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Abstract

Autism Spectrum Disorders (ASD) present a range of symptoms including abnormal social interactions, increased perseverative behaviors, and difficulty with communication. Despite the many studies on these classical symptoms, many patients also display underestimated motor symptoms. Motor symptoms such as impaired motor learning during eyeblink conditioning, as well as difficulty with eye movements and unstable gait or coordination, are strong evidence for cerebellar involvement in the pathology of ASD. Eyeblink conditioning is dependent on an intact, functional cerebellar circuit. Disrupted eyeblink conditioning in the ASD patient population suggests that ASD is a synaptic disorder, and that the cerebellar circuit is impaired and must be interrogated to understand ASD motor symptoms at a synaptic level (Piochon et al 2014). To examine synaptic changes in ASD, I used the mouse model of the human 15q11-13 copy number variation duplication. The human 15q11-13 chromosomal region is syntenic to part of mouse chromosome 7. In these mice, mouse chromosome 7 is duplicated to simulate the autistic-like phenotype. The 15q11-13 model represents the most frequent and penetrant genetic aberration in ASD and is seen in approximately 1-3% of patients. Patients inheriting this duplication maternally show symptoms of ASD, while patients receiving the genetic aberration paternally either do not show symptoms, or show them to a lesser degree. In the mouse model, the opposite is true; mice inheriting the duplication paternally (patDp/+), but not maternally (matDp/+), show autistic-like symptoms. It is known that long-term depression at parallel fiber (PF) – Purkinje cell synapses (PF-LTD), which is a synaptic plasticity mechanism underlying eyeblink conditioning, is impaired in patDp/+ mice (Piochon et al 2014). To determine which part of the PF-LTD induction protocol failed, I measured PF-evoked excitatory postsynaptic potentials (EPSPs) and climbing fiber (CF) – evoked excitatory postsynaptic currents (EPSCs) with patch clamp electrophysiology. I found that in patDp/+ mice, PF-EPSP amplitudes were decreased, while CF-EPSC amplitudes were enhanced, relative to wild-type (WT) littermates. Immunohistochemistry for the vesicular glutamate transporter at CF-Purkinje cell synapses (VGluT2), confirmed that CFs make more synapses onto Purkinje cells in patDp/+ mice than in WT littermates. Importantly, these CF-Purkinje cell synapses, which are typically found on large caliber dendrites, were ectopically located on fine dendrites in patDp/+ mice. Fine dendrites are generally considered territory for PF-Purkinje cell synapses (Palay & Chan-Palay 1974, Ichikawa et al 2002, Watanabe et al 2008, Miyazaki et al 2010). To explore whether this territorial change affected calcium signaling through PF-Purkinje cell and CF-Purkinje cell synapses, I performed spine calcium imaging at PF spines and measured transients resulting from three stimulation protocols: 100 Hz PFb, CF, and 100 Hz PFb+CF (PF-LTD induction protocol). Notably, I found that PF-evoked calcium transients were significantly reduced in amplitude in patDp/+ mice. Weak calcium signaling at PF-Purkinje cell synapses suggests that PF signaling is too weak to induce PF-LTD in patDp/+ mice. To test whether PF-evoked calcium signaling was insufficient to induce PF-LTD, we performed LTD experiments in WT mice, patDp/+ mice – which leads to potentiation instead of depression, and patDp/+ mice with ACSF containing 300 µm CX546. CX546 modulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors to promote prolonged depolarization, which causes voltage-gated calcium channels to open. Effectively, CX546 increases postsynaptic calcium influx. The addition of CX546 sufficiently increased calcium entry to Purkinje cells in patDp/+ mice during the PF-LTD induction protocol and successfully restored PF-LTD in patDp/+ mice. Finally, the 15q11-13 chromosomal region contains approximately 20 genes. I sought to identify the involvement of these genes in ASD symptoms. I explored ubiquitin-ligase E3A, (Ube3a), a gene that when deleted is responsible for Prader-Willi and Angelman Syndromes (Albrecht et al 1997) and found that Ube3a is upregulated in patDp/+ mice relative to WT littermates. Additionally, I explored cytoplasmic FMR1-Interacting Protein 1 (CYFIP1) using mice that globally overexpress (OE) CYFIP1 because the protein is upregulated in ASD patients. CYFIP1 OE mice displayed a slight enhancement in the amplitude of CF-EPSCs. Continued experiments on both mouse models will lead to better understanding of ASD as a synaptic disorder that involves motor pathology. Examination of motor pathology may lead to earlier diagnosis of ASD and earlier behavioral therapeutic interventions.

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