POSITIVE MODULATION OF MGLU5 REVERSES ASD-LIKE BEHAVIORS FOUND IN SHANK3 KNOCK-OUT MICE

Shank proteins are the major scaffold proteins that organize the postsynaptic density at the excitatory synapses. Shank1-3 proteins are associated with type I mGluRs via an interaction with Homer in the proline-rich domain. Phelan-McDermid (PMS) syndrome is characterized by intellectual impairment, absent or delayed speech, and autistic-like behaviors. Loss of Shank3 is now considered to cause the neurobehavioral symptoms of PMS. Furthermore, a significant number of SHANK3 mutations have been identified in patients with Autism Spectrum disorders ASD, and SHANK3 truncating mutations are associated with moderate to profound ID. In this study, we investigated the molecular mechanisms associated with the ASD-like behaviors observed in Shank3\u39411-/- mice in which exon 11 has been deleted. Our results indicate that Shank3 is essential to mediating mGlu5 receptor signaling by recruiting Homer1b/c to the PSD, specifically in the striatum and cortex. Moreover, augmenting mGlu5 receptor activity by administering 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) ameliorated the functional and behavioral defects that were observed in Shank3\u39411-/- mice, suggesting that pharmaceutical treatments that increase mGlu5 activity may represent a new approach for treating patients that are affected by PMS and SHANK3 mutations

3D HDO-CLEM: cellular compartment analysis by correlative light-electron microscopy on cryosections.

Fundamental to obtaining a depth-understanding of the function and structure of cells is the ability to study and correlate their molecular topography with the ultrastructural morphology, for example, to visualize the position of a given protein relative to a given cell compartment and its morphology. Standard fluorescence light microscopy (FLM) relies on simple sample preparations, and localizes proteins in living or fixed cells with a resolution in the range of few hundred nanometers, allowing large field of view. However, FLM is unable to visualize the unlabeled cellular context. On the other hand, electron microscopy (EM) techniques reveal protein topology with the resolution in a range of a few tens of nanometer, retains the cellular context, but can only be applied on a limited field of view. Therefore, both approaches present shortcomings, in terms of field of view, statistical output, resolution, sample preparation, and context analysis, that can likely complement each other. To bridge the gap between FLM imaging and EM, several laboratories have developed methods for correlative light-electron microscopy (CLEM). In a nutshell, CLEM enables one to investigate the same exact region of interest utilizing the two microscope platforms, and thereby virtually combine their capabilities. We describe a protocol based on immunolabeling of Tokuyasu cryosections that allows correlation of LM and EM images with excellent preservation of cellular ultrastructure. We will refer to this method as high-data-output CLEM (HDO-CLEM). The major benefits of HDO-CLEM are the possibility to (1) correlate several hundreds of events at the same time, (2) perform three-dimensional (3D) correlation, (3) immunolabel both endogenous and recombinantly tagged proteins at the same time, and (4) combine the high data analysis capability of FLM with the high precision of transmission EM in a CLEM hybrid morphometric analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D FLM reconstruction and set the basis for a hybrid light/EM morphometry approach

A new filtering technique for removing anti-Stokes emission background in gated CW-STED microscopy

Stimulated emission depletion (STED) microscopy is a prominent approach of super-resolution optical microscopy, which allows cellular imaging with so far unprecedented unlimited spatial resolution. The introduction of time-gated detection in STED microscopy significantly reduces the (instantaneous) intensity required to obtain sub-diffraction spatial resolution. If the time-gating is combined with a STED beam operating in continuous wave (CW), a cheap and low labour demand implementation is obtained, the so called gated CW-STED microscope. However, time-gating also reduces the fluorescence signal which forms the image. Thereby, background sources such as fluorescence emission excited by the STED laser (anti-Stokes fluorescence) can reduce the effective resolution of the system. We propose a straightforward method for subtraction of anti-Stokes background. The method hinges on the uncorrelated nature of the anti-Stokes emission background with respect to the wanted fluorescence signal. The specific importance of the method towards the combination of two-photon-excitation with gated CW-STED microscopy is demonstrated. © 2014 The Authors. J. Biophotonics

High Data Output and Automated 3D Correlative Light–Electron Microscopy Method

Correlative light/electron microscopy (CLEM) allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy (FLM) and electron microscopy. The use of this approach is becoming increasingly frequent in cell biology. In this study, we report on a new high data output CLEM method based on the use of cryosections. We successfully applied the method to analyze the structure of rough and smooth Russell bodies used as model systems. The major advantages of our method are (i) the possibility to correlate several hundreds of events at the same time, (ii) the possibility to perform three-dimensional (3D) correlation, (iii) the possibility to immunolabel both endogenous and recombinantly expressed proteins at the same time and (iv) the possibility to combine the high data analysis capability of FLM with the high precision–accuracy of transmission electron microscopy in a CLEM hybrid morphometry analysis. We have identified and optimized critical steps in sample preparation, defined routines for sample analysis and retracing of regions of interest, developed software for semi/fully automatic 3D reconstruction and defined preliminary conditions for an hybrid light/electron microscopy morphometry approach

Intensity Weighted Subtraction Microscopy Approach for Image Contrast and Resolution Enhancement

We propose and demonstrate a novel subtraction microscopy algorithm, exploiting fluorescence emission difference or switching laser mode and their derivatives for image enhancement. The key novelty of the proposed approach lies in the weighted subtraction coefficient, adjusted pixel-by-pixel with respect to the intensity distributions of initial images. This method produces significant resolution enhancement and minimizes image distortions. Our theoretical and experimental studies demonstrate that this approach can be applied to any optical microscopy techniques, including label free and non-linear methods, where common super-resolution techniques cannot be used

Subdiffraction, Luminescence-Depletion Imaging of Isolated, Giant, CdSe/CdS Nanocrystal Quantum Dots

Subdiffraction spatial resolution luminescence depletion imaging was performed with giant CdSe/14CdS nanocrystal quantum dots (g-NQDs) dispersed on a glass slide. Luminescence depletion imaging used a Gaussian shaped excitation laser pulse overlapped with a depletion pulse, shaped into a doughnut profile, with zero intensity in the center. Luminescence from a subdiffraction volume is collected from the central portion of the excitation spot, where no depletion takes place. Up to 92% depletion of the luminescence signal was achieved. An average full width at half-maximum of 40 ± 10 nm was measured in the lateral direction for isolated g-NQDs at an air interface using luminescence depletion imaging, whereas the average full width at half-maximum was 450 ± 90 nm using diffraction-limited, confocal luminescence imaging. Time-gating of the luminescence depletion data was required to achieve the stated spatial resolution. No observable photobleaching of the g-NQDs was present in the measurements, which allowed imaging with a dwell time of 250 ms per pixel to obtain images with a high signal-to-noise ratio. The mechanism for luminescence depletion is likely stimulated emission, stimulated absorption, or a combination of the two. The g-NQDs fulfill a need for versatile, photostable tags for subdiffraction imaging schemes where high laser powers or long exposure times are used

Genomics in neurodevelopmental disorders: an avenue to personalized medicine

Despite the remarkable number of scientific breakthroughs of the last 100 years, the treatment of neurodevelopmental disorders (e.g., autism spectrum disorder, intellectual disability) remains a great challenge. Recent advancements in genomics, such as whole-exome or whole-genome sequencing, have enabled scientists to identify numerous mutations underlying neurodevelopmental disorders. Given the few hundred risk genes that have been discovered, the etiological variability and the heterogeneous clinical presentation, the need for genotype — along with phenotype- based diagnosis of individual patients has become a requisite. In this review we look at recent advancements in genomic analysis and their translation into clinical practice

Fluorescent Labeling of SNAP-Tagged Proteins in Cells

One of the most prominent self-labeling tags is SNAP-tag. It is an in vitro evolution product of the human DNA repair protein O6 -alkylguanine-DNA alkyltransferase (hAGT) that reacts specifically with benzylguanine (BG) and benzylchloropyrimidine (CP) derivatives, leading to covalent labeling of SNAP-tag with a synthetic probe (Gronemeyer et al., Protein Eng Des Sel 19:309–316, 2006; Curr Opin Biotechnol 16:453–458, 2005; Keppler et al., Nat Biotechnol 21:86–89, 2003; Proc Natl Acad Sci U S A 101:9955– 9959, 2004). SNAP-tag is well suited for the analysis and quantification of fused target protein using fluorescence microscopy techniques. It provides a simple, robust, and versatile approach to the imaging of fusion proteins under a wide range of experimental conditions. © Springer Science+Business Media New York 2015