High-content neurite development study using optically patterned substrates.
Ontology highlight
ABSTRACT:
SUBMITTER: Belisle JM
PROVIDER: S-EPMC3338543 | biostudies-literature | 2012
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Neurons, with their long axons and elaborate dendritic arbour, establish the complex circuitry that is essential for the proper functioning of the nervous system. Whereas a catalogue of structural, molecular, and functional differences between axons and dendrites is accumulating, the mechanisms involved in early events of neuronal differentiation, such as neurite initiation and elongation, are less well understood, mainly because the key molecules involved remain elusive. Here we describe the establishment and application of a microscopy-based approach designed to identify novel proteins involved in neurite initiation and/or elongation. We identified 21 proteins that affected neurite outgrowth when ectopically expressed in cells. Complementary time-lapse microscopy allowed us to discriminate between early and late effector proteins. Localization experiments with GFP-tagged proteins in fixed and living cells revealed a further 14 proteins that associated with neurite tips either early or late during neurite outgrowth. Coexpression experiments of the new effector proteins provide a first glimpse on a possible functional relationship of these proteins during neurite outgrowth. Altogether, we demonstrate the potential of the systematic microscope-based screening approaches described here to tackle the complex biological process of neurite outgrowth regulation.
| S-EPMC1751310 | biostudies-literature
Project description:Multiple lines of evidence indicate a strong relationship between ?? peptide-induced neurite degeneration and the progressive loss of cognitive functions in Alzheimer disease (AD) patients and in AD animal models. This prompted us to develop a high content screening assay (HCS) and Neurite Image Quantitator (NeuriteIQ) software to quantify the loss of neuronal projections induced by A? peptide neurons and enable us to identify new classes of neurite-protective small molecules, which may represent new leads for AD drug discovery. We identified thirty-six inhibitors of A?-induced neurite loss in the 1,040-compound National Institute of Neurological Disorders and Stroke (NINDS) custom collection of known bioactives and FDA approved drugs. Activity clustering showed that non-steroidal anti-inflammatory drugs (NSAIDs) were significantly enriched among the hits. Notably, NSAIDs have previously attracted significant attention as potential drugs for AD; however their mechanism of action remains controversial. Our data revealed that cyclooxygenase-2 (COX-2) expression was increased following A? treatment. Furthermore, multiple distinct classes of COX inhibitors efficiently blocked neurite loss in primary neurons, suggesting that increased COX activity contributes to A? peptide-induced neurite loss. Finally, we discovered that the detrimental effect of COX activity on neurite integrity may be mediated through the inhibition of peroxisome proliferator-activated receptor ? (PPAR?) activity. Overall, our work establishes the feasibility of identifying small molecule inhibitors of A?-induced neurite loss using the NeuriteIQ pipeline and provides novel insights into the mechanisms of neuroprotection by NSAIDs.
| S-EPMC3308767 | biostudies-literature
Project description:Toward the goal of reproducible live neuronal networks, we investigated the influence of substrate patterns on neuron compliance and network activity. We optimized process parameters of micro-contact printing for reproducible geometric patterns of 10 ?m wide lines of polylysine with 4, 6, or 8 connections at a constant square array of nodes overlying the recording electrodes of a multielectrode array (MEA). We hypothesized that an increase in node connections would give the network more inputs resulting in higher neuronal outputs as network spike rates. We also chronically stimulated these networks during development and added astroglia to enhance network activity. Our results show that despite frequent localization of neuron somata over the electrodes, the number of spontaneously active electrodes was reduced 3-fold compared to random networks, independent of pattern complexity. Of the electrodes active, the overall spike rate was independent of pattern complexity, consistent with homeostasis of activity. Lower mean burst rates were seen with higher levels of pattern complexity; however, burst durations increased 1.6-fold with pattern complexity (n=6027 bursts, p<0.001). Inter-burst interval and percentage of active electrodes displaying bursts also increased with pattern complexity. The extra-burst (non-burst or isolated) spike rate increased 4-fold with pattern complexity, but this relationship was reversed with either chronic stimulation or astroglia addition. These studies suggest for the first time that patterns which limit the distribution of branches and inputs are deleterious to activity in a hippocampal network, but that higher levels of pattern complexity promote non-burst activity and favor longer lasting, but fewer bursts.
| S-EPMC3246106 | biostudies-literature
Project description:Block copolymer (BCP) self-assembly is a low-cost means to nanopattern surfaces. Here, we use these nanopatterns to directly print arrays of nanodots onto a conducting substrate (Indium Tin Oxide (ITO) coated glass) for application as an electrochemical sensor for ethanol (EtOH) and hydrogen peroxide (H2O2) detection. The work demonstrates that BCP systems can be used as a highly efficient, flexible methodology for creating functional surfaces of materials. Highly dense iron oxide nanodots arrays that mimicked the original BCP pattern were prepared by an 'insitu' BCP inclusion methodology using poly(styrene)-block-poly(ethylene oxide) (PS-b-PEO). The electrochemical behaviour of these densely packed arrays of iron oxide nanodots fabricated by two different molecular weight PS-b-PEO systems was studied. The dual detection of EtOH and H2O2 was clearly observed. The as-prepared nanodots have good long term thermal and chemical stability at the substrate and demonstrate promising electrocatalytic performance.
| S-EPMC4542519 | biostudies-other
Project description:Perhaps one of the most astounding characteristics of the gecko adhesive system is its versatility. Geckos can locomote across complex substrates in a variety of conditions with apparent ease. In contrast, many of our synthetic pressure sensitive adhesives fail on substrates that are dirty, wet or rough. Although many studies have investigated the effect of environmental challenges on performance, the interaction of multiple, potentially compromising variables is studied less often. Here we focus on substrate structure and surface water, both of which are highly relevant to the biological system and to synthetic design. To do this we utilized a highly controlled, patterned substrate (Sharklet®, by Sharklet® Technologies Inc.). This allowed us to test independently and jointly the effects of reduced surface area substrates, with a defined pattern, on adhesion in both air and water. Our results show that adhesion is not significantly impaired in air, whereas surface area and pattern significantly affect adhesion in water. These findings highlight the need to study multiple parameters that are relevant to the gecko adhesive system to further improve our understanding of the biological system and to design better, more versatile synthetics.
| S-EPMC4687937 | biostudies-literature
Project description:Matrix molecules convey biochemical and physical guiding signals to neurons in the central nervous system (CNS) and shape the trajectory of neuronal fibers that constitute neural networks. We have developed recombinant human IgMs that bind to epitopes on neural cells, with the aim of treating neurological diseases. Here we test the hypothesis that recombinant human IgMs (rHIgM) can guide neurite outgrowth of CNS neurons. Microcontact printing was employed to pattern rHIgM12 and rHIgM22, antibodies that were bioengineered to have variable regions capable of binding to neurons or oligodendrocytes, respectively. rHIgM12 promoted neuronal attachment and guided outgrowth of neurites from hippocampal neurons. Processes from spinal neurons followed grid patterns of rHIgM12 and formed a physical network. Comparison between rHIgM12 and rHIgM22 suggested the biochemistry that facilitates anchoring the neuronal surfaces is a prerequisite for the function of IgM, and spatial properties cooperate in guiding the assembly of neuronal networks.
| S-EPMC3721078 | biostudies-literature
Project description:Cardiac side effects are one of the major causes of drug candidate failures in preclinical drug development or in clinical trials and are responsible for the retraction of several already marketed therapeutics. Thus, the development of a relatively high-throughput, high information content tool to screen drugs and toxins would be important in the field of cardiac research and drug development. In this study, recordings from commercial multielectrode arrays were combined with surface patterning of cardiac myocyte monolayers to enhance the information content of the method; specifically, to enable the measurement of conduction velocity, refractory period after action potentials and to create a functional re-entry model. Two drugs, 1-Heptanol, a gap junction blocker, and Sparfloxacin, a fluoroquinone antibiotic, were tested in this system. 1-Heptanol administration resulted in a marked reduction in conduction velocity, whereas Sparfloxacin caused rapid, irregular and unsynchronized activity, indicating fibrillation. As shown in these experiments, patterning of cardiac myocyte monolayers solved several inherent problems of multielectrode recordings, increased the temporal resolution of conduction velocity measurements, and made the synchronization of external stimulation with action potential propagation possible for refractory period measurements. This method could be further developed as a cardiac side effect screening platform after combination with human cardiomyocytes.
| S-EPMC3102308 | biostudies-literature
Project description:Fabrication of ultrathin films of dielectric (with particular reference to materials with high dielectric constants) materials has significance in many advanced technological applications including hard protective coatings, sensors, and next-generation logic devices. Current state-of-the-art in microelectronics for fabricating these thin films is a combination of atomic layer deposition and photolithography. As feature size decreases and aspect ratios increase, conformality of the films becomes paramount. Here, we show a polymer brush template-assisted deposition of highly conformal, ultrathin (sub 5 nm) high-κ dielectric metal oxide films (hafnium oxide and zirconium oxide) on topographically patterned silicon nitride substrates. This technique, using hydroxyl terminated poly-4-vinyl pyridine (P4VP-OH) as the polymer brush, allows for conformal deposition with uniform thickness along the trenches and sidewalls of the substrate. Metal salts are infiltrated into the grafted monolayer polymer brush films via solution deposition. Tailoring specific polymer interfacial chemistries for ion infiltration combined with subsequent oxygen plasma treatment enabled the fabrication of high-quality sub 5 nm metal oxide films.
| S-EPMC9305981 | biostudies-literature
Project description:Multi-modal imaging, by light-microscopy (LM) and Magnetic Resonance Imaging (MRI), holds promise for examining the brain across various resolutions and scales. While MRI acquires images in three dimensions, acquisition of intact whole-brain by LM requires a process of tissue clearing that renders the brain transparent. Removal of lipids (delipidation) is a critical step in the tissue clearing process, and was previsouly suggested to be the cause for absence of MRI contrast in cleared brains. Yet, the association between MRI contrast, delipidation and the different clearing techniques is debatable. Here, we provide datasets concerning lipid-content in cleared brain tissues obtained by various approaches. Fixed mouse and rat brains were cleared by CLARITY, Scale, uDISCO and ECi clearing techniques. Lipid-content was assessed at various intermediate steps of the different clearing methods, as well as at the end of the processes. Methods employed included whole brain MRI acquisition, Oil Red O (ORO)- and carbocyanine DiI-staining of cryosections, and DiI-washout assay from brain slices. MRI contrast-to-noise ratio, staining intensities and integrity of tissue were systematically analyzed. We demonstrate that lipid electrophoresis, an essential step of the CLARITY approach, engenders progressive reduction in MRI contrast in non-cleared (PFA-fixed) control brains, as well as strongly reduces contrast from uDISCO and ECi-cleared brains. ORO minimally stained CLARITY-cleared brains, however efficiently labelled uDISCO and ECi-cleared brains. Conversely, and in contrast to ORO-staining, DiI equally stained control, CLARITY, ECi and uDISCO-cleared brains. Both ORO- and DiI-staining demonstrated impairment in brain tissue integrity following CLARITY, but less so in uDISCO and ECi brains. DiI-washout assay demonstrated that each of the solvents employed along the process of uDISCO and ECi are highly delipidating, as well as the SDS-electrophoresis employed during CLARITY clearing. However, Scale treatment preserved most of the DiI dye. These data emphasize the variability in lipid assessment of cleared tissues by common techniques, and may help to resolve the contribution of lipids in brain MRI contrast.
| S-EPMC10749242 | biostudies-literature
Project description:Natural tissues can have complex architectures, which arise in part from spatial patterns in gene expression. Regenerative strategies for damaged tissue must recreate these architectures to restore function. In this article, we demonstrate spatially controlled gene delivery from a substrate for directing cellular processes. Non-viral vectors were immobilized to substrates in linear patterns using microfluidic techniques, and cells cultured on the surface had localized gene expression within the cell population. Transfection was achieved in pattern widths as low as 100 mum, with efficiencies dependent on the microchannel treatment and vector concentration. The ability of patterned expression to localize cellular processes was investigated using a neuronal co-culture model. Patterned expression of the diffusible neurotrophic factor nerve growth factor initiated neuron survival and neurite out-growth primarily within the pattern, which decreased significantly in regions directly adjacent to the pattern. Primary neurite density was significantly greater on patterned substrates than on surfaces without patterns. This approach demonstrates the basic technology to create patterns of gene expression that can direct tissue formation and could be employed in regenerative strategies to recreate the complex cellular architectures observed in tissues.
| S-EPMC2648834 | biostudies-other