Abstract:
Touch is essential for interacting with the world, and atypical tactile experience is a core feature of autism that profoundly affects daily life. However, we do not know the neural mechanisms of low-level tactile perception and their alterations in autism. Using a translational forepaw-based perceptual task, we recapitulate the multifaceted tactile features of autistic individuals in the Fmr1(-/y) mouse model of autism, showing reduced detection of low-level vibrotactile stimuli, interindividual variability, and unreliable responses. We reveal that impaired detection decoding in Fmr1(-/y)-hyposensitive mice stems from diminished single-neuron signal-to-noise ratio within layers 2/3 of the primary somatosensory cortex that contributes to weak population encoding of the tactile stimulus and its detection. This manifests as reduced stimulus-dependent neural recruitment, impaired response precision, and disrupted ensemble dynamics. Decreasing neuronal excitability strengthens sensory encoding and restores tactile perception. This work provides a translational framework for probing neuronal-perceptual changes in neurodevelopmental conditions, reveals inter-individual variability in preclinical models, and uncovers the neural basis of tactile hyposensitivity in autism.

Abstract:
Neocortical memory engrams are thought to mature via strengthened interconnectivity, yet synaptic plasticity alone cannot explain the dynamic vividness of enduring memories. Neuronal intrinsic excitability (IE) plasticity has been touted as an early priming mechanism that renders engram neurons susceptible to ongoing plastic processes and later encoding events. Here, we reveal that learning-induced IE plasticity of nascent anterior cingulate cortex (ACC) engram neurons is a permissive mechanism for the formation and specificity of remote memories. Using c-fos-dependent genetic/viral targeting in mice, we found that contextual fear learning triggered a time-limited increase in ACC engram IE during the early phase of memory formation. Remarkably, chemogenetically hyperpolarizing these neurons within-but not outside-the IE plasticity window strengthened consolidated memories, enhanced their context-precision, and prevented interference-induced engram reallocation. Thus, IE plasticity in nascent ACC engram neurons acts as an essential tagging mechanism that determines the fate and dynamic content of remote memories.

Abstract:
Excessive neural variability of sensory responses is a hallmark of atypical sensory processing in autistic individuals with cascading effects on other core autism symptoms but unknown neurobiological substrate. Here, by recording neocortical single neuron activity in a well-established mouse model of Fragile X syndrome and autism, we characterized atypical sensory processing and probed the role of endogenous noise sources in exaggerated response variability in males. The analysis of sensory stimulus evoked activity and spontaneous dynamics, as well as neuronal features, reveals a complex cellular and network phenotype. Neocortical sensory information processing is more variable and temporally imprecise. Increased trial-by-trial and inter-neuronal response variability is strongly related to key endogenous noise features, and may give rise to behavioural sensory responsiveness variability in autism. We provide a novel preclinical framework for understanding the sources of endogenous noise and its contribution to core autism symptoms, and for testing the functional consequences for mechanism-based manipulation of noise.

Abstract:
Metabotropic glutamate receptor subtype 5 (mGluR5) is crucially implicated in the pathophysiology of Fragile X Syndrome (FXS); however, its dysfunction at the sub-cellular level, and related synaptic and cognitive phenotypes are unexplored. Here, we probed the consequences of mGluR5/Homer scaffold disruption for mGluR5 cell-surface mobility, synaptic N-methyl-D-aspartate receptor (NMDAR) function, and behavioral phenotypes in the second-generation Fmr1 knockout (KO) mouse. Using single-molecule tracking, we found that mGluR5 was significantly more mobile at synapses in hippocampal Fmr1 KO neurons, causing an increased synaptic surface co-clustering of mGluR5 and NMDAR. This correlated with a reduced amplitude of synaptic NMDAR currents, a lack of their mGluR5-activated long-term depression, and NMDAR/hippocampus dependent cognitive deficits. These synaptic and behavioral phenomena were reversed by knocking down Homer1a in Fmr1 KO mice. Our study provides a mechanistic link between changes of mGluR5 dynamics and pathological phenotypes of FXS, unveiling novel targets for mGluR5-based therapeutics.

Abstract:
In fragile X syndrome (FXS), sensory hypersensitivity and impaired habituation is thought to result in attention overload and various behavioral abnormalities in reaction to the excessive and remanent salience of environment features that would normally be ignored. This phenomenon, termed sensory defensiveness, has been proposed as the potential cause of hyperactivity, hyperarousal, and negative reactions to changes in routine that are often deleterious for FXS patients. However, the lack of tools for manipulating sensory hypersensitivity has not allowed the experimental testing required to evaluate the relevance of this hypothesis. Recent work has shown that BMS-204352, a BKCa channel agonist, was efficient to reverse cortical hyperexcitability and related sensory hypersensitivity in the Fmr1-KO mouse model of FXS. In the present study, we report that exposing Fmr1-KO mice to novel or unfamiliar environments resulted in multiple behavioral perturbations, such as hyperactivity, impaired nest building and excessive grooming of the back. Reversing sensory hypersensitivity with the BKCa channel agonist BMS-204352 prevented these behavioral abnormalities in Fmr1-KO mice. These results are in support of the sensory defensiveness hypothesis, and confirm BKCa as a potentially relevant molecular target for the development of drug medication against FXS/ASD.Neuropsychopharmacology advance online publication, 16 August 2017; doi:10.1038/npp.2017.149.

Abstract:
Fragile X syndrome (FXS) is a developmental disorder caused by a mutation in the X-linked FMR1 gene, coding for the FMRP protein which is largely involved in synaptic function. FXS patients present several behavioral abnormalities, including hyperactivity, anxiety, sensory hyper-responsiveness, and cognitive deficits. Autistic symptoms, e.g., altered social interaction and communication, are also often observed: FXS is indeed the most common monogenic cause of autism. Mouse models of FXS are therefore of great interest for research on both FXS and autistic pathologies. The Fmr1-KO2 mouse line is the most recent FXS model, widely used for brain studies; surprisingly, little is known about the face validity of this model, i.e., its FXS-like behavioral phenotype. Furthermore, no data are available for the age-related expression of the pathological phenotypes in this mouse line, a critical issue for modelling neurodevelopmental disorders. Here we performed an extensive behavioral characterization of the KO2 model at infancy, adolescent and adult ages. Hyperactivity, altered emotionality, sensory hyper-responsiveness and memory deficits were already present in KO mice at adolescence and remained evident at adulthood. Alterations in social behaviors were instead observed only in young KO animals: during the first 2 weeks of life, KOs emitted longer ultrasonic vocalizations compared to their WT littermates and as adolescents they displayed more aggressive behaviors towards a conspecific. These results strongly support the face validity of the KO2 mouse as a model for FXS, at the same time demonstrating that its ability to recapitulate social autistic-relevant phenotypes depends on early testing ages. Autism Res 2017. (c) 2017 International Society for Autism Research, Wiley Periodicals, Inc.

Abstract:
Current large-scale approaches in neuroscience aim to unravel the complete connectivity map of specific neuronal circuits, or even the entire brain. This emerging research discipline has been termed connectomics. Recombinant glycoprotein-deleted rabies virus (RABV G) has become an important tool for the investigation of neuronal connectivity in the brains of a variety of species. Neuronal infection with even a single RABV G particle results in high-level transgene expression, revealing the fine-detailed morphology of all neuronal features-including dendritic spines, axonal processes, and boutons-on a brain-wide scale. This labeling is eminently suitable for subsequent post-hoc morphological analysis, such as semiautomated reconstruction in 3D. Here we describe the use of a recently developed anterograde RABV G variant together with a retrograde RABV G for the investigation of projections both to, and from, a particular brain region. In addition to the automated reconstruction of a dendritic tree, we also give as an example the volume measurements of axonal boutons following RABV G-mediated fluorescent marker expression. In conclusion RABV G variants expressing a combination of markers and/or tools for stimulating/monitoring neuronal activity, used together with genetic or behavioral animal models, promise important insights in the structure-function relationship of neural circuits.

Abstract:
Fragile X syndrome (FXS), the most common inherited form of intellectual disability disorder and a frequent cause of autism spectrum disorder (ASD), is characterized by a high prevalence of sensory symptoms. Perturbations in the anatomical connectivity of neocortical circuits resulting in their functional defects have been hypothesized to contribute to the underlying etiology of these disorders. We tested this idea by probing alterations in the functional and structural connectivity of both local and long-ranging neocortical circuits in the Fmr1 (-/y) mouse model of FXS. To achieve this, we combined in vivo ultrahigh-field diffusion tensor magnetic resonance imaging (MRI), functional MRI, and viral tracing approaches in adult mice. Our results show an anatomical hyperconnectivity phenotype for the primary visual cortex (V1), but a disproportional low connectivity of V1 with other neocortical regions. These structural data are supported by defects in the structural integrity of the subcortical white matter in the anterior and posterior forebrain. These anatomical alterations might contribute to the observed functional decoupling across neocortical regions. We therefore identify FXS as a 'connectopathy,' providing a translational model for understanding sensory processing defects and functional decoupling of neocortical areas in FXS and ASD.

Abstract:
Hypersensitivity in response to sensory stimuli and neocortical hyperexcitability are prominent features of Fragile X Syndrome (FXS) and autism spectrum disorders, but little is known about the dendritic mechanisms underlying these phenomena. We found that the primary somatosensory neocortex (S1) was hyperexcited in response to tactile sensory stimulation in Fmr1-/y mice. This correlated with neuronal and dendritic hyperexcitability of S1 pyramidal neurons, which affect all major aspects of neuronal computation, from the integration of synaptic input to the generation of action potential output. Using dendritic electrophysiological recordings, calcium imaging, pharmacology, biochemistry and a computer model, we found that this defect was, at least in part, attributable to the reduction and dysfunction of dendritic h- and BKCa channels. We pharmacologically rescued several core hyperexcitability phenomena by targeting BKCa channels. Our results provide strong evidence pointing to the utility of BKCa channel openers for the treatment of the sensory hypersensitivity aspects of FXS.

Abstract:
Glycoprotein-deleted rabies virus (RABV G) is a powerful tool for the analysis of neural circuits. Here, we demonstrate the utility of an anterograde RABV G variant for novel neuroanatomical approaches involving either bulk or sparse neuronal populations. This technology exploits the unique features of RABV G vectors, namely autonomous, rapid high-level expression of transgenes, and limited cytotoxicity. Our vector permits the unambiguous long-range and fine-scale tracing of the entire axonal arbor of individual neurons throughout the brain. Notably, this level of labeling can be achieved following infection with a single viral particle. The vector is effective over a range of ages (>14 months) aiding the studies of neurodegenerative disorders or aging, and infects numerous cell types in all brain regions tested. Lastly, it can also be readily combined with retrograde RABV G variants. Together with other modern technologies, this tool provides new possibilities for the investigation of the anatomy and physiology of neural circuits.