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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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 MOmega . 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)
<IT>P</IT><SUB><IT>Y</IT></SUB>/<IT>P</IT><SUB><IT>X</IT></SUB><IT>=</IT>[<IT>X</IT>]<SUB>i</SUB>/[<IT>Y</IT>]<SUB>o</SUB> ∗ exp(<IT>E</IT><SUB>rev</SUB> ∗ <IT>F/RT</IT>)
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
P<SUB>NMDG</SUB>/<IT>P</IT><SUB>Na</SUB><IT>=</IT>1<IT>/</IT>0.8<IT> ∗ </IT>{exp[(<IT>E</IT><SUB>NMDG80Na20</SUB><IT>−E</IT><SUB>Na</SUB>) ∗ <IT>F/RT</IT>]<IT>−</IT>0.2}


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (tau ) 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 alpha -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.

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.

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.

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 (tau  = 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 tau  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.

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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Table 1.   Summary of inhibitors in extracellular solution showing no effect on cholinergic current

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) (GDPbeta 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.

                              
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Table 2.   Summary of inhibitors in extracellular solution showing no effect on purinergic metabotropic current

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha ,beta -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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