Contribution of changes in the chloride driving force to the
fading of IGABA in frog melanotrophs
Frank
le Foll,
Olivier
Soriani,
Hubert
Vaudry, and
Lionel
Cazin
European Institute for Peptide Research (Institut
Fédératif de Recherches Multidisciplinaires sur les
Peptides no. 23), Laboratory of Cellular and Molecular
Neuroendocrinology, Institut National de la Santé et de la
Recherche Médicale Unité 413, Unité Associée au
Centre National de la Recherche Scientifique, University of Rouen,
76821 Mont-Saint-Aignan, France
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ABSTRACT |
Chloride redistribution during type A
-aminobutyric
acid (GABAA) currents (IGABA) has been
investigated in cultured frog pituitary melanotrophs with imposed
intracellular chloride concentration ([Cl
]i) in the whole cell
configuration or with unaltered
[Cl
]i using the
gramicidin-perforated patch approach. Prolonged GABA exposures elicited
reproducible decaying currents. The decay of IGABA
was associated with both a transient fall of conductance (gGABA) and shift of current reversal potential
(EGABA). The shift of EGABA
appeared to be time and driving force dependent. In the gramicidin-perforated patch configuration, repeated GABA exposures induced currents that gradually vanished. The fading of
IGABA was due to persistent shifts of
EGABA as a result of gGABA
recovering from one GABA application to another. In cells
alternatively clamped at potentials closely flanking resting potential
and submitted to a train of brief GABA pulses, a reversal of
IGABA was observed after 150 s recording. It is
demonstrated that, in intact frog melanotrophs, shifts of
EGABA combine with genuine receptor desensitization to depress IGABA. These findings strongly suggest
that shifts of EGABA may act as a negative
feedback, reducing the bioelectrical and secretory responses induced by
an intense release of GABA in vivo.
desensitization; intracellular chloride concentration; gramicidin-perforated patch recording
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INTRODUCTION |
IN THE MELANOTROPHS of the frog pituitary pars
intermedia,
-aminobutyric acid (GABA) or GABAA-receptor
agonists exert a transient stimulation followed by a prolonged
inhibition of
-melanocyte-stimulating hormone (
-MSH) release in
vitro (7). By using the gramicidin-perforated patch technique, which
avoids artifactual alterations of the physiological intracellular
chloride concentration
([Cl
]i; see Ref. 8), we have
recently demonstrated that GABA induces an outward chloride flux from
the intracellular compartment containing unusually high
[Cl
]i. The depolarization
that results from the outward current triggers the activation of
voltage-operated sodium and calcium channels, leading to the initial
stimulation of
-MSH secretion. The delayed inhibitory phase of the
secretory response likely originates from the strong shunting effect
that accompanies the depolarization induced by high micromolar GABA
concentrations and causes cessation of firing (17). Factors affecting
the efficacy of the GABAergic transmission are of outstanding
importance in the regulation of
-MSH secretion because they
determine which of the effects, i.e., the depolarization or the
resistive shunt, predominantly controls the hormone release. In the
frog melanotrophs, the GABAA receptor is subject to
extracellular positive or negative modulation by benzodiazepines,
barbiturates, and neuroactive steroids (18-20). In addition, in
this cell model, the GABAA receptor function is also
controlled, at the intracellular level, by the balance between phosphatase and kinase activities (4).
An alternative or concomitant mechanism of regulation of the GABAergic
transmission is the fading of the response induced by the agonist
itself. In this regard, the time scale of the synaptic events has to be
considered. For agonist exposure persisting for minutes to hours, a
downregulation of receptors is responsible for a reduction of the
number of [3H]muscimol or
[3H]flunitrazepam binding sites (23, 28) due to
a removal of GABAA receptors from the cell surface (5).
During short-term applications of GABA, electrophysiological
experiments have demonstrated that the GABAA current
(IGABA) declines despite the continuous presence of
the agonist (3, 10, 18, 26). This early decay of responsiveness is
commonly called desensitization. Accumulating evidence indicates that
desensitization may involve distinct processes. A time-dependent
decrease of the membrane chloride conductance in the presence of GABA
has been observed in neurons and human embryonic kidney (HEK 293) cells
expressing recombinant GABAA receptors (9, 26, 34, 35).
This phenomenon is a general feature of ligand-gated ion channels that
possess nonconducting desensitized states (13, 21) and seems to be
functionally equivalent to the inactivation of voltage-sensitive
channels. The current decay could also result from a gradual passive
redistribution of chloride between both sides of the cell membrane,
leading to a shift of the IGABA reversal potential
and a reduction of the chloride driving force
(EGABA). Such changes of EGABA
have been described from experiments using suction pipette (2), sharp microelectrode (32), or whole cell patch-clamp (12) recordings in rat
or frog neurons. However, these recording configurations bring some
drawbacks because of the rapid chloride exchange between the pipette
and cell compartments (17), which likely introduces errors in the
evaluation of the magnitude of EGABA displacements. The recently developed gramicidin-perforated patch technique, allowing
patch-clamp recordings while preserving intact the physiological [Cl
]i (1, 8, 16), represents
a valuable method to investigate the ionic mechanisms responsible for
the fading of the IGABA.
Herein, we have combined the standard whole cell and the
gramicidin-perforated patch approaches to assess the relative
contribution of genuine GABAA receptor desensitization and
shifts in EGABA to the fading of the
IGABA in the cultured frog melanotrophs.
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MATERIALS AND METHODS |
Animals and cell cultures.
Adult male frogs (Rana ridibunda; Couétard, Saint-Hilaire
de Riez, France) were used as tissue donors. Frogs were housed in a
temperature-controlled room (8°C) under an established 12:12-h light-day photoperiod (lights on from 6:00 AM to 6:00 PM). The animals
had free access to running water and were maintained in these
conditions for at least 1 wk before use. Animal manipulations were
performed according to the recommendations of the French Ethical
Committee and under the supervision of authorized investigators. Cell
cultures of frog pituitary melanotrophs were prepared as previously
described (18). After anesthetization by immersion in 1% MS-222, the
animals (body weight 30-40 g) were killed by cervical dislocation
and decapitated. Eight neurointermediate lobes were carefully dissected
out under sterile conditions. The tissues were rinsed in Leibowitz' 15 mammalian culture medium diluted 1.5× to match amphibian
osmolality and were supplemented with 1% (vol/vol) of the kanamycin
and the antibiotic-antimycotic solutions. Cells were then enzymatically
dissociated (15 min) in the same medium to which 0.15% protease type
IX and 0.15% collagenase type IA were added. After mechanical
dispersion, cells were centrifuged (60 g) for 5 min and
resuspended four times in the culture medium containing 10% FCS and
antibiotics. Dispersed cells were then plated at a density of 10,000 cells/35-mm culture dish. Cultured cells were incubated at 22°C in
a humidified atmosphere and were used 5-10 days after plating.
Electrophysiological recordings and analysis.
Conventional tight-seal whole cell and gramicidin perforated-patch (8,
17) recordings were carried out at room temperature (20-22°C).One-half hour before
electrophysiological experiments, the culture medium was replaced
by one of the external solutions described in Table
1. During the recording
session, the culture dish was superfused constantly with saline (3 ml/min) via a flow pipe aimed at the cell under study. A bath volume of
500 µl was maintained, with the excess bathing solution being
continuously aspired via a suction needle. Soft glass patch electrodes
were pulled from thin-wall microhematocrit capillaries on a two-step vertical pipette puller (L/M-3P-A; List Medical, Darmstadt, Germany). When filled with one of the pipette solutions (Table 1), the electrodes
had 3-5 M
resistances. For each combination of external and
pipette solutions used, liquid junction potentials were measured and
corrected before gaining the whole cell access (25). In the
gramicidin-perforated patch configuration, because of the complete lack
of chloride permeability, no Donnan equilibrium junction potential was
expected to develop (16). Stock solution of gramicidin was prepared by
dissolving the antibiotic in methanol (10 mg/ml). Pipette tip filling
was accomplished by a brief immersion (1-2 s) in a gramicidin-free
pipette solution. The remainder of the pipette was then backfilled with
the same solution to which gramicidin (100 µg/ml) was added. The
progress of perforation was assayed by monitoring the access resistance
deduced from the amplitude of the capacitive transients in response to
repetitive 10-mV hyperpolarizing steps. The series resistance,
typically 12 M
in the whole cell configuration and 14 M
after 10 min perforation (17), was partially (60-70%) compensated using
the on-board circuitry of the amplifier. GABA was dissolved in the
standard extracellular solution and pressure ejected (2-4 psi)
through a micropipette (2-4 µm tip diameter). To avoid
uncontrolled drug leakage, the ejection pipette was kept away and
brought in close proximity (10-20 µm) to the cell soma just
before microejection. Currents were recorded using an Axopatch 200A
current-to-voltage converter (Axon Instruments, Foster City, CA)
interfaced to a Digidata 1200 (Axon instruments) and were directly
digitized with pCLAMP 6 software for further off-line analysis.
Theoretical equilibrium potentials were computed with the use of the
Nernst equation. Current-voltage (I-V) curves were plotted
after subtracting the baseline obtained just before the agonist
application from the IGABA.
EGABA was defined as the x-intercept of the
I-V curve or of the instantaneous current in a voltage step or
in a voltage-ramp protocol. Slope conductance was determined by linear
regression of the less-rectifying more-conducting part of the
I-V plot. Nonlinear regressions were performed using the
Marquardt-Levenberg algorithm of SigmaPlot 5.0 (Jandel Scientific,
Sausalito, CA). Quantitative data are expressed as means ± SE.
Reagents.
GABA, MS-222, protease type IX, collagenase type IA, Leibovitz L-15
medium, gramicidin D, HEPES, EGTA, ATP potassium salt, TTX, and
tetraethylammonium were purchased from Sigma (St. Louis, MO).
Kanamycin, the antibiotic-antimycotic solutions, and FCS were obtained
from Boehringer Mannheim (Meylan, France). Tissue culture dishes were
supplied by Costar (Cambridge, MA).
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RESULTS |
Whole cell IGABA were recorded from cultured frog
melanotrophs by using either the tight-seal ruptured-patch or the
gramicidin-perforated patch techniques in the voltage-clamp mode. All
of the melanotrophs examined (n = 109) responded to GABA.
Decay of the IGABA in melanotrophs with imposed
[Cl
]i.
In experiments carried out in the whole cell configuration, a prolonged
ejection of GABA (10 µM, 15 s) elicited a current that
rapidly peaked and then gradually decayed to 26.2 ± 1.9% (n = 25) of the maximum amplitude within 15 s (Fig.
1A). The late amplitude was defined
as the residual current at the end of the GABA application. Repeated
15-s GABA ejections at 2-min intervals generated currents with
reproducible peak and late amplitudes (Fig. 1B). Successive
I-V relationships established at 2-min intervals from a single
melanotroph displayed similar EGABA. Moreover, the slope conductances remained unchanged (Fig. 1, C and
D). To monitor the rate at which IGABA
recovered from the fading provoked by a prolonged (15-s) GABA
application, brief test pulses (600 ms) of GABA were delivered at
incremental intervals (5-s steps, Fig. 2A). These test pulses were
supposed to induce no additional current decrease. The exponential
curve fitted to the data yielded a time constant (
) = 12.9 s for
recovery in the present experimental conditions (Fig. 2B).

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Fig. 1.
Currents evoked by repeated administrations of -aminobutyric acid
(GABA) in the whole cell configuration. Recordings were carried out
using a low-chloride solution in the patch pipette [External 1 (E1)/Whole Cell 1 (WC1), chloride equilibrium potential
(ECl) = 75 mV]. A: currents
evoked in a single melanotroph by 5 successive ejections of GABA (10 µM, 15 s; horizontal bars) at 2-min intervals. Holding potential was
0 mV. B: time course of the peak ( ) and late ( ) currents
shown in A. C: three families of currents (1,
2, and 3) evoked by GABA (10 µM, 5 s) at 2-min
intervals from another cell. In each family, membrane potential was set
for 2 min at values ranging from 95 to +65 mV by 20-mV steps.
D: superimposed current-voltage (I-V) curves
corresponding to C. Peak current amplitude was plotted against
the membrane potential. Reversal potentials for families of
currents 1, 2, and 3 were 73 ( ),
82 ( ), and 76 mV (X), respectively.
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Fig. 2.
Fading and recovery from fading of the GABAA current
(IGABA). Currents were recorded in the whole
cell configuration with low internal chloride (E1/WC1,
ECl = 75 mV). Holding potential was 0 mV.
A: superimposed IGABA traces recorded from
a single cell. Series of three successive GABA (10 µM) ejections were
repeated 10 times at 2-min intervals. In each series, a brief pulse of
GABA (600 ms, a) eliciting a control response was followed
within 10 s by a prolonged GABA application (15 s,
b), provoking a current decay. Recovery was monitored by a
brief test pulse of GABA (600 ms, c) applied at incremental
intervals (5-s steps) after the end of the 15-s GABA ejection.
B: time course of recovery. Relative amplitude of the current
evoked by pulse c was expressed as a fraction of the control
current induced by pulse a and was plotted against the
interpulse interval between b and c (n = 6-11). Data were fitted to a monoexponential function, yielding a
time constant of 12.9 s for recovery of the control current.
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In another set of experiments, a protocol was developed to assess the
relative contribution of the decrease of the GABA-dependent membrane
conductance (gGABA) and the shift of
EGABA to the decay of IGABA.
Both parameters were followed throughout a prolonged (25-s) GABA
exposure, by applying repeated voltage ramps at 3.5-s intervals. A very
fast sweep rate (800 mV/s) was employed to minimize the influence of
the ramp on the global current decay. The resulting I-V
relationships were used to determine EGABA and the
slope gGABA (Fig. 3,
A and B). In these experiments, the intrapipette
chloride concentration (33 mM) was close to the physiological
[Cl
]i (27 mM) previously
measured in the frog melanotrophs (17). To verify that the voltage
ramps did not influence the values of
EGABA and
gGABA, control experiments were performed on single cells. In all cells studied (n = 8), the values of
EGABA and
gGABA obtained using voltage ramps
did differ from that measured with voltage steps (data not shown). In
the presence of GABA, a time-dependent decrease of
gGABA occurred. Concomitantly, a gradual shift of
EGABA toward 0 mV was observed (Fig. 3, B
and C). Although IGABA fell down to 33 ± 6% of the peak current, gGABA dropped to 44 ± 6% of the control, and EGABA markedly drifted from
31.5 ± 0.9 to
20.0 ± 3.2 mV (n = 8, Fig.
3, D-F).

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Fig. 3.
Redistribution of chloride ions during the response to GABA in the
whole cell configuration. Melanotrophs were held at 0 mV with a
physiological internal chloride concentration (E2/WC3,
ECl = 31 mV). A: current evoked
by a prolonged GABA application (10 µM, 25 s; horizontal bar).
High-speed depolarizing triangular voltage ramps (800 mV/s, from 0 to
+75 mV and from +75 to 0 mV) followed by mirror-image hyperpolarizing
ramps were applied before (a) and during (b-h) the GABA
application. B: superimposed instantaneous I-V
relationships corresponding to A. The leak current (a)
has been subtracted from the currents recorded during GABA exposure
(b-h). C: enlarged detail of the I-V curves
shown in B. Arrows indicate the zero-current potential for each
I-V curve. D-F: changes in IGABA
(D), GABA reversal potential (EGABA;
E), and GABA conductance (gGABA; F)
occurring during the prolonged GABA administration. Relative current
amplitude and slope conductance in c-h were expressed as a
fraction of their respective values in b. Each point
corresponds to the mean ± SE of data obtained as in A from 8 cells.
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The role of the chloride flux in the decay of IGABA
was further studied for various chloride driving forces. Therefore, the above-described repeated-ramp protocol was used at three different holding potentials (Fig. 4, A-C).
At
80 mV, the decay phase of the GABA-evoked inward current was
accompanied by a decrease of gGABA, which was
associated with a slight shift of EGABA toward more
negative potentials. At
35 mV, a value close to the chloride equilibrium potential (ECl) in the
present recording conditions, GABA failed to elicit any current or
noticeable change in EGABA. Nevertheless, a
time-dependent decrease of gGABA was still
detected. At +30 mV, GABA generated a robust outward current
characterized by a high initial conductance compared with that measured
at negative potentials. Thereafter, gGABA markedly
decayed during GABA exposure. In addition, EGABA
sharply drifted toward 0 mV (Fig. 4, D and E).

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Fig. 4.
Dependence of the changes in EGABA and
gGABA on the holding potential
(Vh) in a single melanotroph. A-C:
recordings were performed in the whole cell configuration with a
physiological internal chloride concentration (E2/WC3,
ECl = 31 mV). Currents were evoked by
prolonged GABA applications (10 µM, 25 s; horizontal bars) at holding
potentials of 80 (A), 35 (B), or +30
(C) mV. High-speed triangular voltage ramps were applied before
and during GABA exposure, as described in Fig. 3. Voltage
ramp pulses ran from 5 to 155 mV (A), +35 to
115 mV (B), and +105 to 45 mV (C).
Resulting instantaneous I-V curves are presented below the
current traces. D-E: changes in ggaba
(D) and EGABA (E) accompanying the
IGABA, at holding potentials of 80 ( ),
35 ( ), or +30 ( ) mV. Relative slope conductance measured
at the different times was expressed as a fraction of the initial
conductance at 80 mV.
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Fading of the IGABA in melanotrophs with
unaltered physiological
[Cl
]i.
In cells studied in the whole cell configuration, chloride exchange
between pipette and cell compartments could distort the time course of
the current. In this respect, the gramicidin-perforated patch technique
provides an alternative approach that allows recordings of
IGABA without imposing
[Cl
]i (1, 16, 17). In
recordings performed in the gramicidin-perforated patch configuration,
a prolonged GABA ejection (10 µM) µM, 15 s) elicited a
decaying current. Repeated 15-s GABA ejections at 2-min intervals
generated currents that gradually vanished (Fig. 5A). Peak and late amplitudes of
IGABA declined with similar time courses (Fig
5B). To establish successive I-V relationships from a
single melanotroph, families of IGABA at increasing
membrane potentials were recorded at 2-min intervals (Fig 5C).
Interestingly, from one family of currents to another, the I-V
curves exhibited an irreversible drift of EGABA
from
25 to 0 mV, without any marked modification in the slope
conductances (Fig. 5D).

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Fig. 5.
Currents evoked by repeated administrations of GABA in the
gramicidin-perforated patch configuration. Recordings were carried out
using a KCl solution in the patch pipette (E1/patch pipette 1 (PP1),
ECl = 0.6 mV), to which gramicidin D (100 µg/ml) was added. GABA was applied after a complete cell membrane
perforation, indicated by stabilization of the capacitive transients
generated by 10-mV hyperpolarizing pulses. A: currents evoked
from a single melanotroph by successive ejections of GABA (10 µM, 15 s; horizontal bars) at 2-min intervals. Holding potential was 80
mV. B: time course of the peak ( ) and late ( ) currents
shown in A. C: three families of currents (1,
2, and 3) evoked by GABA (10 µM, 5 s) at 2-min
intervals from another cell. In each family, membrane potential was set
for 2 min at values ranging from 80 to +80 mV by 20-mV steps.
D: superimposed I-V curves corresponding to C.
Peak current amplitude was plotted against the membrane potential.
Reversal potentials for families of currents 1, 2 and
3 were 31 ( ), 9 ( ), and 0 (X) mV,
respectively.
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To evaluate the relative contribution of changes in
gGABA and EGABA to the decay of
IGABA recorded in the gramicidin-perforated patch
configuration, the repeated-ramp protocol described in Fig. 3 was used.
Under these conditions, the late current amplitude decreased to
28 ± 3% of the peak during a 25-s GABA (10 µM) ejection. Concomitantly, gGABA diminished to 45 ± 2% of
its control value, and EGABA shifted from
34.8 ± 2.2 to
20.9 ± 2.3 mV (n = 8, Fig. 6, A-C). In addition, in all
melanotrophs (n = 5) submitted to three consecutive
repeated-ramp protocols at 2-min intervals, long-lasting and additive
shifts of EGABA were observed. As shown in the
representative example of Fig. 6, D and E, the
EGABA drifted from
39.6 to
29.3 mV,
31.9 to
22.7 mV, and
24.4 to
17.2 mV
throughout the first, the second, and the third GABA ejection, respectively.

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Fig. 6.
Redistribution of chloride ions during the response to GABA in the
gramicidin-perforated patch configuration. Currents were recorded using
a CsCl and gramicidin D solution in the patch pipette (E2/PP2,
ECl = 0.6 mV). Holding potential was 0 mV. High-speed triangular voltage ramps were applied before and during
25-s GABA (10 µM) exposures, as described in Fig. 3. A-C, with changes in IGABA (A),
EGABA (B), and gGABA
(C) occurring during the prolonged GABA administrations.
Relative current amplitude and slope conductance were expressed as a
fraction of their initial values. Each point indicates the mean ± SE
of data obtained from 8 cells. Only one GABA pulse was delivered per
cell under study. D: three currents (1, 2, and
3) were evoked by prolonged GABA applications at 2-min
intervals in a single melanotroph. The corresponding instantaneous
I-V curves focus on the current reversal potentials. E:
changes in EGABA observed during successive
IGABA 1 ( ), 2( ), and
3 ( ) shown in D.
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The persistent drift of EGABA toward more
depolarized potentials observed in intact melanotrophs could be
accounted for by a continuous accumulation of chloride in the
intracellular compartment, through specific transport systems and/or
chloride channels in addition to the GABAA receptor. To
address this issue, a protocol was designed and applied to single cells
(n = 5, Fig. 7). First, three
successive families of IGABA were recorded at 2-min
intervals. EGABA, deduced from the corresponding
I-V relationships, shifted from its native value (
38 mV)
to a more depolarized and stable level (
11 mV). Thereafter, to
lower [Cl
]i, the cell was
submitted to a series of five GABA ejections at
80 mV.
Subsequently, EGABA measured from a first family of IGABA was
35 mV, a value close to that
obtained initially. In a second determination,
EGABA shifted to
10 mV, as already observed at the beginning of the recording session. Finally, after another series of five GABA ejections at
80 mV, again
EGABA (
36 mV) recovered (Fig. 7, A
and C).

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Fig. 7.
Reversibility of the shifts of EGABA in the
gramicidin-perforated patch configuration. A: recordings were
carried out using a KCl and gramicidin D solution in the patch pipette
(E1/PP1, ECl = 0.6 mV). A single
melanotroph was submitted to the voltage-clamp protocol indicated above
the current traces. In this representation of the voltage protocol, the
time scale is not constant. Repeated GABA (10 µM, 5 s) ejections were
performed at 2-min intervals, as indicated by the vertical arrows. The
stimulation procedure included 6 I-V protocols (noted 1 to 6) and two series of 5 successive GABA ejections at
80 mV. B: superimposed I-V curves obtained from
the I-V protocols shown in A. , , , , ,
and correspond to the families of currents 1, 2,
3, 4, 5, and 6, respectively.
C: changes in EGABA during the stimulation
procedure followed in A. The values of
EGABA were deduced from the I-V curves in
B.
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The possible influence of EGABA changes on the
postsynaptic response of melanotrophs to GABA was investigated by using
an experimental procedure closer to physiological conditions. The cell
was alternatively clamped at potentials (
50 and
40 mV) closely flanking the mean resting potential of the melanotrophs (
46.3 ± 1.5 mV, n = 29). A train of brief GABA pulses
(10 µM, 100 ms, 1.1 Hz) was delivered. At
50 mV, the
GABA-evoked inward current was initially large and gradually diminished
over the course of the experiment. In contrast, at
40 mV, the
inward current was markedly weaker and reversed after 150 s recording
(Fig. 8).

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Fig. 8.
Inversion of IGABA elicited by a train of GABA
pulses in a cell alternatively clamped at 2 membrane potentials in the
gramicidin-perforated patch configuration. Current recordings were
performed using a KCl and gramicidin D solution in the patch pipette
(E1/PP1, ECl = 0.6 mV). A single
melanotroph was alternatively held at 50 or 40 mV at 15-s
intervals (top). IGABA (bottom)
were evoked by a train of brief GABA pulses (10 µM, 100 ms) applied
at the frequency of 1.1 Hz (middle). Extended current traces
shown at bottom display inversion of IGABA
at 40 mV in the second part of the recording. Horizontal dashed
lines superimposed to the current traces indicate the zero-current
levels at 40 and 50 mV.
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DISCUSSION |
In the frog melanotrophs bathed in HEPES-buffered media, chloride is
the exclusive charge carrier of the IGABA (17, 20). By using the gramicidin-perforated patch technique, we have recently found that, in this cell model, EGABA is ~10 mV
positive to the resting potential. As a consequence, GABA provokes a
depolarization, triggering action potentials. The voltage change due to
GABA is underlaid by a dramatic increase of the membrane conductance, which shunts the voltage-activated currents. Thus the initial burst of
activity is followed by a shunt-induced abolition of firing, with GABA
clamping membrane potential at EGABA (17). We now
demonstrate that, in frog melanotrophs with unaltered
[Cl
]i,
EGABA is unexpectedly a labile parameter. It
appears that long-lasting shifts of EGABA can
account for a GABAA receptor use-dependent fading of the
response to GABA.
The main purpose of our study was to decipher, in the frog
melanotrophs, the mechanisms involved in the fading of
IGABA occurring in the presence of the agonist. In
the whole cell configuration, GABA generated a decaying current that
was reproducible over repeated agonist applications at 2-min intervals.
This indicates that the parameters (EGABA and
gGABA) controlling the current flow were modified
throughout the agonist exposure and recovered within the delay between
the GABA pulses. The kinetics of recovery (
= 12.9 s) were found to
be similar to that described in neurons (3, 13, 15). Recovery may
involve both true resensitization, i.e., removal of desensitized states
(13), and restoration of the initial transmembrane chloride gradient by
ion diffusion between the patch pipette and the cell interior (17). The
observation that successive I-V curves were superimposed
confirmed that neither the chloride driving force nor the conductance
varied from one GABA application to another. In contrast, by using the
repeated-ramp protocol to follow EGABA throughout a
prolonged GABA exposure, it appeared that EGABA
gradually and transiently drifted during the chloride flow. The shift
of EGABA toward potentials corresponding to lower
driving forces reflects a passive redistribution of chloride, which
leads, together with the decrease of gGABA, to the
fading of IGABA in the continued presence of the
agonist. This observation is in good agreement with previous findings
in rat and frog neurons (2, 12, 32). To verify the role of the chloride
flux in the current decay, the variations of EGABA
and gGABA have been monitored at various holding
potentials. The data reveal that the displacement of
EGABA was more pronounced at increasing chloride driving forces and was inverted when IGABA
reversed. In these experiments, the initial increase or decrease of
gGABA observed at holding potentials of +30 or
80 mV, respectively, was attributed to the voltage sensitivity
of the GABAA receptor channel. Altogether, the present
results demonstrate that, in frog melanotrophs, the GABA-evoked
chloride flux induces transient time- and driving force-dependent
shifts of EGABA.
Because of the relatively rapid ion equilibration between the pipette
and cell compartments, the whole cell recordings may lead to an
underestimation of the magnitude of EGABA changes
during the response to GABA. To avoid such a limitation, the fading of IGABA has been examined using the
gramicidin-perforated patch approach (16, 17). In the
gramicidin-perforated patch recordings, the decay of
IGABA observed during a single 25-s GABA exposure was underlaid by a fall of gGABA and a shift of
EGABA, which were characterized by magnitudes and
time courses very similar to those obtained from whole cell recordings.
Thus we conclude that, in the whole cell configuration, the chemical
access to the intracellular compartment did not alter the transient
changes of EGABA caused by the chloride flux
itself. This probably originates from the fact that chloride exchanges
between pipette and cell compartments are much less intense than
chloride fluxes evoked by GABA through membrane channels. However, when
GABA was repeatedly ejected while the physiological
[Cl
]i remained unaltered,
IGABA gradually vanished from one GABA application
to the next. This response pattern markedly differed from that obtained
in the whole cell recordings in which repeated IGABA were reproducible. Interestingly, in the
gramicidin-perforated patch configuration, successive GABA ejections
resulted in increased shifts of EGABA that
persisted during washout while gGABA recovered. Moreover, the present work demonstrates that the long-lasting redistributions of chloride ions were highly reproducible and mostly
depended on the direction of the evoked chloride flux. Hence the
possible contribution of a resting chloride permeability to the
regulation of [Cl
]i is
likely negligible. Taken together, the above-described observations indicate that, in intact frog melanotrophs,
[Cl
]i is severely altered by
the passive chloride flux through the GABAA receptor
channels and seems not to be subject to any other short-term regulation.
To influence the response to GABA in vivo, the shifts of
EGABA must be driven by deviations of membrane
potential from the native ECl to maintain a
significant chloride flux throughout the presence of GABA in the
synaptic cleft. This implies that GABA released in synapses formed
between hypothalamic nerve endings and melanotrophs activates only a
few channels so that the resistive shunt still remains negligible and
does not result in a voltage clamp of the membrane at
ECl. Accumulating evidence now supports the
occurrence of such a situation in vivo (6, 17, 24, 27, 29). Moreover,
it has recently been proposed that, during an inhibitory postsynaptic
current, a substantial number of postsynaptic receptors
must be exposed to subsaturating concentrations of GABA because of the
time course of GABA release, the action of transmitter clearance
mechanisms, the cleft configuration, and the "spillover" of GABA,
which leads to activation of extrasynaptic GABAA receptors (11). As a result, GABA must actually open only a part of the postsynaptic GABAA receptor channels for relatively long
periods, contrary to that often obtained by rapid jumps of saturating
GABA concentrations (1 mM, 0.5-3 ms) in patch experiments. In
addition, modification of the membrane potential is a very common
mechanism shared by a panoply of neurotransmitters and neuropeptides in frog melanotrophs (for review, see Ref. 33). It has also been shown
that inhibitory postsynaptic potentials are often associated with
depolarizations in neurons (22, 30, 31). These voltage fluctuations
likely contribute to move the membrane potential beyond
ECl. Herein, we demonstrate that variations of
membrane potential in a restricted range (10 mV) spanning the resting
potential, imposed during a train of 100-ms GABA pulses, were
sufficient to induce the reversal of IGABA. This
indicates that crucial changes of EGABA may
occur after small variations of membrane potential and in response to
GABA pulses, which are both relevant to physiological conditions.
The findings of the present work are schematically illustrated in Fig.
9. In the whole cell configuration, the
transient shift of EGABA induced by the chloride
flow combines with the gradual decline of gGABA to
entail the decay of IGABA. The patch pipette solution does not instantaneously clamp but rather slowly buffers the
[Cl
]i. In the
gramicidin-perforated patch configuration, the physiological [Cl
]i remains unaltered, and
the redistribution of chloride persists over the successive GABA
ejections. As a result, the shift of EGABA leads to
a tachyphylaxis phenomenon.

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|
Fig. 9.
Schematic interpretation of the contribution of shifts in
EGABA to the fading of the
IGABA. Decrease of IGABA in the
presence of GABA is ascribed to a "true" desensitization, i.e., a
fall of gGABA, associated with a shift of
EGABA. Because IGABA = gGABA (membrane potential EGABA) and EGABA = ECl, local variations of intracellular chloride
concentration ([Cl ]i) due to
the chloride flux are responsible for a transient reduction of the
chloride driving force, leading to a fading of the current. In whole
cell recordings, the chemical pipette/cell exchange results in a fast,
but not instantaneous, buffering of
[Cl ]i (shaded bands) so that
repeated GABA applications elicit reproducible responses. In contrast,
in gramicidin-perforated patch recordings, redistribution of
chloride between intra- and extracellular compartments
persists and can account for a tachyphylaxis phenomenon. This latter
observation suggests that, in melanotrophs,
[Cl ]i is not subject to a
short-term regulation. ICl, chloride current.
|
|
Among these lines, it must be mentioned that, so far, no information is
available concerning the regulation of
[Cl
]i by pumps, channels, or
any metabolically dependent active transport systems in melanotrophs.
Nevertheless, because [Cl
]i
is less than extracellular chloride concentration, mechanisms responsible for a net chloride extrusion may exist in these cells. In
addition, in the present work, we have intentionally employed HEPES-buffered saline without CO2 equilibration precisely
to eliminate any involvement of HCO
3
in the dynamics of the responses. Thus the shifts of
EGABA can exclusively be attributed to
chloride movements. However, in other studies using different cell
types, GABAA anion channels were shown to display a
permeability ratio of 0.2 to 0.3 for
HCO
3 vs. chloride. In addition, it has
also been reported that HCO
3 is
involved in various transmembrane exchange systems (for review, see
Ref. 14). Then, to study more accurately the physiological role of
EGABA changes, further investigations need to be
carried out using a preparation closer to the in vivo situation, i.e., in the presence of HCO
3 in the
extracellular solution. A detailed comparison between
HCO
3- buffered gramicidin-perforated
patch recordings and the above-reported results would provide
interesting information concerning the role of chloride,
HCO
3, internal pH, and ion exchange systems in the homeostasis of the response to GABA in melanotrophs.
The regulations of IGABA reported herein have some
functional significance. It is speculated that, in physiological
conditions, the melanotrophs can be switched between excitatory and
inhibitory modes of GABA signaling. For instance, the shift of
EGABA could be considered as a negative feedback of
the GABAergic inputs, regulating the amplitude of the response as a
function of the intensity and duration of previous chloride fluxes.
 |
ACKNOWLEDGEMENTS |
We thank Catherine Buquet for excellent technical assistance.
 |
FOOTNOTES |
This research was supported by grants from Institut National de la
Santé et de la Recherche Médicale (U 413), the European Union (Human Capital and Mobility Program; ERBCHRXCT920017), and the
Conseil Régional de Haute-Normandie. F. Le Foll was a recipient of a doctoral fellowship from La Direction Générale de la
Recherche et de la Technologie.
Present addresses: F. Le Foll, Laboratory of Ecotoxicology, University
of Le Havre, 25 rue Philippe Lebon, 76058 Le Havre Cedex, France; O. Soriani: CNRS ERS 1253, Université de Nice Sophia-Antipolis,
Laboratoire Jean Maetz, La Darse BP68, 06238 Villefranche-sur-Mer Cedex
1, France.
Present address and address for reprint requests and other
correspondence: L. Cazin, Laboratoire de Microbiologie du Froid, UPRESS
2123, University of Rouen, 76821 Mont-Saint-Aignan, Cedex, France
(E-mail: lionel.cazin{at}univ-rouen.fr).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 8 June 1999; accepted in final form 27 September 1999.
 |
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