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Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  • 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 GABAA depolarizing response in pyramidal cell dendrites.

Although brief activation of dendritic GABAA receptors hyperpolarizes the neuronal membrane, prolonged activation of dendritic GABAA receptors is depolarizing (Barker & Ransom, 1978; Alger & Nicoll, 1979, 1982; Andersen et al. 1980; Wong & Watkins, 1982; Scharfman & Sarvey, 1987; Avoli & Perreault, 1987; Ben-Ari et al. 1989; Lambert et al. 1991; Davies & Collingridge, 1993; Grover et al. 1993; Bonnet & Bingmann, 1995; Staley et al. 1995; Perkins & Wong, 1996; Kaila et al. 1997; Dallwig et al. 1999). This activity-dependent, GABA-mediated depolarization is affected by presynaptic (Grover et al. 1993) and postsynaptic (Alger & Nicoll, 1979, 1982; Thalmann, 1988) manipulations of the GABAA conductance. The depolarizing response is seen in structures with low ratios of volume to receptor density, so that it is easily elicited in the dendrites, but not at the soma (Barker & Ransom, 1978; Andersen et al. 1980; Alger & Nicoll, 1982; Scharfmann & Sarvey, 1987). The GABA-mediated dendritic depolarization has a much slower rise time than both the hyperpolarizing response and the depolarizing GABA response seen in neurons that maintain a depolarized resting Cl reversal potential (ECl) (Nicoll, 1978; Alvarez-Leefmans et al. 1988; Staley et al. 1996). Several hypotheses regarding the mechanism for the slow GABAA receptor-mediated depolarizing response have been developed (Alger & Nicoll, 1982; Müller et al. 1989; Perkins & Wong, 1996; Kaila et al. 1997). We have proposed a mechanism for the depolarizing response based on the following ionic characteristics of the GABAA ionophore (Staley et al. 1995).

The direction of current through the GABAA ionophore is determined by the relative permeabilities and transmembrane electrochemical gradients of Cl and HCO3 (reviewed in Alvarez-Leefmans, 1990 and Kaila, 1994). Pyramidal neurons utilize outwardly directed KCl cotransport so that at resting membrane potential (RMP), the electrochemical gradient for Cl favours hyperpolarizing Cl influx (Misgeld et al. 1986; Thompson & Gähwiler, 1989a; Thompson, 1994). In contrast, HCO3 flux through the GABAA ionophore is outward and depolarizing, because the HCO3 reversal potential (EHCO3) is 50 mV more positive than RMP (Kaila, 1994; Staley et al. 1995). GABAA receptor-mediated net anionic flux is inward and hyperpolarizing initially, since both the GABAA channel permeability and the concentration of Cl at the GABAA ionophore entrance are 4-5 times that of HCO3 (Bormann et al. 1987). However, the bidirectional flow of anions through the GABAA receptor results in a short-circuit condition such that even when the membrane potential has settled at the GABAA reversal potential (EGABA), large Cl and HCO3 currents continue to flow, because the membrane potential cannot reach either ECl or EHCO3. If the GABAA current is larger than the local anionic transport capacity, a significant shift in the concentrations of the permeant ions can occur (Ballanyi & Grafe, 1985; Huguenard & Alger, 1986; Kaila et al. 1989; Thompson & Gähwiler, 1989a). This shift will occur more rapidly in structures such as dendrites with a high ratio of GABAA receptors to volume (Qian & Sejnowski, 1990).

Thus one possible mechanism for the depolarizing response is an activity-dependent alteration of the dendritic EGABA (Staley et al. 1995): during intense GABAA receptor activation, Cl on both sides of the membrane comes to equilibrium so that ECl is driven towards the membrane potential and Cl flux decreases (Wong & Watkins, 1982; Ballanyi & Grafe, 1985; Huguenard & Alger, 1986; Thompson & Gähwiler, 1989a). If HCO3 does not come to equilibrium, the remaining GABAA current would consist primarily of depolarizing HCO3 efflux. Here we demonstrate the validity of key assumptions of this model: (1) the rate at which Cl can be transported out of the neuron is fixed and saturable; (2) GABAA receptor-mediated Cl influx can exceed dendritic transport capacity; (3) the driving force for transmembrane HCO3 flux is only minimally affected during maximal GABAA receptor activation; and (4) Cl accumulation sufficient to alter ECl occurs in the dendrites but not the soma during intense GABAA receptor activation.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Recording techniques

In accordance with the guidelines of our institutional animal welfare committee, following pentobarbitone anaesthesia (60 mg kg−1i.p.), adult Sprague-Dawley rats were decapitated and hemibrain slices were cut in the coronal plane. Whole-cell recordings from the CA1 subfield of the hippocampi of these slices were performed as described previously (Staley, 1994). HCO3-buffered artificial cerebrospinal fluid (ACSF) saturated with 95 % O2-5 % CO2 (pH 7.4) contained (mM): 126 NaCl, 2.5 KCl, 26 NaHCO3, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4 and 10 glucose. Nominally HCO3-free ACSF was saturated with 100 % O2, 26 mM Hepes acid replaced NaHCO3 and pH was titrated to 7.4 at 35°C with NaOH. Recording pipette solution contained (mM): 123 K+, 2 MgCl2, 8 Na+ (gluconate and Cl salts were combined as necessary to produce the desired Cl concentration, usually 10 mM), 1 K2EGTA, 4 K2ATP, 0.3 Na2GTP and 16 KHCO3; pH was 7.2 when solution was saturated with 95 % O2-5 % CO2. In HCO3-free experiments, the electrodes were buffered with 10 mM Hepes instead, and the pH titrated to 7.2 with KOH. In low-Cl experiments, 1-4 mM Cl was kept in the media to stabilize the Ag/AgCl2 electrodes, and the balance of the extracellular Cl was replaced with isethionate. Where noted, caesium, which is a substrate for the outward cation-Cl transporter (Aickin et al. 1982), was used as the cation in electrode solutions for tetanic stimulation experiments in which somatic and proximal dendritic GABAA receptors were blocked (Fig. 5) in order to improve the voltage clamp of the distal dendrites (Staley & Mody, 1992).

image

Figure 5. The change in [Cl]ivs. HCO3 due to large, synaptic GABAA receptor-mediated currents

GABAA 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 GABAB receptor antagonists. The rates of anion flux following single and tetanic stimuli were manipulated by changing the test potential. A, GABAA PSCs evoked by single vs. 10 stimuli at -50 and -30 mV. The GABAA 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 GABAA 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 GABAA receptor-mediated HCO3 currents. GABAA PSCs were evoked by electrical stimulation in s. moleculare of CA1 in the presence of ionotropic glutamate and GABAB receptor antagonists. Intra- and extracellular Cl was decreased to 4 mM. Somatic GABAA 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 GABAA 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 EHCO3. The large size of the HCO3 currents was due to the positive reversal potential of GABAA currents in low-Cl media; under these conditions, GABAA receptor activation is excitatory, and polysynaptic GABAA receptor activation will occur with strong stimuli (Avoli et al. 1990).

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Recordings were performed with an Axopatch-1D amplifier, and were digitized at 1-5 kHz using routines written in AxoBasic (Axon Instruments). Only recordings with stable access resistances < 15 MΩ were analysed. Approximately 80 % of the electrode access resistance was compensated in voltage clamp recordings. Junction potentials were corrected, and saline-bridged earths used. Low-frequency synaptic activation of dendritic GABAA receptors was accomplished using single 20-80 μs stimuli via bipolar tungsten electrodes in stratum moleculare (s. moleculare); high-frequency activation utilized trains of 10-40 of the same stimuli delivered at 200 Hz.

Pharmacological agents

Synaptic GABAA receptor activity was isolated using the glutamate receptor blockers 6,7-dinitro-quinoxaline-2,3(1H,4H)-dione (DNQX, 20 μm) and dl-amino-5-phosphonovaleric acid (APV, 50 μm), and the GABAB receptor blockers CGP-35348 (100 μm) and CGP-55845A (1 μm) (Ciba-Geigy). In some experiments, GABAA receptors were selectively and non-synaptically activated by pressure application of 50-250 μm muscimol or GABA in ACSF using pulses of 5-20 ms × 10 p.s.i. through a 1 μm diameter whole-cell pipette. GABAB receptors were blocked when GABA was applied. Tetrodotoxin and pentobarbitone were applied by bath. When measuring dendritic Cl transport, somatic GABAA receptors were blocked, where noted, by local application of 100 μm picrotoxin to stratum pyramidale (s. pyramidale).

Data analysis

Current-voltage plots were obtained by fitting the GABAA receptor-mediated currents to the Goldman-Hodgkin-Katz constant field equation, rewritten as:

  • image(1)

where g is the GABAA conductance, V is the membrane potential, F is Faraday's constant (96 487 C mol−1), R is the gas constant (8.315 J mol−1 K−1) and T is temperature (K). Ao, the extracellular concentration of permeant anions, was calculated as:

  • image(2)

where [Cl]o and [HCO3]o are the extracellular concentrations of Cl and HCO3, respectively, and PermHCO3 and PermCl are the permeabilities for the individual ion species HCO3 and Cl through the GABAA channel. V0 represents the membrane potential corresponding to the zero-current condition for the constant field current equation:

  • image(3)

(Hodgkin & Katz, 1949), where [Cl]i and [HCO3]i are the cytoplasmic concentrations of Cl and HCO3, respectively. Charge transfer by GABAA receptor-mediated post-synaptic currents (PSCs) was calculated by numerical integration of the PSC waveform. Time-averaged currents were obtained by dividing this integral by the duration of the current; 500 ms time intervals were used for averaging.

Kinetics of cation-Cl transport

In these experiments, the change in direction and amplitude of the transmembrane Cl current was assumed to be due to alterations in [Cl]i and ECl as a consequence of GABAA receptor-mediated Cl flux. Under the experimental conditions used in this study, we did not find evidence for substantial alterations in ECl or EK based on interneuron network activity (Kaila et al. 1997; Fig. 1A). Cytoplasmic Cl loading and extrusion via voltage-dependent Cl channels (Staley, 1994; Smith et al. 1995; Fig. 1B) were minimized by the selection of holding potentials: holding potentials positive to -25 mV were not used to avoid activation of ClC-3 chloride channels (Smith et al. 1995) and holding potentials negative to ECl were not used to avoid activation of ClC-2 channels (Staley, 1994; Staley et al. 1996).

image

Figure 1. Activity-dependent alterations in EGABA 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 EGABA. 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. GABAA currents evoked under conditions 1 and 2 overlapped, indicating that holding potentials in this voltage range had no effect on EGABA in the absence of a conditioning GABA application. The inward current evoked under condition 3 demonstrates that GABAA currents, but not holding potentials of -30 mV, are sufficient to shift ECl.

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We used the following three methods to characterize neuronal Cl transport.

1. When the dendritic transport rate was calculated from the rate of recovery of the outward currents evoked by exogenous GABA application (Figs 3 and 4), the resting ECl was calculated from the I-V relationship of the currents evoked at 20 s intervals between GABA applications, using eqn (3). In experiments in which both Cl and HCO3 were present (Fig. 4), we used a value of 0.25 for the HCO3:Cl GABAA permeability ratio (Bormann et al. 1987; Kaila, 1994). The GABAA conductance was calculated from the slope of the I-V plot at potentials near ECl. [Cl]i at the time of repeat GABA application was estimated from the change in EGABA necessary to account for the initial current resulting from the repeat GABA application (eqn (1)). [Cl]i was then calculated from EGABA using eqn (3). Data for the rate of change of [Cl]i were fitted to a monoexponential curve using least-squares fit, where t= 0 was the time at which the initial evoked current decayed to 0 or when the test potential was changed to a value near ECl.

image

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

GABAA currents were evoked by pressure application of 100 μm GABA to s. moleculare of CA1. The electrode Cl concentration was 10 mM, GABAB 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 GABAA 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 GABAA 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 ECl. 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 ECl 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.

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image

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 EGABA 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 ECl= -55 mV.

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2. Large-amplitude dendritic Cl currents were elicited either by brief tetanic stimuli (10-20 stimuli at 200 Hz) during block of GABAB and ionotropic glutamate receptors in s. moleculare or by applicaton of GABA to s. moleculare. These Cl currents did not increase in amplitude at depolarized membrane potentials as predicted based on the slope of the I-V plot at potentials close to ECl (Figs 3C and 5B). The largest PSC evoked at depolarized potentials that could be predicted from the slope of the I-V curve near ECl was assumed to represent the maximum Cl transport rate at the steady-state [Cl]i, i.e. this PSC equalled the largest Cl influx rate at which the Cl driving force did not deteriorate as a consequence of Cl accumulation due to Cl influx exceeding the Cl transport rate. The difference in charge transferred by the PSCs at depolarized membrane potentials vs. the charge transfer expected based on extrapolation from the slope of the I-V curve near ECl was used to estimate the charge lost due to Cl accumulation. The GABAA conductance was obtained from the slope of the I-V curve near ECl. The shift in ECl was calculated as (GABAA conductance)−1× lost current (= lost charge/PSC duration). The volume of the dendrites into which the GABAA currents flowed was calculated as Cl influx (measured as the charge transferred by PSCs at depolarized potentials, converted from coulombs to mM)/change in [Cl]i necessary to account for the calculated change in ECl (eqn (3)). This allowed calculation of maximum Cl transport per unit volume.

3. The neuronal cytoplasm was dialysed with an isotonic high-Cl solution in the whole-cell electrode (Fig. 7). Under these conditions, the neuronal Cl concentration increases to a steady state that is determined by the influx of Cl (determined by the electrode Cl concentration and rate of diffusion) and the rate of neuronal Cl efflux (Staley, 1994). The neuronal cytoplasmic Cl concentration was estimated from the reversal potential of the GABAA receptor-mediated PSCs in HCO3-free media. The maximum cation-Cl cotransport rate is a function of the transport capacity and the gradients for K+ and Cl (Läuger, 1987). When either K+ or Cl gradients are held constant, the rate of transport of the other ion as a function of its cytoplasmic concentration can be characterized using the Michaelis-Menten kinetic model (Tas et al. 1987; Gasbjerg & Brahm, 1991; Lauf et al. 1992). If spontaneous GABAA receptor fluxes are neglected, then at steady state, the rate of Cl influx (from the electrode) equals the rate of efflux via transport (v). From the diffusion equation, Cl influx is proportional to the difference between the electrode Cl concentration ([Cl]E) and the cytoplasmic Cl concentration ([Cl]i), so that v= ([Cl]E - [Cl]i)DEC, where DEC is the diffusion coefficient that characterizes diffusion of Cl from the electrode solution to the cytoplasm for a given concentration gradient. The maximum Cl efflux rate and the Cl affinity of the cation-Cl cotransporter (for a specified K+ gradient) were derived from the intercepts of the Lineweaver-Burke equation:

  • image(4)

where KD is the neuronal Cl concentration at which the extrusion rate is half-maximal and vmax is the maximum rate of Cl extrusion. Data for Lineweaver-Burke plots were fitted using a least-squares algorithm. GABA responses were evoked shortly after whole-cell recording was established, and repeated until the calculated reversal potential reached a steady value; this required between 10 and 30 min (Staley, 1994). In these experiments, Cl diffusion from the electrode will set up a somatic-dendritic Cl gradient. Although proximal stimulation was used to elicit primarily somatic GABA responses, we cannot determine the subcellular location of the activated GABA receptors, thus our measurements represent a weighted mean of both somatic and dendritic concentrations.

image

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. GABAB 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. KD, 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 EK) minus the driving force for Cl influx (the difference between the membrane potential and ECl.); this simplifies to EK - ECl. The data fitted equally well using either the model in D or E.

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Membrane potential model of the depolarizing GABAA response

We utilized a standard model of membrane potential (Finkelstein & Mauro, 1977) in which the membrane potential is considered to be generated by a group of electrochemical potentials, each representing one ionic species, in series with time-varying conductances (Fig. 8B). The currents flowing through the conductances were considered to flow into a structure of specified volume. HCO3 was assumed to be replaced as rapidly as it flowed out, but Cl could accumulate or decrease in this volume depending on the amplitude of the Cl current vs. the Cl pump velocity. Cationic conductances were lumped into a time-invariant leak conductance. Voltage-dependent conductances and membrane capacitance were not included in the model.

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

A, schematic of the ionic currents and pumps underlying the GABAA membrane potential response. GABAA 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 + GABAA conductance) used in the calculations in this figure. D, voltage response to the GABAA 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 GABAA response increases sharply with decreasing radii. E, the corresponding change in [Cl]i during the GABAA conductance. [Cl]i initially increases when Cl influx exceeds the vmax of the transport systems. As the GABAA 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, vmax. 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.

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The equation governing the membrane potential is derived from the principal that the sum of all membrane currents In equals the sum of the products of all conductances and the respective driving forces (Finkelstein & Mauro, 1977):

  • image(5)

so that after rearranging,

  • image(6)

where gn and V0n are the conductance and reversal potential for each ionic species, here restricted to ICl, IHCO3, and a cationic Irest.

The constant field equation was used for GABAA currents, using intracellular anion concentrations rather than V0 as in eqn (1), and setting g=gGABA× fractional ionic permeability. Cl permeability was assumed to be 0.8, and HCO3 permeability was 0.2 (sum of fractional permeabilities, 1.0; HCO3:Cl permeability ratio, 0.25; Kaila, 1994).

  • image(7)
  • image(8)

[Cl]i was calculated as:

  • image(9)

where F is Faraday's constant, vol is the volume of the structure into which the GABAA current flows, v is the pump veolcity and dt is the time increment.

The pump velocity (v) was set according to the Michaelis-Menten model (eqn (4)). KD and vmax were taken from the data; KD was 15 mM and vmax was ∼5 mmol l−1 s−1. [Cl]i(0) in eqn (9) is the steady-state [Cl]i. This was assumed to be 2 mmol l−1 and was achieved by including a Cl leak of 0.3 mmol l−1 s−1, in which case the Cl pump reached a steady-state [Cl]i of 2 mM. The source of this Cl leak could include the electrode Cl as well as Cl components of the input conductance and spontaneous GABAA receptor-mediated activity.

The GABAA conductance was modelled using a two-term exponential function, using values of gmax= 40 nS, τ1= 0.05 s, τ2= 1 s:

  • image(10)

Currents other than GABAA receptor-mediated Cl and HCO3 currents were lumped together as a rest current:

  • image(11)

The rest (input) conductance was set to 10 nS, corresponding to a 100 MΩ input resistance, and the resting membrane potential was set to -65 mV, i.e. V0rest= -65 mV.

When circular references were encountered (for instance, ICl depends on [Cl]i and [Cl]i changes according to ICl× dt), the value of [Cl]i at the previous time point, T - dt, was used. The source code (Quickbasic 4.5 and Visual Basic 6.0, Microsoft) is available on request.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

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.

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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.

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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.

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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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DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

These experiments demonstrate that neuronal transmembrane Cl transport is rate limited, that there is significant alteration of dendritic [Cl]i and ECl as a result of intense GABAA receptor activation, and that the transmembrane gradients for Cl at the soma and HCO3 in the dendrites are significantly less affected by large GABAA currents. Incorporation of these observations into a standard model of membrane potential accurately reproduces both the hyperpolarizing and depolarizing GABAA responses.

Experimental limitations

It would be conceptually simpler to perform the experiments demonstrating the stability of ECl at the soma (Fig. 2) using gramicidin recordings in order to eliminate the possibility that the somatic Cl concentration was buffered by the electrode solution ‘reservoir’. However, gramicidin recordings were not sufficiently stable during the steps to and from -30 mV to be useful. Conductive channels in the membrane are formed from gramicidin dimers, and large voltage steps are likely to cause alterations in gramicidin dimerization in the membrane, as a consequence of either voltage-dependent electrostatic protein interactions or changes in intracellular Ca2+ during voltage steps (Lundbaek et al. 1997). Fortunately such recordings are not necessary to demonstrate stability of somatic ECl. KCl cotransport decreased the somatic Cl concentration to a steady-state value that was less than that of the electrode solution: 7 mM in cytoplasm based on ECl (Fig. 2D) vs. 10 mM in the pipette solution. Thus the direction of Cl diffusion is from the electrode to the cytoplasm, so that the stability of the somatic ECl cannot be ascribed to Cl flux from the soma to the electrode.

Underestimation of distal dendritic currents could arise due to imperfect voltage control of the distal dendrites. Integrating the current over time to obtain transferred charge or using time-averaged currents limits this error to about 10 % (Staley & Mody, 1992). When Cl transport is measured by the rate of change of ECl after a Cl load (Figs 3, 4 and 6), the Cl load will be underestimated, but the rate of change in ECl will not be underestimated as long as the ∼10 % error affects the amplitudes of each of the subsequent GABAA receptor-mediated currents equally. This could produce a 10 % underestimate of the maximum transport rate, since the actual Cl load was larger than measured. When Cl transport is measured from the decline in actual vs. expected current at positive test potentials (Figs 3C and 5B), space clamp errors will also lead to an underestimate of the maximum transport rate, because the current and the corresponding Cl influx rate at which the response deviates from expected is larger than measured. If both distal and proximal dendritic GABAA receptors are activated at the same time, the space clamp error will be larger because of the decreased membrane resistance of the proximal dendrites: the amplitude of the depolarizing response in distal dendrites will be shunted by GABAA conductances in larger, more proximal structures (Staley & Mody, 1992). We addressed this problem by utilizing either focal application of GABA or in some cases by prior application of GABAA antagonists to the soma and proximal dendritic regions.

Cl efflux via the ClC-2 Cl channel may have added to KCl cotransport in some experiments, even though we had set the holding potentials to minimize activation of ClC-2 (Staley, 1994; Staley et al. 1996). This is likely to explain the correlation between ECl and the holding potential from which ECl was determined: in Fig. 2, the holding potential was -70 mV and ECl was -78 mV, while in Figs 5 and 6 the holding potential was -80 or -85 mV and ECl was ∼-98 mV; all these experiments were performed using an electrode Cl concentration of 10 mM. The Cl transport rate calculated from the experiments shown in Figs 3C and 5B were not affected by Cl efflux via ClC-2, because the holding and test potentials were too far from the ClC-2 activation potential. The transport rate in these experiments is quite close to the transport rates calculated from Fig. 3D, indicating that Cl efflux via ClC-2 was small during the time interval between the conditioning Cl current and the test currents, reflecting the slow activation of ClC-2 (Staley, 1994).

Assumptions of the gradient collapse model

The basic assumption of the gradient collapse hypothesis is that Cl influx through GABAA receptors can increase the local intracellular Cl concentration so that EGABA is shifted in a positive direction. This seems contrary to the fact that only a tiny fraction of the ions present on either side of the membrane need to cross over in order to produce a charge imbalance sufficient to alter the membrane potential. For instance, the sequential activation of Na+ and K+ conductances during an action potential cause large changes in membrane potential but insignificant alterations in Na+ and K+ concentrations (Hille, 1992). This is because the ionic flux during the action potential is small due to the brevity of the conductances and the sequential activation of gNa and gK, which minimizes ‘short-circuiting’ of one current by the other. In contrast, bidirectional anionic flux through the GABAA receptor results in a severe short-circuit condition. Thus during prolonged activation of GABAA receptors, even though the membrane potential is at EGABA, a substantial fraction of the dendritic transmembrane Cl gradient is being reduced by ongoing Cl currents because the membrane potential cannot reach either EHCO3 or ECl.

A related assumption of the gradient collapse hypothesis is that the rate of Cl transport can be exceeded by the rate of GABA-mediated Cl influx (Wong & Watkins, 1982; Ballanyi & Grafe, 1985; Huguenard & Alger, 1986; Thompson & Gähwiler, 1989a). The experiments using high-intensity synaptic and exogenous activation of dendritic GABAA receptors (Figs 2-6) demonstrate that synaptic Cl influx through the GABAA ionophore can exceed the maximum transport rate. The experiments illustrated in Fig. 2 support the assumption that at the soma, even if the rate of synaptic Cl influx exceeds the transport rate, the volume of the soma precludes a significant change in [Cl]i and the corresponding shift in ECl for at least several seconds (Fig. 8E and F; Ballanyi & Grafe, 1985), so that the GABAA current remains hyperpolarizing.

The final assumption of the gradient collapse hypothesis is that the dendritic HCO3 transmembrane gradient is more stable than the corresponding Cl gradient. This is reasonable based on the pH alterations that would accompany a dissipation of the HCO3 gradient: at a constant pCO2, if EHCO3 was driven to RMP, the intracellular pH would be 6.3, which would denature proteins and damage the neuron. To avoid this, the intracellular HCO3 concentration is stabilized by diffusion and hydration of CO2 (Pasternak et al. 1993), while the pH is supported by intracellular buffers and H+ extrusion (Kaila & Voipio, 1987; Chen & Chesler, 1992; Staley, 1995). The data in Figs 2E and F and 5C-E confirm that the dendritic HCO3 concentration is maintained so that EHCO3 does not vary significantly during maximal dendritic GABAA receptor activation.

We also assumed in our calculations that changes in ECl are due to changes in [Cl]i, because similar changes in the extracellular Cl concentration produce insignificant changes in ECl (eqn (3)) and because activity-dependent changes in ECl have been demonstrated in cultured cells, where the supply of extracellular Cl is essentially unlimited (Barker & Ransom, 1978; Dallwig et al. 1999). Although we have described the recovery of [Cl]i as a function of active transport, the recovery could be due to both active transport and diffusion of Cl away from the intracellular regions closest to the open GABAA ionophores. In the modelling studies of the GABA depolarizing response, the amount of Cl entering the dendrite during large GABAA currents was sufficient to raise the [Cl]i of the entire volume of the dendritic cytoplasm, so that there was no need to include a radial variation in [Cl]i. Thus Cl diffusion would only be significant at the ends of the active dendritic volume, and was neglected in the model.

Alternative mechanisms

Several explanations for the activity-dependent GABAA depolarizing response have been proposed (Alger & Nicoll, 1982; Staley et al. 1995; Perkins & Wong, 1996; Kaila et al. 1997). An intriguing recent proposal is that the GABA depolarizing response is due to the depolarizing effects of increased [K+]o which arises from activation of an interneuron network that is dependent on both GABA and HCO3. However, increases in [K+]o are not necessary for the GABA depolarizing response, since responses with identical kinetics have been described in cultured cells, where [K+]o is stabilized by the large size of the extracellular space (Dallwig et al. 1999). The study by Dallwig et al. also supports the Cl gradient collapse hypothesis by demonstrating the predicted increase in intracellular Cl using Cl-sensitive fluorescent dyes. A second difficulty with the K+o accumulation hypothesis is the lack of effect of TTX on the depolarizing response (Fig. 1A), which is difficult to reconcile with activation of an interneuron network. The increases in [K+]o of several millimolar occurred in response to prolonged tetanic stimulation (Kaila et al. 1997), whereas focal application of GABA agonists (Figs 1-4) produce much smaller changes in [K+]o (Müller et al. 1989). The increase in [K+]o generated by focal GABAA agonist application is similar in the dendrites and the soma (Müller et al. 1989); thus a direct membrane depolarization by [K+]o is not consistent with the lack of depolarizing response at the soma (Barker & Ransom, 1978; Andersen et al. 1980; Alger & Nicoll, 1982; Scharfman & Sarvey, 1987; Fig. 2). We suggest that GABA-dependent increases in [K+]o arise from KCl exchange (Müller et al. 1989; Payne, 1997) and that activity-dependent increases in [K+]o augment the depolarizing response primarily by decreasing the KCl transport rate, thereby enhancing Cl accumulation (Fig. 9).

An argument against the Cl gradient collapse hypothesis is that the stability of the HCO3 gradient appears to require both extra- and intracellular carbonic anhydrase activity (Fig. 8A) but benzolamide, a carbonic anhydrase inhibitor that is confined to the extracellular space, does not block the depolarizing GABA response (Kaila et al. 1997). However, if extracellular HCO3 is removed by other means such as diffusion or glial transport then inhibition of extracellular carbonic anhydrase will not limit HCO3 efflux. In addition, because the concentrations of HCO3 on both sides of the membrane are nearly symmetrical, a very large change in extracellular HCO3 would be required to significantly alter the HCO3 reversal potential (eqn (3)). For example, even if extracellular HCO3 increased by the same amount as intracellular Cl (e.g. ∼7 mM calculated in Fig. 4), EHCO3 will only change by 7 mV, vs. 28 mV for ECl. For a RMP of -70 mV, this would represent a complete abolition of the driving force for Cl, but the driving force for HCO3 would only decrease from 57 to 50 mV. Thus the small effect on the depolarizing GABA response produced by inhibition of extracellular carbonic anhydrase (Kaila et al. 1997), in contrast to the marked effect of inhibition of both intra- and extracellular carbonic anhydrase by acetazolamide (Staley et al. 1995), is entirely consistent with the proposed Cl gradient collapse mechanism.

Our data do not support the existence of a subset of GABAA receptors with high HCO3 or cationic permeabilities as an explanation for depolarizing GABAA responses: such receptors were not found when applying GABA directly to the dendrites (Figs 2-4). Additionally, in Fig. 4A the change in the direction of the initial current as a function of time since the last GABA application cannot be explained on the basis of diffusion of GABA to receptors with unique ionic permeabilities.

A distinction should be made between the phenomenon we have examined in this paper, namely the activity-dependent GABAA dendritic depolarization that occurs in neurons that are normally hyperpolarized by brief GABAA receptor activation, and the rapid activity-independent GABAA depolarizing response that occurs in many neurons during development (Cherubini et al. 1991; Owens et al. 1996), in some adult neurons (Staley, 1996) and following particular experimental manipulations (Van den Pol et al. 1996; Cerne & Spain, 1997). These shifts in EGABA occur under different conditions and on different time scales to the phenomena studied here, are HCO3 independent (Owens et al. 1996; Staley et al. 1996), and are thus likely to be related to long-term regulation of Cl transport mechanisms (Rohrbough & Spitzer, 1996; Staley et al. 1996).

Unanswered questions

Electrical stimulation of GABAA terminals produced variable responses, presumably because variation in the length and distribution of the stimulated GABAergic axon terminals results in the activation of a mix of somatic and distal and proximal dendritic GABAA receptors that cannot be predicted from the location of the stimulating electrode. This variability applies to the effects of activation of individual interneurons: the interaction between the dendritic depolarizing response and somatic hyperpolarizing response will vary substantially as a function of the subcellular distribution of the GABAergic terminals of each interneuron (Freund & Buzsáki, 1996). Related to this problem, it has not been determined whether the discharge of a single interneuron or an interneuron network is required to elicit the depolarizing response.

At the soma, although Cl transport can be saturated, ECl is stable because the volume is large enough to prevent significant alterations in [Cl]i. The analogous question has not been answered for dendritic HCO3 homeostasis: is the rate of H+ transport so high that GABAA receptor-mediated HCO3 efflux does not alter the intracellular pH, or is the intradendritic H+ concentration stabilized by high buffer capacity? Further characterization of the molecular and functional characteristics of neuronal proton and anion transporters are necessary to resolve these issues.

Physiological significance of the GABAA depolarizing response

A large inefficiency in GABAA receptor function results from bi-directional anionic flux and the consequent dissipation of energy stored in the transmembrane ionic gradients (Fig. 8A). We suggest that this energetically inefficient bi-directional anionic flux is worthwhile because it provides a mechanism for frequency modulation of the GABAA response from inhibition to excitation. This activity-dependent change in GABA response is useful for regulating the voltage-dependent Mg2+ block of the NMDA receptor (Staley et al. 1995), and may contribute to the activity-dependent alterations in inhibition and NMDA receptor function that modulate synaptic plasticity (Bliss & Collingridge, 1993).

The dendritic location and activity dependence of the depolarizing GABAA response suggest that the GABA-releasing interneuron that elicits the response would need to fire in rapid bursts and have a significant axonal distribution in the distal dendrites of the pyramidal cells. Regulation of interneuron burst firing by neuromodulators (Freund & Buzsaki, 1996; Bergles et al. 1996) could therefore restrict the occurrence of the GABA depolarization to specific states. Alterations in synaptic inhibition are an important component of the pathophysiology of epilepsy (Prince & Connors, 1984). Thus dysregulation of GABA-mediated depolarization, for instance due to alterations in the anatomical distribution of the efferent and afferent connections of GABAergic interneurons (Babb et al. 1989), could contribute to epileptogenesis. The anticonvulsant action of the carbonic anhydrase inhibitor acetazolamide, an inhibitor of GABA-mediated depolarization (Staley et al. 1995), supports the idea that this phenomenon may contribute to some epileptic conditions, and suggests that manipulation of neuronal anionic homeostasis may be a promising anticonvulsant strategy.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

This study was supported by NIH grants NS34360 and NS34700, the American Heart Association, and the Epilepsy Foundation of America.