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BTBD3 Controls Dendrite Orientation Toward Active Axons in Mammalian Neocortex Asuka Matsui et al. Science 342, 1114 (2013); DOI: 10.1126/science.1244505

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Fig. 4. Place-responsive cell activity during navigation and recall. (Left) Navigation. “In Field” indicates the average place-responsive cell firing rate when navigating within a cell’s place field, whereas “Out Field” indicates the average placeresponsive cell firing rate at locations outside of a place field (*P < 10−5). (Right) Recall. “Near” indicates the average place-responsive cell firing rate in the time period from 1.5 s before to 1 s after recall onset of items that were initially presented in or close to the center of a place field. In contrast, “Far” represents the average place-responsive cell firing rate in the same time window for recall of items that were initially presented far from the center of a place field (*P = 0.03). Error bars indicate SEM.

of intracranial electroencephalography and hemodynamic activity (24, 25). Reinstatement is not specific to an individual item but also activates neighboring items, as would be expected if those neighboring items provide an abstract temporal context for the recalled item (26, 27). Such a temporal context signal may be reflected in the recent discovery of individual neurons in the rodent hip-

pocampus that appear to encode the relative times of behaviorally important events (28, 29). Our finding that spontaneous recall of an item reactivates its spatial context provides direct neural evidence for theories of episodic memory that postulate context reinstatement as the basis for recollection (2, 30). This result also implies that the spatial coding identified with the hippocampal place-cell system is part of a more general engine of episodic memory in which items become associated with their spatiotemporal contexts, and retrieval of items reinstates those contexts to help cue other context-appropriate memories. References and Notes 1. E. Tulving, Elements of Episodic Memory (Oxford Univ. Press, New York, 1983). 2. S. M. Polyn, M. J. Kahana, Trends Cogn. Sci. 12, 24–30 (2008). 3. W. B. Scoville, B. Milner, J. Neurol. Neurosurg. Psychiatry 20, 11–21 (1957). 4. H. Eichenbaum, Neuron 44, 109–120 (2004). 5. L. Davachi, Curr. Opin. Neurobiol. 16, 693–700 (2006). 6. J. O’Keefe, L. Nadel, The Hippocampus as a Cognitive Map (Oxford Univ. Press, New York, 1978). 7. B. L. McNaughton, F. P. Battaglia, O. Jensen, E. I. Moser, M.-B. Moser, Nat. Rev. Neurosci. 7, 663–678 (2006). 8. A. D. Ekstrom et al., Nature 425, 184–188 (2003). 9. J. Jacobs, M. J. Kahana, A. D. Ekstrom, M. V. Mollison, I. Fried, Proc. Natl. Acad. Sci. U.S.A. 107, 6487–6492 (2010). 10. R. U. Muller, J. L. Kubie, J. Neurosci. 7, 1951–1968 (1987). 11. S. Leutgeb, J. K. Leutgeb, M. B. Moser, E. I. Moser, Curr. Opin. Neurobiol. 15, 738–746 (2005). 12. E. R. Wood, P. A. Dudchenko, R. J. Robitsek, H. Eichenbaum, Neuron 27, 623–633 (2000). 13. J. Ferbinteanu, M. L. Shapiro, Neuron 40, 1227–1239 (2003). 14. H. Eichenbaum, P. Dudchenko, E. Wood, M. Shapiro, H. Tanila, Neuron 23, 209–226 (1999). 15. G. Buzsáki, Hippocampus 15, 827–840 (2005). 16. M. W. Howard, M. S. Fotedar, A. V. Datey, M. E. Hasselmo, Phys. Rev. 112, 75–116 (2005). 17. G. Buzsáki, E. I. Moser, Nat. Neurosci. 16, 130–138 (2013). 18. L. Davachi, J. P. Mitchell, A. D. Wagner, Proc. Natl. Acad. Sci. U.S.A. 100, 2157–2162 (2003).

BTBD3 Controls Dendrite Orientation Toward Active Axons in Mammalian Neocortex Asuka Matsui, May Tran, Aya C. Yoshida, Satomi S. Kikuchi, Mami U, Masaharu Ogawa, Tomomi Shimogori* Experience-dependent structural changes in the developing brain are fundamental for proper neural circuit formation. Here, we show that during the development of the sensory cortex, dendritic field orientation is controlled by the BTB/POZ domain–containing 3 (BTBD3). In developing mouse somatosensory cortex, endogenous Btbd3 translocated to the cell nucleus in response to neuronal activity and oriented primary dendrites toward active axons in the barrel hollow. Btbd3 also directed dendrites toward active axon terminals when ectopically expressed in mouse visual cortex or normally expressed in ferret visual cortex. BTBD3 regulation of dendrite orientation is conserved across species and cortical areas and shows how high-acuity sensory function may be achieved by the tuning of subcellular polarity to sources of high sensory activity.

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roper neural circuit development is important for the newborn animal to receive, process, and respond to information from

the external sensory environment. This process critically depends on the patterning of individual neurons to shape the postsynaptic dendritic field

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19. E. L. Hargreaves, G. Rao, I. Lee, J. J. Knierim, Science 308, 1792–1794 (2005). 20. Materials and methods are available as supplementary materials on Science Online. 21. J. F. Miller, E. M. Lazarus, S. M. Polyn, M. J. Kahana, J. Exp. Psychol. Learn. Mem. Cogn. 39, 773–781 (2013). 22. R. U. Muller, E. Bostock, J. S. Taube, J. L. Kubie, J. Neurosci. 14, 7235–7251 (1994). 23. H. Gelbard-Sagiv, R. Mukamel, M. Harel, R. Malach, I. Fried, Science 322, 96–101 (2008). 24. J. R. Manning, M. R. Sperling, A. Sharan, E. A. Rosenberg, M. J. Kahana, J. Neurosci. 32, 8871–8878 (2012). 25. S. M. Polyn, V. S. Natu, J. D. Cohen, K. A. Norman, Science 310, 1963–1966 (2005). 26. J. R. Manning, S. M. Polyn, G. H. Baltuch, B. Litt, M. J. Kahana, Proc. Natl. Acad. Sci. U.S.A. 108, 12893–12897 (2011). 27. M. W. Howard, I. V. Viskontas, K. H. Shankar, I. Fried, Hippocampus 22, 1833–1847 (2012). 28. E. Pastalkova, V. Itskov, A. Amarasingham, G. Buzsáki, Science 321, 1322–1327 (2008). 29. C. J. MacDonald, K. Q. Lepage, U. T. Eden, H. Eichenbaum, Neuron 71, 737–749 (2011). 30. S. M. Polyn, K. A. Norman, M. J. Kahana, Psychol. Rev. 116, 129–156 (2009). Acknowledgments: This work was sponsored by NIH grant MH-061975, the Brain and Behavior Research Foundation, German Research Foundation (Deutsche Forschungsgemeinschaft) grant SFB 780-TP3, and Federal Ministry of Education and Research (Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Germany) grant BCNT TP B3. We are most grateful to J. Stein and H. Urbach for help with localizing electrode locations in postoperative magnetic resonance imaging. We thank the patients and their families for their participation in this research.

Supplementary Materials www.sciencemag.org/content/342/6162/1111/suppl/DC1 Materials and Methods Supplementary Text Figs. S1 to S3 Tables S1 and S2 References 31 July 2013; accepted 28 October 2013 10.1126/science.1244056

for assembly with presynaptic axons. Dendritic remodeling is a conserved process in which postsynaptic dendrites are pruned in response to presynaptic activity during metamorphosis in Drosophila (1) and during the development of hippocampal CA1, cerebellar Purkinje cells, and retinal ganglion cells in mouse (2). In the rodent primary somatosensory cortex, layer IV spiny stellate neurons are concentrated around barrel walls, forming cell-sparse barrel hollows and pyramidal neuron dense septa that delineate individual barrels. In mouse somatosensory barrel cortex, spiny stellate neurons orient their dendrites toward barrel hollows during the first postnatal week to enable efficient synapse formation with thalamocortical axons from the ventrobasal thalamic nucleus (3). Neuronal activity and monoamine uptake in the synaptic junction contribute to this process (4, 5). We pursued the mechanism of dendrite orientation by RIKEN Brain Science Institute, Laboratory for Molecular Mechanisms of Thalamus Development, 2-1 Hirosawa Wako, Saitama 351-0198, Japan. *Corresponding author. E-mail: tshimogori@brain.riken.jp

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Fig. 1. BTBD3 regulates dendrite orientation in mouse somatosensory cortex. (A) Expression of Btbd3 in developing mouse somatosensory cortex is shown by in situ hybridization in coronal (P0 to P6) and P6 tangential section (right). (B to D) To label single spiny stellate neurons, plasmids carrying CAG-loxP-stop-loxP-YFP (1 mg/ml) and CAG-Cre (0.1 ng/ml) are co-electroporated into E13.5 mouse telencephalon. Tangential section of P6 cortices, stained with antibodies to green fluorescent protein (GFP) and vGlut2 to reveal the location of barrels and sparsely labeled spiny stellate neurons. (B′ to D′ and B′′ to D′′) Confocal images and corresponding schematic traces of neurons indicated by the yellow box in (B) to (D). (E) Quantification of dendritic parameters of spiny stellate cells. Sholl analysis: Histograms represent the mean number of dendrites crossing concentric rings drawn at 10-mm intervals from the cell soma (schema in fig. S2). Dendrite distribution is analyzed by counting primary dendrites in the barrel hollow and the barrel septa, respectively. Quantification of dendritic length is also demonstrating dendrite distribution. Analysis of variance (ANOVA) shows a significant difference in Btbd3 knock-down cells compared with control and rescue cells. *P < 0.05, **P < 0.01, t test. Scale bars are 150 mm in (A); 50 mm in (B), (C), and (D); and 12.5 mm in (B′), (C′), and D′.

Fig. 2. Neuronal activity–dependent nuclear translocation of BTBD3 is required for proper dendritic patterning. (A and B) Tangential sections of P6 cortices of wild-type (WT) (A) and CxNR1KO (B), stained with antibodies to GFP and vGlut2 after sparse labeling of spiny stellate neurons by YFP electroporation at E13.5. (A′ to B′′) Confocal images and corresponding schematic traces of neurons. (C) Dendrite distribution is quantified by Sholl analysis, distribution of primary dendrites, and dendritic length. *P < 0.05, **P < 0.01, t test. (D) Expression of Btbd3 by in situ www.sciencemag.org

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hybridization and immunostaining of antibodies to vGlut2 in tangential sections of WT and CxNR1KO cortices at P6. (E) Western blot analysis of BTBD3 protein from somatosensory cortex. (F) Western blot analysis of BTBD3 in nuclear (Nuc) and cytosolic (Cyto) cellular fractions. Markers for the cytosolic (Raf1) and nuclear fraction (Mef2D) are indicated. (G) Depolarization of the Neuro2A cell line promotes Btbd3-HA nuclear translocation. Scale bars are 150 mm in (A) and (B); 50 mm in (A′), (A′′), (B′), and (B′′); 500 mm in (D); and 10 mm in (G). VOL 342

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REPORTS searching for molecules with selective expression in mouse somatosensory cortex and found that BTB/POZ domain–containing 3 (Btbd3) is localized exclusively in spiny stellate neurons during barrel formation (Fig. 1A). The BTB/POZ (Broad complex, Tramtrack, and Bric-à-brac/Poxvirus and zinc finger) proteins comprise a family of putative transcription factors with essential roles in development, differentiation, and oncogenesis (6). The BTB/POZ domain mediates homo- and/or heterodimerization in a transcriptional repression complex by the recruitment of co-repressors (7). The BTB domain is also contained within proteins such as Abrupt, which controls dendrite formation in Drosophila (8, 9). We hypothesized that the observed Btbd3 expression in spiny stellate neurons of the mouse somatosensory cortex could regulate the formation of dendritic field patterning. In early postnatal somatosensory cortex, Btbd3 mRNA is not expressed at postnatal day (P) 0 (Fig. 1A); however, weak expression is observed at P2, coincident with innervation by thalamocortical axons into layer IV cortex, followed by a barrel-like expression pattern established over the next few days (Fig. 1A and fig. S1). To test the function of BTBD3 in somatosensory neurons, we transfected a Btbd3 short hairpin–mediated RNA (shRNA) construct by in utero electroporation, resulting in ~70% reduction in gene expression compared with control brain (fig. S2). We imaged wild-type spiny stellate neurons with confocal microscopy at P6, which is electroporated with yellow fluorescent protein (YFP)–expressing plasmid at embryonic day 13.5 (E13.5), and observed on average 3 to 4 primary dendrites (Fig. 1E) with a unidirectional orientation (Fig. 1, B to B′′). With Btbd3 knockdown, there was an increase in the number of primary dendrites and misoriented dendrites (Fig. 1, C to C′′ and E). The altered dendritic morphology was rescued by a human BTBD3 construct, the expression of which is not suppressed by the shRNA (fig. S2), suggesting that the observed defects are due to a specific suppression of Btbd3 by the shRNA (Fig. 1, D to D′′ and E). In early postnatal mouse brain development, spiny stellate neuron morphology undergoes a transient increase in the number of primary dendrites without a preferred orientation (fig. S2), and the time at which the greatest number of nonpolarized dendrites is evident (P2) correlates with the onset of Btbd3 expression in the barrel (Fig. 1A). Neuronal activity from thalamocortical axons is required for morphological changes in dendrites of spiny stellate cells in the somatosensory cortex (10–12). Therefore, we examined Btbd3 expression in the NMDAR1 (N-methyl-D-aspartate receptor) cortex-specific conditional mutant mouse, where spiny stellate neurons have disoriented dendritic fields (Fig. 2, A to C) (13). In the somatosensory cortex of these mice at P6, although thalamocortical axons had extensive arbors compared with wild type, their terminals in cortical layer IV were arranged in normal, barrel-like clus-

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Fig. 3. Ectopic BTBD3 orients the dendritic field in mouse visual cortex. (A) In situ hybridization of Btbd3 in the mouse somatosensory and visual cortexes at P6. (B) Schema of the experimental procedure. In utero electroporation was conducted at E13.5 in the occipital lobe. Pups were raised to P14, and the contralateral eye was enucleated for monocular enucleation (ME). (C) GFP antibody staining of the binocular region of the YFP-transfected brain. The binocular region is indicated by the red bracket, and analyzed neurons are indicated by the yellow box. (D and E) Confocal images and corresponding schematic traces of neurons (red arrowhead) in control (D) and Btbd3-transfected neurons (E). (F) Dendrite distribution was quantified by Sholl analysis, distribution of primary dendrites, and dendritic length. *P < 0.05, **P < 0.01, t test. m, monocular region; b, binocular region. Scale bars are 1 mm in (A), 200 mm in (C), and 20 mm in (D) and (E).

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REPORTS ters (14) containing normal Btbd3 expression (Fig. 2D). Total BTBD3 protein levels in somatosensory cortex did not differ between control and

NMDAR1 mutant mice (Fig. 2E). However, the subcellular localization of BTBD3 in wild-type brain partitioned to the nuclear fraction, whereas

Fig. 4. BTBD3 controls dendrite orientation in ferret visual cortex. (A) In situ hybridization of BTBD3 in the ferret brain at P16. (B) Dorsal view of P16 ferret brain that shows transfected neurons in the visual cortex (yellow arrows, left). The WGA-positive axon terminus is observed in the primary visual cortex (yellow bracket, middle). Positions of analyzed YFP-positive layer IV neurons are indicated by red arrows (right). (C) Reduction of BTBD3 mRNA by shRNA electroporation was tested by in situ hybridization (top, asterisks), and by quantitative real-time fluorescence polymerase chain reaction, (bottom). Lane 1, BTBD3 in control brain; lane 2, BTBD3 in shRNA electroporated knock-down brain; lane 3, B-ACTIN in control brain; lane www.sciencemag.org

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BTBD3 in mutant brain was found selectively in the cytosolic fraction (Fig. 2F). To study subcellular localization dynamics, we tagged Btbd3 with

4, B-ACTIN in shRNA electroporated knock-down brain. (D to F) Confocal images and schematic traces of neurons in control (D), 3 days after monocular enucleation (E) and monocular enucleation brain after BTBD3 knock-down (F). (G) Dendrite distribution was quantified by Sholl analysis, distribution of primary dendrites, and dendritic length. *P < 0.05, **P < 0.01, t test. (H) Dendrite morphology in control brain and monocular enucleation brain were observed at P51. (I) Dendrite distribution was quantified by Sholl analysis, distribution of primary dendrites, and dendritic length. *P < 0.05, **P < 0.01, t test. Scale bars are 1 mm in (A), 250 mm in (B) and (C), 25 mm in (D) to (F), and 50 mm in (H). VOL 342

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REPORTS hemagglutinin (HA) and transfected Neuro2A cells (Fig. 2G). Under control, nonstimulating conditions, Btbd3-HA was localized in the cytoplasm, but after KCl depolarization, Btbd3HA had translocated to the nucleus (Fig. 2G). Based on these results, we conclude that neuronal activity triggers the translocation of BTBD3 from the cytosol to the nucleus, where it may control transcriptional programs to trim excess dendrites in developing mouse somatosensory cortex. We adopted a gain-of-function approach to investigate whether BTBD3 can remodel dendrite orientation toward active axons by ectopic expression of BTBD3 in mouse primary visual cortex, which does not express BTBD3 nor mediate high-acuity vision (Fig. 3A). After electroporating Btbd3 into primary visual cortex at E13.5, the contrast of neuronal activity was increased in the binocular region with monocular enucleation and eyelid suturing to effect visual deprivation at P14 before eye opening (Fig. 3B). After 4 days, we analyzed cortical organization by trans-synaptic wheat germ agglutinin (WGA) injected into the ipsilateral eye (Fig. 3C). To determine whether BTBD3 could reorient the dendritic field in visual cortex toward the binocular region, which contains active ipsilateral inputs, YFP-positive neurons close to the WGA-positive binocular region were observed (Fig. 3C). In control YFPexpressing neurons, basal dendrites showed a symmetrical branched pattern (Fig. 3D). In contrast, Btbd3-electroporated cortical neurons showed a highly polarized, unidirectional dendrite pattern directed toward the binocular region (Fig. 3E). We analyzed the number of primary dendrites and the distribution of the dendrites on the right and left side in YFP-positive neurons (Fig. 3F). In control brain (15 neurons from three animals), the average number of primary dendrites was significantly higher than in Btbd3electroporated neurons (24 neurons from four animals), suggesting that dendrite removal was stimulated in these cells (Fig. 3F). To show that neurons had a unidirectional dendritic field, we quantified the number of dendrites in the monocular region (silenced area) and binocular region (active region) and showed that neurons ectopically expressing BTBD3 have fewer dendrites in the low neural activity area (Fig. 3F). These results support the conclusion that BTBD3 preferentially orients dendritic processes toward active axon terminals in vivo. BTBD3 is expressed strongly in the primary visual cortex of neonatal common marmoset (Callithrix jacchus), a brain region that mediates high-acuity vision via ocular dominance columns (15). This observation suggested a conserved function for BTBD3 in dendritic remodeling in the visual cortex of animals with high-resolution vision mediated by ocular dominance columns (16, 17). To test this hypothesis, we analyzed BTBD3 expression in developing ferret primary visual cortex (18, 19) where Btbd3 is observed in visual cortex layer IV neurons (Fig. 4A). High-

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resolution dendrite morphology in the ferret visual cortex was achieved by targeted electroporation (Fig. 4B, yellow arrows). WGA was injected into the contralateral eye to label ocular dominance columns (Fig. 4B, yellow bracket) and their peripheral neurons (Fig. 4B, red arrows). Btbd3 shRNA was transfected into the ferret visual cortex to reduce endogenous BTBD3 expression (Fig. 4C). The basal dendrites of control cortical neurons showed a symmetrical pattern, but after monocular enucleation, an asymmetric morphology was observed, with neurons observed to orient their dendrites toward the open eye column (Fig. 4, D and E). To determine whether BTBD3 regulates dendrite orientation, Btbd3 shRNA was co-electroporated with YFP into visual cortex, followed by monocular enucleation. There were no morphological differences observed, similar to the control non–monocular enucleation group, suggesting that BTBD3 is required for dendrite remodeling toward active axon terminals (Fig. 4, F and G). To address whether the changes in dendrite orientation in the early postnatal developmental critical period were transient or persistent, we assessed dendritic morphology at P51 (20). In control brain, apical dendrites were shorter and difficult to distinguish from basal dendrites (21), and the complexity of dendritic branching was increased. However, the number of primary dendrites and the unilateral morphology of the basal dendrites remained similar to those during P16 (Fig. 4, G and I). These results further support a role for BTBD3 in dendrite orientation toward afferents with high neuronal activity. Dendritic refinement is known to operate by cytoskeletal modification and membrane-initiated activity-dependent pathways through molecules including neurexin-neuroligin, Wnt, and EphBephrinB (22–24). Here, we define a role for Btbd3 as a nuclear regulator of activity-dependent dendritic refinement. In mouse, Btbd3 is also expressed in the mouse olfactory bulb, piriform cortex, and hippocampus (Fig. 3), where dendritic field formation of mitral cells, piriform pyramidal cells, and CA1 neurons are altered or delayed when presynaptic input is altered (25, 26). This observation suggests that BTBD3 or a related family member may control dendrite remodeling in different areas of the brain. We propose that the acquisition of BTBD3 expression in a specific cortical area could provide the ability to streamline the flow of sensory information in neuronal circuits by remodeling dendritic fields to optimally respond to sensory activity. In consequence, high-acuity sensory function may have been enabled by the evolution of BTBD3 and related BTB/POZ family members in cortical development. This idea is supported by the selective expression of BTBD3 in the visual and auditory cortex of the common marmoset, a species that relies heavily on high-acuity vocal and visual communication for survival (17), and in the mouse, where it is expressed in high-acuity tactile and olfactory areas but not in low-acuity visual cortex. Thus, BTBD3 expression and func-

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tion may help to index the evolution of precise columnar organization in many cortical regions of the mammalian brain. References and Notes 1. Y. N. Jan, L. Y. Jan, Nat. Rev. Neurosci. 11, 316–328 (2010). 2. R. O. L. Wong, A. Ghosh, Nat. Rev. Neurosci. 3, 803–812 (2002). 3. T. A. Woolsey, H. Van der Loos, Brain Res. 17, 205–242 (1970). 4. C. Lebrand et al., J. Comp. Neurol. 401, 506–524 (1998). 5. T. Iwasato et al., Nature 406, 726–731 (2000). 6. O. M. Siggs, B. Beutler, Cell Cycle 11, 3358–3369 (2012). 7. S.-U. Lee, T. Maeda, Immunol. Rev. 247, 107–119 (2012). 8. K. Sugimura, D. Satoh, P. Estes, S. Crews, T. Uemura, Neuron 43, 809–822 (2004). 9. W. Li, F. Wang, L. Menut, F. B. Gao, Neuron 43, 823–834 (2004). 10. T. Iwasato et al., Neuron 19, 1201–1210 (1997). 11. J. S. Espinosa, D. G. Wheeler, R. W. Tsien, L. Luo, Neuron 62, 205–217 (2009). 12. N. Narboux-Nême et al., J. Neurosci. 32, 6183–6196 (2012). 13. A. Datwani, T. Iwasato, S. Itohara, R. S. Erzurumlu, Mol. Cell. Neurosci. 21, 477–492 (2002). 14. L. J. Lee, T. Iwasato, S. Itohara, R. S. Erzurumlu, J. Comp. Neurol. 485, 280–292 (2005). 15. H. Mashiko et al., J. Neurosci. 32, 5039–5053 (2012). 16. A. Kossel, S. Löwel, J. Bolz, J. Neurosci. 15, 3913–3926 (1995). 17. Y. Nakagami, A. Watakabe, T. Yamamori, Front. Neural Circuits 7, 43 (2013). 18. H. Kawasaki, L. Iwai, K. Tanno, Mol. Brain 5, 24 (2012). 19. J. C. Crowley, L. C. Katz, Science 290, 1321–1324 (2000). 20. M. Sur, C. A. Leamey, Nat. Rev. Neurosci. 2, 251–262 (2001). 21. E. M. Callaway, V. Borrell, J. Neurosci. 31, 7456–7470 (2011). 22. M. Packard et al., Cell 111, 319–330 (2002). 23. S. X. Chen, P. K. Tari, K. She, K. Haas, Neuron 67, 967–983 (2010). 24. N.-J. Xu, S. Sun, J. R. Gibson, M. Henkemeyer, Nat. Neurosci. 14, 1421–1429 (2011). 25. B. Bathellier, T. W. Margrie, M. E. Larkum, J. Neurosci. 29, 12641–12652 (2009). 26. D. M. Lin et al., Neuron 26, 69–80 (2000). Acknowledgments: We thank C. Yokoyama, A. Kania, T. Young, and A. Terashima for critical reading. We also thank Y. Takahashi, S. Tonegawa, S. Blackshaw, and S. Itohara for providing plasmids and mouse line, and F. Matsuzaki, D. Konno, T. Suetsugu, H. Mashiko, and Y. Maeda for technical assistance. This work was supported by the RIKEN Brain Science Institute, MEXT Strategic Research Program for Brain Science (SRPBS), Center of Novel Science Initiative Brain Science Project, Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST), and the Human Frontier Science Program (HFSP). M.T. is funded by the Japan-U.S. Educational Commission (Fulbright Japan).

Supplementary Materials www.sciencemag.org/content/342/6162/1114/suppl/DC1 Materials and Methods Figs. S1 and S2 References (27–30) 9 August 2013; accepted 17 October 2013 Published online 31 October 2013; 10.1126/science.1244505

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