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Cntnap4 differentially contributes to GABAergic and dopaminergic synaptic transmission

This article has been updated


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.

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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.


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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




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.

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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 (bd, 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). (be: 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 ( 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). df, 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).

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Karayannis, T., Au, E., Patel, J. et al. Cntnap4 differentially contributes to GABAergic and dopaminergic synaptic transmission. Nature 511, 236–240 (2014).

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