Oxidative stress differentially induces tau dissociation from neuronal microtubules in neurites of neurons cultured from different regions of the embryonic Gallus domesticus brain.

Abnormal phosphorylation of microtubule-associated proteins such as tau has been shown to play a role in neurodegenerative disorders. It is hypothesized that oxidative stress-induced aggregates of hyperphosphorylated tau could lead to the microtubule network degradation commonly associated with neurodegeneration. We investigated whether oxidative stress induced tau hyperphosphorylation and focused on neurite degradation using cultured neurons isolated from the embryonic chick brain as a model system. Cells were isolated from the cerebrum, cerebellum, and tectum of 14-day-old chicks, grown separately in culture, and treated with tert-Butyl hydroperoxide (to simulate oxidative stress) for 48 hr. Relative expression and localization of tau or phospho-tau and ß-tubulin III in neurites were determined using quantitative immunocytochemistry and confocal microscopy. In untreated cells, tau was tightly colocalized with ß-tubulin III. Increasing levels of oxidative stress induced an increase in overall tau expression in neurites of cerebral and tectal but not the cerebellar neurons, coupled with a decrease in phospho-tau expression in tectal but not the cerebral or cerebellar neurons. In addition, oxidative stress induced the degeneration of the distal ends of the neurites and redistribution of phospho-tau toward the neuronal soma in the cerebral but not the tectal and cerebellar neurons. These results suggest that oxidative stress induces changes in tau protein that precede cytoskeletal degradation and neurite retraction. Additionally, there is a differential susceptibility of neuronal subpopulations to oxidative stress, which may offer potential avenues for investigation of the cellular mechanisms underlying the differential manifestations of neurodegenerative disorders in different regions of the brain.

Primary Cultures of Olfactory Neurons from the Avian Olfactory Epithelium.

The culture of the olfactory epithelium has been a useful model for the study of neurogenesis since olfactory neurons regenerate continuously throughout the adult lifespan. Structurally and functionally mature olfactory neurons are generated in vitro from non-neuronal precursors in a process that resembles the in vivo counterparts. This chapter describes a technique for culture of olfactory neurons from the avian olfactory epithelium of embryonic chickens; this enables the controlled laboratory study of a critical sensory system that is unstudied in this major vertebrate class. The techniques described here are broadly applicable to other endothermic vertebrate species.

The Developmental Process of the Growing Motile Ciliary Tip Region.

Eukaryotic motile cilia/flagella play vital roles in various physiological processes in mammals and some protists. Defects in cilia formation underlie multiple human disorders, known as ciliopathies. The detailed processes of cilia growth and development are still far from clear despite extensive studies. In this study, we characterized the process of cilium formation (ciliogenesis) by investigating the newly developed motile cilia of deciliated protists using complementary techniques in electron microscopy and image analysis. Our results demonstrated that the distal tip region of motile cilia exhibit progressive morphological changes as cilia develop. This developmental process is time-dependent and continues after growing cilia reach their full lengths. The structural analysis of growing ciliary tips revealed that B-tubules of axonemal microtubule doublets terminate far away from the tip end, which is led by the flagellar tip complex (FTC), demonstrating that the FTC might not directly mediate the fast turnover of intraflagellar transport (IFT).

Sub-micromolar concentrations of retinoic acid induce morphological and functional neuronal phenotypes in SK-N-SH neuroblastoma cells.

Neuroblastoma cells are neural crest derivatives that can differentiate into neuron-like cells in response to exogenous agents, and are known to be particularly sensitive to retinoic acid. The spectrum of neuroblastoma responses, ranging from proliferation, migration, differentiation, or apoptosis, is difficult to predict due to the heterogeneity of these tumors and to the broad effective range of retinoic acid. Our study focused on the effects of nanomolar concentrations of retinoic acid on neuroblastoma differentiation in two cell lines cells: SK-N-SH (HTB-11) and IMR-32. Each cell line was treated with retinoic acid from 1 to 100 nM for up to 6 d. Morphological changes were quantified; immunocytochemistry was used to observe changes in neuronal protein expression and localization, while live-cell calcium imaging utilizing pharmacological agents was conducted to identify neuron-like activity. Retinoic acid-treated HTB-11 but not IMR-32 cells developed specific neuronal phenotypes: acquisition of long neurite-like processes, expression of neurofilament-200, increased responsiveness to acetylcholine, and decreased responsiveness to nicotine and epinephrine. In addition, nanomolar levels of retinoic acid elicited increased nuclear trafficking of the CRABP2, which is traditionally associated with gene expression of cellular pathways related to neuronal differentiation. Collectively, these results show that nanomolar concentrations of retinoic acid are capable of inducing both structural and functional neuron-like features in HTB-11 cells using CRABP2, suggesting differentiation in neuroblastoma cells into neuronal phenotypes. These have important implications for both chemotherapeutic design and for the use of neuroblastomas as in vitro models for neuron differentiation.

Inhibition of exendin-4-induced steatosis by protein kinase A in cultured HepG2 human hepatoma cells.

Nonalcoholic fatty liver is characterized by the abnormal accumulation of triglycerides within hepatocytes, resulting in a steatotic liver. Glucagon-like peptide 1 and its analog exendin-4 can ameliorate certain aspects of this syndrome by inducing weight loss and reducing hepatic triglyceride accumulation, but it is unclear whether these effects result from the effects of glucagon-like peptide 1 on the pancreas, or from direct action on the liver. This study investigated the direct action and putative cellular mechanism of exendin-4 on steatotic hepatocytes in culture. Steatosis was induced in cultured HepG2 human hepatoma cells by incubation in media supplemented with 2 mM each of linoleic acid and oleic acid. Steatotic hepatocytes were then pre-incubated in the protein kinase A inhibitor H89 for 30 min, then treated with exendin-4 over a period of 24 h. Cell viability and triglyceride content were characterized by a TUNEL assay and AdipoRed staining, respectively. Our results showed that steatotic cells maintained high levels of intracellular triglycerides (80%) compared to lean controls (25%). Exendin-4 treatment caused a significant reduction in intracellular triglyceride content after 12 h that persisted through 24 h, while protein kinase A inhibitors abolished the effects of exendin-4. The results demonstrate the exendin-4 induces a partial reduction in triglycerides in steatotic hepatocytes within 12 h via the GLP-1 receptor-mediated activation of protein kinase A. Thus, the reduction in hepatocyte triglyceride accumulation is likely driven primarily by downregulation of lipogenesis and upregulation of ß-oxidation of free fatty acids.

Chronic odorant exposure upregulates acquisition of functional properties in cultured embryonic chick olfactory sensory neurons.

Neuronal development and differentiation is modulated by activity-dependent mechanisms that stimulate endogenous neurogenesis and differentiation to promote adaptive survival of the organism. Studies on bird odor imprinting have shown how sensory stimuli or environmental influences can affect neonatal behavior, presumably by remodeling the developing nervous system. It is unclear whether these changes originate from the sensory neurons themselves or from the brain. Thus, we attempted to address this by using an in vitro system to separate the peripheral neurons from their central connections. Olfactory neurons from embryonic day 17 Gallus domesticus chicks were isolated, cultured, and exposed to 100 µM amyl acetate or phenethyl alcohol in 12-hr bouts, alternated with periods of no-odor exposure. On days 4 and 5 in vitro, cells were immunostained for olfactory marker protein, neuron-specific tubulin, and olfactory GTP-binding protein, and tested for odorant sensitivity using calcium imaging. While odorant exposure did not result in a significant increase in the overall number of neurons, it promoted neuron differentiation: a larger proportion of odorant-exposed cells expressed olfactory marker protein and the olfactory GTP-binding protein. When cell responsiveness was tested using calcium imaging, a greater proportion of odorant-exposed cells responded to stimulation with 100 µM amyl acetate or phenethyl alcohol. Thus, odorant exposure during development modulated the developmental trajectories of individual neurons, resulting in changes in protein expression associated with odorant signaling. This suggests that the neuronal changes in the periphery have an important contribution to the overall long-term functional changes associated with odor imprinting. © 2016 Wiley Periodicals, Inc.

Extracellular glutathione promotes migration of hydrogen peroxide-stressed cultured chick embryonic skin cells.

The ability of glutathione to affect melanocyte survival has fostered its use in a variety of applications related to epithelial cells. Our study focused on fibroblast migration and the effects of oxidative stress. We used scratch assays to measure cell migration: fibroblasts were harvested from embryonic chicks, grown to confluence in a monolayer, and the layer was scratched to initiate migration. Migration rates were measured over 8 h using photomicrographs, and vinculin expression as an indicator focal adhesion formation was measured using immunofluorescence. Addition of 200 µM glutathione to the culture media in which the cells grew resulted in a significantly increased rate of scratch closure. When the scratch assays were performed in the presence of 100 µM H2O2 (to simulate oxidative stress), the cells ceased to migrate. Addition of 200 µM glutathione to the H2O2-treated scratched layers resulted in a restoration of the scratch closure capabilities. At the subcellular level, addition of extracellular glutathione resulted in a redistribution of vinculin into fewer but larger aggregates. In cells at the edge of scratched monolayers that were treated with H2O2, vinculin particles were distributed throughout the cell in smaller aggregates; addition of glutathione resulted in vinculin aggregates that were larger and closer to the edges of the cell, indicating that these cells were more migratory. Our results suggest that glutathione promotes fibroblast migration, possibly via a mechanism that promotes the formation of focal adhesions.

Differential effects of hypoxic and hyperoxic stress-induced hypertrophy in cultured chick fetal cardiac myocytes.

The adult heart responds to contraction demands by hypertrophy, or enlargement, of cardiac myocytes. Adaptive hypertrophy can occur in response to hyperoxic conditions such as exercise, while pathological factors that result in hypoxia ultimately result in heart failure. The difference in the outcomes produced by pathologically versus physiologically induced hypertrophy suggests that the cellular signaling pathways or conditions of myocytes may be different at the cellular level. The structural and functional changes in myocytes resulting from hyperoxia (simulated using hydrogen peroxide) and hypoxia (using oxygen deprivation) were tested on fetal chick cardiac myocytes grown in vitro. Structural changes were measured using immunostaining for α-sarcomeric actin or MyoD, while functional changes were assessed using immunostaining for calcium/calmodulin-dependent kinase (CaMKII) and by measuring intracellular calcium fluxes using live cell fluorescence imaging. Both hypoxic and hyperoxic stress resulted in an upregulation of actin and MyoD expression. Similarly, voltage-gated channels governing myocyte depolarization and the regulation of CaMK were unchanged by hyperoxic or hypoxic conditions. However, the dynamic features of calcium fluxes elicited by caffeine or epinephrine were different in cells subjected to hypoxia versus hyperoxia, suggesting that these different conditions differentially affect components of ligand-activated signaling pathways that regulate calcium. Our results suggest that changes in signaling pathways, rather than structural organization, may mediate the different outcomes associated with hyperoxia-induced versus hypoxia-induced hypertrophy, and these changes are likely initiated at the cellular level.

Time course of structural and functional maturation of human olfactory epithelial cells in vitro.

The unique ability of olfactory neurons to regenerate in vitro has allowed their use for the study of olfactory function, regeneration, and neurodegenerative disorders; thus, characterization of their properties is important. This present study attempts to establish the timeline of structural (protein expression) and functional (odorant sensitivity) maturation of human olfactory epithelial cells (hOE) in vitro using biopsy-derived cultured tissue. Cells were grown for 7 days; on each day, cells were tested for odorant sensitivity using calcium imaging techniques and then protein expression of each cell was tested using immunocytochemistry for proteins typically used for characterizing olfactory cells. Previous studies have shown that mature olfactory neurons in vitro attain a unique "phase-bright" morphology and express the olfactory marker protein (OMP). By day 3 in vitro, a variety of cells were odorant-sensitive, including both "phase-bright" and "phase-dark" cells that have previously been considered glial-like cells. The functional maturation of these hOEs appears to take place within 4 days. Interestingly, the emergence of an odorant sensitivity profile of both phase-bright and phase-dark cells preceded the expression of marker protein expression for OMP (which is expressed only by mature neurons in vivo). This structural maturation took 5 days, suggesting that the development of odorant sensitivity is not coincident with the expression of marker molecules that are hallmarks of structural maturation. These results have important implications for the use of hOEs as in vitro models of olfactory and neuronal function.

Age-associated loss of selectivity in human olfactory sensory neurons.

We report a cross-sectional study of olfactory impairment with age based on both odorant-stimulated responses of human olfactory sensory neurons (OSNs) and tests of olfactory threshold sensitivity. A total of 621 OSNs from 440 subjects in 2 age groups of younger (≤ 45 years) and older (≥ 60 years) subjects were investigated using fluorescence intensity ratio fura-2 imaging. OSNs were tested for responses to 2 odorant mixtures, as well as to subsets of and individual odors in those mixtures. Whereas cells from younger donors were highly selective in the odorants to which they responded, cells from older donors were more likely to respond to multiple odor stimuli, despite a loss in these subjects' absolute olfactory sensitivity, suggesting a loss of specificity. This degradation in peripheral cellular specificity may impact odor discrimination and olfactory adaptation in the elderly. It is also possible that chronic adaptation as a result of reduced specificity contributes to observed declines in absolute sensitivity.

Altered olfactory epithelial structure and function in feline models of mucopolysaccharidoses I and VI.

The mucopolysaccharidoses (MPS) are a family of lysosomal storage diseases resulting in developmental defects and, in some types, mental retardation and other neurological symptoms. To gain insight into the neurological dysfunction in MPS, we examined the morphology of olfactory epithelia (OE) and physiology of olfactory receptor neurons (ORNs) in cat models of MPS I, a type in which neuronal lesions are prominent, and MPS VI, in which they are essentially absent. Histopathology showed that both groups of MPS-affected cats had significantly thinner OE than controls. Although immature and mature ORNs were present in both MPS I and VI affected OE, the OE of MPS I-affected cats was structurally disorganized. ORN function was assessed with calcium imaging and patch-clamp recording. Few viable ORNs were recovered from MPS VI cats, but these exhibited normal responses to odors and pharmacological stimuli. In contrast, viable ORNs were as prevalent in MPS I as in controls but were significantly less likely to respond to odor stimuli, although other responses were normal. Disrupted OE organization and impaired ORN function in MPS I, but not MPS VI, corresponds to the central nervous system lesions found in MPS I but not MPS VI. These data represent the first neurophysiological correlate of this correspondence and have implications both for understanding the role of glycosaminoglycans in maintenance of the OE and for targeting further research into the basis for and treatment of the neurological consequences of MPS disorders.

The peripheral olfactory system of the domestic chicken: physiology and development.

Olfaction is a ubiquitous sensory system found in all terrestrial vertebrates. Birds use olfaction for several important activities such as feeding and mating; thus, understanding bird biology would also require the systematic study olfaction. In addition, the olfactory system has several unique features that are useful for the study of nervous system function and development, including a large multigene family for olfactory receptor expression, peripheral neurons that regenerate, and a complex system for sensory innervation of the olfactory bulb. We focused on physiological, anatomical and behavioral approaches to study the chick olfactory neurons and the olfactory bulb. Chick olfactory neurons displayed some properties similar to those found in mature neurons of other vertebrate species, and other properties that were unique. Since information from these neurons is initially processed in the olfactory bulb, we also conducted preliminary studies on the developmental timeline of this structure and showed that glomerular structures are organized in ovo during a critical time period, during which embryonic chicks can form behavioral associations with odorants introduced in ovo. Lastly, we have shown that chick olfactory neurons can grow and mature in vitro, allowing their use in cell culture studies. These results collectively demonstrate some of the features of the olfactory system that are common to all vertebrates, and some that are unique to birds. These highlight the potential for the use of the physiology and development of the olfactory system as a model system for avian brain neurobiology.

Evidence for multiple calcium response mechanisms in mammalian olfactory receptor neurons.

Olfactory receptor neurons employ a diversity of signaling mechanisms for transducing and encoding odorant information. The simultaneous activation of subsets of receptor neurons provides a complex pattern of activation in the olfactory bulb that allows for the rapid discrimination of odorant mixtures. While some transduction elements are conserved among many species, some species-specificity occurs in certain features that may relate to their particular physiology and ecological niche. However, studies of olfactory transduction have been limited to a relatively small number of vertebrate and invertebrate species. To better understand the diversity and evolution of olfactory transduction mechanisms, we studied stimulus-elicited calcium fluxes in olfactory neurons from a previously unstudied mammalian species, the domestic cat. Isolated cells from cat olfactory epithelium were stimulated with odorant mixtures and biochemical agents, and cell responses were measured with calcium imaging techniques. Odorants elicited either increases or decreases in intracellular calcium; odorant-induced calcium increases were mediated either by calcium fluxes through the cell membrane or by mobilization of intracellular stores. Individual cells could employ multiple signaling mechanisms to mediate responses to different odorants. The physiological features of these olfactory neurons suggest greater complexity than previously recognized in the role of peripheral neurons in encoding complex odor stimuli. The investigation of novel and unstudied species is important for understanding the mechanisms of odorant signaling that apply to the olfactory system in general and suggests both broadly conserved and species-specific evolutionary adaptations.

Characteristics of odorant elicited calcium fluxes in acutely-isolated chick olfactory neurons.

To understand avian olfaction, it is important to characterize the peripheral olfactory system of a representative bird species. This study determined the functional properties of olfactory receptor neurons of the chicken olfactory epithelium. Individual neurons were acutely isolated from embryonic day-18 to newborn chicks by dissection and enzymatic dissociation. We tested single olfactory neurons with behaviorally relevant odorant mixtures and measured their responses using ratiometric calcium imaging; techniques used in this study were identical to those used in other studies of olfaction in other vertebrate species. Chick olfactory neurons displayed properties similar to those found in other vertebrates: they responded to odorant stimuli with either decreases or increases in intracellular calcium, calcium increases were mediated by a calcium influx, and responses were reversibly inhibited by 100 microM L: -cis-diltiazem, 1 mM Neomycin, and 20 microM U73122, which are biochemical inhibitors of second messenger signaling. In addition, some cells showed a complex pattern of responses, with different odorant mixtures eliciting increases or decreases in calcium in the same cell. It appears that there are common features of odorant signaling shared by a variety of vertebrate species, as well as features that may be peculiar to chickens.

Aberrant intracellular calcium signaling in olfactory neurons from patients with bipolar disorder.

OBJECTIVE: The authors examined the feasibility of using olfactory receptor neurons from living patients to test whether calcium signaling is altered in a neuronal cell population in bipolar disorder. METHOD: Ratiometric fluorescence photomicroscopy was used to assess basal and stimulus-induced changes in intracellular calcium levels in biopsy-derived olfactory receptor neurons from seven euthymic patients with bipolar disorder who were medication-free, 10 euthymic patients with bipolar disorder who were treated with mood stabilizers, and 17 age- and sex-matched comparison subjects without bipolar disorder. RESULTS: Olfactory receptor neurons from the seven medication-free patients responded to stimuli predominantly with decreases in intracellular calcium, unlike those from the seven matched healthy subjects. Olfactory receptor neurons from patients treated with mood stabilizers were less likely to respond to stimulation than olfactory receptor neurons from medication-free patients. CONCLUSIONS: This study demonstrates the feasibility of using olfactory receptor neurons to examine alterations in intracellular signaling in neuronal cells from living patients. Our results, although based on a small number of subjects, suggest that altered intracellular calcium signaling in olfactory receptor neurons may be a trait of bipolar disorder.

Cell and molecular biology of human olfaction.

Progress in our understanding of olfactory receptor physiology has progressed greatly over the past 10 years. It has become clear that many anatomical and molecular features of the peripheral aspect of the olfactory system have remained highly conserved across diverse species. Yet, this structure is responsible for conveying a wide variety of information about the environment that is necessary to the successful location of food, mates, and avoidance of danger, and it is thus not surprising that specializations have also evolved to suit the differing needs of different species. While the basic anatomical features reflect those of other mammals, functional studies of human olfactory receptor neurons have revealed physiological features both similar to and differing from those of other mammalian species. This review presents an overview of both the anatomical and physiological data describing the cell and molecular biology of the peripheral human olfactory system and how it functions in health and disease.

The superior turbinate as a source of functional human olfactory receptor neurons.

OBJECTIVES: The function of human olfactory receptor neurons (ORNs) remains incompletely understood, in part because of the difficulty of obtaining viable olfactory tissue for study. During endoscopic sphenoidotomy, a portion of the superior turbinate is often removed to achieve wide and safe access to the sphenoid sinus. The purpose of this study was to determine whether functional olfactory mucosa could be obtained from such superior turbinate tissue. STUDY DESIGN/METHODS: Superior turbinate tissue was resected from 4 patients undergoing transnasal endoscopic approaches to the sphenoid sinus. The gross appearance of the turbinate mucosa was normal at the time of surgery. The specimens were placed directly into cold cell culture media and transferred to the laboratory. A portion of the mucosa was fixed and embedded for histology and immunohistochemistry. The remaining tissue was enzymatically dissociated, and the resulting cell suspension was either prepared for immediate calcium imaging or placed into cell culture. Cultured ORNs underwent calcium imaging after several weeks to assess their ability to respond to odorants. RESULTS: Histologic analysis of superior turbinate tissue revealed the presence of patchy olfactory neuroepithelium staining positive for olfactory marker protein. Acutely dissociated ORNs were capable of generating calcium responses to odorant mixtures. ORNs could be maintained in mixed culture and retained their ability to respond to odorants. CONCLUSIONS: Superior turbinate tissue removed during endoscopic sphenoidotomy can provide a valuable source of human olfactory neuroepithelium for functional or histologic study. Superior turbinate tissue yields stem cells and immature neurons capable of differentiating into ORNs that retain many of their functional characteristics even after growth in culture.