Cortical interneuron-mediated inhibition delays the onset of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a fatal disease resulting from motor neuron degeneration in the cortex and spinal cord. Cortical hyperexcitability is a hallmark feature of amyotrophic lateral sclerosis and is accompanied by decreased intracortical inhibition. Using electrophysiological patch-clamp recordings, we revealed parvalbumin interneurons to be hypoactive in the late pre-symptomatic SOD1*G93A mouse model of amyotrophic lateral sclerosis. We discovered that using adeno-associated virus-mediated delivery of chemogenetic technology targeted to increase the activity of the interneurons within layer 5 of the primary motor cortex, we were able to rescue intracortical inhibition and reduce pyramidal neuron hyperexcitability. Increasing the activity of interneurons in the layer 5 of the primary motor cortex was effective in delaying the onset of amyotrophic lateral sclerosis-associated motor deficits, slowing symptom progression, preserving neuronal populations, and increasing the lifespan of SOD1*G93A mice. Taken together, this study provides novel insights into the pathogenesis and treatment of amyotrophic lateral sclerosis.

In vivo histology of the myelin g-ratio with magnetic resonance imaging.

The myelin g-ratio, defined as the ratio between the inner and the outer diameter of the myelin sheath, is a fundamental property of white matter that can be computed from a simple formula relating the myelin volume fraction to the fiber volume fraction or the axon volume fraction. In this paper, a unique combination of magnetization transfer, diffusion imaging and histology is presented, providing a novel method for in vivo magnetic resonance imaging of the axon volume fraction and the myelin g-ratio. Our method was demonstrated in the corpus callosum of one cynomolgus macaque, and applied to obtain full-brain g-ratio maps in one healthy human subject and one multiple sclerosis patient. In the macaque, the g-ratio was relatively constant across the corpus callosum, as measured by both MRI and electron microscopy. In the human subjects, the g-ratio in multiple sclerosis lesions was higher than in normal appearing white matter, which was in turn higher than in healthy white matter. Measuring the g-ratio brings us one step closer to fully characterizing white matter non-invasively, making it possible to perform in vivo histology of the human brain during development, aging, disease and treatment.

Resolution and contrast enhancement in coherent anti-Stokes Raman-scattering microscopy.

We implement switching laser mode coherent anti-Stokes Raman-scattering (SLAM-CARS) microscopy to enhance the spatial resolution and contrast in label-free vibrational microscopy. The method, based on the intensity difference between two images obtained with Gaussian and doughnut-shaped modes, does not depend on the specimen and relies on minimal modifications of the typical CARS setup. We demonstrate subdiffraction resolution imaging of myelin sheaths in a mouse brainstem. A lateral resolution of 0.36λ(p) is achieved.

Investigation of molecular and protein crystals by three photon polarization resolved microscopy.

We implement three photon fluorescence polarization resolved microscopy to optically investigate molecular and protein crystals. The availability of UV transitions using IR pulses allows analyses without fluorescence staining. Polarization resolved studies indicate that high-order symmetry structures can be revealed and that strong fluorescent energy transfer occurs between molecules. We show how this permits identification of a monocrystalline nature for a sample at the subwavelength resolution scale.

Influence of birefringence on polarization resolved nonlinear microscopy and collagen SHG structural imaging.

We analyze the influence of the anisotropy of molecular and biological samples on polarization resolved nonlinear microscopy imaging. We show in particular the detrimental influence of birefringence on Second Harmonic Generation (SHG) and Two-Photon Excited Fluorescence (TPEF) polarization resolved microscopy imaging, which, if not accounted for, can lead to an erroneous determination of the sample properties and thus to a misinterpretation of the read-out information. We propose a method to measure this birefringence and account for this effect in nonlinear polarization resolved experiments.

Anisotropic light scattering from myelinated axons in the spinal cord.

Optogenetics has become an integral tool for studying and dissecting the neural circuitries of the brain using optical control. Recently, it has also begun to be used in the investigation of the spinal cord and peripheral nervous system. However, information on these regions' optical properties is sparse. Moreover, there is a lack of data on the dependence of light propagation with respect to neural tissue organization and orientation. This information is important for effective simulations and optogenetic planning, particularly in the spinal cord where the myelinated axons are highly organized. To this end, we report experimental measurements for the scattering coefficient, validated with three different methods in both the longitudinal and radial directions of multiple mammalian spinal cords. In our analysis, we find that there is indeed a directional dependence of photon propagation when interacting with organized myelinated axons. Specifically, light propagating perpendicular to myelinated axons in the white matter of the spinal cord produced a measured reduced scattering coefficient ( µ s ' ) of 3.52 ± 0.1 mm - 1 , and light that was propagated along the myelinated axons in the white matter produced a measured µ s ' of 1.57 ± 0.03 mm - 1 , across the various species considered. This 50% decrease in scattering power along the myelinated axons is observed with three different measurement strategies (integrating spheres, observed transmittance, and punch-through method). Furthermore, this directional dependence in scattering power and overall light attenuation did not occur in the gray matter regions where the myelin organization is nearly random. The acquired information will be integral in preparing future light-transport simulations and in overall optogenetic planning in both the spinal cord and the brain.

Quantitative imaging of molecular order in lipid membranes using two-photon fluorescence polarimetry.

We present a polarimetric two-photon microscopy technique to quantitatively image the local static molecular orientational behavior in lipid and cell membranes. This approach, based on a tunable excitation polarization state complemented by a polarized readout, is easily implementable and does not require hypotheses on the molecular angular distribution such as its mean orientation, which is a main limitation in traditional fluorescence anisotropy measurements. The method is applied to the investigation of the molecular angular distribution in giant unilamellar vesicles formed by liquid-ordered and liquid-disordered micro-domains, and in COS-7 cell membranes. The highest order contrast between ordered and disordered domains is obtained for dyes locating within the membrane acyl chains.

Polarization distortion effects in polarimetric two-photon microscopy.

We present a global analysis of experimental factors affecting polarization responses in two-photon inverted microscopy. The role of reflection optics and high numerical aperture focusing is investigated in two-photon fluorescence, which can be extended to other nonlinear processes. We show that both effects strongly distort polarization responses and can lead to misleading extraction of molecular order information from polarimetric measurements. We describe a model accounting for these effects and develop a calibration technique for the determination of polarization parameters in the sample plane using two-photon fluorescence polarimetry in liquids.

Association of a History of Child Abuse With Impaired Myelination in the Anterior Cingulate Cortex: Convergent Epigenetic, Transcriptional, and Morphological Evidence.

OBJECTIVE: Child abuse has devastating and long-lasting consequences, considerably increasing the lifetime risk of negative mental health outcomes such as depression and suicide. Yet the neurobiological processes underlying this heightened vulnerability remain poorly understood. The authors investigated the hypothesis that epigenetic, transcriptomic, and cellular adaptations may occur in the anterior cingulate cortex as a function of child abuse. METHOD: Postmortem brain samples from human subjects (N=78) and from a rodent model of the impact of early-life environment (N=24) were analyzed. The human samples were from depressed individuals who died by suicide, with (N=27) or without (N=25) a history of severe child abuse, as well as from psychiatrically healthy control subjects (N=26). Genome-wide DNA methylation and gene expression were investigated using reduced representation bisulfite sequencing and RNA sequencing, respectively. Cell type-specific validation of differentially methylated loci was performed after fluorescence-activated cell sorting of oligodendrocyte and neuronal nuclei. Differential gene expression was validated using NanoString technology. Finally, oligodendrocytes and myelinated axons were analyzed using stereology and coherent anti-Stokes Raman scattering microscopy. RESULTS: A history of child abuse was associated with cell type-specific changes in DNA methylation of oligodendrocyte genes and a global impairment of the myelin-related transcriptional program. These effects were absent in the depressed suicide completers with no history of child abuse, and they were strongly correlated with myelin gene expression changes observed in the animal model. Furthermore, a selective and significant reduction in the thickness of myelin sheaths around small-diameter axons was observed in individuals with history of child abuse. CONCLUSIONS: The results suggest that child abuse, in part through epigenetic reprogramming of oligodendrocytes, may lastingly disrupt cortical myelination, a fundamental feature of cerebral connectivity.

Direct imaging of molecular symmetry by coherent anti-stokes Raman scattering.

Nonlinear optical methods, such as coherent anti-Stokes Raman scattering and stimulated Raman scattering, are able to perform label-free imaging, with chemical bonds specificity. Here we demonstrate that the use of circularly polarized light allows to retrieve not only the chemical nature but also the symmetry of the probed sample, in a single measurement. Our symmetry-resolved scheme offers simple access to the local organization of vibrational bonds and as a result provides enhanced image contrast for anisotropic samples, as well as an improved chemical selectivity. We quantify the local organization of vibrational bonds on crystalline and biological samples, thus providing information not accessible by spontaneous Raman and stimulated Raman scattering techniques. This work stands for a symmetry-resolved contrast in vibrational microscopy, with potential application in biological diagnostic.

AxonSeg: Open Source Software for Axon and Myelin Segmentation and Morphometric Analysis.

Segmenting axon and myelin from microscopic images is relevant for studying the peripheral and central nervous system and for validating new MRI techniques that aim at quantifying tissue microstructure. While several software packages have been proposed, their interface is sometimes limited and/or they are designed to work with a specific modality (e.g., scanning electron microscopy (SEM) only). Here we introduce AxonSeg, which allows to perform automatic axon and myelin segmentation on histology images, and to extract relevant morphometric information, such as axon diameter distribution, axon density and the myelin g-ratio. AxonSeg includes a simple and intuitive MATLAB-based graphical user interface (GUI) and can easily be adapted to a variety of imaging modalities. The main steps of AxonSeg consist of: (i) image pre-processing; (ii) pre-segmentation of axons over a cropped image and discriminant analysis (DA) to select the best parameters based on axon shape and intensity information; (iii) automatic axon and myelin segmentation over the full image; and (iv) atlas-based statistics to extract morphometric information. Segmentation results from standard optical microscopy (OM), SEM and coherent anti-Stokes Raman scattering (CARS) microscopy are presented, along with validation against manual segmentations. Being fully-automatic after a quick manual intervention on a cropped image, we believe AxonSeg will be useful to researchers interested in large throughput histology. AxonSeg is open source and freely available at: https://github.com/neuropoly/axonseg.

Quantitative analysis of the myelin g-ratio from electron microscopy images of the macaque corpus callosum.

We provide a detailed morphometric analysis of eight transmission electron micrographs (TEMs) obtained from the corpus callosum of one cynomolgus macaque. The raw TEM images are included in the article, along with the distributions of the axon caliber and the myelin g-ratio in each image. The distributions are analyzed to determine the relationship between axon caliber and g-ratio, and compared against the aggregate metrics (myelin volume fraction, fiber volume fraction, and the aggregate g-ratio), as defined in the accompanying research article entitled 'In vivo histology of the myelin g-ratio with magnetic resonance imaging' (Stikov et al., NeuroImage, 2015).