Modulation of mammalian dendritic GABA(A) receptor function by the kinetics of Cl- and HCO3- transport

Abstract

1. During prolonged activation of dendritic GABAA receptors, the postsynaptic membrane response changes from hyperpolarization to depolarization. One explanation for the change in direction of the response is that opposing HCO3- and Cl- fluxes through the GABAA ionophore diminish the electrochemical gradient driving the hyperpolarizing Cl- flux, so that the depolarizing HCO3- flux dominates. Here we demonstrate that the necessary conditions for this mechanism are present in rat hippocampal CA1 pyramidal cell dendrites. 2. Prolonged GABAA receptor activation in low-HCO3- media decreased the driving force for dendritic but not somatic Cl- currents. Prolonged GABAA receptor activation in low-Cl- media containing physiological HCO3- concentrations did not degrade the driving force for dendritic or somatic HCO3- gradients. 3. Dendritic Cl- transport was measured in three ways: from the rate of recovery of GABAA receptor-mediated currents between paired dendritic GABA applications, from the rate of recovery between paired synaptic GABAA receptor-mediated currents, and from the predicted vs. actual increase in synaptic GABAA receptor-mediated currents at progressively more positive test potentials. These experiments yielded estimates of the maximum transport rate (vmax) for Cl- transport of 5 to 7 mmol l-1 s-1, and indicated that vmax could be exceeded by GABAA receptor-mediated Cl- influx. 4. The affinity of the Cl- transporter was calculated in experiments in which the reversal potential for Cl- (ECl) was measured from the GABAA reversal potential in low-HCO3- media during Cl- loading from the recording electrode solution. The calculated KD was 15 mM. 5. Using a standard model of membrane potential, these conditions are demonstrated to be sufficient to produce the experimentally observed, activity-dependent GABA(A) depolarizing response in pyramidal cell dendrites.

Figure 5. The change in [Cl⁻]_ivs. HCO₃⁻ due to large, synaptic GABA_A receptor-mediated currents

GABA_A PSCs were evoked in the distal dendrites (s. moleculare) of a CA1 pyramidal cell in either nominally HCO₃⁻-free (A and B) or low-Cl⁻ (C-E) intra- and extracellular media in the presence of glutamate and GABA_B receptor antagonists. The rates of anion flux following single and tetanic stimuli were manipulated by changing the test potential. A, GABA_A PSCs evoked by single vs. 10 stimuli at -50 and -30 mV. The GABA_A PSC evoked by a single stimulus at -50 mV could be scaled by a factor of 1.8 to match the PSC evoked at -30 mV. However, the PSC evoked at -50 mV by a more intense stimulus (10 stimuli at 200 Hz) and scaled by 1.8 was larger than the corresponding PSC evoked at -30 mV, indicating that the PSC evoked by a tetanic stimulus did not increase as much at positive potentials as the PSC evoked by a single stimulus. B, I-V curves for the GABA_A PSCs shown in A. The amplitude of the PSCs (averaged over 500 ms) evoked by single stimuli (•) increased uniformly with the test potential, but the corresponding amplitude of the PSCs evoked by tetanic stimuli (○) did not increase as much as expected at positive potentials, based on linear extrapolation from the corresponding PSCs evoked at more negative potentials (dashed line). The reversal potential for PSCs evoked by both single and multiple stimuli was -70 mV. The electrode solution contained caesium gluconate and 12 mM Cl⁻. Test potentials were limited to values negative to -25 mV, because at more positive membrane potentials, activation of an outwardly rectifying Cl⁻ conductance resulted in non-synaptic Cl⁻ loading of the dendrites (Smith et al. 1995). C, stability of dendritic GABA_A receptor-mediated HCO₃⁻ currents. GABA_A PSCs were evoked by electrical stimulation in s. moleculare of CA1 in the presence of ionotropic glutamate and GABA_B receptor antagonists. Intra- and extracellular Cl⁻ was decreased to 4 mM. Somatic GABA_A currents were blocked by pressure application of 100 μm picrotoxin to s. pyramidale. The rate of HCO₃⁻ flux was manipulated by changing the stimulus intensity and test potential. Single stimuli evoked small GABA_A receptor-mediated dendritic HCO₃⁻ currents. D, 20 stimuli at 200 Hz evoked much larger dendritic HCO₃⁻ currents. Biphasic response near the reversal potential reflects the residual current carried by the 4 mM Cl⁻. E, the amplitude of the HCO₃⁻ currents evoked using 20 stimuli (the current amplitude was averaged over the initial 500 ms of the response) are plotted together with linearly scaled HCO₃⁻ currents evoked using single stimuli (scale factor × 30) as a function of the test potential. The deviation from the fit line (eqn (8)) for the test potentials farthest from the reversal potential are no greater for the currents elicited using single vs. 20 stimuli, indicating that activity-dependent diminution of the HCO₃⁻ gradient does not limit inward HCO₃⁻ currents. The amplitudes of the currents evoked in this experiment are 5 times larger than the currents evoked to collapse the Cl⁻ gradient (A and B). The size of the largest HCO₃⁻ current was not significantly different (4 ± 9 % larger) from the current predicted by extrapolation from the smaller currents evoked at test potentials closer to E_HCO3. The large size of the HCO₃⁻ currents was due to the positive reversal potential of GABA_A currents in low-Cl⁻ media; under these conditions, GABA_A receptor activation is excitatory, and polysynaptic GABA_A receptor activation will occur with strong stimuli (Avoli et al. 1990).

Figure 1. Activity-dependent alterations in E_GABA are independent of the activation of interneuron networks and voltage-dependent Cl⁻ channels

A, application of 100 μm GABA to the distal apical dendrites of a CA1 pyramidal cell in media containing physiological concentrations of Cl⁻ and HCO₃⁻ results in an initial hyperpolarization, followed by a slow depolarizing response. These responses were not altered by application of 1 μm tetrodotoxin (TTX) (n = 6 cells), indicating that polysynaptic activation of interneuron networks by GABA does not contribute to the response evoked under these conditions. Block of voltage-dependent sodium channels by TTX was assayed by periodically testing for triggering of action potentials using +1 nA current injections in the pyramidal cell. GABA was applied for 120 ms, the RMP for all conditions was -66 mV and 3-8 consecutive responses were averaged for each condition. B, holding the membrane potential of a CA1 pyramidal cell at -30 mV does not result in significant Cl⁻ loading in nominally HCO₃⁻-free media. Currents were evoked by dendritic application of 100 μm GABA at a test potential of -85 mV, which was just positive to E_GABA. Three test conditions were compared: (1) holding potential kept at -85 mV, (2) membrane potential held at -30 mV until just prior to the GABA application at -85 mV, or (3) GABA also applied at the holding potential of -30 mV prior to stepping to -85 mV. GABA_A currents evoked under conditions 1 and 2 overlapped, indicating that holding potentials in this voltage range had no effect on E_GABA in the absence of a conditioning GABA application. The inward current evoked under condition 3 demonstrates that GABA_A currents, but not holding potentials of -30 mV, are sufficient to shift E_Cl.

Figure 3. Estimating the rate of recovery of [Cl⁻]_i from the amplitude of GABA_A currents elicited at varying intervals after a large conditioning GABA_A current

GABA_A currents were evoked by pressure application of 100 μm GABA to s. moleculare of CA1. The electrode Cl⁻ concentration was 10 mM, GABA_B receptor antagonists were added to the bath and nominally HCO₃⁻-free media were used. A, top trace, is a schematic of the voltage clamp protocol. An initial GABA_A current was produced by GABA application at a HP of -30 or -85 mV, and the rate of recovery of [Cl⁻]_i was monitored by evoking a subsequent GABA_A current at -85 mV. The timing of the second GABA application was varied sequentially between repetitions of the experiment, as indicated by the arrowheads. Lower traces: the leftmost currents are the initial currents evoked by GABA application; the larger current was evoked at -30 mV, the smaller at -85 mV. The currents to the right were evoked by the second GABA application, all at -85 mV. After the large initial current evoked at -30 mV, the GABA currents evoked at short intervals after the initial current are inward; outward currents are produced when GABA is applied at a delay of more than 2 s after the end of the initial large current. Currents evoked after the smaller initial current at -85 mV are always outward, although the amplitude increases as the delay between the initial and subsequent GABA application is increased. B, I-V relationship for currents evoked at low frequency (0.05 Hz) by GABA applications as in A. Currents were evoked by GABA application to s. moleculare in CA1 at the test potentials indicated in the I-V curve in C. C, I-V plot of the time-averaged amplitudes of the currents shown in B. The continuous line is the constant field equation fitted to the 4 points nearest E_Cl. The points evoked at more positive potentials were smaller than predicted from the constant field equation; the rate of Cl⁻ transport could be estimated from this difference (see Fig. 5B). D, the calculated [Cl⁻]_i at the time of the subsequent GABA applications in A (•). [Cl⁻]_i was estimated from eqn (7) from the amount by which [Cl⁻]_i must have shifted (from the value calculated from the steady-state E_Cl shown in B) to explain the amplitude and direction of the currents evoked after the large initial current in A. The continuous line represents fitted exponential decay: the time constant for the change in [Cl⁻]_i was 3.5 s, and the maximum rate of recovery of [Cl⁻]_i was 8.1 mmol l⁻¹ s⁻¹. ○, calculated values of [Cl⁻]_i at various times after the initial current after correcting for receptor desensitization (estimated from the fractional recovery of the amplitude of the currents evoked after the smaller initial current evoked at -85 mV). Dashed line, corresponding exponential fit: the time constant was 0.8 s, and the initial rate of recovery of [Cl⁻]_i was 22 mmol l⁻¹ s⁻¹.

Figure 4. Estimating the rate of recovery of dendritic [Cl⁻]_i after currents evoked near RMP in physiological media

Media were buffered with HCO₃⁻/CO₂ and the electrode Cl⁻ concentration was 12 mM. A, currents were evoked by dendritic GABA application in a CA1 pyramidal cell voltage clamped at -55 mV. The time interval between dendritic GABA applications was varied between 2 and 20 s as indicated. The brief initial current was outward at 20 s intervals, but inward at 2 s intervals. B, currents were evoked in the same cell at the indicated test potentials using 20 s intervals between GABA applications. C, I-V relationship for the initial currents shown in B. Initial current was the time average of the first 100 ms after GABA application. D, estimate of [Cl⁻]_i at the time of GABA application for the currents shown in A.[Cl⁻]_i was determined from the shift in E_GABA necessary to explain the direction and amplitude of the initial currents, using a Cl⁻:HCO₃⁻ permeability ratio of 0.25. Initial current amplitude was the time average of the current during the first 100 ms after GABA application. Continuous line: after the evoked current ended (1 s after GABA application), [Cl⁻]_i decreased monoexponentially with a time constant of 2.4 s from a value of 17 mM, which corresponded to E_Cl= -55 mV.

Figure 7. Kinetics of pyramidal cell cation-Cl⁻ exchange in nominally HCO₃⁻-free media

A, postsynaptic currents elicited at test potentials ranging from -95 mV (lower trace) to -45 mV (upper trace) by stimulation in the proximal s. radiatum of CA1. GABA_B and ionotropic glutamate receptors were blocked. The electrode solution contained 25 mM Cl⁻. Stimulus artifacts are blanked. B, fit of the time-averaged current amplitudes to the Goldman-Hodgkin-Katz constant field current equation. C, plot of [Cl⁻]_ivs. the electrode Cl⁻ concentration. [Cl⁻]_i was calculated from the the I-V curves (eqn (1)). Each point represents 4-7 cells; error bars are ±s.e.m. The dashed line represents complete dialysis of the cytoplasm by the electrode Cl⁻. D, the relationship between [Cl⁻]_i and electrode Cl⁻ concentration ([Cl⁻]_E) is plotted using the Michaelis-Menten kinetic model of enzyme activity. The rate of transport is represented as the difference between [Cl⁻]_i and [Cl⁻]_E (see Methods) The maximum rate of transport, calculated as 1/(fitted y-intercept), was 38 mM. K_D, the [Cl⁻]_i at which transport was half-maximal, was calculated from 1/(fitted x-intercept) as 15 mM. E, the difference between electrode and cytoplasmic Cl⁻ achieved by KCl transport is plotted against the energy available for KCl cotransport. The free energy available for transport was calculated as the driving force for K⁺ efflux (the difference between the membrane potential and E_K) minus the driving force for Cl⁻ influx (the difference between the membrane potential and E_Cl.); this simplifies to E_K - E_Cl. The data fitted equally well using either the model in D or E.

Figure 8. Model of the effect of activity-dependent Cl⁻ accumulation on the GABA_A postsynaptic potential

A, schematic of the ionic currents and pumps underlying the GABA_A membrane potential response. GABA_A receptor-mediated Cl⁻ and HCO₃⁻ currents dissipate the resting ionic gradients, which are replenished by ion transport mechanisms. The hydration and dehydration of CO₂ is catalysed by carbonic anhydrase, and can be inhibited by acetazolamide. EC, extracellular; IC, intracellular. B, kinetic model to describe the effects of the ion movements shown in A. The model is based on a linear summation of ionic potentials (Finkelstein & Mauro, 1977) as described in Methods. It assumes that the HCO₃⁻ gradient is stable (Figs 2E and F and 5C-E), so that HCO₃⁻ replenishment mechanisms are not included. C, time course of the total neuronal conductance (a constant resting conductance + GABA_A conductance) used in the calculations in this figure. D, voltage response to the GABA_A conductance shown in C in 3 structures with different radii (as labelled; length = 100 μm for the 0.5 and 1 μm radii; length lowered to 10 μm for the 10 μm radius to approximate the soma). Each structure had the same initial potential and chemical gradients. The depolarizing component of the GABA_A response increases sharply with decreasing radii. E, the corresponding change in [Cl⁻]_i during the GABA_A conductance. [Cl⁻]_i initially increases when Cl⁻ influx exceeds the v_max of the transport systems. As the GABA_A conductance decreases, Cl⁻ influx no longer exceeds the transport rate, and [Cl⁻]_i begins to return to the steady-state value. F, plot of the rate at which the Cl⁻ gradient collapses vs. the rate of Cl⁻ influx and the local volume. The rate of change of the intracellular Cl⁻ concentration, [Cl⁻]_i, is plotted on the z-axis vs. two variables, the radius of the neuronal process and the amount by which the Cl⁻ influx exceeds the maximum rate of Cl⁻ transport, v_max. Because the change in concentration is dependent on influx/volume and volume is proportional to the square of the radius, the rate of increase of [Cl⁻]_i is linearly related to the rate of influx of Cl⁻, and to the square of the radius of the dendrite.

Figure 2. Stability of neuronal anion gradients

Anion gradients were challenged with a high-amplitude GABA_A receptor-mediated current induced by local GABA application, and the effect on the GABA_A reversal potential was assessed by a second GABA applicaton at various test potentials. By changing the holding potential (HP) at which the first current was evoked, the size of the anionic flux could be made large or small without changing the population of activated GABA_A receptors. All experiments were performed in 1 μm CGP 55845A to block GABA_B currents; A-D were performed in low-HCO₃⁻ media, and E-F were performed in low-Cl⁻ media. A, stability of the dendritic Cl⁻ gradient was tested in nominally HCO₃⁻-free media saturated with 100 % O₂. Top traces are schematics of the voltage clamp protocols; thick lines indicate the voltage steps for the currents shown. Bottom, currents evoked by the first and second GABA applications to the distal apical dendrites of a CA1 pyramidal cell (arrowheads). For each test potential, a step was performed with and without a second GABA application in order to obtain an accurate baseline for current subtraction. The current evoked at -30 mV by the second GABA application is larger when the first GABA application evoked a small current (at a HP of -70 mV, right) than when the first GABA application evoked a large current (at a HP of -30 mV, left). B, for the experiment shown in A, subtracted currents evoked by the second GABA application are shown for the test potentials listed on the right. Some currents are biphasic; this may result from large Cl⁻ currents flowing into dendrites of different diameters, producing different rates of change of [Cl⁻]_i and the Cl⁻ driving force (cf. Fig. 8D and E). C, change in E_Cl, measured as the GABA_A reversal potential, in the dendrites as a result of the first GABA_A current. Data are from the experiment shown in A and B. D, stability of the somatic Cl⁻ gradient. Using the same protocol as in A, 800 pA currents evoked by the first GABA application to the soma of a CA1 pyramidal cell produced no change in E_Cl. E, stability of the dendritic HCO₃⁻ gradient. Extracellular Cl⁻ was reduced to 4 mM, the electrode solution contained 1 mM Cl⁻, and physiological HCO₃⁻/CO₂ concentrations were used. The same protocol as in A was employed, with the exception that the first GABA_A currents were evoked at HPs of -20 and -70 mV. GABA was applied to the distal apical dendrites of a CA1 pyramidal cell. The size of the second GABA_A receptor-mediated current, evoked at a test potential of -50 mV, was not affected by the amplitude of the first current. The current carried by the residual Cl⁻ was outward at the -20 mV test potential and decayed as expected based on activity-dependent gradient shifts. F, plot of the average amplitude of the current evoked by the second GABA application vs. the test potential. E_HCO3 was not changed by the largest dendritic GABA_A receptor-mediated currents that could be evoked.

Figure 6. Estimating the rate of recovery of [Cl⁻]_i from the amplitude of GABA_A PSCs elicited at varying intervals after a large conditioning GABA_A PSC

Same protocol as Fig. 3, except that the negative test potential was -80 mV and GABA_A currents were evoked by electrical stimuli in s. moleculare of the CA1 subfield. The electrode Cl⁻ concentration was 10 mM, ionotropic glutamate and GABA_B receptor antagonists and 50 μm pentobarbitone were added to the bath, and nominally HCO₃⁻-free medium was used. A, top trace, schematic of the voltage clamp protocol. An initial GABA_A current was produced by an 80 V, 80 μs electrical stimulation at a HP of -30 mV, and the rate of recovery of [Cl⁻]_i was monitored by evoking a subsequent GABA_A current at -80 mV. The timing of the second GABA current was varied sequentially between repetitions of the experiment as indicated by the arrowheads. The leftmost currents are the initial currents evoked at -30 mV. The currents to the right were evoked by the second stimulation at -80 mV. Stimulus artifacts are blanked. After the large initial current evoked at -30 mV, the GABA currents evoked at short intervals are small and become progressively larger at longer stimulus intervals (note different scale bars for currents at -30 and -80 mV). B, when the amplitude of the initial GABA_A receptor-mediated current was decreased by changing the test potential to -80 mV, the amplitude of the subsequent currents does not vary with the stimulus interval. Currents in A and B were evoked in the same cell by alternating the intial test potential for each stimulus interval. Interval between trials was 30 s. C, I-V relationship for synaptic currents evoked as in A and B at 0.033 Hz. Inset: currents evoked by electrical stimulation in s. moleculare at the test potentials indicated in the I-V curve. The continuous line is the constant field eqn (7) fitted to all points. D, the calculated [Cl⁻]_i at the time of the subsequent GABA applications in A.[Cl⁻]_i was estimated from eqn (7) from the amount by which [Cl⁻]_i must have shifted (from the value calculated from the steady-state E_Cl derived in C) to explain the amplitude and direction of the currents evoked after the large initial current in A. The line represents fitted exponential decay: the time constant for the change in [Cl⁻]_i is 1.1 s, and the maximum rate of recovery of [Cl⁻]_i is 3 mmol l⁻¹ s⁻¹. Despite the large initial current, [Cl⁻]_i did not increase as much as in Fig. 3, suggesting that the GABA_A currents evoked by electrical stimulation were distributed more widely in the dendrites than the currents evoked by GABA application.

Figure 9. Effect of altering E_K on the direction and kinetics of GABA_A receptor-mediated synaptic responses

The left panel demonstrates the maximal GABA_A receptor-mediated IPSP that could be elicited by a single stimulus in s. moleculare under control conditions. When the same stimulus is delivered after increasing [K⁺]_o from 2.5 to 8.5 mM, the amplitude of the hyperpolarizing component is decreased and a late depolarizing response is seen (n = 4). The right panel shows the output of the computer model. The waveform of the control response was matched to the control IPSP by optimizing the dendritic volume, the Cl⁻ transport parameters, and the time course of the GABA conductance. The same parameters were then used to produce the GABA response in 8.5 mM ${\text{K}}_0^{+}$; the RMP was set to the experimentally observed values, the v_max of the transport rate was decreased (Fig. 7E;Thompson et al. 1989b) from 5 to 1 mmol l⁻¹ s⁻¹, and the amplitude of the GABA conductance was increased by 20 % to reflect increased excitability of the interneuron in elevated [K⁺]_o (McBain 1994).