Clampfit 10

Manufactured by Molecular Devices

Sourced in United States, United Kingdom, Canada

Clampfit 10 is a software application designed for the analysis of electrophysiological data. It provides tools for data acquisition, visualization, and signal processing.

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Clampfit (397 citations) Clampfit version 10.2 (15 citations) Clampfit software version 10.7 (11 citations) PClamp Clampfit 10.4 (10 citations) Clampfit 10 program (8 citations) Clampfit software 10.4 (3 citations) Clampfit 10.7 program (2 citations) Clampfit of pClamp 10.7 (2 citations) Clampfit 10 functions (2 citations) Clampfit 10 Data Analysis Module of the pCLAMP™ 10 Electrophysiology Data Acquisition & Analysis Software (2 citations)

854 protocols using clampfit 10

Intracellular Recordings of LES Muscle Oscillations

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LES muscles (see Fig. 1A) were isolated from C57BL/6 mice (2-3 months old). The final LES muscle strips used for electrophysiological experiments were 8 x 2mm. The muscle strips were pinned down in a recording chamber lined with Sylgard elastomer 184 (Dow, USA), and incubated at 37 ± 0.5°C with continuously flowing, oxygenated KRB solution for 1hr before beginning intracellular recordings. Cells in the LES were impaled with glass microelectrodes filled with 3 M KCl and having resistance 50-100 MΩ. Transmembrane potentials were measured using a high input impedance amplifier (Axopatch 2B, Molecular Devices Corp., Sunnyvale, CA, USA) and recorded with Axoscope 10.3 software. The data were analyzed by Clampfit 10.4 (Molecular Devices). All recordings were made in the presence of nicardipine (100 nM) to reduce movements and extend the durations of impalements.
LES muscles displayed continuous random oscillations of resting membrane potentials (RMP). Therefore, membrane potentials (referred to in this paper as membrane potential oscillations; MPOs) were analyzed by generating amplitude histograms from 1 min recordings using Clampfit 10.4 software (Molecular Devices). The median values and standard errors (SE) were calculated by a Gaussian Function for RMP and MPOs, respectively.

Drumm B.T., Hannigan K.I., Lee J.Y., Rembetski B.E., Baker S.A., Koh S.D., Cobine C.A, & Sanders K.M. (2022). Ca2+ signalling in interstitial cells of Cajal contributes to generation and maintenance of tone in mouse and monkey lower esophageal sphincters. The Journal of physiology, 600(11), 2613-2636.

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Slow Wave Event Characterization

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A stable impalement for a ~30 second time period consisting of at least 10 slow wave events was considered for data analysis. All the raw slow wave recordings were low-pass filtered using a Butterworth (8-pole) digital filter in Clampfit 10.3.1.5 software (Molecular Devices Corp., Sunnyvale, CA) with a cut-off of 60 Hz. Data were analyzed off-line using Clampfit 10.3.1.5 software (Molecular Devices Corp., Sunnyvale, CA) for the following slow wave parameters: (i) resting membrane potential (E_m, mV), (ii) peak slow wave amplitude (mV), (iii) membrane potential (MP) at peak amplitude (E_m-Pk, mV); (iv) half-width (ms), (v) rise slope 10%–90% (mV/ms), (vi) rise time 10%–90% (ms) and (vii) instantaneous frequency (Hz). In 8 out of the 81 cells studied, the RMPs were adjusted for the electrode pullout potential upon withdrawal of the electrode from the cell. Data are expressed as means ± standard errors of the mean (SEM). Statistical significance was determined by Graphpad Prism using the appropriate statistical test with corrections for multiple comparisons where necessary, as indicated in the results section. P values of less than 0.05 were considered statistically significant. The values from different cells (n) were analyzed and reported as means ± SEM. The ‘n’ value refers to the count of separate cells recorded from tissue strips; and the ‘N’ value refers to the animal count.

Beyder A., Gibbons S.J., Mazzone A., Strege P.R., Saravanaperumal S.A., Sha L., Higgins S., Eisenman S.T., Bernard C.E., Geurts A., Kline C.F., Mohler P.J, & Farrugia G. (2015). Expression and Function of the Scn5a-Encoded Voltage-Gated Sodium Channel NaV1.5 in the Rat Jejunum. Neurogastroenterology and motility : the official journal of the European Gastrointestinal Motility Society, 28(1), 64-73.

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Electrophysiological Analysis of Synaptic Transmission

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Post-synaptic current frequencies, amplitudes, inter-event intervals and decay time constants were measured using Clampfit 10.7 (Molecular Devices) and Axograph (AxoGraph, Inc). I/V curves from perforated patch recordings were calculated in Clampfit 10.7 (Molecular Devices). Statistical outliers were identified using the ROUT method (Q=1% cutoff threshold) as implemented in GraphPad Prism 9. Group differences were analyzed with two-way ANOVA followed by Tukey's multiple comparisons post hoc test using Prism 8 and 9 (GraphPad). When required, three-way ANOVA was performed using the aov function in R (v3.6.3) using the R package r/emmeans for post hoc Tukey's multiple comparison testing with Bonferroni's correction. For repeated measures analysis, group differences were analyzed by two-way repeated measures-ANOVA using SPSS (IBM) or R. Data visualization was performed using Prism or r/ggplot2. For all statistical tests, a value of p < 0.05 was considered significant. Data are presented as the mean ± SEM; violin plots are presented as median ± quartile.

Korgan A.C., Wei W., Martin S.L.A., Kaczorowski C.C., & O’Connell K.M. (2021). High-fat diet induced loss of GABAergic inhibition decouples intrinsic and synaptic excitability in AgRP neurons.

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Electrophysiological Analysis of sEPSCs and mEPSCs

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Data were obtained using Clampfit 10.5 (Molecular Devices, San Jose, CA, USA) via a DIGIDATA 1550B series A/D board (Molecular Devices, San Jose, CA, USA) at a sampling frequency of 10 kHz. Data from sEPSCs and mEPSCs were analyzed using Clampfit 10.5 (Molecular Devices, San Jose, CA, USA), Mini 6.0 (Synaptosoft Inc., Fort Lee, NJ, USA) and Origin Pro 8.0 (OriginLab, Northampton, MA, USA) on a PC. Average values of inter-event interval (IEI) and amplitude for events in the control and drug administered group were compared. The mean of the averaged values for each group was then statistically compared using paired t-test unless otherwise stated. Cumulative probability distributions of IEI and amplitude were assessed to determine the significance of the shifts using the nonparametric Kolmogorov–Smirnoff (K-S) test. To analyze the kinetic properties of the sEPSCs/mEPSCs, 100 s EPSCs’ recordings for each cell were selected and the EPSCs in the time course were averaged. Approximately 100–500 events were recorded for each cell. The decay of the average current was fitted using a mono-exponential function with a single decay time constant. The rise time was defined as the average current rise time from 10% to 90% of the peak (Simonyan et al., 2012 (link)). All data were presented as mean ± SEM unless otherwise stated (Wang et al., 2015b (link)).

Wang S., Liu S., Wang Q., Sun Y., Yao L., Li D, & Meng W. (2020). Dopamine Modulates Excitatory Synaptic Transmission by Activating Presynaptic D1-like Dopamine Receptors in the RA Projection Neurons of Zebra Finches. Frontiers in Cellular Neuroscience, 14, 126.

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Single-Channel Recording and Analysis

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Single‐channel recordings were digitized at 20 kHz and low‐pass filtered at 800 Hz. Po was determined over 3 min of continuous recording at 0 mV using 50% threshold analysis (Colquhoun and Sigworth, 1995) in Clampfit 10.6 (Molecular Devices, USA) as previously described (Sitsapesan and Williams, 1990; Sitsapesan et al.,1991). Where Po values are shown in figures, the Po above each trace refers to the value determined over 3 min for that particular channel. Where more than one channel was incorporated into the bilayer, Po is reported as an average (total Po divided by number of channels). Lifetime distributions were constructed using Clampfit 10.6 (Molecular Devices, USA) and fitted to a probability density function by the method of maximum likelihood (Colquhoun and Sigworth, 1995) according to the following equation:

f (t) = \sum_{i}^{N} a_{i} f_{0} (t - ln τ_{i})

where i is the number of exponential components of the distribution, τ_i are the time constants and a_i are the fractions of the total events represented by the ith component. The set of parameters were adjusted by the maximum likelihood iterative algorithm until the optimum value of the log of likelihood L was reached (Blatz and Magleby, 1986).

Venturi E., Lindsay C., Lotteau S., Yang Z., Steer E., Witschas K., Wilson A.D., Wickens J.R., Russell A.J., Steele D., Calaghan S, & Sitsapesan R. (2018). Simvastatin activates single skeletal RyR1 channels but exerts more complex regulation of the cardiac RyR2 isoform. British Journal of Pharmacology, 175(6), 938-952.

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Whole-Cell Patch Clamp Electrophysiology

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Electrophysiological recordings were performed in whole-cell patch clamping mode as described previously (Gong et al., 2016 (link)). Micropipettes were pulled from borosilicate glass capillary tubes (World Precision Instruments, Inc.) by using a P-97 micropipette puller (Sutter Instruments). The micropipette solution for recording mEPSC contained 140 mM CsCl, 5 mM NaCl, 10 mM HEPES, 5 mM EGTA, 0.3 mM Na2GTP, and 3 mM Mg2ATP. Adjusted pH to 7.2–7.4 with CsOH. Adjusted osmotic pressure to about 305 mOsm with dd H₂O. For recording eEPSC, add 5 mM QX-314 to the micropipette solution before use. The bath solution contained 150 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 10 mM glucose. Adjusted pH of bath solution to 7.2–7.4 with NaOH and adjust osmotic pressure to about 315 mOsm with dd H₂O. For mEPSC recording, 50 μM AP-5, 100 μM picrotoxin, and 1 μM tetrodotoxin were added to the bath solution before use. Add 50 μM AP-5 and 100 μM picrotoxin to the bath solution for eEPSC recording.
Synaptic currents were monitored with an EPC10 amplifier (HEKA). Miniature events were analyzed in Clampfit 10 (Molecular Devices) using the template matching search function. Single extracellular stimulus pulses (90 μA, 1 ms) were controlled with a Model 2100 Isolated Pulse Stimulator (A-M Systems, Inc.) for eEPSCs measurements. All data were analyzed in Clampfit 10 (Molecular Devices).

Mo X., Liu M., Gong J., Mei Y., Chen H., Mo H., Yang X, & Li J. (2022). PTPRM Is Critical for Synapse Formation Regulated by Zinc Ion. Frontiers in Molecular Neuroscience, 15, 822458.

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Spinal Cord Stimulation EMG Responses

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The EMG responses elicited by spinal cord stimulation were divided into early (ER, latency 1e4 ms), middle (MR, latency 5e10 ms) and late responses (LR, latency 11e15 ms) relative to the stimulation pulse [19] . The amplitude and time to peak of each EMG response was determined using Clampfit® 10.3 software (Molecular Devices, LLC, CA, USA) to plot time course graphs. Responses were statistically compared by individually calculating the peak of 20 (for single DS) or 100 (for repetitive DSs) consecutive sweeps immediately before and after the application of each protocol. Then, single peaks were averaged for statistical comparison. The ratio between the standard deviation and mean amplitude provided the amplitude coefficient of variation (CV), which is an index of consistency of EMG evoked responses (the lower the CV, the less variable the responses [29] ). The power spectrum of the patterns composing DS was obtained through Clampfit® 10.3 software (Molecular Devices, LLC, CA, USA).

Taccola G., Gad P., Culaclii S., Ichiyama R.M., Liu W, & Edgerton V.R. (2020). Using EMG to deliver lumbar dynamic electrical stimulation to facilitate cortico-spinal excitability. Brain stimulation, 13(1).

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Quantifying Synaptic AMPAR Properties

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To measure mean AMPAR channel conductance (γ) and the number of channels exposed to glutamate (N) per synapse, peak scaled noise analysis of AMPAR-miniature EPSCs (mEPSCs) was performed using Clampfit 10.2 (Molecular Devices). The peak of each mEPSC in a recording was scaled to the average mEPSC waveform for that recording, and the variance of current around the mean for each time point was calculated. The data were fit with the following parabolic equation:
where σ² = variance, I = mean current, i = single-channel current, N = number of open channels at peak current, and σ_b² = background variance. From this equation, γ was calculated by dividing i by the driving force (-70 mV; AMPAR reversal potential was ∼0 mV with the recording solutions used). Recordings were discarded if the parabolic fits of the current variance plots had R² <0.5 (Traynelis et al., 1993 (link); Hartveit and Veruki, 2007 (link)).

Smith-Dijak A.I., Nassrallah W.B., Zhang L.Y., Geva M., Hayden M.R, & Raymond L.A. (2019). Impairment and Restoration of Homeostatic Plasticity in Cultured Cortical Neurons From a Mouse Model of Huntington Disease. Frontiers in Cellular Neuroscience, 13, 209.

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Quantification of GABA-A Receptor Tonic Current

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GABA_A receptor-mediated tonic current was measured as the resulting shift of the holding current when the GABA_A receptor antagonist gabazine (20 μM) was applied. For quantification, 5 ms long samples of the holding current were taken every 100 ms before and at the maximum effect of gabazine. The subtraction of the minor from the maximal current value was taken as the tonic current (Drasbek and Jensen, 2006 (link)). The resulting values were then normalized and used for statistical analysis. Offline analysis of the data was performed using Clampfit 10.2 (Molecular Devices) and graphing and statistical software (Origin 8, Microcal, Northampton MA). Data are expressed as means ± S.E.M. For each experimental group a minimum of 5 cells were recorded, 1 cell per slice. Statistical analysis was performed with GraphPad Prism 6 software (San Jose CA, USA) using Wilcoxon's or Mann-Whitney's tests for paired or non-paired samples respectively (p < 0.05 was taken as significant).

Trujeque-Ramos S., Castillo-Rolón D., Galarraga E., Tapia D., Arenas-López G., Mihailescu S, & Hernández-López S. (2018). Insulin Regulates GABAA Receptor-Mediated Tonic Currents in the Prefrontal Cortex. Frontiers in Neuroscience, 12, 345.

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Eye and Electrophysiology Data Analysis

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Analysis of eye tracking and in vivo electrophysiology was performed using custom-written codes in Matlab. Analysis of in vitro electrophysiology was done with Clampfit 10.2 (Molecular Devices).

Liu B.H., Huberman A.D, & Scanziani M. (2016). Cortico-fugal output from visual cortex promotes plasticity of innate motor behaviour. Nature, 538(7625), 383-387.

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