scholarly journals Optogenetic feedback control of neural activity

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Jonathan P Newman ◽  
Ming-fai Fong ◽  
Daniel C Millard ◽  
Clarissa J Whitmire ◽  
Garrett B Stanley ◽  
...  
Keyword(s):  
Feedback Control ◽  
Dynamic Range ◽  
Neuronal Firing ◽  
Optical Control ◽  
Optical Stimulation ◽  
Gabaergic Neurotransmission ◽  
Neuronal Populations ◽  
Neuronal Control

Optogenetic techniques enable precise excitation and inhibition of firing in specified neuronal populations and artifact-free recording of firing activity. Several studies have suggested that optical stimulation provides the precision and dynamic range requisite for closed-loop neuronal control, but no approach yet permits feedback control of neuronal firing. Here we present the ‘optoclamp’, a feedback control technology that provides continuous, real-time adjustments of bidirectional optical stimulation in order to lock spiking activity at specified targets over timescales ranging from seconds to days. We demonstrate how this system can be used to decouple neuronal firing levels from ongoing changes in network excitability due to multi-hour periods of glutamatergic or GABAergic neurotransmission blockade in vitro as well as impinging vibrissal sensory drive in vivo. This technology enables continuous, precise optical control of firing in neuronal populations in order to disentangle causally related variables of circuit activation in a physiologically and ethologically relevant manner.

scholarly journals ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250265
Author(s):  
Hubert Hayden ◽  
Nahla Ibrahim ◽  
Johannes Klopf ◽  
Branislav Zagrapan ◽  
Lisa-Marie Mauracher ◽  
...  
Keyword(s):  
Human Plasma ◽  
Dynamic Range ◽  
Neutrophil Extracellular Traps ◽  
Plasma Samples ◽  
Dna Complexes ◽  
High Background ◽  
Isotype Control ◽  
Extracellular Traps

Over the past years, neutrophil extracellular traps (NETs) were shown to contribute to states of acute and chronic inflammatory disease. They are composed of expelled chromatin and decorated by neutrophil-derived proteins. Therefore, the analysis of DNA complexes with myeloperoxidase (MPO) by ELISA has become an attractive tool to measure NET formation in in vitro and in vivo samples. When we used a published MPO-DNA ELISA protocol and included an isotype control for the anti-MPO coating antibody, we observed high assay specificity for in vitro prepared NET samples, whereas the specificity for in vivo plasma samples was low. In addition, the assay failed to detect in vitro generated MPO-DNA complexes when spiked into plasma. Therefore, we set out to improve the specificity of the MPO-DNA ELISA for plasma samples. We found that the use of Fab fragments or immunoglobulins from different species or reversal of the antibody pair led to either a high background or a low dynamic range of detection that did not improve the specificity for plasma samples. Also, the use of higher plasma dilutions or pre-clearing of plasma immunoglobulins were ineffective. Finally, we found that a commercial reagent designed to block human anti-mouse antibodies and multivalent substances increased the detection window between the MPO antibody and isotype control for highly diluted plasma. We applied this modified ELISA protocol to analyze MPO-DNA complexes in human blood samples of acute and chronic inflammatory conditions. While markers of neutrophil activation and NET formation such as MPO, elastase and citrullinated histone H3 correlated significantly, we observed no correlation with the levels of MPO-DNA complexes. Therefore, we conclude that ELISA measurements of MPO-DNA complexes in human plasma are highly questionable regarding specificity of NET detection. In general, plasma analyses by ELISA should more frequently include isotype controls for antibodies to demonstrate target specificity.

scholarly journals SUN-238 Estrogen Modulates Expression Levels of Gonadotropin-Releasing Hormone Receptor (GNRHR) in Immortalized Kisspeptin Neurons in Vitro

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Noa Rayzmann ◽  
Hiruni Aponso ◽  
Christopher Y Markgraf ◽  
Patrick Everett Chappell
Keyword(s):  
Negative Feedback ◽  
Rna Isolation ◽  
Dependent Manner ◽  
Neuronal Populations ◽  
Feedback Inhibitor ◽  
Concentration Changes ◽  
Arcuate Nuclei ◽  
Exposure Conditions

Abstract In female animals, ovarian estradiol (E2) can act as both a negative feedback inhibitor of GnRH secretion, as well as a positive feedback stimulator at the time of ovulation. Both of these E2-regulated mechanisms work via stimulation or repression of two distinct neuronal populations of Kisspeptin (KP)-synthesizing neurons. While it is clear that AVPV KP neurons increase kiss1 expression during the preovulatory surge on proestrus, subsequent secretory mechanisms required for potentiation of GnRH surge release remain unclear. Two KP-secreting cell lines, KTaV-3, which demonstrate increased kiss1 expression under high E2 exposure, and KTaR-1, which exhibit kiss1 suppression under low E2 exposure, were used to probe the presence of GnRH receptor (GnRHR) expression under different E2 exposure conditions. KTaV-3 and KtaR-1 cells were treated with a range of doses of E2 (5-100pM) and/or progesterone (20nM) for varying durations (4-96h), exposed to steroid hormones either constitutively or via modulating levels over time, approximating concentration changes found during the murine estrous cycle. Following RNA isolation, cDNAs were probed with primers for gnrhr. Preliminary results in KTaV-3 cells reveal the expression of gnrhr is induced only following elevated (50-100pM) E2 treatment for 18-24h. These same E2 exposure conditions were also found to increase expression of the homeobox protein dlx3, a transcription factor required for GnRHR expression in pituitary gonadotropes. In Arc-derived KTaR-1 cells, gnrhr expression was observed only following decreases in E2 concentration, while dlx3 remained constitutively elevated in this cell line. While reciprocal GnRH-Kisspeptin connections have not yet been observed in vivo, these observations suggest the potential for Kisspeptin neurons to respond to GnRH secretory changes under particular E2 exposure conditions, by modulating receptivity to GnRH at the level of the AVPV and/or Arcuate nuclei. We are continuing to explore the temporal parameters of this induction of GnRHR in KP cells, and if exposure of immortalized KP neurons to GnRH in vitro elicits expression and signaling changes in a time- and E2-dependent manner. Results will provide a more complete understanding of positive and negative feedback mechanisms required for normal neuroendocrine regulation of reproduction.

scholarly journals Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor

Sensors ◽  
2019 ◽  
Vol 19 (16) ◽  
pp. 3502 ◽  
Author(s):  
Se-Hong Min ◽  
Alexander R. French ◽  
Keelan J. Trull ◽  
Kiet Tat ◽  
S. Ashley Varney ◽  
...  
Keyword(s):  
Luminescence Intensity ◽  
High Throughput Screening ◽  
Dynamic Range ◽  
Expression Level ◽  
Mouse Tissue ◽  
Photon Flux ◽  
Color Palette ◽  
Animal Imaging

Luciferase-based reporters provide a key measurement approach in a broad range of applications, from in vitro high-throughput screening to whole animal imaging. For example, luminescence intensity is widely used to measure promoter activity, protein expression levels, and cell growth. However, luminescence intensity measurements are subject to quantitative irregularities caused by luminescence decay and variation in reporter expression level. In contrast, bioluminescence resonance energy transfer (BRET) sensors provide the advantages of luciferase-based reporters but overcome the aforementioned irregularities because of the inherently ratiometric readout. Here, we generated a new ratiometric BRET sensor of ATP (ARSeNL—ATP detection with a Ratiometric mScarlet-NanoLuc sensor), and we demonstrated that it provides a stable and robust readout across protein, cell, and whole animal tissue contexts. The ARSeNL sensor was engineered by screening a color palette of sensors utilizing variants of the high photon flux NanoLuc luciferase as donors and a panel of red fluorescent proteins as acceptors. We found that the novel combination of NanoLuc and mScarlet exhibited the largest dynamic range, with a 5-fold change in the BRET ratio upon saturation with ATP. Importantly, the NanoLuc-mScarlet BRET pair provided a large spectral separation between luminescence emission channels that is compatible with green and red filter sets extensively used in typical biological microscopes and animal imaging systems. Using this new sensor, we showed that the BRET ratio was independent of luminescence intensity decay and sensor expression level, and the BRET ratio faithfully reported differences in live-cell energy metabolism whether in culture or within mouse tissue. In particular, BRET analyte sensors have not been used broadly in tissue contexts, and thus, in principle, our sensor could provide a new tool for in vivo imaging of metabolic status.

scholarly journals In vivo Interictal Neuronal Signatures of Human Periventricular Nodular Heterotopia

2019 ◽  
Author(s):  
Valerio Frazzini ◽  
Stephen Whitmarsh ◽  
Katia Lehongre ◽  
Pierre Yger ◽  
Jean-Didier Lemarechal ◽  
...  
Keyword(s):  
Epileptic Activity ◽  
Neuronal Firing ◽  
Periventricular Nodular Heterotopia ◽  
Firing Rates ◽  
Neuronal Populations ◽  
Malformation Of Cortical Development ◽  
Nodular Heterotopia ◽  
Epileptic Spikes ◽  
Drug Resistant Epilepsy

AbstractPeriventricular nodular heterotopia (PNH) is a common type of malformation of cortical development and a cause of drug-resistant epilepsy. In contrast with other cortical malformations, no consistent interictal patterns have yet been identified in PNH, and it remains controversial whether epileptic activity originates within nodules. The current study addresses the heterogeneity in LFP signatures, as well as the question of epileptogenicity of nodules, by means of microelectrode recordings implanted within PNH tissues in two epileptic patients. Microelectrodes also allowed the first in vivo recording of heterotopic neurons in humans, permitting the investigation of neuronal firing rates during patterns of interictal activity. Highly consistent interictal patterns (IPs) were identified within PNH: 1) trains of periodic slow waves and 2) isolated slow deflections, both with superimposed fast activity, and 3) epileptic spikes. Neuron firing rates were significantly modulated during all IPs, suggesting that different IPs were generated by the same local neuronal populations. To conclude, this study presents the first in vivo microscopic description of local PNH microcircuits in humans and their organization into multiple epileptic neurophysiological patterns, providing a first pathognomonic signature of human PNH.

scholarly journals A versatile genetic tool for post-translational control of gene expression in Drosophila melanogaster

2017 ◽  
Author(s):  
Sachin Sethi ◽  
Jing W. Wang
Keyword(s):  
Gene Expression ◽  
Side Effects ◽  
Translational Control ◽  
High Efficiency ◽  
Developmental Stages ◽  
Dynamic Range ◽  
Control Of Gene Expression ◽  
Neuronal Populations ◽  
Regulate Protein

AbstractSeveral techniques have been developed to manipulate gene expression temporally in intact neural circuits. However, the applicability of current tools developed for in vivo studies in Drosophila is limited by their incompatibility with existing GAL4 lines and side effects on physiology and behavior. To circumvent these limitations, we adopted a strategy to reversibly regulate protein degradation with a small molecule by using a destabilizing domain (DD). We show that this system is effective across different tissues and developmental stages. We further show that this system can be used to control in vivo gene expression levels with low background, large dynamic range, and in a reversible manner without detectable side effects on the lifespan or behavior of the animal. Additionally, we engineered tools for chemically controlling gene expression (GAL80-DD) and recombination (FLP-DD). We demonstrate the applicability of this technology in manipulating neuronal activity and for high-efficiency sparse labeling of neuronal populations.

scholarly journals Optical Control of Invertebrate nAChR and Behaviors with Dithienylethene-Imidacloprid

2021 ◽  
Author(s):  
Chao Zhang ◽  
Qi Xu ◽  
Zhiping Xu ◽  
Long Wang ◽  
Zewen Liu ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Behavioral Responses ◽  
Optical Control ◽  
Mosquito Larvae ◽  
Spatiotemporal Resolution ◽  
Neuron Membrane ◽  
Nicotinic Acetylcholine ◽  
Highly Active

Photopharmacology has changed established methods of studying receptor functions, allowing for increasing spatiotemporal resolution. However, no photopharmacological tools are available for the invertebrate nicotinic acetylcholine receptor (nAChR). Here, we report a photochromic ligand, dithienylethene-imidacloprid (DitIMI), targeting invertebrate nAChR. We demonstrated that DitIMI has low spontaneous in vivo and in vitro activity but can be photoisomerized to a highly active closed-form. This photoisomerization can further be translated to photomodulation of neuron membrane potential and behavioral responses of living mosquito larvae and American cockroaches. Furthermore, we discovered that DitIMI is a specific reporter for fluorescence polarization based high-throughput screening of nAChR ligands.

scholarly journals FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity

2020 ◽  
Vol 21 (8) ◽  
pp. 3012 ◽  
Author(s):  
Natalia V. Barykina ◽  
Vladimir P. Sotskov ◽  
Anna M. Gruzdeva ◽  
You Kure Wu ◽  
Ruben Portugues ◽  
...  
Keyword(s):  
Neuronal Activity ◽  
Dynamic Range ◽  
Fluorescent Proteins ◽  
Confocal Imaging ◽  
Ion Concentration ◽  
Neuronal Cultures ◽  
Two Phase ◽  
Intracellular Environment

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging.

scholarly journals High resolution biosensor to test the capping level and integrity of mRNAs

2020 ◽  
Vol 48 (22) ◽  
pp. e129-e129
Author(s):  
Ignacio Moya-Ramírez ◽  
Clement Bouton ◽  
Cleo Kontoravdi ◽  
Karen Polizzi
Keyword(s):  
Single Molecule ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Chimeric Protein ◽  
Single Step ◽  
Quality Analysis ◽  
Eukaryotic Mrnas ◽  
In Vitro Capping

Abstract 5′ Cap structures are ubiquitous on eukaryotic mRNAs, essential for post-transcriptional processing, translation initiation and stability. Here we describe a biosensor designed to detect the presence of cap structures on mRNAs that is also sensitive to mRNA degradation, so uncapped or degraded mRNAs can be detected in a single step. The biosensor is based on a chimeric protein that combines the recognition and transduction roles in a single molecule. The main feature of this sensor is its simplicity, enabling semi-quantitative analyses of capping levels with minimal instrumentation. The biosensor was demonstrated to detect the capping level on several in vitro transcribed mRNAs. Its sensitivity and dynamic range remained constant with RNAs ranging in size from 250 nt to approximately 2700 nt and the biosensor was able to detect variations in the capping level in increments of at least 20%, with a limit of detection of 2.4 pmol. Remarkably, it also can be applied to more complex analytes, such mRNA vaccines and mRNAs transcribed in vivo. This biosensor is an innovative example of a technology able to detect analytically challenging structures such as mRNA caps. It could find application in a variety of scenarios, from quality analysis of mRNA-based products such as vaccines to optimization of in vitro capping reactions.

scholarly journals A Membrane-Targeted Photoswitch Potently Modulates Neuronal Firing

2019 ◽  
Author(s):  
Mattia L. DiFrancesco ◽  
Francesco Lodola ◽  
Elisabetta Colombo ◽  
Luca Maragliano ◽  
Giuseppe M. Paternò ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Neural Activity ◽  
Neuronal Firing ◽  
Physiological Effects ◽  
Action Potential Firing ◽  
Polar Groups ◽  
Potential Firing ◽  
Spatio Temporal

ABSTRACTOptical technologies allowing modulation of neuronal activity at high spatio-temporal resolution are becoming paramount in neuroscience. We engineered novel light-sensitive molecules by adding polar groups to a hydrophobic backbone containing azobenzene and azepane moieties. We demonstrate that the probes stably partition into the plasma membrane, with affinity for lipid rafts, and cause thinning of the bilayer through their trans-dimerization in the dark. In neurons pulse-labeled with the compound, light induces a transient hyperpolarization followed by a delayed depolarization that triggers action potential firing. The fast hyperpolarization is attributable to a light-dependent decrease in capacitance due to membrane relaxation that follows disruption of the azobenzene dimers. The physiological effects are persistent and can be evoked in vivo after labeling the mouse somatosensory cortex. These data demonstrate the possibility to trigger neural activity in vitro and in vivo by modulating membrane capacitance, without directly affecting ion channels or local temperature.

scholarly journals α-Synuclein-induced Kv4 channelopathy in mouse vagal motoneurons causes non-motor parkinsonian symptoms

2019 ◽  
Author(s):  
Wei-Hua Chiu ◽  
Lora Kovacheva ◽  
Ruth E. Musgrove ◽  
Hadar Arien-Zakay ◽  
James B. Koprich ◽  
...  
Keyword(s):  
Rational Design ◽  
Causal Chain ◽  
Prodromal Symptom ◽  
Motor Nucleus ◽  
Prodromal Phase ◽  
Neuronal Populations ◽  
Clinical Biomarkers ◽  
Dorsal Motor Nucleus

AbstractNo disease modifying therapy is currently available for Parkinson’s disease (PD), the second most common neurodegenerative disease. The long non-motor prodromal phase of PD is a window of opportunity for early detection and intervention. However, we lack the pathophysiological understanding to develop selective biomarkers and interventions. By developing a mutant α-synuclein selective-overexpression mouse model of prodromal PD, we identified a cell-autonomous selective Kv4 channelopathy in dorsal motor nucleus of the vagus (DMV) neurons. This functional remodeling of intact DMV neurons leads to impaired pacemaker function in vitro and in vivo, which in turn reduces gastrointestinal motility which is a common, very early symptom of prodromal PD. We show for the first time a causal chain of events from α-synuclein via a biophysical dysfunction of specific neuronal populations to a clinically relevant prodromal symptom. These findings can facilitate the rational design of clinical biomarkers to identify people at risk for PD.

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