Non-Spontaneous Firing

DAngelo et al. 1995

sample_size	43
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 0 +/- 0 Hz
mean	0.0
units	Hz
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
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.
standard_deviation	0.0
validation_info	This is the experimental data demonstrating no spontaneous firing of in-vitro 43 GranularCells from P21-31 Wistar rats.

Brickley, Cull-Candy and Farrant 1996

reference	Brickley SG, Cull-Candy SG, Farrant M (1996) Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors. J Physiol (Lond) 497:753-759. PMID 9003560
data_collection	There is no direct description in the paper. But this paper is cited in D'Angelo et al 2001 10.1523/JNEUROSCI.21-03-00759.2001 and this data collection is based off Fig3A in Brikley et al's paper. Focussing on the voltage trace of current clamp on control rats (not when blockers were used, lower trace is with Bisculline) you do not see any spiking prior to the first current injection of 6pA (increased in steps). From this figure and the paragraph preceeding the Discussion section we get, n = 4 cells, P21 rats and set 0 +/- 0 Hz
units	Hz
validation_info	This is the experimental data demonstrating no spontaneous firing of in-vitro 4 GranularCells from P14 Sprague-Dawley rats.
mean	0.0
protocol	Whole-cell recording: Sprague-Dawley rats were killed by decapitation and parasaggital slices (150-200 um) cut from the cerebellum. Patch-pipettes were pulled from thick-walled glass coated with Sylgard and had 5-10 Mohm resistance. The pipette solution contained (mM): CsCl, 140; NaCl, 4; CaCl2, 0.5; Hepes, 10; EGTA, 5; Mg-ATP, 2 (adjusted to pH 7.3 with CsOH). The bathing solution contained (mM): NaCl, 125; KCl, 2.5; CaCl2, 2; MgCl2, 1; NaHCO3, 26; NaH2PO4, 1.25; glucose, 25 (pH 7.4 with 95% O2 and 5% CO2). No correction was made for the estimated liquid junction potential of 3.9 mV (calculated from the Generalised Henderson equation using Axoscope 1.1; Axon Instruments). Current recordings were obtained at 22-25 C. Input resistance, series resistance and cell capacitance were determined from the current response to a 10 mV hyperpolarizing voltage step; currents were filtered at 50 kHz (-3 dB, 4-pole Bessel filter) and digitized at 200 kHz. Series resistance (25-40 Mohm) and capacitance (2.6-3.7pF) were similar at all ages. Input resistance decreased significantly with age, from 11.2 +/- 1.9 Gohm at P7 (n = 28) to 4.6 +/- 1.4 Gohm at P21 (n = 10). All other data were stored on digital audiotape (DC to 20 kHz). For analysis, currents were filtered at 2 kHz (-3 dB, 8-pole Bessel filter) and digitized at 10 kHz. Synaptic currents were identified visually. The holding current and current variance for each record was calculated from a 200 ms epoch containing no synaptic events. Charge transfer resulting from tonic GABA_A receptor activation was calculated from the measured conductance in the absence and presence of bicuculline. Charge transfer via PSCs (post synaptic currents) was calculated from the integral of their averaged waveforms and their mean frequency. Perforated-patch recording with gramicidin: To avoid altering the normal intracellular Cl- concentration during current-clamp recordings these were made using the gramicidinperforated-patch technique. The pipette solution contained (mM): KCl, 145; Hepes, 10; EGTA, 5; CaCl2, 0.1 (pH 7-3 with KOH). Pipettes were tip-filled with this solution then back-filled with one containing 6-9 ug/ml gramicidin. Series resistance was continuously monitored in voltageclamp mode and the switch to current clamp (I_fast setting, Axopatch 200A) was made once this was < 100 Mohm (20-60 min after forming seal). No correction was made for the estimated liquid junction potential of 3.3 mV. Except where stated, all current-clamp recordings were obtained in the absence of glutamate and glycine receptor antagonists. All values are expressed as the mean + S.E.M. Statistical comparisons were made using Student's t test and nonparametric randomization or sign tests; differences were considered significant at P < 0 05.
standard_deviation	0.0
sample_size	4

DAngelo et al. 1997

protocol	Cerebellar slices (250 uM thick) were obtained from 4 to 21 day-old rats (Wistar strain, day of birth = P1). The rats were anesthetized with halothane before being killed by decapitation. Krebs solution for slice cutting and recovery contained (in mM) 120 NaCl, 2 KCl, 1.2 MgSO4, 26 NaHCO3, 1.2 KH2PO4, 2CaCl2, and 11 glucose. This solution was equilibrated with 95% O2 - 5% CO2 (pH 7.4). Slices were maintained at room temperature before being transferred to the recording chamber (1.5 ml) 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 glycine and 10 uM bicuculline were added, and maintained at 30 C with a feed-back Peltier device. Granule cells in lobules IV-IX were recorded in the whole cell patch-clamp configuration using the blind-patch approach. Recordings were performed with fast current-clamp mode amplifier. The data were sampled with sampling time = 250 us for current-clamp recordings, 10 us for voltage-clamp recordings and analyzed with pClamp software. Mossy fiber stimulation was performed with a bipolar tungsten electrode via a stimulus isolation unit. The stimulating electrode was placed over the mossy fiber bundle, and stimuli were applied at the frequency of 0.1 Hz or in 500-ms trains of 5, 10, or 50 Hz. In some experiments, a second stimulating electrode was placed in proximity of the Purkinje cell layer to test granule cell antidromic excitation. Patch pipettes were pulled from borosilicate glass capillaries 8-12 Mohm resistance before a seal was formed with a filling solution containing (in mM) 126 K-gluconate, 4 NaCl, 1 MgSO4, 0.02 CaCl2, 0.1 bis-(o-aminophenoxy)-N,N,N',N'tetraacetic acid, 15 glucose, 3 ATP, 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH adjusted to 7.2 with KOH). This solution buffered intracellular Ca2+ at 100 nM, similar to the resting Ca2+ concentration measured in granule cells. After a giga-seal was formed (seal resistance was usually >20 Gohm), the electrode capacitance was canceled carefully before rupturing the patch to allow for the electronic compensation of pipette charging during subsequent current-clamp recordings. Once in the whole cell configuration, the current transients elicited by 10-mV hyperpolarizing pulses from the holding potential of -70 mV in voltage-clamp mode showed a mono-exponential relaxation (time constant = 81 +/- 27 us, n = 40), and were used to estimate series resistance (21.1 +/- 8.7 Mohm, n = 40) and input resistance and capacitance. Depending on the high-input-to-series resistance ratio, bridge balancing in current-clamp recordings proved of little effect and was not routinely used. Membrane potential was measured relative to an agar-bridge reference electrode. Reported membrane potential values have been adjusted off-line for liquid-junction potentials (usually <= 5 mV). In the current-clamp mode, the granule cell input resistance (Rm) was monitored repeatedly by measuring the steady state membrane potential change generated by hyperpolarizing current pulses from -70 to -80 mV. Experiments in which Rm changed by more than +/- 10% during recordings were rejected. Experimental tracings were analyzed using pClamp software. HW denotes duration of excitatory postsynaptic potentials (EPSPs) or spikes at half-amplitude. Data are reported as means +/- SD, and statistical comparisons were done using Student's t-test (NS, not significant).
sample_size	40
units	Hz
reference	D'Angelo E, De Filippi G, Rossi P, Taglietti V (1997) Synaptic activation of Ca2+ action potentials in immature rat cerebellar granule cells in situ. J Neurophysiol 78:1631-1642. doi: 10.1152/jn.1998.80.2.493
standard_deviation	0.0
data_collection	There is no direct description in the paper. But this paper is cited in D'Angelo et al 2001 10.1523/JNEUROSCI.21-03-00759.2001 Based on Fig1 of D'Angelo et al 1997 (10.1152/jn.1997.78.3.1631) and the first few sentences under results with says: Patch-clamp recordings were performed on 183 neurons in the internal granular layer of rat cerebellar slices from P4 to P21. The neurons did not display spontaneous firing either during seal formation or after having established the whole cell recording configuration and had the low membrane capacitance and high-input resistance typical of granule cells. Thus based on this we get, n = 40 cells, P4-21 rats and set 0 +/- 0 Hz
mean	0.0
validation_info	This is the experimental data demonstrating no spontaneous firing of in-vitro 40 GranularCells from P4-21 Wistar rats.