scholarly journals A calibrated optogenetic toolbox of stable zebrafish opsin lines

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Paride Antinucci ◽  
Adna Dumitrescu ◽  
Charlotte Deleuze ◽  
Holly J Morley ◽  
Kristie Leung ◽  
...  
Keyword(s):  
High Throughput ◽  
Neural Activity ◽  
Ion Selectivity ◽  
Transgenic Zebrafish ◽  
Substantial Variation ◽  
Opsin Expression ◽  
Electrophysiological Recordings ◽  
Subcellular Resolution ◽  
Kinetics Of

Optogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.

scholarly journals A calibrated optogenetic toolbox of stable zebrafish opsin lines

2020 ◽  
Author(s):  
P Antinucci ◽  
AS Dumitrescu ◽  
C Deleuze ◽  
HJ Morley ◽  
K Leung ◽  
...  
Keyword(s):  
High Throughput ◽  
Neural Activity ◽  
Ion Selectivity ◽  
Transgenic Zebrafish ◽  
Substantial Variation ◽  
Opsin Expression ◽  
Electrophysiological Recordings ◽  
Subcellular Resolution ◽  
Kinetics Of

AbstractOptogenetic actuators with diverse spectral tuning, ion selectivity and kinetics are constantly being engineered providing powerful tools for controlling neural activity with subcellular resolution and millisecond precision. Achieving reliable and interpretable in vivo optogenetic manipulations requires reproducible actuator expression and calibration of photocurrents in target neurons. Here, we developed nine transgenic zebrafish lines for stable opsin expression and calibrated their efficacy in vivo. We first used high-throughput behavioural assays to compare opsin ability to elicit or silence neural activity. Next, we performed in vivo whole-cell electrophysiological recordings to quantify the amplitude and kinetics of photocurrents and test opsin ability to precisely control spiking. We observed substantial variation in efficacy, associated with differences in both opsin expression level and photocurrent characteristics, and identified conditions for optimal use of the most efficient opsins. Overall, our calibrated optogenetic toolkit will facilitate the design of controlled optogenetic circuit manipulations.

Ex Vivo Brain Preparation to Analyze Vocal Pathways of Xenopus Frogs

2021 ◽  
Vol 2021 (9) ◽  
pp. pdb.prot106872
Author(s):  
Ayako Yamaguchi
Keyword(s):  
Neural Activity ◽  
Ex Vivo ◽  
Neural Circuitry ◽  
Neural Basis ◽  
Vocal Production ◽  
Basic Principles ◽  
Electrophysiological Recordings ◽  
The Brain

Understanding the neural basis of behavior is a challenging task for technical reasons. Most methods of recording neural activity require animals to be immobilized, but neural activity associated with most behavior cannot be recorded from an anesthetized, immobilized animal. Using amphibians, however, there has been some success in developing in vitro brain preparations that can be used for electrophysiological and anatomical studies. Here, we describe an ex vivo frog brain preparation from which fictive vocalizations (the neural activity that would have produced vocalizations had the brain been attached to the muscle) can be elicited repeatedly. When serotonin is applied to the isolated brains of male and female African clawed frogs, Xenopus laevis, laryngeal nerve activity that is a facsimile of those that underlie sex-specific vocalizations in vivo can be readily recorded. Recently, this preparation was successfully used in other species within the genus including Xenopus tropicalis and Xenopus victorianus. This preparation allows a variety of techniques to be applied including extracellular and intracellular electrophysiological recordings and calcium imaging during vocal production, surgical and pharmacological manipulation of neurons to evaluate their impact on motor output, and tract tracing of the neural circuitry. Thus, the preparation is a powerful tool with which to understand the basic principles that govern the production of coherent and robust motor programs in vertebrates.

scholarly journals The BioLuminescent-OptoGenetic in vivo Response to Coelenterazine is Proportional, Sensitive and Specific in Neocortex

2019 ◽  
Author(s):  
Manuel Gomez-Ramirez ◽  
Alexander I. More ◽  
Nina G. Friedman ◽  
Ute Hochgeschwender ◽  
Christopher I. Moore
Keyword(s):  
Neural Activity ◽  
Bioluminescence Imaging ◽  
Photon Emission ◽  
Gaussia Luciferase ◽  
Light Production ◽  
Electrophysiological Recordings ◽  
Dynamic Concentration ◽  
And Behavior ◽  
The Relationship

ABSTRACTBioLuminescent (BL) light production can modulate neural activity and behavior through coexpressed OptoGenetic (OG) elements, an approach termed ‘BL-OG’. Yet, the relationship between BL-OG effects and bioluminescent photon emission has not been characterized in vivo. Further, the degree to which BL-OG effects strictly depend on optogenetic mechanisms driven by bioluminescent photons is unknown. Crucial to every neuromodulation method is whether the activator shows a dynamic concentration range driving robust, selective, and non-toxic effects. We systematically tested the effects of four key components of the BL-OG mechanism (luciferin, oxidized luciferin, luciferin vehicle, and bioluminescence), and compared these against effects induced by the Luminopsin-3 (LMO3) BL-OG molecule, a fusion of slow burn Gaussia luciferase (sbGLuc) and Volvox ChannelRhodopsin-1 (VChR1). We performed combined bioluminescence imaging and electrophysiological recordings while injecting specific doses of Coelenterazine (substrate for sbGluc), Coelenteramide (CTM, the oxidized product of CTZ), or CTZ vehicle. CTZ robustly drove activity in mice expressing LMO3, with photon production proportional to firing rate. In contrast, low and moderate doses of CTZ, CTM, or vehicle did not modulate activity in mice that did not express LMO3. We also failed to find bioluminescence effects on neural activity in mice expressing an optogenetically non-sensitive LMO3 variant. We observed weak responses to the highest dose of CTZ in control mice, but these effects were significantly smaller than those observed in the LMO3 group. These results show that in neocortex in vivo, there is a large CTZ range wherein BL-OG effects are specific to its active chemogenetic mechanism.

scholarly journals Zebrafish Models of Cancer Therapy-Induced Cardiovascular Toxicity

2021 ◽  
Vol 8 (2) ◽  
pp. 8
Author(s):  
Sarah Lane ◽  
Luis Alberto More ◽  
Aarti Asnani
Keyword(s):  
Cancer Therapy ◽  
High Throughput ◽  
Early Developmental Stage ◽  
Transgenic Zebrafish ◽  
Cardiovascular Toxicity ◽  
Zebrafish Model ◽  
In Vivo Models ◽  
Mechanisms Of Toxicity

Purpose of review: Both traditional and novel cancer therapies can cause cardiovascular toxicity in patients. In vivo models integrating both cardiovascular and cancer phenotypes allow for the study of on- and off-target mechanisms of toxicity arising from these agents. The zebrafish is the optimal whole organism model to screen for cardiotoxicity in a high throughput manner, while simultaneously assessing the role of cardiotoxicity pathways on the cancer therapy’s antitumor effect. Here we highlight established zebrafish models of human cardiovascular disease and cancer, the unique advantages of zebrafish to study mechanisms of cancer therapy-associated cardiovascular toxicity, and finally, important limitations to consider when using the zebrafish to study toxicity. Recent findings: Cancer therapy-associated cardiovascular toxicities range from cardiomyopathy with traditional agents to arrhythmias and thrombotic complications associated with newer targeted therapies. The zebrafish can be used to identify novel therapeutic strategies that selectively protect the heart from cancer therapy without affecting antitumor activity. Advances in genome editing technology have enabled the creation of several transgenic zebrafish lines valuable to the study of cardiovascular and cancer pathophysiology. Summary: The high degree of genetic conservation between zebrafish and humans, as well as the ability to recapitulate cardiotoxic phenotypes observed in patients with cancer, make the zebrafish an effective model to study cancer therapy-associated cardiovascular toxicity. Though this model provides several key benefits over existing in vitro and in vivo models, limitations of the zebrafish model include the early developmental stage required for most high-throughput applications.

scholarly journals Proximal and distal modulation of neural activity by spatially confined optogenetic activation with an integrated high-density optoelectrode

2018 ◽  
Vol 120 (1) ◽  
pp. 149-161 ◽  
Author(s):  
Sarah Libbrecht ◽  
Luis Hoffman ◽  
Marleen Welkenhuysen ◽  
Chris Van den Haute ◽  
Veerle Baekelandt ◽  
...  
Keyword(s):  
Neural Activity ◽  
Large Scale ◽  
Heat Dissipation ◽  
Extracellular Recording ◽  
Cell Types ◽  
Cortical Layer ◽  
Neural Firing ◽  
Optical Stimulation ◽  
Electrophysiological Recordings

Optogenetic manipulations are widely used for investigating the contribution of genetically identified cell types to behavior. Simultaneous electrophysiological recordings are less common, although they are critical for characterizing the specific impact of optogenetic manipulations on neural circuits in vivo. This is at least in part because combining photostimulation with large-scale electrophysiological recordings remains technically challenging, which also poses a limitation for performing extracellular identification experiments. Currently available interfaces that guide light of the appropriate wavelength into the brain combined with an electrophysiological modality suffer from various drawbacks such as a bulky size, low spatial resolution, heat dissipation, or photovoltaic artifacts. To address these challenges, we have designed and fabricated an integrated ultrathin neural interface with 12 optical outputs and 24 electrodes. We used the device to measure the effect of localized stimulation in the anterior olfactory cortex, a paleocortical structure involved in olfactory processing. Our experiments in adult mice demonstrate that because of its small dimensions, our novel tool causes far less tissue damage than commercially available devices. Moreover, optical stimulation and recording can be performed simultaneously, with no measurable electrical artifact during optical stimulation. Importantly, optical stimulation can be confined to small volumes with approximately single-cortical layer thickness. Finally, we find that even highly localized optical stimulation causes inhibition at more distant sites. NEW & NOTEWORTHY In this study, we establish a novel tool for simultaneous extracellular recording and optogenetic photostimulation. Because the device is built using established microchip technology, it can be fabricated with high reproducibility and reliability. We further show that even very localized stimulation affects neural firing far beyond the stimulation site. This demonstrates the difficulty in predicting circuit-level effects of optogenetic manipulations and highlights the importance of closely monitoring neural activity in optogenetic experiments.

scholarly journals Evaluation of a high-throughput deorphanization strategy to identify cytochrome p450s important for odor degradation in Drosophila

2021 ◽  
Author(s):  
Shane R Baldwin ◽  
Pratyajit Mohapatra ◽  
Monica Nagalla ◽  
Rhea Sindvani ◽  
Desiree Amaya ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
High Throughput ◽  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Neuronal Cells ◽  
Cytochrome P450s ◽  
Olfactory Neuron ◽  
Plant Volatile ◽  
Electrophysiological Recordings

Members of the cytochrome p450 (CYP) enzyme family are abundantly expressed in insect olfactory tissues, where they are thought to act as Odorant Degrading Enzymes (ODEs). However, their contribution to olfactory signaling in vivo is poorly understood. This is due in part to the challenge of identifying which of the dozens of antennal-expressed CYPs might inactivate a given odorant. Here, we tested a high-throughput deorphanization strategy in Drosophila to identify CYPs that are transcriptionally induced by exposure to a plant volatile. We discovered three CYPs selectively upregulated by the odorant using transcriptional profiling. Although these CYPs are shown to be broadly expressed in the antenna in non-neuronal cells, electrophysiological recordings from CYP mutants did not reveal any changes in olfactory neuron responses to the odorant. Neurons were desensitized by pre-exposing flies to the odorant, but this effect was similar in CYP mutants. Together, our data suggest that this transcriptomic approach may not be useful for identifying CYPs that contribute to olfactory signaling. We go on to show that some CYPs have highly restricted expression patterns in the antenna, and suggest that such CYPs may be useful candidates for further studies on olfactory CYP function.

scholarly journals Immobilization of C. elegans on cultivation plates by thermoelectric cooling for high-throughput subcellular-resolution microscopy

2021 ◽  
Author(s):  
Yao L. Wang ◽  
Erik L. Jaklitsch ◽  
Noa W. F. Grooms ◽  
Leilani G. Schulting ◽  
Samuel H. Chung
Keyword(s):  
High Throughput ◽  
Animal Movement ◽  
Biological Research ◽  
Nematode Caenorhabditis Elegans ◽  
C Elegans ◽  
Cooling Method ◽  
Subcellular Resolution ◽  
High Resolution Microscopy

Imaging, visual screens, and optical surgery are frequently applied to the nematode Caenorhabditis elegans at subcellular resolution for in vivo biological research. However, these approaches remain low-throughput and require significant manual effort. To improve throughput and enable automation in these techniques, we implement a novel cooling method to immobilize C. elegans directly on their cultivation plate. Previous studies cooled animals in microfluidics or flooded wells to 1-4 C. Counterintuitively, we find that cooling to 5-7 C immobilizes animals more effectively than lower temperatures. At 6 C, animal movement consists of bouts of submicron nose tip movement occurring at a sufficiently low magnitude and frequency to permit clear imaging. We demonstrate the ability to perform subcellular-resolution fluorescence imaging, including 64x magnification 3D image stacks and 2-min long timelapse recordings of the ASJ neuron without blurring from animal motion. We also observe no long-term side effects from cooling immobilization on animal lifespan or fecundity. We believe our cooling method enables high-throughput and high-resolution microscopy with no chemical or mechanical interventions.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

Sign in / Sign up

Export Citation Format

Share Document