The ribbon synapses of auditory inner hair cells (IHCs) undergo morphological and electrophysiological transitions during cochlear development. Here we report that myosin VI (Myo6), an actin-based motor protein involved in genetic forms of deafness, is necessary for some of these changes to occur. By using post-embedding immunogold electron microscopy, we showed that Myo6 is present at the IHC synaptic active zone. In Snell's waltzer mutant mice, which lack Myo6, IHC ionic currents and ribbon synapse maturation proceeded normally until at least post-natal day 6. In adult mutant mice, however, the IHCs displayed immature potassium currents and still fired action potentials, as normally only observed in immature IHCs. In addition, the number of ribbons per IHC was reduced by 30%, and 30% of the remaining ribbons were morphologically immature. Ca²⁺-dependent exocytosis probed by capacitance measurement was markedly reduced despite normal Ca²⁺ currents and the large proportion of morphologically mature synapses, which suggests additional defects, such as loose Ca²⁺-exocytosis coupling or inefficient vesicular supply. Finally, we provide evidence that Myo6 and otoferlin, a putative Ca²⁺ sensor of synaptic exocytosis also involved in a genetic form of deafness, interact at the IHC ribbon synapse, and we suggest that this interaction is involved in the recycling of synaptic vesicles. Our findings thus uncover essential roles for Myo6 at the IHC ribbon synapse, in addition to that proposed in membrane turnover and anchoring at the apical surface of the hair cells.

Gastrointestinal (GI) problems constitute an important comorbidity in many patients with autism. Multiple mutations in the neuroligin family of synaptic adhesion molecules are implicated in autism, however whether they are expressed and impact GI function via changes in the enteric nervous system is unknown. We report the GI symptoms of two brothers with autism and an R451C mutation in Nlgn3 encoding the synaptic adhesion protein, neuroligin‐3. We confirm the presence of an array of synaptic genes in the murine GI tract and investigate the impact of impaired synaptic protein expression in mice carrying the human neuroligin‐3 R451C missense mutation (NL3^R451C). Assessing in vivo gut dysfunction, we report faster small intestinal transit in NL3^R451C compared to wild‐type mice. Using an ex vivo colonic motility assay, we show increased sensitivity to GABA_A receptor modulation in NL3^R451C mice, a well‐established Central Nervous System (CNS) feature associated with this mutation. We further show increased numbers of small intestine myenteric neurons in NL3^R451C mice. Although we observed altered sensitivity to GABA_A receptor modulators in the colon, there was no change in colonic neuronal numbers including the number of GABA‐immunoreactive myenteric neurons. We further identified altered fecal microbial communities in NL3^R451C mice. These results suggest that the R451C mutation affects small intestinal and colonic function and alter neuronal numbers in the small intestine as well as impact fecal microbes. Our findings identify a novel GI phenotype associated with the R451C mutation and highlight NL3^R451C mice as a useful preclinical model of GI dysfunction in autism. Autism Res 2019, 12: 1043–1056. © 2019 International Society for Autism Research, Wiley Periodicals, Inc.Lay Summary: People with autism commonly experience gastrointestinal problems, however the cause is unknown. We report gut symptoms in patients with the autism‐associated R451C mutation encoding the neuroligin‐3 protein. We show that many of the genes implicated in autism are expressed in mouse gut. The neuroligin‐3 R451C mutation alters the enteric nervous system, causes gastrointestinal dysfunction, and disrupts gut microbe populations in mice. Gut dysfunction in autism could be due to mutations that affect neuronal communication.

Understanding neuronal mechanisms underlying aggression in patients with autism spectrum disorder (ASD) could lead to better treatments and prognosis. The Neuroligin-3 (NL3)^R451C mouse model of ASD has a heightened aggressive phenotype, however the biological mechanisms underlying this behavior are unknown. It is well established that NL3^R451C mice have imbalanced excitatory and inhibitory synaptic activity in the hippocampus and somatosensory cortex. The amygdala plays a role in modulating aggressive behavior, however potential changes in synaptic activity in this region have not previously been assessed in this model. We investigated whether aggressive behavior is robustly present in mice expressing the R451C mutation, following back-crossing onto a congenic background strain. Endocannabinoids influence social interaction and aggressive behavior, therefore we also studied the effects of cannabinoid receptor 1 (CB1) agonist on NL3^R451C mice. We report that NL3^R451C mice have increased amplitude of miniature excitatory postsynaptic currents (EPSCs) with a concomitant decrease in the amplitude of inhibitory postsynaptic currents (IPSCs) in the basolateral amygdala. Importantly, we demonstrated that NL3^R451C mice bred on a C57Bl/6 background strain exhibit an aggressive phenotype. Following non-sedating doses (0.3 and 1.0 mg/kg) of the CB1 receptor agonist WIN55,212-2 (WIN), we observed a significant reduction in aggressive behavior in NL3^R451C mice. These findings demonstrate altered synaptic activity in the basolateral amygdala and suggest that the NL3^R451C mouse model is a useful preclinical tool to understand the role of CB1 receptor function in aggressive behavior.

Individuals diagnosed with neurodevelopmental conditions such as autism spectrum disorder (ASD; autism) often experience tissue inflammation as well as gastrointestinal dysfunction, yet their underlying causes remain poorly characterised. Notably, the largest components of the body’s immune system, including gut-associated lymphoid tissue (GALT), lie within the gastrointestinal tract. A major constituent of GALT in humans comprises secretory lymphoid aggregates known as Peyer’s patches that sense and combat constant exposure to pathogens and infectious agents. Essential to the functions of Peyer’s patches is its communication with the enteric nervous system (ENS), an intrinsic neural network that regulates gastrointestinal function. Crosstalk between these tissues contribute to the microbiota-gut-brain axis that altogether influences mood and behaviour. Increasing evidence further points to a critical role for this signalling axis in neurodevelopmental homeostasis and disease. Notably, while the neuroimmunomodulatory functions for Peyer’s patches are increasingly better understood, functions for tissues of analogous function, such as caecal patches, remain less well characterised. Here, we compare the structure, function and development of Peyer’s patches, as well as caecal and appendix patches in humans and model organisms including mice to highlight the roles for these essential tissues in health and disease. We propose that perturbations to GALT function may underlie inflammatory disorders and gastrointestinal dysfunction in neurodevelopmental conditions such as autism.

A missense mutation (R43Q) in the γ2 subunit of the γ‐aminobutyric acid (GABA)_A receptor is associated with generalized (genetic) epilepsy with febrile seizures plus (GEFS+). Heterozygous GABA_Aγ2(R43Q) mice displayed a lower temperature threshold for thermal seizures as compared to wild‐type littermates. Temperature‐dependent internalization of GABA_Aγ2(R43Q)–containing receptors has been proposed as a mechanism underlying febrile seizure genesis in patients with this mutation. We tested this idea using the GABA_Aγ2(R43Q) knockin mouse model and analyzed GABAergic miniature postsynaptic inhibitory currents (mIPSCs) in acute brain slices after exposure to varying temperatures. Incubation of slices at an elevated temperature increased mIPSC amplitude in neurons from heterozygous mice, with no change seen in wild‐type controls. [³H]Flumazenil binding measured in whole‐brain homogenates from mutant and control mice following elevation of body temperature showed no temperature‐dependent differences in γ2‐containing receptor density. Therefore, in vivo mouse data do not support earlier in vitro observations that proposed temperature‐dependent internalization of γ2 R43Q containing GABA_A receptors as the cellular mechanism underlying febrile seizure genesis in patients with the GABA_Aγ2(R43Q) mutation.