Inhibition of AKT Signaling Alters ßIV Spectrin Distribution at the AIS and Increases Neuronal Excitability.

The axon initial segment (AIS) is a highly regulated subcellular domain required for neuronal firing. Changes in the AIS protein composition and distribution are a form of structural plasticity, which powerfully regulates neuronal activity and may underlie several neuropsychiatric and neurodegenerative disorders. Despite its physiological and pathophysiological relevance, the signaling pathways mediating AIS protein distribution are still poorly studied. Here, we used confocal imaging and whole-cell patch clamp electrophysiology in primary hippocampal neurons to study how AIS protein composition and neuronal firing varied in response to selected kinase inhibitors targeting the AKT/GSK3 pathway, which has previously been shown to phosphorylate AIS proteins. Image-based features representing the cellular pattern distribution of the voltage-gated Na+ (Nav) channel, ankyrin G, ßIV spectrin, and the cell-adhesion molecule neurofascin were analyzed, revealing ßIV spectrin as the most sensitive AIS protein to AKT/GSK3 pathway inhibition. Within this pathway, inhibition of AKT by triciribine has the greatest effect on ßIV spectrin localization to the AIS and its subcellular distribution within neurons, a phenotype that Support Vector Machine classification was able to accurately distinguish from control. Treatment with triciribine also resulted in increased excitability in primary hippocampal neurons. Thus, perturbations to signaling mechanisms within the AKT pathway contribute to changes in ßIV spectrin distribution and neuronal firing that may be associated with neuropsychiatric and neurodegenerative disorders.

Automated sorting of neuronal trees in fluorescent images of neuronal networks using NeuroTreeTracer.

Fluorescence confocal microscopy has become increasingly more important in neuroscience due to its applications in image-based screening and profiling of neurons. Multispectral confocal imaging is useful to simultaneously probe for distribution of multiple analytes over networks of neurons. However, current automated image analysis algorithms are not designed to extract single-neuron arbors in images where neurons are not separated, hampering the ability map fluorescence signals at the single cell level. To overcome this limitation, we introduce NeuroTreeTracer - a novel image processing framework aimed at automatically extracting and sorting single-neuron traces in fluorescent images of multicellular neuronal networks. This method applies directional multiscale filters for automated segmentation of neurons and soma detection, and includes a novel tracing routine that sorts neuronal trees in the image by resolving network connectivity even when neurites appear to intersect. By extracting each neuronal tree, NeuroTreetracer enables to automatically quantify the spatial distribution of analytes of interest in the subcellular compartments of individual neurons. This software is released open-source and freely available with the goal to facilitate applications in neuron screening and profiling.

Multiscale Analysis of Neurite Orientation and Spatial Organization in Neuronal Images.

The spatial organization of neurites, the thin processes (i.e., dendrites and axons) that stem from a neuron's soma, conveys structural information required for proper brain function. The alignment, direction and overall geometry of neurites in the brain are subject to continuous remodeling in response to healthy and noxious stimuli. In the developing brain, during neurogenesis or in neuroregeneration, these structural changes are indicators of the ability of neurons to establish axon-to-dendrite connections that can ultimately develop into functional synapses. Enabling a proper quantification of this structural remodeling would facilitate the identification of new phenotypic criteria to classify developmental stages and further our understanding of brain function. However, adequate algorithms to accurately and reliably quantify neurite orientation and alignment are still lacking. To fill this gap, we introduce a novel algorithm that relies on multiscale directional filters designed to measure local neurites orientation over multiple scales. This innovative approach allows us to discriminate the physical orientation of neurites from finer scale phenomena associated with local irregularities and noise. Building on this multiscale framework, we also introduce a notion of alignment score that we apply to quantify the degree of spatial organization of neurites in tissue and cultured neurons. Numerical codes were implemented in Python and released open source and freely available to the scientific community.

Automated detection of soma location and morphology in neuronal network cultures.

Automated identification of the primary components of a neuron and extraction of its sub-cellular features are essential steps in many quantitative studies of neuronal networks. The focus of this paper is the development of an algorithm for the automated detection of the location and morphology of somas in confocal images of neuronal network cultures. This problem is motivated by applications in high-content screenings (HCS), where the extraction of multiple morphological features of neurons on large data sets is required. Existing algorithms are not very efficient when applied to the analysis of confocal image stacks of neuronal cultures. In addition to the usual difficulties associated with the processing of fluorescent images, these types of stacks contain a small number of images so that only a small number of pixels are available along the z-direction and it is challenging to apply conventional 3D filters. The algorithm we present in this paper applies a number of innovative ideas from the theory of directional multiscale representations and involves the following steps: (i) image segmentation based on support vector machines with specially designed multiscale filters; (ii) soma extraction and separation of contiguous somas, using a combination of level set method and directional multiscale filters. We also present an approach to extract the soma's surface morphology using the 3D shearlet transform. Extensive numerical experiments show that our algorithms are computationally efficient and highly accurate in segmenting the somas and separating contiguous ones. The algorithms presented in this paper will facilitate the development of a high-throughput quantitative platform for the study of neuronal networks for HCS applications.

3-D discrete shearlet transform and video processing.

In this paper, we introduce a digital implementation of the 3-D shearlet transform and illustrate its application to problems of video denoising and enhancement. The shearlet representation is a multiscale pyramid of well-localized waveforms defined at various locations and orientations, which was introduced to overcome the limitations of traditional multiscale systems in dealing with multidimensional data. While the shearlet approach shares the general philosophy of curvelets and surfacelets, it is based on a very different mathematical framework, which is derived from the theory of affine systems and uses shearing matrices rather than rotations. This allows a natural transition from the continuous setting to the digital setting and a more flexible mathematical structure. The 3-D digital shearlet transform algorithm presented in this paper consists in a cascade of a multiscale decomposition and a directional filtering stage. The filters employed in this decomposition are implemented as finite-length filters, and this ensures that the transform is local and numerically efficient. To illustrate its performance, the 3-D discrete shearlet transform is applied to problems of video denoising and enhancement, and compared against other state-of-the-art multiscale techniques, including curvelets and surfacelets.