Resting Membrane Voltage

DAngelo et al. 1995

units	mV
reference	D'Angelo E, De Filippi G, Rossi P, Taglietti V (1995) Synaptic excitation of individual rat cerebellar granule cells in situ: evidence for the role of NMDA receptors. J Physiol (Lond) 484:397-413. doi: 10.1113/jphysiol.1995.sp020673
data_collection	Based on Figure 2 and under Results section and sub-heading Passive membrane properties of rat cerebellar granule cells: No spontaneous extracellular action currents were observed during seal formation, nor was any spontaneous firing seen after patch disruption. Granule cell resting membrane potential measured soon after gaining access to the cell was -57.8 +/- 6.4 mV (n = 43; range from -45 to -72 mV). Therefore, from this, n = 43 cells, and set -57.8 +/- 6.4 mV
confidence	0.95
validation_info	This is the experimental data is from in-vitro 43 GranularCells from P21-31 Wistar rats.
protocol	Whole-cell patch-clamp recordings were made from granule cells in the internal granular layer of rat cerebellar slices. Cerebellar slices were obtained from 21- to 31-day-old Wistar rats (day of birth = 1). RECORDING AND ANALYSIS: Patch pipettes were pulled from thick-walled borosilicate glass capillaries and had 8-12 Mohm resistance before seal formation. Electrical stimulation of afferent fibres was performed with a bipolar tungsten electrode via a stimulus insulation unit. In some experiments, a second stimulating electrode was located at the edge of the molecular layer. Stimulating pulses lasting 100-200 us were usually delivered at 0.25 Hz. Tight-seal whole-cell recordings were performed conventionally using the 'blind patch' approach. Membrane current and voltage were recorded using an Axopatch-ID patch-clamp amplifier with output cut-off frequency = 10 kHz. Signals were simultaneously stored on a DAT recorder and fed to a PC at sampling frequency = 250 us for current-clamp recordings, 10-50 us for voltage-clamp recordings. Data were reported as means + S.D. and the number of observations is indicated in parentheses. Statistical comparisons were done using Student's t test. Analysis of current transients activated by 10-20 mV hyperpolarizing steps from the holding potential of -70 mV in voltage-clamp mode gave the following values (n = 79 for all measures): membrane input resistance Rm = 2.3 +/- 1.1 Gohm, membrane input capacitance Cm = 3.1 +/- 1.5 pF, membrane time constant Tm= RmCm = 6.7 +/- 3.3 ms, decay time constant of the current transient tau_s = 57.9 +/- 24 us, series resistance Rs, = tau_s/Cm = 18.5 +/- 15.6 Mohm. In current-clamp mode, electrode capacitance compensation was critical in recording membrane voltage changes reliably, since granule cell membrane capacitance (3 pF) was comparable to electrode capacitance (5 pF). Electrode capacitance was compensated electronically using the value matched during current transient cancellation in cell-attached configuration. Cancellation achieved by maintaining the immersion depth to 1 mm or less and holding the electrode at a rather steep angle (45-60 deg) was virtually complete and no effective improvement was obtained after having reduced the electrode capacitive current with Sylgard coating. The current charging the patch-pipette was provided by the feed-back 'negative capacitance' compensation circuit in the Axopatch-ID amplifier. Note that capacitive compensation currents, due to their dependence on the rate of membrane potential change, are much greater during action potentials than during the EPSPs. Nearly maximal compensation was usually achieved, since <10% overcompensation invariably produced oscillations. On the other hand, decreasing negative capacitance compensation slowed membrane charging considerably. ERROR ESTIMATES: Pipette offset was compensated electronically. Since liquid junction potential was <2 mV, membrane potential values were left uncorrected. Field potentials produced by neighbouring granule cell activity, which were measured after removing the pipette from the cell and cleaning its tip by gently applying positive pressure, were smaller than 0.5 mV (n = 8) and therefore did not appreciably modify the transmembrane potential. Attenuation of a constant command potential across the electrode resistance (Re = 10 Mohm) was calculated by considering current partitioning between the seal leak resistance (R1 >20 Gohm) and cell input resistance (either Rm = 0.5 Gohm or Rm = 5 Gohm to simulate inward rectification), according to the equation Vp/ Ve = 1 - [Re(Rm + Ri)/RmR]. Attenuation of Vp caused by the voltage drop across the access resistance (Ra = R, - Re = 10 Mohm) was obtained as Vm/Vp = 1 - [Ra/Rm]. With the resistance values given, the membrane potential ratio Vm/Vc was >0.94 for Rm = 0.5 Gohm and > 0.996 for Rm= 5 Gohm. Consistent with negligible voltage attenuation, bridge compensation did not produce any appreciable changes in EPSPs or passive voltage transients (n = 11). Attenuation of a potential generated by mossy fibre synapses at the end of the dendrites and measured from the soma was estimated using a neuron model consisting of a spherical soma (diameter = 6 um) connected to four identical unbranched dendrites (diameter = 1 um, length = 10 um) and an axon (diameter = 0.1 um). The procedure used is based on cable's equations. Computations were carried out using a specific axoplasmic resistance of 80 ohm cm and a specific membrane resistance of either 1500 ohm cm2 or 15000 ohm cm2 to simulate inward rectification. In the absence of synaptic inputs, soma-to-dendritic membrane potential ratios were 0.981 and 0.998, respectively. When an active load of 500 pS (which is a reasonable estimate of synaptic conductance) was applied to one to four dendrites, calculations yielded soma-to-dendritic membrane potential ratios of 0.977, 0.972, 0.967 and 0.962, respectively (Rm = 0.5 Gohm), and 0.993, 0.988, 0.983 and 0.978, respectively (Rm = 5 Gohm). Therefore, in current-clamp as well as in voltage-clamp conditions, the granule cells tend to behave like a single electrical compartment. SOLUTIONS AND DRUGS: Krebs solution for slice cutting and recovery contained (mM): NaCl, 120; KCl, 2; MgSO4.7H20, 12; NaHCO3, 26; KH2PO4, 12; CaCl2, 2; glucose, 11. This solution was equilibrated with 95% 02 and 5% CO2 (pH 7.4) and was perfused at a rate of 2.4 ml/min. The recording chamber had a volume of 1P5 ml and was maintained at 30 +/- 1 C. The intracellular solution contained (mM): potassium gluconate, 122; KCl, 4; NaCl, 4; MgCl2, 1; CaCl2, 002; BAPTA, 0-1; glucose, 15; ATP, 3; Hepes, 5; pH was adjusted to 7.2 with KOH. This solution buffered intracellular Ca2+ at 100 nm. Drugs dissolved in extracellular Krebs solution were applied locally through a multi-barrelled pipette. Unless stated otherwise, all solutions perfused during recordings contained 10 M glycine and 10 uM bicuculline.
sample_size	43
mean	-57.8
margin_of_error	6.4

DAngelo et al. 1998

reference	D'Angelo E, De Filippi G, Rossi P, Taglietti V (1998) Ionic mechanism of electroresponsiveness in cerebellar granule cells implicates the action of a persistent sodium current. J Neurophysiol 80:493-503. doi 10.1152/jn.1998.80.2.493
mean	-62.4
units	mV
sample_size	36
protocol	Cerebellar slices (250 uM thick) were obtained from 21 to 26 day old rats (Wistar strain, day of birth = P1). The rats were decapitated after halothane anesthesia. Krebs solution for slice cutting and recovery contained (in mM) 120 NaCl, 2 KCl, 1.2 MgSO4, 26 NaHCO3, 1.2 KH2PO4, 2 CaCl2, and 11 glucose. This solution was equilibrated with 95% O2 - 5% CO2 (pH 7.4). The slices were maintained at room temperature before being transferred to a 1.5ml recording chamber mounted on the stage of an upright microscope. The preparations were superfused at a rate of 5-10 ml/min with a Krebs solution to which 10 uM bicuculline had been added and were maintained at 30 C with a Peltier feedback device. Bicuculline increased input resistance by 1.5 - 2.5 times and enhanced spike activation by current injection (n = 8, not shown), consistent with tonic gamma-aminobutyric acid-A (GABA_A) receptor activation by ambient GABA. More importantly for this study, bicuculline reduced spontaneous inhibitory synaptic activity. Patch-clamp recordings were taken from granule cells in the internal granular layer of rat cerebellar slices using the blind-patch approach. Most of these recordings were made by using an Axopatch 200-A amplifier for current-clamp recordings in the fast mode. Patch pipettes were pulled from borosilicate glass capillaries having 8-12 Mohm resistance before a seal was formed (seal resistance was usually >20 Gohm). The pipette solution used for whole cell recordings contained (in mM) 126 K-gluconate, 4 KCl, 4 NaCl, 1 MgSO4, 0.02 CaCl2, 0.1 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), 15 glucose, 3 ATP, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH adjusted to 7.2 with KOH). In some cases Cs+ was used instead of K+ to reduce permeation through K+ channels [it contained (in mM) 78 Cs2SO4, 4 CsCl, 4 NaCl, 1 MgSO4, 0.02 CaCl2, 0.1 BAPTA, 15 glucose, 3 ATP, and 15 HEPES; pH adjusted to 7.2 with CsOH]. These solutions reproduced the cytoplasmic Ca2+ buffer concentration (0.03-0.3 mM), and the resting Ca2+ levels measured in central neurons (usually 50-100 nM) (for the granule cells). In other experiments, the perforated-patch technique was used to prevent cytoplasmic washout and maintain the endogenous Ca2+ buffer. The pipette solution used for perforated-patch recordings contained (in mM) 80 K2SO4, 8 NaCl, 15 glucose, and 5 HEPES (pH adjusted to 7.2 with KOH) and nystatin 100 ug/ml. Solutions for both whole cell and perforated-patch recordings maintained a chloride reversal potential of -65 mV as recently measured in mature rat cerebellar granule cells. Because patch-clamp amplifiers may influence the excitable response, patch-clamp current-clamp recordings performed using Axopatch-200A amplifier in fast mode were compared with those obtained with a conventional microelectrode amplifier in bridge mode. The action potentials recorded with the patch-clamp amplifier did not significantly differ from those recorded using the microelectrode amplifier. It also should be noted that firing remained unchanged when using the perforated-patch instead of whole cell recording configuration. In the cell-attached configuration, electrode capacitance was cancelled carefully before obtaining electrical access to the cell to allow for electronic compensation of pipette charging during subsequent current-clamp recordings. In those experiments in which the patch-clamp amplifier was used, the current transients elicited by 10mV hyperpolarizing pulses from the holding potential of -70 mV in voltage-clamp mode showed a monoexponential relaxation [with time constant tau_s = 66 +/- 27 us in whole-cell recordings (mean +/- SD), n = 14; tau_s = 93 +/- 31 us in perforated-patch recordings, n = 10] and were used to estimate series resistance (Rs = 21.1 ± 8.7 Mohm in whole cell recordings, n = 14; Rs = 35 +/- 12 Mohm in perforated-patch recordings, n = 10), input resistance (Rin = 2 +/- 0.7 Gohm in whole cell recordings, n = 14; Rin = 2.2 +/- 0.8 Gohm in perforated-patch recordings, n = 10), and input capacitance (Cin = 3 +/- 0.5 pF in whole cell recordings, n = 14; Cin = 2.8 +/- 0.6 pF in perforated-patch recordings, n = 10). Depending on the high Rin:Rs ratio (Rin:Rs > 60), bridge balancing in current-clamp recordings proved to be of little effect and was not routinely used either in the whole cell or the perforated-patch recording configuration. The data were sampled with a TL-1 DMA Interface (sampling time = 50-250 us for current-clamp recordings, 10 us for voltage-clamp recordings) and analyzed with pClamp software. Membrane potential was measured relative to an agar-bridge reference electrode and was not adjusted for liquid-junction potentials (usually < 5 mV). HW denotes duration of action potentials at half-amplitude. Data are reported as means +/- SD, and statistical comparisons were done using Student's t-test. The control and test solutions were applied locally through a multibarrel pipette. The perfusion of the control solution was commenced before seal formation, and was maintained until switching to the test solutions. Stock solutions were prepared for all drugs and stored frozen at -20 C. The drugs were diluted to their final concentration in the appropriate Krebs solution before use.
data_collection	Based on Figure 2 and under Results section and sub-heading Passive membrane properties of rat cerebellar granule cells: No spontaneous extracellular action currents were observed during seal formation, nor was any spontaneous firing seen after patch disruption. Granule cell resting membrane potential measured soon after gaining access to the cell was -57.8 +/- 6.4 mV (n = 43; range from -45 to -72 mV). The granule cells showed a resting membrane potential of -62.4 +/- 11.1 mV (n = 36). Therefore, from this, n = 36 cells, and set -62.4 +/- 11.1 mV
margin_of_error	11.1
validation_info	This is the experimental data is from in-vitro 43 GranularCells from P21-31 Wistar rats.
confidence	0.95