A failure to discriminate social from non-social touch at the circuit level may underlie social avoidance in autism.

Social touch is critical for communication and to impart emotions and intentions. However, certain autistic individuals experience aversion to social touch, especially when it is unwanted. We used a novel social touch assay and Neuropixels probes to compare neural responses to social vs. non-social interactions in three relevant brain regions: vibrissal somatosensory cortex, tail of striatum, and basolateral amygdala. We find that wild type (WT) mice showed aversion to repeated presentations of an inanimate object but not of another mouse. Cortical neurons cared most about touch context (social vs. object) and showed a preference for social interactions, while striatal neurons changed their preference depending on whether mice could choose or not to interact. Amygdalar and striatal neurons were preferentially modulated by forced object touch, which was the most aversive. In contrast, the Fmr1 knockout (KO) model of autism found social and non-social interactions equally aversive and displayed more aversive facial expressions to social touch when it invaded their personal space. Importantly, when Fmr1 KO mice could choose to interact, neurons in all three regions did not discriminate social valence. Thus, a failure to differentially encode social from non-social stimuli at the circuit level may underlie social avoidance in autism.

The NKCC1 inhibitor bumetanide restores cortical feedforward inhibition and lessens sensory hypersensitivity in early postnatal fragile X mice.

BACKGROUND: Exaggerated responses to sensory stimuli, a hallmark of Fragile X syndrome (FXS), contribute to anxiety and learning challenges. Sensory hypersensitivity is recapitulated in the Fmr1 knockout (KO) mouse model of FXS. Recent studies in Fmr1 KO mice have demonstrated differences in activity of cortical interneurons and a delayed switch in the polarity of GABA signaling during development. Previously, we reported that blocking the chloride transporter NKCC1 with the diuretic bumetanide, could rescue synaptic circuit phenotypes in primary somatosensory cortex (S1) of Fmr1 KO mice. However, it remains unknown whether bumetanide can rescue earlier circuit phenotypes or sensory hypersensitivity in Fmr1 KO mice. METHODS: We used acute and chronic systemic administration of bumetanide in Fmr1 KO mice and performed in vivo 2-photon calcium imaging to record neuronal activity, while tracking mouse behavior with high-resolution videos. RESULTS: We demonstrate that layer (L) 2/3 pyramidal neurons in S1 of Fmr1 KO mice show a higher frequency of synchronous events at postnatal day (P) 6 compared to wild-type controls. This was reversed by acute administration of bumetanide. Furthermore, chronic bumetanide treatment (P5-P14) restored S1 circuit differences in Fmr1 KO mice, including reduced neuronal adaptation to repetitive whisker stimulation, and ameliorated tactile defensiveness. Bumetanide treatment also rectified the reduced feedforward inhibition of L2/3 neurons in S1 and boosted the circuit participation of parvalbumin interneurons. CONCLUSIONS: This further supports the notion that synaptic, circuit, and sensory behavioral phenotypes in Fmr1 KO can be mitigated by inhibitors of NKCC1, such as the FDA-approved diuretic bumetanide.

Network state transitions during cortical development.

Mammalian cortical networks are active before synaptogenesis begins in earnest, before neuronal migration is complete, and well before an animal opens its eyes and begins to actively explore its surroundings. This early activity undergoes several transformations during development. The most important of these is a transition from episodic synchronous network events, which are necessary for patterning the neocortex into functionally related modules, to desynchronized activity that is computationally more powerful and efficient. Network desynchronization is perhaps the most dramatic and abrupt developmental event in an otherwise slow and gradual process of brain maturation. In this Review, we summarize what is known about the phenomenology of developmental synchronous activity in the rodent neocortex and speculate on the mechanisms that drive its eventual desynchronization. We argue that desynchronization of network activity is a fundamental step through which the cortex transitions from passive, bottom-up detection of sensory stimuli to active sensory processing with top-down modulation.

Hypersensitivity to Distractors in Fragile X Syndrome from Loss of Modulation of Cortical VIP Interneurons.

Attention deficit is one of the most prominent and disabling symptoms in Fragile X syndrome (FXS). Hypersensitivity to sensory stimuli contributes to attention difficulties by overwhelming and/or distracting affected individuals, which disrupts activities of daily living at home and learning at school. We find that auditory or visual distractors selectively impair visual discrimination performance in humans and mice with FXS but not in typically developing controls. In both species, males and females were examined. Vasoactive intestinal polypeptide (VIP) neurons were significantly modulated by incorrect responses in the poststimulus period during early distractor trials in WT mice, consistent with their known role as error signals. Strikingly, however, VIP cells from Fmr1 -/- mice showed little modulation in error trials, and this correlated with their poor performance on the distractor task. Thus, VIP interneurons and their reduced modulatory influence on pyramidal cells could be a potential therapeutic target for attentional difficulties in FXS.SIGNIFICANCE STATEMENT Sensory hypersensitivity, impulsivity, and persistent inattention are among the most consistent clinical features of FXS, all of which impede daily functioning and create barriers to learning. However, the neural mechanisms underlying sensory over-reactivity remain elusive. To overcome a significant challenge in translational FXS research we demonstrate a compelling alignment of sensory over-reactivity in both humans with FXS and Fmr1 -/- mice (the principal animal model of FXS) using a novel analogous distractor task. Two-photon microscopy in mice revealed that lack of modulation by VIP cells contributes to susceptibility to distractors. Implementing research efforts we describe here can help identify dysfunctional neural mechanisms associated not only with sensory issues but broader impairments, including those in learning and cognition.

A Novel Head-Fixed Assay for Social Touch in Mice Uncovers Aversive Responses in Two Autism Models.

Social touch, an important aspect of social interaction and communication, is essential to kinship across animal species. How animals experience and respond to social touch has not been thoroughly investigated, in part because of the lack of appropriate assays. Previous studies that examined social touch in freely moving rodents lacked the necessary temporal and spatial control over individual touch interactions. We designed a novel head-fixed assay for social touch in mice, in which the experimenter has complete control to elicit highly stereotyped bouts of social touch between two animals. The user determines the number, duration, context, and type of social touch interactions, while monitoring an array of complex behavioral responses with high resolution cameras. We focused on social touch to the face because of its high translational relevance to humans. We validated this assay in two different models of autism spectrum disorder (ASD), the Fmr1 knock-out (KO) model of Fragile X syndrome (FXS) and maternal immune activation (MIA) mice. We observed higher rates of avoidance running, hyperarousal, and aversive facial expressions (AFEs) to social touch than to object touch, in both ASD models compared with controls. Fmr1 KO mice showed more AFEs to mice of the same sex but whether they were stranger or familiar mice mattered less. Because this new social touch assay for head-fixed mice can be used to record neural activity during repeated bouts of social touch it could be used to uncover underlying circuit differences.SIGNIFICANCE STATEMENT Social touch is important for communication in animals and humans. However, it has not been extensively studied and current assays to measure animals' responses to social touch have limitations. We present a novel head-fixed assay to quantify how mice respond to social facial touch with another mouse. We validated this assay in autism mouse models since autistic individuals exhibit differences in social interaction and touch sensitivity. We find that mouse models of autism exhibit more avoidance, hyperarousal, and aversive facial expressions (AFEs) to social touch compared with controls. Thus, this novel assay can be used to investigate behavioral responses to social touch and the underlying brain mechanisms in rodent models of neurodevelopmental conditions, and to evaluate therapeutic responses in preclinical studies.

Improvement of sensory deficits in fragile X mice by increasing cortical interneuron activity after the critical period.

Changes in the function of inhibitory interneurons (INs) during cortical development could contribute to the pathophysiology of neurodevelopmental disorders. Using all-optical in vivo approaches, we find that parvalbumin (PV) INs and their immature precursors are hypoactive and transiently decoupled from excitatory neurons in postnatal mouse somatosensory cortex (S1) of Fmr1 KO mice, a model of fragile X syndrome (FXS). This leads to a loss of parvalbumin INs (PV-INs) in both mice and humans with FXS. Increasing the activity of future PV-INs in neonatal Fmr1 KO mice restores PV-IN density and ameliorates transcriptional dysregulation in S1, but not circuit dysfunction. Critically, administering an allosteric modulator of Kv3.1 channels after the S1 critical period does rescue circuit dynamics and tactile defensiveness. Symptoms in FXS and related disorders could be mitigated by targeting PV-INs.

A novel head-fixed assay for social touch in mice uncovers aversive responses in two autism models.

Social touch, an important aspect of social interaction and communication, is essential to kinship across animal species. How animals experience and respond to social touch has not been thoroughly investigated, in part due to the lack of appropriate assays. Previous studies that examined social touch in freely moving rodents lacked the necessary temporal and spatial control over individual touch interactions. We designed a novel head-fixed assay for social touch in mice, in which the experimenter has complete control to elicit highly stereotyped bouts of social touch between two animals. The user determines the number, duration, context, and type of social touch interactions, while monitoring with high frame rate cameras an array of complex behavioral responses. We focused on social touch to the face because of their high translational relevance to humans. We validated this assay in two different models of autism spectrum disorder (ASD), the Fmr1 knockout model of Fragile X Syndrome and maternal immune activation mice. We observed increased avoidance, hyperarousal, and more aversive facial expressions to social touch, but not to object touch, in both ASD models compared to controls. Because this new social touch assay for head-fixed mice can be used to record neural activity during repeated bouts of social touch it should be of interest to neuroscientists interested in uncovering the underlying circuits.

Hypersensitivity to distractors in Fragile X syndrome from loss of modulation of cortical VIP interneurons.

Attention deficit is one of the most prominent and disabling symptoms in Fragile X Syndrome (FXS). Hypersensitivity to sensory stimuli contributes to attention difficulties by overwhelming and/or distracting affected individuals, which disrupts activities of daily living at home and learning at school. We find that auditory or visual distractors selectively impair visual discrimination performance in both humans and mice with FXS, but not their typically developing controls. Vasoactive intestinal polypeptide (VIP) neurons were significantly modulated by incorrect responses in the post-stimulus period during early distractor trials in WT mice, consistent with their known role as 'error' signals. Strikingly, however, VIP cells from Fmr1-/- mice showed little modulation in error trials, and this correlated with their poor performance on the distractor task. Thus, VIP interneurons and their reduced modulatory influence on pyramidal cells, could be a potential therapeutic target for attentional difficulties in FXS.

aGABRacadabra: A surprising new role for GABAA receptors in cortical development.

In this issue of Neuron, Babij, Ferrer, and colleagues provide new evidence that ß3 subunit of GABAA receptors is critical for the maturation of functional networks in the neonatal somatosensory cortex.

Two-photon calcium imaging of neuronal activity.

In vivo two-photon calcium imaging (2PCI) is a technique used for recording neuronal activity in the intact brain. It is based on the principle that, when neurons fire action potentials, intracellular calcium levels rise, which can be detected using fluorescent molecules that bind to calcium. This Primer is designed for scientists who are considering embarking on experiments with 2PCI. We provide the reader with a background on the basic concepts behind calcium imaging and on the reasons why 2PCI is an increasingly powerful and versatile technique in neuroscience. The Primer explains the different steps involved in experiments with 2PCI, provides examples of what ideal preparations should look like and explains how data are analysed. We also discuss some of the current limitations of the technique, and the types of solutions to circumvent them. Finally, we conclude by anticipating what the future of 2PCI might look like, emphasizing some of the analysis pipelines that are being developed and international efforts for data sharing.

Cortical interneurons in autism.

The mechanistic underpinnings of autism remain a subject of debate and controversy. Why do individuals with autism share an overlapping set of atypical behaviors and symptoms, despite having different genetic and environmental risk factors? A major challenge in developing new therapies for autism has been the inability to identify convergent neural phenotypes that could explain the common set of symptoms that result in the diagnosis. Although no striking macroscopic neuropathological changes have been identified in autism, there is growing evidence that inhibitory interneurons (INs) play an important role in its neural basis. In this Review, we evaluate and interpret this evidence, focusing on recent findings showing reduced density and activity of the parvalbumin class of INs. We discuss the need for additional studies that investigate how genes and the environment interact to change the developmental trajectory of INs, permanently altering their numbers, connectivity and circuit engagement.

Barrel cortex plasticity after photothrombotic stroke involves potentiating responses of pre-existing circuits but not functional remapping to new circuits.

Recovery after stroke is thought to be mediated by adaptive circuit plasticity, whereby surviving neurons assume the roles of those that died. However, definitive longitudinal evidence of neurons changing their response selectivity after stroke is lacking. We sought to directly test whether such functional "remapping" occurs within mouse primary somatosensory cortex after a stroke that destroys the C1 barrel. Using in vivo calcium imaging to longitudinally record sensory-evoked activity under light anesthesia, we did not find any increase in the number of C1 whisker-responsive neurons in the adjacent, spared D3 barrel after stroke. To promote plasticity after stroke, we also plucked all whiskers except C1 (forced use therapy). This led to an increase in the reliability of sensory-evoked responses in C1 whisker-responsive neurons but did not increase the number of C1 whisker-responsive neurons in spared surround barrels over baseline levels. Our results argue against remapping of functionality after barrel cortex stroke, but support a circuit-based mechanism for how rehabilitation may improve recovery.

EZcalcium: Open-Source Toolbox for Analysis of Calcium Imaging Data.

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.

Spectro-temporal encoded multiphoton microscopy and fluorescence lifetime imaging at kilohertz frame-rates.

Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration. In terms of imaging speeds, however, mechanical scanners still limit the acquisition rates to typically 10-100 frames per second. Here we present a high-speed non-linear microscope achieving kilohertz frame rates by employing pulse-modulated, rapidly wavelength-swept lasers and inertia-free beam steering through angular dispersion. In combination with a high bandwidth, single-photon sensitive detector, this enables recording of fluorescent lifetimes at speeds of 88 million pixels per second. We show high resolution, multi-modal - two-photon fluorescence and fluorescence lifetime (FLIM) - microscopy and imaging flow cytometry with a digitally reconfigurable laser, imaging system and data acquisition system. These high speeds should enable high-speed and high-throughput image-assisted cell sorting.

Autism in the Balance: Elevated E-I Ratio as a Homeostatic Stabilization of Synaptic Drive.

In this issue of Neuron, Antoine et al. (2019) find reduced feedforward inhibition in cortical neurons in four genetic mouse models of autism but without evidence of increased spontaneous or sensory-evoked activity.

Critical period inhibition of NKCC1 rectifies synapse plasticity in the somatosensory cortex and restores adult tactile response maps in fragile X mice.

Sensory perturbations in visual, auditory and tactile perception are core problems in fragile X syndrome (FXS). In the Fmr1 knockout mouse model of FXS, the maturation of synapses and circuits during critical period (CP) development in the somatosensory cortex is delayed, but it is unclear how this contributes to altered tactile sensory processing in the mature CNS. Here we demonstrate that inhibiting the juvenile chloride co-transporter NKCC1, which contributes to altered chloride homeostasis in developing cortical neurons of FXS mice, rectifies the chloride imbalance in layer IV somatosensory cortex neurons and corrects the development of thalamocortical excitatory synapses during the CP. Comparison of protein abundances demonstrated that NKCC1 inhibition during early development caused a broad remodeling of the proteome in the barrel cortex. In addition, the abnormally large size of whisker-evoked cortical maps in adult Fmr1 knockout mice was corrected by rectifying the chloride imbalance during the early CP. These data demonstrate that correcting the disrupted driving force through GABAA receptors during the CP in cortical neurons restores their synaptic development, has an unexpectedly large effect on differentially expressed proteins, and produces a long-lasting correction of somatosensory circuit function in FXS mice.

Author Correction: Impaired perceptual learning in a mouse model of Fragile X syndrome is mediated by parvalbumin neuron dysfunction and is reversible.

The original and corrected Acknowledgements are shown in the accompanying Author Correction.

Dendritic Spines in Early Postnatal Fragile X Mice Are Insensitive to Novel Sensory Experience.

Autism spectrum disorders are often associated with atypical sensory processing and sensory hypersensitivity, which can lead to maladaptive behaviors, such as tactile defensiveness. Such altered sensory perception in autism spectrum disorders could arise from disruptions in experience-dependent maturation of circuits during early brain development. Here, we tested the hypothesis that synaptic structures of primary somatosensory cortex (S1) neurons in Fragile X syndrome (FXS), which is a common inherited cause of autism, are not modulated by novel sensory information during development. We used chronic in vivo two-photon microscopy to image dendritic spines and axon "en passant" boutons of layer 2/3 pyramidal neurons in S1 of male and female WT and Fmr1 KO mice, a model of FXS. We found that a brief (overnight) exposure to dramatically enhance sensory inputs in the second postnatal week led to a significant increase in spine density in WT mice, but not in Fmr1 KO mice. In contrast, axon "en passant" boutons dynamics were impervious to this novel sensory experience in mice of both genotypes. We surmise that the inability of Fmr1 KO mice to modulate postsynaptic dynamics in response to increased sensory input, at a time when sensory information processing first comes online in S1 cortex, could play a role in altered sensory processing in FXS.SIGNIFICANCE STATEMENT Very few longitudinal in vivo imaging studies have investigated synaptic structure and dynamics in early postnatal mice. Moreover, those studies tend to focus on the effects of sensory input deprivation, a process that rarely occurs during normal brain development. Early postnatal imaging experiments are critical because a variety of neurodevelopmental disorders, including those characterized by autism, could result from alterations in how circuits are shaped by incoming sensory inputs during critical periods of development. In this study, we focused on a mouse model of Fragile X syndrome and demonstrate how dendritic spines are insensitive to a brief period of novel sensory experience.

Impaired perceptual learning in a mouse model of Fragile X syndrome is mediated by parvalbumin neuron dysfunction and is reversible.

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.