scholarly journals Creating Custom Neural Circuits on Multiple Electrode Arrays Utilizing Optical Tweezers for Precise Nerve Cell Placement

2020 ◽  
Vol 3 (2) ◽  
pp. 44
Author(s):  
Frank H. Kung ◽  
Ellen Townes-Anderson
Keyword(s):  
Optical Tweezers ◽  
Neuronal Development ◽  
Neural Circuits ◽  
Electrode Array ◽  
Bipolar Cells ◽  
Electrode Arrays ◽  
Cell Placement ◽  
Multiple Electrode

Precise creation, maintenance, and monitoring of neuronal circuits would facilitate the investigation of subjects such as neuronal development or synaptic plasticity, or assist in the development of neuronal prosthetics. Here we present a method to precisely control the placement of multiple types of neuronal retinal cells onto a commercially available multiple electrode array (MEA), using custom-built optical tweezers. We prepared the MEAs by coating a portion of the MEA with a non-adhesive substrate (Poly (2-hydroxyethyl methacrylate)), and the electrodes with an adhesive cell growth substrate. We then dissociated the retina of adult tiger salamanders, plated them onto prepared MEAs, and utilized the optical tweezers to create retinal circuitry mimicking in vivo connections. In our hands, the optical tweezers moved ~75% of photoreceptors, bipolar cells, and multipolar cells, an average of ~2000 micrometers, at a speed of ~16 micrometers/second. These retinal circuits were maintained in vitro for seven days. We confirmed electrophysiological activity by stimulating the photoreceptors with the MEA and measuring their response with calcium imaging. In conclusion, we have developed a method of utilizing optical tweezers in conjunction with MEAs that allows for the design and maintenance of custom neural circuits for functional analysis.

scholarly journals Force generation by protein–DNA co-condensation

2021 ◽  
Author(s):  
Thomas Quail ◽  
Stefan Golfier ◽  
Maria Elsner ◽  
Keisuke Ishihara ◽  
Vasanthanarayan Murugesan ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Self Assembly ◽  
Spider Silk ◽  
Regulatory Elements ◽  
Heterochromatin Formation ◽  
First Order Phase Transition ◽  
Dna Strand ◽  
First Order Phase

AbstractInteractions between liquids and surfaces generate forces1,2 that are crucial for many processes in biology, physics and engineering, including the motion of insects on the surface of water3, modulation of the material properties of spider silk4 and self-assembly of microstructures5. Recent studies have shown that cells assemble biomolecular condensates via phase separation6. In the nucleus, these condensates are thought to drive transcription7, heterochromatin formation8, nucleolus assembly9 and DNA repair10. Here we show that the interaction between liquid-like condensates and DNA generates forces that might play a role in bringing distant regulatory elements of DNA together, a key step in transcriptional regulation. We combine quantitative microscopy, in vitro reconstitution, optical tweezers and theory to show that the transcription factor FoxA1 mediates the condensation of a protein–DNA phase via a mesoscopic first-order phase transition. After nucleation, co-condensation forces drive growth of this phase by pulling non-condensed DNA. Altering the tension on the DNA strand enlarges or dissolves the condensates, revealing their mechanosensitive nature. These findings show that DNA condensation mediated by transcription factors could bring distant regions of DNA into close proximity, suggesting that this physical mechanism is a possible general regulatory principle for chromatin organization that may be relevant in vivo.

Regulation of expression of mRNAs encoding the nerve growth factor receptors p75 and trkA in developing sensory neurons

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 635-648 ◽  
Author(s):  
S. Wyatt ◽  
A.M. Davies
Keyword(s):  
Neuronal Development ◽  
Polymerase Chain Reaction Amplification ◽  
Mrna Levels ◽  
Regulation Of Expression ◽  
Trigeminal Neurons ◽  
Nerve Growth Factor Receptors ◽  
Reaction Amplification ◽  
Amplification Technique

We have used a quantitative reverse transcription/polymerase chain reaction amplification technique to study the regulation of p75 mRNA and trkA mRNA expression in developing NGF-dependent trigeminal neurons. Before becoming NGF dependent, these neurons express low levels of p75 and trkA mRNAs in vivo. At this stage in vitro, the level of p75 mRNA is maintained and up-regulated by BDNF, whereas the level of trkA mRNA is sustained independently of neurotrophins and is down-regulated by BDNF. With the acquisition of NGF dependence, p75 and trkA mRNA levels increase markedly in vivo. At this stage in vitro, the level of p75 mRNA is up-regulated by NGF, but this response is lost at later stages. The level of trkA mRNA is sustained in neurons grown with NGF but is not up-regulated by concentrations of NGF above those required to support survival. At no stage during the early development of trigeminal neurons do depolarising levels of potassium ions affect the expression of either p75 mRNA or trkA mRNA. These findings suggest that the expression of p75 and trkA mRNAs are differentially regulated by BDNF and NGF at successive early stages of neuronal development.

scholarly journals KBP interacts with SCG10, linking Goldberg–Shprintzen syndrome to microtubule dynamics and neuronal differentiation

2010 ◽  
Vol 19 (18) ◽  
pp. 3642-3651 ◽  
Author(s):  
Maria M. Alves ◽  
Grzegorz Burzynski ◽  
Jean-Marie Delalande ◽  
Jan Osinga ◽  
Annemieke van der Goot ◽  
...  
Keyword(s):  
Nervous System ◽  
Enteric Nervous System ◽  
Neuronal Differentiation ◽  
Cortical Neurons ◽  
Neuronal Development ◽  
Direct Interaction ◽  
Clinical Disorder ◽  
Neurite Development

Abstract Goldberg–Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and enteric nervous system defects. This syndrome is caused by inactivating mutations in the Kinesin Binding Protein (KBP) gene, which encodes a protein of which the precise function is largely unclear. We show that KBP expression is up-regulated during neuronal development in mouse cortical neurons. Moreover, KBP-depleted PC12 cells were defective in nerve growth factor-induced differentiation and neurite outgrowth, suggesting that KBP is required for cell differentiation and neurite development. To identify KBP interacting proteins, we performed a yeast two-hybrid screen and found that KBP binds almost exclusively to microtubule associated or related proteins, specifically SCG10 and several kinesins. We confirmed these results by validating KBP interaction with one of these proteins: SCG10, a microtubule destabilizing protein. Zebrafish studies further demonstrated an epistatic interaction between KBP and SCG10 in vivo . To investigate the possibility of direct interaction between KBP and microtubules, we undertook co-localization and in vitro binding assays, but found no evidence of direct binding. Thus, our data indicate that KBP is involved in neuronal differentiation and that the central and enteric nervous system defects seen in GOSHS are likely caused by microtubule-related defects.

The zebrafish brain in research and teaching: a simple in vivo and in vitro model for the study of spontaneous neural activity

2011 ◽  
Vol 35 (2) ◽  
pp. 188-196 ◽  
Author(s):  
R. Vargas ◽  
I. þ. Jóhannesdóttir ◽  
B. Sigurgeirsson ◽  
H. þorsteinsson ◽  
K. Æ. Karlsson
Keyword(s):  
Neural Development ◽  
Neural Circuits ◽  
Brain Slices ◽  
Functional Roles ◽  
Zebrafish Brain ◽  
Vitro Model ◽  
Immunohistochemical Techniques ◽  
Neuronal Groups

Recently, the zebrafish ( Danio rerio ) has been established as a key animal model in neuroscience. Behavioral, genetic, and immunohistochemical techniques have been used to describe the connectivity of diverse neural circuits. However, few studies have used zebrafish to understand the function of cerebral structures or to study neural circuits. Information about the techniques used to obtain a workable preparation is not readily available. Here, we describe a complete protocol for obtaining in vitro and in vivo zebrafish brain preparations. In addition, we performed extracellular recordings in the whole brain, brain slices, and immobilized nonanesthetized larval zebrafish to evaluate the viability of the tissue. Each type of preparation can be used to detect spontaneous activity, to determine patterns of activity in specific brain areas with unknown functions, or to assess the functional roles of different neuronal groups during brain development in zebrafish. The technique described offers a guide that will provide innovative and broad opportunities to beginner students and researchers who are interested in the functional analysis of neuronal activity, plasticity, and neural development in the zebrafish brain.

scholarly journals Graphene-based carbon-layered electrode array technology for neural imaging and optogenetic applications

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Dong-Wook Park ◽  
Amelia A. Schendel ◽  
Solomon Mikael ◽  
Sarah K. Brodnick ◽  
Thomas J. Richner ◽  
...  
Keyword(s):  
Soft Tissues ◽  
Electrode Array ◽  
Electrode Arrays ◽  
Neural Imaging ◽  
Brain Surface ◽  
Array Technology ◽  
Micro Electrode Arrays ◽  
The Brain

Abstract Neural micro-electrode arrays that are transparent over a broad wavelength spectrum from ultraviolet to infrared could allow for simultaneous electrophysiology and optical imaging, as well as optogenetic modulation of the underlying brain tissue. The long-term biocompatibility and reliability of neural micro-electrodes also require their mechanical flexibility and compliance with soft tissues. Here we present a graphene-based, carbon-layered electrode array (CLEAR) device, which can be implanted on the brain surface in rodents for high-resolution neurophysiological recording. We characterize optical transparency of the device at >90% transmission over the ultraviolet to infrared spectrum and demonstrate its utility through optical interface experiments that use this broad spectrum transparency. These include optogenetic activation of focal cortical areas directly beneath electrodes, in vivo imaging of the cortical vasculature via fluorescence microscopy and 3D optical coherence tomography. This study demonstrates an array of interfacing abilities of the CLEAR device and its utility for neural applications.

Design of an Experimental Test System to Investigate Parameters Affecting Distal Tip Loads of Pacemaker and Defibrillator Leads

2015 ◽  
Vol 9 (1) ◽  
Author(s):  
Elizabeth A. Stephen ◽  
Donna L. Walsh ◽  
Nandini Duraiswamy ◽  
Oleg Vesnovsky ◽  
L. D. Timmie Topoleski
Keyword(s):  
Cardiac Tissue ◽  
Test System ◽  
Load Cell ◽  
Test Protocol ◽  
Minimal Impact ◽  
Test Configuration ◽  
Cell Placement ◽  
Ventricular Dysrhythmias

The purpose of this study was to design and evaluate a system to test the mechanical behavior of pacemaker and defibrillator leads. Over 300,000 pacemaker and implantable cardioverter defibrillator (ICD) procedures are performed every year in the U.S. for the treatment of cardiac arrhythmias, ventricular dysrhythmias, and congestive heart failure. These procedures require implanting transvenous leads into the interior wall of the heart. A serious and sometimes fatal complication that may occur during or after lead implantation is perforation of the lead tip through the heart wall. The factors that lead to perforation are not fully understood. This illustrates that the mechanical interactions between the lead tip and the cardiac tissue need to be further investigated to improve the outcome for pacemaker and ICD patients. To improve the performance of lead tips, the testing protocols must reproduce physiological and clinically relevant tip-tissue interactions. As a first step toward this goal, testing parameters that influence those interactions must be identified. We investigated the effect of test system parameters, which reproduce potentially critical physiological constraints, on the load experienced at the distal tip of thirteen pacemaker and defibrillator active-fixation leads. We evaluated the use of a constraint to simulate the effect of the right ventricle (RV constraint) in vivo, how and where the lead was fixed in the test configuration, location of the load cell in the test system, rotation and frequency of the test protocol, and the effect of stylets. Results showed the RV constraint and load cell placement had the largest impact on lead tip load, while rotation of the test setup and test frequency had a minimal impact. Recommendations are made for a test system and protocol for in vitro testing of leads that take into consideration in vivo conditions. Better approximations of the in vivo environment may lead to improved product development. The potential of this system to more effectively evaluate new pacemaker and defibrillator lead designs will require further study.

Modulation of voltage-dependent K+ currents (IK(V)) in retinal bipolar cells by ascorbate is mediated by dopamine D1 receptors

1999 ◽  
Vol 16 (5) ◽  
pp. 923-931 ◽  
Author(s):  
SHIH-FANG FAN ◽  
STEPHEN YAZULLA
Keyword(s):  
Receptor Antagonist ◽  
Glutamate Uptake ◽  
Sch 23390 ◽  
D2 Receptor ◽  
Bipolar Cells ◽  
D1 Receptors ◽  
Voltage Dependent ◽  
Average Latency

Ascorbic acid (AA), a neuromodulator in the vertebrate CNS, is released from glutamatergic neurons in exchange with glutamate uptake and, in turn, modulates the release of both glutamate and dopamine. We have reported that voltage-gated K+ currents (IK(V)) in ON-mixed rod/cone bipolar cells (Mb) were suppressed 60% by 100–200 μM AA when added to an ascorbate-free solution. However, as the in vivo [AA]o in retina is about 200 μM, we studied the effects of changes in [AA]o on IK(V) when [AA]o was varied around a baseline concentration of 200 μM. Whole-cell currents were recorded with patch-clamp methods from goldfish Mb cells in retinal slices, bathed in a solution containing 200 μM AA. We found that (1) IK(V) was enhanced (180 ± 36%, n = 9) by increases of [AA]o less than 40 μM with an average latency of 8 min. (2) However, IK(V) was suppressed without an appreciable latent period by two conditions: increases more than 40 μM [AA]o and decreases by any amount greater than 10 μM. (3) Effects of Δ[AA]o on IK(V) were blocked by a D1 dopamine receptor antagonist, SCH 23390, but not by a D2 receptor antagonist, spiperone. Increased concentrations of a D1 agonist (SKF 38390) and dopamine had similar concentration-dependent effects on IK(V) as did AA, even in the presence of 200 μM ascorbate. Ascorbate has complicated concentration-dependent effects on IK(V) of Mb cells in vitro that were mediated by D1 dopamine receptors, suggesting that dopamine and ascorbate may be involved reciprocally in modulating IK(V), with consequences on the transmission of rod signals to the inner retina.

scholarly journals Spontaneous and Perturbational Complexity in Cortical Cultures

2021 ◽  
Vol 11 (11) ◽  
pp. 1453
Author(s):  
Ilaria Colombi ◽  
Thierry Nieus ◽  
Marcello Massimini ◽  
Michela Chiappalone
Keyword(s):  
Cortical Neurons ◽  
Low Frequency ◽  
Initial Response ◽  
Moderate Increase ◽  
Electrode Arrays ◽  
In Vivo Experiments ◽  
Cortical Cultures ◽  
Cortical Slices

Dissociated cortical neurons in vitro display spontaneously synchronized, low-frequency firing patterns, which can resemble the slow wave oscillations characterizing sleep in vivo. Experiments in humans, rodents, and cortical slices have shown that awakening or the administration of activating neuromodulators decrease slow waves, while increasing the spatio-temporal complexity of responses to perturbations. In this study, we attempted to replicate those findings using in vitro cortical cultures coupled with micro-electrode arrays and chemically treated with carbachol (CCh), to modulate sleep-like activity and suppress slow oscillations. We adapted metrics such as neural complexity (NC) and the perturbational complexity index (PCI), typically employed in animal and human brain studies, to quantify complexity in simplified, unstructured networks, both during resting state and in response to electrical stimulation. After CCh administration, we found a decrease in the amplitude of the initial response and a marked enhancement of the complexity during spontaneous activity. Crucially, unlike in cortical slices and intact brains, PCI in cortical cultures displayed only a moderate increase. This dissociation suggests that PCI, a measure of the complexity of causal interactions, requires more than activating neuromodulation and that additional factors, such as an appropriate circuit architecture, may be necessary. Exploring more structured in vitro networks, characterized by the presence of strong lateral connections, recurrent excitation, and feedback loops, may thus help to identify the features that are more relevant to support causal complexity.

scholarly journals Vaping Perpetuates Cardiac Electrical Instability

2019 ◽  
Author(s):  
Obada Abouassali ◽  
Mengmeng Chang ◽  
Michelle Reiser ◽  
Manasa Kanithi ◽  
Ravi Soni ◽  
...  
Keyword(s):  
Cigarette Smoking ◽  
Cardiac Toxicity ◽  
Optical Mapping ◽  
Cellular Respiration ◽  
Electrode Arrays ◽  
Tobacco Cigarette ◽  
Nicotine Delivery ◽  
Beating Frequency

ABSTRACTBackgroundTobacco cigarette smoking is on the decline, but the usage of electronic nicotine delivery systems (ENDS) is gaining popularity, specifically in the teen and young adult age groups. While the cardiac toxicity of tobacco cigarette smoking has been widely studied and is well established, the possible cardiac toxicity of ENDS products and their design characteristics, such as added flavorings, are largely underexplored. Vaping, a form of electronic nicotine delivery, uses “e-liquid” to generate “e-vapor”, a smoke-like aerosolized mixture of nicotine and flavors. Here, we tested the hypothesis that vaping results in cardiac electrophysiological instability and arrhythmogenesis. We thus investigated how e-liquids with different flavors affect cardiac in-vitro and in-vivo toxicity, in cell culture and in animals.MethodsThree e-liquids with vanilla, cinnamon or fruit flavors were studied. We quantified apoptosis and oxygen consumption rate in HL-1 cells cultured with e-vapors extracts. In human iPSC derived ventricular cardiomyocytes (hiPSC-CM) cultured with e-vapor extract, beating frequency and repolarization duration were measured using multiple electrode arrays (MEA). Mass spectrometry (GC-MS) was used to analyze the composition of the e-vapors. Telemetric ECGs were obtained in freely moving C57BL/6J mice exposed to vanilla flavored e-vapor for 10 weeks and heart rate variability was analyzed (HRV). In-vivo inducibility of ventricular tachycardia as well as optical mapping of voltage in isolated Langendorff-perfused hearts were also carried out.ResultsE-vapor caused a dose dependent increase in toxicity in Hl-1 myocytes and e-vapors containing vanilla and cinnamon flavorings, as indicated by GC-MS, were more toxic, and inhibited cellular respiration more than the fruit flavored e-vapor. In hiPSC-CM cultured with 25% cinnamon flavored e-vapor for 24 hours, beating frequency increased, and the field potential duration significantly increased compared to air control. Inhalation exposure to vanilla flavored e-vapor for 10 weeks caused significant effects on HRV. Additionally, inducible VT was significantly longer, and in optical mapping, the magnitude of ventricular action potential duration alternans was significantly larger in the exposed mice compared to controlConclusionThe widely popular flavored ENDS are not harm free, and they show potential toxicity to the heart, in-vitro, and in vivo. Further studies are needed to further assess their cardiac safety profile, and long-term health effects.

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