The development of mouse models of ASD is crucial to study the disorder at the molecular level, gain insight into disease mechanisms, and test potential pharmacological interventions. Here, we show that the consequences of CNTNAP2 deficiency in the mouse resemble many of the behavioral and cognitive features observed in patients with idiopathic autism and of the pathological features observed in patients with recessive CNTNAP2
mutations that cause a Mendelian form of syndromic autism (Strauss et al., 2006
mice have normal anxiety related responses, visual spatial memory, and sensorimotor integration, but show abnormal vocal communication, repetitive and restrictive behaviors, and abnormal social interactions. In addition, they also show hyperactivity and epileptic seizures, both features described in CDFE patients and in many patients with ASD. Heterozygous animals did not show any of the behavioral (Figure S5
) or neuropathological (data not shown) abnormalities observed in homozygote knockouts, nor did they develop epileptic seizures, consistent with the recessive nature of the pathology in humans.
Autism and epilepsy are neurodevelopmental syndromes with a high frequency of co-occurrence (Geschwind, 2009
), which suggests shared underlying mechanisms. At a molecular level, CNTNAP2 is a single pass transmembrane protein with a short cytoplasmic region involved in clustering K+
channels at juxtaparanodes in myelinated axons (Horresh et al., 2008
), and a long extracellular region that forms a neuron-glia cell adhesion complex with contactin 2 (TAG-1), which is necessary for the proper localization of K+
channels in this structure (Poliak et al., 2003
). Thus, defects in myelination and K+
channel mislocalization at the nodes of Ranvier could theoretically lead to epilepsy in Cntnap2−/−
mice. However, this is not likely the case, since both light microscopic and ultra-structural analysis using electron microscopy in the peripheral and central nerves in CNTNAP2 deficient mice (Poliak et al., 2003
) showed that nodal morphology and myelination were normal. In addition, electrophysiological investigation of nerve conductance revealed no abnormalities in conduction velocity, refractory period or excitability (Poliak et al., 2003
). In the current study, neuropathological analysis of Cntnap2−/−
mice revealed two major mechanisms that have been shown to lead to epilepsy in humans, cortical neuronal migration abnormalities and a reduction in the number of GABAergic interneurons. Whereas neuronal migration abnormalities might have been expected based on observations in patients with CDFE syndrome, the reduction in GABAergic neurons was unexpected, as CNTNAP2 has not been previously associated with GABAergic neuronal function and no such deficit has been demonstrated in CDFE. These data suggest that assessment of interneurons in patients with CDFE would be worthwhile. Further, whether this reduction in interneurons is also due to a migration defect or rather to a defect in neurogenesis, differentiation and/or survival, remains to be elucidated in future work. Nevertheless, the embryonic expression of the gene in the ganglionic eminences and in migrating interneurons supports a role for CNTNAP2 in the early development and migration of these cells.
The large extracellular domain of CNTNAP2 is composed of a number of protein-protein interaction domains common to cell adhesion molecules including laminin G, EGF repeats, and discoidin-like domains (Poliak et al., 1999
). During myelination, CNTNAP2 localizes to the developing nodes of Ranvier where, as previously mentioned, it interacts extracellularly with TAG-1. Interestingly, TAG-1 is also expressed embryonically and blockade of its function results in migration abnormalities of cortical pioneer neurons and GABAergic interneurons (Denaxa et al., 2001
; Morante-Oria et al., 2003
), although normal numbers of interneurons were reported in TAG-1 deficient mice, likely due to compensatory mechanisms (Denaxa et al., 2005
). Thus, analysis of CNTNAP2 interactors, including TAG-1, during embryogenesis could provide insight to the role of CNTNAP2 in neuronal migration.
One physiological consequence of the observed neuropathology caused by Cntnap2
knockout is significantly reduced neuronal synchronization. It is generally accepted that most cognitive functions are based on the coordinated interactions of large neuronal ensembles within and across different specialized brain areas (Uhlhaas and Singer, 2006
). Synchronization determines the pattern of neuronal interactions in a way that effective neuronal connectivity would diminish when synchronization is less precise (Womelsdorf et al., 2007
). A number of functional neuroimaging studies have reported reduced connectivity in ASD (Just et al., 2004
; Villalobos et al., 2005
; Cherkassky et al., 2006
; Damarla, 2010
), supporting the notion that the deficits in cognition and behavior associated with ASD are most likely the result of a developmental disconnection (Geschwind and Levitt, 2007
). Interestingly, we have recently associated common genetic-risk variants in CNTNAP2
with abnormal functional brain connectivity in humans (Scott-Van Zeeland et al., 2010
). Our observations of migration abnormalities and reduced number of GABAergic interneurons in Cntnap2−/−
mice, together with the normal global neuronal activity, as measured by the firing rate and amplitude, suggest an abnormal neuronal circuit architecture as the cause of the asynchronous firing pattern, rather than abnormalities in neuronal function per se
. There are a number of factors that contribute to the precise timing of neural activity (reviewed in Wang, 2010
). Interestingly, GABAergic interneurons, in particular PAVLB+, have been reported to play a crucial role in the rhythmic pacing of cortical neuronal activity (Sohal et al., 2009
). Therefore, further studies analyzing the structure of neuronal networks and interneuron function in Cntnap2−/−
mice may have important implications both for the potential understanding and treatment of ASD.
The ultimate goal of understanding the pathophysiology of the disorder is to develop therapeutic interventions that improve or restore normal brain activity and, ultimately, the associated cognitive and behavioral deficits. Recent studies in mouse models are very encouraging in this regard, including Rett syndrome (Guy et al., 2007
), fragile X syndrome (Dolen et al., 2007
), neurofibromatosis type 1 (Costa et al., 2002
), Down syndrome (Fernandez et al., 2007
) and tuberous sclerosis (Ehninger et al., 2008
). Here, we have shown that risperidone efficiently reduces hyperactivity, motor stereotypies and perseveration in Cntnap2−/−
mice, while having no effect on sociability. This observation provides evidence that different pathways lead to the ASD associated core domains of social and repetitive behavior observed in this mouse model, parallel to the situation in humans, and supports the validity of this mouse model for testing new pharmacological treatments.
Repetitive behavior is recognized to reflect a disruption of the coordinated function of the cortico-striatal circuit (Albin et al., 1989
). Briefly, two main pathways compose this system: the direct pathway, which promotes motor behavior, and the indirect pathway, which inhibits it (Gerfen et al., 1990
). In general, stereotypies indicate an unbalanced activity of this network favoring the direct pathway (Lewis et al. 2007
). At the neuronal level, the ultimate firing output to the direct and indirect pathways is determined by excitatory inputs from cortex and thalamus, and inhibitory inputs from local interneurons within the striatum (Kreitzer and Malenka, 2008
). Interestingly, PALVB+ fast spiking interneurons, which are the main source of inhibitory input in the cortico-striatal circuit, have recently been shown to target mainly the direct pathway (Gittis et al., 2010
). Therefore, the reduced number of this type of interneuron in Cntnap2−/−
mice likely results in over-activation of this pathway, leading to hyperactivity and repetitive behavior. Indeed, risperidone is known to potentiate the indirect pathway, which likely re-balances the activity of this network alleviating these behaviors.
Nest-building has also been shown to be related to the dopaminergic pathway (Szczypka et al., 2001
), and has been reported disrupted in mice with hyperactivity (Kwon et al., 2006
; Zhou et al., 2010
). That risperidone also normalized the nesting ability in KO mice provides additional evidence for abnormal striatal dopaminergic function in this model Exploration of cortico-striatal function in Cntnap2−/−
mice will provide a better understanding of the neural basis of repetitive behavior in ASD. In addition, the dissociation between repetitive and social behavior with regards to treatment response, suggests that Cntnap2−/−
mice will be useful for dissecting the distinct circuitries involved in these core components of autistic related abnormal behavior. Finally, since understanding of CNTNAP2 function was previously focused primarily on postnatal development, these data set a new direction for investigation of CNTNAP2’s role during development and in the formation and function of neuronal circuits.