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Results: 11

1.
Figure 7

Figure 7. From: Post-depolarization potentiation of GABAA receptors: A novel mechanism regulating tonic conductance in hippocampal neurons.

PDP of tonic currents due to endogenous GABA. A: Tonic currents due to endogenous GABA before and after depolarization to +40 mV. Period of depolarization is clipped to display the PDP of tonic current. Upward deflections (e.g. the one indicated by arrow) are the response to 10 µM bicuculline (Bic). B: Time course of normalized tonic current from panel A. Solid line is a single exponential fit with indicated time constant. C: Summary of peak PDP for tonic current due to endogenous GABA. Depolarization in the presence of 100 µM SR95531 (SR) attenuated PDP of endogenous tonic currents. PDP of endogenous tonic currents was increased by a 5 s application of exogenous GABA (1 µM) during the period of depolarization only (GABA). ** - p<0.01, * - p<0.05.

Christopher B. Ransom, et al. J Neurosci. ;30(22):7672-7684.
2.
Figure 4

Figure 4. From: Post-depolarization potentiation of GABAA receptors: A novel mechanism regulating tonic conductance in hippocampal neurons.

Post-depolarization potentiation (PDP) of GABA currents. A: Current recordings during repeated GABA applications (1 µM, 5 s) at indicated times (holding potential −60 mV). At t=0 GABA was applied at +40 mV (note the slow activation of the GABA response similar to that seen in Fig. 3). Both peak and “steady-state” current at −60 mV were increased when measured 30 s after GABAA receptor activation at +40 mV. PDP of “steady-state” currents was 46% in this neuron. Baselines are adjusted to allow comparison of currents. B: Time course of normalized “steady-state” current amplitudes for data in panel A. Solid line represents a single exponential function fit to the data. Time constant for recovery of PDP was 25 s. C: Mean data from experiments on 5 cells as in panel A. The time constant for recovery was also 25 s.

Christopher B. Ransom, et al. J Neurosci. ;30(22):7672-7684.
3.
Figure 10

Figure 10. From: Post-depolarization potentiation of GABAA receptors: A novel mechanism regulating tonic conductance in hippocampal neurons.

PDP was independent of intracellular Cl accumulation. A: GABA currents measured every 30 s before and after depolarization to +40 mV. At t=0 GABA was co-applied to the depolarized neuron in a low extracellular [Cl] bath solution (Na-methanesulfonate substitution for NaCl, [Cl]o=3 mM). The net charge movement induced by GABA in low [Cl] solution was −2.37 nC, indicating a net outward Cl flux. PDP was still seen despite this reduction in intracellular Cl concentration. No baseline adjustment was made to the displayed records. B: Mean normalized current from 7 experiments as in A (open circles). Filled circle represents the mean PDP produced by transient depolarization alone without GABA application. Solid line is a single exponential function.

Christopher B. Ransom, et al. J Neurosci. ;30(22):7672-7684.
4.
Figure 11

Figure 11. From: Post-depolarization potentiation of GABAA receptors: A novel mechanism regulating tonic conductance in hippocampal neurons.

GABA current was potentiated by an epileptiform burst. A: Membrane potential of a hippocampal neuron in the presence of 4-aminopyridine (100 µM). Bursts of high frequency action potentials occur spontaneously. Data enclosed in the dashed box was used to create the stimulus waveform used in voltage-clamp experiments. B: Responses to GABA (1 or 10 µM) at a holding potential of −60 mV with and without epileptiform depolarization. Current amplitude was measured 0.2 and 2 s after the stimulus (see dashed lines in right panel) and compared to control currents at the same time points. C: Mean potentiation of GABA current by epileptiform depolarization at 0.2 s and 2 s after repolarization (n=4). D: Tonic current before and after epileptiform depolarization. Illustrated currents are the average of 3 trials from this cell recorded in presence of 0.3 µM exogenous GABA. E: Normalized tonic current amplitude from experiment in panel D. F: Mean potentiation of tonic current by epileptiform depolarization (n=7).

Christopher B. Ransom, et al. J Neurosci. ;30(22):7672-7684.
5.
Figure 3

Figure 3. From: Post-depolarization potentiation of GABAA receptors: A novel mechanism regulating tonic conductance in hippocampal neurons.

Currents evoked with exogenous GABA were outwardly-rectifying. A: Current traces during GABA application (1 µM) at different holding potentials (as indicated). Baselines are adjusted to allow comparison of currents. Note the acceleration of desensitization at hyperpolarized potentials and the slow, continuous activation of currents at +40 mV. B: I–V plot of current from neuron in panel A. Currents were measured early during GABA application (near “peak” current at −100 mV; closed circle) and prior to offset of GABA (“steady-state”; open circle). Outward rectification is most pronounced for currents measured near steady-state. C: Mean current density (pA/pF) as a function of voltage from 5 cells studied as in panel A. D: Rectification ratio for endogenous tonic currents and currents evoked with exogenous GABA. Rectification ratio with exogenous GABA is for steady-state currents. No significant difference in rectification ratio was seen for currents evoked with exogenous GABA and endogenous tonic currents under control conditions (p=0.38). Rectification ratios of tonic currents with SKF or zero Ca2+ were not significantly different than control conditions (p=0.11 and p=0.90, respectively). Numbers in parentheses indicate number of cells in this and subsequent figures.

Christopher B. Ransom, et al. J Neurosci. ;30(22):7672-7684.
6.
Figure 2

Figure 2. From: Post-depolarization potentiation of GABAA receptors: A novel mechanism regulating tonic conductance in hippocampal neurons.

Voltage-dependent increases in tonic conductance were insensitive to inhibition of transporter-mediated or vesicular GABA release. A: Current responses to bicuculline application (Bic, 10 µM) under control conditions (left panel) and in the presence of SKF89976A (SKF, 40 µM) (right panel). Tonic current was increased by SKF. Holding potential was −40 mV. B: Mean tonic current density (pA/pF) vs. voltage under control conditions and in presence of SKF (n=6). Outward rectification was unaffected by SKF. C: Mean capacitance-specific conductance vs. voltage (n=6). There was a shift in the conductance curve, but the voltage-dependence of tonic conductance was unaffected by SKF, the slopes of the solid lines are 0.31 pS/pF*mV−1 and 0.33 pS/pF*mV−1 for control and SKF, respectively. Symbols are same as panel B. D: Current responses to Bic under control conditions (left panel) and with zero extracellular Ca2+/1 mM EGTA (right panel). As seen previously (Wu et al., 2006), phasic currents were reduced by zero extracellular Ca2+ but tonic currents were not (amplitudes of illustrated tonic currents were −31 pA for control and −38 pA for zero Ca2+). Holding potential was −60 mV. E: Tonic current density vs. voltage for experiments with zero Ca2+/1 mM EGTA (n=6). F: Capacitance-specific conductance vs. voltage for all neurons tested in zero Ca2+/1 mM EGTA. Same symbols as E. The voltage-dependent increase in tonic conductance was independent of extracellular Ca2+. Slopes of the solid lines are 0.30 pS/pF*mV−1 and 0.23 pS/pF*mV−1 for control and zero Ca2+, respectively.

Christopher B. Ransom, et al. J Neurosci. ;30(22):7672-7684.
7.
Figure 1

Figure 1. From: Post-depolarization potentiation of GABAA receptors: A novel mechanism regulating tonic conductance in hippocampal neurons.

Tonic GABAA conductance in rat hippocampal neurons increased with depolarization. A: Tonic GABAA receptor-mediated currents at −40 mV and +40 mV. Horizontal bars indicate period of bicuculline application (10 µM, Bic) in this and subsequent panels. B: Quantification of tonic current amplitude. All-points histograms were created from current data before and after Bic application (gray bars in right hand panel). The peaks of these histograms were fit with a Gaussian equation (solid lines in right hand panel) to determine mean current amplitudes (dashed lines). Tonic current was defined as change in mean holding current produced by bicuculline. C: Current-voltage (I–V) plot from the neuron in (A). Values are means of 2–3 measurements at each potential. Tonic currents were outwardly-rectifying. D: Tonic chord conductance as a function of voltage from data illustrated in panels A–C. Tonic conductance increased in a near-linear fashion with membrane depolarization. Solid line represents a linear fit of the data (slope = 13 pS/mV). E: Mean current density as a function of voltage for all neurons tested (n=12). F: Mean capacitance-specific conductance as a function of voltage for all neurons tested (n=12). Linear fit of the data had a slope of 0.32 pS*pF−1*mV−1.

Christopher B. Ransom, et al. J Neurosci. ;30(22):7672-7684.
8.
Figure 5

Figure 5. From: Post-depolarization potentiation of GABAA receptors: A novel mechanism regulating tonic conductance in hippocampal neurons.

PDP of GABA current was time- and voltage-dependent. A: Current responses to GABA (1 µM) with step depolarizations of increasing duration. Duration of initial depolarization was 1 s and increased by 0.5 s with each subsequent trial (not all traces shown, illustrated traces are for 0, 1.5, 2.5, 3.5, and 4.5 s of depolarization). A progressive increase in current amplitude was seen upon repolarization as the duration of the preceding depolarization increased. Lower panel shows command potentials. Note that the current after 2.5 s of depolarization was larger than the peak current produced by GABA alone. B: Mean potentiation of current as a function of depolarization duration. Current was measured 1.5 s after repolarization and normalized to current values at corresponding time point of control trace (−60 mV) from experiments as in panel A (n=4). C: GABA-evoked currents (3 µM) with step depolarization to a range of potentials from −30 to +30 mV. Current at −60 mV was potentiated by transient depolarization to −30 mV (compared to control current recorded at −60 mV) and increased progressively as the magnitude of the preceding depolarization increased. D: Data from panel C on expanded time scale illustrating the current increase seen upon repolarization. Labels refer to value of preceding depolarization (−60 mV is control). E: Mean normalized current at −60 mV as a function of prepulse potential. Current was measured 1.5 s after repolarization and normalized to corresponding time point of control trace (n=4). Data from experiments as in panel C. Solid line represents a Boltzmann equation fit to the data; the apparent half-maximal voltage (V0.5) for PDP of GABA current was +10 mV.

Christopher B. Ransom, et al. J Neurosci. ;30(22):7672-7684.
9.
Figure 8

Figure 8. From: Post-depolarization potentiation of GABAA receptors: A novel mechanism regulating tonic conductance in hippocampal neurons.

PDP of GABA current was due to increased conductance. A: Membrane currents in response to voltage ramps before (control) and after GABA application (1 µM). The GABA-evoked ramp current (“difference current”) was obtained from point-by-point subtraction of these data for determination of reversal potential (i.e. EGABA) and slope conductance. B: Schematic of experimental protocol. Difference currents were obtained with voltage ramps 30 s before (t =−30 s) GABA was applied to a neuron depolarized to +40 mV (t=0) and then 30 s after this depolarization (t=30 s). C: GABA-evoked ramp currents during PDP. Difference currents are plotted as a function of voltage in the left- (t=−30 s) and right-hand (t=30 s) panels. Middle panel shows GABA response at +40 mV (t=0). Slope conductance was determined from a linear fit to current between −60 and −10 mV (solid lines in left-hand and right-hand panels). Chord conductance was calculated using the measured EGABA (indicated in figure) and current at −60 mV. Voltage ramps were given ~2 s into GABA applications. Slope and chord conductance were both increased by depolarization (values in figure). D: GABA-evoked (3 µM) ramp currents from a neuron recorded with pipette solution containing 15 mM Cl. Same protocol as B-C. EGABA shifted from −57 mV at baseline (t=−30 s) to −32 mV after depolarization (t=30 s). Slope conductances are indicated in figure. E: Mean values of slope and chord conductance for experiments with [Cl]in = 135 mM. The slope and chord conductances were increased by 60% and 56%, respectively (n=5, p<0.05). Legend applies to this panel and panel F. Data with [Cl]in = 135 mM were pooled from experiments with 1–3 µM GABA. F: Mean values of slope and chord conductance for experiments with [Cl]in = 15 mM. During PDP with [Cl]in = 15 mM, conductance was increased by 91–132% (n=5, p<0.05). Data with [Cl]in = 15 mM were obtained with 3 µM GABA.

Christopher B. Ransom, et al. J Neurosci. ;30(22):7672-7684.
10.
Figure 6

Figure 6. From: Post-depolarization potentiation of GABAA receptors: A novel mechanism regulating tonic conductance in hippocampal neurons.

PDP of tonic currents. A: Tonic currents in the presence of low levels of exogenous GABA (0.3 µM). Upward deflections (e.g. the one indicated by solid arrow) represent response to 10 µM bicuculline (Bic) used to measure tonic current amplitude. Tonic current at −60 mV was increased by transient depolarization to +40 mV (large upward deflections). This increase was attenuated when depolarization occurred in the presence of the GABAA receptor antagonists SR95531 (SR, 100 µM) or Bic (10 µM). B: Tonic current amplitude from experiment in panel A. Solid lines represent single exponential fits, time constants (τ) for recovery after each depolarization are indicated. Lower case letters refer to sequentially labeled depolarizations in panel A. Note the progressive rundown of tonic current over duration of experiment (dashed line), which was seen in 11/11 cells studied with 0.3 µM GABA. C: PDP of tonic currents under control conditions (left panel) and when depolarization occurred in the presence of SR (right panel) on expanded time scale (different neuron than in A–B). Wash off of SR occurred in <10 s and was complete prior to measuring tonic current with Bic. D: Normalized tonic current from data in C. Data was normalized to value immediately preceding each depolarization and aligned so that repolarization occurred at t=0. Gray bar indicates period of depolarization to +40 mV. PDP in the presence of SR is reduced compared to control for 1 min after repolarization, much longer than the time required for wash of SR. Solid lines are single exponential fits to data with indicated time constants. E: Potentiation of first three measurements of tonic current after repolarization for experiments with and without SR. Peak PDP with SR was significantly reduced compared to both the first and second measurements under control conditions (p<0.01, n=5). F: Potentiation of tonic current vs. time after repolarization for experiments with and without Bic. PDP induced in Bic was significantly reduced compared to each corresponding control measurement (p<0.01, n=6).

Christopher B. Ransom, et al. J Neurosci. ;30(22):7672-7684.
11.
Figure 9

Figure 9. From: Post-depolarization potentiation of GABAA receptors: A novel mechanism regulating tonic conductance in hippocampal neurons.

Time course for PDP of tonic conductance. A: Bicuculline-sensitive ramp currents. Ramp currents were recorded every 10 s, first at a holding potential of −60 mV, during depolarization to +40 mV, and then after repolarization to −60 mV. Voltage ramps were from −30 to +30 mV over 0.5 s. Illustrated currents are difference currents obtained by subtracting ramp currents recorded in the presence of bicuculline from control currents. Bicuculline (10 µM) was applied every 30–50 s, the mean holding current at −60 mV in bicuculline was subtracted from control holding current prior to each voltage ramp to yield tonic current amplitude. Times at which currents were measured are indicated. Reversal potentials (i.e. EGABA) shifted towards more positive values during depolarization and recovered slowly after repolarization. Currents were recorded in the presence of 0.3 µM exogenous GABA. B: Double y-axis plot of tonic conductance (open circles) and EGABA (solid line) showing time course of changes produced by depolarization to +40 mV (horizontal bar). Chord conductance was calculated with EGABA and tonic current measured as in panel A. Conductance continued to increase during depolarization to +40 mV even after shifts in EGABA approached a plateau. Conductance dropped to 8.7 nS immediately upon repolarization to −60 mV, prior to substantial recovery of EGABA, but remained above baseline values. Solid line represents a single exponential fit to the recovery portion of conductance data. C: Time course for tonic current (open circles) from experiment in panels A–B. Theoretical changes in tonic current that would occur solely due to changes in driving force produced by shifts in EGABA, assuming no change in conductance from baseline, are plotted as a solid line for comparison to experimental data. PDP of tonic current in this situation would be only 27%, which is small compared to the 257% potentiation observed experimentally. Conversely, the theoretical current amplitude using the conductance values from panel B but assuming EGABA remained constant at −7 mV throughout the experiment is illustrated as triangles. In this case PDP would be 178% at its peak. Based on these calculations, changes in electromotive driving force from anion redistribution contributed no more than 10–30% to PDP of tonic current.

Christopher B. Ransom, et al. J Neurosci. ;30(22):7672-7684.

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