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    A multistep deep learning framework for the automated detection and segmentation of astrocytes in fuorescent images of brain tissue
    (Nature Publishing Group, 2020) Kayasandık, Cihan Bilge; Ru, Wenjuan; Labate, Demetrio
    While astrocytes have been traditionally described as passive supportive cells, studies during the last decade have shown they are active players in many aspects of CNS physiology and function both in normal and disease states. However, the precise mechanisms regulating astrocytes function and interactions within the CNS are still poorly understood. This knowledge gap is due in large part to the limitations of current image analysis tools that cannot process astrocyte images efficiently and to the lack of methods capable of quantifying their complex morphological characteristics. To provide an unbiased and accurate framework for the quantitative analysis of fluorescent images of astrocytes, we introduce a new automated image processing pipeline whose main novelties include an innovative module for cell detection based on multiscale directional filters and a segmentation routine that leverages deep learning and sparse representations to reduce the need of training data and improve performance. Extensive numerical tests show that our method performs very competitively with respect to state-of-the-art methods also in challenging images where astrocytes are clustered together. Our code is released open source and freely available to the scientific community.
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    Advances in quantitative analysis of astrocytes using machine learning
    (Wolters Kluwer Medknow Publications, 2023) Labate, Demetrio; Kayasandık, Cihan Bilge
    Astrocytes, a subtype of glial cells, are starshaped cells that are involved in the homeostasis and blood flow control of the central nervous system (CNS). They are known to provide structural and functional support to neurons, including the regulation of neuronal activation through extracellular ion concentrations, the regulation of energy dynamics in the brain through the transfer of lactate to neurons, and the modulation of synaptic transmission via the release of neurotransmitters such as glutamate and adenosine triphosphate. In addition, astrocytes play a critical role in neuronal reconstruction after brain injury, including neurogenesis, synaptogenesis, angiogenesis, repair of the blood-brain barrier, and glial scar formation after traumatic brain injury (Zhou et al., 2020). The multifunctional role of astrocytes in the CNS with tasks requiring close contact with their targets is reflected by their morphological complexity, with processes and ramifications occurring over multiple scales where interactions are plastic and can change depending on the physiological conditions. Another major feature of astrocytes is reactive astrogliosis, a process occurring in response to traumatic brain injury, neurological diseases, or infection which involves substantial morphological alterations and is often accompanied by molecular, cytoskeletal, and functional changes that ultimately play a key role in the disease outcome (Schiweck et al., 2018). Because morphological changes in astrocytes correlate so significantly with brain injury and the development of pathologies of the CNS, there is a major interest in methods to reliably detect and accurately quantify such morphological alterations. We review below the recent progress in the quantitative analysis of images of astrocytes. We remark that, while our discussion is focused on astrocytes, the same methods discussed below can be applied to other types of complex glial cells.
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    Automated detection of GFAP-labeled astrocytes in micrographs using YOLOv5
    (Nature Research, 2022) Huang, Yewen; Kruyer, Anna; Syed, Sarah; Kayasandık, Cihan Bilge; Papadakis, Manos; Labate, Demetrio
    Astrocytes, a subtype of glial cells with a complex morphological structure, are active players in many aspects of the physiology of the central nervous system (CNS). However, due to their highly involved interaction with other cells in the CNS, made possible by their morphological complexity, the precise mechanisms regulating astrocyte function within the CNS are still poorly understood. This knowledge gap is also due to the current limitations of existing quantitative image analysis tools that are unable to detect and analyze images of astrocyte with sufficient accuracy and efficiency. To address this need, we introduce a new deep learning framework for the automated detection of GFAP-immunolabeled astrocytes in brightfield or fluorescent micrographs. A major novelty of our approach is the applications of YOLOv5, a sophisticated deep learning platform designed for object detection, that we customized to derive optimized classification models for the task of astrocyte detection. Extensive numerical experiments using multiple image datasets show that our method performs very competitively against both conventional and state-of-the-art methods, including the case of images where astrocytes are very dense. In the spirit of reproducible research, our numerical code and annotated data are released open source and freely available to the scientific community.
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    Imaging of the axon initial segment
    (Blackwell Publishing, Inc., 2019) Di Re, Jessica; Kayasandık, Cihan Bilge; Botello-Lins, Gonzalo; Labate, Demetrio; Laezza, Fernanda M.
    The axon initial segment (AIS) is the first 20- to 60-?m segment of the axon proximal to the soma of a neuron. This highly specialized subcellular domain is the initiation site of the action potential and contains a high concentration of voltage-gated ion channels held in place by a complex nexus of scaffolding and regulatory proteins that ensure proper electrical activity of the neuron. Studies have shown that dysfunction of many AIS channels and scaffolding proteins occurs in a variety of neuropsychiatric and neurodegenerative diseases, raising the need to develop accurate methods for visualization and quantification of the AIS and its protein content in models of normal and disease conditions. In this article, we describe methods for immunolabeling AIS proteins in cultured neurons and brain slices as well as methods for quantifying protein expression and pattern distribution using fluorescent labeling of these proteins.
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    Inhibition of AKT signaling alters beta IV spectrin distribution at the AIS and increases neuronal excitability
    (Frontiers Media S.A., 2021) Di Re, Jessica; Hsu, Wei-Chun J.; Kayasandık, Cihan Bilge; Fularczyk, Nickolas; James, Thomas F.; Nenov, Miroslav N.; Negi, Pooran; Marosi, Mate; Scala, Federico; Prasad, Saurabh; Labate, Demetrio; Laezza, Fernanda
    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, beta IV spectrin, and the cell-adhesion molecule neurofascin were analyzed, revealing beta IV 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 beta IV 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 beta IV spectrin distribution and neuronal firing that may be associated with neuropsychiatric and neurodegenerative disorders.