Automatic Robust Neurite Detection and Morphological Analysis of Neuronal Cell Cultures in High-content Screening

Cell-based high content screening (HCS) is becoming an important and increasingly favored approach in therapeutic drug discovery and functional genomics. In HCS, changes in cellular morphology and biomarker distributions provide an information-rich profile of cellular responses to experimental treatments such as small molecules or gene knockdown probes. One obstacle that currently exists with such cell-based assays is the availability of image processing algorithms that are capable of reliably and automatically analyzing large HCS image sets. HCS images of primary neuronal cell cultures are particularly challenging to analyze due to complex cellular morphology. Here we present a robust method for quantifying and statistically analyzing the morphology of neuronal cells in HCS images. The major advantages of our method over existing software lie in its capability to correct non-uniform illumination using the contrast-limited adaptive histogram equalization method; segment neuromeres using Gabor-wavelet texture analysis; and detect faint neurites by a novel phase-based neurite extraction algorithm that is invariant to changes in illumination and contrast and can accurately localize neurites. Our method was successfully applied to analyze a large HCS image set generated in a morphology screen for polyglutaminemediated neuronal toxicity using primary neuronal cell cultures derived from embryos of a Drosophila Huntington’s Disease (HD) model.National Institutes of Health (U.S.) (Grant

Image informatics strategies for deciphering neuronal network connectivity

Brain function relies on an intricate network of highly dynamic neuronal connections that rewires dramatically under the impulse of various external cues and pathological conditions. Among the neuronal structures that show morphologi- cal plasticity are neurites, synapses, dendritic spines and even nuclei. This structural remodelling is directly connected with functional changes such as intercellular com- munication and the associated calcium-bursting behaviour. In vitro cultured neu- ronal networks are valuable models for studying these morpho-functional changes. Owing to the automation and standardisation of both image acquisition and image analysis, it has become possible to extract statistically relevant readout from such networks. Here, we focus on the current state-of-the-art in image informatics that enables quantitative microscopic interrogation of neuronal networks. We describe the major correlates of neuronal connectivity and present workflows for analysing them. Finally, we provide an outlook on the challenges that remain to be addressed, and discuss how imaging algorithms can be extended beyond in vitro imaging studies

NeuriteQuant: An open source toolkit for high content screens of neuronal Morphogenesis

<p>Abstract</p> <p>Background</p> <p>To date, some of the most useful and physiologically relevant neuronal cell culture systems, such as high density co-cultures of astrocytes and primary hippocampal neurons, or differentiated stem cell-derived cultures, are characterized by high cell density and partially overlapping cellular structures. Efficient analytical strategies are required to enable rapid, reliable, quantitative analysis of neuronal morphology in these valuable model systems.</p> <p>Results</p> <p>Here we present the development and validation of a novel bioinformatics pipeline called NeuriteQuant. This tool enables fully automated morphological analysis of large-scale image data from neuronal cultures or brain sections that display a high degree of complexity and overlap of neuronal outgrowths. It also provides an efficient web-based tool to review and evaluate the analysis process. In addition to its built-in functionality, NeuriteQuant can be readily extended based on the rich toolset offered by ImageJ and its associated community of developers. As proof of concept we performed automated screens for modulators of neuronal development in cultures of primary neurons and neuronally differentiated P19 stem cells, which demonstrated specific dose-dependent effects on neuronal morphology.</p> <p>Conclusions</p> <p>NeuriteQuant is a freely available open-source tool for the automated analysis and effective review of large-scale high-content screens. It is especially well suited to quantify the effect of experimental manipulations on physiologically relevant neuronal cultures or brain sections that display a high degree of complexity and overlap among neurites or other cellular structures.</p

Sustained synchronized neuronal network activity in a human astrocyte co-culture system

Impaired neuronal network function is a hallmark of neurodevelopmental and neurodegenerative disorders such as autism, schizophrenia, and Alzheimer's disease and is typically studied using genetically modified cellular and animal models. Weak predictive capacity and poor translational value of these models urge for better human derived in vitro models. The implementation of human induced pluripotent stem cells (hiPSCs) allows studying pathologies in differentiated disease-relevant and patient-derived neuronal cells. However, the differentiation process and growth conditions of hiPSC-derived neurons are non-trivial. In order to study neuronal network formation and (mal) function in a fully humanized system, we have established an in vitro co-culture model of hiPSC-derived cortical neurons and human primary astrocytes that recapitulates neuronal network synchronization and connectivity within three to four weeks after final plating. Live cell calcium imaging, electrophysiology and high content image analyses revealed an increased maturation of network functionality and synchronicity over time for co-cultures compared to neuronal monocultures. The cells express GABAergic and glutamatergic markers and respond to inhibitors of both neurotransmitter pathways in a functional assay. The combination of this co-culture model with quantitative imaging of network morphofunction is amenable to high throughput screening for lead discovery and drug optimization for neurological diseases

High-Content Chemical and RNAi Screens for Suppressors of Neurotoxicity in a Huntington's Disease Model

To identify Huntington's Disease therapeutics, we conducted high-content small molecule and RNAi suppressor screens using a Drosophila primary neural culture Huntingtin model. Drosophila primary neurons offer a sensitive readout for neurotoxicty, as their neurites develop dysmorphic features in the presence of mutant polyglutamine-expanded Huntingtin compared to nonpathogenic Huntingtin. By tracking the subcellular distribution of mRFP-tagged pathogenic Huntingtin and assaying neurite branch morphology via live-imaging, we identified suppressors that could reduce Huntingtin aggregation and/or prevent the formation of dystrophic neurites. The custom algorithms we used to quantify neurite morphologies in complex cultures provide a useful tool for future high-content screening approaches focused on neurodegenerative disease models. Compounds previously found to be effective aggregation inhibitors in mammalian systems were also effective in Drosophila primary cultures, suggesting translational capacity between these models. However, we did not observe a direct correlation between the ability of a compound or gene knockdown to suppress aggregate formation and its ability to rescue dysmorphic neurites. Only a subset of aggregation inhibitors could revert dysmorphic cellular profiles. We identified lkb1, an upstream kinase in the mTOR/Insulin pathway, and four novel drugs, Camptothecin, OH-Camptothecin, 18β-Glycyrrhetinic acid, and Carbenoxolone, that were strong suppressors of mutant Huntingtin-induced neurotoxicity. Huntingtin neurotoxicity suppressors identified through our screen also restored viability in an in vivo Drosophila Huntington's Disease model, making them attractive candidates for further therapeutic evaluation.National Institutes of Health (U.S.) (grant R01 EB007042)National Institutes of Health (U.S.

Spiral Ganglion Neurite Outgrowth and Pathfinding on Electrospun Microfibrous Piezoelectric Nanocomposite Polymer Scaffolds

Sensorineural hearing loss (SNHL) can be caused by hair cell loss and spiral ganglion neurone (SGN) degeneration. Cochlear implants (CIs), the only means of restoring residual hearing to profoundly deaf people, stimulate possible preserved SGNs electrically. Thus, SGN degeneration dictates the efficacy of CIs. SGN degeneration reduces sensitivity and frequency selectivity. In addition, stimulation thresholds increase due to SGN degeneration consequently increasing power demands. The replacement of auditory neurones with proper functional spatial alignment is an important step in the attempt to restore auditory function. This study adopts a tissue-engineering approach. We examined the viability of polyvinylidene fluoride (PVDF) and polyvinylidene trifluoroethylene (P(VDF-TrFE)). P(VDF-TrFE) was chosen to add directional growth cues through electrospinning aligned microfibrous scaffolds. The effects of the scaffolds on the length and orientation of re-growing SGN neurites and glia were tested in vitro using primary murine cultures. Two methods of SGN preparation were compared; explants and dissociated cultures. Primary SGNs showed preferential affinity to P(VDF-TrFE) microfibres and the microfibrous scaffolds were found to promote aligned SGN neurite regrowth compared to glass coverslips. Subsequently, we doped the electrospun P(VDF-TrFE) microfibres with carbon nanotubes (CNT) to optimise the scaffold mechanically and electrically. The CNT addition was found to be biocompatible and promoted aligned SGN neurite regrowth. The CNT doping enhanced the mechanical properties of the microfibres and improved scaffold handling. Moreover, the scaffolds could be biofunctionalized with neurone modulating drugs. Preliminary testing of gamma-secretase inhibitor (LY411575) showed promising regenerative effects on SGNs in vitro. In conclusion, electrospun aligned microfibrous P(VDF-TrFE)-CNT nanocomposite scaffolds can modulate glial and SGN neurite and axon organization in vitro. Combined with a specific protocol of electrical induction in the first weeks of implantation, the piezoelectric fibrous scaffold could significantly improve cochlear implantation results, frequency selectivity and minimize power demands

A Rapid, Inexpensive High Throughput Screen Method for Neurite Outgrowth

Neurite outgrowth assays are the most common phenotypic screen to assess chemical effects on neuronal cells. Current automated assays involve expensive equipment, lengthy sample preparation and handling, costly reagents and slow rates of data acquisition and analysis. We have developed a high throughput screen (HTS) for neurite outgrowth using a robust neuronal cell model coupled to fast and inexpensive visualization methods, reduced data volume and rapid data analysis. Neuroscreen-1 (NS-1) cell, a subclone of PC12, possessing rapid growth and enhanced sensitivity to NGF was used as a model neuron. This method reduces preparation time by using cells expressing GFP or native cells stained with HCS CellMask™ Red in a multiplexed 30 min fixation and staining step. A 2x2 camera binning process reduced both image data files and analysis times by 75% and 60% respectively, compared to current protocols. In addition, eliminating autofocus steps during montage generation reduced data collection time. Pharmacological profiles for stimulation and inhibition of neurite outgrowth by NGF and SU6656 were comparable to current standard method utilizing immunofluorescence detection of tubulin. Potentiation of NGF-induced neurite outgrowth by members of a 1,120-member Prestwick compound library as assayed using this method identified six molecules, including etoposide, isoflupredone acetate, fludrocortisone acetate, thioguanosine, oxyphenbutazone and gibberellic acid, that more than doubled the neurite mass primed by 2 ng/ml NGF. This simple procedure represents an important routine approach in high throughput screening of large chemical libraries using the neurite outgrowth phenotype as a measure of the effects of chemical molecules on neuronal cells

Effect of space conditions on neuronal plasticity and connectivity

Looking for opportunities to explore new frontiers and developing new technologies have always been in the nature of mankind. In 1957, the first rocket in space opened a new era for space traveling towards other planets. Concomitantly, a wide range of concerns related to human health risks that could occur during spaceflight was raised. Up to now, a large number of experiments has been performed to determine the biological effects of space conditions on human health, in order to develop appropriate countermeasures. However, extensive investigations still need to be performed before considering long-term spaceflight towards other planets such as Mars. Since the first human space flight, it has been observed that in weightlessness conditions, equilibrium sense organs can send misleading inputs to the central nervous system which is forced to develop new strategies and adapt to adequately translate these messages. Furthermore, cosmic radiations are known to induce oxidative stress as well as genomic damages. In this thesis, we studied concomitant microgravity and radiation exposures as models for space conditions and developed various methods to analyse their specific and combined effects on in vitro neuronal network models. In vitro primary neuronal network cultures were established and exposed to simulated space conditions to investigate neuronal network remodelling (plasticity and connectivity) as well as genomic damage/repair dynamics. This work was performed to address questions on neuronal network disorders occurring during spaceflights and, in the future, to develop strategies against these effects