Nonselective cation conductance activated by muscarinic and
purinergic receptors in rat spiral ganglion neurons
Ken
Ito1,2 and
Didier
Dulon1
1 Laboratoire de Biologie Cellulaire et
Moléculaire de l'Audition, Institut National de la
Santé et de la Recherche Médicale EMI 99-27, Université de Bordeaux 2, Hôpital Pellegrin,
33076 Bordeaux, France; and
2 Department of Otolaryngology, Faculty of Medicine,
University of Tokyo, Tokyo 113-8655, Japan
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ABSTRACT |
The present study
characterizes the ionic conductances activated by acetylcholine (ACh)
and ATP, two candidate neuromodulators, in isolated spiral ganglion
neurons (SGNs). Brief application (1 s) of ACh evoked in a
dose-dependent manner (EC50 = 4.1 µM) a reversible
inward current with a long latency (average 1.3 s), at holding
potential (Vh) =
50 mV. This current was
reversibly blocked by atropine and mimicked by muscarine. Application
of ATP also evoked a reversible inward current at
Vh =
50 mV, but the current showed two
components. A fast component with a short latency was largely reduced
when N-methyl-D-glucamine (NMDG) replaced extracellular sodium, implying a P2X-like ionotropic conductance. The
second component had a longer latency (average 1.1 s) and was
presumably activated by metabotropic P2Y-like receptors. The second
component of ATP-evoked current shared similar characteristics with the
responses evoked by ACh: the current reversed near 0 mV, displayed
inward rectification, could be carried by NMDG, and was insensitive to
extracellular and intracellular calcium. This ACh-/ATP-evoked
conductance was reversibly inhibited by preapplication of ionomycin.
These results suggest that muscarinic receptors and purinergic
metabotropic receptors activate a similar large nonselective cation
conductance via a common intracellular pathway in SGNs, a candidate
mechanism to regulate neuronal excitability of SGNs.
acetylcholine; adenosine 5'-triphosphate; cochlear neuron
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INTRODUCTION |
SPIRAL GANGLION
NEURONS (SGNs) are peripheral bipolar neurons located in the
cochlea that convey to the brain stem the acoustic information arising
from the mechanoelectrical transduction of the inner hair cells (IHCs).
The dendrites of SGNs below IHCs are contacted by efferent fibers that
originate from small central neurons in the lateral superior olive.
This lateral efferent innervation from the brain stem has long been
suggested as a central control of the auditory nerve activity at the
periphery [for review, see Warr (70)]. However, efferent
regulation of SGN excitability still remains largely unexplored.
Several immunocytochemical studies have suggested that acetylcholine
(ACh) could be one of the main neurotransmitters present at these
lateral efferent synapses [for review, see Eybalin
(15)]. On the other hand, microiontophoresis of ACh in
the subsynaptic area below IHCs in vivo has been shown to increase the
subsynaptic spiking activity of the afferent nerve fibers
(18), thus suggesting an excitatory regulation by ACh.
However, the type of cholinergic receptors involved at these lateral
efferent synapses is still debated, because mRNA expression of both
muscarinic (12, 52) and nicotinic (44)
receptors has been reported in SGNs. Recently, our laboratory
demonstrated (50) the presence of functional muscarinic
receptors (mAChRs) that mobilize intracellular calcium in isolated
SGNs, but we still do not know whether mAChRs or ligand-gated cholinergic receptors (nAChRs) can suppress or activate ionic conductances in these neurons. ATP is another potential
neuromodulator of the electrical activity of SGNs. Indeed, experimental
perfusion of the cochlea in vivo with ATP or analogs has also
shown a significant influence on neural thresholds (5, 34,
60). Calcium mobilization via metabotropic P2Y receptors
(7) and membrane depolarization via ionotropic P2X
receptors have been shown in isolated SGNs (53). However,
the influence of the P2Y metabotropic receptors in the regulation of
the electrical activity of SGNs remains largely unclear.
The main objective of the present study was to investigate the overall
ionic conductances activated by ATP and ACh in freshly isolated rat
SGNs with the whole cell patch-clamp technique and to characterize the
type of cholinergic and purinergic receptors involved.
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METHODS |
Preparation of neurons.
SGNs were isolated from rat cochleae as previously described
(50). Animals were treated in accordance with French
Ministry of Agriculture guidelines in agreement with EEC regulations.
Briefly, newborn rats [1-7 days old (P1-P7)] were deeply
anesthetized with an intraperitoneal injection of 0.1-0.2 ml of a
solution consisting of 1 vol of 50 mg/ml ketamine and 1 vol of 2%
xylazine. The animals were then decapitated. Extracted cochleae were
bathed in Dulbecco's phosphate-buffered saline (DPBS) containing (in
mM) 136.9 NaCl, 6.5 Na2HPO4, 2.7 KCl, 1.5 KH2PO4, 0.9 CaCl2, and 0.5 MgCl2, with pH adjusted to 7.3. The shell, which is not yet
completely ossified at this age (P1-P7), was opened, and the stria
vascularis and the organ of Corti were removed. Spiral ganglions were
extracted from the spiral lamina of the two basal turns, and the
neurons were then isolated by mechanical dissociation after treatment with trypsin (type III, 100 µg/ml; 37°C, 30 min). Before the
experiments started, SGNs were left at rest for at least 2 h at
room temperature. SGNs were easily identified under the microscope by
their shape and grouping and remnants of dendrites or axons [see Fig.
1 in Rome et al. (50)]. Our results are essentially based
on recordings obtained from type I neurons, which represented >95% of
the cells in our preparation. Type I neurons from newborn rats could be distinguished from the rare type II neurons by their larger size and
the presence of a soft, thin myelin shield.

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Fig. 1.
Acetylcholine (ACh) activates a depolarizing current in spiral
ganglion neuron (SGN). A: top, a brief
application (1 s) of ACh (100 µM) evoked a reversible inward current
with small oscillations under voltage-clamp conditions at 50 mV.
Bottom, a magnification of the beginning of the same current
indicates a long latency of the cholinergic response. B:
dose-response relationship of the cholinergic current measured at
holding potential (Vh) = 50 mV.
Average ± SD peak amplitudes of ACh-evoked currents are expressed
relative to the reference response obtained at 100 µM for each neuron
(n = 3-6 for each concentration). With the
empirical Hill equation, the sigmoidal dose-response curve was best
fitted with half-effective concentration (EC50) of 4.1 µM
and a Hill coefficient (nH) value of 2.0. C: example of a typical current-voltage (I-V)
relationship obtained by the ramp protocol in an SGN stimulated with
100 µM ACh. Traces 1, 2, and 3 are
the currents obtained before, during, and 120 s after the
application of ACh, respectively. Trace 4 displays the
cholinergic current obtained by subtraction.
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Electrophysiological recordings.
Neurons were recorded under a whole cell patch-clamp configuration with
electrodes pulled from borosilicate glass capillaries (GC150TF-10;
Clark Electromedical). "Standard" internal solution consisted of
(in mM) 158 KCl, 3.5 KOH, 2.0 MgCl2, 1.1 EGTA, and 5.0 HEPES, with pH adjusted to 7.2 and osmolality adjusted to 300 mosmol/kgH2O. The other internal solutions used
were "CsCl" solution consisting of (in mM) 100 CsCl, 50 KCl, 2.0 NaCl, 2.0 MgCl2, 1.1 EGTA, HEPES 5.0, and 4.0 glucose, with
pH adjusted to 7.2; "B10Glu" solution consisting of (in mM) 125 K-gluconate, 20 KCl, 35 NaOH, 2.0 MgCl2, 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA), and 5.0 HEPES, with pH adjusted to 7.2; and "B10Na" solution consisting of (in mM) 100 KCl, 39 KOH, 28.5 NaCl, 2.0 MgCl2, 0.1 CaCl2, 10 BAPTA, 5.0 HEPES, and 8.0 glucose, with pH adjusted to 7.2. Patch-clamp recordings were performed
by means of an Axopatch-1D amplifier (Axon Instruments, Foster City,
CA). Axotape and pCLAMP software (Axon Instruments) and IGOR Pro
software (Wavemetrics, Lake Oswego, OR) were used for data collection
and analysis. Junction potentials were set to zero immediately before gigaseal formation. Electrode resistance ranged from 3 to 6 M
. The
resting potential of the tested neurons was
36.2 ± 11.9 mV. Electrode capacitance and series resistance were not corrected at the
time of experimentation. Current-voltage (I-V) relationships were obtained from voltage ramps and corrected for series
resistance-induced voltage errors. The ramp protocol started from a
holding potential (Vh) of
50 mV, jumped to
80 mV for 20 ms, and then swept linearly to +70 mV in 900 ms. To
measure the I-V relationship during agonist stimulation, we
started the ramp protocol at the peak current, therefore, ~4 s after
the ligand application. For ATP currents, neurons lacking the first
component or those with a negligibly small first component were chosen
to isolate and measure the I-V characteristics of the second
component (for description of the 2 components, see
RESULTS). Furthermore, to minimize errors due to an overlap
of the two components, only brief applications of ATP (1 s) were used
(indeed, the first component rapidly inactivated at the end of the 1-s
ligand application whereas the second component generally peaked in
4 s after the beginning of the ligand application).
The correct diffusion of internal solution within the cell body was
verified by using a fluorescent dye (indo 1) in some neurons. All
experiments were performed at room temperature (20-22°C).
Drug application.
Test solutions were applied to SGNs by means of a Picospritzer puffer
system (Picospritzer II; General Valve, Fairfield, NJ) as previously
described (3). Puff pipettes were pulled similarly to the
recording patch-clamp pipettes and were placed ~20-40 µm from
the neurons. The delay for drugs to reach the cell ranged from 20 to 50 ms (3). To change the external bath solution, we used a
peristaltic pump system at a perfusion rate of 300 µl/min (MasterFlex, Barnant, Barrington, IL). In experiments with the low
Ca2+ (0 mM)-containing solution, calcium chloride was
omitted in the preparation of DPBS and 0.5 mM EGTA was added to make a
nominally calcium-free solution. In experiments with the
high-Ca2+-containing solution, the concentration of calcium
chloride was raised to 30 mM in a Hanks' balanced salt solution (HBSS)
rather than DPBS to avoid calcium-phosphate precipitation. The original constitution of HBSS was (in mM) 136.9 NaCl, 5.4 KCl, 0.8 MgSO4, 1.3 CaCl2, 0.4 KH2PO4, 0.3 Na2HPO4,
4.2 NaHCO3, and 5.5 glucose, with pH adjusted to 7.3. The
characteristics of the ACh- and ATP-evoked currents in SGNs were the
same in DPBS and in HBSS. In experiments with the
low-Na+-containing solution, 80% of sodium in DPBS was
replaced by equimolar N-methyl-D-glucamine
(NMDG), which reduced the extracellular concentration of sodium from
137 to 27.4 mM. When repetitive applications of drugs were made on the
same SGN, we waited at least 2 min between applications to reduce desensitization.
Chemicals.
KN-62, SK&F-96365, K-252a, and aristolochic acid were purchased from
Biomol. Suramin was purchased from Calbiochem. All other chemicals were
purchased from Sigma.
Data analysis.
Data are expressed as means ± SD unless specifically noted.
Student's t-tests were used for data comparison. Relative
ion permeabilities were calculated based on the constant field theory (17)
where P, X, Y, and
Erev represent permeability, internal cation,
external cation, and reversal potential, respectively. F, R, and T have their usual meanings. When
80% of the extracellular sodium was replaced by NMDG, the equation was
as follows
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RESULTS |
Cholinergic response.
Under whole cell voltage-clamp conditions, pressure application of ACh
(100 µM, 1 s) evoked an inward current in 72% of tested neurons
between P1 and P7 (n = 354; Fig.
1A). The amplitude of the
cholinergic current was dose dependent (Fig. 1B), averaging 408 ± 375 pA when ACh was applied at a concentration of 100 µM for 1 s at Vh =
50 mV. Corresponding
depolarization in current-clamp conditions was also observed, averaging
32 mV from an initial Vh =
50 mV. The
dose-response relationship was constructed by normalizing for each
neuron the peak amplitude of the cholinergic currents obtained at a
test concentration to the response obtained with 100 µM ACh. The
normalized responses were plotted on a semilogarithmic scale and fitted
with the empirical Hill equation. Best fit yielded an apparent
half-effective concentration (EC50) of 4.1 µM and a Hill
coefficient (nH) of 2.0.
Analysis of the I-V relationship of the cholinergic
response, tested with a voltage-ramp protocol, indicated a large
increase in membrane conductance (Fig. 1C). The
I-V curve showed inward rectification at negative membrane
potentials (maximum slope conductance averaged 10 nS) and was reversed
near 0 mV. These results demonstrated that ACh activated a nonselective
cation conductance and suggested the involvement of nAChRs. However,
for all neurons tested, the onset of the inward current showed a rather
long delay on ACh application (Fig. 1A). Indeed, the
cholinergic response developed after a latency averaging 1.3 ± 1.0 s, activated with a mean time constant (
) of 0.7 s
(single exponential fit), and reversed slowly within 15-60 s, even
with sustained application of the ligand. This long latency argued
against the direct activation of nAChRs, which usually show a very
short latency of less than a few milliseconds in outer hair cells, for
example (3). Interestingly, the waveforms in the
deactivation phase of the current were rather variable. During the
decay of the cholinergic response in SGNs, large oscillations of
membrane current could be observed in ~30% of tested neurons (median
peak-to-peak interval 3.3 s, i.e., 0.3 Hz; Fig. 1A).
These oscillations were reminiscent of the involvement of a
metabotropic process in which diffusion of intracellular messengers is
involved in repeated activation of the effector channel.
The application of 250 µM muscarine to SGNs also activated a large
inward current at negative membrane potentials, mimicking those evoked
by ACh (Fig. 2A;
n = 3). On the other hand, the application of 100 µM
nicotine did not trigger any significant change in membrane conductance, whereas the neurons were responsive to ACh
(n = 3; Fig. 2C). The absence of response
with nicotine does not necessarily preclude the presence of functional
nAChRs, because
-9 receptors are not activated by nicotine
(14). However, atropine, a competitive antagonist of
mAChRs, reversibly blocked the ACh-evoked membrane currents at a
concentration of 10 µM (Fig. 2B). The atropine block was
another strong argument suggesting the involvement of mAChRs rather
than nAChRs.

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Fig. 2.
Evidence for the involvement of muscarinic receptors. A:
brief application (1 s) of muscarine (250 µM) evoked a reversible
inward current with small oscillations under voltage-clamp conditions
at 50 mV. B: ACh-evoked inward currents were reversibly
blocked by coapplying ACh (100 µM) with atropine (1 µM). Note that
at the 2nd application of ACh + atropine, the cholinergic response
was completely suppressed (middle trace). A complete
recovery was obtained at the second application of ACh alone
(bottom trace). C: application of nicotine (100 µM) did not evoke a current, whereas the neuron was responsive to ACh
before and after the test with nicotine (Vh = 50 mV). D: example of desensitization of the cholinergic
current during repeated ACh application. Full recovery of the
first ACh current was generally obtained after 2 min
(Vh = 50 mV). Dotted lines indicate zero
current levels.
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During a continued ACh application (5-10 s) or repetitive brief
applications, the cholinergic currents showed desensitization and the
time required for complete recovery of the initial response varied from
60 to 120 s (Fig. 2D). The amplitude, kinetics, and desensitization of the cholinergic responses were not significantly changed when we added 1 mM ATP in the intracellular recording solution
(n = 3). To overcome desensitization of the response when repetitive ACh applications were needed, the applications of ACh
with or without the antagonist were separated by at least 120 s.
Purinergic response.
Under voltage clamp at
50 mV, a brief application of ATP also evoked
a reversible inward current in a dose-dependent manner (Fig.
3). Such ATP-evoked inward current was
observed in 75% of tested cochlear neurons between P1 and P7
(n = 254). The current evoked by ATP displayed two
components: a first fast component with a latency <50 ms and a second
slow component with a latency averaging 1.0 ± 0.4 s. The
second component was often larger in amplitude, taking 3-4 s to
peak, and reversed slowly within 15-30 s even with sustained
application of the ligand. During the decay of the ATP-evoked current,
several slow oscillations resembling those observed with ACh were also
observed in a small number of neurons (Fig. 3B).

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Fig. 3.
ATP evokes a depolarizing current in SGNs. A:
top, brief application (1 s) of ATP (100 µM) evoked a
reversible inward current under voltage-clamp conditions at 50 mV.
Bottom, magnification of the beginning of the same current.
Note that the current displayed 2 components. B: examples
from 2 different neurons of current responses to ATP with small
oscillations. ATP (100 µM) was applied for 1 s to neurons held
at 50 mV. C: dose-response relationship of the second
component of ATP-evoked current measured at
Vh = 50 mV. Average ± SD peak
amplitudes of ATP-evoked currents are expressed relative to the
reference response obtained at 100 µM for each neuron
(n = 5-6 for each concentration). With the
empirical Hill equation, the sigmoidal dose-response curve was best
fitted with an EC50 of 33 µM and an
nH value of 0.6. D: example of a
typical I-V relationship obtained by the ramp protocol in an
SGN stimulated with 100 µM ATP. Traces 1, 2,
and 3 are the currents obtained before, during and 120 s after the application of ATP, respectively. Trace 4 displays the purinergic current obtained by subtraction.
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At Vh =
50 mV, the peak amplitude of the
larger current (the second component) averaged 760 ± 530 pA
(n = 135) for a concentration of 100 µM ATP applied
for 1 s. The apparent peak amplitude of the first component was
21.4 ± 22.2% of the second component with this stimulation (100 µM, 1 s). Corresponding depolarization, from initial
Vh =
50 mV, averaged 38 mV in
current-clamp conditions. To build the dose-response curve, the peak
current obtained at various concentrations of ATP was also normalized
for each neuron to the peak current obtained with 100 µM ATP. The
normalized responses were plotted on a semilogarithmic scale and fitted
with the empirical Hill equation. Best fit yielded an apparent
EC50 of 33 µM and nH of
0.60.
Analysis of the I-V relationship tested with a voltage-ramp
protocol also indicated a large increase in membrane conductance during
ATP application (Fig. 3D). Similarly to the cholinergic currents, ATP-evoked currents displayed a strong inward rectification at negative potentials (mean maximum slope conductance of 18 nS) and
were reversed near 0 mV, a finding also indicating the activation of a
nonselective cation conductance. Suramin, a well-known P2-purinergic antagonist (13, 24), reversibly inhibited a large
proportion of ATP-evoked inward currents (Fig.
4A). The current responses also displayed desensitization during repeated applications of ATP
(Fig. 4B). After the neuron was left at rest for 2-5
min, full recovery of the original response was generally attained. The
presence of 1 mM ATP in the recording intracellular solution did not
affect the amplitude, kinetics, and desensitization of the purinergic
responses (n = 3).

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Fig. 4.
Some characteristics of the ATP-evoked conductance. A:
the purinergic currents were reversibly blocked by coapplying ATP (100 µM) with suramin (1 µM). Note that after the 2nd application of
ATP + suramin, the response was diminished. A complete recovery
was obtained at the 2nd application of ATP alone. B: example
of desensitization of the purinergic current during repeated ATP
application. Note that the response started to decrease only at the 3rd
ATP application and desensitization mainly affected the 2nd component.
Full recovery of the current was generally obtained after 2 min
(Vh = 50 mV). Dotted lines indicate zero
current levels.
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Interestingly, three recorded neurons displayed only the first fast
ATP-evoked component (Fig.
5A), whereas 44 other neurons (representing 23% of the neurons responsive to ATP) displayed only the
second ATP-evoked component (Fig. 5B), thus suggesting that
the two purinergic components arise from different conductances that
can desensitize independently. The first component was sustained during
ATP application and inactivated rapidly (
= 3 s) at the end of the ATP application (Fig. 5A). The second component
of the ATP response showed a long latency similar to that observed with
ACh and inactivated slowly. The activation time constant of the second
component was similar to that of the cholinergic response, with
averaging 0.7 s (single exponential fit). However, the
deactivating time course of this current was also rather variable, as
in ACh-evoked currents. The two components of the purinergic response could be more easily separated and identified by using a very
brief ATP application (Fig. 5C). Replacement of 80% of the
extracellular Na+ by NMDG+, a much larger
organic cation than Na+, reduced only the first component
of the ATP-evoked current without affecting the second slow
current (Fig. 5D). Overall, these results suggested that the
ATP-evoked currents were a combination of the activation of P2X
ionotropic receptors (the first fast current blocked in NMDG medium)
and the activation of P2Y metabotropic receptors that secondarily
activated nonselective cation channels.

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Fig. 5.
Additional evidence for 2 distinct components in ATP-evoked
currents. A: example of a neuron expressing only the 1st
rapid component of the ATP current. The current displayed a short
latency, was sustained during ligand application (ATP 100 µM), and
inactivated rapidly at the end of ligand application. The neuron was
held at 50 mV. B: example of another neuron that showed
only the 2nd component of the current. The magnification of the
beginning of the current displays the long latency. C: the 2 purinergic components could be isolated in the time domain by using a
very short ATP (100 µM) application (5 ms). The neuron was held at
50 mV. D: replacing 80% of extracellular sodium by NMDG
largely reduced the 1st fast component of the ATP-current without
affecting the 2nd large component. The same neuron was consecutively
stimulated by a 1-s application of ATP [100 µM in normal Dulbecco's
phosphate-buffered saline (DPBS)] and by another 1-s application of
ATP (100 µM prepared in DPBS in which 80% of sodium was replaced by
NMDG). Dotted lines indicate zero current levels.
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In the rest of the present study, we focused our investigations on the
characterization of the second purinergic-activated conductance and
compared it to the ACh-evoked nonselective cation conductance. The main
technical problem that prevented a detailed investigation of adult rat
SGNs was that it was increasingly more difficult to patch SGNs after 7 days from birth (>P7), presumably because the myelin sheath became
more solid, making its removal more difficult with our dissociation
method. However, because similar ACh- and ATP-induced currents were
also observed in some more mature neurons (P12-P14,
n = 19; at that age hearing becomes functional in the
rat) and even in older cochlear neurons of P17 or more
(n = 3 for each agonist), we believe that these
cholinergic and purinergic receptors were not only present during
postnatal development but also maintained during adulthood.
Common characteristics of ACh- and ATP-evoked currents.
As seen in Purinergic response, although the ATP-evoked
current (slow component) showed a somewhat larger inward conductance compared with the ACh-evoked currents, the responses shared numerous common properties such as a similar long latency of activation, inward
rectification, and reversion near 0 mV. In this part of the
study we compared their ionic permeability and calcium sensitivity. Both currents were insensitive to the presence of the fast
calcium-chelating agent BAPTA (10 mM) in the internal recording
solution (Fig. 6). Indeed, with the B10Na
internal solution, the ACh-evoked current and the second ATP-evoked
current averaged 342 ± 106 (n = 3) and 806 ± 491 (n = 8) pA, respectively, values not
significantly different from those obtained with the internal EGTA
solution. This indicated that the nonselective cation current
was not activated consecutively to an increase in intracellular
calcium. Furthermore, replacement of Cl
with gluconate in
the internal solution (n = 5) did not significantly affect ATP- or ACh-evoked currents (Fig. 6), indicating a slight participation, if any, of a Cl
conductance. Replacing
K+ with Cs+ (n = 8) in the
internal solution (Fig. 6) or adding 100 µM linopirdine in the
extracellular solution (Table 1) also did
not affect the purinergic or cholinergic currents, suggesting that the
change in membrane conductance evoked by ACh or ATP in rat SGNs was not due to the suppression of an M-like K+ current as shown in
cultured chick cochlear ganglion neurons (73).

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Fig. 6.
Changing the intracellular Ca2+ buffering capacity and
replacing intracellular K+ by Cs+ or
Cl by gluconate did not affect the currents evoked by ACh
and ATP. A: examples of current responses evoked by ACh (100 µM, 1 s) in 3 different neurons. The internal solutions
were B10Na [10 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA); see METHODS], B10Glu (10 mM BAPTA + K-gluconate), and CsCl (top, middle, and
bottom, respectively). These internal solutions were
used to prevent calcium-related intracellular processes, to block
anionic (Cl ) conductances, and to block outward potassium
currents, respectively. B: examples of current responses
evoked by ATP (100 µM, 1 s) in 3 different neurons using the
same 3 different internal solutions described in A. All
neurons were voltage-clamped at 50 mV.
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Neither reducing the extracellular Ca2+ concentration by
changing the bath solution nor application of ACh in a pipette solution containing a higher Ca2+ concentration reduced or augmented
the ATP- or ACh-evoked current responses (Fig.
7). Replacing 80% of extracellular
Na+ with NMDG+ also did not significantly
affect the ATP- or ACh-evoked currents (Fig.
8). Indeed, the I-V
relationships of the currents in NMDG medium did not differ
greatly from those recorded in normal extracellular medium. In
ACh-evoked conductances, Erev averaged 7.3 ± 12.5 mV in normal solution (n = 15) and 2.3 ± 10.0 mV in NMDG solution (n = 6), indicating a slightly
smaller permeability of the channel to NMDG+ compared with
Na+
(PNMDG/PNa = 0.78).
In ATP-evoked conductance, Erev averaged 4.4 ± 11 mV in normal solution (n = 17) and
3.4 ± 12.3 mV in NMDG solution (n = 5), also
indicating a slightly smaller permeability of NMDG+
compared with Na+
(PNMDG/PNa = 0.67).
Furthermore, in neurons in which both ligands were sequentially tested,
90% of the neurons responsive to ACh (n = 123) also
responded to ATP and 92% of those responsive to ATP (n = 121) also responded to ACh.

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Fig. 7.
Changing the concentration of external calcium did not affect the
currents evoked by ACh or ATP. Figure indicates responses of the same
neuron in 2 different calcium solutions. A: top,
examples of currents evoked by ACh (100 µM, 1 s) in normal
DPBS (left) and in calcium-free DPBS (right) in 2 neurons. In such conditions, the mean relative current size in the
low-calcium medium compared with normal DPBS was 103 ± 13%
(n = 9). Bottom, example of currents evoked
by application of ACh (100 µM, 5 s) in normal Hanks' balanced
salt solution (HBSS; left) and in HBSS that contained 30 mM
calcium (right). The relative current size compared with
normal HBSS was 97 ± 16% (n = 10). B:
experiments similar to those described in A but using ATP
instead of ACh. The relative current size in the low-calcium medium
compared with normal DPBS was 97 ± 4.9% (n = 5).
The relative current size in high-calcium medium compared with normal
HBSS was 101 ± 15% (n = 6). Neurons
were voltage-clamped at 50 mV. Dotted lines indicate zero current
levels.
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Fig. 8.
I-V relationships evoked by ACh and ATP in neurons
bathed in a NMDG+ solution (replacing 80% of
Na+) and in a normal Na+ extracellular
solution. A: top, current responses to ACh (100 µM, 1 s) in the same neuron bathed in normal DPBS and
low-Na+-containing (20%) DPBS. The neuron was
voltage-clamped at 50 mV. The relative current size in the
low-Na+ medium compared with normal DPBS was 117 ± 25% (n = 9). Bottom, I-V
relationships in ACh (100 µM)-evoked conductances obtained by the
ramp protocol. Because of the relatively large variance in current
size, currents were normalized for each neuron to the current measured
at 50 mV (corresponding to 100%). Averages ± SE are shown
for the conductance in normal DPBS (left; n = 15) and in low-Na+-containing DPBS (right,
Na+ 20% NMDG+ 80%; n = 6).
B: experiments similar to those described in A
with ATP (100 µM). The relative current size in the
low-Na+ medium compared with normal DPBS was 97 ± 12% (n = 4). In the I-V relationships of
ATP (100 µM)-evoked conductances, averages ± SE are shown for
the conductance in normal DPBS (Na+ 100%,
n = 17) and low-Na+-containing (20%) DPBS
(n = 5).
|
|
Inhibition study of currents evoked by ACh and ATP.
Tables 1 and 2 summarize our attempts to
block the ACh- and ATP-evoked conductances with various molecules known
to affect certain channels or intracellular processes. The drugs were
selected empirically and on a trial-and-error basis with regard to
their previously reported action on nonselective cation conductance: staurosporine inhibits protein kinase C (54, 65);
trifluoperazine is widely used as an inhibitor of calmodulin-dependent
stimulation of cyclic nucleotide phosphodiesterases (25, 27, 28,
67, 74); U-73122 inhibits G protein-mediated phospholipase C
activation (75); linopirdine blocks so-called M currents
(1, 10); N-ethylmaleimide is an uncoupling
agent that is used as an inhibitor of G protein-related processes
(21, 47); ruthenium red is a blocker of various
calcium-related channels (8, 71), and it also blocks
nonselective cation conductance evoked by capsaicin (39);
aristolochic acid inhibits phospholipase A2 (51,
69); KN-62 inhibits calcium/calmodulin-dependent protein kinase
II(43) and blocks porelike nonselective cation
conductances evoked by P2Z purinoceptors in nonneurons (26,
42); SK&F-96365 is an inhibitor of receptor-mediated calcium
channels (41) and blocked nonselective cation channels in
a nonneuron cell line (20); octanol is a blocker of gap
junctional conductance (62); and K-252a is a general
protein kinase inhibitor (31). The intracellularly loaded
inhibitors
phalloidin (7.5 µM), which interferes with processes concerning actin (48); heparin (1.0 mg/ml), which inhibits
the phosphatidylinositol cascade (19); guanosine
5'-O-(2-thiodiphosphate) (GDP
S, 2.0 mM), which is a
nonactive analog of GTP and blocks intracellular processes associated
with G protein activation (23, 33, 45); and aristolochic
acid (100 µM)
did not have significant effects on either ACh- or
ATP-evoked currents (n = 3-8 for each condition).
The fact that none of these reagents inhibited the nonselective cation
conductance evoked by ACh or ATP in SGNs suggested that the activation
of these conductances may involve a novel metabolic pathway.
The only agent that we could identify with a significant blocking
effect on the cholinergic and the purinergic-evoked nonselective cation
conductance was the calcium ionophore ionomycin (38). Ionomycin is also known to be an activator of phospholipase
A2 and C (49). Interestingly, a brief
application via a puff pipette of 50 µM ionomycin evoked the
activation of a transient inward current when SGNs were voltage-clamped
at
50 mV (n = 10). The I-V relationship of
the ionomycin-evoked current also suggested activation of nonselective
cation conductance resembling that observed with ACh or ATP (not
shown). The subsequent application of ACh after ionomycin (ACh was
applied at least 1 min after the ionomycin-evoked conductance has
returned to prestimulation level) no longer triggered an inward current
(100% inhibition; n = 3). Similarly, the second
component of the ATP-evoked current largely decreased after ionomycin
application (inhibition 75-100%; n = 3). The
cholinergic and purinergic responses recovered slowly after ionomycin
exposure, generally within 20-90 min (Fig.
9). It should be noted that the first
component of the ATP-evoked current attributable to a P2X current
persisted after ionomycin treatment.

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|
Fig. 9.
Ionomycin inhibits the currents evoked by ACh and ATP.
A: consecutive current recordings from the same neuron
voltage-clamped at Vh = 50 mV. The neuron
was first stimulated by ACh (100 µM, 1 s), then by 50 µM
ionomycin (1 s), and after 1, 40, and 90 min with ACh (100 µM, 1 s) again. Note that ACh-evoked currents completely disappeared after
the application of ionomycin but slowly reappeared after 40 min.
B: Similar experiment with another neuron stimulated
consecutively with ATP and ionomycin. Note that only the 2nd component
of the current was inhibited. Dotted lines indicate zero current
levels.
|
|
A mutual inhibition or desensitization was also observed between the
ACh- and ATP-evoked currents (Fig. 10).
The ACh-evoked current was inhibited by 45-100%
(n = 3) after repeated application of ATP and recovered
to 70-110% of the initial response after 10 min. The second
component of the ATP-evoked current was inhibited by 25-45%
(n = 3) after repeated application of ACh and recovered to 85-115% of the initial response after 10 min. This mutual
inhibition by ACh and ATP, although partial, was another argument for a
common metabolic pathway to activate the nonselective cation
conductance. Furthermore, the current evoked by a mixed solution of ACh
and ATP (each 100 µM) was significantly smaller (60-70%;
n = 3) than the sum of the currents evoked by ATP and
ACh separately (Fig. 10C), thus reinforcing our hypothesis
of the involvement of similar channels.

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|
Fig. 10.
Mutual inhibition and nonadditivity of the ACh- and
ATP-evoked currents (agonists were applied at 100 µM for 1 s at
constant Vh = 50 mV). A: recorded on the
same neuron, repeated applications of ATP (ATP train) reversibly
diminished the size of the ACh-evoked currents. B: in
another neuron, repeated application of ACh (ACh train) reversibly
reduced the 2nd component of the ATP-evoked current. C: in
another neuron, the combined application of ACh and ATP (each at 100 µM) generated an inward current smaller than the sum of 2 currents
evoked separately by each ligand (ACh only and ATP only). Dotted lines
indicate zero current levels.
|
|
 |
DISCUSSION |
Nonselective cation conductance activated by muscarinic and
metabotropic purinergic receptors.
The present study demonstrates for the first time the presence of a
nonselective cation conductance in SGNs. The novelty of our findings is
that this nonselective cation conductance can be activated by
muscarinic receptors and metabotropic purinergic receptors (P2Y-like).
Indeed, several lines of evidence suggest the involvement of a
diffusible second messenger. First, the long time delay of the evoked
currents supports this hypothesis as in previous reports (16, 30,
36). The first component of the ATP-evoked current, presumably
involving P2X receptors, served here as a comparison with a direct
ionotropic process that displayed a much shorter latency. In addition,
the involvement of mAChRs in the cholinergic responses is also clearly
reinforced by the effects of muscarine as agonist and atropine as
antagonist of the response. On the other hand, the second component of
the ATP-evoked current was mimicked by UTP, a rather selective agonist
of P2Y metabotropic receptors, but not by
,
-methylene ATP or
2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), which are almost
exclusively agonists of ionotropic receptors (data not shown).
Nonselective conductance permeable to large cations such as
NMDG+.
One of the important characteristics of the nonselective cation
conductance evoked in SGNs is its permeability to large cations such as
NMDG+. NMDG+ is an organic cation that has been
used widely to verify the degree of nonselectivity of cationic channels
(16, 29, 32, 46, 55, 68, 72). In some other neurons, not
mentioned in RESULTS, the replacement of 80% of
extracellular Na+ by another large cation,
tetraethylammonium (TEA+), did not block the ACh- or
ATP-evoked conductances and maintained an Erev
near 0 mV (3.8 ± 7.2 mV for ACh,
PTEA/PNa = 0.83, n = 4; 0.4 ± 2.5 mV for ATP,
PTEA/PNa = 0.81, n = 3; data not shown). Although most of the
nonselective cation conductances are not permeable to such large
cations, the activation of a similar nonselective large conductance has
been described with substance P (29) and with P2Z/P2X
receptors for ATP. The latter channels have been reported to be
membrane "pores" in neurons (32, 63, 68). However, the
nonselective conductance evoked by ATP or ACh in the present study
appears somewhat different from the ionotropic pore evoked via P2X/P2Z
receptors, because much longer (10 s to 1 min) application of ATP was
necessary to form these pores (32, 68) and the pore
conductance increased with repeated application of ATP in these studies
(6). This was not the case in the present study with SGNs.
Although the slope conductance of the ATP current was larger, the
nonselective conductance activated by ATP and ACh appeared to be due to
similar channels, as suggested by their similar latency, time course,
and I-V relationship, mutual desensitization, and nonlinear
summation criteria also adopted in previous reports (22, 59, 61,
64). The difference in current amplitude may simply be due to
the activation of a larger number of channels with ATP. In dissociated
hippocampal CA3 neurons, interaction among multiple G protein-coupled
receptors has also been shown in neurotransmitter activation of G
protein-activated inwardly rectifying potassium (GIRK) channels
(61). In these central neurons, although showing
differences in slope conductance and kinetics, the same GIRK channels
were shown to be activated by GABAB receptors and
metabotropic glutamate receptors. In the present study, the similar
inhibition of both currents by ionomycin was another good argument that
they involve similar channels. Moreover, the pores of the channels
activated by ATP or ACh appeared similarly wide enough for large
cations as NMDG+ and TEA+ to pass freely.
Although sharing of the GIRK channel by a large number of
neurotransmitters is well known (2, 11, 40, 61, 64), activation of a nonselective cation conductance by different ligands on
the same cell has been mentioned only in a few reports in neurons (4, 22, 39, 59). The porelike nonselective conductance activated by the marine toxin maitotoxin and P2Z purinergic receptor stimulation by BzATP have also been shown to share a common cytolytic pore in nonneuronal cells (56). Our present study is the
first to describe the sharing of a porelike effector channel between two different receptors such as P2Y-like receptors and mAChRs in
peripheral neurons such as cochlear SGNs.
Calcium ions are not involved in signal transduction pathway.
Muscarinic receptors and P2Y-like purinergic receptors are known to be
associated with an elevation of intracellular calcium in SGNs (7,
50), so the nonselective cation conductance observed in the
present study could be activated via a calcium-dependent process. This
could also have been suggested by the effect of the calcium ionophore
ionomycin, which activated a conductance similar to that activated by
ACh or ATP. However, the presence of 10 mM intracellular BAPTA, a fast
calcium-chelating buffer known to prevent the activation of numerous
membranous Ca2+-sensitive channels, did not affect the
cholinergic or purinergic response in SGNs. This suggested that a rise
in intracellular calcium concentration was not responsible for the
direct activation of the nonselective cation conductance in SGNs. This
idea was reinforced by the effects of 10 mM caffeine, a molecule known to act directly at internal calcium stores (66). Caffeine,
although raising intracellular Ca2+ in SGNs (as measured by
indo spectrofluorometry; data not shown), did not evoke significant
depolarizing currents, whereas the same SGNs were seen to respond to
100 µM ACh (n = 3). The ACh- or ATP-evoked nonselective conductances were also insensitive to both a decrease and
an increase in extracellular calcium concentration, in contrast to most
nonselective cation conductances sensitive to extracellular divalent
cations (6, 9, 16, 22, 32, 57, 63). Such channels
insensitive to divalent cations are rather rare (58, 72).
Although improbable, the possibility remains that the internal calcium
stores in SGNs are situated just beneath the cytoplasmic membrane where
the internal solution is not reachable by BAPTA, making the
nonselective cation channels still possibly activated by intracellular calcium.
Signal transduction pathway can be activated by ionomycin.
Surprisingly, none of the potential blockers/inhibitors of various
intracellular second messenger pathways tested in the present study
were effective in blocking the nonselective cation current in SGNs, so
the signal transduction pathway involved remains undetermined. Because
the ATP- or ACh-evoked conductances observed in this study may be due
to the activation of membrane pores and not ordinary ion channels, like
those described with P2X/P2Z receptors in central neurons (32,
63, 68), we also tested some agents that influence gap junctions
(octanol) and cell skeletons (phalloidin). None of these agents
affected the cholinergic or purinergic depolarization, suggesting that
the activated conductance was not due to the formation of membrane
cytolytic pores.
The calcium ionophore ionomycin (38) was the only agent
that could interfere reversibly with the ACh- and ATP-evoked currents. As discussed above, the action of ionomycin in the membrane conductance of SGNs was probably not related to an increase in intracellular calcium. Besides its calcium ionophoric effects, ionomycin is also
known to activate the phopholipase C and phospholipase A2 pathways in various cell types (49), suggesting that these
metabolic pathways could be involved in our study. However, the
inhibitors of phospholipases A2 and C, aristolochic acid
and U-73122, respectively, were ineffective in blocking the ACh or ATP
response. It is therefore possible that another unknown metabolic
pathway evoked by ionomycin is involved. Unknown intracellular
metabolic processes have also been reported in the activation of
nonselective cationic conductance by metabotropic glutamate and
muscarinic receptors in C3 pyramidal central neurons (22).
Another hypothesis is that ionomycin activates the same channels as in
ACh or ATP and blocks the ACh and ATP currents by desensitization.
The oscillation in membrane currents after ACh application in SGNs does
not appear to be related to oscillations of intracellular calcium
reported in other systems (35). The essential arguments are that current oscillations were also observed when BAPTA (10 mM) was
added to the internal solution and that oscillation of calcium signals
was not observed in our previous study with ACh (50). On
the other hand, the oscillation of membrane currents in SGNs was more
frequently encountered with ACh responses than with ATP responses, thus
suggesting a certain difference in the mediating intracellular
processes between mAChRs and P2Y-like receptors.
Physiological implications.
The present study suggests that two different neurotransmitters, ACh
and ATP, share the activation of a similar nonselective cation
conductance in SGNs. The postsynaptic receptors involved are mAChRs and
P2Y-like receptors. It is always difficult to extrapolate for a
physiological role in cochlear function from a cellular study, but we
believe that one of the roles of these depolarizing responses would be
to increase the neural excitability of SGNs on cholinergic or
purinergic stimulation. This hypothesis fits well with the study of
Felix and Ehrenberger (18) showing that intracochlear
application of ACh below IHCs increases afferent nerve fiber spiking
activity. It has yet to be confirmed that such functional coupling of
metabotropic receptors and nonspecific cation channels is present at
the dendrites below IHCs, the area where lateral efferent synapses
occur. Another possible role of such porelike channels is cell
autolysis, i.e., apoptosis, because the conductance allows the
passage of large molecules, which can cause cell death
(63). However, in the present study, repeated application
of ligands (ACh or ATP) did not lead to cell death, diminishing the
pertinence of this second hypothesis. Finally, because the ATP- and
ACh-activated nonselective conductance was also observed at the
earliest stage of postnatal development, it is possible that these
responses have an important role in SGN development and survival
(37).
 |
ACKNOWLEDGEMENTS |
This work was supported by the Canon Foundation Europe (Leiden, The
Netherlands), Fondation pour la Recherche Médicale
(Paris, France), and Conseil Régional
d'Aquitaine (Bordeaux, France).
 |
FOOTNOTES |
Address for reprint requests and other correspondence: D. Dulon, Laboratoire de Biologie Cellulaire et Moléculaire de
l'Audition, INSERM EMI 99-27, Hôpital Pellegrin,
Université de Bordeaux 2, 33076 Bordeaux, France (E-mail:
didier.dulon{at}bordeaux.inserm.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. Section 1734 solely to indicate this fact.
First published December 19, 2001;10.1152/ajpcell.00364.2001
Received 2 August 2001; accepted in final form 17 December 2001.
 |
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