 |
INTRODUCTION |
-Aminobutyric acid (GABA) transporters are sodium dependent and electrogenic (Kavanaugh et al. 1992
; Mager et al. 1993
; Malchow and Ripps 1990)
, and as with other passive transporters, they will operate in either direction depending on which is most thermodynamically favorable (Cammack et al. 1994
; Levi and Raiteri 1993
; Nicholls and Attwell 1990)
. Under normal conditions, GABA transporters remove GABA from the extracellular space. Antagonists of GABA transporters enhance the inhibitory action of exogenous GABA (Krogsgaard-Larsen 1980)
, prolong inhibitory postsynaptic currents (IPSCs) (Rekling et al. 1990
; Thompson and Gahwiler 1992)
and have anticonvulsant properties (Schousboe et al. 1991
; Suzdak et al. 1992)
. However, under some conditions "reverse operation" of GABA transporters can result in nonvesicular GABA release. For example, depolarization in the absence of extracellular Ca2+ induces release of GABA from retinal horizontal cells of some fish (Cammack and Schwartz 1993
; Schwartz 1987
; Yazulla and Kleinschmidt 1983)
and toads (Schwartz 1982)
and from growth cones of neurons isolated from rat forebrain (Taylor and Gordon-Weeks 1991)
. GABA also is released from cultured striatal neurons in response to 56 mM [K+], veratridine, and glutamate receptor agonists in the absence of calcium or after treatment with tetanus toxin (Pin and Bockaert 1989)
and in response to electrical stimulation in rat striatal slices in the absence of calcium (Bernath and Zigmond 1988)
. This nonvesicular GABA release is Na+ dependent and can be blocked by antagonists of GABA transporters (Belhage et al. 1993)
.
In most previous studies, carrier-mediated GABA release has been measured biochemically, in which case it is not clear whether the GABA that is released causes functional effects. Postsynaptic responses due to carrier-mediated release have been measured in response to depolarization in catfish retinal neurons (Schwartz 1987)
and by nipecotate (NPA) application in rat brain slices (Honmou et al. 1995
; Solis and Nicoll 1992)
. However, there is currently no evidence that carrier-mediated GABA release induced by physiologically relevant stimuli results in significant electrophysiological effects in mammalian tissue. Here we report that a brief increase in [K+]o from 3 to only 12 mM and/or a decrease in [Na+]o, induced large GABAA-receptor-mediated responses in cultured rat hippocampal neurons as a result of carrier-mediated GABA release from neighboring cells. A preliminary report of this work has been published in abstract form (Gaspary and Richerson 1996)
.
 |
METHODS |
Sources of chemicals and reagents
Picrotoxin, bicuculline methiodide(
), GABA, tetrodotoxin (TTX), ethylene glycol-bis (
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), EDTA, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), cytosine
-D-arabino-furanoside hydrochloride (Ara C), tetraethyl ammonium (TEA) chloride, TEA hydroxide, CdCl2, choline, CsOH, CsCl, papain, cysteine, trypsin inhibitor, bovine serum albumin (BSA), streptomycin, penicillin, and all salts and chemicals not otherwise listed were purchased from Sigma Chemical (St. Louis, MO). (±)-2-Amino-5-phosphonopentanoic acid (AP-5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), RS(±)-nipecotic acid (NPA), and 1-(2-{[(diphenylmethylene)amino]oxy}ethy) - 1, 2, 5, 6 - tetrahydro - 3 - pyridine-carboxylic acid hydrochloride (NO-711 or NNC-711) were purchased from Research Biochemicals (RBI, Natick, MA). N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium chloride (QX-314) and tetanus toxin were purchased from Alomone Labs (Jerusalem, Israel). 1-(4,4-Diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride (SKF89976A) was a generous gift of SmithKline Beecham Pharmaceuticals (King of Prussia, PA). Fetal bovine serum (FBS), Neurobasal medium, and B27 supplement were purchased from Gibco BRL Products (Gaithersburg, MD). Dulbecco's modified Eagle's medium (DMEM) and F12 supplement were purchased from JRH Biosciences (Lenexa, KS).
Cell culture
Primary cultures of hippocampus were prepared from embryonic or fetal (E19-P1) Sprague-Dawley rats using aseptic technique. Hippocampi were dissected under direct microscopic visualization (Stemi 2000-C; Carl Zeiss, Thornwood, NY). Tissue was placed in oxygenated, HEPES-buffered Ringer solution, which contained (in mM) 130 NaCl, 4 KCl, 1 MgCl2, 1.5 CaCl2, 10 HEPES, and 10 dextrose, pH 7.3, and incubated in digestion solution for 10 min (HEPES-buffered Ringer solution with 9 U/ml papain, 0.2 mg/ml cysteine, 1.5 mM extra CaCl2, and 0.5 mM EDTA, pre-activated for 30 min at 37°C). After digestion, tissue was washed three times with culture medium (70% MEM/10% FBS, with 3.6 gm/l glucose, 100 U/ml penicillin, and 100 µg/ml streptomycin), to which trypsin inhibitor (1.5 mg/ml) and BSA (1.5 mg/ml) were added, then triturated five to eight times with a Pasteur pipette. Hippocampal cells were plated at a density of 2.5-5·105 cells/ml on 12 mm round, poly-L-ornithine and laminin-coated coverslips in 12-well culture dishes (Corning Glass Works, Corning, NY) and incubated in culture medium at 37°C with 5% CO2 in room air. At 4 days, the medium was switched to Neurobasal medium with B27 supplement (1:50). Although Neurobasal medium can inhibit glial growth (Brewer et al. 1993)
, we found that Ara-C also was required to control glial growth after ~7 days in vitro (DIV). Cultures were fed with half-medium changes on day 7 and then weekly. In most cases, cultures were grown for 10-21 days before recording because most cells <10 DIV had undetectable responses to elevated [K+]. These responses first developed between 10 and 16 DIV, possibly because of maturation of neurons, increased expression of transporters, or formation of cell to cell contacts.
Electrophysiology
For recording, coverslips were placed in a chamber on the stage of an inverted light microscope (Axiovert 100, Carl Zeiss), and superfused (at 3-4 ml/min) with artificial cerebrospinal fluid containing (in mM) 128 NaCl, 3.0 KCl, 2.0 MgCl2, 2.0 CaCl2, 1.3 NaH2PO4, 22 NaHCO3, and 10 dextrose. During recording, the bath solution was changed to one of the solutions described below. All bath solutions were bubbled with 5% CO2-95% O2 at pH 7.4. All experiments were performed at room temperature (24°C).
Whole cell, patch-clamp recordings were performed in voltage clamp mode using a patch-clamp amplifier (Axopatch 1D, Axon Instruments, Foster City, CA). Recording electrodes (2.5-4.0 M
) were fabricated from thin-walled, borosilicate glass tubing (Diamond General, Ann Arbor, MI) with a micropipette puller (Sutter Instruments, Model P-87, Novato, CA). Electrodes were filled with one of the following solutions (in mM): 1) ECl
= 0 mV: 94 CsCl, 40 TEA (chloride salt), 5 HEPES, 10 EGTA, and 15 CsOH; 2) ECl
=
60 mV: 13.4 CsCl, 40 TEA-OH, 105 methanesulfonic acid, 85 CsOH, 10 EGTA, and 10 HEPES; or 3) QX-314: 114 CsCl, 20 QX-314 (chloride salt), 20 CsOH, 10 HEPES, and 10 EGTA.
Electrode solutions were adjusted to pH 7.2 with CsOH and to an osmolarity of 270 ± 5 mOsm with ultrafiltered, deionized H2O (Milli-Q Plus Water System, Millipore, Bedford, MA). For all recordings, initial seal resistance was
1 G
. Recordings were included in analyses if initial access resistance was <20 M
(mean ± SD = 9.6 ± 2.8, n = 90) and changed <30% during the recording and if input resistance remained
100 M
.
Data were digitized and stored on computer using a commercially available data-acquisition system (TL-1 DMA interface, and pClamp software, Axon Instruments) and on tape with a VCR (SLV-441, Sony, Park Ridge, NJ) using a digitizing unit (Neurocorder DR-484, Neurodata Instruments, New York, NY).
Experimental paradigm
For each recording, holding current and/or whole cell conductance was measured in hippocampal pyramidal neurons, while the extracellular solution was exchanged rapidly with a "test" solution locally applied using a microejection pipette. Two approaches were used. In the first, the test solution was used to fill a pressure ejection micropipette (tip diameter
10 µm) that was connected to a pressure microejector (Picospritzer II, General Valve, Fairfield, NJ) and was positioned close to the recorded neuron. In the second, the "micropuffer" technique for the application of multiple solutions developed by Greenfield and Macdonald (1996)
was used to systematically vary the bicuculline concentration. In both cases, trypan blue (0.2 mM) was added to the test solution to allow visualization of the stream to ensure that the soma and proximal dendrites of recorded neurons were bathed completely by test solution (Oyelese et al. 1995)
. Application of Ringer solution alone, with or without trypan blue, did not result in a change in conductance in recorded neurons (n = 6).
Solutions were designed to block all currents in the recorded neuron except those mediated by GABAA receptors. Thus the standard solutions blocked all nonchloride currents in the following way (Table 1): 1) TTX was added to the bath solution to block Na+ currents; 2) the bath solution contained no Ca2+ but did contain EGTA; 3) Cs+ and TEA were included in the electrode solution to block outward K+ currents; 4) TEA (2 mM) was included in the bath solution to block inward K+ currents; and 5) CNQX and AP-5 were included in the bath solution to block glutamate receptors.
In some individual experiments (described in the figure legends), this recipe was modified in the following ways: 1) QX-314 (20 mM) was included in the electrode solution, in place of TTX in the bath solution (Connors and Prince 1982)
; 2) Cd2+ was included in the bath solution with 0.5 mM Ca2+ to block Ca2+ influx (Cd2+ solution); 3) TEA was not included in the bath solution in those experiments that increased [K+]o to 12 mM without a change in [Na+]o; or 4) in experiments using tetanus toxin pretreatment, 2 mM CaCl2 was included in the Ringer solution (nl Ca2+). We assumed that these solutions would block GABAB-receptor-mediated responses because GABAB receptors modulate calcium and potassium conductances, which were blocked as just described.
To block vesicular GABA release, parallel experiments were performed in the following ways: the bath solution contained no Ca2+, but did contain EGTA (0 Ca2+ solution), cadmium (100 µM) was added to the bath solution (0.5 mM Ca2+), or cultured neurons were incubated with tetanus toxin (1 µg/ml or
60 nM in culture medium) for 18-30 h before recording (Albus and Habermann 1983
; Monyer et al. 1992
; Pin and Bockaert 1989)
.
Microejection (test) solutions were identical to the bath solution at the time, except for an increase in [K+] and/or a decrease in [Na+]. The osmolarity of the test solution was routinely measured and was always within 1% of the bath solution. The GABAA receptor antagonist picrotoxin (50 µM) and the GABA transporter inhibitors, SKF89776A (20-100 µM) and NO-711 (10 µM), were added only to the bath solution and not to the microejection solution. Bicuculline (1-50 µM) was added to both the test and bath solutions during experiments with the multipuffer technique. In some experiments before adoption of the multipuffer technique in this laboratory, bicuculline was added only to the bath solution. In those experiments, higher concentrations of bicuculline (500 µM) were necessary to achieve comparable degrees of GABA blockade as when lower doses (20 µM) were added to both bath and test solutions.
Data analysis
Responses were analyzed by recording changes in either holding current or whole cell conductance. When measuring holding current, the amplitude of the response (GABA-induced current; iGABA) was calculated as the difference between the holding current at baseline and at the peak of the response. The area under the curve (AUC) of the current response was calculated as the integral of the absolute value of the difference between the measured current and the baseline current
where i(0) = baseline current before pressure microejection and i(
) was the current at the last measured point (at 15 or 30 s, at which time the current had not always returned to baseline).
To estimate the change in conductance when current was only measured at one potential, it was assumed that the current induced by GABA was zero at the calculated ECl
of 0 mV. Thus when the holding potential was
60 mV, the estimated GABA conductance was calculated as
In some experiments, whole cell conductance was determined directly every 0.5-1 s for 15-30 s by voltage clamping neurons at a holding potential of
60 mV and applying 50-ms test pulses to
80 and
100 mV. The slope conductance was calculated from the steady-state current measured at the end of each pulse (Oyelese et al. 1995)
. Conductances induced by test solutions were calculated as the difference between the conductance at the peak of the induced response and the baseline conductance. In some experiments, voltage-clamp pulses to +60 mV were used to directly measure the reversal potential of the induced response. In other cases, reversal potential was determined by extrapolation of the I-V curve of the peak GABA-induced current. The 10-90% rise time was calculated for induced responses using plots of either slope conductance versus time or holding current versus time.
For all results, values are means ± SD unless otherwise stated. Probability values were determined using the Mann-Whitney U test. Data were analyzed using commercially available software, (Excel, Microsoft, Redmond, WA; Origin, Microcal, Northhampton, MA).
 |
RESULTS |
Nipecotate acid induced currents in hippocampal cultures due to carrier-mediated GABA release
In hippocampal slices, rapid application of NPA induces heteroexchange release of GABA via the GABA transporter (Szerb 1982)
, which then activates GABA receptors on neighboring neurons (Honmou et al. 1995
; Solis and Nicoll 1992)
. Measurement of these responses requires the following conditions to be true: neurons must be present with GABAA receptors, neurons with GABAA receptors must lie in close proximity to other neurons and/or glia that possess GABA transporters, free cytosolic GABA, available for release, must be present in neurons and/or glia that have GABA transporters, and the extracellular space must be restricted enough to prevent rapid diffusion of released GABA so that it reaches a sufficient concentration to activate GABAA receptors. These conditions exist in slices (Honmou et al. 1995
; Solis and Nicoll 1992)
, but it was not clear whether they would exist in tissue culture. We wanted to use tissue culture to study carrier-mediated GABA release, because it permitted better control of extracellular ion concentrations, and neurons could be incubated with tetanus toxin for 24 h to block vesicular release. However, to use this approach, it was first necessary to demonstrate that the above conditions existed in culture.
In Ringer solution containing TTX, AP-5, and CNQX, there was spontaneous miniature synaptic activity. When QX-314 was used in the recording electrode in place of TTX in the bath, frequent, large postsynaptic potentials also were recorded. Both forms of spontaneous activity were blocked completely by bicuculline, 0 Ca2+ solution, or tetanus toxin pretreatment. When solutions were used with symmetric [Cl
] (ECl
= 0 mV) and a holding potential of
60 mV, this activity was depolarizing. However, under physiological conditions in adult hippocampal neurons, this GABAergic input would be expected to be hyperpolarizing and inhibitory.
When GABA (100 µM) was applied by pressure microejection to cultured hippocampal neurons for 0.5 s in 0 Ca2+ solution, a large inward current was induced in neurons voltage clamped at
60 mV (ECl
= 0 mV; n = 6; Fig. 1A). This current resulted from a rapid increase in whole cell conductance of 36.4 ± 14 nS (10-90% rise time = 0.47 ± 0.26 s; n = 6), which quickly returned to baseline (Fig. 1B). Smaller currents were induced by application of GABA (10 µM) and were blocked reversibly by bicuculline (50 µM; n = 2). The reversal potential of the response to GABA was approximately equal to the calculated Nernst potential for chloride (0 ± 1.8 mV; ECl
= 0; n = 5; Fig. 1C).

View larger version (18K):
[in this window]
[in a new window]
| FIG. 1.
Pressure microejection of -aminobutyric acid (GABA) or nipecotate (NPA) activated GABAA receptors on hippocampal neurons in culture. A: GABA (100 µM; 0.5 s; bar) induced an inward current. Shown is holding current at the potential of 60 mV, with ECl = 0 mV. (Same conditions were used in all current traces in subsequent figures). B: GABA (100 µM; 0.5 s; top bar) and NPA (10 mM; 3.0 s; bottom bar) both induced an increase in whole cell conductance. Conductance was measured every 0.5 s for GABA and every 1.0 s for NPA. C: I-V relationship for GABA response when ECl = 0 mV. D: I-V relationship for NPA response when ECl = 0 mV. E: I-V relationship for NPA response when ECl = 60 mV. Lines in C-E are least squares fit. Experiments performed in 0 Ca2+ solution.
|
|
Rapid application of NPA also activated GABAA receptors. Pressure microejection of NPA (10 mM) for 1-3 seconds in either 0 Ca2+ (n = 6) or Cd2+ (n = 2) solution induced an inward current. This current was also due to an increase in whole cell conductance, but in this case it was slower in onset than with GABA (10-90% rise time = 3.37 ± 1.98 s, n = 6; Fig. 1B) and had a more gradual return to baseline. The mean baseline conductance in these cells was 4.8 ± 2.7 nS, with a mean increase of 12.7 ± 5.7 nS in response to NPA application (n = 8). The response to NPA was reversibly blocked by picrotoxin (50 µM, n = 6), and the reversal potential of the NPA response was close to the Nernst potential for chloride (Fig. 1D: 4.0 ± 8.5 mV when ECl
= 0 mV, n = 2; Fig. 1E:
56.2 ± 4.3 mV when ECl
=
60 mV, n = 6). These responses were similar to those previously obtained in hippocampal slices using the same concentration of NPA (10 mM) (Honmou et al. 1995
; Solis and Nicoll 1992)
. NPA-induced responses are due to activation of GABAA receptors from heteroexchange release of GABA and not due to direct effects on the GABAA receptor (Solis and Nicoll 1992)
. These results indicated that cell culture is a useful model system for detecting electrophysiological responses due to carrier-mediated GABA release.
GABAA receptors were activated in response to elevation of [K+]o in the absence of extracellular Ca2+
During high-frequency neuronal firing, extracellular potassium rises. Even modest firing rates can lead to an increase in [K+]o to >12 mM (Krnjevic et al. 1980
; Somjen and Giacchino 1985)
, which may contribute to the pathophysiology of seizures (Fisher et al. 1976)
. We hypothesized that depolarization of neurons and/or glia caused by a rise in [K+]o to 12 mM would be sufficient to induce significant amounts of carrier-mediated release of GABA.
When [K+]o was increased near the soma and proximal dendrites of a recorded neuron to 12 mM for 3 s with microejection of test solution in the absence of extracellular calcium (n = 16), a large inward current was induced of 1.14 ± 0.4 nA (holding potential
60 mV; ECl
= 0 mV; Fig. 2A, left). Assuming the reversal potential of this response was equal to the calculated Nernst potential for Cl
, the magnitude of the induced current would correspond to an increase in whole cell conductance of 18.9 ± 6.8 nS. Similar responses were obtained in the presence of 100 µM Cd2+ (n = 12). In these cells, the rise time of the response to 12 mM [K+]o was slower than that produced by direct application of GABA (mean 10-90% rise time = 5.90 ± 2.80 s; n = 12) but was similar to that produced by NPA.

View larger version (19K):
[in this window]
[in a new window]
| FIG. 2.
GABAA receptors were activated on hippocampal neurons by an increase in [K+]o to 12 mM. A, left: pressure microejection of 12.0 mM [K+]o (3 s bar) induced an inward current after block of vesicular GABA release by removal of extracellular calcium. Middle: bicuculline (20 µM) blocked the response. Right: bicuculline washout. B: concentration-response curve for bicuculline block of the response to 12 mM [K+]o. Error bars = SD (1 µM, n = 5; 10 µM, n = 7; 20 µM, n = 11; 50 µM, n = 4). C: I-V relationship for response to 12 mM [K+]o. Top: I-V curve when ECl = 0 mV. Bottom: I-V curve when ECl = 60 mV. Lines in C are least squares fit. All experiments performed in 0 Ca2+ solution.
|
|
The response to increased [K+]o was blocked reversibly by bicuculline (1-50 µM) in a dose-dependent manner (Fig. 2, A and B). The reversal potential of the response induced by 12 mM [K+]o was approximately equal to the calculated Nernst potential for chloride (-3.3 ± 2.9 mV when ECl
= 0 mV, n = 3;
56.5 ± 4.2 mV when ECl
=
60 mV, n = 8; Fig. 2C). These results indicated that elevated [K+]o, in the absence of extracellular calcium or presence of cadmium, induced nonvesicular release of GABA, which then activated GABAA receptors.
Elevation of [K+]o also activated GABAA receptors after tetanus toxin treatment
Tetanus toxin blocks synaptic vesicle exocytosis due to proteolytic cleavage of synaptobrevin (Schiavo et al. 1992)
. To provide further evidence that GABA-receptor-mediated responses induced by 12 mM [K+]o were a result of nonvesicular GABA release, cultured cells were preincubated with tetanus toxin (1 µg/ml) for 18-30 h before recording (Albus and Habermann 1983
; Monyer et al. 1992
; Pearce et al. 1983
; Pin and Bockaert 1989)
. This protocol resulted in complete elimination of spontaneous large and miniature IPSCs and excitatory postsynaptic currents (EPSCs; n = 3; data not shown).
When we used tetanus toxin to block vesicular exocytosis, the responses were similar to those when Ca2+ influx was blocked. These experiments were performed using normal Ca2+ Ringer solution. In neurons preincubated with tetanus toxin, application of 12 mM [K+]o for 3 s induced large inward currents (590 ± 240 pA, Em =
60 mV, ECl
= 0 mV, n = 12; Fig. 3B). In contrast, currents induced by 12 mM [K+]o in neurons that had not been treated with tetanus toxin (Fig. 3A, sister culture dishes) were 2.4 times larger (1.4 ± 5.3 nA, n = 5) than those in tetanus toxin-treated neurons. The response to 12 mM [K+]o was blocked by bicuculline (500 µM in bath only) in control (n = 3; Fig. 3A) and in tetanus toxin-treated (n = 4; Fig. 3B) neurons. Comparison of the responses after blockade of vesicular exocytosis with the responses resulting from both vesicular and nonvesicular release (Fig. 3C) suggested that nonvesicular release makes up a significant fraction of the total release induced by depolarization.

View larger version (16K):
[in this window]
[in a new window]
| FIG. 3.
Nonvesicular GABA release comprised a large fraction of total GABA release in response to 12 mM [K+]o. A: application of 12 mM [K+]o (3 s bar) in "normal Ringer" solution containing 2 mM CaCl2, plus tetrodotoxin (TTX), (±)-2-amino-5-phosphonopentanoic acid (AP-5), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). This elicited a response in control (without tetanus treatment) hippocampal neurons. In this case, both vesicular, and nonvesicular GABA release would occur. Responses (1.4 ± 0.53 nA; n = 5) were reversibly blocked by bicuculline (500 µM in bath only). B: GABAA-receptor-mediated currents (0.59 ± 0.24 nA; n = 12) also were induced in cells pretreated with tetanus toxin and were reversibly inhibited by bicuculline (500 µM in bath only). C: comparison of response to GABA release induced by 12 mM [K+]o in tetanus toxin-treated cells and in controls. Control cells: 20.1 ± 4.3 (SE) nA·s; n = 5. Tetanus toxin-treated cells: 7.5 ± 0.9 (SE) nA·s; n = 12. All data were collected using paired recordings from cells of the same age in culture. *P < 0.05, Mann Whitney U test.
|
|
GABAA receptors were activated by reversal of the transmembrane sodium gradient
Because the GABA transporter is both electrogenic and sodium dependent, we hypothesized that a change in the transmembrane sodium gradient also would induce sufficient carrier-mediated GABA release to produce measurable postsynaptic responses. During high-frequency firing and seizures, [Na+]i increases while [Na+]o decreases. There is evidence that efflux of GABA through the transporter can be induced by a rise in [Na+]i (Cammack et al. 1994)
. However, using pressure microejection of solutions to the outside of cells, it is not possible to replicate experimentally the actual changes in transmembrane sodium gradient that occur because [Na+]i can not be rapidly changed. Therefore we instead applied a test solution of 0 mM [Na+]o and 13 mM [K+]o to produce the largest possible driving force for sodium efflux. When this solution was applied for 3 s in the presence of cadmium (100 µM), an inward current of 810 ± 270 pA (n = 23) was induced at a holding potential of
60 mV (ECl
= 0 mV). Similar responses were obtained in 0 Ca2+ solution (Fig. 4A). Typical of responses induced by application of 12 mM [K+]o alone, the response was relatively slow in onset (10-90% rise time = 4.88 ± 1.60 s). Assuming the reversal potential was equal to the Nernst potential for Cl
, the induced current would correspond to an increase in whole cell conductance of 13.0 ± 4.8 nS.

View larger version (15K):
[in this window]
[in a new window]
| FIG. 4.
Reversal of the transmembrane Na+ gradient alone or combined with K+-induced depolarization also resulted in activation of GABAA receptors. A: comparison of response to GABA (10 µM, 1 s; top bar), 12 mM [K+]o alone (4 s, bottom bar), application of 0 mM [Na+]o with 13 mM [K+]o (4 s, bottom bar), and 1.0 mM [Na+]o alone (4 s, bottom bar). Recordings were obtained from different neurons that were not aged-matched. All responses were obtained in 0 Ca2+ Ringer solution. B: inward current induced by 0 [Na+]o/13 mM [K+]o was due to an increase in whole cell conductance. Experiment performed in 0 Ca2+ Ringer solution. Response to 0 [Na+]o/13 mM [K+]o was reversibly blocked by bicuculline (500 µM in bath only). C: reversal potential of the induced response was approximately equal to ECl . I-V relationship for response to 0 [Na+]o/13 mM [K+]o solution. Top: I-V curve with ECl = 0 mV; bottom: I-V curve with ECl = 60 mV.
|
|
The conductance change induced by 0 [Na+]o and increased [K+]o also was measured directly by using voltage-clamp pulses from
60 to
80 mV and
100 mV. When a solution with 0 mM [Na+]o and 13 mM [K+]o was applied by pressure microejection for 1 s in 0 Ca2+ bath solution (n = 18), an increase in whole cell conductance of 7.08 ± 6.74 nS occurred (Fig. 4B) from a baseline conductance of 6.27 ± 3.70 nS. The increase in conductance was blocked reversibly by bath application of picrotoxin (50 µM; n = 4) or bicuculline (500 µM in the bath only; n = 2; Fig. 4B). The reversal potential of the response to 0 [Na+]o and increased [K+]o was near the Nernst potential for Cl
(1.9 ± 9.23 mV when ECl
= 0 mV, n = 8;
56 ± 3.5 mV when ECl
=
60 mV, n = 8; Fig. 4C).
Responses also were induced when cells were exposed to 1.0 mM [Na+]o alone (n = 5). The responses to 1.0 mM [Na+]o were smaller than those to 12 mM [K+]o, and slower in onset (Fig. 4A). These results suggested that depolarization was a more potent stimulus for reversal of the GABA transporter than a decrease in [Na+]o. In retrospect, because of the effect of a rise in [Na+]i on carrier-mediated GABA efflux, a decrease in [Na+]o to 0 mM may not be the maximal Na+ gradient stimulus. Instead, a sudden rise in [Na+]i might be more effective but could not be accomplished with the pressure microejection approach used here.
GABA receptor activation was prevented by blockade of the GABA transporter
The responses obtained with changes in [K+]o and [Na+]o were apparently due to release of GABA from neighboring neurons and/or glia with subsequent activation of GABAA receptors on the recorded neuron. Thus the recorded neuron served as a sensitive, functionally relevant biological assay of GABA release (Fig. 5A). Vesicular release is calcium dependent and sensitive to tetanus toxin and would be blocked under the conditions of these experiments. To provide further evidence that the release of GABA was due to reversal of the GABA transporter, we examined the effect of GABA transporter antagonists on the evoked responses.

View larger version (36K):
[in this window]
[in a new window]
| FIG. 5.
GABA-mediated responses induced by 12 mM [K+]o alone or 0 [Na+]o/13 mM [K+]o were blocked by the GABA transport inhibitors 1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride (SKF89976A) and 1-(2-{[(diphenylmethylene)amino]oxy}ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NO-711). A: schematic model for the mechanism of induced responses. Recorded neuron acted as a functionally relevant, biological assay of GABA released from neighboring neurons. B: schematic of the theoretical effect of GABA transporter blockade on alternative mechanisms of GABA release. If the response was due to carrier-mediated release, transporter antagonists would decrease synaptic GABA concentrations and postsynaptic responses. In contrast, if recorded GABA responses were due to incomplete blockade of vesicular release or nonspecific leakage, synaptic GABA levels and postsynaptic responses would increase in the presence of GABA transporter blockers. C: 2 responses to application of 0 [Na+]o/13 mM [K+]o Ringer solution obtained in the same neuron. Bottom: control response. Top: response in same cell after application of SKF89976A (40 µM) for 8 min. D: superimposed recordings from 2 different neurons from a single culture dish. Response to 12 mM [K+]o in a control neuron is compared with that in a neuron exposed to NO-711 for 15 min. E: SKF89976A decreased the response to 0 [Na+]o/13 mM [K+]o. Plotted is the ratio, expressed as a percentage, of the area under the curve (AUC) of the response at 8 min to the response at baseline (t = 0) for control cells (108 ± 28%; n = 10), and for SKF89976A-treated cells (54 ± 15%; n = 12). ***P = 0.005, Mann Whitney U test. Data pooled for all 3 concentrations of SKF89976A. F: mean response (AUC) in 5 control neurons (9.0 ± 1.0 nA·s; mean ± SE) vs. 6 cells pretreated with NO-711 (10 µM; 5.1 ± 0.7 nA·s; mean ± SE). Each recording was paired with a matched control neuron from the same culture dish. **P < 0.025, Mann Whitney U test. Experiments in C and E performed in Cd2+ Ringer solution. Data in D and F from tetanus toxin-treated cells using modified Ringer solution with 2 mM CaCl2 and with QX-314 (20 mM) in the patch electrode instead of TTX in the bath.
|
|
If the observed responses to changes in [K+]o and [Na+]o resulted from carrier-mediated GABA release, it would be predicted that application of GABA transporter antagonists would decrease the responses. Alternatively, if the observed responses resulted from residual, unblocked vesicular release, nonspecific leakage, or some other undefined form of GABA release, then blockade of the GABA transporter would lead to an increase in the response because the GABA transporter would clear the extracellular space of this released GABA (Fig. 5B).
SKF89976A is a highly specific, noncompetitive GABA transporter antagonist that previously has been used to study the electrophysiological response to carrier-mediated GABA release induced by NPA application (Solis and Nicoll 1992)
. SKF89976A previously has been used at concentrations from 10 to 100 µM (Belhage et al. 1993
; Cammack et al. 1994
; Mager et al. 1993
; Solis and Nicoll 1992
; Taylor and Gordon-Weeks 1991)
to obtain block of GABA transport. SKF89976A and the other noncompetitive GABA transporter antagonists tiagabine and NO-711 are highly lipid-soluble drugs and have a slow time to peak effect (Solis and Nicoll 1992)
and are difficult to reverse after application (Thompson and Gahwiler 1992)
. Therefore, the following approach was used. Recordings were made in Cd2+ Ringer solution. A baseline response was induced by pressure microejection of 0 [Na+]o, 13 mM [K+]o solution. Then the GABA transport inhibitor SKF89976A (20 µM, n = 1; 40 µM, n = 5; or 100 µM, n = 6) was applied for 8 min, and a second response was induced. When this was done, the area under the curve of the induced current response was reduced to 54% ± 15% of the initial response (n = 12; Fig. 5, C and E). Rundown of GABAA receptors of approximately this amount can occur during whole cell recording but typically does not begin until >15 min of recording (Chen et al. 1990
; Honmou et al. 1995)
. However, to be certain that the observed reduction of the response was not a result of rundown, we used paired recordings from different neurons in the same dishes as controls before using the same dish for recording from neurons to which SKF89976A was applied. When this was done, the response in these control recordings to the second application of 0 [Na+]o, 13 mM [K+]o was 108% ± 28% (P > 0.1; n = 10) of the first response 8 min earlier (Fig. 5E). When the change in response over the first 8 min of each recording was compared between the SKF89976A-treated and control groups, the decrease of the response in the treated cells was significantly different than the control group (P = 0.005). The mean response at eight minutes for only those cells treated with 40 µM SKF89976A was 53.6 ± 28% of the first response (n = 5), which also was significantly different than for the control group (P = 0.004), as was the mean response for only those cells treated with 100 µM SKF89976A (58.2% ± 12%; n = 6; P = 0.004). The response decreased to 36% of control in one cell treated with 20 µM SKF89976A. There was no significant difference in the response between cells treated with 40 µM or 100 µM SKF89976A.
A different approach was used to verify these results using a second GABA transporter antagonist, NO-711. NO-711 is a noncompetitive GABA transporter antagonist that has been suggested to be relatively selective for neuronal GABA transporters (Suzdak et al. 1992)
. Responses were induced by 12 mM [K+]o in tetanus toxin-treated cultures with normal Ringer solution with 2 mM Ca2+. The responses to 12 mM [K+]o were measured immediately after establishing stable whole cell recordings (n = 5). These responses were compared with those of neurons in the same culture dishes after NO-711 (10 µM) had been applied for
15 min before beginning whole cell recording (n = 6). In cells treated with tetanus toxin in which NO-711 had been applied, the response to 12 mM [K+]o was reduced significantly compared with controls (57.6 ± 4.6 % of control; Fig. 5, D and F).
Prolonged exposure to high potassium led to decay of the GABA response
Brief exposure to rapid increases in [K+]o induced large, calcium-independent, GABAA-receptor-mediated responses in cultured hippocampal neurons, which peaked over ~5-6 s and gradually returned to baseline during the next 20-30 s. To characterize the response to sustained increases in [K+]o, experiments were repeated with application of 12 mM [K+]o for 30 s to 1 min. This induced a peak response within 5-10 s, but despite continued exposure to 12 mM [K+]o, the response would rapidly decrease before the end of the stimulus (n = 6; Fig. 6). Note the difference in holding current at the end of the pulse and the baseline at the end of the trace.

View larger version (13K):
[in this window]
[in a new window]
| FIG. 6.
Time course of the response to prolonged (1 min) application of 12 mM [K+]o. Experiment in 0 Ca2+ solution. Note the fading of the response with prolonged application, such that the end of the response is only ~200 pA greater than the baseline.
|
|
 |
DISCUSSION |
Mechanism of response induced by elevated [K+]o
We have demonstrated that an increase in [K+]o with or without a decrease in [Na+]o induced a large increase in conductance in cultured hippocampal neurons with a reversal potential equal to the Nernst potential for chloride. Antagonists of GABAA receptors reversibly blocked the conductance, indicating that it was due to evoked release of GABA. The response occurred after blockade of vesicular release by removal of Ca2+, addition of Cd2+, or preincubation with tetanus toxin. Specific antagonists of the GABA transporter decreased the response. We conclude that this GABAA-receptor-mediated response was due to carrier-mediated GABA release from neighboring neurons and/or glia induced by changing the electrochemical gradient to favor reverse operation of the GABA transporter.
Blockade of calcium influx (Del Castillo and Engbaek 1954
; Pin and Bockaert 1989)
and tetanus toxin (Albus and Habermann 1983
; Monyer et al. 1992
; Pin and Bockaert 1989)
commonly are used to prevent vesicular release. In our hands, the loss of spontaneous IPSCs and EPSCs indicated that these manipulations were effective. However, one could interpret the incomplete block of responses by NO-711, and SKF89976A as consistent with a mixture of carrier-mediated release and residual vesicular GABA release. We considered this possibility, but others have shown incomplete block by these agents. For example, the GABA transport inhibitor SKF89976A (20 µM) only produces 50-60% inhibition of postsynaptic responses to carrier-mediated GABA release induced by NPA (Solis and Nicoll 1992)
. SKF89976A (10-100 µM) is also only partially effective in blocking calcium-independent release of GABA from rat forebrain synaptosomes (70-80% inhibition), and rat forebrain growth cones (60-75% inhibition) (Taylor and Gordon-Weeks 1991)
. Although SKF89976A can result in nearly complete blockade of GABA transporter currents mediated by GAT-1 expressed in Xenopus oocytes (Quick et al. 1997)
, hippocampal cultures contain multiple isoforms of the GABA transporter and multiple neuronal and glial cell types, which have different sensitivities to antagonists (Clark and Amara 1994
; Clark et al. 1992
; Larsson et al. 1988
; Schousboe and Westergaard 1995)
. In addition, our electrophysiological assay detected GABA released near postsynaptic receptors and may be very sensitive to small amounts of GABA released locally. This portion of released GABA may be less sensitive to GABA transporter antagonists and not be easily detected by biochemical assays of GABA in the bulk bath solution.
If the responses we observed were a combination of carrier-mediated release and vesicular release, blockade of the transporter would lead to enhancement of the vesicular portion due to blockade of reuptake (Krogsgaard-Larsen 1980
; Rekling et al. 1990
; Thompson and Gahwiler 1992)
, and inhibition of the carrier-mediated portion due to blockade of reverse transport. The minimum possible contribution from carrier-mediated release then would be approximately half of the observed 7-19 nS responses, still a substantial effect. However, we favor the conclusion that the entire response to 12 mM [K+]o resulted from carrier-mediated release and that incomplete block by the GABA transporter antagonists SKF89976A and NO-711 was consistent with what is expected from these agents (Fichter et al. 1996
; Solis and Nicoll 1992
; Taylor and Gordon-Weeks 1991)
.
We also considered the possibility that the responses evoked by [K+]o resulted from direct measurement of transporter currents. The kinetics were similar to those of transporter currents during uptake of exogenously applied GABA in skate retinal horizontal cells (Malchow and Ripps 1990)
. However, our results were not consistent with direct measurement of transporter currents for the following reasons. 1) Bicuculline and picrotoxin do not block transporter currents (Malchow and Ripps 1990)
. 2) Although transporter currents are dependent on [Cl
], their I-V curve is usually not linear, and their reversal potential does not follow ECl
(Cammack et al. 1994)
. 3) Transporter currents can be measured when present in high abundance, such as after transfection into HEK293 cells (Cammack et al. 1994)
or in skate retinal horizontal cells (Malchow and Ripps 1990)
, but even in those cases the currents are small (<100 pA). The currents we measured were too large to be transporter currents in hippocampal neurons. 4) The recorded neuron was voltage clamped, making it unresponsive to the depolarizing effect of 12 mM [K+]o, and eliminating any change in driving force for the transporter. 5) The whole cell patch-clamp electrode solution contained no Na+ and no GABA, which are required for the GABA transporter to carry an outward current (Cammack et al. 1994)
. This was not consistent with our results showing reversal of the current. Thus the similarity of the kinetics of our responses and transporter currents likely reflect the fact that our responses were an indirect measure of GABA efflux via the transporter. This observation also provides further support for the hypothesis that these responses were due to carrier-mediated GABA release.
Source of carrier-mediated GABA release
The carrier-mediated GABA release occurred in the immediate vicinity of GABAA receptors on the recorded neurons. It is theoretically possible that GABA released from a cell could stimulate GABA receptors on itself. However, for the reasons mentioned above, GABA would not have been released from the recorded neuron using our experimental approach. Thus under these experimental conditions, GABA came from neighboring cells. The current data do not differentiate between neurons, glia, or both as the source of this GABA release. Carrier-mediated GABA release can occur from glia (Gallo et al. 1991)
as well as from neurons, so it is possible that either cell type is involved. The intracellular store of GABA is presumably a cytosolic pool, rather than the vesicular pool.
Magnitude of responses induced by carrier-mediated GABA release
The postsynaptic conductance change induced by 12 mM [K+]o was often >10 nS. These were very large responses for these neurons with mean resting conductance of 6.3 nS. In comparison, direct application of 100 µM GABA for 0.5 s resulted in an increase in conductance of 36.4 ± 14 nS, which is comparable with previous studies where application of 10 µM to 1 mM GABA for 0.5 s induced a conductance of 5-50 nS in hippocampal neurons (Huguenard and Alger 1986)
. In contrast to the conductance induced by direct application of GABA, the conductance change due to carrier-mediated GABA release was much longer in duration. These large and long conductance changes would be expected to have major functional effects.
The response to 12 mM [K+]o in tetanus toxin-treated cells was 42% of the response in control cells. This indicates that nonvesicular GABA release was a significant fraction of total (vesicular + nonvesicular) release. Previous biochemical assays of calcium-independent and tetanus toxin-insensitive GABA release have indicated that ~25% of release induced by 56 mM [K+]o was nonvesicular (Pin and Bockaert 1989)
. Whereas those results were assumed to reflect GABA released diffusely, without necessarily having electrophysiological effects, the current results suggest that is not the case. Thus nonvesicular release may play a more important functional role in response to elevations in [K+]o than was recognized previously.
Threshold for reversal of the GABA transporter
It generally is accepted that GABA transporters carry two Na+ ions and one Cl
ion (Erecinska 1987
; Lester et al. 1994
; Mager et al. 1993)
with each molecule of GABA. Because GABA is a zwitterion with no net charge at physiological pH, GABA transporters are electrogenic, carrying one net positive charge with each translocation cycle. Thus it would be predicted that GABA transporters would operate in reverse during depolarization or when the inward Na+ gradient is decreased. That this is the case has been demonstrated in a variety of preparations (Belhage et al. 1993
; Bernath and Zigmond 1988
; Cammack et al. 1994
; Mager et al. 1993
; Moscowitz and Cutler 1980
; Pin and Bockaert 1989
; Schwartz 1982)
.
It is difficult to theoretically predict just how easily GABA transporters can be induced to operate in reverse. In the case of GAT-1, transport is affected by both membrane potential and the Na+ gradient, but the behavior of the transporter is more complex than suggested above. Recent work demonstrates that the stoichiometry is not fixed (Cammack et al. 1994)
. An "uncoupled" current can flow in the absence of GABA, and the ratio of Na+, Cl
, and GABA can be variable. Reversal of the GABA transporter is also more sensitive to [Na+]i than would be predicted based on linear dependence on the sodium gradient.
The sensitivity of the GABA transporter to K+-induced depolarization was surprising. In previous studies, 25-56 mM [K+]o was used to induce Ca2+-independent release (Belhage et al. 1993
; Pin and Bockaert 1989
; Taylor and Gordon-Weeks 1991
; Yazulla and Kleinschmidt 1983)
. In contrast, we found that carrier-mediated release also can be induced with a physiologically relevant increase in [K+]o to only 12 mM. The depolarization of surrounding cells by this increase in [K+]o would not result in a significant increase in [Na+]i in those cells because most experiments were performed in the presence of TTX and antagonists of glutamate receptors. Thus the response was apparently a result of only a change in membrane potential. Complete removal of [Na+]o, which might be expected to lead to an even greater amount of GABA release, had a relatively small effect. Our current knowledge of the properties of the transporter does not allow us to predict these experimental observations in advance. To understand these effects of depolarization and changes in [Na+], it will be important to more fully define the relationship between GABA flux, GABA transporter currents, membrane potential, [Na+]o, and [Na+]i among the different isoforms of the transporter, and in different cell types.
Physiologic relevance
Previous studies of carrier-mediated release have primarily used biochemical assays of GABA efflux (Belhage et al. 1993
; Bernath and Zigmond 1988
; Larsson et al. 1983
; Moscowitz and Cutler 1980
; Pin and Bockaert 1989
; Schwartz 1982
; Yazulla and Kleinschmidt 1983)
or direct measurement of GABA transporter currents (Cammack and Schwartz 1993
; Cammack et al. 1994
; Mager et al. 1993
; Malchow and Ripps 1990)
. However, assaying efflux of GABA or measuring transporter currents leaves two questions unresolved. Is GABA released at a site where it can activate GABA receptors? Is the concentration of GABA released near receptors sufficiently high to have an electrophysiological effect?
Although release of GABA by reverse transport has been demonstrated clearly, it has not been widely recognized as a form of release that is physiologically relevant. Increases in [K+]o to levels of ~12 mM have been measured both during seizures, as well as high-frequency neuronal firing (Fisher et al. 1976
; Krnjevic et al. 1980
; Somjen and Giacchino 1985)
. Under these conditions, it would be predicted that an increase in [Na+]i also would occur, which would facilitate reverse transport. Such a mechanism for GABA release may offer advantages during periods of high energy utilization, such as burst firing or seizures, because it relies only on the Na+ and K+ gradients and not on a continuous supply of ATP. Under these conditions, GABAergic vesicular exocytosis might be reduced, whereas carrier-mediated GABA release would be enhanced, possibly serving an autoprotective mechanism.
The mechanism of the decrease in GABA response with prolonged exposure to 12 mM [K+]o (Fig. 6) is unclear but may be due to desensitization of the GABAA receptor or depletion of the cytosolic pool of GABA available for carrier-mediated release. This result suggests that carrier-mediated release may be greatest during brief periods of depolarization, as in bursts of high-frequency firing. Thus carrier-mediated release may contribute less to GABA-mediated inhibition during conditions of prolonged depolarization, as in stroke or status epilepticus.
Carrier-mediated release potentially could occur under physiological conditions for other neurotransmitter transporters. Glutamate transporters are also electrogenic and sodium dependent, and have been shown to reverse in vitro (Eliasof and Werblin 1993
; Nicholls and Attwell 1990)
. However, the stoichiometry is different for the two types of transporter, and there may be a differential threshold for reverse operation. Elucidation of the relative contributions of reverse transport of these two neurotransmitters is important because reversal of GABA transporters could play a protective role, whereas reversal of glutamate transporters could be neurotoxic.
In human patients with temporal lobe epilepsy, electrophysiologic studies have suggested deficiencies in GABAergic inhibition (Knowles et al. 1992
; Williamson et al. 1995)
. Similarly, in vivo microdialysis studies have demonstrated that GABA levels are decreased during seizures in the abnormal hippocampus (During and Spencer 1993)
, possibly due to deficient carrier-mediated GABA release (During et al. 1995)
. The novel anticonvulsant gabapentin enhances carrier-mediated release of GABA induced by NPA in hippocampal slices (Honmou et al. 1995)
. This agent also increases brain GABA levels (Petroff et al. 1996)
, which, if cytosolic, would enhance the driving force for carrier-mediated GABA release. The data presented here are consistent with the hypothesis that carrier-mediated release of GABA plays an inhibitory role during pathophysiological conditions and may be enhanced by some anticonvulsants (Richerson and Gaspary 1997)
.