Autism spectrum disorder (ASD) is a behaviorally defined developmental disorder with a strong genetic component [1, 2]. The identification of genetic risk factors such as common genetic variants, rare inherited and de novo mutations have lead to the implication of hundreds of different genes [3, 4]. These findings illustrate the complexity and heterogeneity of the genetic architecture of ASD.

Subsequently, mouse models are being used to unravel the functional impact of implicated genes on ASD phenotypes in a controlled genetic and environmental background. However, knowledge of the impact on cognitive and behavioral development of the majority of these genes is missing or incomplete [5], and most behavioral characterizations of animal models are limited to adult phenotypes disregarding the developmental nature of ASD [6].

Here, we characterize the impact of contactin 4 (Cntn4) null mutation on behavioral development, using our recently developed longitudinal test battery for mice that tests a wide array of neurological, cognitive, and behavioral parameters across development starting from 3 weeks of age [6].

CNTN4 is an axonal glycoprotein belonging to the contactin family, a six-member subgroup of the immunoglobulin superfamily of cell adhesion molecules [7]. CNTN4 is known to act as an axon guidance molecule in the establishment of olfactory neural circuitry during neural development [8] and promotes target-specific axon arborization of a subset of retinal ganglion cells onto the nucleus of the optic tract [9]. Knowledge of the neurobiological functions of CNTN4 in normal and abnormal development of brain systems are far from complete.

The CNTN4 gene has been implicated in ASD due to its presence in the genetic locus of the 3p-deletion syndrome, a mental retardation syndrome [10]. Subsequently, evidence for a role of CNTN4 has been accumulated [11–14] but has also been questioned [15, 16]. Three cases carrying a copy number variant (CNV) in the CNTN4 gene were reported by the Autism Genome Project Consortium (AGP) [12]. Deletions and duplications in the CNTN4 gene or its promoter region were found in 10 families of the Autism Genetic Resource Exchange (AGRE) collection [13]. In the Autism Case-Control cohort (ACC), deletion in the promoter region of CNTN4 was found in three cases but not in controls [13]. Association of CNTN4 with developmental disorders such as ASD further seems supported by the protein’s neurobiological functions [2, 7].

Follwoing these observations, we performed a careful longitudinal functional characterization of homozygous and heterozygous disruptions of Cntn4 to resolve the impact of this gene on behavioral and cognitive development.

We present a comprehensive assessment of the impact of the ASD candidate gene CNTN4 on a variety of neurological, behavioral, and cognitive aspects of development. We found no effect of Cntn4 deficiency on ASD-related behavioral mouse paradigms such as the juvenile social interaction test, the three-chamber test, grooming behavior, and sensorimotor coordination. We also did not observe developmental neurological, behavioral, or cognitive abnormalities in the extended SHIRPA screen.

The reversal-learning phase of the set-shifting task yielded an inconclusive result, as no shift cost was observed in wild-type animals for the number of trials to reach criterion. Indeed, the genotype difference that was observed in the number of trials to reach the criterion in the reversal-learning phase was rather caused by lack of shift cost in wild-type animals than a reversal-learning deficit in the Cntn4 ^−/− mice. In line with this reasoning, when we compared the outcome of Cntn4 ^−/− mice in the reversal-learning phase with the results obtained for C57BL/6J mice obtained in a previous study, we find no genotype difference in this phase of the set-shifting task [6]. In the Barnes maze reversal-learning paradigm, we also did not find a reversal-learning deficit. Based on the summary of these data, we conclude that Cntn4 disruption has no effect on cognitive flexibility.

Non-ASD-specific effects at adult ages were indicated by increased startle response to auditory stimuli of different high amplitudes and by faster escape hole finding during subsequent days of acquisition in the Barnes maze.

Hyperresponsivity to acoustic stimuli is related to many neurodevelopmental disorders and has also been reported in ASD [22]. In addition, acoustic hyperresponsivity in patients with fragile X syndrome is known to be consistent with animal model data [23]. Similar to our findings, an increased startle response to acoustic stimuli was recently described in children with ASD [24], although these were in response to weak stimuli in contrast to the high amplitudes we found. The observed hyperresponsivity in Cntn4-deficient mice was unrelated to anxiety levels, as mice showed similar exploratory behaviors in classical anxiety behavioral tests, such as the elevated plus maze and open field. Although speculative, the increased startle response together with the enhanced acquisition in the Barnes maze could indicate that Cntn4 deficiency in the C57BL/6J background leads to a state of increased behavioral responsiveness without overt anxiety or avoidance behavior in mice [25].

Together, the findings show that in the C57BL/6J background, disruption of Cntn4 does not lead to substantial behavioral defects related to autistic development. A limited behavioral penetrance of CNTN4 mutations on autistic development may be consistent with a recent study that revisited the association of contactins, including CNTN4, with ASD [16]. The behavioral phenotype of Cntn4 mice contrasts other previously studied genetic ASD models [18]. For instance, Shank3 or Pten mice show extensive impairments in social interaction and sensorimotor phenotypes and are therefore regarded as translational models for ASD [26–28].

Although ASD-related phenotypes in Cntn4-deficient mice were observed, our findings do have relevance for neurodevelopmental disorder research. The specific phenotypes observed in Cntn4-deficient mice may be used to study the mechanisms underlying increased responsiveness or vigilance, a trait observed across many different human disorders such as attention-deficit hyperactivity disorder, post-traumatic stress disorder, and schizophrenia [29–31]. Cntn4-deficient mice may serve as a model to study the mechanistic underpinnings of behavioral states in which vigilance is altered. Indeed, common single-nucleotide variants in the CNTN4 locus have recently been associated with other neuropsychiatric disorders, such as schizophrenia [32], perhaps pointing to a non-disorder specific contribution of this cell adhesion gene in neuropsychiatric pathogenesis. In addition, a role for Cntn4, was recently shown in target-specific arborization during development of the accessory optic system [9]. Our study shows the importance of detailed developmental neurological, behavioral, and cognitive characterization of genetic animal models to complement human genetic studies in ASD and related disorders.

In our test battery, disruption of Cntn4, a prominent ASD candidate gene, had no effect on cognitive and behavioral development or ASD-specific phenotypes. At adult age, we could detect an effect of Cntn4 disruption on an adult sensory behavioral and a spatial cognitive feature.