Stability of the Cl− and HCO3− gradients
To assess the stability of the dendritic Cl− and HCO3− gradients, we applied GABA (100 μm) directly to the distal dendrites of voltage-clamped CA1 pyramidal cells in media in which only Cl− or HCO3− was present in physiological concentration; the concentration of the other anion was minimized as described in Methods. GABAB conductances were blocked with 1 μm CGP 55845A. Paired GABA applications were utilized. The first GABA application was designed to collapse the anion gradient by evoking a large current at a test potential far from the reversal potential. To assess the impact of this current on the anion gradient, the membrane was then stepped to various test potentials and GABA was applied 2.5 s after the first application. The experiment was repeated with the first GABA application performed at a holding potential very close to the steady-state anionic reversal potential in order to minimize the initial anion flux. By comparing the anionic reversal potentials after large and small anion fluxes, effects due to the collapse of the anion gradient could be separated from other effects such as receptor desensitization (Huguenard & Alger, 1986; Thompson & Gähwiler, 1989a). Cl− gradients were assessed in nominally HCO3−-free ACSF containing 136.5 mM Cl− using a 10 mM Cl− electrode solution. For eight dendritic (s. moleculare) and four somatic (s. pyramidale) locations of GABA application in six CA1 pyramidal cells, the difference in the first dendritic Cl− current evoked at -30 vs. -70 mV was 646 ± 117 pA (Fig. 2A and B). The average shift in dendritic ECl due to the large Cl− current evoked at -30 mV was 12.6 ± 4 mV (Fig. 2C). When GABA was applied to the soma, the average difference in the first GABAA receptor-mediated Cl− currents elicited at the different holding potentials was larger (1180 ± 525 pA) than at the dendrites because the maximum response at the soma was larger. The corresponding shift in ECl at the soma was 1.5 ± 1 mV (Fig. 2D). HCO3− gradients were assessed using ACSF and electrode solutions containing physiological HCO3−/CO2 and 4 mM extracellular Cl−, and 1 mM electrode Cl−. For seven dendritic and two somatic locations in four cells, the average difference in the first dendritic HCO3− current evoked at -20 vs. -70 mV was 238 ± 91 pA, and the average shift in EHCO3 due to the first HCO3− current evoked at -70 mV was -0.5 ± 1 mV at the dendrites (Fig. 2E and F). At the soma, the average difference in the first HCO3− current was 304 pA, and the corresponding shift in EHCO3 was 0 mV. These data confirm that the dendritic Cl− gradient can collapse significantly during GABAA receptor activation (Ballanyi & Grafe, 1985; Huguenard & Alger, 1986; Thompson & Gähwiler, 1989a), and that dendritic and somatic HCO3− gradients and somatic Cl− gradients are stable under the same conditions.
Figure 2. Stability of neuronal anion gradients
Anion gradients were challenged with a high-amplitude GABAA receptor-mediated current induced by local GABA application, and the effect on the GABAA 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 GABAA receptors. All experiments were performed in 1 μm CGP 55845A to block GABAB 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 ECl, measured as the GABAA reversal potential, in the dendrites as a result of the first GABAA 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 ECl. 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 GABAA 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 GABAA 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. EHCO3 was not changed by the largest dendritic GABAA receptor-mediated currents that could be evoked.
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The first GABA application produced HCO3− currents that were smaller than the corresponding Cl− currents, and the ratio of these maximum Cl− and HCO3− currents is consistent with the reported permeabilities of Cl− and HCO3− through the GABAA receptor ionophore (Bormann et al. 1987). While searching in low-Cl− media for the dendritic location at which GABA application resulted in the largest possible inward current at -70 mV, we never found a dendritic region with a higher amplitude current. This argues against the existence of a population of GABAA receptors with a uniquely high HCO3− or cationic permeability.
The rate of recovery of the dendritic transmembrane Cl− gradient
The rate at which the dendritic Cl− gradient recovered was assessed using the protocol described above, except that the timing of the second GABAA application was varied, and the amplitude of the second GABAA current was plotted as a function of the time elapsed since the end of the first GABAA current. The amplitude of the second GABAA current was used to estimate ECl and [Cl−]i. Recordings were performed in nominally HCO3−-free media, and the electrode solutions contained 10 mM Cl−.
A single GABA application at -85 mV produced an outward current, but inward currents were produced when GABA was applied at -85 mV within 2 s of a preceding GABA application at a test potential of -30 mV. At larger time intervals the second GABA application produced outward currents at -85 mV (Fig. 3A). The steady-state ECl in these experiments, obtained from GABA applications at 0.05 Hz, was -98 ± 5 mV (n= 7; Fig. 3B and C). To account for the inward currents evoked 500 ms after the voltage step, ECl would need to shift 28 ± 4.7 mV (n= 7); this corresponds to an increase in the calculated dendritic [Cl−]i from 4.1 ± 0.9 mM at steady state to 10.8 ± 1.8 mM (n= 7). The [Cl−]i was calculated in this manner for each time interval between the loading and test GABAA currents. As shown in Fig. 3D, the rate of recovery of [Cl−]i following the loading current was monoexponential, with a time constant of 3.3 ± 0.2 s (n= 7). The maximum rate of decrease in [Cl−]i, calculated from the initial 100 ms of the fitted exponential function, was 6.1 ± 2.7 mmol l−1 s−1 (n= 7).
Desensitization of the dendritic GABAA receptors during the first GABAA application would reduce the conductance evoked by the second GABA application at short time intervals more than those at longer intervals; this would lead to an underestimate of the initial change in ECl and also the rate at which ECl recovers to the steady-state value. As in the previous experiments, to provide an estimate of the degree to which desensitization may alter our estimate of the activity-dependent change in ECl and the subsequent recovery, a smaller initial GABAA receptor-mediated current was evoked using the same GABA application at -85 mV rather than -30 mV (Fig. 3A, lower traces). If the amplitudes of these currents are assumed to be limited by receptor desensitization, then the GABAA receptor-mediated conductances evoked after the -30 mV initial current can be corrected by a factor proportional to the fractional recovery of the amplitude of the corresponding current evoked after the -85 mV initial GABAA current. This correction would increase by approximately 2 the estimated Cl− accumulation due to the initial current at -30 mV, as well as the rate at which [Cl−]i recovers (Fig. 3D, ○). However, the initial current evoked at -85 mV is likely to increase [Cl−]i to some degree (compare with Fig. 6B), and even a 2 mM increase would account for the change in amplitude of the subsequent currents.
Figure 6. Estimating the rate of recovery of [Cl−]i from the amplitude of GABAA PSCs elicited at varying intervals after a large conditioning GABAA PSC
Same protocol as Fig. 3, except that the negative test potential was -80 mV and GABAA currents were evoked by electrical stimuli in s. moleculare of the CA1 subfield. The electrode Cl− concentration was 10 mM, ionotropic glutamate and GABAB 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 GABAA 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 GABAA 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 GABAA 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 ECl 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 GABAA currents evoked by electrical stimulation were distributed more widely in the dendrites than the currents evoked by GABA application.
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These data indicate that intense activation of dendritic GABAA receptors produces currents sufficient to increase the dendritic Cl− concentration such that ECl is shifted far enough to explain the depolarizing response. To establish whether the net anionic current would change direction as predicted by our estimates of the shift in ECl, we performed voltage clamp experiments at potentials close to RMP using media containing physiological concentrations of HCO3− and Cl−.
CA1 pyramidal neurons were voltage clamped at -55 mV. The current evoked by dendritic application of 100 μm GABA consisted of a brief initial outward current followed by an inward current (Fig. 4A). As in Fig. 3, when the interval between GABA applications was decreased, the initial current was inward, rather than outward; the longer the interval between GABA applications, the more outward the initial current became, reaching a minimum by 10-20 s (Fig. 4A). We considered the large inward currents and the change in direction of the initial current at short application intervals to be due to collapse of the dendritic Cl− gradient in the face of ongoing HCO3− efflux; the outward current was re-established at long application intervals due to recovery of the Cl− gradient. As in Fig. 3, the resting EGABA was established from the initial amplitudes of currents evoked at different test potentials at 0.05 Hz (Fig. 4B and C). We then estimated the shift from the resting ECl and [Cl−]i necessary to explain the change in direction of the GABA currents, assuming EHCO3 remained constant. [Cl−]i following dendritic GABA application is plotted as a function of time in Fig. 4D. [Cl−]i decreased exponentially from the peak concentration at the end of the current evoked by GABA application to the steady-state value with a time constant of 2.3 ± 0.5 s (n= 3) and an average initial rate of decrease of 3.3 ± 0.8 mmol l−1 s−1. The calculated Cl− recovery rate was slower in these experiments compared to those shown in Fig. 3. This is probably due to innaccuracies related to the calculation of ECl and [Cl−]i from mixed anionic currents: we assumed here that the steady-state [Cl−]i= the electrode Cl− concentration, but in the experiments with Cl− as the only permeant anion, [Cl−]i was substantially less than the electrode Cl− concentration due to ongoing Cl− transport (see Fig. 7).
Synaptic anion fluxes
The experiments illustrated in Figs 3 and 4 provide an estimate of the rate of recovery of dendritic [Cl−]i following large Cl− influx. However, the Cl− currents used to increase [Cl−]i were induced by application of exogenous GABA. To determine whether Cl− currents resulting from synaptic activity could exceed transport capacity and thereby increase [Cl−]i, we elicited pharmacologically isolated GABAA postsynaptic currents by brief tetanic stimulation in the s. moleculare in nominally HCO3−-free media (Fig. 5A). The amplitude of the dendritic GABAA PSCs was increased by clamping the membrane at more depolarized test potentials. In this way the amplitude of the GABAA receptor-mediated current could be increased without altering the amount of GABA released at the activated synapses, which was important in order to avoid interpretational difficulties related to possible differences in the ionic permeabilities of synaptic vs. extrasynaptic GABAA receptors, or receptor desensitization. For large GABAA receptor-mediated conductances, as the test potential is made more positive, Cl− influx will increase to the point where it exceeds the vmax of Cl− transport. This will lead to Cl− accumulation during large PSCs with a consequent shift in ECl and decrease in the driving force. Thus at more positive test potentials, the GABAA current should be smaller than predicted based on the GABAA conductance and the initial driving force, so that the I-V relationship would become more shallow at test potentials substantially more positive than the resting ECl. The current amplitude at the point where the slope of the I-V curve decreases equals the maximum Cl− transport rate at the resting [Cl−]i.
In the initial series of synaptic Cl− loading experiments, the effect of Cl− accumulation on the GABAA PSCs was quite variable (n= 3). This variation was decreased substantially when GABAA receptors at the soma and proximal dendrites were blocked by local pressure application of 100 μm picrotoxin, suggesting (1) that tetanic stimulation was activating axons that were presynaptic to both dendritic and somatic GABAA receptors (Freund & Buzsaki, 1996), and (2) that large somatic GABAA currents were more stable than the dendritic currents (Fig. 2). When proximal GABAA receptors were blocked, the amplitude of PSCs evoked at depolarized membrane potentials by intense synaptic activation of dendritic GABAA receptors was smaller than predicted based on extrapolation from PSCs evoked at test potentials closer to resting ECl (decrease in actual vs. predicted charge transfer at -30 mV = -29 ± 8 % when resting ECl= -70 mV; n= 4; Fig. 5B; compare with similar effect demonstrated in Fig. 3C). In contrast, smaller PSCs evoked concurrently using single stimuli did increase as expected at the same test potentials, so that the depression of the large GABA currents was unlikely to be due to voltage-dependent alterations in GABAA receptor function or deterioration of the pre- or postsynaptic neurons. Further, when the experiment was repeated in bicarbonate-buffered, low-Cl− media to test the stability of the HCO3− transmembrane gradient, the HCO3− currents elicited by tetanic stimuli changed with the test potential as predicted by extrapolation from the currents elicited by single stimuli (maximum difference = 4 ± 9 %, n= 3; Fig. 5C-E). The simplest way to explain the nonlinear increase in the amplitude of the large vs. small PSCs carried by Cl− at depolarized test potentials is that at high current densities, Cl− influx exceeded the maximum Cl− cotransport rate, resulting in accumulation of [Cl−]i and a deterioration in the driving force and therefore the Cl− current. In contrast, the PSC carried by HCO3− shows no evidence of alteration in the transmembrane HCO3− gradient.
We obtained the GABAA conductance from the slope of the I-V relationship near ECl. The amount of Cl− influx was estimated by integrating the PSC waveform, and the shift in the Cl− driving force at depolarized test potentials was estimated from the difference between the extrapolated and the actual current amplitude (see Methods). Using these estimates, several parameters can be derived from the experiment shown in Fig. 5A and B, including the time-averaged depolarizing shift in ECl (+7.7 ± 1.2 mV; n= 4), the corresponding increase in [Cl−]i (4 ± 0.6 mM), the volume of the dendrites activated by the large IPSC (4 × 10−13± 1.5 × 10−13 l, which for a sense of scale corresponds to a 500 μm length of dendrite 1 μm in diameter), and the maximal rate of dendritic Cl− cotransport, 7 ± 1.5 mmol l−1 s−1. This value, which is not affected by receptor desensitization, is quite close to the estimate of 6.1 mmol l−1 s−1 obtained from the experiments shown in Fig. 3. However, there are several limitations to this experiment: first, only a few tetanic stimuli could be delivered before the postsynaptic response degraded. This limited the number of test potentials at which we could elicit tetanic responses, and thus the accuracy with which we could determine the maximum transport rate. The degradation after tetanic stimulation was presumably due to injury of nerve terminals arising from the high current densities required to elicit a large GABA release when ionotropic glutamate receptors were blocked. To control for this effect (a) smaller PSCs were evoked using single stimuli to assess the stability of the response, (b) tetanic responses at positive potentials were evoked before evoking the responses near ECl, and (c) tetanic stimuli were applied at 10 min intervals to avoid alterations in GABA release or Cl− transport due to changes in [K+]o (Kaila et al. 1997). The second experimental limitation was the accuracy with which the distal dendritic membrane potential could be controlled during the tetanic stimulation at positive test potentials. For the experiments shown in Fig. 5, to improve control of the dendritic membrane potential, Cs+ rather than K+ had to be used in the electrode solution (see Methods). Finally, tetanic stimulation releases a variety of transmitters and modulators besides GABA. For instance, glutamate release will activate both metabotropic glutamate receptors and electrogenic glutamate reuptake.
To avoid these experimental limitations, we repeated the protocol shown in Fig. 3, using single synaptic activation rather than tetanic stimulation or exogenous GABA application. Recordings were performed using the same low-HCO3− media as in Fig. 3. Ionotropic glutamate receptors and GABAB receptors were blocked, and pentobarbitone (50 μm) was added to the bath to enhance the GABAA receptor-mediated postsynaptic conductance (Alger & Nicoll, 1982; Thallman, 1988). Under these conditions, large (80 V for 80 μs) single stimuli in the distal dendritic layer produced enough Cl− loading at -30 mV to decrease the amplitude of subsequent GABAA receptor-mediated currents elicited by the same stimuli at -80 mV (Fig. 6). The average initial rate of decrease of dendritic Cl− concentration was calculated as for Fig. 3, and in these experiments was 2.8 ± 0.2 mmol l−1 s−1 (n= 6). Comparison of Fig. 6D with Fig. 3D indicates that the lower rate of decrease of Cl− appears to be related to the smaller increase in [Cl−]i in these experiments, suggesting that the GABAA current evoked by electrical stimulation was distributed more widely among the dendrites than the current evoked by local GABA application. Thus in these experiments it is less likely that we were measuring the vmax of the transporter.
Calculation of the dendritic transport rate from the decrease in the amplitude of the Cl− current evoked by GABA application at depolarized potentials relative to that predicted from the slope of the I-V curve near ECl (Fig. 3C) in recordings utilizing K+ electrode solutions led to a value of 5.4 ± 2.3 mmol l−1 s−1 (n= 7, same cells and experiments as shown in Fig. 3). This suggests that the value of 6.1 mmol l−1 s−1 calculated from the rate of recovery of the dendritic Cl− currents after exogenous GABA application, and the value of 7 mmol l−1 s−1 calculated from the tetanic stimulation experiments are reasonable estimates for the vmax of dendritic KCl cotransport, and that factors such as receptor desensitization (Fig. 3D) do not substantially reduce the accuracy of these calculated values.
Cl− affinity of the transport process
In order to model the Cl− transport, it was necessary to know the affinity of the transport process for [Cl−]i. In the next experiments, we loaded neurons with Cl− via the recording electrode. If Cl− is actively transported out of the neuronal cytoplasm by a saturable KCl cotransporter (Misgeld, 1986; Kaila, 1994), then loading neurons with Cl− via the recording electrode should result in a steady state between the rate of Cl− influx from the electrode and the rate of outward Cl− cotransport. Although the rate of Cl− influx from the electrode cannot be quantified, [Cl−]i can be estimated from the reversal potential of the GABAA response (Fig. 7A and B), so that the activity of the cotransporter can be characterized in terms of [Cl−]i. Figure 7C illustrates this approach: for any given electrode Cl− concentration, the calculated [Cl−]i is lower than the electrode Cl− concentration, and the difference between the electrode Cl− concentration and [Cl−]i is proportional to [Cl−]i. In these experiments, nominally HCO3−-free intra- and extracellular solutions were used to minimize HCO3− flux, and the holding potential was set equal or positive to the predicted EGABA to minimize conductive Cl− efflux through ClC-2 (Staley, 1994; Staley et al. 1996). The increase in error in calculated [Cl−]i at higher electrode Cl− concentrations in Fig. 7C may reflect the combined effects of an increase in the somatic-to-dendritic Cl− gradient and the experimental variation in the subcellular distribution of activated GABAA receptors. When the transport velocity was expressed as the difference between the electrode Cl− and the cytoplasmic Cl−, and the concentration of the transported species (Cl−) was expressed as the cytoplasmic Cl− concentration, then the kinetics of Cl− transport could be expressed using Michaelis-Menten kinetics, with a vmax of 38 mM DEC−1 and a KD of 15 mM (Fig. 7D). The vmax is not a physiologically relevant value, since it reflects the difference between electrode and cytoplasmic Cl− concentration when Cl− cotransport was maximal, and as calculated here (see Methods) included the term DEC, the electrode-to-cytoplasm diffusion coefficient whose value is unknown. The vmax of 5-7 mmol l−1 s−1 obtained from the experiments in Figs 3-5 is more useful. However, this experiment does provide a useful KD of 15 mM, which represents the cytoplasmic Cl− concentration at which somatic Cl− cotransport is half-maximal.
One problem with characterizing Cl− cotransport using enzyme kinetic models is that the concentration of the substrate, [Cl−]i, affects not only the probability of binding to the transporter, but also the amount of energy required to transport Cl− across the neuronal membrane. The Michaelis-Menten model assumes that increasing Cl− affects only the probability of binding to the transporter, but changing [Cl−]i also changes ECl. The free energy gained by Cl− during transmembrane transport is proportional to the difference between the membrane potential and ECl, thus changing [Cl−]i also changes the amount of work done by the transporter. The source of this additional energy is the transmembrane K+ gradient, and is therefore proportional to the difference between the membrane potential and EK (Thompson & Gähwiler, 1989b; Alvarez-Leefmans, 1990). Thus for any given membrane potential, the difference between EK and ECl represents the free energy available for KCl cotransport. The data used in Fig. 7D can also be expressed as in Fig. 7E to show that the potential difference generated by the cation-Cl− cotransporter is proportional to the free energy available for transport. This indicates that at physiological concentrations of K+ and Cl−, either the affinity of the transporter for Cl− or the free energy gradient for K+vs. Cl− (Thompson et al. 1988; Thompson & Gähwiler, 1989b) may have limited Cl− cotransport measured in these experiments.
Use of anionic transport data to model the GABAA depolarizing response
The data characterizing dendritic and somatic anionic homeostasis are sufficient to test the hypothesis that large GABAA currents cause a rundown in the transmembrane Cl− gradient and thereby shift EGABA towards EHCO3 (Fig. 8A). We used a standard model of membrane potential (Fig. 8B; Finkelstein & Mauro, 1977; see Methods) incorporating a GABAA conductance (Fig. 8B and C), a leak conductance responsible for the resting membrane potential and input conductance, a Cl− transport system with a vmax of 5 mmol l−1 s−1 and a KD of 15 mM, a resting Cl− leak sufficient to keep the resting Cl− at 2 mM in the face of outward transport, a stable HCO3− gradient, and a variable volume into which the GABAA current flowed. This simple model accurately predicts the duration and waveform of the hyperpolarizing and depolarizing GABAA responses in structures with volumes similar to dendrites and soma (Fig. 8D; compare with Fig. 1A). The kinetics of the hyperpolarizing and depolarizing response vary substantially depending on the volume into which the Cl− current flows; Cl− accumulation results in much smaller changes in [Cl−]i in large structures due to dilution, so ECl is more stable in larger structures such as the soma (Alger & Nicoll, 1982; Staley et al. 1995). The time courses of Cl− accumulation and extrusion (Fig. 8E) are consistent with observations regarding the direction and amplitude of a second GABAA current evoked shortly after a conditioning GABAA current (Figs 2-4 and 6). The accuracy of this simple model suggests that the proposed mechanism of anionic gradient collapse is a sufficient explanation of the depolarizing GABAA response.
Recent findings suggest that activity-dependent increases in [K+]o may contribute to GABAA receptor-mediated, HCO3−-dependent, post-tetanic membrane depolarization (Kaila et al. 1997). Although the increase in [K+]o may directly depolarize the cell membrane, increased [K+]o will also decrease KCl cotransport (Thompson & Gähwiler, 1989b). We tested the effects of increased [K+]o in the Cli− accumulation model by altering the maximum velocity of the KCl cotransporter (by decreasing EK; Thompson & Gähwiler, 1989b). GABAA receptor-mediated responses were elicited by single electrical stimuli in s. moleculare of CA1. The stimulus intensity was increased until the late membrane potential response was depolarizing, and then the stimulus was decreased until no depolarizing component was present. At this stimulus intensity, the late depolarizing component reappeared when the extracellular [K+] was increased from 2.5 to 8.5 mM (n= 5; Fig. 9). In each case the control and high-[K+]o waveforms could be well approximated using the model shown in Fig. 8. The only model parameters altered between the control and high-[K+]o responses were gGABA (McBain, 1994) and vmax. Thus the Cli− accumulation model is sufficient to predict the effects of physiologically relevant alterations in GABAA receptor-mediated Cl− flux or Cl− transport on the kinetics and direction of the GABAA response.
Figure 9. Effect of altering EK on the direction and kinetics of GABAA receptor-mediated synaptic responses
The left panel demonstrates the maximal GABAA 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 K+o; the RMP was set to the experimentally observed values, the vmax 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).
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