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Inhibition of voltage-gated channel currents in rat auditory cortex neurons by salicylate
Neuropharmacology 53 (2007) 870e880 www.elsevier.com/locate/neuropharm
Inhibition of voltage-gated channel currents in rat auditory cortex neurons by salicylate Yanxing Liu a,b,*, Hailin Zhang a, Xuepei Li b, Yongli Wang a, Hong Lu c, Xiang Qi d, Changsheng Ma a, Junxiu Liu b a
Department of Pharmacology and Neurobiology, Hebei Medical University, No. 361 Zhongshan East Road, Shijiazhuang 050017, Hebei Province, P.R. China b Department of Otorhinolaryngology, Peking University Third Hospital, No. 49 Huayuan Road, Beijing 100083, P.R. China c Department of Otorhinolaryngology, Hebei Medical University Second Hospital, Shijiazhuang 050000, Hebei Province, P.R. China d Department of Anesthesiology, Hebei Medical University Second Hospital, Shijiazhuang 050000, Hebei Province, P.R. China Received 1 March 2007; received in revised form 10 August 2007; accepted 16 August 2007
Abstract Salicylate is a medicine for anti-inflammation with a side effect of tinnitus. To understand the mechanisms of tinnitus induced by salicylate, we studied the effects of salicylate on voltage-gated ion channels and action potential firing rates in freshly dissociated rat pyramidal neurons in auditory cortex (AC) using the whole-cell patch technique. We found that salicylate reduced the voltage-gated sodium current (INa), the delayed rectifier potassium current (IK(DR)) and the L-type voltage-gated calcium current (ICa,L) in concentration-dependent manner. An amount of 1 mM salicylate shifted the steady-state inactivation curve of INa negatively by about 5 mV, shifted the steady-state activation and inactivation curve of IK(DR) negatively by approximately 14 mV and 17 mV, respectively, and shifted the steady-state activation curve of ICa,L negatively by about 10 mV. 1 mM salicylate significantly increased the action potential firing rates, ultimately. From the results, we speculated that through affecting the voltage-gated ion channels in AC, an important position in auditory system, salicylate increased the firing rate of neurons and enhanced neuronal excitability on the one hand, increased the excitatory transmitters release and reduced the inhibitory transmitter release on the other hand, thus finally induced tinnitus. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Salicylate; Voltage-gated sodium channel current; Delayed rectifier potassium channel current; L-type voltage-gated calcium channel current; Auditory cortex
Abbreviations: AC, auditory cortex; ACSF, artificial cerebrospinal fluid; 4AP, 4-aminopyridine; ATP, adenosine-50 -triphosphate; EGTA, ethyleneglycolbis-(2-aminoethyl)-tetraacetic acid; GABA, g-aminobutyric acid; GABA(A), g-aminobutyric acid type A receptor; GTP, guanosine-50 -triphosphate; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; IC50, halfblocking concentration; ICa,L, L-type voltage-gated calcium channel current; IK(A), transient outward potassium channel current; IK(DR), delayed rectifier potassium channel current; INa, voltage-gated sodium channel current; MOPS, 3-[N-morpholino]propane-sulfonic acid; TEA, tetraethylammonium; TTX, tetrodotoxin. * Corresponding author. Department of Pharmacology and Neurobiology, Hebei Medical University, No. 361 Zhongshan East Road, Shijiazhuang 050017, Hebei Province, P.R. China. Tel.: þ86 (0)311 8626 6291; fax: þ86 (0)311 8605 7291. E-mail address: [email protected] (Y. Liu). 0028-3908/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2007.08.015
1. Introduction Tinnitus is a phantom auditory experience in the absence of an external or internal, physically quantifiable, sound. It is a frequent and devastating symptom of disorders of the auditory system. Salicylate (the active component of aspirin) overdose induces tinnitus in humans (Myers and Berstein, 1965) and in animals (Penner and Jastreboff, 1996), but the neural origins and mechanisms underlying salicylate-induced tinnitus are still obscure. The most widely used animal models of tinnitus are rats receiving salicylate (Jastreboff et al., 1988; Ru¨ttiger et al., 2003).
Y. Liu et al. / Neuropharmacology 53 (2007) 870e880
In previous studies, we found that salicylate affected the electrophysiological properties of the voltage-gated ion channels in rat inferior colliculus neurons (Liu and Li, 2004a,b; Liu et al., 2005). It is well known that the auditory cortex (AC) is also an important site to study tinnitus because it is the most advantaged center in performing sense of hearing. Injections of salicylate (350 mg/kg body weight) led to accumulation of arg3.1 and c-fos immunoreactive neurons in gerbil AC (Wallhausser-Franke et al., 2003; Mahlke and Wallhausser-Franke, 2004). When it induced tinnitus, salicylate significantly increased the spontaneous discharge rate and the local field potentials recorded from the multiunit cluster in the auditory cortex of awake rats (Yang et al., 2007). A positron emission tomography (PET) study in 11 patients suffering from chronic disabling tinnitus showed that metabolic activity in primary AC of 10 tinnitus patients significantly increased. This study gave objective evidence of tinnitus sensation and localization for the first time (Arnold et al., 1996). The salicylate concentrations in rat cerebrospinal fluid over 8 h after salicylate injection (460 mg/kg body weight) were studied (Jastreboff et al., 1986). The salicylate concentrations in cerebrospinal fluid rose to approximately 200e300 mg/l (equivalent to 1.25e1.87 mM) within 1 h, remained at this level for the next 3 h, and then decreased slowly to 150e 200 mg/l (equivalent to 0.94e1.25 mM) in the next 4 h. Another study of induced phantom auditory sensations in rats showed that tinnitus was induced by the administration of salicylate (350 mg/kg body weight) given 3 h before testing (Ru¨ttiger et al., 2003). Thus, when tinnitus develops, the salicylate concentration in their cerebrospinal fluid is about 1 mM. Electrical and chemical signaling in the nervous system is of fundamental importance to the integration of external and internal stimuli. Proper signaling depends on a finely tuned series of ion-channel-mediated events mediating electrical activity and transduction at synapses. Slight changes in ion channel properties will cause drastic changes in normal physiological functions of neurons. Voltage-gated sodium channels mediate the very rapid rising phase and initial component of the falling phase of action potentials in the excitable cells (Hodgkin and Huxley, 1952; Hille, 1992). Voltage-gated sodium channels also play a critical role in action potential propagation as well as transduction of electrical currents to transmitter release from the nerve terminal. Voltage-gated potassium channels are widely expressed throughout the brain, and they play crucial roles in regulating a variety of cellular processes in neurons, such as setting the resting membrane potential, repolarization, action potential duration, discharge patterns, and delay between a stimulus and the first action potential (Storm, 1990). Voltage-gated calcium currents play an important role in regulating neurotransmitter, such as g-aminobutyric acid (GABA), release. Behavioral studies suggested that nimodipine, an L-type Ca-channel blocker, could totally abolish salicylate-induced tinnitus (Jastreboff and Brennan, 1988; Wang et al., 2000). L-type calcium channels mediate long-lasting calcium currents in response to depolarization in excitable cells. Calcium influx through L-type calcium channels leads
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to activation of a cascade of intracellular signals and is also responsible for the afterhyperpolarization phase following action potentials in neurons (Marrion and Tavalin, 1998). These particular features of L-type calcium channel-dependent calcium influx play important role in neuronal excitability and plasticity. In all, changes in properties of voltage-gated sodium, potassium channels and/or calcium channels would directly affect neuronal excitability and activities. The aim of the present study was to obtain insight into the actions of salicylate on voltage-gated sodium, potassium channels, L-type calcium channels and action potential firing rates in freshly dissociated AC pyramidal neurons by use of the whole-cell patch clamp method. 2. Materials and methods 2.1. Cell preparation Fourteen-day-old Wistar rats (either sex) were anesthetized with pentobarbital (50 mg/kg body weight, i.p.) and decapitated. The AC was rapidly removed and cut into slices (400e500 mm thick) with a vibratome in ice-cold artificial cerebrospinal fluid (ACSF) bubbled continuously with a 95% O2/ 5% CO2 gas mixture. The AC slices were incubated in oxygenated ACSF for at least 60 min at room temperature. After incubation, AC slices were digested with Pronase E (0.75 mg/ml) in oxygenated ACSF at room temperature for 35 min. After enzyme treatment, the AC slices were rinsed twice with ACSF and kept in oxygenated ACSF at room temperature. Subsequently, AC neurons were mechanically dissociated by gentle trituration with firepolished Pasteur pipettes of decreasing diameter. The neuron suspension was transferred to a chamber, and neurons were allowed to settle to the bottom of the chamber before recording. The chamber was continuously perfused with an extracellular solution. The pyramidal neurons in AC were selected for our experiments by their shapes. To avoid possible phenotypic changes of currents due to culture conditions, AC neurons were used within 10 h following dissection. Animals were cared for and killed according to the principles of animal care outlined by the Chinese Academy of Sciences. All efforts were made to minimize animal suffering and to use the minimal number of animals in these studies.
2.2. Solutions and drugs The ACSF contained (in mM): NaCl 125, KCl 2.5, NaH2PO4 1.25, NaHCO3 26, CaCl2 2, MgCl2 1, D-glucose 10, with pH 7.4. The standard extracellular solution for recording of voltage-gated sodium channel currents (INa) contained (in mM): NaCl 50, choline chloride 90, MgCl2 1, CaCl2 1, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) 5, D-glucose 5, tetraethylammonium (TEA) chloride 25, 4-aminopyridine (4-AP) 5, and LaCl3 0.01, with pH 7.4. The internal solution for recording of INa contained (in mM): CsCl 125, NaF 20, HEPES 10, ethyleneglycol-bis-(2-aminoethyl)tetraacetic acid (EGTA) 10, TEA chloride 20, Mg adenosine-50 -triphosphate (ATP) 5, Na guanosine-50 -triphosphate (GTP) 0.1, and leupeptin 0.1, with pH 7.2. For recording of delayed rectifier potassium channel currents (IK(DR)), the standard extracellular solution contained (in mM): NaCl 125, KCl 5, MgCl2 2, CaCl2 2, D-glucose 10, HEPESeNaOH 10, and tetrodotoxin (TTX) 0.001, with pH 7.4. The pipette solution containing (in mM): KF 110, Trisebase 20, HEPES 10, EGTA 10, and Mg-ATP 2, adjusted to pH 7.4. For recording of L-type voltage-gated calcium channel currents (ICa,L), the extracellular solution contained (in mM): NaCl 80.4, KCl 5.4, TEA 25.0, BaCl2 5.0, 4-AP 10.0, MgCl2 0.8, NaHCO3 0.9, D-glucose 25, NaH2PO4 0.9, CaCl2 1.8, sodium pyruvate 0.23, 3-[N-morpholino]propane-sulfonic acid (MOPS) 10, TTX 0.001, and u-conotoxin GVIA 0.001, with pH 7.25. The internal solution contained (in mM): NaCl 5, cesium methanesulfonate
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115.6, MgCl2 2, CaCl2 0.5, EGTA 5, HEPES 10, and Mg-ATP 2, with pH 7.25. For recording of action potential firing rates, the extracellular solution was similar to ACSF. The pipette solution contained (in mM): K-methylsulfate 140, MgCl2 5, CaCl2 1, EGTA 10, HEPES 10, Na2-ATP 0.3 and Mg-ATP 2.2, with pH 7.2. Pronase E, HEPES, EGTA, Mg-ATP, Na-GTP, TEA, 4-AP, TTX and salicylate were purchased from Sigma Chemical Co. (St. Louis, MO). Other chemicals were obtained from Beijing Chemical Factory. For evaluation of salicylate effects, sodium salicylate was dissolved in extracellular solution and locally applied onto cells by use of a perfusion application system. Even at the highest concentration used in the study (10 mM) salicylate did not change the pH of the solution (pH 7.38 0.05 in control, 7.38 0.08 in the presence of salicylate at 1 mM, 7.37 0.07 in the presence of salicylate at 10 mM, respectively; n ¼ 5; P > 0.05). For evaluation the effects of salicylate on voltage-gated ion currents or action potential firing rates, cells were perfused for at least 2 min with control or salicylate solution before initiating the channel opening or the action potential.
2.3. Electrophysiological recording For the whole-cell patch clamp recordings, we used an EPC-9 amplifier, together with the corresponding PULSE 8.02 software (HEKA Elektronik, Germany). Patch pipettes were pulled from thin-walled borosilicate glass on a P 87 micropipette puller (Sutter Instruments, USA). The resistance of the fire-polished pipette was 4e6 MU when filled with the internal solution. Evoked currents were filtered at 3 kHz, digitized at 10 kHz and stored on a computer. The liquid junction potential between internal and external solutions was 5 mV on average and was used to correct for the recorded membrane potential. Capacity transients were canceled and series resistance compensated (>70%) by use of the internal circuitry of EPC-9. The leak current was digitally subtracted by use of the p/n protocol. All experiments were performed at room temperature. When the effects of salicylate on INa were studied, step depolarizations could activate inward currents. They were completely and reversibly blocked by 0.5 mM TTX, so these inward currents were attributed to INa. There are two main kind of voltage-gated potassium channels in cortex neurons: transient outward potassium channels and delayed rectifier potassium channels (Zhou and Hablitz, 1996). Their activation and inactivation voltage ranges and kinetics and pharmacological sensitivities are completely different. Step depolarizations of AC neurons from rest potential could mainly activate IK(DR), while the transient outward potassium current (IK(A)) was not obvious. When IK(DR) was measured, the 50-ms interval (during which the membrane potential was held at 40 mV) was inserted between the conditioning prepulse and test pulse to exclude IK(A). Therefore, IK(DR) could be measured at 50 ms after the onset of depolarization, because IK(A) was almost completely inactivated at a holding potential of 40 mV and to a considerable extent 50 ms after the onset of depolarization. 30 mM TEA could reversibly block the current amplitude of IK(DR) by 90.82 6.76% (n ¼ 9, P < 0.01). When ICa,L was measured, various blockers were used to inhibit the channels other than L-type channels. For example, TTX was used to block sodium channels. 4-AP and TEA were used to block transient outward and delayed rectifier potassium channels, separately. The voltage protocols used in this study could inhibit low threshold voltageactivated calcium channels. u-conotoxin GVIA was used here to block N-type calcium channels, the other main high threshold voltage-activated calcium channels. Therefore, the currents recorded in this condition were the ICa,L. 10 mM nimodipine could reversibly inhibit the current amplitude of ICa,L by 91.16 7.33% (n ¼ 8, P < 0.01). Action potentials were evoked by brief current injection (800 ms, 1 nA) through the patch pipette.
2.4. Data analysis Data were analyzed by use of PULSEFIT software (Heka Elektronik, Germany) and Origin 6.0 software (MS, USA). Results were expressed as mean S.D., and n represented the number of the cells examined. Statistical significance was assessed by use of a 2-way ANOVA; a P value of <0.05 was considered significant.
3. Results 3.1. Effects of salicylate on INa The freshly dissociated AC neurons were held at 80 mV and depolarized to 30 mV for 20 ms at 0.1 Hz to activate INa. Upon the administration of salicylate (in mM: 0.1, 0.3, 1, 3 and 10), the amplitudes of INa decreased with increments of concentrations from 0.1 to 10 mM. The normalized current, obtained from normalizing the INa after salicylate application to the control INa, was plotted as a function of the salicylate concentration (Fig. 1A). The concentrationeresponse curve was fitted with the Hill equation: I/I0 ¼ 1/[1 þ (x/Ki)h], where x is the concentration of the salicylate, h is the Hill coefficient, and Ki is the half-blocking concentration (IC50). The curve was best fitted when IC50 was 3.52 mM and h was 0.76 (n ¼ 9). The results indicate that salicylate decreased the amplitudes of INa in a concentration-dependent manner. In this study, we used the concentration of 1 mM for the experimental concentration of salicylate. In this experiment, the current amplitude decreased rapidly and reached a steady state within 2 min after salicylate application. After washout with salicylate-free solution, the current amplitude was soon restored. At a concentration of 1 mM, salicylate application for 2 min reduced the sodium current to 71.42 9.39% of control (n ¼ 9, P < 0.05). Upon washout with salicylate-free solution for 2 min, the sodium current was restored to 95.67 2.07% of control (n ¼ 9, P > 0.05). Our results showed that the block effect of salicylate could be reversible. The current densityevoltage curve of INa was obtained by depolarizing steps from a holding potential of 80 to þ50 mV at 10-mV steps. The maximal peak current was divided by cell capacitance to obtain peak current density (pA/ pF ) for each cell. Shown in Fig. 1 are representative families of INa responses in controls (Fig. 1B) and in 1 mM salicylate (Fig. 1C). Fig. 1D shows the current densityevoltage curve for INa before and after salicylate application. Salicylate attenuated the current amplitude of INa in a voltage-dependent manner. The effects of salicylate on the conductanceevoltage curve of voltage-gated sodium channels are illustrated in Fig. 1E. The INa was induced by the step pulses (10-mV increments) from a holding potential of 80 to 30 mV. Peak current amplitude was converted into conductance by use of the equation G ¼ I/(V VNa), where V is the membrane potential, and VNa is the sodium equilibrium potential obtained as the reversal potential from the IeV curve. The sodium conductance was normalized to the respective maximal value and plotted as a function of membrane potential. With use of a least-squares fit procedure, the normalized conductance was well fitted with a Boltzmann equation: G/Gmax ¼ 1/{1 þ exp[(V V1/2)/k]}, where G is conductance, Gmax is maximum conductance, V is test potential, V1/2 is the membrane potential at half-activation, and k is a slope factor. Before and after application of 1 mM salicylate, the values of V1/2 were 48.06 2.37 and 48.03 1.81 mV (n ¼ 9, P > 0.05), with k of 3.48 0.93
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Fig. 1. (A) Concentrationeresponse relationship for the blockade of INa by salicylate. The current was evoked by depolarization to 30 mV for 20 ms from a holding potential of 80 mV. The current amplitude was measured before and after salicylate treatment, and the fraction of control current amplitudes was plotted as a function of salicylate concentration. Each point represents the mean S.D. (n ¼ 9). Data were fitted well with the Hill equation. The IC50 was 3.52 mM and the h was 0.76. (BeD) Effect of 1 mM salicylate on the current densityevoltage relation of peak INa. A series of 20 ms of depolarizing steps from a holding potential of 80 to þ50 mV (at 10-mV increments) was applied at 0.1 Hz. (B) Original traces of INa in control; (C) original traces of INa after 1 mM salicylate application; (D) peak current densityevoltage curves for control and 1 mM salicylate. Each point represents mean S.D. (n ¼ 9). *P < 0.05, **P < 0.01. (E) Effects of 1 mM salicylate on the steady-state activation curves for INa. A series of 20-ms depolarizing steps from a holding potential of 80 to 30 mV (at 10-mV increments) were used. Interpulse interval was 1 s. Normalized peak conductance was fitted with a Boltzmann function. For the control, V1/2 and k were 48.06 2.37 mV and 3.48 0.93, respectively, while for 1 mM salicylate V1/2 and k were 48.03 1.81 mV (n ¼ 9, P > 0.05) and 3.40 1.22 (n ¼ 9, P > 0.05), respectively. Each point represents mean S.D.
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and 3.40 1.22 (n ¼ 9, P > 0.05), which indicated that the GeV curve did not shift toward the positive or negative potential and the activation process was not affected by salicylate. A test pulse to 30 mV for 20 ms was given immediately following a 1-s conditioning pulse (120 to 40 mV in 10mV increments) from a holding potential of 80 mV. Fig. 2 shows the steady-state inactivation curves for INa under control conditions and after exposure to 1 mM salicylate. The peak amplitude for INa was normalized and plotted versus prepulse potentials. Sodium channels were inactivated at a holding potential above 50 mV and relieved from inactivation at a holding potential below 100 mV. With use of a least-squares fit method, the curves were well fitted with Boltzmann equation I/Imax ¼ 1/{1 þ exp[(V V1/2)/k]}, where I is current amplitude, Imax is maximum current amplitude, V is holding potential, V1/2 is the potential at which the current becomes half maximum, and k is a slope factor. Salicylate at 1 mM significantly shifted the steady-state inactivation curve in the hyperpolarizing direction. Before salicylate application, V1/2 was estimated to be 69.99 2.64 mV and k 4.90 1.25. After 1 mM salicylate application, V1/2 was 74.82 1.72 mV (n ¼ 9, P < 0.01) and k 6.49 0.52 (n ¼ 9, P > 0.05). The results showed that salicylate significantly changed the V1/2 by about 5 mV without affecting k of the steady-state inactivation curve. 3.2. Effects of salicylate on IK(DR) Fig. 3A shows the concentrationeresponse curves for the action of salicylate on IK(DR). IK(DR) was evoked by a 150ms test pulse to þ60 mV following a 150-ms conditioning pulse to 110 mV and a 50-ms insertion at 40 mV from a holding potential of 40 mV. Salicylate blocked IK(DR) in a concentration-dependent manner. When the IC50 values of salicylate on IK(DR) were 2.13 mM, and the h was 1.06 (n ¼ 9), the curve was well fitted with the Hill equation. Fig. 3B and C show the original current of IK(DR) in controls and in 1 mM salicylate, respectively. Fig. 3D shows the current densityevoltage relationship for IK(DR) before and after salicylate application. The voltage protocol is that a 150-ms conditioning pulse to 110 mV was followed by a 150-ms test pulse (30 to þ60 mV in 10-mV increments) applied after 50 ms at 40 mV from a holding potential of 40 mV. Salicylate decreased the current amplitude of IK(DR) in a voltage-dependent manner. In addition, in the blank control without salicylate (time-matched control), IK(DR) were decreased by 4.08 1.79% (n ¼ 9, P > 0.05) after 15 min current recording. Thus, the run-down of IK(DR) was ruled out. We studied the effect of salicylate on the activation kinetics of IK(DR). Fig. 3E shows the conductanceevoltage curve of delayed rectifier potassium channels. In the absence of salicylate, IK(DR) was half activated at 2.13 1.95 mV and k was 24.28 1.58. In the presence of salicylate, IK(DR) was half activated at 16.22 3.07 mV (n ¼ 9, P < 0.01) and k was 18.22 1.26 (n ¼ 9, P > 0.05). Thus, the application of 1 mM salicylate produced a 14-mV negative shift of GeV curve. The results suggest that salicylate modified the activation process of IK(DR) significantly.
Fig. 2. Effects of 1 mM salicylate on the steady-state inactivation curves for INa. The pre-pulse potential changed from 120 to 40 mV in 10-mV increments for 1 s and was immediately followed by a 20-ms step depolarization to 30 mV. The interpulse interval was 10 s. (A) Original traces of INa in control. (B) Original traces of INa after 1 mM salicylate application. (C) Steady-state inactivation curves for control and 1 mM salicylate. The normalized current of steady-state inactivation was fitted with a Boltzmann function. For the control, V1/2 was 69.99 2.64 mV and k 4.90 1.25. For 1 mM salicylate, V1/2 was 74.82 1.72 mV (n ¼ 9, P < 0.01) and k 6.49 0.52 (n ¼ 9, P > 0.05). Each point represents mean S.D.
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Fig. 3. (A) Concentrationeresponse relationship for the blocking of IK(DR) by salicylate. The current was evoked by a 150-ms test pulse to þ60 mV following a 150-ms conditioning pulse to 110 mV and a 50-ms insertion at 40 mV from a holding potential of 40 mV. The current amplitude was measured before and after salicylate treatment, and the fraction of control current amplitudes was plotted as a function of salicylate concentration. Each point represents the mean S.D. (n ¼ 9). Data were fitted well with the Hill equation. The IC50 was 2.13 mM and the h was 1.06. (BeD) Effect of 1 mM salicylate on the current densityevoltage relation of peak IK(DR). The voltage protocol is that a 150-ms conditioning pulse to 110 mV was followed by a 150-ms test pulse (30 to þ60 mV in 10-mV increments) applied after 50 ms at 40 mV from a holding potential of 40 mV. (B) Original traces of IK(DR) in control; (C) original traces of IK(DR) after 1 mM salicylate application; (D) current densityevoltage curves for control and 1 mM salicylate. Each point represents mean S.D. (n ¼ 9). *P < 0.05, **P < 0.01. (E) Effects of 1 mM salicylate on the steady-state activation curves for IK(DR). The voltage protocol is that a 150-ms conditioning pulse to 110 mV was followed by a 150-ms test pulse (30 to þ60 mV in 10-mV increments) applied after 50 ms at 40 mV from a holding potential of 40 mV. Normalized peak conductance was fitted with a Boltzmann function. For the control, V1/2 and k were 2.13 1.95 mV and 24.28 1.58, respectively, while for 1 mM salicylate V1/2 and k were 16.22 3.07 mV (n ¼ 9, P > 0.05) and 18.22 1.26 (n ¼ 9, P > 0.05), respectively. Each point represents mean S.D.
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immediately following a 1600-ms conditioning pulse (180 to 40 mV in 10-mV increments) from a holding potential of 30 mV. Fig. 4C shows the steady-state inactivation curve of IK(DR) before and after salicylate application. In the absence of salicylate, IK(DR) was half inactivated at 80.76 3.47 mV and k was 22.78 2.03. In the presence of salicylate, IK(DR) was half activated at 97.58 3.09 mV (n ¼ 9, P < 0.01) and k was 22.38 1.82 (n ¼ 9, P > 0.05). Thus, the application of 1 mM salicylate produced a 17-mV hyperpolarizing shift of steady-state inactivation curve. The results suggest that salicylate also significantly modified the inactivation properties of IK(DR). 3.3. Effects of salicylate on ICa,L
Fig. 4. Effects of 1 mM salicylate on the steady-state inactivation curves for IK(DR). The current was evoked by a test pulse to þ30 mV for 450 ms given immediately following a 1600-ms conditioning pulse (180 to 40 mV in 10-mV increments) from a holding potential of 30 mV. The interpulse interval was 10 s. (A) Original traces of IK(DR) in control. (B) Original traces of IK(DR) after 1 mM salicylate application. (C) Steady-state inactivation curves for control and 1 mM salicylate. The normalized current of steady-state inactivation was fitted with a Boltzmann function. For the control, V1/2 and k were 80.76 3.47 mV and 22.78 2.03, respectively, while for 1 mM salicylate V1/2 and k were 97.58 3.09 mV (n ¼ 9, P < 0.01) and 22.38 1.82 (n ¼ 9, P > 0.05), respectively. Each point represents mean S.D.
We also studied the effect of salicylate on the inactivation kinetics of IK(DR). Fig. 4A and B show the original current of IK(DR) in controls and in 1 mM salicylate, respectively. IK(DR) was evoked by a test pulse to þ30 mV for 450 ms given
To study the effects of salicylate on ICa,L, the AC neurons were held at 40 mV and depolarized to 0 mV for 200 ms to activate ICa,L. The current amplitudes decreased with increments of salicylate concentrations. Fig. 5A shows the concentrationeresponse curve for effects of salicylate on ICa,L. The curve was best fitted with the Hill equation when IC50 was 3.76 mM and h was 0.71 (n ¼ 8). Thus, the decreased amplitudes of ICa,L by salicylate were concentration dependent. In the blank control without salicylate (time-matched control), ICa,L were decreased by 4.77 1.92% (n ¼ 8, P > 0.05), after 15 min current recording. So the run-down of calcium current was ruled out. In addition, the current amplitude of ICa,L decreased rapidly and reached a steady state within 2 min after salicylate application, and was soon restored after washout with salicylate-free solution. Salicylate application (1 mM) for 2 min reduced ICa,L to 68.61 8.43% of control (n ¼ 8, P < 0.05). Upon washout with salicylate-free solution for 2 min, the ICa,L was restored to 93.87 3.72% of control (n ¼ 8, P > 0.05). Our results showed that the block effect of salicylate on ICa,L could be reversible. Fig. 5B and C show the original current of ICa,L in controls and in 1 mM salicylate. ICa,L was obtained by depolarizing steps from a holding potential of 40 to þ40 mV (200 ms) at 10-mV steps. Fig. 5D shows the current density-potential curve of ICa,L before and after salicylate application. 1 mM salicylate attenuated ICa,L and significantly changed the potential of maximum peak ICa,L in a voltage-dependent manner. The effects of salicylate on the conductanceevoltage curve of ICa,L are shown in Fig. 5E. ICa,L was induced by the step pulses (10-mV increments) from a holding potential of 40 to þ10 mV. Before and after application of 1 mM salicylate, the values of V1/2 were 6.82 1.91 and 16.74 3.25 mV (n ¼ 8, P < 0.01), with k 4.53 1.14 and 2.21 0.86 (n ¼ 8, P < 0.05). Thus, salicylate significantly changed the V1/2 by approximately 10 mV toward the hyperpolarizing direction with affecting k of the steady-state activation curve. Therefore, salicylate significantly alter the steady-state activation kinetics of ICa,L. To study the effects of salicylate on the inactivation kinetics of ICa,L, a test pulse to 0 mV for 200 ms was given immediately following a 1-s conditioning pulse (70 to þ20 mV in 10-mV increments) from a holding potential of 40 mV.
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Fig. 5. (A) Concentrationeresponse relationship for the blockade of ICa,L by salicylate. The current was evoked by depolarization to 0 mV for 200 ms from a holding potential of 40 mV. The current amplitude was measured before and after salicylate treatment, and the fraction of control current amplitudes was plotted as a function of salicylate concentration. Each point represents the mean S.D. (n ¼ 8). Data fit well with the Hill equation. The IC50 was 3.76 mM and the h was 0.71. (BeD) Effects of 1 mM salicylate on the current densityevoltage relation of peak ICa,L. A series of 200 ms of depolarizing steps from a holding potential of 40 to þ40 mV (at 10-mV increments) was applied at 0.1 Hz. (B) Original traces of ICa,L in control; (C) original traces of ICa,L after 1 mM salicylate application; (D) peak current densityevoltage curves for control and 1 mM salicylate. Each point represents mean S.D. (n ¼ 10). *P < 0.05, **P < 0.01. (E) Effects of 1 mM salicylate on the steady-state activation curves for L-type calcium channels. A series of 200-ms depolarizing steps were applied at 1-s intervals from a holding potential of 40 to þ10 mV (at 10-mV increments). Normalized peak conductance was fitted with a Boltzmann function. For the control, V1/2 and k were 6.82 1.91 mV and 4.53 1.14 (n ¼ 8), respectively, and for 1 mM salicylate 16.74 3.25 mV (P < 0.01) and 2.21 0.86 (P < 0.05), respectively.
Fig. 6A and B show the original traces of in controls and in 1 mM salicylate, respectively. Fig. 6C shows the steady-state inactivation curve before and after salicylate application. Before salicylate application, V1/2 was estimated to be15.23 2.83 mV and k 8.35 0.96. After 1 mM
salicylate application, V1/2 was 14.16 2.37 mV (n ¼ 8, P > 0.05) and k 7.97 0.71 (n ¼ 8, P > 0.05). Thus, salicylate did not shift the steady-state inactivation curve toward either direction and had no significant effect on the steady-state inactivation kinetics of ICa,L.
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number of spikes generated by a 800-ms depolarizing current pulse was 8.31 0.85 under control conditions, and 14.76 0.92 in the presence of 1 mM salicylate (n ¼ 7, P < 0.01). The results showed us that salicylate significantly increased the action potential firing rates of AC neurons. 4. Discussion
Fig. 6. Effects of 1 mM salicylate on the steady-state inactivation curves for ICa,L. The pre-pulse potential changed from 70 to þ20 mV in 10-mV increments at 1-s intervals and was immediately followed by a 200-ms step depolarization to 0 mV. (A) Original traces of ICa,L in control. (B) Original traces of ICa,L after 1-mM salicylate application. (C) Steady-state inactivation curves for control and 1 mM salicylate. The normalized current of steady-state inactivation was fitted with a Boltzmann function. For the control, V1/2 was 15.23 283 mV and k 8.35 0.96 (n ¼ 8). For 1 mM salicylate, V1/2 was 14.16 2.37 mV (P > 0.05) and k 7.97 0.71 (P > 0.05).
3.4. Effects of salicylate on action potential firing rates Because the number of acutely isolated AC neurons that firing spontaneously is very low, we injected an 800-ms current pulse (1.0 nA) to evoke their action potential firing rates under control conditions and in the presence of 1 mM salicylate. The
Both tinnitus loudness and tinnitus incidence increase with plasma salicylate level (Jager and Always, 1946; Graham and Parker, 1948). These studies are consistent with our results that the blockades of salicylate on INa, IK(DR), ICa,L were concentration or dose dependent. In humans with chronic overdose of salicylate, salicylate-induced tinnitus may level off and reverse within a few days after the cessation of drug use (McCabe and Dey, 1965; Myers and Berstein, 1965; Cazals, 2000). These studies are also consistent with our results that the blockades of salicylate on INa, IK(DR), and ICa,L could be reversed with the salicylate-free solution. Thus, the reduction of INa, IK(DR), and ICa,L by salicylate that we found are consistent with its effect on tinnitus. Our results showed that salicylate inhibited the current amplitudes of INa, IK(DR) and ICa,L in a concentration-dependent manner, and the IC50 values of inhibition were 3.52 mM, 2.13 mM and 3.76 mM, respectively. From these data, we could conclude that the inhibition of salicylate on IK(DR) was a little stronger than its inhibition on INa or ICa,L. Our results also showed that: 1 mM salicylate shifted the steady-state inactivation curve negatively by about 5 mV without affecting the steady-state activation curve of INa; 1 mM salicylate produced a 14-mV negative shift of the steady-state activation curve and a 17-mV negative shift of the steady-state inactivation curve of IK(DR). 1 mM salicylate shifted the steady-state activation curve negatively by about 10 mV without affecting the steady-state inactivation curve of ICa,L. Thus, 1 mM salicylate significantly affected both activation and inactivation kinetics of IK(DR), while only affected inactivation kinetics of INa and activation kinetics of ICa,L. Furthermore, the effect of 1 mM salicylate on the activation and inactivation kinetics of IK(DR) was more stronger than the effect on those of INa and ICa,L. Our results showed that 1 mM salicylate shifted the voltage dependence of inactivation for INa by 5 mV in the hyperpolarizing direction without shifting the activation toward any direction. Therefore, salicylate interacted with the inactive state of the AC sodium channels and had a higher affinity to the inactivated sodium channels than to the resting ones. Functionally, the activity of voltage-gated sodium channels is essential for the conductivity and excitability of all neurons, as they mediate the very rapid rising phase and initial component of the falling phase of action potentials in many excitable cells (Hodgkin and Huxley, 1952; Hille, 1992). Sodium channels on the axon initial segment of neurons determine the threshold for the action potential and affect the duration and frequency of repetitive firings. Also, the release of neurotransmitters from the presynaptic nerve terminal is influenced by sodium channel activity. Considering the importance of sodium currents in action potential
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generation, their modulations cause significant changes in membrane excitability (Catterall, 1997; Shrager et al., 1998). The shift of the steady-state inactivation curve of INa to hyperpolarized potentials could result in a reduction of the number of action potentials during depolarization. Besides inhibiting the current amplitude of IK(DR), 1 mM salicylate significantly affected its kinetic properties, including shifting the steady-state activation curve by about 14 mV and inactivation curve by about 17 mV to the negative direction, respectively. The changes in the kinetics of IK(DR) were probably due to electrostatic and/or allosteric effects on the activation and inactivation gating of the delayed rectifier potassium channels. There are several potential explanations for our results: The number or the properties of delayed rectifier potassium channels in AC neurons is altered by salicylate. The hyperpolarizing shifts of half-activation and half-inactivation potential after salicylate application are consistent with the hypothesis that alterations in potassium channel biophysics, rather than channel number, are responsible for the observed increment. IK(DR) is responsible for the neuronal firing pattern and influences neuronal excitability and synaptic transmission. The inhibition of IK(DR), by, for example, TEA, might depolarize the neuron and result in enhancing the release of neurotransmitters such as ACh (Sanz et al., 2000). The effect of salicylate on 5-HT system in AC has been monitored by microdialysis in salicylate-induced tinnitus animal models. The 5-HT level increased significantly after application (Liu et al., 2003). Therefore, salicylate might increase neurotransmitter release, and modify the neuronal synaptic transmission and neuronal excitability through blocking the potassium channels. In addition, IK(DR) is crucial to repolarizing the action potential. Salicylate’s block of IK(DR) retards the repolarization, which contributes to an increase in firing rates (Sumners et al., 2002). Salicylate at 1 mM significantly shifted the activation curve of the ICa,L of the AC neurons to more hyperpolarized potentials by about 10 mV, while it did not affect the inactivation curve to either direction. These blocking effects of salicylate could be explained by salicylate interacting with the resting state of L-type calcium channels and having a higher affinity to the resting state than to the inactivated state. Calcium movements through L-type calcium channels and calcium concentration changes contribute to many cellular activities, normal or pathological. L-type calcium channels mediate long-lasting calcium currents in response to depolarization in excitable cells. Calcium influx through L-type calcium channels leads to activation of a cascade of intracellular signals and is also responsible for the afterhyperpolarization phase following action potentials in neurons (Marrion and Tavalin, 1998). These particular features of L-type calcium currents are thought to play an important role in neuronal excitability. Thus, effects of salicylate on ICa,L would result in the changes of the neuronal excitability. Although salicylate decreased or increased the membrane excitability of AC neurons through affecting INa, IK(DR) or ICa,L, the ultimate effects of salicylate on the neuronal excitability showed us that 1 mM salicylate significantly increased the action potential firing rates of AC neurons.
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Another study (Yang et al., 2007) suggested that salicylate significantly increased the spontaneous discharge rate and the local field potentials recorded from the multiunit cluster in AC. Voltage-gated calcium channels are known to contribute to the release of various neurotransmitters, including GABA. The actions of salicylate on g-aminobutyric acid type A receptor (GABA(A)) current in cultured rat spinal dorsal horn neurons were investigated. Salicylate reduced GABA(A) current in a reversible and concentration-dependent manner, and salicylate was effective only when GABA and sodium salicylate were applied together. This results suggested that GABA(A) receptors are pharmacological targets of salicylate (Xu et al., 2005). Salicylate reduced both the evoked and the miniature inhibitory postsynaptic currents in AC, and these two currents could be completely blocked by bicuculline, a selective GABA(A) antagonist. The results suggested that the alteration of inhibitory neural circuits might participate in the cellular mechanisms for tinnitus induced by salicylate in AC (Wang et al., 2006). Behavioral studies in rats suggested that tinnitus induced by salicylate could be totally abolished by nimodipine, the L-type calcium channel blocker. These studies showed us that there is a close correlation between L-type calcium current and salicylate-induced tinnitus (Jastreboff and Brennan, 1988; Wang et al., 2000). When a focal stimulation was applied to a single bouton, L-type channels played a significant role in generation of an action potential, which subsequently activated high threshold voltage-activated P/Q- and N-type channels at GABA release sites to provide the calcium influx necessary for synchronous transmitter release (Murakami et al., 2002). Thus, salicylate-induced attenuation of ICa,L might decrease the GABA release within the AC by inhibiting the second messenger system and/or reducing calcium concentration in the neurons. Salicylate alters auditory processing within the AC by depressing the L-type calcium channels, which results in loss of GABA-mediated inhibition and which might underlie the development of tinnitus. Therefore, L-type voltage-gated calcium channels may play an important role in salicylate-induced tinnitus. Taken together, besides inferior colliculus, AC is also an important site in the development of salicylate-induced tinnitus. The voltage-gated sodium channels, delayed rectifier potassium channels, L-type calcium channels in AC neurons were all affected by salicylate. Thus, we speculated that through affecting the voltage-gated ion channels in AC, salicylate ultimately increased the firing rate of neurons and enhanced neuronal excitability on the one hand, increased the excitatory transmitters release and reduced the inhibitory transmitter release on the other hand, thus finally induced tinnitus. The mechanism of salicylate-induced tinnitus might be related to its effect on voltage-gated channels in AC neurons. Acknowledgements This work was supported by National Science Foundation of China (Grant 30600125, Grant 30270361 and Grant 30325038) and China Postdoctoral Science Foundation (Grant 2005038512).
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References Arnold, W., Bartenstein, P., Oestreicher, E., Romer, W., Schwaiger, M., 1996. Focal metabolic activation in the predominant left auditory cortex in patients suffering from tinnitus: a PET study with [18F] deoxyglucose. ORL J. Otorhinolarynolaryngol. Relat. Spec. 58, 195e199. Catterall, W.A., 1997. Modulation of sodium and calcium channels by protein phosphorylation and G proteins. Adv. Second Messenger Phosphoprotein Res. 31, 159e181. Cazals, Y., 2000. Auditory sensori-neural alterations induced by salicylate. Prog. Neurobiol. 62, 583e631. Graham, J.D.P., Parker, W.A., 1948. The toxic manifestations of sodium salicylate therapy. Q. J. Med. 17, 153e163. Hille, B., 1992. Ionic Channels of Excitable Membranes, second ed. Sinauer Press, Sunderland, MA. Hodgkin, A.L., Huxley, A.F., 1952. Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo. J. Physiol. 116, 176e195. Jager, B.V., Always, R., 1946. The treatment of acute rheumatic fever with large doses of sodium salicylate. Am. J. Med. Sci. 211, 273e285. Jastreboff, P.J., Brennan, J.F., 1988. Specific effects of nimodipine on the auditory system. Ann. N.Y. Acad. Sci. 522, 716e718. Jastreboff, P.J., Hansen, R., Sasaki, P.G., Sasaki, C.T., 1986. Differential uptake of salicylate in serum, cerebrospinal fluid, and perilymph. Arch. Otolaryngol. Head Neck Surg. 112, 1050e1053. Jastreboff, P.J., Brennan, J.F., Sasaki, C.T., 1988. An animal model for tinnitus. Laryngoscope 98, 280e286. Liu, Y., Li, X., 2004a. Effects of salicylate on voltage-gated sodium channels in rat inferior colliculus neurons. Hear. Res. 193, 68e74. Liu, Y., Li, X., 2004b. Effects of salicylate on transient outward and delayed rectifier potassium channels in rat inferior colliculus neurons. Neurosci. Lett. 369, 115e120. Liu, J., Li, X., Wang, L., Dong, Y., Han, H., Liu, G., 2003. Effects of salicylate on serotoninergic activities in rat inferior colliculus and auditory cortex. Hear. Res. 175, 45e53. Liu, Y., Li, X., Ma, C., Liu, J., Lu, H., 2005. Salicylate blocks L-type calcium channels in rat inferior colliculus neurons. Hear. Res. 205, 271e276. Mahlke, C., Wallhausser-Franke, E., 2004. Evidence for tinnitus-related plasticity in the auditory and limbic system, demonstrated by arg3.1 and c-fos immunocytochemistry. Hear. Res. 195, 17e34. Marrion, N.V., Tavalin, S.J., 1998. Selective activation of Ca2þ-activated Kþ channels by co-localized Ca2þ channels in hippocampal neurons. Nature 395, 900e905.
McCabe, P.A., Dey, F.L., 1965. The effect of aspirin upon auditory sensitivity. Ann. Otol. Rhinol. Laryngol. 74, 312e324. Murakami, N., Ishibashi, H., Katsurabayashi, S., Akaike, N., 2002. Calcium channel subtypes on single GABAergic presynaptic terminal projecting to rat hippocampal neurons. Brain Res. 951, 121e129. Myers, E., Berstein, J.M., 1965. Salicylate ototoxicity a clinical and experimental study. Arch. Otolarygol. 82, 483e493. Penner, M.J., Jastreboff, P.J., 1996. Tinnitus: psychophysical observations in humans and an animal model. In: Van de Water, T.R., Popper, A.N., Fax, R.R. (Eds.), Clinical Aspects of Hearing. Springer, New York. Ru¨ttiger, L., Ciuffani, J., Zenner, H.P., Knipper, M., 2003. A behavioral paradigm to judge acute sodium salicylate-induced sound experience in rats: a new approach for an animal model on tinnitus. Hear. Res. 180, 39e50. Sanz, A.G., Hospital, S., Badia, A., Clos, M.V., 2000. Presynaptic effect of 7-OH-DPAT on evoked [3H]-acetylcholine release in rat striatal synaptosomes. Brain Res. 874, 116e122. Shrager, P., Custer, A.W., Kazarinova, K., Rasband, M.N., Mattson, D., 1998. Nerve conduction block by nitric oxide that is mediated by the axonal environment. J. Neurophysiol. 79, 529e536. Storm, J.F., 1990. Potassium currents in hippocampal pyramidal cells. Prog. Brain Res. 83, 161e187. Sumners, C., Fleegal, M.A., Zhu, M., 2002. Angiotensin AT1 receptor signaling pathways in neurons. Clin. Exp. Pharmacol. Physiol. 29, 483e490. Wallhausser-Franke, E., Mahlke, C., Oliva, R., Braum, S., Wenz, G., Langner, G., 2003. Expression of c-fos in auditory and non-auditory brain regions of the gerbil after manipulations that induce tinnitus. Exp. Brain Res. 153, 649e654. Wang, H., Jiang, S., Yang, W., Han, D., 2000. Evaluating effects of some medicine on tinnitus with animal behavioral model in rats. Zhonghua Er Bi Yan Hou Ke Za Zhi 35, 331e334. Wang, H.T., Luo, B., Zhou, K.Q., Xu, T.L., Chen, L., 2006. Sodium salicylate reduces inhibitory postsynaptic currents in neurons of rat auditory cortex. Hear. Res. 215, 77e83. Xu, H., Gong, N., Chen, L., Xu, T.L., 2005. Sodium salicylate reduces gamma aminobutyric acid-induced current in rat spinal dorsal horn neurons. Neuroreport 16, 813e816. Yang, G., Lobarinas, E., Zhang, L., Turner, J., Stolzberg, D., Salvi, R., Sun, W., 2007. Salicylate induced tinnitus: behavioral measures and neural activity in auditory cortex of awake rats. Hear. Res. 226, 244e 253. Zhou, F.M., Hablitz, J.J., 1996. Layer I neurons of rat neocortex. II. Voltagedependent outward currents. J. Neurophysiol. 76, 668e682.
Inhibition of voltage-gated channel currents in rat auditory cortex neurons by salicylate Yanxing Liu a,b,*, Hailin Zhang a, Xuepei Li b, Yongli Wang a, Hong Lu c, Xiang Qi d, Changsheng Ma a, Junxiu Liu b a
Department of Pharmacology and Neurobiology, Hebei Medical University, No. 361 Zhongshan East Road, Shijiazhuang 050017, Hebei Province, P.R. China b Department of Otorhinolaryngology, Peking University Third Hospital, No. 49 Huayuan Road, Beijing 100083, P.R. China c Department of Otorhinolaryngology, Hebei Medical University Second Hospital, Shijiazhuang 050000, Hebei Province, P.R. China d Department of Anesthesiology, Hebei Medical University Second Hospital, Shijiazhuang 050000, Hebei Province, P.R. China Received 1 March 2007; received in revised form 10 August 2007; accepted 16 August 2007
Abstract Salicylate is a medicine for anti-inflammation with a side effect of tinnitus. To understand the mechanisms of tinnitus induced by salicylate, we studied the effects of salicylate on voltage-gated ion channels and action potential firing rates in freshly dissociated rat pyramidal neurons in auditory cortex (AC) using the whole-cell patch technique. We found that salicylate reduced the voltage-gated sodium current (INa), the delayed rectifier potassium current (IK(DR)) and the L-type voltage-gated calcium current (ICa,L) in concentration-dependent manner. An amount of 1 mM salicylate shifted the steady-state inactivation curve of INa negatively by about 5 mV, shifted the steady-state activation and inactivation curve of IK(DR) negatively by approximately 14 mV and 17 mV, respectively, and shifted the steady-state activation curve of ICa,L negatively by about 10 mV. 1 mM salicylate significantly increased the action potential firing rates, ultimately. From the results, we speculated that through affecting the voltage-gated ion channels in AC, an important position in auditory system, salicylate increased the firing rate of neurons and enhanced neuronal excitability on the one hand, increased the excitatory transmitters release and reduced the inhibitory transmitter release on the other hand, thus finally induced tinnitus. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Salicylate; Voltage-gated sodium channel current; Delayed rectifier potassium channel current; L-type voltage-gated calcium channel current; Auditory cortex
Abbreviations: AC, auditory cortex; ACSF, artificial cerebrospinal fluid; 4AP, 4-aminopyridine; ATP, adenosine-50 -triphosphate; EGTA, ethyleneglycolbis-(2-aminoethyl)-tetraacetic acid; GABA, g-aminobutyric acid; GABA(A), g-aminobutyric acid type A receptor; GTP, guanosine-50 -triphosphate; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; IC50, halfblocking concentration; ICa,L, L-type voltage-gated calcium channel current; IK(A), transient outward potassium channel current; IK(DR), delayed rectifier potassium channel current; INa, voltage-gated sodium channel current; MOPS, 3-[N-morpholino]propane-sulfonic acid; TEA, tetraethylammonium; TTX, tetrodotoxin. * Corresponding author. Department of Pharmacology and Neurobiology, Hebei Medical University, No. 361 Zhongshan East Road, Shijiazhuang 050017, Hebei Province, P.R. China. Tel.: þ86 (0)311 8626 6291; fax: þ86 (0)311 8605 7291. E-mail address: [email protected] (Y. Liu). 0028-3908/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2007.08.015
1. Introduction Tinnitus is a phantom auditory experience in the absence of an external or internal, physically quantifiable, sound. It is a frequent and devastating symptom of disorders of the auditory system. Salicylate (the active component of aspirin) overdose induces tinnitus in humans (Myers and Berstein, 1965) and in animals (Penner and Jastreboff, 1996), but the neural origins and mechanisms underlying salicylate-induced tinnitus are still obscure. The most widely used animal models of tinnitus are rats receiving salicylate (Jastreboff et al., 1988; Ru¨ttiger et al., 2003).
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In previous studies, we found that salicylate affected the electrophysiological properties of the voltage-gated ion channels in rat inferior colliculus neurons (Liu and Li, 2004a,b; Liu et al., 2005). It is well known that the auditory cortex (AC) is also an important site to study tinnitus because it is the most advantaged center in performing sense of hearing. Injections of salicylate (350 mg/kg body weight) led to accumulation of arg3.1 and c-fos immunoreactive neurons in gerbil AC (Wallhausser-Franke et al., 2003; Mahlke and Wallhausser-Franke, 2004). When it induced tinnitus, salicylate significantly increased the spontaneous discharge rate and the local field potentials recorded from the multiunit cluster in the auditory cortex of awake rats (Yang et al., 2007). A positron emission tomography (PET) study in 11 patients suffering from chronic disabling tinnitus showed that metabolic activity in primary AC of 10 tinnitus patients significantly increased. This study gave objective evidence of tinnitus sensation and localization for the first time (Arnold et al., 1996). The salicylate concentrations in rat cerebrospinal fluid over 8 h after salicylate injection (460 mg/kg body weight) were studied (Jastreboff et al., 1986). The salicylate concentrations in cerebrospinal fluid rose to approximately 200e300 mg/l (equivalent to 1.25e1.87 mM) within 1 h, remained at this level for the next 3 h, and then decreased slowly to 150e 200 mg/l (equivalent to 0.94e1.25 mM) in the next 4 h. Another study of induced phantom auditory sensations in rats showed that tinnitus was induced by the administration of salicylate (350 mg/kg body weight) given 3 h before testing (Ru¨ttiger et al., 2003). Thus, when tinnitus develops, the salicylate concentration in their cerebrospinal fluid is about 1 mM. Electrical and chemical signaling in the nervous system is of fundamental importance to the integration of external and internal stimuli. Proper signaling depends on a finely tuned series of ion-channel-mediated events mediating electrical activity and transduction at synapses. Slight changes in ion channel properties will cause drastic changes in normal physiological functions of neurons. Voltage-gated sodium channels mediate the very rapid rising phase and initial component of the falling phase of action potentials in the excitable cells (Hodgkin and Huxley, 1952; Hille, 1992). Voltage-gated sodium channels also play a critical role in action potential propagation as well as transduction of electrical currents to transmitter release from the nerve terminal. Voltage-gated potassium channels are widely expressed throughout the brain, and they play crucial roles in regulating a variety of cellular processes in neurons, such as setting the resting membrane potential, repolarization, action potential duration, discharge patterns, and delay between a stimulus and the first action potential (Storm, 1990). Voltage-gated calcium currents play an important role in regulating neurotransmitter, such as g-aminobutyric acid (GABA), release. Behavioral studies suggested that nimodipine, an L-type Ca-channel blocker, could totally abolish salicylate-induced tinnitus (Jastreboff and Brennan, 1988; Wang et al., 2000). L-type calcium channels mediate long-lasting calcium currents in response to depolarization in excitable cells. Calcium influx through L-type calcium channels leads
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to activation of a cascade of intracellular signals and is also responsible for the afterhyperpolarization phase following action potentials in neurons (Marrion and Tavalin, 1998). These particular features of L-type calcium channel-dependent calcium influx play important role in neuronal excitability and plasticity. In all, changes in properties of voltage-gated sodium, potassium channels and/or calcium channels would directly affect neuronal excitability and activities. The aim of the present study was to obtain insight into the actions of salicylate on voltage-gated sodium, potassium channels, L-type calcium channels and action potential firing rates in freshly dissociated AC pyramidal neurons by use of the whole-cell patch clamp method. 2. Materials and methods 2.1. Cell preparation Fourteen-day-old Wistar rats (either sex) were anesthetized with pentobarbital (50 mg/kg body weight, i.p.) and decapitated. The AC was rapidly removed and cut into slices (400e500 mm thick) with a vibratome in ice-cold artificial cerebrospinal fluid (ACSF) bubbled continuously with a 95% O2/ 5% CO2 gas mixture. The AC slices were incubated in oxygenated ACSF for at least 60 min at room temperature. After incubation, AC slices were digested with Pronase E (0.75 mg/ml) in oxygenated ACSF at room temperature for 35 min. After enzyme treatment, the AC slices were rinsed twice with ACSF and kept in oxygenated ACSF at room temperature. Subsequently, AC neurons were mechanically dissociated by gentle trituration with firepolished Pasteur pipettes of decreasing diameter. The neuron suspension was transferred to a chamber, and neurons were allowed to settle to the bottom of the chamber before recording. The chamber was continuously perfused with an extracellular solution. The pyramidal neurons in AC were selected for our experiments by their shapes. To avoid possible phenotypic changes of currents due to culture conditions, AC neurons were used within 10 h following dissection. Animals were cared for and killed according to the principles of animal care outlined by the Chinese Academy of Sciences. All efforts were made to minimize animal suffering and to use the minimal number of animals in these studies.
2.2. Solutions and drugs The ACSF contained (in mM): NaCl 125, KCl 2.5, NaH2PO4 1.25, NaHCO3 26, CaCl2 2, MgCl2 1, D-glucose 10, with pH 7.4. The standard extracellular solution for recording of voltage-gated sodium channel currents (INa) contained (in mM): NaCl 50, choline chloride 90, MgCl2 1, CaCl2 1, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) 5, D-glucose 5, tetraethylammonium (TEA) chloride 25, 4-aminopyridine (4-AP) 5, and LaCl3 0.01, with pH 7.4. The internal solution for recording of INa contained (in mM): CsCl 125, NaF 20, HEPES 10, ethyleneglycol-bis-(2-aminoethyl)tetraacetic acid (EGTA) 10, TEA chloride 20, Mg adenosine-50 -triphosphate (ATP) 5, Na guanosine-50 -triphosphate (GTP) 0.1, and leupeptin 0.1, with pH 7.2. For recording of delayed rectifier potassium channel currents (IK(DR)), the standard extracellular solution contained (in mM): NaCl 125, KCl 5, MgCl2 2, CaCl2 2, D-glucose 10, HEPESeNaOH 10, and tetrodotoxin (TTX) 0.001, with pH 7.4. The pipette solution containing (in mM): KF 110, Trisebase 20, HEPES 10, EGTA 10, and Mg-ATP 2, adjusted to pH 7.4. For recording of L-type voltage-gated calcium channel currents (ICa,L), the extracellular solution contained (in mM): NaCl 80.4, KCl 5.4, TEA 25.0, BaCl2 5.0, 4-AP 10.0, MgCl2 0.8, NaHCO3 0.9, D-glucose 25, NaH2PO4 0.9, CaCl2 1.8, sodium pyruvate 0.23, 3-[N-morpholino]propane-sulfonic acid (MOPS) 10, TTX 0.001, and u-conotoxin GVIA 0.001, with pH 7.25. The internal solution contained (in mM): NaCl 5, cesium methanesulfonate
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115.6, MgCl2 2, CaCl2 0.5, EGTA 5, HEPES 10, and Mg-ATP 2, with pH 7.25. For recording of action potential firing rates, the extracellular solution was similar to ACSF. The pipette solution contained (in mM): K-methylsulfate 140, MgCl2 5, CaCl2 1, EGTA 10, HEPES 10, Na2-ATP 0.3 and Mg-ATP 2.2, with pH 7.2. Pronase E, HEPES, EGTA, Mg-ATP, Na-GTP, TEA, 4-AP, TTX and salicylate were purchased from Sigma Chemical Co. (St. Louis, MO). Other chemicals were obtained from Beijing Chemical Factory. For evaluation of salicylate effects, sodium salicylate was dissolved in extracellular solution and locally applied onto cells by use of a perfusion application system. Even at the highest concentration used in the study (10 mM) salicylate did not change the pH of the solution (pH 7.38 0.05 in control, 7.38 0.08 in the presence of salicylate at 1 mM, 7.37 0.07 in the presence of salicylate at 10 mM, respectively; n ¼ 5; P > 0.05). For evaluation the effects of salicylate on voltage-gated ion currents or action potential firing rates, cells were perfused for at least 2 min with control or salicylate solution before initiating the channel opening or the action potential.
2.3. Electrophysiological recording For the whole-cell patch clamp recordings, we used an EPC-9 amplifier, together with the corresponding PULSE 8.02 software (HEKA Elektronik, Germany). Patch pipettes were pulled from thin-walled borosilicate glass on a P 87 micropipette puller (Sutter Instruments, USA). The resistance of the fire-polished pipette was 4e6 MU when filled with the internal solution. Evoked currents were filtered at 3 kHz, digitized at 10 kHz and stored on a computer. The liquid junction potential between internal and external solutions was 5 mV on average and was used to correct for the recorded membrane potential. Capacity transients were canceled and series resistance compensated (>70%) by use of the internal circuitry of EPC-9. The leak current was digitally subtracted by use of the p/n protocol. All experiments were performed at room temperature. When the effects of salicylate on INa were studied, step depolarizations could activate inward currents. They were completely and reversibly blocked by 0.5 mM TTX, so these inward currents were attributed to INa. There are two main kind of voltage-gated potassium channels in cortex neurons: transient outward potassium channels and delayed rectifier potassium channels (Zhou and Hablitz, 1996). Their activation and inactivation voltage ranges and kinetics and pharmacological sensitivities are completely different. Step depolarizations of AC neurons from rest potential could mainly activate IK(DR), while the transient outward potassium current (IK(A)) was not obvious. When IK(DR) was measured, the 50-ms interval (during which the membrane potential was held at 40 mV) was inserted between the conditioning prepulse and test pulse to exclude IK(A). Therefore, IK(DR) could be measured at 50 ms after the onset of depolarization, because IK(A) was almost completely inactivated at a holding potential of 40 mV and to a considerable extent 50 ms after the onset of depolarization. 30 mM TEA could reversibly block the current amplitude of IK(DR) by 90.82 6.76% (n ¼ 9, P < 0.01). When ICa,L was measured, various blockers were used to inhibit the channels other than L-type channels. For example, TTX was used to block sodium channels. 4-AP and TEA were used to block transient outward and delayed rectifier potassium channels, separately. The voltage protocols used in this study could inhibit low threshold voltageactivated calcium channels. u-conotoxin GVIA was used here to block N-type calcium channels, the other main high threshold voltage-activated calcium channels. Therefore, the currents recorded in this condition were the ICa,L. 10 mM nimodipine could reversibly inhibit the current amplitude of ICa,L by 91.16 7.33% (n ¼ 8, P < 0.01). Action potentials were evoked by brief current injection (800 ms, 1 nA) through the patch pipette.
2.4. Data analysis Data were analyzed by use of PULSEFIT software (Heka Elektronik, Germany) and Origin 6.0 software (MS, USA). Results were expressed as mean S.D., and n represented the number of the cells examined. Statistical significance was assessed by use of a 2-way ANOVA; a P value of <0.05 was considered significant.
3. Results 3.1. Effects of salicylate on INa The freshly dissociated AC neurons were held at 80 mV and depolarized to 30 mV for 20 ms at 0.1 Hz to activate INa. Upon the administration of salicylate (in mM: 0.1, 0.3, 1, 3 and 10), the amplitudes of INa decreased with increments of concentrations from 0.1 to 10 mM. The normalized current, obtained from normalizing the INa after salicylate application to the control INa, was plotted as a function of the salicylate concentration (Fig. 1A). The concentrationeresponse curve was fitted with the Hill equation: I/I0 ¼ 1/[1 þ (x/Ki)h], where x is the concentration of the salicylate, h is the Hill coefficient, and Ki is the half-blocking concentration (IC50). The curve was best fitted when IC50 was 3.52 mM and h was 0.76 (n ¼ 9). The results indicate that salicylate decreased the amplitudes of INa in a concentration-dependent manner. In this study, we used the concentration of 1 mM for the experimental concentration of salicylate. In this experiment, the current amplitude decreased rapidly and reached a steady state within 2 min after salicylate application. After washout with salicylate-free solution, the current amplitude was soon restored. At a concentration of 1 mM, salicylate application for 2 min reduced the sodium current to 71.42 9.39% of control (n ¼ 9, P < 0.05). Upon washout with salicylate-free solution for 2 min, the sodium current was restored to 95.67 2.07% of control (n ¼ 9, P > 0.05). Our results showed that the block effect of salicylate could be reversible. The current densityevoltage curve of INa was obtained by depolarizing steps from a holding potential of 80 to þ50 mV at 10-mV steps. The maximal peak current was divided by cell capacitance to obtain peak current density (pA/ pF ) for each cell. Shown in Fig. 1 are representative families of INa responses in controls (Fig. 1B) and in 1 mM salicylate (Fig. 1C). Fig. 1D shows the current densityevoltage curve for INa before and after salicylate application. Salicylate attenuated the current amplitude of INa in a voltage-dependent manner. The effects of salicylate on the conductanceevoltage curve of voltage-gated sodium channels are illustrated in Fig. 1E. The INa was induced by the step pulses (10-mV increments) from a holding potential of 80 to 30 mV. Peak current amplitude was converted into conductance by use of the equation G ¼ I/(V VNa), where V is the membrane potential, and VNa is the sodium equilibrium potential obtained as the reversal potential from the IeV curve. The sodium conductance was normalized to the respective maximal value and plotted as a function of membrane potential. With use of a least-squares fit procedure, the normalized conductance was well fitted with a Boltzmann equation: G/Gmax ¼ 1/{1 þ exp[(V V1/2)/k]}, where G is conductance, Gmax is maximum conductance, V is test potential, V1/2 is the membrane potential at half-activation, and k is a slope factor. Before and after application of 1 mM salicylate, the values of V1/2 were 48.06 2.37 and 48.03 1.81 mV (n ¼ 9, P > 0.05), with k of 3.48 0.93
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Fig. 1. (A) Concentrationeresponse relationship for the blockade of INa by salicylate. The current was evoked by depolarization to 30 mV for 20 ms from a holding potential of 80 mV. The current amplitude was measured before and after salicylate treatment, and the fraction of control current amplitudes was plotted as a function of salicylate concentration. Each point represents the mean S.D. (n ¼ 9). Data were fitted well with the Hill equation. The IC50 was 3.52 mM and the h was 0.76. (BeD) Effect of 1 mM salicylate on the current densityevoltage relation of peak INa. A series of 20 ms of depolarizing steps from a holding potential of 80 to þ50 mV (at 10-mV increments) was applied at 0.1 Hz. (B) Original traces of INa in control; (C) original traces of INa after 1 mM salicylate application; (D) peak current densityevoltage curves for control and 1 mM salicylate. Each point represents mean S.D. (n ¼ 9). *P < 0.05, **P < 0.01. (E) Effects of 1 mM salicylate on the steady-state activation curves for INa. A series of 20-ms depolarizing steps from a holding potential of 80 to 30 mV (at 10-mV increments) were used. Interpulse interval was 1 s. Normalized peak conductance was fitted with a Boltzmann function. For the control, V1/2 and k were 48.06 2.37 mV and 3.48 0.93, respectively, while for 1 mM salicylate V1/2 and k were 48.03 1.81 mV (n ¼ 9, P > 0.05) and 3.40 1.22 (n ¼ 9, P > 0.05), respectively. Each point represents mean S.D.
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and 3.40 1.22 (n ¼ 9, P > 0.05), which indicated that the GeV curve did not shift toward the positive or negative potential and the activation process was not affected by salicylate. A test pulse to 30 mV for 20 ms was given immediately following a 1-s conditioning pulse (120 to 40 mV in 10mV increments) from a holding potential of 80 mV. Fig. 2 shows the steady-state inactivation curves for INa under control conditions and after exposure to 1 mM salicylate. The peak amplitude for INa was normalized and plotted versus prepulse potentials. Sodium channels were inactivated at a holding potential above 50 mV and relieved from inactivation at a holding potential below 100 mV. With use of a least-squares fit method, the curves were well fitted with Boltzmann equation I/Imax ¼ 1/{1 þ exp[(V V1/2)/k]}, where I is current amplitude, Imax is maximum current amplitude, V is holding potential, V1/2 is the potential at which the current becomes half maximum, and k is a slope factor. Salicylate at 1 mM significantly shifted the steady-state inactivation curve in the hyperpolarizing direction. Before salicylate application, V1/2 was estimated to be 69.99 2.64 mV and k 4.90 1.25. After 1 mM salicylate application, V1/2 was 74.82 1.72 mV (n ¼ 9, P < 0.01) and k 6.49 0.52 (n ¼ 9, P > 0.05). The results showed that salicylate significantly changed the V1/2 by about 5 mV without affecting k of the steady-state inactivation curve. 3.2. Effects of salicylate on IK(DR) Fig. 3A shows the concentrationeresponse curves for the action of salicylate on IK(DR). IK(DR) was evoked by a 150ms test pulse to þ60 mV following a 150-ms conditioning pulse to 110 mV and a 50-ms insertion at 40 mV from a holding potential of 40 mV. Salicylate blocked IK(DR) in a concentration-dependent manner. When the IC50 values of salicylate on IK(DR) were 2.13 mM, and the h was 1.06 (n ¼ 9), the curve was well fitted with the Hill equation. Fig. 3B and C show the original current of IK(DR) in controls and in 1 mM salicylate, respectively. Fig. 3D shows the current densityevoltage relationship for IK(DR) before and after salicylate application. The voltage protocol is that a 150-ms conditioning pulse to 110 mV was followed by a 150-ms test pulse (30 to þ60 mV in 10-mV increments) applied after 50 ms at 40 mV from a holding potential of 40 mV. Salicylate decreased the current amplitude of IK(DR) in a voltage-dependent manner. In addition, in the blank control without salicylate (time-matched control), IK(DR) were decreased by 4.08 1.79% (n ¼ 9, P > 0.05) after 15 min current recording. Thus, the run-down of IK(DR) was ruled out. We studied the effect of salicylate on the activation kinetics of IK(DR). Fig. 3E shows the conductanceevoltage curve of delayed rectifier potassium channels. In the absence of salicylate, IK(DR) was half activated at 2.13 1.95 mV and k was 24.28 1.58. In the presence of salicylate, IK(DR) was half activated at 16.22 3.07 mV (n ¼ 9, P < 0.01) and k was 18.22 1.26 (n ¼ 9, P > 0.05). Thus, the application of 1 mM salicylate produced a 14-mV negative shift of GeV curve. The results suggest that salicylate modified the activation process of IK(DR) significantly.
Fig. 2. Effects of 1 mM salicylate on the steady-state inactivation curves for INa. The pre-pulse potential changed from 120 to 40 mV in 10-mV increments for 1 s and was immediately followed by a 20-ms step depolarization to 30 mV. The interpulse interval was 10 s. (A) Original traces of INa in control. (B) Original traces of INa after 1 mM salicylate application. (C) Steady-state inactivation curves for control and 1 mM salicylate. The normalized current of steady-state inactivation was fitted with a Boltzmann function. For the control, V1/2 was 69.99 2.64 mV and k 4.90 1.25. For 1 mM salicylate, V1/2 was 74.82 1.72 mV (n ¼ 9, P < 0.01) and k 6.49 0.52 (n ¼ 9, P > 0.05). Each point represents mean S.D.
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Fig. 3. (A) Concentrationeresponse relationship for the blocking of IK(DR) by salicylate. The current was evoked by a 150-ms test pulse to þ60 mV following a 150-ms conditioning pulse to 110 mV and a 50-ms insertion at 40 mV from a holding potential of 40 mV. The current amplitude was measured before and after salicylate treatment, and the fraction of control current amplitudes was plotted as a function of salicylate concentration. Each point represents the mean S.D. (n ¼ 9). Data were fitted well with the Hill equation. The IC50 was 2.13 mM and the h was 1.06. (BeD) Effect of 1 mM salicylate on the current densityevoltage relation of peak IK(DR). The voltage protocol is that a 150-ms conditioning pulse to 110 mV was followed by a 150-ms test pulse (30 to þ60 mV in 10-mV increments) applied after 50 ms at 40 mV from a holding potential of 40 mV. (B) Original traces of IK(DR) in control; (C) original traces of IK(DR) after 1 mM salicylate application; (D) current densityevoltage curves for control and 1 mM salicylate. Each point represents mean S.D. (n ¼ 9). *P < 0.05, **P < 0.01. (E) Effects of 1 mM salicylate on the steady-state activation curves for IK(DR). The voltage protocol is that a 150-ms conditioning pulse to 110 mV was followed by a 150-ms test pulse (30 to þ60 mV in 10-mV increments) applied after 50 ms at 40 mV from a holding potential of 40 mV. Normalized peak conductance was fitted with a Boltzmann function. For the control, V1/2 and k were 2.13 1.95 mV and 24.28 1.58, respectively, while for 1 mM salicylate V1/2 and k were 16.22 3.07 mV (n ¼ 9, P > 0.05) and 18.22 1.26 (n ¼ 9, P > 0.05), respectively. Each point represents mean S.D.
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immediately following a 1600-ms conditioning pulse (180 to 40 mV in 10-mV increments) from a holding potential of 30 mV. Fig. 4C shows the steady-state inactivation curve of IK(DR) before and after salicylate application. In the absence of salicylate, IK(DR) was half inactivated at 80.76 3.47 mV and k was 22.78 2.03. In the presence of salicylate, IK(DR) was half activated at 97.58 3.09 mV (n ¼ 9, P < 0.01) and k was 22.38 1.82 (n ¼ 9, P > 0.05). Thus, the application of 1 mM salicylate produced a 17-mV hyperpolarizing shift of steady-state inactivation curve. The results suggest that salicylate also significantly modified the inactivation properties of IK(DR). 3.3. Effects of salicylate on ICa,L
Fig. 4. Effects of 1 mM salicylate on the steady-state inactivation curves for IK(DR). The current was evoked by a test pulse to þ30 mV for 450 ms given immediately following a 1600-ms conditioning pulse (180 to 40 mV in 10-mV increments) from a holding potential of 30 mV. The interpulse interval was 10 s. (A) Original traces of IK(DR) in control. (B) Original traces of IK(DR) after 1 mM salicylate application. (C) Steady-state inactivation curves for control and 1 mM salicylate. The normalized current of steady-state inactivation was fitted with a Boltzmann function. For the control, V1/2 and k were 80.76 3.47 mV and 22.78 2.03, respectively, while for 1 mM salicylate V1/2 and k were 97.58 3.09 mV (n ¼ 9, P < 0.01) and 22.38 1.82 (n ¼ 9, P > 0.05), respectively. Each point represents mean S.D.
We also studied the effect of salicylate on the inactivation kinetics of IK(DR). Fig. 4A and B show the original current of IK(DR) in controls and in 1 mM salicylate, respectively. IK(DR) was evoked by a test pulse to þ30 mV for 450 ms given
To study the effects of salicylate on ICa,L, the AC neurons were held at 40 mV and depolarized to 0 mV for 200 ms to activate ICa,L. The current amplitudes decreased with increments of salicylate concentrations. Fig. 5A shows the concentrationeresponse curve for effects of salicylate on ICa,L. The curve was best fitted with the Hill equation when IC50 was 3.76 mM and h was 0.71 (n ¼ 8). Thus, the decreased amplitudes of ICa,L by salicylate were concentration dependent. In the blank control without salicylate (time-matched control), ICa,L were decreased by 4.77 1.92% (n ¼ 8, P > 0.05), after 15 min current recording. So the run-down of calcium current was ruled out. In addition, the current amplitude of ICa,L decreased rapidly and reached a steady state within 2 min after salicylate application, and was soon restored after washout with salicylate-free solution. Salicylate application (1 mM) for 2 min reduced ICa,L to 68.61 8.43% of control (n ¼ 8, P < 0.05). Upon washout with salicylate-free solution for 2 min, the ICa,L was restored to 93.87 3.72% of control (n ¼ 8, P > 0.05). Our results showed that the block effect of salicylate on ICa,L could be reversible. Fig. 5B and C show the original current of ICa,L in controls and in 1 mM salicylate. ICa,L was obtained by depolarizing steps from a holding potential of 40 to þ40 mV (200 ms) at 10-mV steps. Fig. 5D shows the current density-potential curve of ICa,L before and after salicylate application. 1 mM salicylate attenuated ICa,L and significantly changed the potential of maximum peak ICa,L in a voltage-dependent manner. The effects of salicylate on the conductanceevoltage curve of ICa,L are shown in Fig. 5E. ICa,L was induced by the step pulses (10-mV increments) from a holding potential of 40 to þ10 mV. Before and after application of 1 mM salicylate, the values of V1/2 were 6.82 1.91 and 16.74 3.25 mV (n ¼ 8, P < 0.01), with k 4.53 1.14 and 2.21 0.86 (n ¼ 8, P < 0.05). Thus, salicylate significantly changed the V1/2 by approximately 10 mV toward the hyperpolarizing direction with affecting k of the steady-state activation curve. Therefore, salicylate significantly alter the steady-state activation kinetics of ICa,L. To study the effects of salicylate on the inactivation kinetics of ICa,L, a test pulse to 0 mV for 200 ms was given immediately following a 1-s conditioning pulse (70 to þ20 mV in 10-mV increments) from a holding potential of 40 mV.
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Fig. 5. (A) Concentrationeresponse relationship for the blockade of ICa,L by salicylate. The current was evoked by depolarization to 0 mV for 200 ms from a holding potential of 40 mV. The current amplitude was measured before and after salicylate treatment, and the fraction of control current amplitudes was plotted as a function of salicylate concentration. Each point represents the mean S.D. (n ¼ 8). Data fit well with the Hill equation. The IC50 was 3.76 mM and the h was 0.71. (BeD) Effects of 1 mM salicylate on the current densityevoltage relation of peak ICa,L. A series of 200 ms of depolarizing steps from a holding potential of 40 to þ40 mV (at 10-mV increments) was applied at 0.1 Hz. (B) Original traces of ICa,L in control; (C) original traces of ICa,L after 1 mM salicylate application; (D) peak current densityevoltage curves for control and 1 mM salicylate. Each point represents mean S.D. (n ¼ 10). *P < 0.05, **P < 0.01. (E) Effects of 1 mM salicylate on the steady-state activation curves for L-type calcium channels. A series of 200-ms depolarizing steps were applied at 1-s intervals from a holding potential of 40 to þ10 mV (at 10-mV increments). Normalized peak conductance was fitted with a Boltzmann function. For the control, V1/2 and k were 6.82 1.91 mV and 4.53 1.14 (n ¼ 8), respectively, and for 1 mM salicylate 16.74 3.25 mV (P < 0.01) and 2.21 0.86 (P < 0.05), respectively.
Fig. 6A and B show the original traces of in controls and in 1 mM salicylate, respectively. Fig. 6C shows the steady-state inactivation curve before and after salicylate application. Before salicylate application, V1/2 was estimated to be15.23 2.83 mV and k 8.35 0.96. After 1 mM
salicylate application, V1/2 was 14.16 2.37 mV (n ¼ 8, P > 0.05) and k 7.97 0.71 (n ¼ 8, P > 0.05). Thus, salicylate did not shift the steady-state inactivation curve toward either direction and had no significant effect on the steady-state inactivation kinetics of ICa,L.
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number of spikes generated by a 800-ms depolarizing current pulse was 8.31 0.85 under control conditions, and 14.76 0.92 in the presence of 1 mM salicylate (n ¼ 7, P < 0.01). The results showed us that salicylate significantly increased the action potential firing rates of AC neurons. 4. Discussion
Fig. 6. Effects of 1 mM salicylate on the steady-state inactivation curves for ICa,L. The pre-pulse potential changed from 70 to þ20 mV in 10-mV increments at 1-s intervals and was immediately followed by a 200-ms step depolarization to 0 mV. (A) Original traces of ICa,L in control. (B) Original traces of ICa,L after 1-mM salicylate application. (C) Steady-state inactivation curves for control and 1 mM salicylate. The normalized current of steady-state inactivation was fitted with a Boltzmann function. For the control, V1/2 was 15.23 283 mV and k 8.35 0.96 (n ¼ 8). For 1 mM salicylate, V1/2 was 14.16 2.37 mV (P > 0.05) and k 7.97 0.71 (P > 0.05).
3.4. Effects of salicylate on action potential firing rates Because the number of acutely isolated AC neurons that firing spontaneously is very low, we injected an 800-ms current pulse (1.0 nA) to evoke their action potential firing rates under control conditions and in the presence of 1 mM salicylate. The
Both tinnitus loudness and tinnitus incidence increase with plasma salicylate level (Jager and Always, 1946; Graham and Parker, 1948). These studies are consistent with our results that the blockades of salicylate on INa, IK(DR), ICa,L were concentration or dose dependent. In humans with chronic overdose of salicylate, salicylate-induced tinnitus may level off and reverse within a few days after the cessation of drug use (McCabe and Dey, 1965; Myers and Berstein, 1965; Cazals, 2000). These studies are also consistent with our results that the blockades of salicylate on INa, IK(DR), and ICa,L could be reversed with the salicylate-free solution. Thus, the reduction of INa, IK(DR), and ICa,L by salicylate that we found are consistent with its effect on tinnitus. Our results showed that salicylate inhibited the current amplitudes of INa, IK(DR) and ICa,L in a concentration-dependent manner, and the IC50 values of inhibition were 3.52 mM, 2.13 mM and 3.76 mM, respectively. From these data, we could conclude that the inhibition of salicylate on IK(DR) was a little stronger than its inhibition on INa or ICa,L. Our results also showed that: 1 mM salicylate shifted the steady-state inactivation curve negatively by about 5 mV without affecting the steady-state activation curve of INa; 1 mM salicylate produced a 14-mV negative shift of the steady-state activation curve and a 17-mV negative shift of the steady-state inactivation curve of IK(DR). 1 mM salicylate shifted the steady-state activation curve negatively by about 10 mV without affecting the steady-state inactivation curve of ICa,L. Thus, 1 mM salicylate significantly affected both activation and inactivation kinetics of IK(DR), while only affected inactivation kinetics of INa and activation kinetics of ICa,L. Furthermore, the effect of 1 mM salicylate on the activation and inactivation kinetics of IK(DR) was more stronger than the effect on those of INa and ICa,L. Our results showed that 1 mM salicylate shifted the voltage dependence of inactivation for INa by 5 mV in the hyperpolarizing direction without shifting the activation toward any direction. Therefore, salicylate interacted with the inactive state of the AC sodium channels and had a higher affinity to the inactivated sodium channels than to the resting ones. Functionally, the activity of voltage-gated sodium channels is essential for the conductivity and excitability of all neurons, as they mediate the very rapid rising phase and initial component of the falling phase of action potentials in many excitable cells (Hodgkin and Huxley, 1952; Hille, 1992). Sodium channels on the axon initial segment of neurons determine the threshold for the action potential and affect the duration and frequency of repetitive firings. Also, the release of neurotransmitters from the presynaptic nerve terminal is influenced by sodium channel activity. Considering the importance of sodium currents in action potential
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generation, their modulations cause significant changes in membrane excitability (Catterall, 1997; Shrager et al., 1998). The shift of the steady-state inactivation curve of INa to hyperpolarized potentials could result in a reduction of the number of action potentials during depolarization. Besides inhibiting the current amplitude of IK(DR), 1 mM salicylate significantly affected its kinetic properties, including shifting the steady-state activation curve by about 14 mV and inactivation curve by about 17 mV to the negative direction, respectively. The changes in the kinetics of IK(DR) were probably due to electrostatic and/or allosteric effects on the activation and inactivation gating of the delayed rectifier potassium channels. There are several potential explanations for our results: The number or the properties of delayed rectifier potassium channels in AC neurons is altered by salicylate. The hyperpolarizing shifts of half-activation and half-inactivation potential after salicylate application are consistent with the hypothesis that alterations in potassium channel biophysics, rather than channel number, are responsible for the observed increment. IK(DR) is responsible for the neuronal firing pattern and influences neuronal excitability and synaptic transmission. The inhibition of IK(DR), by, for example, TEA, might depolarize the neuron and result in enhancing the release of neurotransmitters such as ACh (Sanz et al., 2000). The effect of salicylate on 5-HT system in AC has been monitored by microdialysis in salicylate-induced tinnitus animal models. The 5-HT level increased significantly after application (Liu et al., 2003). Therefore, salicylate might increase neurotransmitter release, and modify the neuronal synaptic transmission and neuronal excitability through blocking the potassium channels. In addition, IK(DR) is crucial to repolarizing the action potential. Salicylate’s block of IK(DR) retards the repolarization, which contributes to an increase in firing rates (Sumners et al., 2002). Salicylate at 1 mM significantly shifted the activation curve of the ICa,L of the AC neurons to more hyperpolarized potentials by about 10 mV, while it did not affect the inactivation curve to either direction. These blocking effects of salicylate could be explained by salicylate interacting with the resting state of L-type calcium channels and having a higher affinity to the resting state than to the inactivated state. Calcium movements through L-type calcium channels and calcium concentration changes contribute to many cellular activities, normal or pathological. L-type calcium channels mediate long-lasting calcium currents in response to depolarization in excitable cells. Calcium influx through L-type calcium channels leads to activation of a cascade of intracellular signals and is also responsible for the afterhyperpolarization phase following action potentials in neurons (Marrion and Tavalin, 1998). These particular features of L-type calcium currents are thought to play an important role in neuronal excitability. Thus, effects of salicylate on ICa,L would result in the changes of the neuronal excitability. Although salicylate decreased or increased the membrane excitability of AC neurons through affecting INa, IK(DR) or ICa,L, the ultimate effects of salicylate on the neuronal excitability showed us that 1 mM salicylate significantly increased the action potential firing rates of AC neurons.
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Another study (Yang et al., 2007) suggested that salicylate significantly increased the spontaneous discharge rate and the local field potentials recorded from the multiunit cluster in AC. Voltage-gated calcium channels are known to contribute to the release of various neurotransmitters, including GABA. The actions of salicylate on g-aminobutyric acid type A receptor (GABA(A)) current in cultured rat spinal dorsal horn neurons were investigated. Salicylate reduced GABA(A) current in a reversible and concentration-dependent manner, and salicylate was effective only when GABA and sodium salicylate were applied together. This results suggested that GABA(A) receptors are pharmacological targets of salicylate (Xu et al., 2005). Salicylate reduced both the evoked and the miniature inhibitory postsynaptic currents in AC, and these two currents could be completely blocked by bicuculline, a selective GABA(A) antagonist. The results suggested that the alteration of inhibitory neural circuits might participate in the cellular mechanisms for tinnitus induced by salicylate in AC (Wang et al., 2006). Behavioral studies in rats suggested that tinnitus induced by salicylate could be totally abolished by nimodipine, the L-type calcium channel blocker. These studies showed us that there is a close correlation between L-type calcium current and salicylate-induced tinnitus (Jastreboff and Brennan, 1988; Wang et al., 2000). When a focal stimulation was applied to a single bouton, L-type channels played a significant role in generation of an action potential, which subsequently activated high threshold voltage-activated P/Q- and N-type channels at GABA release sites to provide the calcium influx necessary for synchronous transmitter release (Murakami et al., 2002). Thus, salicylate-induced attenuation of ICa,L might decrease the GABA release within the AC by inhibiting the second messenger system and/or reducing calcium concentration in the neurons. Salicylate alters auditory processing within the AC by depressing the L-type calcium channels, which results in loss of GABA-mediated inhibition and which might underlie the development of tinnitus. Therefore, L-type voltage-gated calcium channels may play an important role in salicylate-induced tinnitus. Taken together, besides inferior colliculus, AC is also an important site in the development of salicylate-induced tinnitus. The voltage-gated sodium channels, delayed rectifier potassium channels, L-type calcium channels in AC neurons were all affected by salicylate. Thus, we speculated that through affecting the voltage-gated ion channels in AC, salicylate ultimately increased the firing rate of neurons and enhanced neuronal excitability on the one hand, increased the excitatory transmitters release and reduced the inhibitory transmitter release on the other hand, thus finally induced tinnitus. The mechanism of salicylate-induced tinnitus might be related to its effect on voltage-gated channels in AC neurons. Acknowledgements This work was supported by National Science Foundation of China (Grant 30600125, Grant 30270361 and Grant 30325038) and China Postdoctoral Science Foundation (Grant 2005038512).
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