Tactile Defensiveness and Impaired Adaptation of Neuronal Activity in the Fmr1 Knock-Out Mouse Model of Autism.

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.SIGNIFICANCE STATEMENT We use a novel paradigm of repetitive whisker stimulation and in vivo calcium imaging to assess tactile defensiveness and barrel cortex activity in young and adult Fmr1 knock-out mice, the mouse model of fragile X syndrome (FXS). We describe evidence of tactile defensiveness, as well as a lack of L2/3 neuronal adaptation in barrel cortex, during whisker stimulation. We propose that a defect in sensory adaptation within local neuronal networks, beginning at a young age and continuing into adulthood, likely contributes to sensory overreactivity in FXS and perhaps other ASDs.

The Crypto-Apartheid of Race, Class, and Safety Arbitration on the Inpatient Psychiatric Unit.

Inpatient psychiatric treatment and violence assessment are strongly influenced by a patient's race and class identity. The authors argue that psychiatrists enact a crypto-apartheid wherein they recognize and condemn the structural racism and classism disadvantaging many patients, but through violence risk assessments and dispositional decisions, psychiatrists also function as arbiters of public safety and repeatedly disadvantage less-privileged patients to further symptomatic decline or even subsequent incarceration.

Effects of zinc deuteroporphyrin bis glycol on newborn mice after heme loading.

Infants with hemolytic diseases frequently develop hyperbilirubinemia and are treated with phototherapy, which only eliminates bilirubin after its production. A better strategy might be to directly inhibit heme oxygenase (HO), the rate-limiting enzyme in bilirubin production. Metalloporphyrins (Mps) are heme analogs that competitively inhibit HO activity in vitro and in vivo and suppress plasma bilirubin levels in vivo. A promising Mp, zinc deuteroporphyrin bis glycol (ZnBG), is orally absorbed and effectively inhibits HO activity at relatively low doses. We determined the I(50) (the dose needed to inhibit HO activity by 50%) of orally administered ZnBG in vivo and then evaluated ZnBG's effects on in vivo bilirubin production, HO activity, HO protein levels, and HO-1 gene expression in newborn mice after heme loading, a model analogous to a hemolytic infant. The I(50) of ZnBG was found to be 4.0 µmol/kg body weight (BW). At a dose of 15 µmol/kg BW, ZnBG reduced in vivo bilirubin production, inhibited heme-induced liver HO activity and spleen HO activity to and below baseline, respectively, transiently induced liver and spleen HO-1 gene transcription, and induced liver and spleen HO-1 protein levels. We conclude that ZnBG may be an attractive compound for treating severe neonatal hyperbilirubinemia caused by hemolytic disease.

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.

A Versatile Method for Viral Transfection of Calcium Indicators in the Neonatal Mouse Brain.

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.

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells.

Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-ß plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.