To uncover the circuit-level alterations that underlie atypical sensory processing associated with autism, we adopted a symptom-to-circuit approach in the Fmr1-knockout (Fmr1) mouse model of Fragile X syndrome. Using a go/no-go task and in vivo two-photon calcium imaging, we find that impaired visual discrimination in Fmr1 mice correlates with marked deficits in orientation tuning of principal neurons and with a decrease in the activity of parvalbumin interneurons in primary visual cortex. Restoring visually evoked activity in parvalbumin cells in Fmr1 mice with a chemogenetic strategy using designer receptors exclusively activated by designer drugs was sufficient to rescue their behavioral performance. Strikingly, human subjects with Fragile X syndrome exhibit impairments in visual discrimination similar to those in Fmr1 mice. These results suggest that manipulating inhibition may help sensory processing in Fragile X syndrome.

Sensory hypersensitivity is a common symptom in autism spectrum disorders (ASDs), including fragile X syndrome (FXS), and frequently leads to tactile defensiveness. In mouse models of ASDs, there is mounting evidence of neuronal and circuit hyperexcitability in several brain regions, which could contribute to sensory hypersensitivity. However, it is not yet known whether or how sensory stimulation might trigger abnormal sensory processing at the circuit level or abnormal behavioral responses in ASD mouse models, especially during an early developmental time when experience-dependent plasticity shapes such circuits. Using a novel assay, we discovered exaggerated motor responses to whisker stimulation in young Fmr1 knock-out (KO) mice (postnatal days 14 -16), a model of FXS. Adult Fmr1 KO mice actively avoided a stimulus that was innocuous to wild-type controls, a sign of tactile defensiveness. Using in vivo two-photon calcium imaging of layer 2/3 barrel cortex neurons expressing GCaMP6s, we found no differences between wild-type and Fmr1 KO mice in overall whisker-evoked activity, though 45% fewer neurons in young Fmr1 KO mice responded in a time-locked manner. Notably, we identified a pronounced deficit in neuronal adaptation to repetitive whisker stimulation in both young and adult Fmr1 KO mice. Thus, impaired adaptation in cortical sensory circuits is a potential cause of tactile defensiveness in autism.

Fluorescence calcium imaging using a range of microscopy approaches, such as two-photon excitation or head-mounted "miniscopes," is one of the preferred methods to record neuronal activity and glial signals in various experimental settings, including acute brain slices, brain organoids, and behaving animals. Because changes in the fluorescence intensity of genetically encoded or chemical calcium indicators correlate with action potential firing in neurons, data analysis is based on inferring such spiking from changes in pixel intensity values across time within different regions of interest. However, the algorithms necessary to extract biologically relevant information from these fluorescent signals are complex and require significant expertise in programming to develop robust analysis pipelines. For decades, the only way to perform these analyses was for individual laboratories to write their custom code. These routines were typically not well annotated and lacked intuitive graphical user interfaces (GUIs), which made it difficult for scientists in other laboratories to adopt them. Although the panorama is changing with recent tools like CaImAn, Suite2P, and others, there is still a barrier for many laboratories to adopt these packages, especially for potential users without sophisticated programming skills. As two-photon microscopes are becoming increasingly affordable, the bottleneck is no longer the hardware, but the software used to analyze the calcium data optimally and consistently across different groups. We addressed this unmet need by incorporating recent software solutions, namely NoRMCorre and CaImAn, for motion correction, segmentation, signal extraction, and deconvolution of calcium imaging data into an open-source, easy to use, GUI-based, intuitive and automated data analysis software package, which we named EZcalcium.

The first three postnatal weeks in rodents are a time when sensory experience drives the maturation of brain circuits, an important process that is not yet well understood. Alterations in this critical period of experience-dependent circuit assembly and plasticity contribute to several neurodevelopmental disorders, such as autism, epilepsy, and schizophrenia. Therefore, techniques for recording network activity and tracing neuronal connectivity over this time period are necessary for delineating circuit refinement in typical development and how it deviates in disease. Calcium imaging with GCaMP6 and other genetically encoded indicators is rapidly becoming the preferred method for recording network activity at the single-synapse and single-cell level in vivo, especially in genetically identified neuronal populations. We describe a protocol for intracortical injection of recombinant adeno-associated viruses in P1 neonatal mice and demonstrate its use for longitudinal imaging of GCaMP6s in the same neurons over several weeks to characterize the developmental desynchronization of cortical network activity. Our approach is ideally suited for chronic in vivo two-photon calcium imaging of neuronal activity from synapses to entire networks during the early postnatal period.

Fluorescence calcium imaging using a range of microscopy approaches, such as 2-photon excitation or head-mounted 'miniscopes', is one of the preferred methods to record neuronal activity and glial signals in various experimental settings, including acute brain slices, brain organoids, and behaving animals. Because changes in the fluorescence intensity of genetically encoded or chemical calcium indicators correlate with action potential firing in neurons, data analysis is based on inferring such spiking from changes in pixel intensity values across time within different regions of interest. However, the algorithms necessary to extract biologically relevant information from these fluorescent signals are complex and require significant expertise in programming to develop robust analysis pipelines. For decades, the only way to perform these analyses was for individual laboratories to write their own custom code. These routines were typically not well annotated and lacked intuitive graphical user interfaces (GUIs), which made it difficult for scientists in other laboratories to adopt them. Although the panorama is changing with recent tools like CaImAn, Suite2P and others, there is still a barrier for many laboratories to adopt these packages, especially for potential users without sophisticated programming skills. As 2-photon microscopes are becoming increasingly affordable, 2 the bottleneck is no longer the hardware, but the software used to analyze the calcium data in an optimal manner and consistently across different groups. We addressed this unmet need by incorporating recent software solutions for motion correction, segmentation, signal extraction and deconvolution of calcium imaging data into an open-source, easy to use, GUI-based, intuitive and automated data analysis software, which we named EZcalcium.

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The authors thank K. Cohan, S. Cohen, and M. Hong for help with early behavioral experiments; P. Golshani and M. Einstein for advice on mouse behavior; the Janelia GENIE project (for providing GCaMP6s); P. Yu for building custom lick ports; and D. Buonomano, A. Contractor, and J. Sweeney for feedback on the manuscript. The authors also thank P. Pellionisz and A. Cheng for help assembling the two-photon microscope. K. Battista created the illustration in Fig. 1b.

Finally, we found that human subjects with FXS exhibit strikingly similar impairments in visual discrimination as Fmr1 -/-mice. We conclude that manipulating orientation tuning in autism could improve visually guided behaviors that are critical for playing sports, driving or judging emotions.For these studies, we focused on FXS because it is the leading inherited cause of autism 5 , because there are no associated major neuroanatomical defects, and because a single, wellcharacterized animal model, the Fmr1 -/-mouse, is widely used. Although many human psychophysical studies have demonstrated deficits in visual perception in individuals with autism, including those with FXS 6, 7 , whether animal models of autism also exhibit impaired peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/217414 doi: bioRxiv preprint first posted online Nov. 13, 2017; ! 3 visual processing is not known.Thus, we first sought to determine whether Fmr1 -/-mice manifest perceptual learning deficits associated with abnormal visual sensory discrimination.We trained male and female Fmr1 knockout (Fmr1 -/-; n= 21) and wild-type (WT; n= 19) mice (FVB strain) on a go/no-go visual discrimination task 8, 9 . Following water deprivation, awake head-restrained young adult mice (2-4 months old) were allowed to run on an air-suspended polystyrene ball while they performed the task ( Fig. 1a, b; see Materials and Methods). Mice were presented with sinusoidal gratings drifting in two orthogonal directions, 45° (preferred, 'go') vs. 135° (non-preferred, 'no-go') at 100% contrast. Incorrect behavioral responses resulted in a 6.5 s 'time-out' period ( Fig.1c). Task performance, as determined by the discriminability index statistic d' (Materials and methods), was dependent on primary visual cortex (V1), because pharmacological silencing of V1 with bilateral infusions of muscimol, a GABA-A receptor agonist, reversibly disrupted perceptual learning in WT mice (Fig. 1d).WT mice learned quickly (3-4 sessions) to lick in response to the preferred orientation for a water reward and withhold licking when presented with the non-preferred orientation (Fig. 1e).In contrast, Fmr1 -/-mice exhibited a significantly delayed learning curve, compared to the WT mice ( Fig. 1e and Suppl. Fig. 1 caused by hyperactivity or abnormal locomotion, as the average running speed was similar between mice of both genotypes (not shown) and there was no change in running speed in mice of either genotype during the course of each trial on session 1 (Suppl. Fig. 3c). However, by session 4, we observed significantly more slowing down in WT than in Fmr1 -/-mice, towards the end of each trial (Suppl. Fig. 3c). The delay of Fmr1 -/-mice in learning the visual discrimination task was evident in female and male mice alike (Suppl . Fig. 4). Importantly, both WT and peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copy...