scholarly journals Scaled, high fidelity electrophysiological, morphological, and transcriptomic cell characterization

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Brian R Lee ◽  
Agata Budzillo ◽  
Kristen Hadley ◽  
Jeremy A Miller ◽  
Tim Jarsky ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Mammalian Species ◽  
Membrane Integrity ◽  
Brain Slices ◽  
Brain Regions ◽  
Quality Data ◽  
Key Factors ◽  
Patch Clamp Technique ◽  
Online Quality Control ◽  
Patch Clamp Electrophysiology

The Patch-seq approach is a powerful variation of the patch-clamp technique that allows for the combined electrophysiological, morphological, and transcriptomic characterization of individual neurons. To generate Patch-seq datasets at scale, we identified and refined key factors that contribute to the efficient collection of high-quality data. We developed patch-clamp electrophysiology software with analysis functions specifically designed to automate acquisition with online quality control. We recognized the importance of extracting the nucleus for transcriptomic success and maximizing membrane integrity during nucleus extraction for morphology success. The protocol is generalizable to different species and brain regions, as demonstrated by capturing multimodal data from human and macaque brain slices. The protocol, analysis and acquisition software are compiled at https://github.com/AllenInstitute/patchseqtools. This resource can be used by individual labs to generate data across diverse mammalian species and that is compatible with large publicly available Patch-seq datasets.

scholarly journals Scaled, high fidelity electrophysiological, morphological, and transcriptomic cell characterization

2020 ◽  
Author(s):  
Brian R. Lee ◽  
Agata Budzillo ◽  
Kristen Hadley ◽  
Jeremy A. Miller ◽  
Tim Jarsky ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Large Scale ◽  
Mammalian Species ◽  
Membrane Integrity ◽  
Substantial Improvement ◽  
Quality Data ◽  
Patch Clamp Technique ◽  
High Quality ◽  
Transcriptomic Data ◽  
Online Quality Control

The Patch-seq approach is a powerful variation of the standard patch clamp technique that allows for the combined electrophysiological, morphological, and transcriptomic characterization of individual neurons. To generate Patch-seq datasets at a scale and quality that can be integrated with high-throughput dissociated cell transcriptomic data, we have optimized the technique by identifying and refining key factors that contribute to the efficient collection of high-quality data. To rapidly generate high-quality electrophysiology data, we developed patch clamp electrophysiology software with analysis functions specifically designed to automate acquisition with online quality control. We recognized a substantial improvement in transcriptomic data quality when the nucleus was extracted following the recording. For morphology success, the importance of maximizing the neuron’s membrane integrity during the extraction of the nucleus was much more critical to success than varying the duration of the electrophysiology recording. We compiled the lab protocol with the analysis and acquisition software at https://github.com/AllenInstitute/patchseqtools. This resource can be used by individual labs to generate Patch-seq data across diverse mammalian species and that is compatible with recent large-scale publicly available Allen Institute Patch-seq datasets.

scholarly journals Single-neuron identification of chemical constituents, physiological changes, and metabolism using mass spectrometry

2017 ◽  
Vol 114 (10) ◽  
pp. 2586-2591 ◽  
Author(s):  
Hongying Zhu ◽  
Guichang Zou ◽  
Ning Wang ◽  
Meihui Zhuang ◽  
Wei Xiong ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Patch Clamp ◽  
Single Cell ◽  
Single Neuron ◽  
Biological Activities ◽  
Chemical Constituents ◽  
Cell Mass ◽  
Brain Slices ◽  
Patch Clamp Technique

The use of single-cell assays has emerged as a cutting-edge technique during the past decade. Although single-cell mass spectrometry (MS) has recently achieved remarkable results, deep biological insights have not yet been obtained, probably because of various technical issues, including the unavoidable use of matrices, the inability to maintain cell viability, low throughput because of sample pretreatment, and the lack of recordings of cell physiological activities from the same cell. In this study, we describe a patch clamp/MS-based platform that enables the sensitive, rapid, and in situ chemical profiling of single living neurons. This approach integrates modified patch clamp technique and modified MS measurements to directly collect and detect nanoliter-scale samples from the cytoplasm of single neurons in mice brain slices. Abundant possible cytoplasmic constituents were detected in a single neuron at a relatively fast rate, and over 50 metabolites were identified in this study. The advantages of direct, rapid, and in situ sampling and analysis enabled us to measure the biological activities of the cytoplasmic constituents in a single neuron, including comparing neuron types by cytoplasmic chemical constituents; observing changes in constituent concentrations as the physiological conditions, such as age, vary; and identifying the metabolic pathways of small molecules.

Patch-Clamp Technique in Brain Slices

2003 ◽  
pp. 233-258 ◽  
Author(s):  
T. D. Plant ◽  
J. Eilers ◽  
A. Konnerth
Keyword(s):  
Patch Clamp ◽  
Brain Slices ◽  
Patch Clamp Technique ◽  
Clamp Technique

scholarly journals Synthesis and Evaluation of Novel α-Aminoamides Containing Benzoheterocyclic Moiety for the Treatment of Pain

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1716
Author(s):  
Kun Tong ◽  
Ruotian Zhang ◽  
Fengzhi Ren ◽  
Tao Zhang ◽  
Junlin He ◽  
...  
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Formalin Test ◽  
Sodium Ion ◽  
Inhibitory Potency ◽  
Voltage Gated ◽  
Patch Clamp Electrophysiology ◽  
Sodium Ion Channels

Novel α-aminoamide derivatives containing different benzoheterocyclics moiety were synthesized and evaluated as voltage-gated sodium ion channels blocks the treatment of pain. Compounds 6a, 6e, and 6f containing the benzofuran group displayed more potent in vivo analgesic activity than ralfinamide in both the formalin test and the writhing assay. Interestingly, they also exhibited potent in vitro anti-Nav1.7 and anti-Nav1.8 activity in the patch-clamp electrophysiology assay. Therefore, compounds 6a, 6e, and 6f, which have inhibitory potency for two pain-related Nav targets, could serve as new leads for the development of analgesic medicines.

scholarly journals Patch-clamp and multi-electrode array electrophysiological analysis in acute mouse brain slices

2021 ◽  
Vol 2 (2) ◽  
pp. 100442
Author(s):  
Kevin M. Manz ◽  
Justin K. Siemann ◽  
Douglas G. McMahon ◽  
Brad A. Grueter
Keyword(s):  
Patch Clamp ◽  
Mouse Brain ◽  
Brain Slices ◽  
Electrode Array ◽  
Electrophysiological Analysis ◽  
Multi Electrode Array

Physiology, Pharmacology, and Topography of Cholinergic Neocortical Oscillations In Vitro

1997 ◽  
Vol 77 (5) ◽  
pp. 2427-2445 ◽  
Author(s):  
Heath S. Lukatch ◽  
M. Bruce Maciver
Keyword(s):  
Current Source ◽  
Brain Slices ◽  
Receptor Activation ◽  
Brain Regions ◽  
Oscillatory Activity ◽  
Cortical Layers ◽  
Eeg Activity ◽  
Layer 2

Lukatch, Heath S. and M. Bruce MacIver. Physiology, pharmacology, and topography of cholinergic neocortical oscillations in vitro. J. Neurophysiol. 77: 2427–2445, 1997. Rat neocortical brain slices generated rhythmic extracellular field [microelectroencephalogram (micro-EEG)] oscillations at theta frequencies (3–12 Hz) when exposed to pharmacological conditions that mimicked endogenous ascending cholinergic and GABAergic inputs. Use of the specific receptor agonist and antagonist carbachol and bicuculline revealed that simultaneous muscarinic receptor activation and γ-aminobutyric acid-A (GABAA)-mediated disinhibition werenecessary to elicit neocortical oscillations. Rhythmic activity was independent of GABAB receptor activation, but required intact glutamatergic transmission, evidenced by blockade or disruption of oscillations by 6-cyano-7-nitroquinoxaline-2,3-dione and (±)-2-amino-5-phosphonovaleric acid, respectively. Multisite mapping studies showed that oscillations were localized to areas 29d and 18b (Oc2MM) and parts of areas 18a and 17. Peak oscillation amplitudes occurred in layer 2/3, and phase reversals were observed in layers 1 and 5. Current source density analysis revealed large-amplitude current sinks and sources in layers 2/3 and 5, respectively. An initial shift in peak inward current density from layer 1 to layer 2/3 indicated that two processes underlie an initial depolarization followed by oscillatory activity. Laminar transections localized oscillation-generating circuitry to superficial cortical layers and sharp-spike-generating circuitry to deep cortical layers. Whole cell recordings identified three distinct cell types based on response properties during rhythmic micro-EEG activity: oscillation-on (theta-on) and -off (theta-off) neurons, and transiently depolarizing glial cells. Theta-on neurons displayed membrane potential oscillations that increased in amplitude with hyperpolarization (from −30 to −90 mV). This, taken together with a glutamate antagonist-induced depression of rhythmic micro-EEG activity, indicated that cholinergically driven neocortical oscillations require excitatory synaptic transmission. We conclude that under the appropriate pharmacological conditions, neocortical brain slices were capable of producing localized theta frequency oscillations. Experiments examining oscillation physiology, pharmacology, and topography demonstrated that neocortical brain slice oscillations share many similarities with the in vivo and in vitro theta EEG activity recorded in other brain regions.

Vasorelaxing Activity of R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol from Dalbergia tonkinensis: Involvement of Smooth Muscle CaV1.2 Channels

Planta Medica ◽  
2020 ◽  
Vol 86 (04) ◽  
pp. 284-293 ◽  
Author(s):  
Nguyen Manh Cuong ◽  
Ninh The Son ◽  
Ngu Truong Nhan ◽  
Pham Ngoc Khanh ◽  
Tran Thu Huong ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Antihypertensive Drugs ◽  
Docking Simulation ◽  
Mechanical Activity ◽  
Dependent Manner ◽  
Vascular Effects ◽  
Patch Clamp Technique ◽  
Spasmolytic Activity ◽  
Cav1.2 Channels ◽  
Dalbergia Tonkinensis

Abstract Dalbergia species heartwood, widely used in traditional medicine to treat various cardiovascular diseases, might represent a rich source of vasoactive agents. In Vietnam, Dalbergia tonkinensis is an endemic tree. Therefore, the aim of the present work was to investigate the vascular activity of R-(−)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol isolated from the heartwood of D. tonkinensis and to provide circular dichroism features of its R absolute configuration. The vascular effects of R-(−)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol were assessed on the in vitro mechanical activity of rat aorta rings, under isometric conditions, and on whole-cell Ba2+ currents through CaV1.2 channels (IBa1.2) recorded in single, rat tail main artery myocytes by means of the patch-clamp technique. R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol showed concentration-dependent, vasorelaxant activity on both endothelium-deprived and endothelium intact rings precontracted with the α 1 receptor agonist phenylephrine. Neither the NO (nitric oxide) synthase inhibitor Nω-nitro-L-arginine methyl ester nor the cyclooxygenase inhibitor indomethacin affected its spasmolytic activity. R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol-induced vasorelaxation was antagonized by (S)-(−)-Bay K 8644 and unaffected by tetraethylammonium plus glibenclamide. In patch-clamp experiments, R-(−)-3′-hydroxy-2,4,5-trimethoxydalbergiquinol inhibited IBa1.2 in a concentration-dependent manner and significantly decreased the time constant of current inactivation. R-(−)-3′-Hydroxy-2,4,5-trimethoxydalbergiquinol likely stabilized the channel in its closed state, as suggested by molecular modelling and docking simulation to the CaV1.2 channel α 1c subunit. In conclusion, D. tonkinensis species may represent a source of agents potentially useful for the development of novel antihypertensive drugs.

Sign in / Sign up

Export Citation Format

Share Document