Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Cntnap4 differentially contributes to GABAergic and dopaminergic synaptic transmission

Nature volume 511, pages 236–240 (2014)Cite this article

Subjects

This article has been updated

Abstract

Although considerable evidence suggests that the chemical synapse is a lynchpin underlying affective disorders, how molecular insults differentially affect specific synaptic connections remains poorly understood. For instance, Neurexin 1a and 2 (NRXN1 and NRXN2) and CNTNAP2 (also known as CASPR2), all members of the neurexin superfamily of transmembrane molecules, have been implicated in neuropsychiatric disorders. However, their loss leads to deficits that have been best characterized with regard to their effect on excitatory cells1,2. Notably, other disease-associated genes such as BDNF and ERBB4 implicate specific interneuron synapses in psychiatric disorders3,4. Consistent with this, cortical interneuron dysfunction has been linked to epilepsy, schizophrenia and autism5,6. Using a microarray screen that focused upon synapse-associated molecules, we identified Cntnap4 (contactin associated protein-like 4, also known as Caspr4) as highly enriched in developing murine interneurons. In this study we show that Cntnap4 is localized presynaptically and its loss leads to a reduction in the output of cortical parvalbumin (PV)-positive GABAergic (γ-aminobutyric acid producing) basket cells. Paradoxically, the loss of Cntnap4 augments midbrain dopaminergic release in the nucleus accumbens. In Cntnap4 mutant mice, synaptic defects in these disease-relevant neuronal populations are mirrored by sensory-motor gating and grooming endophenotypes; these symptoms could be pharmacologically reversed, providing promise for therapeutic intervention in psychiatric disorders.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Cellular and subcellular localization of Cntnap4.
Figure 2: Cntnap4 mutant mice show increased dopamine but decreased GABA signalling.
Figure 3: Loss of Cntnap4 results in ultrastructural deficits in perisomatic inhibitory synapses.
Figure 4: Aberrant behaviour exhibited by Cntnap4 mutant mice can be rescued by specific pharmacological intervention.

Change history

  • 09 July 2014

    The affiliation number for author A. Gordon was updated, text was added to the Acknowledgements, and asterisks and P values were updated in Figure 4d and in the Methods.

References

  1. Peñagarikano, O. et al. Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities, and core autism-related deficits. Cell 147, 235–246 (2011)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Etherton, M. R., Blaiss, C. A., Powell, C. M. & Sudhof, T. C. Mouse neurexin-1alpha deletion causes correlated electrophysiological and behavioral changes consistent with cognitive impairments. Proc. Natl Acad. Sci. USA 106, 17998–18003 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  3. Fazzari, P. et al. Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling. Nature 464, 1376–1380 (2010)

    Article  ADS  CAS  PubMed  Google Scholar 

  4. Berghuis, P. et al. Brain-derived neurotrophic factor controls functional differentiation and microcircuit formation of selectively isolated fast-spiking GABAergic interneurons. Eur. J. Neurosci. 20, 1290–1306 (2004)

    Article  PubMed  Google Scholar 

  5. Lewis, D. A. Cortical circuit dysfunction and cognitive deficits in schizophrenia - implications for preemptive interventions. Eur. J. Neurosci. 35, 1871–1878 (2012)

    Article  PubMed  PubMed Central  Google Scholar 

  6. Blatt, G. J. & Fatemi, S. H. Alterations in GABAergic biomarkers in the autism brain: research findings and clinical implications. Anat. Rec. 294, 1646–1652 (2011)

    Article  CAS  Google Scholar 

  7. Ashrafi, S. et al. Neuronal Ig/Caspr recognition promotes the formation of axoaxonic synapses in mouse spinal cord. Neuron 81, 120–129 (2014)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Spiegel, I., Salomon, D., Erne, B., Schaeren-Wiemers, N. & Peles, E. Caspr3 and Caspr4, two novel members of the Caspr family are expressed in the nervous system and interact with PDZ domains. Mol. Cell. Neurosci. 20, 283–297 (2002)

    Article  CAS  PubMed  Google Scholar 

  9. Ho, A., Morishita, W., Hammer, R. E., Malenka, R. C. & Sudhof, T. C. A role for Mints in transmitter release: Mint 1 knockout mice exhibit impaired GABAergic synaptic transmission. Proc. Natl Acad. Sci. USA 100, 1409–1414 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Atasoy, D. et al. Deletion of CASK in mice is lethal and impairs synaptic function. Proc. Natl Acad. Sci. USA 104, 2525–2530 (2007)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. Patel, J. C. & Rice, M. E. Monitoring axonal and somatodendritic dopamine release using fast-scan cyclic voltammetry in brain slices. Methods Mol. Biol. 964, 243–273 (2013)

    Article  CAS  PubMed  Google Scholar 

  12. Li, X. et al. Enhanced striatal dopamine transmission and motor performance with LRRK2 overexpression in mice is eliminated by familial Parkinson’s disease mutation G2019S. J. Neurosci. 30, 1788–1797 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Doischer, D. et al. Postnatal differentiation of basket cells from slow to fast signaling devices. J. Neurosci. 28, 12956–12968 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Hefft, S. & Jonas, P. Asynchronous GABA release generates long-lasting inhibition at a hippocampal interneuron-principal neuron synapse. Nature Neurosci. 8, 1319–1328 (2005)

    Article  CAS  PubMed  Google Scholar 

  15. Labasque, M. & Faivre-Sarrailh, C. GPI-anchored proteins at the node of Ranvier. FEBS Lett. 584, 1787–1792 (2010)

    Article  CAS  PubMed  Google Scholar 

  16. Krueger, D. D., Tuffy, L. P., Papadopoulos, T. & Brose, N. The role of neurexins and neuroligins in the formation, maturation, and function of vertebrate synapses. Curr. Opin. Neurobiol. 22, 412–422 (2012)

    Article  CAS  PubMed  Google Scholar 

  17. Chang, M. C. et al. Narp regulates homeostatic scaling of excitatory synapses on parvalbumin-expressing interneurons. Nature Neurosci. 13, 1090–1097 (2010)

    Article  CAS  PubMed  Google Scholar 

  18. Sylwestrak, E. L. & Ghosh, A. Elfn1 regulates target-specific release probability at CA1-interneuron synapses. Science 338, 536–540 (2012)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Cathala, L., Holderith, N. B., Nusser, Z., DiGregorio, D. A. & Cull-Candy, S. G. Changes in synaptic structure underlie the developmental speeding of AMPA receptor-mediated EPSCs. Nature Neurosci. 8, 1310–1318 (2005)

    Article  CAS  PubMed  Google Scholar 

  20. Peça, J. et al. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature 472, 437–442 (2011)

    Article  ADS  PubMed  PubMed Central  CAS  Google Scholar 

  21. Chao, H. T. et al. Dysfunction in GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes. Nature 468, 263–269 (2010)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Geyer, M. A., Krebs-Thomson, K., Braff, D. L. & Swerdlow, N. R. Pharmacological studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review. Psychopharmacol. 156, 117–154 (2001)

    Article  CAS  Google Scholar 

  23. Stark, K. L., Burt, R. A., Gogos, J. A. & Karayiorgou, M. Analysis of prepulse inhibition in mouse lines overexpressing 22q11.2 orthologues. Int. J. Neuropsychopharmacol. 12, 983–989 (2009)

    Article  CAS  PubMed  Google Scholar 

  24. Champagne, F. A. et al. Variations in nucleus accumbens dopamine associated with individual differences in maternal behavior in the rat. J. Neurosci. 24, 4113–4123 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Grados, M. A. The genetics of obsessive-compulsive disorder and Tourette's syndrome: what are the common factors? Curr. Psychiatry Rep. 11, 162–166 (2009)

    Article  PubMed  Google Scholar 

  26. Crowley, J. J. et al. Antipsychotic-induced vacuous chewing movements and extrapyramidal side effects are highly heritable in mice. Pharmacogenomics J. 12, 147–155 (2012)

    Article  CAS  PubMed  Google Scholar 

  27. Foster, A. C. et al. In vivo pharmacological characterization of indiplon, a novel pyrazolopyrimidine sedative-hypnotic. J. Pharmacol. Exp. Ther. 311, 547–559 (2004)

    Article  CAS  PubMed  Google Scholar 

  28. Petroski, R. E. et al. Indiplon is a high-affinity positive allosteric modulator with selectivity for alpha1 subunit-containing GABAA receptors. J. Pharmacol. Exp. Ther. 317, 369–377 (2006)

    Article  CAS  PubMed  Google Scholar 

  29. Klausberger, T., Roberts, J. D. B. & Somogyi, P. Cell type- and input-specific differences in the number and subtypes of synaptic GABAA receptors in the hippocampus. J. Neurosci. 22, 2513–2521 (2002)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Castellanos, F. X. et al. Sensorimotor gating in boys with Tourette’s syndrome and ADHD: preliminary results. Biol. Psychiatry 39, 33–41 (1996)

    Article  CAS  PubMed  Google Scholar 

  31. Glessner, J. T. et al. Autism genome-wide copy number variation reveals ubiquitin and neuronal genes. Nature 459, 569–573 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Elia, J. et al. Genome-wide copy number variation study associates metabotropic glutamate receptor gene networks with attention deficit hyperactivity disorder. Nature Genet. 44, 78–84 (2012)

    Article  CAS  Google Scholar 

  33. Glessner, J. T. et al. Strong synaptic transmission impact by copy number variations in schizophrenia. Proc. Natl Acad. Sci. USA 107, 10584–10589 (2010)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. Karayiorgou, M. et al. Phenotypic characterization and genealogical tracing in an Afrikaner schizophrenia database. Am. J. Med. Genet. B. Neuropsychiatr. Genet. 124B, 20–28 (2004)

    Article  PubMed  Google Scholar 

  35. Abecasis, G. R. et al. Genomewide scan in families with schizophrenia from the founder population of Afrikaners reveals evidence for linkage and uniparental disomy on chromosome 1. Am. J. Hum. Genet. 74, 403–417 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Xu, B. et al. Strong association of de novo copy number mutations with sporadic schizophrenia. Nature Genet. 40, 880–885 (2008)

    Article  CAS  PubMed  Google Scholar 

  37. Xu, B. et al. Elucidating the genetic architecture of familial schizophrenia using rare copy number variant and linkage scans. Proc. Natl Acad. Sci. USA 106, 16746–16751 (2009)

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  38. Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, 559–575 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Li, Y., Willer, C. J., Ding, J., Scheet, P. & Abecasis, G. R. MaCH: using sequence and genotype data to estimate haplotypes and unobserved genotypes. Genet. Epidemiol. 34, 816–834 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  40. Li, M., Boehnke, M. & Abecasis, G. R. Joint modeling of linkage and association: identifying SNPs responsible for a linkage signal. Am. J. Hum. Genet. 76, 934–949 (2005)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Li, M., Boehnke, M. & Abecasis, G. R. Efficient study designs for test of genetic association using sibship data and unrelated cases and controls. Am. J. Hum. Genet. 78, 778–792 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Feinberg, K. et al. A glial signal consisting of gliomedin and NrCAM clusters axonal Na+ channels during the formation of nodes of Ranvier. Neuron 65, 490–502 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Spiegel, I. et al. A central role for Necl4 (SynCAM4) in Schwann cell-axon interaction and myelination. Nature Neurosci. 10, 861–869 (2007)

    Article  CAS  PubMed  Google Scholar 

  44. Spiegel, I., Salomon, D., Erne, B., Schaeren-Wiemers, N. & Peles, E. Caspr3 and Caspr4, two novel members of the Caspr family are expressed in the nervous system and interact with PDZ domains. Mol. Cell. Neurosci. 20, 283–297 (2002)

    Article  CAS  PubMed  Google Scholar 

  45. Jordan, B. A. et al. Identification and verification of novel rodent postsynaptic density proteins. Mol. Cell. Proteomics 3, 857–871 (2004)

    Article  CAS  PubMed  Google Scholar 

  46. Phillips, G. R. et al. The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution. Neuron 32, 63–77 (2001)

    Article  CAS  PubMed  Google Scholar 

  47. Restituito, S. et al. Synaptic autoregulation by metalloproteases and gamma-secretase. J. Neurosci. 31, 12083–12093 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Patel, J. C. & Rice, M. E. Monitoring axonal and somatodendritic dopamine release using fast-scan cyclic voltammetry in brain slices. Methods Mol. Biol. 964, 243–273 (2013)

    Article  CAS  PubMed  Google Scholar 

  49. Patel, J. C., Rossignol, E., Rice, M. E. & Machold, R. P. Opposing regulation of dopaminergic activity and exploratory motor behavior by forebrain and brainstem cholinergic circuits. Nature Commun. 3, 1172 (2012)

    Article  ADS  Google Scholar 

  50. Li, X. et al. Enhanced striatal dopamine transmission and motor performance with LRRK2 overexpression in mice is eliminated by familial Parkinson's disease mutation G2019S. J. Neurosci. 30, 1788–1797 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Nicholson, C. & Patel, J. A Simplified Analysis of Dopamine Uptake by Michaelis–Menten Kinetics in Monitoring Molecules in Neuroscience, Proceedings of the 13th International Conference on In Vivo Methods (eds Westerink, B. et al.) 328–330 (Vrije Universiteit Brussel, 2010)

    Google Scholar 

  52. Rice, M. E., Patel, J. C. & Cragg, S. J. Dopamine release in the basal ganglia. Neurosci. 198, 112–137 (2011)

    Article  CAS  Google Scholar 

  53. Osten, P. et al. The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and α- and β-SNAPs. Neuron 21, 99–110 (1998)

    Article  CAS  PubMed  Google Scholar 

  54. MacAskill, A. F. et al. Miro1 is a calcium sensor for glutamate receptor-dependent localization of mitochondria at synapses. Neuron 61, 541–555 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Nikolaev, A., McLaughlin, T., O’Leary, D. D. & Tessier-Lavigne, M. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature 457, 981–989 (2009)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  56. Foster, A. C. et al. In vivo pharmacological characterization of indiplon, a novel pyrazolopyrimidine sedative-hypnotic. J. Pharmacol. Exp. Ther. 311, 547–559 (2004)

    Article  CAS  PubMed  Google Scholar 

  57. Hoeffer, C. A. et al. Removal of FKBP12 enhances mTOR-Raptor interactions, LTP, memory, and perseverative/repetitive behavior. Neuron 60, 832–845 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Thomas, A. et al. Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacol. 204, 361–373 (2009)

    Article  CAS  Google Scholar 

  59. Miyoshi, G., Butt, S. J. B., Takebayashi, H. & Fishell, G. Physiologically distinct temporal cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors. J. Neurosci. 27, 7786–7798 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Spiegel, I., Salomon, D., Erne, B., Schaeren-Wiemers, N. & Peles, E. Caspr3 and Caspr4, two novel members of the Caspr family are expressed in the nervous system and interact with PDZ domains. Mol. Cell. Neurosci. 20, 283–297 (2002)

    Article  CAS  PubMed  Google Scholar 

  61. González, M. I., Cruz Del Angel, Y. & Brooks-Kayal, A. Down-regulation of gephyrin and GABAA receptor subunits during epileptogenesis in the CA1 region of hippocampus. Epilepsia 54, 616–624 (2013)

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Brandon, N. J. et al. A-kinase anchoring protein 79/150 facilitates the phosphorylation of GABAA receptors by cAMP-dependent protein kinase via selective interaction with receptor β subunits. Mol. Cell. Neurosci. 22, 87–97 (2003)

    Article  CAS  PubMed  Google Scholar 

  63. Panzanelli, P. et al. Distinct mechanisms regulate GABAA receptor and gephyrin clustering at perisomatic and axo-axonic synapses on CA1 pyramidal cells. J. Physiol. (Lond.) 589, 4959–4980 (2011)

    Article  CAS  Google Scholar 

  64. Hoon, M. et al. Neuroligin 2 controls the maturation of GABAergic synapse and information processing in the retina. J. Neurosci. 29, 8039–8050 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Miyoshi, G. et al. Genetic fate mapping reveals that the caudual ganglionic eminence produces a large and diverse population of superficial cortical interneurons. J. Neurosci 30, 1582–1594 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Restituito, S. et al. Synaptic autoregulation by metalloproteases and γ-secretase. J. Neurosci. 31, 12083–12093 (2011)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Batista-Brito, R. et al. The cell-intrinsic requirement of Sox6 for cortical interneuron development. Neuron 63, 466–481 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Condé, F., Lund, J. S., Jacobowitz, D. M., Baimbridge, K. G. & Lewis, D. A. Local circuit neurons immunoreactive for calretinin, calbindin D-28k or parvalbumin in monkey prefrontal cortex: distribution and morphology. J. Comp. Neurol. 341, 95–116 (1994)

    Article  PubMed  Google Scholar 

  69. Belzil, C. et al. A Ca2+-dependent mechanism of neuronal survival mediated by the microtubule-associated protein p600. J. Biol. Chem. 288, 24452–24464 (2013)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Miyoshi, G. & Fishell, G. Dynamic FoxG1 expression coordinates the integration of multipolar pyramidal neuron precursors into the cortical plate. Neuron 74, 1045–1058 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Close, J. et al. Satb1 is an activity-modulated transcription factor required for the terminal differentiation and connectivity of medial ganglionic eminence-derived cortical interneurons. J. Neurosci. 32, 17690–17705 (2012)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Haycock, J. W. Stimulation-dependent phosphorylation of tyrosine hydroxylase in rat corpus striatum. Brain Res. Bull. 19, 619–622 (1987)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors are grateful to R. Froemke for critically reading the manuscript, to B. Benedetti, M. McKenzie Chang, L. Cobbs, B. A. Heller, T. Petros and N. Yumoto (all NYU) for help with experiments and analysis and to Charles Nicholson (NYU) for providing specialized software to analyse Vmax. Research in the Fishell laboratory is supported by the NIH (grants R01 NS081297, R01 MH071679, R01 NS074972, P01 NS074972 to B.R. and G.F.) and the Simons Foundation (94534). The Rice laboratory is supported by the NIH (grants R01 NS036362 and R01 DA033811) and the Attilio and Olympia Ricciardi Research Fund. The Rudy laboratory is supported by the NIH (NS30989). The Peles laboratory is supported by the NIH (grant NS50220) and the Israel Science Foundation. T.K. support was provided through postdoctoral fellowships from the Patterson Trust and Roche. E.A. support was provided by New York State through its NYSTEM initiative (C024326) and fellowship from Canadian Institutes of Health Research. J.C.P support was provided by NYU COE Addiction Seed Grant. This work was funded by the Institut Pasteur, INSERM, AP-HP, University Paris Diderot and the Bettencourt-Schueller, Orange, FondaMental, Conny-Maeva, Cognacq-Jay foundations.

Author information

Author notes

  1. T. Karayannis and E. Au: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neuroscience and Physiology, NYU Neuroscience Institute, New York, 10016, New York, USA

    T. Karayannis, E. Au, I. Kruglikov, S. Restituito, N. C. Roy, B. Rudy, C. Hoeffer, R. W. Tsien & G. Fishell

  2. Department of Neurosurgery, Neuroscience and Physiology, New York University Langone Medical Center, New York, 10016, New York, USA

    J. C. Patel & M. E. Rice

  3. Department of Psychiatry, College of Physicians and Surgeons, Columbia University, 1051 Riverside Drive, New York, New York 10032, USA,

    S. Markx, L. Rodriguez-Murillo & M. Karayiorgou

  4. Institut Pasteur, Human Genetics and Cognitive Functions Unit, 75724 Paris, France,

    R. Delorme, H. Hakonarson, G. Huguet & T. Bourgeron

  5. CNRS URA 2182 Genes, Synapses and Cognition, Institut Pasteur, 75724 Paris, France,

    R. Delorme, G. Huguet & T. Bourgeron

  6. Department of Child and Adolescent Psychiatry, Assistance Publique-Hôpitaux de Paris, Robert Debré Hospital, 75019 Paris, France,

    R. Delorme

  7. Département de Génétique et Cytogénétique, Unité Fonctionnelle de Génétique Médicale AP-HP, Centre de Référence, Déficiences intellectuelles de causes rares, CRicm, UMR-S975, Groupe Hospitalier Pitié-Salpêtrière, F-75013, Paris, France,

    D. Héron

  8. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel,

    D. Salomon, A. Gordon & E. Peles

  9. Center for Applied Genomics, The Children’s Hospital of Philadelphia, Philadelphia, 19104, Pennsylvania, USA

    J. Glessner

  10. Section on Synaptic Transmission, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, 20892, Maryland, USA

    N. C. Roy

  11. Departments of Physiology and Cellular Biophysics and Neuroscience, Columbia University Medical Center, New York, 10032, New York, USA

    J. A. Gogos

  12. Département de Génétique et Cytogénétique, Unité Fonctionnelle de Génétique Chromosomique AP-HP, CRicm, UMR-S975, Groupe Hospitalier, Pitié-Salpêtrière, 75013 Paris, France,

    B. Keren

  13. University Paris Diderot, Sorbonne Paris Cité, Human Genetics and Cognitive Functions, 75005 Paris, France,

    G. Huguet & T. Bourgeron

  14. FondaMental Foundation, 94000 Créteil, France,

    T. Bourgeron

Authors
  1. T. Karayannis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E. Au
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. C. Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. I. Kruglikov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S. Markx
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. R. Delorme
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. D. Héron
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. D. Salomon
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. J. Glessner
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. S. Restituito
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. A. Gordon
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. L. Rodriguez-Murillo
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. N. C. Roy
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. J. A. Gogos
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. B. Rudy
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. M. E. Rice
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. M. Karayiorgou
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. H. Hakonarson
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. B. Keren
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. G. Huguet
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. T. Bourgeron
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. C. Hoeffer
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. R. W. Tsien
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. E. Peles
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. G. Fishell
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.K., E.A. and G.F. designed the study and wrote the manuscript. T.K. and E.A. performed all the experiments and analysis except for the following: J.C.P. performed the in vitro voltammetry experiments and analysed the data. I.K. performed the majority of the in vitro paired recordings. M.K., L.R.-M. and S.M. provided the SNP data. J.G. and H.H. provided the intronic CNV data. D.H., B.K., G.H., R.D. and T.B. provided the exonic CNV data. S.R. performed the synaptosome preparation and western blots. D.S. and E.P. made the Cntnap4 mouse. A.G. performed the verification of the Cntnap4 mouse. N.C.R. performed the in vivo electrophysiological experiments. C.H. set up and advised on behavioural experiments. J.A.G., R.W.T., B.R. and M.E.R. advised on experiments and manuscript preparation.

Corresponding author

Correspondence to G. Fishell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Generation of Cntnap4 knockout (KO) mice and localization of the gene and the protein.

a, Schematic representation of targeting strategy to generate mutant eGFP-knock-in knockout Cntnap4 allele. Primers used for genotyping (a–c) are indicated. X, XhoI, H, NheI, B, BahmHI sites. PCR, RT–PCR and western blot show correct gene targeting, disrupted transcription and translation of Cntnap4, respectively. b, Distribution of Cntnap4-positive cells that are also GAD67 positive by layer in a double fluorescent in situ hybridization analysis (n = 2 brains). c, Percentage of overlap between interneuron markers (NPY, reelin, calretinin and VIP) and Cntnap4-eGFP. d, e, Percentage of overlap between PV and Cntnap4-eGFP across all layers (b–d, bars represent the mean; error bars, s.e.m.; n = 4 brains). f, Cntnap4–Fc in vitro labelling and controls. Colorimetric detection of a human Fc-tagged Cntnap4 extracellular domain on live dissociated hippocampal neuronal cultures (top image). No specific binding or signal upon application of human-Fc negative control (middle panel) or with no human Fc present (bottom panel) (n = 4 cultures, 2 replicates each).

Extended Data Figure 2 Presynaptic measures for dopaminergic and GABAergic transmission.

a, Voltammetric monitoring of extracellular [DA]o in the CPu and NAc of striatal slices. Frequency dependence (5 p-to-1 p ratio) of evoked [DA]o in WT and mutant Cntnap4 mice (HET/KO) was not different across frequencies (5, 10, 15, 50, 100 Hz; n = 4 per genotype; two-way ANOVA, post-hoc Bonferroni test). b, Cumulative distributions of spontaneous inhibitory postsynaptic current (sIPSC) amplitude and rise time recorded from P17–P21 control (n = 8 cells) and Cntnap4 KO (n = 7 cells) layer 2/3 pyramidal cells in vitro. Kolmogorov–Smirnov test used for statistical analysis. c, Series of evoked synaptic IPSCs recorded in RS pyramidal cells at 20 Hz (below) in HET (right) versus KO Cntnap4 mice (left). Average trace in red with black individual traces (10 sweeps). d, Plots showing the paired pulse ratios calculated for the first and second and the first and fifth responses in the synaptic train (HET: n = 4 brains, n = 8 cells; KO: n = 4 brains, n = 11 cells; unpaired t-test used to compare groups statistically) e, Loss of Cntnap4 does not alter pre-synaptic calcium channel type dependence for synaptic release. IPSCs completely blocked by the P/Q-type blocker ω−agatoxin (100 μM), not altered by the N-type blocker ω−conotoxin (200 μM) (n = 3 brains, n = 3 cells).

Extended Data Figure 3 FS to pyramidal cell synaptic transmission deficits persist into adulthood in Cntnap4 KO mice by in vitro slice physiology and mild epileptiform-like discharges observed in vivo under anaesthesia.

a, Examples of FS evoked IPSCs from adult Cntnap4 WT and KO mice (P60–P90), showing that the latter remain immature (unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.005. WT: n = 2 brains, n = 10 pairs; KO: n = 1 brain; n = 4 pairs). b, Graph depicting the number of LFP spikes per minute over the time course of the in vivo recordings of 3 wild type (WT: black circle, triangle and diamond) and 3 knock out (KO: red circle, triangle and diamond) adult mice. Time 0 is the time a large injection of ketamine/xylazine was given, bringing the animal back into deep anaesthesia. Example traces from LFP signals of a WT and a KO mouse taken sequentially under deep, light and deep anaesthesia are shown underneath (asterisks mark spikes). Bar graph of the average number of LFP spikes per min shows absence of spikes in WT animals. Calculated relative power (relative power = band power/total power) for delta (0.5–4 Hz) and gamma (20–80 Hz) frequency bands are shown underneath for light and deep anaesthesia. No statistically significant effect of genotype on the relative power in either frequency band was detected (three-way ANOVA performed with animal ID as factor within genotype on gamma and delta power).

Extended Data Figure 4 Intrinsic electrophysiological properties, morphology and localization of postsynaptic GABAA receptors in Cntnap4 WT and KO mice.

a, No differences were detected in the passive or active membrane properties of fast spiking (FS) basket cells in the Cntnap4 HET versus Cntnap4 KO mice. (WT: n = 2 brains, n = 7 cells; HET: n = 6 brains, n = 10 cells; KO: n = 4 brains, n = 9 cells; ANOVA with post hoc Tukey’s test used to compare groups statistically). b, Reconstruction of a layer 5 Cntnap4 KO FS cell. Soma in black, dendrites in blue and axon in red. c, Images of hippocampal CA1 pyramidal cell layer from Cntnap4 HET and KO mice, showing normal perisomatic labelling of parvalbumin-positive terminals. The images were also stained for eGFP (Cntnap4) staining. The closed dotted lines show the position of cell somata. d, Representative blots of GABAA-α1, GABAA-γ2, gephyrin, PSD 95 and N-cadherin of various brain fractions. Bar graphs showing GABAA-α1 levels quantified and normalized to gephyrin, PSD-95 and N-cadherin loading controls. Also shown are GABAA-γ2 levels quantified and normalized to gephyrin, PSD-95 and N-cadherin loading controls (n = 3 biological replicates).

Extended Data Figure 5 Ultrastructural analysis of excitatory synapses between WT and KO animals.

a, Representative electron micrographs of Cntnap4 WT and KO excitatory synapses at ×57 000 magnification. b, c, Postsynaptic density length (b) and cleft width (c) of excitatory synapses in the SSBF1 of Cntnap4 WT and KO mice. A statistically significant difference in cleft was observed in Cntnap4 KO compared to the WT mice (P = 0.0089). PSD length of excitatory synapses however, was unchanged between KO and WT. d, Dot plot comparison of inhibitory versus excitatory synapses across WT and KO. The relative effect of Cntnap4 loss is much more pronounced in inhibitory synapses. e, This effect is also readily apparent when the data sets are represented by culmulative distribution. (d, e, **P < 0.01; ***P < 0.001, Kolmogorov–Smirnov test for width). (b–e: n = 2 brains for each genotype; width: WT n = 93 synapses; KO n = 124 synapses; length: WT n = 119 synapses; KO n = 143 synapses).

Extended Data Figure 6 Human genetics data implicating CNTNAP4 in neuropsychiatric disorders.

a, Novel and published CNVs present in the CNTNAP4 locus on human chromosome 16. We identified eight new cases of human individuals with neuropsychiatric disorders (2 with schizophrenia, 4 with ASD and 2 with ADHD). Six of these individuals had CNVs in the second intron of the gene (top), whereas two had larger exonic deletions in CNTNAP4 (bottom). Previously reported cases of deletions (red striped) or duplications (blue striped) within the gene are presented underneath. Green bars depict CNVs in the CNTNAP4 gene and proximal regions on either side of it found in control non-afflicted individuals. b, Two CASPR4 (CNTNAP4) single nucleotide polymorphisms (SNPs) associating with schizophrenia (SCZ) were found to have gene-wide significant association (rs7185429 and rs7201297). The region containing the two SNPs is shown below in light blue (Schizophrenia-GWAS-SNP). The plot depicts the association P values of SNPs within CNTNAP4 in SCZ families (top). Both SNPs reside in a region predicted to be regulatory by the ESPERR Regulatory Potential program (http://www.genome.ucsc.edu). ESPERR regulatory potential based on 7 species (bottom).

Extended Data Figure 7 Identification of a heterozygous deletion of CNTNAP4.

a, All family members, except the grandfather with Asperger syndrome, were genotyped using the Illumina Human Omni 1 SNP array. The patient with Asperger syndrome (105-001) and his mother were carrying a 191 kb deletion on chromosome 16q13.3 including the 3 last exons of CNTNAP4 and the AK057218 gene. Based on informative SNPs located within the deletion, we ascertained that the deletion was on the grandfather’s chromosome. This grandfather was diagnosed with Asperger syndrome, but DNA was not available to ascertain if he was carrying the deletion or if the deletion appeared de novo in his daughter (105-003). Each dot shows Log R Ratio (LRR; in red), the B allele frequency (BAF; in green) and the copy number (CN; in blue). b, The CNTNAP4 deletion was validated by quantitative PCR. Results obtained on the genomic DNA from the proband (105-001), his parents, and two controls confirmed that the deletion was inherited from the mother and removed the 3 last exons of CNTNAP4. Bars represent mean of RQ ± s.e.m. c, Primers used for the CNTNAP4 CNV validation by quantitative PCR.

Extended Data Figure 8 Identification and validation of CNTNAP4 intronic and exonic deletions in individuals with neuropsychiatric disorders.

a, CNTNAP4 chromosome 16 (chr16): 74,482,036–75,589,757 with Illumina Infinium Human 550K SNPs coverage displayed as dark blue lines across the top. CNVs are shown in red for hemizygous deletions. All intron II deletions in CNTNAP4 in the six cases are listed first, followed by two larger duplications and two deletions affecting CNTNAP4 previously reported in the literature. b, All CNV calls in cases shown in a were positively validated by TaqMan copy number assay. c, An heterozygous CNTNAP4 deletion was identified with the cytoSNP array from Illumina in family II. An individual diagnosed with autism and mild intellectual disability possesses a heterozygous deletion, maternally inherited, which spans 916.2 kb on chromosome 16q23.1 (hg19, 75,766,089–76,682,263), and includes all exons of CNTNAP4 (delineated by the orange square). The upper plot shows B allele frequency (in blue) and the lower plot shows Log R Ratio (in red).

Extended Data Figure 9 Behavioural tests in Cntnap4 mice.

a, b, No major changes in anxiety levels were observed in the mutant mice as indicated by time and distance spent in periphery versus centre in open field arena (OFA) (WT, n = 14; HET, n = 13; KO, n = 10). c, No major changes observed in extent of marble burying in the mutant versus control mice (WT, n = 17; HET, n = 33; KO, n = 14). d–f, No difference was found in the time spent in the open or closed arms, total distance travelled or centre crossings in the elevated plus maze (EPM) between mutants and control animals (WT, n = 14; HET, n = 12; KO, n = 11). g, Grooming tracks with the Cntnap4 mutant allele. A series of representative images of Cntnap4 mutant mice (Het and KO) cross-fostered by Swiss Webster wild type (SW WT) dam or SW WT mice cross-fostered onto Cntnap4 mutant dam. Grooming status documented at two different ages: just after weaning (P22) and at P76. At P22, presence or absence of allo-grooming by the mother is apparent, whereas at P76 the presence or lack of whiskers depends on the mouse’s genotype. See Methods for more detail.

Extended Data Figure 10 Drug effects on spontaneous IPSCs, as well as PPI and locomotion.

a, Haloperidol administration does not lead to a significant reduction in locomotion in Cntnap4 HET mice as measured by the total distance travelled in an open field arena (OFA) (vehicle n = 6 mice; low haloperidol n = 6 mice; high haloperidol n = 3 mice; ANOVA used for statistical analysis). b, Percentage of pre-pulse inhibition (PPI) for each genotype in control and under haloperidol and indiplon administration for a series of pre-pulses (74,82,90 dB). Below, same data re-organized by drug regimen in each genotype group. c, Effect of indiplon on amplitude, charge and half-width of proximal and perisomatic spontaneous IPSCs recorded from layer 2/3 P23–25 pyramidal cells of KO (n = 3 brains, n = 6 cells) and HET (n = 1 brain, n = 3) mice in vitro, unpaired t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

Supplementary information

Supplementary Information

This file contains Supplementary Results and Supplementary Table 1. (PDF 261 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Karayannis, T., Au, E., Patel, J. et al. Cntnap4 differentially contributes to GABAergic and dopaminergic synaptic transmission. Nature 511, 236–240 (2014). https://doi.org/10.1038/nature13248

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature13248

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing