Page 1. Computer Vision Algorithms for Quantifying the Growth and Behavior of Neurons Cultured on... more Page 1. Computer Vision Algorithms for Quantifying the Growth and Behavior of Neurons Cultured on Nanofabricated Surfaces Muhammad-Amri Abdul Karim, Khalid Al-Kofahi, and Badrinath Roysam Electrical, Computer & ...
IEEE Journal of Selected Topics in Signal Processing, Feb 1, 2016
The morphologies of microglial arbors in brain tissue are important indicators of their state of ... more The morphologies of microglial arbors in brain tissue are important indicators of their state of activation, and their spatial distribution indicates patterns of tissue perturbation. This paper presents a non-parametric Bayesian approach to unsupervised quantitative profiling of microglial activation states, and mapping their 3-D spatial distributions across extended brain tissue regions imaged by mosaiced confocal microscopy. Our approach is to perform large-scale automated reconstruction of all the microglia using a learned label-consistent dictionary model for the microglial processes, and extract 136 arbor features per cell. We then fit an infinite Gaussian mixture model with collapsed Gibbs sampling to the arbor features, to discover arbor-morphological classes, their morphological `signatures', and the associated covariance matrices all in an unsupervised manner. This nonparametric approach can handle clusters with arbitrarily shaped covariance matrices, perform automatic model selection, handle unbalanced clusters, and detect overlapping classes, without the need to specify the number of clusters. The results can be used for diverse investigational purposes, including: population-scale modeling, automated discovery of arbor classes and their distinguishing quantitative signatures and features, quantifying the nature and extent of the cellular heterogeneity, and spatial mapping of cellular arbor morphologies, and mapping tissue alterations.
... on low-level image-based cues, such as coherence of brightness, color, texture or motion, top... more ... on low-level image-based cues, such as coherence of brightness, color, texture or motion, top ... described an algorithm that uses a statistical model-based approach to combine the attractive ...intensity-gradient-based cues, and the knowledge of the expected anatomic shape of the ...
Publisher Summary This chapter discusses the mechanism of synaptic modulation. Synaptic modulatio... more Publisher Summary This chapter discusses the mechanism of synaptic modulation. Synaptic modulation is an observed change in the synaptic function. This change can occur by summation of a variety of physiological and biochemical cellular responses. Thus, the term, “synaptic modulation” does not make a precise statement and in this sense, is not analogous to the term, “synaptic transmission.” Synaptic transmission describes the transfer of a signal from one cell to another using mechanisms that are similar at all synapses. Synaptic transmission occurs at a particular location that can be described by morphologically pre- and postsynaptic specializations, which involves vesicular release of a neurotransmitter, and elicits a response from the postsynaptic cell through receptors that recognize the specific transmitter released. There are data inconsistent with this description, that is, release of amino acid transmitters that may not be vesicular, and adrenergic synapses that are difficult to describe morphologically. However, the term, “synaptic transmission” conveys both the description of the event as well as the mechanism involved.
International review of experimental pathology, 1996
1. Int Rev Exp Pathol. 1996;36:53-72. Three-dimensional confocal light and electron microscopy of... more 1. Int Rev Exp Pathol. 1996;36:53-72. Three-dimensional confocal light and electron microscopy of central nervous system tissue and neurons and glia in culture. Turner JN, Swann JW, Szarowski DH, Smith KL, Shain W, Carpenter ...
Antiserums prepared against hybrid cell strains formed between freshly isolated rodent neural ret... more Antiserums prepared against hybrid cell strains formed between freshly isolated rodent neural retina cells and a human fibroblast cell line W.I. 18, VA-2 recognize cell surface antigens that are 1) restricted to the neural retina, 2) present in both embryonic and adult retina, and 3) localized to groups of cells within the retina. Normal segregants (i.e., those lines that had retained rodent chromosomes and lost human chromosomes) were used as immunogens in rabbits to produce the antiserums. Antiserums against both whole cells and purified plasma membranes were adsorbed with human parent VA-2 cells and nonneural and neural rodent tissue to remove cross-reacting specificities. All six antiserums studied continued to react with embryonic retina after brain cross-reactivity was removed.
ABSTRACTProteins often deform, dehydrate or otherwise denature when adsorbed or patterned directl... more ABSTRACTProteins often deform, dehydrate or otherwise denature when adsorbed or patterned directly onto an inorganic substrate, thus losing specificity and biofunctionality. One method used to maintain function is to pattern the protein of interest directly onto another underlying protein or polypeptide that acts as a buffer layer between the substrate and the desired protein. We have used microcontact printing (μcp) to cross-stamp orthogonal linear arrays of two different proteins (e.g., IgG, poly-lysine, protein A) onto glass substrates. This created three separate types of protein-substrate microenvironments, including crossover structures of protein one on protein two. We report preliminary fluorescent microscopy and scanning force microscopy characterization of these structures, including commonly encountered structural defects.
Stellation is the process by which astrocytes change from epithelial-like to process-bearing cell... more Stellation is the process by which astrocytes change from epithelial-like to process-bearing cells. Stellation occurs following activation of either cyclic AMP-dependent protein kinase or protein kinase C. This process occurs through tubulin-dependent rearrangement of the cytoskeleton. We have evaluated the ability of phorbol, 12-myristate, 13-acetate (PMA) to induce astrocyte stellation. Astrocytes from five brain regions (cerebellum, cerebral cortex, hippocampus, diencephalon, and brain-stem) were examined to determine if all astrocytes would exhibit similar responses to this activator of protein kinase C. Stellation was evaluated following cell fixation by either phase optics using conventional light microscopy, or scanning laser confocal light microscopy of cultures prepared using immunocytochemistry for tubulin and glial fibrillary acidic protein. Both the number of cells responding to PMA and the sensitivity to PMA varied for astrocytes from each brain region. PMA-induced stellation was most robust in cerebellar and brainstem astrocytes, with greater than 70% responding. Less than 40% of hippocampal and diencephalic astrocytes responded to PMA at the maximum dose (10(-5) M). PMA also induced different numbers of processes or branching patterns of processes on astrocytes from different brain regions. The protein kinase C induced stellation response in astrocytes supports the hypothesis that astrocytes contribute to neural plasticity.
The endothelial cells comprising brain capillaries have extremely tight intercellular junctions w... more The endothelial cells comprising brain capillaries have extremely tight intercellular junctions which form an essentially impermeable barrier to passive transport of water soluble molecules between the blood and brain. Several in vitro models of the blood-brain barrier (BBB) have been studied, most utilizing commercially available polymer membranes affixed to plastic inserts. There is mounting evidence that direct contact between endothelial cells and astrocytes, another cell type found to have intimate interaction with the brain side of BBB capillaries, is at least partially responsible for the development of the tight intercellular junctions between BBB endothelial cells. However, the membranes commonly used for BBB in vitro models are lacking certain attributes that would permit a high degree of direct contact between astrocytes and endothelial cells cultured on opposing sides. This work is based on the hypothesis that co-culturing endothelial and astrocyte cells on opposite sides of an ultra-thin, highly porous membrane will allow for increased direct interaction between the two cell types and therefore result in a better model of the BBB. We used standard nanofabrication techniques to make membranes from low-stress silicon nitride that are at least an order of magnitude thinner and at least two times more porous than commercial membrane inserts. An experimental survey of pore sizes for the silicon nitride membranes suggested pores approximately 400 nm in diameter are adequate for restricting astrocyte cell bodies to the seeded side while allowing astrocyte processes to pass through the pores and interact with endothelial cells on the opposite side. The inclusion of a spun-on, cross-linked collagen membrane allowed for astrocyte attachment and culture on the membranes for over two weeks. Astrocytes and endothelial cells displayed markers specific to their cell types when grown on the silicon nitride membranes. The transendothelial electrical resistances, a measure of barrier tightness, of endothelial and astrocyte co-cultures on the silicon nitride membranes were comparable to the commercial membranes, but neither system showed synergy between the two cell types in forming a tighter barrier. This lack of synergy may have been due to the loss of ability of commercially available primary bovine brain microvascular endothelial cells to respond to astrocyte differentiating signals.
Abstract: Activation of β‐adrenergic receptors on astrocytes in primary cell culture results in t... more Abstract: Activation of β‐adrenergic receptors on astrocytes in primary cell culture results in the release of taurine, an inhibitory transmitter. Taurine release occurs via a cyclic AMP‐mediated intracellular pathway, because (a) taurine release and intracellular cyclic AMP accumulation have similar pharmacologies and time courses of activation and (b) N6,O2′‐dibutyryl cyclic AMP stimulates release with a time course similar to that observed with the β‐adrenergic agonist isoproterenol. These results describe a previously unrecognized physiological function of astrocytes in the CNS—receptor‐mediated release of the neuroactive amino acid taurine. This observation indicates that astrocytes may function as local regulators of neuronal activity.
Page 1. Computer Vision Algorithms for Quantifying the Growth and Behavior of Neurons Cultured on... more Page 1. Computer Vision Algorithms for Quantifying the Growth and Behavior of Neurons Cultured on Nanofabricated Surfaces Muhammad-Amri Abdul Karim, Khalid Al-Kofahi, and Badrinath Roysam Electrical, Computer & ...
IEEE Journal of Selected Topics in Signal Processing, Feb 1, 2016
The morphologies of microglial arbors in brain tissue are important indicators of their state of ... more The morphologies of microglial arbors in brain tissue are important indicators of their state of activation, and their spatial distribution indicates patterns of tissue perturbation. This paper presents a non-parametric Bayesian approach to unsupervised quantitative profiling of microglial activation states, and mapping their 3-D spatial distributions across extended brain tissue regions imaged by mosaiced confocal microscopy. Our approach is to perform large-scale automated reconstruction of all the microglia using a learned label-consistent dictionary model for the microglial processes, and extract 136 arbor features per cell. We then fit an infinite Gaussian mixture model with collapsed Gibbs sampling to the arbor features, to discover arbor-morphological classes, their morphological `signatures', and the associated covariance matrices all in an unsupervised manner. This nonparametric approach can handle clusters with arbitrarily shaped covariance matrices, perform automatic model selection, handle unbalanced clusters, and detect overlapping classes, without the need to specify the number of clusters. The results can be used for diverse investigational purposes, including: population-scale modeling, automated discovery of arbor classes and their distinguishing quantitative signatures and features, quantifying the nature and extent of the cellular heterogeneity, and spatial mapping of cellular arbor morphologies, and mapping tissue alterations.
... on low-level image-based cues, such as coherence of brightness, color, texture or motion, top... more ... on low-level image-based cues, such as coherence of brightness, color, texture or motion, top ... described an algorithm that uses a statistical model-based approach to combine the attractive ...intensity-gradient-based cues, and the knowledge of the expected anatomic shape of the ...
Publisher Summary This chapter discusses the mechanism of synaptic modulation. Synaptic modulatio... more Publisher Summary This chapter discusses the mechanism of synaptic modulation. Synaptic modulation is an observed change in the synaptic function. This change can occur by summation of a variety of physiological and biochemical cellular responses. Thus, the term, “synaptic modulation” does not make a precise statement and in this sense, is not analogous to the term, “synaptic transmission.” Synaptic transmission describes the transfer of a signal from one cell to another using mechanisms that are similar at all synapses. Synaptic transmission occurs at a particular location that can be described by morphologically pre- and postsynaptic specializations, which involves vesicular release of a neurotransmitter, and elicits a response from the postsynaptic cell through receptors that recognize the specific transmitter released. There are data inconsistent with this description, that is, release of amino acid transmitters that may not be vesicular, and adrenergic synapses that are difficult to describe morphologically. However, the term, “synaptic transmission” conveys both the description of the event as well as the mechanism involved.
International review of experimental pathology, 1996
1. Int Rev Exp Pathol. 1996;36:53-72. Three-dimensional confocal light and electron microscopy of... more 1. Int Rev Exp Pathol. 1996;36:53-72. Three-dimensional confocal light and electron microscopy of central nervous system tissue and neurons and glia in culture. Turner JN, Swann JW, Szarowski DH, Smith KL, Shain W, Carpenter ...
Antiserums prepared against hybrid cell strains formed between freshly isolated rodent neural ret... more Antiserums prepared against hybrid cell strains formed between freshly isolated rodent neural retina cells and a human fibroblast cell line W.I. 18, VA-2 recognize cell surface antigens that are 1) restricted to the neural retina, 2) present in both embryonic and adult retina, and 3) localized to groups of cells within the retina. Normal segregants (i.e., those lines that had retained rodent chromosomes and lost human chromosomes) were used as immunogens in rabbits to produce the antiserums. Antiserums against both whole cells and purified plasma membranes were adsorbed with human parent VA-2 cells and nonneural and neural rodent tissue to remove cross-reacting specificities. All six antiserums studied continued to react with embryonic retina after brain cross-reactivity was removed.
ABSTRACTProteins often deform, dehydrate or otherwise denature when adsorbed or patterned directl... more ABSTRACTProteins often deform, dehydrate or otherwise denature when adsorbed or patterned directly onto an inorganic substrate, thus losing specificity and biofunctionality. One method used to maintain function is to pattern the protein of interest directly onto another underlying protein or polypeptide that acts as a buffer layer between the substrate and the desired protein. We have used microcontact printing (μcp) to cross-stamp orthogonal linear arrays of two different proteins (e.g., IgG, poly-lysine, protein A) onto glass substrates. This created three separate types of protein-substrate microenvironments, including crossover structures of protein one on protein two. We report preliminary fluorescent microscopy and scanning force microscopy characterization of these structures, including commonly encountered structural defects.
Stellation is the process by which astrocytes change from epithelial-like to process-bearing cell... more Stellation is the process by which astrocytes change from epithelial-like to process-bearing cells. Stellation occurs following activation of either cyclic AMP-dependent protein kinase or protein kinase C. This process occurs through tubulin-dependent rearrangement of the cytoskeleton. We have evaluated the ability of phorbol, 12-myristate, 13-acetate (PMA) to induce astrocyte stellation. Astrocytes from five brain regions (cerebellum, cerebral cortex, hippocampus, diencephalon, and brain-stem) were examined to determine if all astrocytes would exhibit similar responses to this activator of protein kinase C. Stellation was evaluated following cell fixation by either phase optics using conventional light microscopy, or scanning laser confocal light microscopy of cultures prepared using immunocytochemistry for tubulin and glial fibrillary acidic protein. Both the number of cells responding to PMA and the sensitivity to PMA varied for astrocytes from each brain region. PMA-induced stellation was most robust in cerebellar and brainstem astrocytes, with greater than 70% responding. Less than 40% of hippocampal and diencephalic astrocytes responded to PMA at the maximum dose (10(-5) M). PMA also induced different numbers of processes or branching patterns of processes on astrocytes from different brain regions. The protein kinase C induced stellation response in astrocytes supports the hypothesis that astrocytes contribute to neural plasticity.
The endothelial cells comprising brain capillaries have extremely tight intercellular junctions w... more The endothelial cells comprising brain capillaries have extremely tight intercellular junctions which form an essentially impermeable barrier to passive transport of water soluble molecules between the blood and brain. Several in vitro models of the blood-brain barrier (BBB) have been studied, most utilizing commercially available polymer membranes affixed to plastic inserts. There is mounting evidence that direct contact between endothelial cells and astrocytes, another cell type found to have intimate interaction with the brain side of BBB capillaries, is at least partially responsible for the development of the tight intercellular junctions between BBB endothelial cells. However, the membranes commonly used for BBB in vitro models are lacking certain attributes that would permit a high degree of direct contact between astrocytes and endothelial cells cultured on opposing sides. This work is based on the hypothesis that co-culturing endothelial and astrocyte cells on opposite sides of an ultra-thin, highly porous membrane will allow for increased direct interaction between the two cell types and therefore result in a better model of the BBB. We used standard nanofabrication techniques to make membranes from low-stress silicon nitride that are at least an order of magnitude thinner and at least two times more porous than commercial membrane inserts. An experimental survey of pore sizes for the silicon nitride membranes suggested pores approximately 400 nm in diameter are adequate for restricting astrocyte cell bodies to the seeded side while allowing astrocyte processes to pass through the pores and interact with endothelial cells on the opposite side. The inclusion of a spun-on, cross-linked collagen membrane allowed for astrocyte attachment and culture on the membranes for over two weeks. Astrocytes and endothelial cells displayed markers specific to their cell types when grown on the silicon nitride membranes. The transendothelial electrical resistances, a measure of barrier tightness, of endothelial and astrocyte co-cultures on the silicon nitride membranes were comparable to the commercial membranes, but neither system showed synergy between the two cell types in forming a tighter barrier. This lack of synergy may have been due to the loss of ability of commercially available primary bovine brain microvascular endothelial cells to respond to astrocyte differentiating signals.
Abstract: Activation of β‐adrenergic receptors on astrocytes in primary cell culture results in t... more Abstract: Activation of β‐adrenergic receptors on astrocytes in primary cell culture results in the release of taurine, an inhibitory transmitter. Taurine release occurs via a cyclic AMP‐mediated intracellular pathway, because (a) taurine release and intracellular cyclic AMP accumulation have similar pharmacologies and time courses of activation and (b) N6,O2′‐dibutyryl cyclic AMP stimulates release with a time course similar to that observed with the β‐adrenergic agonist isoproterenol. These results describe a previously unrecognized physiological function of astrocytes in the CNS—receptor‐mediated release of the neuroactive amino acid taurine. This observation indicates that astrocytes may function as local regulators of neuronal activity.
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