1Department of Biology,
Sacchi, Oscar,
Maria Lisa Rossi,
Rita Canella, and
Riccardo Fesce.
Participation of a Chloride Conductance in the Subthreshold
Behavior of the Rat Sympathetic Neuron.
J. Neurophysiol. 82: 1662-1675, 1999.
The presence of a novel
voltage-dependent chloride current, active in the subthreshold range of
membrane potential, was detected in the mature and intact rat
sympathetic neuron in vitro by using the two-microelectrode
voltage-clamp technique. Hyperpolarizing voltage steps applied to a
neuron held at Below the threshold potential for spike
generation, the neuronal membrane is generally considered to exhibit a
virtually passive electrical behavior: the concept itself of
"resting" potential implies that a relatively fixed equilibrium
value for membrane potential is set by the relative magnitudes of basal
potassium and leakage conductances, and their respective equilibrium potentials.
We have recently shown that the momentary (and past) value of membrane
potential markedly affects neuronal response to artificially imposed
voltage steps as well as to physiological synaptic activation, mostly
via a modification of the inactivation state of
IA potassium conductance. Although
voltage-dependent inactivation of IA
does not directly contribute to determining the membrane potential, it
considerably modifies the neuronal response to synaptic activation and
the shape of the action potential (Sacchi et al. 1998 In this paper we examine in detail the electrical properties of the
intact and mature rat sympathetic neuron "at rest" and demonstrate
the presence of relevant active chloride conductances in the
under-threshold range of membrane potential values. This novel chloride
current can be readily demonstrated over a wide voltage range provided
that the membrane potential is moved, even by a few millivolts, and
that internal chloride concentration is not clamped, as in whole cell
patch-clamp experiments, but remains free to readjust according to the
new voltage level.
Detailed information on membrane chloride conductance and channels has
been obtained primarily from nonmammalian and nonneuronal systems; in
most cases, the chloride permeability proved to be the dominant resting
ion conductance and to play a special role in stabilizing the membrane
potential and thereby determining the passive characteristics of the
membrane. Additional functions have been attributed to chloride
channels activity in cell volume regulation and transepithelial
transport (for review, see Strange et al. 1996 The macroscopic chloride conductance has been characterized in some
detail by several investigators. A hyperpolarization-activated chloride
current, which exhibits inwardly rectifying properties, has been
described in Aplysia neurons (Chesnoy-Marchais
1983 The particular chloride conductance described here in the mature
sympathetic neuron originates physiologically relevant currents and
chloride ion redistribution, and its activation state slowly changes
when the membrane potential of the neuron is displaced. The resulting
modifications in chloride conductance and equilibrium potential modify,
in turn, the resting potential (and input impedance) of the neuron.
Thus the idea arises that the membrane potential of the resting neuron
is not a predetermined electrical property but rather the dynamic
result of its previous history (both in terms of electrical changes and
chloride regulation), and in turn influences neuronal excitability and responsiveness.
All experiments were performed on superior cervical ganglia
isolated from young rats (5-6 wk old; 120-150 g body weight) during urethan anesthesia (1-1.5 g kg The low chloride solutions were made by replacing 136 mM NaCl with
isoosmolar amounts of sodium isethionate or methanesulfonate and were
applied when both microelectrodes were inside the cell. Potentials
arising between the bath and the reference agar-bridge electrode were
measured by comparing the potential of the 3 M KCl agar bridge with
that of a broken-tip microelectrode filled with 3 M KCl
(Alvarez-Leefmans et al. 1988 When TEA-Cl (tetraethylammonium chloride, Sigma) was used, appropriate
amounts of NaCl were removed from the control solution composition to
maintain isoosmolarity.
9AC (anthracene-9-carboxylic acid, Sigma) and DIDS
(4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, Sigma) were bath applied by exchanging the normal medium with drug-containing medium by
means of a continuous rapid perfusion system.
Neuronal general behavior in the subthreshold range
Representative examples of current recordings under two-electrode
voltage-clamp conditions from a rat sympathetic neuron in response to
hyperpolarizing voltage steps are displayed in Fig. 1A. The neuron was stepped
from a holding potential of
ABSTRACT
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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40/
50 mV elicited inward currents, whose initial
magnitude displayed a linear instantaneous current-voltage
(I-V) relationship; afterward, the currents decayed exponentially with a single voltage-dependent time constant (63.5 s at
40 mV; 10.8 s at
130 mV). The cell input conductance decreased during the command step with the same time course as the current. On
returning to the holding potential, the ensuing outward currents were
accompanied by a slow increase in input conductance toward the initial
values; the inward charge movement during the transient ON
response (a mean of 76 nC in 8 neurons stepped from
50 to
90 mV)
was completely balanced by outward charge displacement during the
OFF response. The chloride movements accompanying voltage modifications were studied by estimating the chloride equilibrium potential (ECl) at different holding
potentials from the reversal of GABA evoked currents.
[Cl
]i was strongly affected by membrane
potential, and at steady state it was systematically higher than
expected from passive ion distribution. The transient current was
blocked by substitution of isethionate for chloride and by
Cl
channel blockers (9AC and DIDS). It proved insensitive
to K+ channel blockers, external Cd2+,
intracellular Ca2+ chelators
[bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic
acid (BAPTA)] and reduction of [Na+]e. It is
concluded that membrane potential shifts elicit a chloride current that
reflects readjustment of [Cl
]i. The cell
input conductance was measured over the
40/
120-mV voltage range, in
control medium, and under conditions in which either the chloride or
the potassium current was blocked. A mix of chloride, potassium, and
leakage conductances was detected at all potentials. The leakage
component was voltage independent and constant at ~14 nS. Conversely,
gCl decreased with hyperpolarization (80 nS at
40 mV, undetectable
below
110 mV), whereas gK displayed a maximum at
80 mV (55.3 nS).
Thus the ratio gCl/gK continuously varied with membrane polarization
(2.72 at
50 mV; 0.33 at
110 mV). These data were forced in a model
of the three current components here described, which accurately
simulates the behavior observed in the "resting" neuron during
membrane migrations in the subthreshold potential range, thereby
confirming that active K and Cl conductances contribute to the genesis
of membrane potential and possibly to the control of neuronal excitability.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
).
). The
relationships among molecular identity, gene superfamilies, and
functional expression have been partly elucidated (reviewed by
Jentsch 1996
; Pusch and Jentsch 1994
).
Comparable information on gCl has been similarly obtained in neurons,
but it remains in doubt which channel types are ubiquitously present and which are expressed in a highly specific manner, and little is
known about their mechanisms of activation and their specific physiological function.
), in hippocampal pyramidal neurons (Madison et al.
1986
; Staley 1994
), and in the dissociated rat
sympathetic neuron (Clark et al. 1998
; see, however,
Lamas 1998
; Selyanko 1984
). A
calcium-dependent chloride current, responsible for a slow
afterdepolarization following spike firing, has been described in rat
sympathetic neurons after axotomy (Sánchez-Vives and
Gallego 1994
) and as a normal complement of the mouse
sympathetic ganglion cell (De Castro et al. 1997
). On
the other hand, chloride currents activated by injected calcium have
been described in several neuron types and produce slow depolarizing aftereffects (reviewed by Mayer et al. 1990
;
Scott et al. 1995
). Single voltage-dependent chloride
channels have been dissected and kinetically characterized in rat
hippocampal neurons in culture (Franciolini and Nonner
1987
), in acutely dissociated rat cerebral cortical neurons
(Blatz 1991
), and in Aplysia neurons
(Chesnoy-Marchais and Evans 1986
).
METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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1 ip) and
maintained in vitro at 37°C. After surgery, the animals were killed
with an overdose of anesthetic. The ganglion was desheathed and pinned
to the bottom of a chamber mounted on the stage of a compound
microscope; individual neurons were identified at a magnification of
×500 by using diffraction interference optics and impaled with two
independent glass microelectrodes filled with neutralized 4 M potassium
acetate (30-40 M
resistance). Recordings were obtained under
two-electrode voltage-clamp conditions as described previously
(Belluzzi et al. 1985
). The preparation was continuously
superfused with a medium (in mM: 136 NaCl, 5.6 KCl, 5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 14.3 NaHCO3, and 5.5 glucose) pregassed with 95%
O2-5% CO2 to a final pH
7.3. Atropine sulfate 10
6 M was systematically
added to the saline. The bath was grounded through an agar-3 M KCl
bridge. The usual protocol was to hold the potential at
40 or
50
mV, and to jump to the test potential in the
40/
130-mV range.
Long-lasting current tracings were filtered at 5 kHz and digitized
continuously on tape (Biologic, DTR-1200; 0-10 kHz). Data were
analyzed on Pentium personal computers (AST) with pCLAMP (Axon
Instruments) and MATLAB 386 (The MathWorks, Natick, MA) software packages.
); values around +1-2 mV
were measured and were not taken into account.
-Aminobutyric acid (GABA) was focally applied to the soma of
identified neurons by 0.1-s pulses of pressure (20 psi; PDES 2 l,
NPI, Tamm, Germany) to the back of micropipettes of 2-4 µm internal
diameter, filled with 1 mM GABA dissolved in the same solution present
in the bath. The pipette was positioned as close as possible to the
neuron and maintained in place, in some experiments, or removed and
repositioned during subsequent applications to avoid the development of
receptor desensitization due to agonist leakage from the pipette; no
differences were apparent between these procedures. GABA applications
were repeated at 30-s intervals over an appropriate range of voltage
commands, while maintaining the neuron under voltage-clamp conditions
at different holding potentials over the
40/
120-mV range.
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50 mV to a series of command potentials
between
60 and
130 mV, in 10-mV increments. The current was probed
at different potentials for a period of 150 s, and the initial
holding potential was thereafter restored and maintained for further
210 s, before starting a new cycle. An inward current developed
with hyperpolarization and instantaneously reached its maximum value.
This value displayed a linear current-voltage (I-V)
relationship (Fig. 1B) as expected for leakage currents, but
was not maintained (Fig. 1, A and C). The
currents decayed toward a new steady-state value with a time course
systematically well fit by a single exponential. The unusually long-lasting traces are differently illustrated with appropriate time
resolution and acquisition bandwidth in Fig. 1, A-C (in
A the superimposed tracings are heavily filtered for
clarity); the figure also shows the stability of the recordings, which
is a must for subsequent analysis. In spite of the linear
I-V relationship observed for the initial values of the
currents, the final steady-state components were scaled by variable
factors with respect to the starting values and were not proportional
to the voltage step, suggesting that the decaying currents were
sustained by (active) conductances that changed during the sojourn at
the new imposed voltage levels. In other words, these currents should
be interpreted as tail currents sustained by a conductance already
active at the holding potential of
50 mV. This was an unexpected
finding, because the electrical properties of the sympathetic neuron
are generally considered to be exclusively "passive" over the
membrane potential range tested (the inward rectifier current of the IQ or IH type is absent or negligible).
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Fig. 1.
Transient currents in the subthreshold voltage region.
A: family of currents evoked in a sympathetic neuron by
long-lasting hyperpolarizing commands in the 60/
130-mV membrane
potential range, repeated in 10-mV steps. Holding potential was
50 mV
and was maintained for 210 s before starting a new cycle.
B and C: currents observed in different
neurons with appropriate time resolution to show the early pattern of
current development. In C the neuron was successively
stepped to
70,
80, and
90 mV from a holding potential of
50 mV.
D: time constant of decay of the transient current
component measured at different membrane potentials during
ON responses (
, n = 4-8) or OFF responses on returning to the holding level
(
, n = 4-13). Bars indicate SE.
On termination of the command steps, the neuron generated large outward transient currents (Figs. 1A and 2, trace a), which also decayed exponentially.
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The time courses of all currents became faster with increasing membrane
polarization; the mean values of the decay time constants in our
experiments (neurons held at 50 or
40 mV) are illustrated in Fig.
1D; they were calculated from the ON responses
in the
60/
130-mV voltage range or from the OFF
responses in the
40/
50-mV region (Fig. 2 illustrates an example of
the whole time course).
The instantaneous cell input conductance at different membrane
potentials was estimated by measuring the currents induced by short (10 ms) hyperpolarizing voltage pulses of 30/
40 mV amplitude,
superimposed on the longer voltage commands. The membrane chord
conductance was measured assuming that the single conductances at
hyperpolarized potentials showed neither fast voltage sensitivity (the
current level during the short step was constant, after the capacity
transient) nor instantaneous rectification (the total instantaneous
I-V relationship was linear down to
130 mV). Figure 2
shows an example of these tests, during a 3-min
50/
100/
50-mV cycle (the current response is illustrated in trace a,
middle panel). The input conductance at
50 mV was constant
(58 nS) during the application of the test pulses at 1 Hz, indicating
that the procedure did not perturb the resting state (trace
a in the bottom panel). At
100 mV the cell input
conductance values were initially confirmed but thereafter slowly
decreased with the same time course of the current decay, reaching a
final steady-state value of 31 nS. The opposite was observed during the
OFF response: the cell input conductance smoothly
increased, reaching within a few minutes the values originally measured
at
50 mV at the beginning of the cycle.
Among the possible current carriers, the chloride ion appeared to be
the most likely candidate when Na-isethionate was isoosmotically substituted for the external NaCl (136 mM). This is illustrated in Fig.
2 (trace b, same neuron as a): the holding inward
current at 50 mV was reduced by 0.68 nA, and both the transient
inward current during the command at
100 mV and the outward transient on returning to
50 mV were completely canceled with a general behavior now constantly passive during the long-lasting pulse. Consistently, the cell input conductance proved to be reduced to 24 nS
at rest and displayed very limited excursions during the voltage
migrations imposed thereafter.
It immediately appeared evident that the excess electric charge
inwardly displaced during the transient phase of the currents (relative
to the final steady state) was balanced out by the outward charge that
left the neuron on returning to the initial membrane potential level.
This was tested in eight neurons during 50/
90/
50-mV commands:
despite the different time courses of the currents, the mean charge
entering the neuron during the transient response at
90 mV was 76 nC
and was balanced out by a symmetrical outward displacement of 77 nC at
50 mV.
Chloride distribution in the sympathetic neuron
The possible role for chloride suggested by the present
observations urged a precise understanding of its movements at rest and
during voltage pulses. Previous work in the rat sympathetic neuron
demonstrated that intracellular chloride activity at rest, as measured
with ion-sensitive microelectrodes, is higher than predicted from a
passive distribution of the ion (aCli = 29.9 mM) (Ballanyi and Grafe 1985). The same
conclusion was reached by directly measuring intracellular chloride
concentration with an electron microprobe
([Cl
]i = 32 mM) (Galvan et al.
1984
). This unbalance ([Cl
]i = 23.3 mM is the expected Nernst's value at
50 mV resting potential)
is maintained by inward pumping of chloride through an active process,
which involves an electroneutral K+:Cl
co-transport and possibly a
Na+:K+:Cl
co-transport (as
suggested for the amphibian dorsal root ganglion neurons by
Alvarez-Leefmans et al. 1988
).
The presence of GABA-sensitive chloride channels in rat sympathetic
neurons has been long demonstrated (Adams and Brown
1975). There is no evidence for a role of GABA as a ganglionic
neurotransmitter; when applied via the bathing medium, however, it
produced a large fall in cell input resistance accompanied by a
clear-cut membrane depolarization, which reversed between
40 and
50
mV. We have repeated here this type of experiment, using the
voltage-clamp technique and perisomatic 1 mM GABA-Cl application, to
indirectly determine the ECl from reversal
potential of the currents evoked over a range of command potentials.
Neurons were maintained at variable holding levels for at least
210 s (long enough to reach a steady-state condition) before the
first application of GABA; successive applications were performed every
30 s to minimize receptor desensitization (Dominguez-Perrot
et al. 1996
). Large currents were recorded with repeated pulse
application of constant amounts of GABA, allowing the desired
ECl to be evaluated. Moreover, a drastic
effect of the holding potential appeared evident both on the intensity
and the direction of flow of the currents, as illustrated in the
typical recordings of Fig.
3A: when GABA was applied
at
40 mV, for example, the response to GABA was an inward chloride
current of relatively small intensity if the holding potential was also
40 mV, whereas a large and outward current followed the sojourn at
90 mV. This behavior was systematically observed. The results of
these tests in three different neurons are summarized in Fig.
3B, in which ECl and the
corresponding [Cl
]i are plotted against the
steady-state holding potential over the
40/
120 mV range. The
internal chloride concentration was confirmed to be higher than
predicted by a passive distribution of Cl
, and this
occurred at all voltage levels tested.
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Figure 3 reflects the static chloride distribution in the sympathetic
neuron, when voltage and ionic gradients are presumably unvarying over
time. The complementary dynamic information is illustrated in Fig.
4, in which the effects of chloride
readjustment during and after a voltage pulse are probed. If the
transient currents of Fig. 1A were actually related to
chloride leaving the cell during hyperpolarization and entering the
cell during depolarization, as indicated by the data of Fig.
3B, then the GABA currents at a constant voltage should
be affected by the chloride driving force smoothly changing between the
initial and final values. This was actually detected when GABA was
applied at different times (small letters of Fig. 4) during repeated
50/
90/
50-mV cycles. During the hyperpolarizing step the chloride
currents evoked by GABA were inward and displayed a major decrease in
amplitude while ECl was changing
(traces a-d); conversely, on returning to
50 mV, Cl
currents were initially outward, decreased in amplitude, and eventually
reversed when [Cl
]i had fully readjusted
(traces e-h). From the data presented in Figs. 2,
3A, and 4, it would appear that the transient inward currents, the loss of internal chloride, and the shifts of
ECl in the negative direction (vice versa
during neuronal depolarization) are parallel aspects of the same
process, namely the redistribution of chloride according to the
transmembrane potential, and are accompanied by time-dependent changes
in input conductance.
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Ionic and pharmacological properties of the transient currents
Discernible chloride currents are also evoked by depolarizing
membrane shifts, even of small amplitude, and in the presence of other
voltage-dependent ionic currents. This is illustrated in Fig.
5A, which shows in
a the response recorded during a 50/
40/
50-mV cycle:
the currents are virtually symmetrical and coherent with the direction
of chloride flow. In this experiment, 170 mM
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA)-4K was dissolved in the K-acetate solution, which filled both
intracellular microelectrodes, and diffused into the neuron, reaching
an internal concentration sufficient to cancel the spike
afterhyperpolarization. The fast Ca2+ chelator
had no apparent effects on the transient ON-OFF
currents. A more complex command sequence generated the tracing of Fig. 5Ab. A very long-lasting pulse to
30 mV was applied to a
neuron from a holding potential of
50 mV: at this potential all the voltage-dependent currents described in this neuron start to be activated (Belluzzi and Sacchi 1991
), and a large and
persistent outward potassium current
(IKV plus the residual
IKCa) was actually recorded. At the
command onset, however, the transient outward chloride-related
component (96.5 nC total outward charge displacement, measured relative
to the noninactivating fraction of potassium current) systematically
appeared superimposed on the delayed current. The chloride
OFF current was recorded in isolation on returning to
50
mV (the potassium channels are rapidly closed by repolarization); at
this potential 84.5 nC were returned as inward charge. A second long-lasting command to
30 mV was applied thereafter, and an intermediate step at
40 mV was applied before returning to the starting level. Chloride OFF currents were recorded at both
levels; the charge that had moved during the ON response
was recovered in a first fraction of 48.8 nC at
40 mV and a second
fraction of 43.9 nC at the final level of
50 mV. As in the case of
Fig. 2, the substitution of isethionate for chloride virtually canceled any transient ON-OFF response within this
voltage range.
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In further experiments the chloride current was isolated as the
difference current between measurements performed during a voltage ramp
(from 50 to +20 mV, relative to the holding level; 200 ms duration)
in control solution and after specifically blocking the chloride
channels or reducing the external chloride complement. In Fig.
5B, traces a-c, 0.5 mM 9AC was used as a chloride channel blocker in a neuron held at
40 mV: the dissected chloride current (trace c) exhibited a linear I-V relationship and
reversed at about
34 mV, in agreement with the GABA experiments of
Fig. 3B. In the same neuron, the chloride current was
virtually canceled when the holding potential was
110 mV (the
chloride channels are closed at this membrane potential; see Fig.
9C). In a different neuron, the isolation procedure
was applied at
50 (trace e) and
70 mV (trace
f) before and after substituting 136 mM Na-methanesulfonate for
NaCl, thus reducing [Cl
]e to ~18 mM. The
reversal potential of chloride currents displayed a strong dependence
on the holding potential but was systematically a few millivolts less
negative than the holding potential (+6 mV in trace e;
+2 mV in f), in line with previous observations.
ICl was insensitive to modifications of
[Ca2+]e in the 2- to 5-mM range, to external
0.5 mM cadmium-Cl2 (and to intracellular BAPTA application,
see the first paragraph of this section) and to reduction in
potassium or sodium concentration of the bathing medium. Similarly, it
was unaffected by 5 mM external caesium-Cl and potassium channel
blockers; this is illustrated in Fig. 7 (see also Fig. 8C), which
shows the cumulative effects of the latter treatments: the
chloride-related inward charge displacement within successive 10-mV
steps of long duration from 40 to
80 mV was unaffected by reducing
external sodium to 60% in the presence of 5 mM caesium and 50 mM
TEA-Cl.
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The chloride current in the sympathetic neuron was quite efficiently
blocked by 0.5 mM 9AC (see Figs. 5B and
8B) or 0.3 mM DIDS. The block, as evaluated by the
reduction in amplitude of the transient current, was usually complete
with 9AC; in two of three neurons tested with DIDS, the block was
complete, whereas in the third, ICl was
reduced to 14.3%. Other drugs, such as 4 × 105 M
ouabain, 20 µM bicuculline, and a benzodiazepine (2 × 10
7 M flunitrazepam-HCl), were devoid of any detectable effect.
Current variance during membrane potential migrations
Although the holding current had to be increased to hyperpolarize the neuron, the current variance was systematically reduced, and conversely the variance increased in depolarized conditions. This finding is consistent with the conductance measurements, indicating that voltage-dependent channels, opened by depolarization, sustain the recorded current.
When the neuron was progressively hyperpolarized from 50 to
100 mV
through steps of 2-min duration, a decrease in current variance
accompanied each hyperpolarizing step (from 0.075 nA2 at
50 mV to 0.014 nA2
at
100 mV), and small further reductions of the variance were often
observed during each step, suggesting that channels were slowly
deactivating during the procedure (Fig.
6).
In response to larger hyperpolarizing voltage steps (e.g., 50 to
90
mV) current variance systematically declined during the command; this
was true even after correcting for the extra variance added by the slow
drift in mean current (especially at early times during the command).
Conversely, on depolarization the current variance slowly increased.
Again, the observations are consistent with the conductance
measurements and confirm that channel activation/deactivation
contributes to the currents. As expected, isethionate substitution for
chloride or the addition of DIDS markedly reduced current variance and
abolished its dependence on voltage (typically, in a neuron exposed to
isethionate, 0.0047 nA2 at
40 mV vs. a stable
value of 0.0035 nA2 at
90 mV).
The current variance was in general too low to permit noise analysis to
characterize single-channel behavior; indeed, the very low
single-channel conductance reported for chloride channels (typically, 1 pS for skeletal muscle chloride channels, ClC-1) (Pusch et al.
1994) is known to heavily hamper such an approach.
Ionic conductances active in the subthreshold membrane potential range
The present data suggest that in the voltage range in which the
neuron exhibits a behavior conventionally indicated as "passive," active conductances do exist, and these are controlled by membrane potential. The contribution by potassium, chloride, and leakage currents was thus systematically evaluated by isolating the individual components at different holding levels within the 40/
120-mV range.
The neuron membrane potential was progressively moved in 10-mV steps,
each level was maintained for 120-180 s before imposing an additional
negative voltage gradient, and during this period the cell input
conductance was measured as illustrated in Fig. 2 (the application rate
of the test pulses was actually minimized, because this procedure might
have been damaging to the neuron with high internal negativity). The
results of these experiments provide the continuous tracings of the
transmembrane current at different levels, represented in Fig. 8, and
the voltage dependence of the steady-state values of cell input
conductance, illustrated in Fig. 9.
Control tracings reproduce a common pattern at each step (Fig.
8A), namely the expected inward transient current and the
input conductance slowly decaying to the steady-state values pertinent
to the imposed potential. The current transients were small because
they now reflected limited fractional changes in [Cl
]i, but they were
systematically evoked even at
120 mV. The total input conductance
(mean values from 10 neurons; Fig. 9A) showed an unexpected
behavior in that it progressively decreased with increasing negativity,
after a small increase around
60 mV, and was down to 37% of its
maximum value at
120 mV. Treatment with 9AC abolished the chloride
current, leaving only the potassium plus leakage fractions in the
tracings. A typical recording is shown in Fig. 8B: the
squared behavior of the trace indicates that any transient component
was actually canceled throughout. The mean steady-state conductance
values (n = 6) exhibit a complex voltage dependence,
consistently observed in each cell, namely a slight decrease at
50
mV, a peak value around
80 mV and a progressive decrease for higher
polarizations with a minimum of 22 nS at
120 mV (Fig. 9B,
). The biphasic nature of the curve is most likely due to the sum of
at least two different conductances, because the addition of 5 mM
caesium to the bath generated a monotonic decay of steady-state
conductance, by apparently subtracting a consistent fraction of the
residual potassium conductance in the
50/
120-mV region (Fig.
9B,
). Caesium is usually considered to be a specific
blocker of the inward rectifier current in this range of membrane
potential, but this current is hardly detected in the rat sympathetic
neuron (and is unknown to inactivate with increasing negativity).
Contributions to the total potassium conductance could arise from
gKV,
gKCa, and
gA. In simulations from a
five-conductance model of the rat sympathetic neuron (Belluzzi
and Sacchi 1991
), a cumulative potassium conductance of 9.1 nS
was calculated at
50 mV; it is questionable, however, whether such
computations, based on extrapolation of curves fitting experimental
data obtained at less negative potentials, might reflect physiological
behavior. Single calcium-activated potassium channels have been shown
in cultured rat sympathetic neurons to be active at negative potentials (
45/
67 mV), although with a low
Popen (Smart 1987
).
Similarly, M-type channels may open, but only at membrane voltages
positive to
60 mV (Stansfeld et al. 1993
). The precise
characterization of K currents at negative potentials, however, was not
the aim of this study and was not further pursued.
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To block all potassium conductances, 50 mM TEA plus 5 mM caesium were
applied to the bath (Fig. 8C). The current tracing
consistently showed the typical transient chloride currents at each
voltage step, whereas the cell input conductance smoothly decayed with voltage toward a final value of 20 nS at 120 mV (Fig. 9C;
n = 5). Finally, the blockers of both potassium and chloride
channels were cumulatively applied, with the result that the current
amplitude became virtually insensitive to the membrane potential (Fig.
8D); the cell input conductance also remained clamped around
a constant value of 11-15 nS (Fig. 9D; n = 3). The
latter values thus represent a pedestal, independent of membrane
potential, which reflects the true passive behavior of the neuron, onto
which voltage-dependent conductances are superimposed. Once this
background is subtracted, the values of Fig. 9B apply to the
voltage-dependent potassium conductance, whereas those of Fig.
9C describe the chloride conductance. Both conductances
exhibit a strong and selective voltage dependence, so that their ratio
continuously varies with membrane potential (gCl/gK = 2.72 at
50
mV, 0.75 at
70 mV and 0.35 at
90 mV); they tend, however, to
decrease with increasing internal negativity, vanishing at
120
mV, where total conductance virtually coincides with that of the
leakage component. This conclusion is confirmed by difference currents
recorded during voltage ramps. The well-defined chloride current
dissected in Fig. 5Bc at
40 mV in the presence and absence
of 9AC is actually canceled when the procedure is repeated at
110 mV
(Fig. 5Bd).
The application of Goldman's equation to the relation
[Cl]i versus membrane
potential (see Fig. 3B) indicates that steady-state [Cl
]i only accounts for
part of the change in steady-state chloride conductance, gCl (Fig.
9C), over the
40/
120-mV range, and does not predict the
observed shape for the gCl versus membrane potential relation. Thus
channel activation-deactivation must also be involved.
The curves of Fig. 9, A-C, were obtained independently from
different cell groups, so that the values are not readily comparable; nevertheless, the summated gK and gCl from Fig. 9, B and
C account for 92-99% of the average total input
conductance in control neurons (A) in the voltage range
50/
90 mV; the recovery is less accurate above and below this
region. The chloride conductance-voltage relationship in the
subthreshold region was described by the following Boltzmann-type
equation (V = membrane potential in mV)
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(1) |
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(2) |
Cell conductances were measured also in three neurons maintained at 22°C. The cell input conductance values proved to be scaled by a constant factor over the whole membrane potential region tested. The temperature sensitivity coefficient (Q10) was 1.55, a value typical of diffusive processes, suggesting that open channel probabilities are temperature independent.
Model for the neuronal subthreshold behavior
The conductances here characterized in the subthreshold range of membrane potential were forced in a mathematical model of the simplified electrical circuit shown in Fig. 10A. Cell capacitance and steady current contributions by active ionic pumps were neglected, in simulating the time courses of the slowly evolving currents of interest.
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The leakage conductance value, gL, was considered to be voltage
independent and constant at 14 nS, whereas the values of potassium and
chloride conductances are voltage dependent. Potassium channel kinetics
are sufficiently fast to make their precise evaluation irrelevant to
the present purposes (activation and deactivation of potassium currents
are not discernible in Figs. 1B and 2, for example), so a
conventional, voltage-independent value of = 4 ms was used in
the computations for potassium channel gating time constant, in line
with the
n of
IKV at the membrane potentials here
considered (Belluzzi and Sacchi 1991
). Thus the compound potassium conductance, gK, is assumed to rapidly settle to the values
predicted by Eq. 2. Chloride conductance, gCl, is considered to relax toward its steady-state value, predicted by Eq. 1,
with a time constant,
, influenced by voltage according to the
equation best fitting the data of Fig. 1D, which presumably
describes the voltage sensitivity of
ICl gating time constant:
= 253.55 exp(0.04 V) + 11.47 ms (V in mV).
The two batteries in the circuit represent the electromotive forces
generated by the Nernstian equilibrium potentials for potassium
(EK, assumed constant at 90 mV) and
chloride (ECl). The steady-state value
of ECl is expected to vary with
voltage according to the linear equation best fitting data points of
Fig. 3B: ECl = 0.6 · Vh
13.82 mV
(Vh = holding potential in mV). However, its detailed time course must be computed from time-dependent changes in [Cl
]i during
chloride redistribution. At the beginning of transients, [Cl
]i can be estimated
from the steady-state values of ECl;
subsequently, changes in
[Cl
]i are determined by
Cl
movements and the cell volume; thus the
latter can be estimated from the ratio between total
Cl
charge transfer and the change in
steady-state [Cl
]i on
displacing membrane potential. Once the cell volume is known, then
[Cl
]i can be updated in
the mathematical model during the transient, based on the momentary
chloride current, and employed to update ECl in turn. Total charge transfers
from the various experiments illustrated in Figs. 8 and 9 point to cell
volumes ranging between 30 and 60 pl (i.e., the volume of spherical
neurons with diameter between 38 and 48 µm). Such values for cell
volume, which are in reasonable agreement with the size of the neurons,
as seen under the microscope, would result in time constants in the
order of 30-100 s (i.e., within a range very similar to the time
constants of gCl changes) for the chloride redistribution process,
depending on chloride conductance and the amplitude of the voltage step.
A noninactivating chloride current is generated, in this model, at
membrane potentials positive to 120 mV; its transient behavior during
voltage jumps is not related to inactivation, but simply reflects the
slow process of channel activation/deactivation and the similarly slow
driving-force shifts that accompany chloride redistribution. Figure
10Ba illustrates the responses of the model neuron to
commands in the subthreshold voltage range typically used in Figs.
1A and 5A. The amplitude and time course of the computed current tracings, as well as the nonlinear voltage dependence of the late component, favorably compare with the experimental data,
suggesting that the equivalent circuit in Fig. 10A is
adequate to provide a correct description of the ganglion cell membrane in the subthreshold region, and that the simulation of chloride redistribution yields valid time-varying estimates of
ECl and [Cl
]i during the
simulation of current transients (Fig. 10B, trace b). Figure
10C shows a plot of the mean values of the steady-state potassium and chloride currents operating at different membrane potentials, together with their standard errors, as estimated from the
variability of experimental measurements of the single conductances.
Although these are not the only currents operating in the neuron "at
rest" (at least leakage and pump current must be considered in
addition), they suggest that the potassium outward current component
dominates for membrane potential values positive to
80 mV, at steady
state. The crucial aspect, however, is that when the membrane potential
is displaced (by sustained activation of the synaptic input, for
example), the currents will depart from this static picture for up to
1-2 min, and chloride conductance and redistribution will determine
the momentary values of membrane potential and net current flow.
The charge transfers predicted by the model in response to successive
10-mV voltage steps from 40 to
120 mV, for a cell volume of 30 pl,
are compared in Fig. 10D (
) to the values measured using
the same protocol in the experiment illustrated in Fig. 8C.
The good fit between the two sets of data is further evidence in
support of the present mode of correlating observed transient currents
and transmembrane chloride movements.
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DISCUSSION |
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The central findings presented in this paper describe the
transmembrane movements of chloride during membrane potential
excursions in the subthreshold range, and the participation of a
chloride conductance in the genesis and control of the actual membrane potential of the neuron at rest. We demonstrate that discernible transient currents are associated with membrane potential modifications in either directions within the 30/
130-mV voltage range and that
the direction and size of these currents are coherent with the chloride
movements during the voltage migrations. Further arguments in favor of
the chloride nature of the current transients include the following:
1) the current reversal potential is coincident with the
momentary chloride equilibrium potential and is influenced by the
ECl shifts related to membrane
potential; 2) the current is blocked by impermeant ions
substituting for external chloride and by treatments, such as 9AC and
DIDS, known to block chloride channels in other systems; 3)
it is unaffected by reduced external sodium concentration and blockers
of the potassium channels; moreover, 4) there is a good
correlation between measured charge movements and the accompanying
modifications of chloride concentration.
Previous work has strongly suggested that the intracellular chloride
concentration in the mammalian sympathetic neuron is higher than
predicted by a passive Nernst distribution, and evidence has been
provided for the existence of an active electroneutral mechanism
sustaining the inward flux of chloride that maintains the ion
unbalanced; in the present paper we demonstrate that this holds over a
wide range of membrane potential, so that at steady-state the chloride
battery sustains an inward current at any membrane potential level.
Membrane potential proves to be the main controller of the
intracellular chloride concentration; membrane potential migrations are
systematically accompanied by measurable chloride currents, which flow
in the inward direction when the potential becomes more negative and
the intracellular chloride content decreases, or in the outward
direction when the neuron acquires chloride during depolarization. The
amount of charge carried by ICl is virtually symmetrical in response to voltage steps of equal amplitude in opposite directions, suggesting that the chloride acquired by the
cell during depolarization is actually returned during hyperpolarization. The final steady state is reached when the chloride
concentrations are close to but not quite at equilibrium, the
intracellular space remaining systematically enriched of chloride. Chloride current might escape detection under many experimental conditions, because at rest it is negligible and mixed with other components, and its presence becomes clearly discernible only during
voltage transients, i.e., under conditions that artificially increase
the momentary chloride driving force; furthermore, its characterization
requires that chloride is allowed to redistribute and intracellular
chloride concentration is not clamped. It might also be of interest to
observe that in our preparation chloride conductance (blocked by
specific antagonists) exceeds ~80 nS at rest and accounts for a major
fraction of total conductance (~100 nS). These values are almost an
order of magnitude higher than usually observed by patch-clamp
technique in isolated neurons (where the cell input impedence often
exceeds 100 M); it is therefore likely that most chloride channels
(as occurs for other voltage-dependent and synaptic channels of the
intact ganglion) disappear after isolation and enzymatic treatment of
the neuron.
The modifications in the instantaneous input conductance of the neuron and in total current variance that accompany the transient currents evoked by voltage steps support the basic premise that the current is due to the ongoing activity of specialized chloride channels, which open and close following a very slow kinetics, on the order of tens of seconds. All transient chloride currents rise instantaneously to initial values linear with the voltage step applied to the neuron, independent of the polarity of the command step used in the experiments: this indicates that the current is actually carried by the channels already open at that time, and not opened (or closed) by the voltage step. Only the subsequent time course of the current will depend on the momentary membrane potential, because the number of open channels will slowly decline during neuron hyperpolarization, whereas it will increase with membrane depolarization. Membrane potential, but not the total amount of charge transfer during a voltage step, controls the duration of the transients: ICl time course becomes faster with increasing internal negativity, suggesting that, although the kinetics of the channel are very slow, channel closure rate constant depends on membrane potential. The simple model discussed above suggests that the shifts of the actual intracellular chloride concentration follow time courses similar to the changes in conductance and that the kinetics of the two processes fully account for the time course of the transient currents.
The basic properties of the chloride current here described markedly
differ from those of the currents referred to in the INTRODUCTION as hyperpolarization-activated inward
rectifier current and calcium-activated current. The conductance
underlying the former is activated, and not shut off, by increased
membrane negativity; the charge movements are not symmetrical, and the
pharmacological profiles are also different. As regards the
calcium-activated current, calcium transmembrane movements are expected
to be negligible, if ever present, in the membrane potential range here
considered; moreover, cadmium treatment and the addition of BAPTA to
the solution filling the microelectrodes proved ineffective in our
experiments. Some functional similarities do exist, on the contrary,
with the well-known chloride current present in skeletal muscle
(Palade and Barchi 1977). The high resting chloride
conductance, its activation with depolarization, and, conversely, its
decrease on hyperpolarization are in fact reminiscent of the behavior
of the ClC-1 channels. The chloride conductance that has been observed
in hippocampal pyramidal neurons shares the properties of the cloned
and functionally expressed chloride channel ClC-2 (Smith et al.
1995
). Recently, mRNA for ClC-2 has been shown to be present in
rat superior cervical ganglia as well and may be functionally expressed
(Clark et al. 1998
); the chloride current described in
that paper, coherently, exhibited the properties of a typical
hyperpolarization-activated current. ClC-1 is considered to be muscle
specific; its presence also in ganglia, if it were detected, would be
consistent with the properties of the chloride current determined here
by electrophysiological techniques.
The resting condition of the mammalian sympathetic neuron is often
interpreted as the result of potassium and leakage current flow, each
of them displaying a defined equilibrium potential and remaining
sufficiently constant over time and independent of membrane potential
to confer stable passive properties to the neuronal behavior at
membrane potentials negative to some 50 mV. The nature of both
conductances, however, is ill-defined because the contribution of each
of at least five different potassium conductances coexisting in the
soma (gKV,
gKCa,
gA,
gAHP,
gM) are not specified, and similarly
the physical nature of the leakage current is poorly understood in
terms of its ionic carrier(s) and equilibrium potential. In an accurate
study on frog sympathetic neurons, Jones (1989)
demonstrated that the resting potential primarily takes origin from a
voltage-insensitive potassium current, not
IM or
IQ (the inwardly rectifying current),
whereas in cultured parasympathetic neurons from rat intracardiac
ganglia the resting potential appeared to be set by a
voltage-insensitive leakage current and a voltage-dependent potassium
current of the delayed rectifier type (Xu and Adams
1992
). These data provide a rather rigid view of the resting
potential, which would appear to be virtually a constant among the
multiple neuronal electrical variables and would be predetermined by
factors insensitive to voltage and time, and potentially modified only
by shifts in the potassium equilibrium potential.
A different picture arises from the present findings in which the
individual potassium, chloride, and leakage conductances have been
isolated and measured over a wide membrane potential range. The values
of each conductance and the ratios between them are continuously
modified by membrane potential, and active conductances only vanish at
membrane potential levels negative to 120 mV; here only will the
neuron exhibit a truly passive behavior. At any membrane potential
level positive to EK, the holding
current in the experiments is built up by the sum of three currents:
one of them (the leakage) possibly reflects the only true passive properties of the neuronal geometry; the second is represented by the
outward voltage-dependent potassium current, fed by a virtually constant potassium battery; the third component is the chloride current, which is obligatorily inward at steady-state, as the consequence of the chloride internal concentration being maintained higher than at equilibrium, and is generated by a voltage-dependent chloride battery that produces a constantly small driving force. When
the holding potential moves, the system is perturbed with the result
that the potassium conductance attains, presumably within milliseconds,
the value appropriate to the new potential, whereas the chloride
current relaxes to the new steady-state value with a time constant of
tens of seconds. Thus in addition to leakage current and potassium
conductances, the electrical properties of the neuron at "rest" are
molded by previously undetected voltage-dependent and slowly
time-varying chloride conductances and currents, at membrane potentials
positive to
120 mV.
As opposed to the classical view of a voltage-insensitive potassium
conductance and a stable potassium battery, which are expected to clamp
the membrane at a fixed level, much more flexible is the situation in
which both chloride conductance and chloride battery are voltage
dependent. The previous activity of the neuron will influence the
momentary status of the chloride battery and this, like any event
acting on the chloride ionic gradient, is expected to modify in turn
the resting potential of the neuron and its excitability. The chloride
conductance may also be specifically regulated; in cortical astrocytes,
for example, it has been shown to depend on intracellular calcium and
ATP concentration and more generally on the metabolic activity of the
brain (Lascola and Kraig 1996). Membrane potential thus
appears to constitute a dynamic link between previous history (and
chloride regulation) and momentary excitability of the neuron, rather
than a predetermined electrical status. In this perspective, the
demonstration of a mechanism capable of sustaining a variable resting
potential in the sympathetic neuron helps appreciating the functional
significance of many previous findings. In fact, in earlier studies we
recurrently observed that the initial membrane potential level is a
crucial parameter in the cell firing strategy. It appears that membrane potential controls the mix of different channel types available on the
cell to generate different firing patterns, and that this effect is so
strong, for example, to completely subvert the repolarization of the
action potential: this process, in fact, depends on the delayed
rectifier current, IKV, and the
calcium-dependent current, IKCa, when
the neuron is depolarized at rest, but these conductances are
completely substituted for by the transient potassium current, IA, when the
IA inactivation is removed by
increased internal negativity (
50 mV) (Belluzzi and
Sacchi 1991
). Even more relevant under a functional
perspective, the spike discharge following synaptic activation is
controlled by the underlying IA, which antagonizes the synaptic current flow in the subthreshold range. The
practical result is that the threshold synaptic conductance (and thus
the degree of activation of the synaptic input which is necessary to
elicit postsynaptic firing) increases with increasing membrane
negativity, mirroring the voltage dependence of
IA inactivation removal (Sacchi et
al. 1998
). The identification of an active ionic mechanism,
such as the chloride conductance here characterized, capable of
controlling the basal membrane potential of the active neuron, yields
new intriguing hints about how the neuron may modify its excitability
properties under different functional conditions.
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ACKNOWLEDGMENTS |
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We thank Dr. Giorgio Rispoli for many helpful comments on the manuscript.
This research was supported by grants from the Ministero della Università e della Ricerca Scientifica e Tecnologica within the "Neurobiological Systems" national research project and from the Italian Consiglio Nazionale delle Ricerche.
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FOOTNOTES |
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Address for reprint requests: O. Sacchi, Dept. of Biology, Sezione di Fisiologia Generale, University of Ferrara, Via Borsari 46, I-44100 Ferrara, Italy.
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. Section 1734 solely to indicate this fact.
Received 16 February 1999; accepted in final form 20 April 1999.
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REFERENCES |
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