Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

1995 ◽  
Vol 53 ◽  
pp. 972-973
Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Time Lapse ◽  
Neuronal Firing ◽  
Neuronal Membrane ◽  
Pituitary Cells ◽  
Patch Clamp Recording ◽  
Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

Dopamine enhancement and depression of glutamate-regulated calcium and electrical activity in hypothalamic neurons

1996 ◽  
Vol 76 (6) ◽  
pp. 3934-3948 ◽  
Author(s):  
A. N. van den Pol ◽  
V. Cao ◽  
A. B. Belousov
Keyword(s):  
Membrane Potential ◽  
Electrical Activity ◽  
Dopamine Receptor ◽  
Glutamate Receptor ◽  
Independent Effect ◽  
Patch Clamp Recording ◽  
Hypothalamic Neurons ◽  
Whole Cell ◽  
Synaptic Release

1. The neurotransmitter dopamine is found throughout the hypothalamus both in cell bodies and in axons originating from intra- and extrahypothalamic sources. To study the mechanisms of action of dopamine on cultured rat hypothalamic neurons, particularly in relation to Ca2+ regulation, we used Ca2+ digital imaging with fura-2 and whole cell patch-clamp recording. We focused on the modulatory actions of dopamine on glutamate. 2. Dopamine administration had little or no independent effect on intracellular Ca2+. However, in the presence of tetrodotoxin to block action potentials and action-potential-dependent transmitter release, dopamine (10 microM for 2-3 min) caused an increase in glutamate-evoked Ca2+ rises in 22% of 64 neurons and depressed glutamate-evoked Ca2+ rises in an equal number of neurons. Shorter exposure to dopamine reduced the number of responding cells. 3. Dopamine application to neurons with an elevated Ca2+ due to synaptic release of glutamate (in the absence of tetrodotoxin) generally caused a decrease in Ca2+ levels (40% of 106 neurons), but sometimes increased cytosolic Ca2+ (10% of 106 neurons). That dopamine influenced cells differently in conditions of spontaneous activity compared with evoked activity may be due to dopamine effects on presynaptic receptors detected under conditions of ongoing synaptic release of glutamate. 4. Dopamine modulation of glutamate responses was detected at early stages of neuronal development (embryonic day 18 after 2 days in vitro) and also after 60 days in vitro. 5. The D1, D2, and D3 dopamine receptor agonists SKF38393, quinpirole, and 7-OH-DPAT (+/- 7 hydroxy-dipropylaminotetralin) caused a reduction in Ca2+ levels raised by endogenous glutamate release or evoked by exogenous glutamate application. 6. To block the actions of dopamine released by hypothalamic neurons, D1 and D2 dopamine receptor antagonists were used. As with dopamine, dopamine antagonists had no effect on intracellular Ca2+ during glutamate receptor blockade. In the absence of glutamate receptor block, the D1 antagonist SCH23390 (1 microM) reduced Ca2+ in responding cells; in contrast, the D2 antagonist eticlopride (1 microM) generated a delayed increase in Ca2+ levels. 7. Dopamine is known to activate second messengers through G proteins independent of changes in membrane potential or input resistance. Whole cell recording was used to demonstrate that, parallel to the modulation of Ca2+, dopamine exerted a dramatic change in glutamate-mediated electrical activity, generally depressing activity and hyperpolarizing the membrane potential (8 of 15 neurons). In a smaller number of neurons (5 of 15), dopamine enhanced glutamate-mediated excitatory activity. 8. Dopamine-evoked changes in membrane potential were in part mediated through modulation of glutamate actions. Dopamine depressed glutamate-evoked currents in a dose-dependent fashion, with Hill slopes in individual neurons ranging from 0.3 to 0.6. Dopamine could also evoke a direct hyperpolarizing action on hypothalamic neurons in the presence of tetrodotoxin or glutamate receptor blockers, at least in part by opening K+ channels. 9. Glutamate plays an important role as a primary excitatory transmitter within the hypothalamus. Our data support the hypothesis that a major mechanism of dopamine's influence on hypothalamic neurons involves the modulation of glutamate's excitatory action, mostly by inhibition. This is consistent with the hypothesis that modulation of glutamate activity may be an important mechanism of dopamine action throughout the nervous system.

scholarly journals Mural propagation of descending vasa recta responses to mechanical stimulation

2013 ◽  
Vol 305 (3) ◽  
pp. F286-F294 ◽  
Author(s):  
Zhong Zhang ◽  
Kristie Payne ◽  
Chunhua Cao ◽  
Thomas L. Pallone
Keyword(s):  
Membrane Potential ◽  
Patch Clamp ◽  
Mechanical Stimulation ◽  
Channel Blockade ◽  
Patch Clamp Recording ◽  
Automated Method ◽  
Whole Cell Patch Clamp ◽  
Vasa Recta ◽  
Gated Channel ◽  
Phosphoinositide 3 Kinase

To investigate the responses of descending vasa recta (DVR) to deformation of the abluminal surface, we devised an automated method that controls duration and frequency of stimulation by utilizing a stream of buffer from a micropipette. During stimulation at one end of the vessel, fluorescent responses from fluo4 or bis[1,3-dibutylbarbituric acid-(5)] trimethineoxonol [DiBAC4(3)], indicating cytoplasmic calcium ([Ca2+]CYT) or membrane potential, respectively, were recorded from distant cells. Alternately, membrane potential was recorded from DVR pericytes by nystatin whole cell patch-clamp. Mechanical stimulation elicited reversible [Ca2+]CYT responses that increased with frequency. Individual pericyte responses along the vessel were initiated within a fraction of a second of one another. Those responses were inhibited by gap junction blockade with 18 β-glycyrrhetinic acid (100 μM) or phosphoinositide 3 kinase inhibition with 2-morpholin-4-yl-8-phenylchromen-4-one (50 μM). [Ca2+]CYT responses were blocked by removal of extracellular Ca2+ or L-type voltage-gated channel blockade with nifedipine (10 μM). At concentrations selective for the T-type channel blockade, mibefradil (100 nM) was ineffective. During mechanostimulation, pericytes rapidly depolarized, as documented with either DiBAC4(3) fluorescence or patch-clamp recording. Single stimuli yielded depolarizations of 22.5 ± 2.2 mV while repetitive stimuli at 0.1 Hz depolarized pericytes by 44.2 ± 4.0 mV. We conclude that DVR are mechanosensitive and that rapid transmission of signals along the vessel axis requires participation of gap junctions, L-type Ca2+ channels, and pericyte depolarization.

scholarly journals Optical spike detection and connectivity analysis with a far-red voltage-sensitive fluorophore reveals changes to network connectivity in development and disease

2020 ◽  
Author(s):  
Alison S. Walker ◽  
Benjamin K. Raliski ◽  
Kaveh Karbasi ◽  
Patrick Zhang ◽  
Kate Sanders ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Patch Clamp ◽  
Amyloid Beta ◽  
Transmembrane Potential ◽  
Network Connectivity ◽  
Neuronal Membrane ◽  
Connectivity Analysis ◽  
Neuronal Connectivity ◽  
Voltage Imaging ◽  
Patch Clamp Electrophysiology

AbstractThe ability to optically record dynamics of neuronal membrane potential promises to revolutionize our understanding of neurobiology. In this study, we show that the far-red voltage sensitive fluorophore, Berkeley Red Sensor of Transmembrane potential −1, or BeRST 1, can be used to monitor neuronal membrane potential changes across dozens of neurons at a sampling rate of 500 Hz. Notably, voltage imaging with BeRST 1 can be implemented with affordable, commercially available illumination sources, optics, and detectors. BeRST 1 is well-tolerated in cultures of rat hippocampal neurons and provides exceptional optical recording fidelity, as judged by dual fluorescence imaging and patch-clamp electrophysiology. We developed a semi-automated spike-picking program to reduce user bias when calling action potentials and used this in conjunction with BeRST 1 to develop an optical spike and connectivity analysis workflow (OSCA) for high-throughput dissection of neuronal activity dynamics in development and disease. The high temporal resolution of BeRST 1 enables dissection of firing rate changes in response to acute, pharmacological interventions with commonly used inhibitors like gabazine and picrotoxin. Over longer periods of time, BeRST 1 also tracks chronic perturbations to neurons exposed to amyloid beta (Aβ1-42), revealing modest changes to spiking frequency but profound changes to overall network connectivity. Finally, we use OSCA to track changes in neuronal connectivity during development, providing a functional readout of network assembly. We envision that use of BeRST 1 and OSCA described here will be of use to the broad neuroscience community.Significance StatementOptical methods to visualize membrane potential dynamics provide a powerful complement to Ca2+ imaging, patch clamp electrophysiology, and multi-electrode array recordings. However, modern voltage imaging strategies often require complicated optics, custom-built microscopes, or genetic manipulations that are impractical outside of a subset of model organisms. Here, we describe the use of Berkeley Red Sensor of Transmembrane potential, or BeRST 1, a far-red voltage-sensitive fluorophore that can directly visualize membrane potential changes with millisecond resolution across dozens of neurons. Using only commercially available components, voltage imaging with BeRST 1 reveals profound changes in neuronal connectivity during development, exposes changes to firing rate during acute pharmacological perturbation, and illuminates substantial increases in network connectivity in response to chronic exposure to amyloid beta.

scholarly journals Feasibility analysis of semiconductor voltage nanosensors for neuronal membrane potential sensing

2019 ◽  
Author(s):  
Anastasia Ludwig ◽  
Pablo Serna ◽  
Lion Morgenstein ◽  
Gaoling Yang ◽  
Omri Bar-Elli ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Electric Fields ◽  
Stark Effect ◽  
High Sensitivity ◽  
Time Lapse ◽  
Neuronal Membrane ◽  
Attractive Alternative ◽  
Wide Field ◽  
Particle Level ◽  
Semiconductor Voltage

AbstractIn the last decade, optical imaging methods have significantly improved our understanding of the information processing principles in the brain. Although many promising tools have been designed, sensors of membrane potential are lagging behind the rest. Semiconductor nanoparticles are an attractive alternative to classical voltage indicators, such as voltage-sensitive dyes and proteins. Such nanoparticles exhibit high sensitivity to external electric fields via the quantum-confined Stark effect. Here we report the development of lipid-coated semiconductor voltage-sensitive nanorods (vsNRs) that self-insert into the neuronal membrane. We describe a workflow to detect and process the photoluminescent signal of vsNRs after wide-field time-lapse recordings. We also present data indicating that vsNRs are feasible for sensing membrane potential in neurons at a single-particle level. This shows the potential of vsNRs for detection of neuronal activity with unprecedentedly high spatial and temporal resolution.

Patch Clamp Recording in Brain Slices

2015 ◽  
Author(s):  
Luke Campagnola ◽  
Paul Manis
Keyword(s):  
Patch Clamp ◽  
Brain Slices ◽  
Neuronal Membrane ◽  
Electrical Noise ◽  
Patch Clamp Recording ◽  
Experimental Conditions ◽  
Neural Substrate ◽  
Hardware Configuration ◽  
Technical Issues

Patch clamp recording in brain slices allows unparalleled access to neuronal membrane signals in a system that approximates the in-vivo neural substrate while affording greater control of experimental conditions. In this chapter we discuss the theory, methodology, and practical considerations of such experiments including the initial setup, techniques for preparing and handling viable brain slices, and patching and recording signals. A number of practical and technical issues faced by electrophysiologists are also considered, including maintaining slice viability, visualizing and identifying healthy cells, acquiring reliable patch seals, amplifier compensation features, hardware configuration, sources of electrical noise and table vibration, as well as basic data analysis issues and some troubleshooting tips.

scholarly journals TRPM4 Expression During Postnatal Developmental of Mouse CA1 Pyramidal Neurons

2021 ◽  
Vol 15 ◽  
Author(s):  
Denise Riquelme ◽  
Oscar Cerda ◽  
Elias Leiva-Salcedo
Keyword(s):  
Membrane Potential ◽  
Patch Clamp ◽  
Postnatal Development ◽  
Pyramidal Neurons ◽  
Brain Slices ◽  
Long Term Potentiation ◽  
Neuronal Firing ◽  
Resting Membrane Potential ◽  
Ca1 Pyramidal Neurons ◽  
Apical Dendrites

TRPM4 is a non-selective cation channel activated by intracellular calcium and permeable to monovalent cations. This channel participates in the control of neuronal firing, neuronal plasticity, and neuronal death. TRPM4 depolarizes dendritic spines and is critical for the induction of NMDA receptor-dependent long-term potentiation in CA1 pyramidal neurons. Despite its functional importance, no subcellular localization or expression during postnatal development has been described in this area. To examine the localization and expression of TRPM4, we performed duplex immunofluorescence and patch-clamp in brain slices at different postnatal ages in C57BL/6J mice. At P0 we found TRPM4 is expressed with a somatic pattern. At P7, P14, and P35, TRPM4 expression extended from the soma to the apical dendrites but was excluded from the axon initial segment. Patch-clamp recordings showed a TRPM4-like current active at the resting membrane potential from P0, which increased throughout the postnatal development. This current was dependent on intracellular Ca2+ (ICAN) and sensitive to 9-phenanthrol (9-Ph). Inhibiting TRPM4 with 9-Ph hyperpolarized the membrane potential at P14 and P35, with no effect in earlier stages. Together, these results show that TRPM4 is expressed in CA1 pyramidal neurons in the soma and apical dendrites and associated with a TRPM4-like current, which depolarizes the neurons. The expression, localization, and function of TRPM4 throughout postnatal development in the CA1 hippocampal may underlie an important mechanism of control of membrane potential and action potential firing during critical periods of neuronal development, particularly during the establishment of circuits.

scholarly journals Characterization of Developmental Changes in Spontaneous Electrical Activity of Medial Superior Olivary Neurons Before Hearing Onset With a Combination of Injectable and Volatile Anesthesia

2021 ◽  
Vol 15 ◽  
Author(s):  
Mariano Nicolás Di Guilmi ◽  
Adrián Rodríguez-Contreras
Keyword(s):  
Patch Clamp ◽  
Electrical Activity ◽  
Volatile Anesthetic ◽  
Auditory Brainstem ◽  
Superior Olivary Complex ◽  
Medial Region ◽  
Spontaneous Electrical Activity ◽  
Rat Pups ◽  
The Impact

In this work the impact of two widely used anesthetics on the electrical activity of auditory brainstem neurons was studied during postnatal development. Spontaneous electrical activity in neonate rats of either sex was analyzed through a ventral craniotomy in mechanically ventilated pups to carry out patch clamp and multi-electrode electrophysiology recordings in the medial region of the superior olivary complex (SOC) between birth (postnatal day 0, P0) and P12. Recordings were obtained in pups anesthetized with the injectable mix of ketamine/xylazine (K/X mix), with the volatile anesthetic isoflurane (ISO), or in pups anesthetized with K/X mix that were also exposed to ISO. The results of patch clamp recordings demonstrate for the first time that olivary and periolivary neurons in the medial region of the SOC fire bursts of action potentials. The results of multielectrode recordings suggest that the firing pattern of single units recorded in K/X mix is similar to that recorded in ISO anesthetized rat pups. Taken together, the results of this study provide a framework to use injectable and volatile anesthetics for future studies to obtain functional information on the activity of medial superior olivary neurons in vivo.

Dorsal unpaired median neurones in the insect central nervous system: towards a better understanding of the ionic mechanisms underlying spontaneous electrical activity

2000 ◽  
Vol 203 (11) ◽  
pp. 1633-1648 ◽  
Author(s):  
F. Grolleau ◽  
B. Lapied
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Ion Channels ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Delayed Rectifier ◽  
Spontaneous Electrical Activity ◽  
Voltage Dependent ◽  
Insect Central Nervous System ◽  
Dorsal Unpaired Median

The efferent dorsal unpaired median (DUM) neurones, which include octopaminergic neurones, are among the most intensively studied neurones in the insect central nervous system. They differ from other insect neurones in generating endogenous spontaneous overshooting action potentials. The second half of the 1980s is certain to be considered a turning point in the study of the ion channels underlying the electrical activity of DUM neurones. Recent advances made using the patch-clamp technique have stimulated an increasing interest in the understanding of the biophysical properties of both voltage-dependent and voltage-independent ion channels. Patch-clamp studies of DUM neurones in cell culture demonstrate that these neurones express a wide variety of ion channels. At least five different types of K(+) channel have been identified: inward rectifier, delayed rectifier and A-like channels as well as Ca(2+)- and Na(+)-activated K(+) channels. Moreover, besides voltage-dependent Na(+) and Ca(2+)-sensitive Cl(−) channels, DUM neurones also express four types of Ca(2+) channel distinguished on the basis of their kinetics, voltage range of activation and pharmacological profile. Finally, two distinct resting Ca(2+) and Na(+) channels have been shown to be involved in maintaining the membrane potential and in regulating the firing pattern. In this review, we have also attempted critically to evaluate these existing ion channels with regard to their specific functions in the generation of the different phases of the spontaneous electrical activity of the DUM neurone.

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