Developmental changes in electrophysiological properties of auditory cortical neurons in the Cntnap2 knockout rat

Disruptions in the CNTNAP2 gene are known to cause language impairments and symptoms associated with autism spectrum disorder (ASD). Importantly, knocking out this gene in rodents results in ASD-like symptoms that include auditory processing deficits. This study used in vitro patch-clamp electrophysiology to examine developmental alterations in auditory cortex pyramidal neurons of Cntnap2-/- rats, hypothesizing that CNTNAP2 is essential for maintaining intrinsic neuronal properties and synaptic wiring in the developing auditory cortex. Whole cell patch-clamp recordings were conducted in wildtype and Cntnap2-/- littermates at three postnatal age ranges (P8-12, P18-21, and P70-90). Consistent changes across age were seen in all measures of intrinsic membrane properties and spontaneous synaptic input. Intrinsic cell properties such as action potential half-widths, rheobase, and action-potential firing frequencies were different between wildtype and Cntnap2-/- rats predominantly during the juvenile stage (P18-21), whereas adult Cntnap2-/- rats showed higher frequencies of spontaneous and mini postsynaptic currents (sPSCs; mPSCs), with lower sPSC amplitudes. These results indicate that intrinsic cell properties are altered in Cntnap2-/- rats during the juvenile age, leading to a hyperexcitable phenotype during this stage of synaptic remodeling and refinement. Although intrinsic properties eventually normalize by reaching adulthood, changes in synaptic input, potentially caused by the differences in intrinsic membrane properties, seem to manifest in the adult age and are presumably responsible for the hyperreactive behavioral phenotype. In conjunction with a previous study, the present results also indicate a large influence of breeding scheme, i.e., pre- or postnatal environment, on the impact of Cntnap2 on cellular physiology. NEW & NOTEWORTHY This study shows that neurons in the auditory cortex of Cntnap2 knockout rats are hyperexcitable only during the juvenile age, whereas resulting changes in synaptic input persist in the adult. In conjunction with a previous study, the present results indicate that it is not the genes alone, but also the influence of pre- and postnatal environment, that shape neuronal function, highlighting the importance of early intervention in neurodevelopmental disorder

Hyperexcitable and immature-like neuronal activity in the auditory cortex of adult rats lacking the language-linked CNTNAP2 gene.

The contactin-associated protein-like 2 gene, CNTNAP2, is a highly penetrant risk gene thought to play a role in the genetic etiology of language-related disorders, such as autism spectrum disorder and developmental language disorder. Despite its candidacy for influencing language development, few preclinical studies have examined the role of CNTNAP2 in auditory processing. Using in vivo and in vitro electrophysiological recordings in a rat model with translational validity, we report that a loss of the Cntnap2 gene function caused immature-like cortical evoked potentials, delayed multiunit response latencies to acoustic stimuli, impaired temporal processing, and led to a pattern of hyperexcitability in both multiunit and single cell recordings in adulthood. These collective results provide direct evidence that a constitutive loss of Cntnap2 gene function in rats can cause auditory processing impairments similar to those seen in language-related human disorders, indicating that its contribution in maintaining cortical neuron excitability may underlie the cortical activity alterations observed in Cntnap2-/- rats

Differences in Startle and Prepulse Inhibition in Contactin-associated Protein-like 2 Knock-out Rats are Associated with Sex-specific Alterations in Brainstem Neural Activity

The contactin-associated protein-like 2 (CNTNAP2) gene encodes for the CASPR2 protein, which plays an essential role in neurodevelopment. Mutations in CNTNAP2 are associated with neurodevelopmental disorders, including autism spectrum disorder and schizophrenia. Rats with a loss of function mutation in the Cntnap2 gene show increased acoustic startle response (ASR) and decreased prepulse inhibition (PPI). The neural basis of this altered auditory processing in Cntnap2 knock-out rats is currently unknown. Auditory brainstem recordings previously revealed no differences between the genotypes. The next step is to investigate brainstem structures outside of the primary auditory pathway that mediate ASR and PPI, which are the pontine reticular nucleus (PnC) and pedunculopontine tegmentum (PPTg), respectively. Multi-unit responses from the PnC and PPTg in vivo of the same rats revealed sex-specific effects of loss of CASPR2 expression on PnC activity, but no effects on PPTg activity. Female Cntnap

Characterizing The Auditory Processing Alterations At The Electrophysiological Neuronal Level In The Cntnap2-/- Rat Model Of Autism

The contactin-associated protein-like 2 (CNTNAP2) gene is an important developmental gene that, when mutated, is known to result in a developmental disorder with the core symptoms of autism spectrum disorder (ASD). In rodents, the deletion of Cntnap2 results in ASD-like phenotypes including hyperreactivity to sensory stimuli, particularly acoustic stimuli. Although auditory information processing is shown to be altered in Cntnap2-/- rats through behavioral tests, there is a lack of understanding of how Cntnap2 deletion affects neuronal and synaptic function in the brain areas responsible for relaying and processing auditory information. Therefore in this thesis, I explored the electrophysiological changes due to Cntnap2 deletion in multiple levels of the auditory system, from the peripheral brainstem level to higher processing at the cortical level. First, I aimed to understand the neuronal changes underlying the increased acoustic startle and reduced habituation in Cntnap2-/- rats, which is also commonly seen in people with ASD. As startle and its habituation involve synaptic plasticity in auditory and trigeminal afferents that innervate giant neurons located in the caudal pontine reticular nucleus (PnC), I hypothesized this synaptic plasticity would be impaired in Cntnap2-/- rats. Indeed, I found a reduction in synaptic depression, which is likely the mechanism for disrupted habituation in Cntnap2-/- rats. Next, changes in the excitatory neurons in the auditory cortex were assessed in Cntnap2-/- rats at multiple developmental timepoints, in order to understand how Cntnap2 deletion impacts the developmental trajectory of higher order auditory processing. Cntnap2-/- neurons were hyperexcitable at the juvenile age (post-natal day 18-21), but most differences were ameliorated by the adult age. Finally, to assess the environmental effect on the penetrance of Cntnap2 deletion, I employed different breeding strategies where the wildtype and Cntnap2-/- rats were obtained through homozygous breeding (Cntnap2-/- x Cntnap2-/-) or heterozygous breeding (Cntnap2+/- x Cntnap2+/-), which influence the rearing environment. Auditory cortical neurons in adult Cntnap2-/- rats from homozygous breeding were more immature and hyperexcitable than those from heterozygous breeding. Overall the findings in my thesis show that Cntnap2 deletion results in neuronal alterations at multiple levels of auditory processing that likely underly the behavioral changes like increased reactivity and reduced habitation to sound. They also show that these neuronal properties are malleable through environmental changes during development

Loss of Cntnap2 in the Rat Causes Autism-Related Alterations in Social Interactions, Stereotypic Behavior, and Sensory Processing

Autism spectrum disorder (ASD) is characterized by social interaction and communication impairments, as well as restrictive/repetitive patterns of behavior, interests or activities, which can coexist with intellectual disability and altered sensory processing. To study the mechanisms underlying these core features of ASD, preclinical research has developed animal models with manipulations in ASD-linked genes, such as CNTNAP2. In order to fully interpret the findings from mechanistic studies, the extent to which these models display behaviors consistent with ASD must be determined. Toward that goal, we conducted an investigation of the consequences of a functional loss of Cntnap2 on ASD-related behaviors by comparing the performance of rats with a homozygous or heterozygous knockout of Cntnap2 to their wildtype littermates across a comprehensive test battery. Cntnap2−/− rats showed deficits in sociability and social novelty, and they displayed repetitive circling and hyperlocomotion. Moreover, Cntnap2−/− rats demonstrated exaggerated acoustic startle responses, increased avoidance to sounds of moderate intensity, and a lack of rapid audiovisual temporal recalibration; indicating changes in sensory processing at both the pre-attentive and perceptual levels. Notably, sensory behaviors requiring learned associations did not reveal genotypic differences, whereas tasks relying on automatic/implicit behaviors did. Ultimately, because these collective alterations in social, stereotypic, and sensory behaviors are phenotypically similar to those reported in individuals with ASD, our results establish the Cntnap2 knockout rat model as an effective platform to study not only the molecular and cellular mechanisms associated with ASD, but also the complex relationship between altered sensory processing and other core ASD-related behaviors. Lay Summary: Autism spectrum disorder (ASD) is characterized by social interaction differences, and restrictive/repetitive patterns of behavior. We studied the behavioral alterations caused by the loss of an autism-linked gene, Cntnap2, in the rat to determine how mutations in this gene contribute to autism-related behaviors. We show the loss of Cntnap2 leads to changes in social, stereotypic, and sensory behaviors, indicating this rat model can be used to better understand the brain changes underlying ASD. Autism Res 2020, 13: 1698–1717. © 2020 International Society for Autism Research and Wiley Periodicals LLC