scale bars represent 5 mm (A) and 0.5 mm (B). Cd, caudate nucleus; GPe, external segment of the globus pallidus; GPi, internal segment of the globus pallidus; HTh, hypothalamus; NA, nucleus accumbens; Put, putamen; Th, thalamus; WH, white matter; 3, primary somatosensory cortex (S1); 4, primary motor cortex (M1); 6, premotor cortex (PM); 9, 46, dorsolateral prefrontal cortex (DLPFC); 11, orbitofrontal cortex (OFC); 24, anterior cingulate cortex (ACC); 32, medial prefrontal cortex (mPFC); 40, secondary somatosensory cortex (S2). See also Figure S8.
Electrophysiology Whole-cell patch-clamp recordings were performed as previously described (Tanimura et al., 2010).
ACKNOWLEDGMENTS
helpful discussions regarding immunohistochemistry. Y. Kiyama and T. Manabe offered valuable insights on behavioral tests. Y. Oikawa and K. Takatsuka provided technical support on superresolution imaging. T. Nakano and J.R. Whickens advised us on stereotaxic surgery. T. Abe and S. Aizawa produced the PCDH17 / mice. J. Miyazaki supplied CAGCre transgenic mice. T. Akagi furnished the pCX4-bsr vector. R.F. Whittier and S.D. Aird gave the manuscript a critical reading, and members of our laboratory offered valuable comments. This work was supported by Grants-in-Aid for Scientific Research 23700411 (N.H.), 20220006 (M.T.), 19100005 (M.W.), 21220006 (M.K.), and 17013021 and 19390070 (T.Y.), the Strategic Research Program for Brain Sciences (Development of Biomarker Candidates for Social Behavior), the Comprehensive Brain Science Network (Development of Molecular Profiling of Brain), and the Global COE Program (Integrative Life Science Based on the Study of Biosignaling Mechanisms) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
We acknowledge the assistance of the following individuals and express our gratitude for their support. H. Takeuchi and H. Sakano provided advice and
Accepted: March 28, 2013 Published: May 16, 2013
Behavioral Tests All behavioral experiments were performed as blind tests. Male mice, 7–9 weeks of age, were analyzed for all experiments as previously described (Taniguchi et al., 2009). SUPPLEMENTAL INFORMATION Supplemental Information includes eight figures, two movies, and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10.1016/j.neuron.2013.03.031.
852 Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc.
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
REFERENCES Alexander, G.E., and Crutcher, M.D. (1990). Functional architecture of basal ganglia circuits: neural substrates of parallel processing. Trends Neurosci. 13, 266–271. Angelucci, A., Clasca´, F., and Sur, M. (1996). Anterograde axonal tracing with the subunit B of cholera toxin: a highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains. J. Neurosci. Methods 65, 101–112. Aoki, E., Kimura, R., Suzuki, S.T., and Hirano, S. (2003). Distribution of OL-protocadherin protein in correlation with specific neural compartments and local circuits in the postnatal mouse brain. Neuroscience 117, 593–614. Arikkath, J., and Reichardt, L.F. (2008). Cadherins and catenins at synapses: roles in synaptogenesis and synaptic plasticity. Trends Neurosci. 31, 487–494. Bamji, S.X. (2005). Cadherins: actin with the cytoskeleton to form synapses. Neuron 47, 175–178. Bamji, S.X., Shimazu, K., Kimes, N., Huelsken, J., Birchmeier, W., Lu, B., and Reichardt, L.F. (2003). Role of beta-catenin in synaptic vesicle localization and presynaptic assembly. Neuron 40, 719–731. Cabin, D.E., Shimazu, K., Murphy, D., Cole, N.B., Gottschalk, W., McIlwain, K.L., Orrison, B., Chen, A., Ellis, C.E., Paylor, R., et al. (2002). Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking alpha-synuclein. J. Neurosci. 22, 8797–8807. Covington, H.E., 3rd, Lobo, M.K., Maze, I., Vialou, V., Hyman, J.M., Zaman, S., LaPlant, Q., Mouzon, E., Ghose, S., Tamminga, C.A., et al. (2010). Antidepressant effect of optogenetic stimulation of the medial prefrontal cortex. J. Neurosci. 30, 16082–16090. Cryan, J.F., and Holmes, A. (2005). The ascent of mouse: advances in modelling human depression and anxiety. Nat. Rev. Drug Discov. 4, 775–790.
Kazmierczak, P., Sakaguchi, H., Tokita, J., Wilson-Kubalek, E.M., Milligan, R.A., Mu¨ller, U., and Kachar, B. (2007). Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells. Nature 449, 87–91. Kim, S.Y., Chung, H.S., Sun, W., and Kim, H. (2007). Spatiotemporal expression pattern of non-clustered protocadherin family members in the developing rat brain. Neuroscience 147, 996–1021. Krishnan, V., and Nestler, E.J. (2008). The molecular neurobiology of depression. Nature 455, 894–902. Lu, X., Miyachi, S., and Takada, M. (2012). Anatomical evidence for the involvement of medial cerebellar output from the interpositus nuclei in cognitive functions. Proc. Natl. Acad. Sci. USA 109, 18980–18984. Luo, L., and Flanagan, J.G. (2007). Development of continuous and discrete neural maps. Neuron 56, 284–300. Maejima, T., Oka, S., Hashimotodani, Y., Ohno-Shosaku, T., Aiba, A., Wu, D., Waku, K., Sugiura, T., and Kano, M. (2005). Synaptically driven endocannabinoid release requires Ca2+-assisted metabotropic glutamate receptor subtype 1 to phospholipase C b4 signaling cascade in the cerebellum. J. Neurosci. 25, 6826–6835. McCracken, C.B., and Grace, A.A. (2009). Nucleus accumbens deep brain stimulation produces region-specific alterations in local field potential oscillations and evoked responses in vivo. J. Neurosci. 29, 5354–5363. McFarland, N.R., and Haber, S.N. (2002). Thalamic relay nuclei of the basal ganglia form both reciprocal and nonreciprocal cortical connections, linking multiple frontal cortical areas. J. Neurosci. 22, 8117–8132. Nakao, S., Platek, A., Hirano, S., and Takeichi, M. (2008). Contact-dependent promotion of cell migration by the OL-protocadherin-Nap1 interaction. J. Cell Biol. 182, 395–410.
Dani, A., Huang, B., Bergan, J., Dulac, C., and Zhuang, X. (2010). Superresolution imaging of chemical synapses in the brain. Neuron 68, 843–856.
Nakazawa, T., Komai, S., Watabe, A.M., Kiyama, Y., Fukaya, M., ArimaYoshida, F., Horai, R., Sudo, K., Ebine, K., Delawary, M., et al. (2006). NR2B tyrosine phosphorylation modulates fear learning as well as amygdaloid synaptic plasticity. EMBO J. 25, 2867–2877.
Draganski, B., Kherif, F., Klo¨ppel, S., Cook, P.A., Alexander, D.C., Parker, G.J., Deichmann, R., Ashburner, J., and Frackowiak, R.S. (2008). Evidence for segregated and integrative connectivity patterns in the human Basal Ganglia. J. Neurosci. 28, 7143–7152.
Nakazawa, T., Kuriu, T., Tezuka, T., Umemori, H., Okabe, S., and Yamamoto, T. (2008). Regulation of dendritic spine morphology by an NMDA receptorassociated Rho GTPase-activating protein, p250GAP. J. Neurochem. 105, 1384–1393.
Dufour, A., Seibt, J., Passante, L., Depaepe, V., Ciossek, T., Frise´n, J., Kullander, K., Flanagan, J.G., Polleux, F., and Vanderhaeghen, P. (2003). Area specificity and topography of thalamocortical projections are controlled by ephrin/Eph genes. Neuron 39, 453–465.
Oshimori, N., Li, X., Ohsugi, M., and Yamamoto, T. (2009). Cep72 regulates the localization of key centrosomal proteins and proper bipolar spindle formation. EMBO J. 28, 2066–2076.
Fukabori, R., Okada, K., Nishizawa, K., Kai, N., Kobayashi, K., Uchigashima, M., Watanabe, M., Tsutsui, Y., and Kobayashi, K. (2012). Striatal direct pathway modulates response time in execution of visual discrimination. Eur. J. Neurosci. 35, 784–797. Goda, Y., and Davis, G.W. (2003). Mechanisms of synapse assembly and disassembly. Neuron 40, 243–264. Goto, J., Tezuka, T., Nakazawa, T., Sagara, H., and Yamamoto, T. (2008). Loss of Fyn tyrosine kinase on the C57BL/6 genetic background causes hydrocephalus with defects in oligodendrocyte development. Mol. Cell. Neurosci. 38, 203–212. Hertel, N., Krishna-K, Nuernberger, M., and Redies, C. (2008). A cadherinbased code for the divisions of the mouse basal ganglia. J. Comp. Neurol. 508, 511–528. Hopf, F.W., Waters, J., Mehta, S., and Smith, S.J. (2002). Stability and plasticity of developing synapses in hippocampal neuronal cultures. J. Neurosci. 22, 775–781. Ju¨ngling, K., Eulenburg, V., Moore, R., Kemler, R., Lessmann, V., and Gottmann, K. (2006). N-cadherin transsynaptically regulates short-term plasticity at glutamatergic synapses in embryonic stem cell-derived neurons. J. Neurosci. 26, 6968–6978. Kano, M., Ohno-Shosaku, T., Hashimotodani, Y., Uchigashima, M., and Watanabe, M. (2009). Endocannabinoid-mediated control of synaptic transmission. Physiol. Rev. 89, 309–380.
Powell, A.W., Sassa, T., Wu, Y., Tessier-Lavigne, M., and Polleux, F. (2008). Topography of thalamic projections requires attractive and repulsive functions of Netrin-1 in the ventral telencephalon. PLoS Biol. 6, e116. Price, J.L., and Drevets, W.C. (2012). Neural circuits underlying the pathophysiology of mood disorders. Trends Cogn. Sci. 16, 61–71. Redgrave, P., Rodriguez, M., Smith, Y., Rodriguez-Oroz, M.C., Lehericy, S., Bergman, H., Agid, Y., DeLong, M.R., and Obeso, J.A. (2010). Goal-directed and habitual control in the basal ganglia: implications for Parkinson’s disease. Nat. Rev. Neurosci. 11, 760–772. Redies, C. (2000). Cadherins in the central nervous system. Prog. Neurobiol. 61, 611–648. Redies, C., Vanhalst, K., and Roy, Fv. (2005). d-Protocadherins: unique structures and functions. Cell. Mol. Life Sci. 62, 2840–2852. Regehr, W.G. (2012). Short-term presynaptic plasticity. Cold Spring Harb. Perspect. Biol. 4, a005702. Sakano, H. (2010). Neural map formation in the mouse olfactory system. Neuron 67, 530–542. Schermelleh, L., Carlton, P.M., Haase, S., Shao, L., Winoto, L., Kner, P., Burke, B., Cardoso, M.C., Agard, D.A., Gustafsson, M.G., et al. (2008). Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320, 1332–1336. Shapiro, L., and Colman, D.R. (1999). The diversity of cadherins and implications for a synaptic adhesive code in the CNS. Neuron 23, 427–430.
Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc. 853
Neuron Protocadherin 17 in Corticobasal Ganglia Circuits
Simpson, E.H., Kellendonk, C., and Kandel, E. (2010). A possible role for the striatum in the pathogenesis of the cognitive symptoms of schizophrenia. Neuron 65, 585–596.
Uemura, M., Nakao, S., Suzuki, S.T., Takeichi, M., and Hirano, S. (2007). OL-Protocadherin is essential for growth of striatal axons and thalamocortical projections. Nat. Neurosci. 10, 1151–1159.
Stan, A., Pielarski, K.N., Brigadski, T., Wittenmayer, N., Fedorchenko, O., Gohla, A., Lessmann, V., Dresbach, T., and Gottmann, K. (2010). Essential cooperation of N-cadherin and neuroligin-1 in the transsynaptic control of vesicle accumulation. Proc. Natl. Acad. Sci. USA 107, 11116–11121.
Utter, A.A., and Basso, M.A. (2008). The basal ganglia: an overview of circuits and function. Neurosci. Biobehav. Rev. 32, 333–342.
Surmeier, D.J., Ding, J., Day, M., Wang, Z., and Shen, W. (2007). D1 and D2 dopamine-receptor modulation of striatal glutamatergic signaling in striatal medium spiny neurons. Trends Neurosci. 30, 228–235. Tai, K., Kubota, M., Shiono, K., Tokutsu, H., and Suzuki, S.T. (2010). Adhesion properties and retinofugal expression of chicken protocadherin-19. Brain Res. 1344, 13–24. Takada, M., Tokuno, H., Hamada, I., Inase, M., Ito, Y., Imanishi, M., Hasegawa, N., Akazawa, T., Hatanaka, N., and Nambu, A. (2001). Organization of inputs from cingulate motor areas to basal ganglia in macaque monkey. Eur. J. Neurosci. 14, 1633–1650. Takeuchi, H., Inokuchi, K., Aoki, M., Suto, F., Tsuboi, A., Matsuda, I., Suzuki, M., Aiba, A., Serizawa, S., Yoshihara, Y., et al. (2010). Sequential arrival and graded secretion of Sema3F by olfactory neuron axons specify map topography at the bulb. Cell 141, 1056–1067. Taniguchi, S., Nakazawa, T., Tanimura, A., Kiyama, Y., Tezuka, T., Watabe, A.M., Katayama, N., Yokoyama, K., Inoue, T., Izumi-Nakaseko, H., et al. (2009). Involvement of NMDAR2A tyrosine phosphorylation in depressionrelated behaviour. EMBO J. 28, 3717–3729. Tanimura, A., Yamazaki, M., Hashimotodani, Y., Uchigashima, M., Kawata, S., Abe, M., Kita, Y., Hashimoto, K., Shimizu, T., Watanabe, M., et al. (2010). The endocannabinoid 2-arachidonoylglycerol produced by diacylglycerol lipase alpha mediates retrograde suppression of synaptic transmission. Neuron 65, 320–327. Togashi, H., Abe, K., Mizoguchi, A., Takaoka, K., Chisaka, O., and Takeichi, M. (2002). Cadherin regulates dendritic spine morphogenesis. Neuron 35, 77–89. Tsai, N.P., Wilkerson, J.R., Guo, W., Maksimova, M.A., DeMartino, G.N., Cowan, C.W., and Huber, K.M. (2012). Multiple autism-linked genes mediate synapse elimination via proteasomal degradation of a synaptic scaffold PSD-95. Cell 151, 1581–1594.
854 Neuron 78, 839–854, June 5, 2013 ª2013 Elsevier Inc.
Warden, M.R., Selimbeyoglu, A., Mirzabekov, J.J., Lo, M., Thompson, K.R., Kim, S.Y., Adhikari, A., Tye, K.M., Frank, L.M., and Deisseroth, K. (2012). A prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge. Nature 492, 428–432. Wiesendanger, E., Clarke, S., Kraftsik, R., and Tardif, E. (2004). Topography of cortico-striatal connections in man: anatomical evidence for parallel organization. Eur. J. Neurosci. 20, 1915–1922. Williams, M.E., de Wit, J., and Ghosh, A. (2010). Molecular mechanisms of synaptic specificity in developing neural circuits. Neuron 68, 9–18. Williams, M.E., Wilke, S.A., Daggett, A., Davis, E., Otto, S., Ravi, D., Ripley, B., Bushong, E.A., Ellisman, M.H., Klein, G., and Ghosh, A. (2011). Cadherin-9 regulates synapse-specific differentiation in the developing hippocampus. Neuron 71, 640–655. Wright, A.G., Demyanenko, G.P., Powell, A., Schachner, M., Enriquez-Barreto, L., Tran, T.S., Polleux, F., and Maness, P.F. (2007). Close homolog of L1 and neuropilin 1 mediate guidance of thalamocortical axons at the ventral telencephalon. J. Neurosci. 27, 13667–13679. Yamasaki, M., Matsui, M., and Watanabe, M. (2010). Preferential localization of muscarinic M1 receptor on dendritic shaft and spine of cortical pyramidal cells and its anatomical evidence for volume transmission. J. Neurosci. 30, 4408– 4418. Yasuda, S., Tanaka, H., Sugiura, H., Okamura, K., Sakaguchi, T., Tran, U., Takemiya, T., Mizoguchi, A., Yagita, Y., Sakurai, T., et al. (2007). Activityinduced protocadherin arcadlin regulates dendritic spine number by triggering N-cadherin endocytosis via TAO2beta and p38 MAP kinases. Neuron 56, 456–471. Zipursky, S.L., and Sanes, J.R. (2010). Chemoaffinity revisited: dscams, protocadherins, and neural circuit assembly. Cell 143, 343–353. Ziv, N.E., and Garner, C.C. (2004). Cellular and molecular mechanisms of presynaptic assembly. Nat. Rev. Neurosci. 5, 385–399.
