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Nitric oxide in the afferent synaptic transmission of the axolotl vestibular system
Nitric oxide in the vestibular system
Pergamon
PII: S0306-4522(00)00587-X
Neuroscience Vol. 103, No. 2, pp. 457±464, 2001 457 q 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/01 $20.00+0.00
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NITRIC OXIDE IN THE AFFERENT SYNAPTIC TRANSMISSION OF THE AXOLOTL VESTIBULAR SYSTEM A. FLORES,* E. SOTO and R. VEGA Instituto de FisiologõÂa, Universidad AutoÂnoma de Puebla, Apartado Postal 406, Puebla, Pue., CP 72000, Mexico
AbstractÐThis study was performed using intracellular and multiunit extracellular recording techniques in order to characterize the role of nitric oxide in the afferent synaptic transmission of the vestibular system of the axolotl (Ambystoma tigrinum). Bath application of nitric oxide synthase inhibitors N G-nitro-l-arginine (0.01 mM to 10 mM) and N-nitro-larginine methyl ester hydrochloride (0.1 mM to 1000 mM) elicited a dose-dependent decrease in the basal discharge of the semicircular canal afferent ®bers. N G-Nitro-l-arginine also diminished the response to mechanical stimuli. Moreover, N G-nitro-l-arginine (1 mM) produced a hyperpolarization associated with a decrease in the spike discharge and diminished the frequency of the excitatory postsynaptic potentials on afferent ®bers recorded intracellularly. Nitric oxide donors were also tested: (i) S-nitroso-N-acetyl-dl-penicillamine (0.1 mM to 100 mM) increased the basal discharge and the response to mechanical stimuli. At the maximum effective concentration (100 mM) this drug affected neither the amplitude nor the frequency of the excitatory postsynaptic potentials. However, it slightly depolarized the afferent neurons and decreased their input resistance. (ii) 3-Morpholino-sydnonimine hydrochloride did not signi®cantly affect the basal discharge or the mechanically evoked peak response of afferent neurons at any of the concentrations used (1 mM to 1000 mM). However, after 10 min of perfusion in the bath, 1 mM and 10 mM 3-morpholino-sydnonimine hydrochloride signi®cantly modi®ed the baseline of the mechanically evoked response, producing an increase in the mean spike discharge of the afferent ®bers. These results indicate that nitric oxide may have a facilitatory role on the basal discharge and on the response to mechanical stimuli of the vestibular afferent ®bers. Thus, nitric oxide probably participates in the sensory coding and adaptative changes of vestibular input in normal and pathological conditions. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: semicircular canal, hair cells, sensory coding, nitric oxide synthase inhibitors, nitric oxide donors, amphibian.
glutamate receptors. 4,9,10,19 Release of endogenous NO may be essential for induction of synaptic plasticity. 13,17,25 NOS enzyme is widely distributed in the body. There are three isoforms of NOS (NOS I, II and III) based on the physical and the biochemical characteristics of the puri®ed enzymes, its subcellular location and its regulation by the intracellular Ca 21 concentration. 20,24 Isoform I of NOS is primarily found in central and peripheral neurons and in some specialized epithelial cells of the lung and the gut. NOS III is mainly found in endothelial cells. The inducible NOS isoform II can be expressed by virtually any cell type when adequately stimulated with cytokines or other agents. 8,21 Recent evidence suggests that NO may play a signi®cant role in the vestibular system, especially in the vestibular periphery. 7,14±16,26 However, only limited information concerning the role of NO in the vestibular system hair cells is available. 3,5 Furthermore, NOS has been morphologically localized in type I and type II hair cells, and in a subpopulation of vestibular efferents and afferents with NADPH diaphorase histochemistry. 7,14,26 Recently, NOS I and III have been detected by immunohistochemical methods in the vestibulocochlear system, 15,16 and so has NOS II expression during inner ear development. 1 Although NOS has been localized in the vestibular system, there is insuf®cient information concerning the
Since the discovery of nitric oxide (NO) as an endotheliumderived relaxing factor, and as a neurotransmitter in both the central and peripheral nervous systems, there has been great interest in the study of its actions in biological systems (for reviews see Refs 9, 27, 32 and 34). NO is produced on demand from l-arginine by nitric oxide synthase (NOS), with concomitant production of l-citrulline, in the presence of several co-factors including nicotinamide adenine dinucleotide phosphate (NADPH), and of calmodulin, to which Ca 21 binds. 19 NO acts both as an intercellular and as an intracellular signaling messenger that controls and in¯uences a number of critical physiological processes. 24 In the brain, NO is released both tonically and in response to activation of N-methyl-d-aspartate (NMDA)-type *Corresponding author. Tel.: 152-22-44-16-57/44-88-11; fax: 15222-33-45-11. E-mail address: a¯[email protected] (A. Flores). Abbreviations: AHP, after-hyperpolarization; cGMP, cyclic guanosine monophosphate; CV, coef®cient of variation of the interspike intervals distribution; EPSPs, excitatory postsynaptic potentials; HEPES, N-2-hydroxyethylpiperazine-N 0 -2-ethanesulphonic acid; l-NAME, N-nitro-l-arginine methyl ester hydrochloride; lNOARG, N G-nitro-l-arginine; NADPH, nicotinamide adenine dinucleotide phosphate; NMDA, N-methyl-d-aspartate; NO, nitric oxide; NOS, nitric oxide synthase; sGC, soluble guanylyl cyclase; SIN-1, 3-morpholino-sydnonimine hydrochloride; SNAP, Snitroso-N-acetyl-dl-penicillamine. 457
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potential role of NO in the regulation of the hair cell response, neurotransmitter release or discharge of the afferent neurons. The present study was performed to determine the role of NO in the excitability of the amphibian vestibular system. For this purpose, we examined the effects of NOS inhibitors (analogues of l-arginine) and of NO donors (compounds that release NO in solution) on the membrane and action potential parameters, excitatory postsynaptic potential (EPSP) characteristics, and ®ring rate of the afferent ®bers from the semicircular canals of the axolotl. EXPERIMENTAL PROCEDURES
Animal preparation Experiments were made using the isolated inner ear of the axolotl (Ambystoma tigrinum, obtained from a local supplier) as reported previously. 31 All efforts were made to minimize animal suffering, and to reduce the number of animals used, as outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institutes of Health. In brief, larval axolotls weighing 30±60 g were decapitated and the otic capsule was immediately opened. The nerve ®bers of the anterior and lateral canals were dissected up to the brainstem. The cartilaginous otic capsule was cut and isolated from the cranium. The isolated inner ear was transferred to a recording chamber and continuously perfused with Mg 21-free Ringer solution of the following composition (in mM): NaCl 111, KCl 2.5, CaCl2 1.8, glucose 10, HEPES 5, pH adjusted to 7.4 with NaOH. Extracellular multiunit recording Extracellular multiunit recordings of the semicircular canal afferent ®bers were obtained using a suction electrode (A-M Systems, WA, USA). This permits the detection of changes in the ®ring rate of much of the afferent ®ber population. The signal was ampli®ed using an AC ampli®er (Grass P-15, Rhode Island, USA) and monitored in an oscilloscope (Tektronix, 5111, Oregon, USA). The signal was also led to a window discriminator (WPI, 121, FL, USA), the output of which was connected to a computer for on-line analysis of discharge rate in the form of spikes/bin vs time plots. In some of the extracellular multiunit recording experiments, the recording chamber, the ampli®er and the manipulators were mounted on a rotating table driven by a d.c. servo-controlled motor (Aerotech, 49179, CA, USA). Sinusoidal accelerations (0.2 Hz) were induced using a function generator (Hewlett-Packard, 8904A, Idaho, USA). Intracellular recording The experiments for intracellular recording of the afferent neurons were performed using quartz glass microelectrodes ®lled with potassium acetate 3 M with a resistance of 100±120 MV. The bridge balance was checked and adjusted previously to each intracellular recording to measure the input resistance. Signals were led to a d.c. ampli®er (Axoprobe 1A, Axon Instruments, CA, USA) and to a computer for the measurement of the amplitude and frequency of EPSPs, and of the action potential parameters. 28 In addition, we used pClamp version 6.02 software (Axon Instruments) for the pulse protocols used to measure the input resistance for each neuron recording. Data were digitized using a DigiData 1200 interface (Axon Instruments). In both types of recording, the signals were also stored on magnetic tapes (Dagan, Unitrade and Vetter, model 3000A, Pennsylvania, USA). Nitric oxide-related drugs Once the basal discharge activity of the preparation was found
to be stable, drugs were applied by bath perfusion. The following drugs were employed: N G-nitro-l-arginine (l-NOARG), N-nitrol-arginine methyl ester hydrochloride (l-NAME), S-nitroso-Nacetyl-dl-penicillamine (SNAP) and 3-morpholino-sydnonimine hydrochloride (SIN-1). l-NOARG and SNAP were purchased from Research Biochemicals (Natick, MA, USA); l-NAME and SIN-1 from Sigma (St Louis, MO, USA). Stock solutions of all drugs were prepared taking extreme care to minimize exposure to light. They were diluted in Ringer's solution immediately before application. The SNAP and SIN-1 stock solutions were kept cold in the dark until just before use. Statistical analysis To determine whether a drug had a signi®cant effect or not, we used a non-parametric Mann±Whitney U-test comparing the control versus the drug electrical discharge. To construct the concentration±effect relationship, the spike discharge was normalized as a percentage of change with respect to control conditions. Comparison of the mechanical responses was done by obtaining the mean of the peak response in at least ®ve cycles of the sinusoidal stimulus period (the ®rst and the last cycles were eliminated). Data are expressed as means ^ S.E.M. Statistical signi®cance in the concentration±effect data was calculated using the paired Student's t-test (P , 0.05). Graphs were obtained 10 min after drug perfusion. For the analysis of the action potential waveform and spike discharge intervals only neurons with stable membrane potentials negative to 240 mV were collected. The following electrophysiological parameters were measured: membrane potential, input resistance, action potential amplitude, action potential duration, amplitude of the after-hyperpolarization (AHP) and the coef®cient of variation of the interspike interval distribution (CV). RESULTS
Recordings of the electrical activity of semicircular canal afferent ®bers were obtained from a total of 137 preparations. Extracellular multiunit recordings Effects of nitric oxide synthase inhibitors. Basal and mechanically evoked activities were recorded in control conditions and after bath perfusion of NOS inhibitors. l-NOARG in a concentration range from 0.1 mM to 10 mM (n 21; Fig. 1A) produced a signi®cant timeand concentration-dependent inhibitory action of the afferent neurons basal and mechanically evoked discharge. After 10 min l-NOARG (10 mM) perfusion (n 5), basal discharge signi®cantly decreased to 78.5 ^ 4.9% and the mechanically evoked response to 48.3 ^ 4.7% of control values (paired Student's t-test, P , 0.05). These effects are illustrated in Fig. 1B. The actions were partially reversible after 45 min washing with Ringer's solution. The inhibitory action of lNOARG 10 mM on the basal and on the mechanically evoked activity increased to 95.8 ^ 1% and 76.9 ^ 1.4%, respectively, 30 min after perfusion with the drug. Application of l-NOARG 0.01 mM (n 6) after 10 min did not produce a signi®cant change in the basal and mechanically evoked discharge of the vestibular afferent neurons (paired Student's t-test, P . 0.05). Perfusion of l-NAME (0.1 mM to 1000 mM; n 24) consistently decreased the basal discharge of the semicircular canal nerve ®bers in a dose-dependent and reversible manner, but with lower potency than l-NOARG.
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Fig. 1. Blockade of basal and mechanically evoked discharge of the afferent ®bers of the semicircular canal by l-NOARG. (A) Concentration±response relationship of l-NOARG (n 21) effects on the basal discharge and in the mechanically evoked peak response. Each point represents the mean ^ S.E.M. of at least ®ve experiments. Asterisk indicates statistical signi®cance using a paired Student's t-test (P , 0.05). The changes are expressed relative to control values (100%, dotted line). (B) The plots represent consecutive records from the same preparation. Top, spikes/bin versus time plot of the basal (1 min) and mechanically evoked discharge of the semicircular canal afferent neurons in control conditions (black bar, sinusoidal 0.2 Hz angular accelerations). Middle, after 10 min of bath perfusion with 10 mM l-NOARG. Bottom, 45 min after washing with Ringer's solution, a partial recovery was observed. Bin 200 ms.
After 10 min of its perfusion l-NAME 100 mM (n 6) signi®cantly diminished the basal discharge to 45 ^ 9% and 1000 mM (n 5) to 53 ^ 10% of the control level (paired Student's t-test, P , 0.05). This is illustrated by Fig. 2A and B, which show the changes in afferent discharge under l-NAME (100 mM) observed in one typical experiment. It was found that the discharge rate was enhanced compared with the initial control activity after washing the l-NAME with Ringer's solution for 10 min.
Fig. 2. (A) Concentration±response relationship for l-NAME (n 24). Each point with bar represents the mean ^ S.E.M. of at least ®ve experiments. The asterisk indicates statistical signi®cance from control value (100%, dotted line) using a paired Student's t-test (P , 0.05). (B) l-NAME 100 mM decreased the basal discharge of the afferent ®bers of the semicircular canal. The horizontal bar above the record indicates the duration of the bath perfusion of the drug. Bin 1 s.
Effects of nitric oxide donors. The effects of bath application of two different nitric oxide donors (SNAP and SIN-1) on the ®ring rates of semicircular canal afferent ®bers were also studied. SNAP (0.1 mM to 100 mM; n 25) produced an increase in the basal discharge, and in the response to mechanical stimuli. For lower concentrations, between 0.1 and 10 mM, the SNAP effect was not concentration dependent (Fig. 3A), and it was not signi®cantly different from the control values (paired Student's t-test, P . 0.05). For concentrations above 10 mM SNAP produced a signi®cant increase in the basal and in the mechanically evoked response (paired Student's t-test, P , 0.05). The maximal effect was obtained with 100 mM SNAP (n 8). In the experiment shown in Fig. 3B, the basal discharge increased to 42 ^ 2.7%. In contrast, SNAP induced a 25.2 ^ 6.7% increment in the response to mechanical stimuli. A complete recovery of the electrical activity was observed after a 45 min washout of the drug. SIN-1 (1 mM to 1000 mM; n 58) was tested on the discharge rate of the afferent ®bers of the semicircular
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Fig. 3. Actions of SNAP in the electrical activity of the semicircular canal afferent neurons. (A) Concentration±response relationship of SNAP (n 25) does not show a concentration-dependent activity in the range between 0.1 and 10 mM; larger concentrations produced a signi®cant excitatory action (paired Student's t-test, P , 0.05). Each point represents the mean ^ S.E.M. of at least ®ve experiments. The asterisk indicates statistical signi®cance from control value (100%, dotted line) using a paired Student's t-test (P , 0.05). (B) Top, spikes/bin versus time plots of the control recording of the basal discharge (1 min) and of mechanically evoked response of the vestibular afferent ®bers. Middle, perfusion of 100 mM SNAP increased the basal and mechanically evoked activity. Bottom, washing (45 min) with Ringer's solution. Bin 200 ms.
Fig. 4. (A) Concentration±response relationship (n 58) between SIN-1 concentration and electrical activity basal (X), peak (7) and the minimum (A) of the response to mechanical stimuli. Each point represents the mean ^ S.E.M. of at least eight experiments. Asterisk indicates statistical signi®cance from control value (100%, dotted line) using a paired Student's t-test (P , 0.05). (B) Top, spikes/bin versus time plots of the basal and mechanically evoked discharge of vestibular afferent ®bers in control conditions. Middle, after 10 min of bath perfusion with 10 mM SIN-1 the baseline of the mechanical response increased signi®cantly by 61.9 ^ 22.5%, consequently the deacceleratory phase of the mechanical stimulus was no longer able to completely inhibit the afferent ®ber discharge (arrow). Bottom, excitatory action of SIN-1 was partially reversible during 20 min of washing. Bin 200 ms.
canal (Fig. 4A). At low concentrations SIN-1 (1 mM and 10 mM, n 8 and n 12, respectively) had no signi®cant effect on the basal discharge and on the mechanically evoked peak response of the afferent neurons from the semicircular canals (paired Student's t-test, P . 0.05). At
a concentration of 1 mM, bath application of SIN-1 (n 8) consistently elevated the basal discharge of these ®bers to 22.6 ^ 18.4%, and the peak response to mechanical stimuli to 8.1 ^ 5.1% above the control discharge, although this action was not statistically
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Fig. 5. (A) Intracellular recording of an afferent ®ber in control conditions. (B) After 10 min of bath administration with l-NOARG 1 mM. Note that l-NOARG produced a decrease in the spike discharge rate and a hyperpolarization (248.4 ^ 2.65 vs 251.4 ^ 2.01 mV) of the mean membrane potential (n 5). The dotted lines indicate 0 mV. (C) Averaged action potentials (10 spikes) in control condition (continuous line), and after 10 min l-NOARG 1 mM perfusion (dotted line). Action potential parameters (amplitude, duration, AHP amplitude) were not signi®cantly affected by l-NOARG, although membrane potential was slightly hyperpolarized (paired Student's t-test, P . 0.05).
signi®cant. It is worth noting that after 10 min of bath perfusion, SIN-1 1 mM signi®cantly modi®ed the baseline (minimum) during the mechanical stimulation, producing a signi®cant increase of 41.7 ^ 16.4% in the mean spike discharge of the afferent ®bers (paired Student's t-test, P , 0.05). With 10 mM SIN-1 (n 12) also signi®cantly increased the baseline (minimum) of the mechanically evoked response to 61.9 ^ 22.5% of the control value (paired Student's t-test, P , 0.05) (Fig. 4B). In contrast, higher concentrations of SIN-1 (100 mM, 300 mM and 1000 mM; n 13, 11 and 14, respectively) diminished the spike discharge rate of the afferent ®bers. Maximal inhibitory effect in this study was attained with 1000 mM SIN-1 (n 14), basal activity signi®cantly decreased in 30 ^ 12.5% (paired Student's t-test, P , 0.05) but not the mechanically evoked peak response (17.2 ^ 13.3%; paired Student's t-test, P . 0.05). The baseline of the mechanical response was not signi®cantly affected. During repeated applications of SIN-1 no obvious hypo- or hypersensitivity was observed. Intracellular recordings To determine whether NO acts at the synaptic level or directly on the afferent ®bers, we studied the capability of NOS inhibition and NO to modulate the action potentials and EPSPs of vestibular nerve ®bers. Spontaneous action
potentials and EPSPs were measured in control conditions and 10 min after bath perfusion with l-NOARG or SNAP. Bath perfusion of l-NOARG 1 mM (n 5) resulted in a 3 mV hyperpolarization from the mean membrane potential (248.4 ^ 2.65 vs 251.4 ^ 2.01 mV) associated with a decrease in the frequency of discharge (Fig. 5A, B). As shown in Fig. 5C, the averaged action potential amplitude (51.4 ^ 3.9 vs 48.6 ^ 6.26 mV), the duration (1.06 ^ 0.08 vs 1.06 ^ 0.09 ms) and the AHP amplitude (4.72 ^ 0.46 vs 4.75 ^ 0.49 mV) were not signi®cantly modi®ed (paired Student's t-test, P . 0.05). Analysis of the frequency and mean amplitude of EPSPs recorded in control conditions and after l-NOARG administration reveals that this NOS inhibitor signi®cantly decreased the frequency of the EPSPs in 32.3 ^ 1.4% (paired Student's t-test, P , 0.05) without changes in their amplitude (1.5 ^ 0.4%). l-NOARG 1 mM did not signi®cantly alter the CV of the neurons recorded (0.72 ^ 0.10 vs 0.77 ^ 0.12) (paired Student's t-test, P . 0.05). SNAP 100 mM (n 4) affected neither the mean amplitude (1.45 ^ 0.55%) nor the frequency of EPSPs (4.55 ^ 2.32%). In contrast, SNAP produced a depolarization of 2.5 mV of the mean membrane potential (259 ^ 2.34 vs 257 ^ 2.48 mV) and decreased the input resistance (88.35 vs 76.41 MV) of afferent neurons. SNAP 100 mM increased the spike discharge rate of all the neurons that were recorded (Fig. 6A, B). The averaged action potential amplitude (59.87 ^ 6.0 vs 56.2 ^ 4.8 mV), duration (1.25 ^ 0.18 vs 1.3 ^ 0.17 ms) and AHP amplitude (3.37 ^ 0.93 vs 3.7 ^ 0.93 mV) were not signi®cantly modi®ed (paired Student's t-test, P . 0.05) (Fig. 6C). Analysis of all the neurons recorded showed that SNAP signi®cantly increased the CV from 0.65 ^ 0.23 to 0.86 ^ 0.53 (paired Student's t-test, P , 0.05). DISCUSSION
These results provide evidence indicating that NOS inhibitors may inhibit, and NO donors may facilitate the electrical activity of afferent neurons. We found that bath application of NOS inhibitors, l-NOARG and l-NAME decreased the basal electrical activity of the afferent neurons of the semicircular canals from the axolotl in a concentration-dependent manner. The preparation showed higher sensitivity to l-NOARG than to lNAME. These results coincide with several reports indicating that l-NOARG is more potent than l-NAME both in vitro and in vivo. 22 In our experiments, the biological effect of l-NOARG was partially reversed by washing with Ringer's solution. In contrast, l-NAME inhibition of the basal electrical activity was completely reversible. The effects of l-NOARG and l-NAME hint at the presence of NOS in the preparation and can be explained by the competitive action with l-arginine for the substrate-binding site. Previous reports described the presence of NOS in the vestibular system. 7,14,26 NADPH diaphorase histochemistry of the axolotl vestibular system showed the afferent ®bers and hair cells predominantly stained. 7 In the chinchilla, NADPH
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Fig. 6. Intracellular recording of an afferent ®ber in control conditions (A), and after 10 min of bath perfusion with SNAP 100 mM (B). Note the increase in the spike discharge rate. SNAP 100 mM produced a small depolarization (259 ^ 2.34 vs 257 ^ 2.48 mV) associated with a decrease in input resistance (88.35 vs 76.41 MV) of the afferent ®bers (n 4). (C) Averaged action potentials (10 spikes) in control condition (continuous line), and after 10 min SNAP 100 mM perfusion (dotted line). Action potential parameters (amplitude, duration, AHP amplitude) were not signi®cantly affected by SNAP, although membrane potential was slightly depolarized (paired Student's t-test, P . 0.05).
diaphorase-positive staining was observed in efferent boutons, rather than in afferent boutons, and in some hair cells predominantly of type I. 26 In the adult rat vestibular system, NOS has been found in ®bers of the vestibular ganglion and boutons throughout the crista ampullaris. 14 Recently, NOS I and III, as well as soluble guanylyl cyclase (sGC), have been detected in the vestibulocochlear system of mice 15 and in the vestibular system of guinea-pigs. 16 The selective expression of the NOS II isoform during mouse vestibulocochlear receptorgenesis has also been described. 1 In the guineapig vestibular system, studies using a ¯uorescent indicator show the production of NO in hair cells. 33 All of these results support the presence of NOS in the vestibular system of several animal models, and are consistent with our present ®ndings indicating that NO signi®cantly contributes to the basal discharge, and to the response of afferent ®bers to mechanical stimuli. In the present study we observed that SNAP produced an excitatory action on the basal and mechanically evoked discharge, but the concentration±reponse curve is ¯at, except for the concentration of 100 mM which produced a signi®cant excitatory action. It should be considered that SNAP is an unstable nitrosothiol; its half-life can vary from seconds to hours at pH 7.4 depending on the experimental conditions. SNAP is also very photosensitive and highly susceptible to many factors, including temperature.6,23 SNAP at concentrations
higher than 100 mM produced an irreversible block of the afferent ®ber discharge. Although NO is an important messenger molecule, when generated in high concentrations it can produce cellular damage, consequently explaining this last effect. 11 SIN-1 is a sydnonimine, highly water-soluble and stable without signi®cant decomposition when stored protected from light at 48C. 6 SIN-1 at concentrations lower than 100 mM increased the basal discharge and the peak response to mechanical stimuli. At higher concentrations SIN-1 decreased the basal and the mechanically evoked activity. In this regard, it has been reported recently that SIN-1 has dual effects: facilitation and suppression on laryngeal motoneurons in the nucleus ambiguous. 35 In our experiments, after 10 min of bath perfusion with SIN-1, it signicantly modi®ed the baseline during the mechanical stimulation, producing an increase in the mean spike discharge of the afferent ®bers. This effect is similar to the previous one reported when glycine was added to the Ringer's solution in which the electrical discharge of the semicircular canal afferent ®bers increased during the deacceleratory phase of sinusoidal mechanical stimulus. 30 This action was dependent on the stimulus duration, and was blocked by NMDA antagonists and also by extracellular magnesium. These results provide support for a signi®cant role of NMDA receptors in mediating the activation of afferent neurons, and also probably participating in some type of adaptative changes in the gain of the afferent synapse mediated by the activation of NOS and NO generation. 29,30 There is evidence that NO leads to an elevation of cyclic guanosine monophosphate (cGMP) levels in the cytoplasm by the activation of the cytosolic enzyme sGC. 18,4,2 cGMP then mediates the physiological effects of the pathway, often by activating a cGMP-dependent protein kinase. Furthermore, it is known that high cGMP levels reduce the inward potassium conductance (IK,1) of the vestibular hair cells in the rat. 3 The reduction in the K 1 current may lead to increased changes in membrane potential due to the mechano-electrical transducer currents, therefore the release of the excitatory neurotransmitter may also increase. Moreover, patch clamp recordings have shown that NO donors, such as sodium nitroprusside and nitroglycerin, inhibit a low voltageactivated potassium current (IK,L) speci®c to mammalian type I vestibular hair cells. 8-Bromo-cGMP, the membrane-permeant analog of cGMP, also inhibited the onset current. 5 Chen and Eatok 5 suggest the possibility that, in vivo, IK,L can be turned off by a retrograde messenger released by the calyx endings from primary afferent neurons. This retrograde messenger could be NO. Considerable data suggest that NO is capable of modifying the electrical activity of neurons. 11 In our preparation the basal ®ring rate and the frequency of EPSPs were decreased by the NOS inhibitor l-NOARG, suggesting that it acts at the presynaptic level, inhibiting neurotransmitter release. It has been argued that NO produced in one cell acts principally on NO targets in other cells. Thus, NO produced in the vestibular hair cells diffuses
Nitric oxide in the vestibular system
to the afferent neurons and acts there. In contrast, perfusion of the NO donor SNAP increased the ®ring rate and decreased the input resistance of the vestibular afferent neurons, thus suggesting a postsynaptic action of SNAP. This compound also increased the CV of the interspike intervals, indicating that NO donors could modify the regularity of discharge of vestibular afferents. The basal and the mechanically evoked electrical activity were differentially affected by l-NOARG and SNAP (the latter at the highest concentration used). This implies that, as has been previously suggested, 12 the mechanisms determining the resting basal discharge and the mechanically evoked response are probably different. Both the release processes 12 and the type of postsynaptic receptors involved 29 can explain the differential effect of NO upon the basal and the mechanically evoked activities.
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Particular interest has revolved around NO production following NMDA receptor activation, making NO an attractive candidate for a retrograde messenger involved in the correlation of pre- and postsynaptic activity during changes in synaptic ef®cacy. Our results indicate that apart from the role that NO may have in blood ¯ow, immune response and metabolic regulation, NO plays a signi®cant role in the sensory coding in the vestibular system, participating as a neuromodulator of the afferent discharge, thus in¯uencing the overall gain of the system. These results imply that NO-related drugs may have potential use in the clinical treatment of vestibular disorders. AcknowledgementsÐThe authors thank Professor Augustine Udeh for proof reading the English manuscript. This work was partially supported by CONACyT grant J28904-N to A. Flores.
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Physiol. 57, 683±706. 12. Guth P. S., Aubert A., Ricci A. J. and Norris C. H. (1991) Differential modulation of spontaneous and evoked neurotransmitter release from hair cells: some novel hypotheses. Hearing Res. 56, 69±78. 13. Haley J. E., Wilcox G. L. and Chapman P. F. (1992) The role of nitric oxide in hippocampal long-term potentiation. Neuron 8, 211±216. 14. Harper A., Blythe W. R., Zdanski C. J., Prazma J. and Pillsbury H. C. (1994) Nitric oxide in the rat vestibular system. Otolaryngol. Head Neck Surg. 4, 430±438. 15. Hess A., Bloch W., Arnhold S., Andressen C., Stennert E., Addicks K. and Michel O. (1998) Nitric oxide synthase in the vestibulocochlear system of mice. Brain Res. 813, 97±102. 16. Hess A., Bloch W., Su J., Stennert E., Addicks K. and Michel O. (1998) Localisation of the nitric oxide (NO)/cGMP-pathway in the vestibular system of guinea pigs. Neurosci. Lett. 251, 185±188. 17. HoÈlscher C. (1997) Nitric oxide, the enigmatic neuronal messenger: its role in synaptic plasticity. Trends Neurosci. 20, 298±302. 18. Ignarro L. J. (1990) Haem-dependent activation of guanylate cyclase and cyclic GMP formation by endogenous nitric oxide: a unique transduction mechanism for transcellular signaling. Pharmac. Toxicol. 67, 1±7. 19. Knowles R. G., Palacios M., Palmer R. M. J. and Moncada S. (1989) Formation of nitric oxide from l-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc. natn. Acad. Sci. USA 86, 5159±5162. 20. Marletta M. A. (1993) Nitric oxide synthase structure and mechanism. J. biol. Chem. 268, 12,231±12,234. 21. Moncada S., Palmer R. M. J. and Higgs E. A. (1991) Nitric oxide: physiology, pathophysiology and pharmacology. Pharmac. Rev. 43, 109±142. 22. Moore P. K. and Handy R. L. C. (1997) Selective inhibitors of neuronal nitric oxide synthaseÐis no NOS really good NOS for the nervous system? Trends pharmac. Sci. 18, 204±211. 23. Ramirez J., Yu L., Ki J., Braunschweiger P. G. and Wang P. G. (1996) Glyco-S-nitrosothiols, a novel class of NO donor compounds. Bio. Med. Chem. Lett. 21, 2575±2580. 24. Schmidt H. H., Lohmann S. M. and Walter U. (1993) The nitric oxide and cGMP signal transduction system: regulation and mechanism of action. Biochim. biophys. Acta 1178, 153±157. 25. Shibuki K. and Okada D. (1991) Endogenous nitric oxide release required for long-term synaptic depression in the cerebellum. Nature 349, 326±328. 26. Singer M. and Lysakowski A. (1996) Nitric oxide synthase localized in a subpopulation of vestibular efferents with NADPH diaphorase histochemistry. Ann. N.Y. Acad. Sci. 781, 658±662. 27. Snyder S. H. (1992) Nitric oxide and neurons. Curr. Opin. Neurobiol. 2, 323±327. 28. Soto E., Manjarrez E. and Vega R. (1997) A microcomputer program for automated neural spike detection and analysis. Int. J. Med. Informatics 44, 203±212. 29. Soto E., Flores A., Erostegui C. and Vega R. (1994) Evidence for NMDA receptor in the afferent synaptic transmission of the vestibular system. Brain Res. 633, 289±296.
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30. Soto E., Flores A. and Vega R. (1994) NMDA-mediated potentiation of inner ear afferent synapse. NeuroReport 5, 1963±1965. 31. Soto E. and Vega R. (1988) Actions of excitatory amino acid agonists and antagonists on the primary afferents of the vestibular system of the axolotl (Ambystoma mexicanum). Brain Res. 462, 104±111. 32. Szabo C. (1996) Physiological and pathophysiological roles of nitric oxide in the central nervous system. Brain Res. Bull. 41, 131±141. 33. Takumida M. and Anniko M. (2000) Direct evidence of nitric oxide production in guinea pig vestibular sensory cells. Acta Otolaryngol. (Stockh.) 120, 34±38. 34. Vincent S. R. (1994) Nitric oxide: a radical neurotransmitter in the central nervous system. Prog. Neurobiol. 42, 129±160. 35. Yajima Y., Hayashi Y. and Hayakawa T. (2000) Paradoxical actions of the NO donor SIN-1 on single laryngeal motoneurons in the nucleus ambiguous. NeuroReport 11, 765±769. (Accepted 19 December 2000)
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Neuroscience Vol. 103, No. 2, pp. 457±464, 2001 457 q 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/01 $20.00+0.00
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NITRIC OXIDE IN THE AFFERENT SYNAPTIC TRANSMISSION OF THE AXOLOTL VESTIBULAR SYSTEM A. FLORES,* E. SOTO and R. VEGA Instituto de FisiologõÂa, Universidad AutoÂnoma de Puebla, Apartado Postal 406, Puebla, Pue., CP 72000, Mexico
AbstractÐThis study was performed using intracellular and multiunit extracellular recording techniques in order to characterize the role of nitric oxide in the afferent synaptic transmission of the vestibular system of the axolotl (Ambystoma tigrinum). Bath application of nitric oxide synthase inhibitors N G-nitro-l-arginine (0.01 mM to 10 mM) and N-nitro-larginine methyl ester hydrochloride (0.1 mM to 1000 mM) elicited a dose-dependent decrease in the basal discharge of the semicircular canal afferent ®bers. N G-Nitro-l-arginine also diminished the response to mechanical stimuli. Moreover, N G-nitro-l-arginine (1 mM) produced a hyperpolarization associated with a decrease in the spike discharge and diminished the frequency of the excitatory postsynaptic potentials on afferent ®bers recorded intracellularly. Nitric oxide donors were also tested: (i) S-nitroso-N-acetyl-dl-penicillamine (0.1 mM to 100 mM) increased the basal discharge and the response to mechanical stimuli. At the maximum effective concentration (100 mM) this drug affected neither the amplitude nor the frequency of the excitatory postsynaptic potentials. However, it slightly depolarized the afferent neurons and decreased their input resistance. (ii) 3-Morpholino-sydnonimine hydrochloride did not signi®cantly affect the basal discharge or the mechanically evoked peak response of afferent neurons at any of the concentrations used (1 mM to 1000 mM). However, after 10 min of perfusion in the bath, 1 mM and 10 mM 3-morpholino-sydnonimine hydrochloride signi®cantly modi®ed the baseline of the mechanically evoked response, producing an increase in the mean spike discharge of the afferent ®bers. These results indicate that nitric oxide may have a facilitatory role on the basal discharge and on the response to mechanical stimuli of the vestibular afferent ®bers. Thus, nitric oxide probably participates in the sensory coding and adaptative changes of vestibular input in normal and pathological conditions. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: semicircular canal, hair cells, sensory coding, nitric oxide synthase inhibitors, nitric oxide donors, amphibian.
glutamate receptors. 4,9,10,19 Release of endogenous NO may be essential for induction of synaptic plasticity. 13,17,25 NOS enzyme is widely distributed in the body. There are three isoforms of NOS (NOS I, II and III) based on the physical and the biochemical characteristics of the puri®ed enzymes, its subcellular location and its regulation by the intracellular Ca 21 concentration. 20,24 Isoform I of NOS is primarily found in central and peripheral neurons and in some specialized epithelial cells of the lung and the gut. NOS III is mainly found in endothelial cells. The inducible NOS isoform II can be expressed by virtually any cell type when adequately stimulated with cytokines or other agents. 8,21 Recent evidence suggests that NO may play a signi®cant role in the vestibular system, especially in the vestibular periphery. 7,14±16,26 However, only limited information concerning the role of NO in the vestibular system hair cells is available. 3,5 Furthermore, NOS has been morphologically localized in type I and type II hair cells, and in a subpopulation of vestibular efferents and afferents with NADPH diaphorase histochemistry. 7,14,26 Recently, NOS I and III have been detected by immunohistochemical methods in the vestibulocochlear system, 15,16 and so has NOS II expression during inner ear development. 1 Although NOS has been localized in the vestibular system, there is insuf®cient information concerning the
Since the discovery of nitric oxide (NO) as an endotheliumderived relaxing factor, and as a neurotransmitter in both the central and peripheral nervous systems, there has been great interest in the study of its actions in biological systems (for reviews see Refs 9, 27, 32 and 34). NO is produced on demand from l-arginine by nitric oxide synthase (NOS), with concomitant production of l-citrulline, in the presence of several co-factors including nicotinamide adenine dinucleotide phosphate (NADPH), and of calmodulin, to which Ca 21 binds. 19 NO acts both as an intercellular and as an intracellular signaling messenger that controls and in¯uences a number of critical physiological processes. 24 In the brain, NO is released both tonically and in response to activation of N-methyl-d-aspartate (NMDA)-type *Corresponding author. Tel.: 152-22-44-16-57/44-88-11; fax: 15222-33-45-11. E-mail address: a¯[email protected] (A. Flores). Abbreviations: AHP, after-hyperpolarization; cGMP, cyclic guanosine monophosphate; CV, coef®cient of variation of the interspike intervals distribution; EPSPs, excitatory postsynaptic potentials; HEPES, N-2-hydroxyethylpiperazine-N 0 -2-ethanesulphonic acid; l-NAME, N-nitro-l-arginine methyl ester hydrochloride; lNOARG, N G-nitro-l-arginine; NADPH, nicotinamide adenine dinucleotide phosphate; NMDA, N-methyl-d-aspartate; NO, nitric oxide; NOS, nitric oxide synthase; sGC, soluble guanylyl cyclase; SIN-1, 3-morpholino-sydnonimine hydrochloride; SNAP, Snitroso-N-acetyl-dl-penicillamine. 457
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potential role of NO in the regulation of the hair cell response, neurotransmitter release or discharge of the afferent neurons. The present study was performed to determine the role of NO in the excitability of the amphibian vestibular system. For this purpose, we examined the effects of NOS inhibitors (analogues of l-arginine) and of NO donors (compounds that release NO in solution) on the membrane and action potential parameters, excitatory postsynaptic potential (EPSP) characteristics, and ®ring rate of the afferent ®bers from the semicircular canals of the axolotl. EXPERIMENTAL PROCEDURES
Animal preparation Experiments were made using the isolated inner ear of the axolotl (Ambystoma tigrinum, obtained from a local supplier) as reported previously. 31 All efforts were made to minimize animal suffering, and to reduce the number of animals used, as outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institutes of Health. In brief, larval axolotls weighing 30±60 g were decapitated and the otic capsule was immediately opened. The nerve ®bers of the anterior and lateral canals were dissected up to the brainstem. The cartilaginous otic capsule was cut and isolated from the cranium. The isolated inner ear was transferred to a recording chamber and continuously perfused with Mg 21-free Ringer solution of the following composition (in mM): NaCl 111, KCl 2.5, CaCl2 1.8, glucose 10, HEPES 5, pH adjusted to 7.4 with NaOH. Extracellular multiunit recording Extracellular multiunit recordings of the semicircular canal afferent ®bers were obtained using a suction electrode (A-M Systems, WA, USA). This permits the detection of changes in the ®ring rate of much of the afferent ®ber population. The signal was ampli®ed using an AC ampli®er (Grass P-15, Rhode Island, USA) and monitored in an oscilloscope (Tektronix, 5111, Oregon, USA). The signal was also led to a window discriminator (WPI, 121, FL, USA), the output of which was connected to a computer for on-line analysis of discharge rate in the form of spikes/bin vs time plots. In some of the extracellular multiunit recording experiments, the recording chamber, the ampli®er and the manipulators were mounted on a rotating table driven by a d.c. servo-controlled motor (Aerotech, 49179, CA, USA). Sinusoidal accelerations (0.2 Hz) were induced using a function generator (Hewlett-Packard, 8904A, Idaho, USA). Intracellular recording The experiments for intracellular recording of the afferent neurons were performed using quartz glass microelectrodes ®lled with potassium acetate 3 M with a resistance of 100±120 MV. The bridge balance was checked and adjusted previously to each intracellular recording to measure the input resistance. Signals were led to a d.c. ampli®er (Axoprobe 1A, Axon Instruments, CA, USA) and to a computer for the measurement of the amplitude and frequency of EPSPs, and of the action potential parameters. 28 In addition, we used pClamp version 6.02 software (Axon Instruments) for the pulse protocols used to measure the input resistance for each neuron recording. Data were digitized using a DigiData 1200 interface (Axon Instruments). In both types of recording, the signals were also stored on magnetic tapes (Dagan, Unitrade and Vetter, model 3000A, Pennsylvania, USA). Nitric oxide-related drugs Once the basal discharge activity of the preparation was found
to be stable, drugs were applied by bath perfusion. The following drugs were employed: N G-nitro-l-arginine (l-NOARG), N-nitrol-arginine methyl ester hydrochloride (l-NAME), S-nitroso-Nacetyl-dl-penicillamine (SNAP) and 3-morpholino-sydnonimine hydrochloride (SIN-1). l-NOARG and SNAP were purchased from Research Biochemicals (Natick, MA, USA); l-NAME and SIN-1 from Sigma (St Louis, MO, USA). Stock solutions of all drugs were prepared taking extreme care to minimize exposure to light. They were diluted in Ringer's solution immediately before application. The SNAP and SIN-1 stock solutions were kept cold in the dark until just before use. Statistical analysis To determine whether a drug had a signi®cant effect or not, we used a non-parametric Mann±Whitney U-test comparing the control versus the drug electrical discharge. To construct the concentration±effect relationship, the spike discharge was normalized as a percentage of change with respect to control conditions. Comparison of the mechanical responses was done by obtaining the mean of the peak response in at least ®ve cycles of the sinusoidal stimulus period (the ®rst and the last cycles were eliminated). Data are expressed as means ^ S.E.M. Statistical signi®cance in the concentration±effect data was calculated using the paired Student's t-test (P , 0.05). Graphs were obtained 10 min after drug perfusion. For the analysis of the action potential waveform and spike discharge intervals only neurons with stable membrane potentials negative to 240 mV were collected. The following electrophysiological parameters were measured: membrane potential, input resistance, action potential amplitude, action potential duration, amplitude of the after-hyperpolarization (AHP) and the coef®cient of variation of the interspike interval distribution (CV). RESULTS
Recordings of the electrical activity of semicircular canal afferent ®bers were obtained from a total of 137 preparations. Extracellular multiunit recordings Effects of nitric oxide synthase inhibitors. Basal and mechanically evoked activities were recorded in control conditions and after bath perfusion of NOS inhibitors. l-NOARG in a concentration range from 0.1 mM to 10 mM (n 21; Fig. 1A) produced a signi®cant timeand concentration-dependent inhibitory action of the afferent neurons basal and mechanically evoked discharge. After 10 min l-NOARG (10 mM) perfusion (n 5), basal discharge signi®cantly decreased to 78.5 ^ 4.9% and the mechanically evoked response to 48.3 ^ 4.7% of control values (paired Student's t-test, P , 0.05). These effects are illustrated in Fig. 1B. The actions were partially reversible after 45 min washing with Ringer's solution. The inhibitory action of lNOARG 10 mM on the basal and on the mechanically evoked activity increased to 95.8 ^ 1% and 76.9 ^ 1.4%, respectively, 30 min after perfusion with the drug. Application of l-NOARG 0.01 mM (n 6) after 10 min did not produce a signi®cant change in the basal and mechanically evoked discharge of the vestibular afferent neurons (paired Student's t-test, P . 0.05). Perfusion of l-NAME (0.1 mM to 1000 mM; n 24) consistently decreased the basal discharge of the semicircular canal nerve ®bers in a dose-dependent and reversible manner, but with lower potency than l-NOARG.
Nitric oxide in the vestibular system
459
Fig. 1. Blockade of basal and mechanically evoked discharge of the afferent ®bers of the semicircular canal by l-NOARG. (A) Concentration±response relationship of l-NOARG (n 21) effects on the basal discharge and in the mechanically evoked peak response. Each point represents the mean ^ S.E.M. of at least ®ve experiments. Asterisk indicates statistical signi®cance using a paired Student's t-test (P , 0.05). The changes are expressed relative to control values (100%, dotted line). (B) The plots represent consecutive records from the same preparation. Top, spikes/bin versus time plot of the basal (1 min) and mechanically evoked discharge of the semicircular canal afferent neurons in control conditions (black bar, sinusoidal 0.2 Hz angular accelerations). Middle, after 10 min of bath perfusion with 10 mM l-NOARG. Bottom, 45 min after washing with Ringer's solution, a partial recovery was observed. Bin 200 ms.
After 10 min of its perfusion l-NAME 100 mM (n 6) signi®cantly diminished the basal discharge to 45 ^ 9% and 1000 mM (n 5) to 53 ^ 10% of the control level (paired Student's t-test, P , 0.05). This is illustrated by Fig. 2A and B, which show the changes in afferent discharge under l-NAME (100 mM) observed in one typical experiment. It was found that the discharge rate was enhanced compared with the initial control activity after washing the l-NAME with Ringer's solution for 10 min.
Fig. 2. (A) Concentration±response relationship for l-NAME (n 24). Each point with bar represents the mean ^ S.E.M. of at least ®ve experiments. The asterisk indicates statistical signi®cance from control value (100%, dotted line) using a paired Student's t-test (P , 0.05). (B) l-NAME 100 mM decreased the basal discharge of the afferent ®bers of the semicircular canal. The horizontal bar above the record indicates the duration of the bath perfusion of the drug. Bin 1 s.
Effects of nitric oxide donors. The effects of bath application of two different nitric oxide donors (SNAP and SIN-1) on the ®ring rates of semicircular canal afferent ®bers were also studied. SNAP (0.1 mM to 100 mM; n 25) produced an increase in the basal discharge, and in the response to mechanical stimuli. For lower concentrations, between 0.1 and 10 mM, the SNAP effect was not concentration dependent (Fig. 3A), and it was not signi®cantly different from the control values (paired Student's t-test, P . 0.05). For concentrations above 10 mM SNAP produced a signi®cant increase in the basal and in the mechanically evoked response (paired Student's t-test, P , 0.05). The maximal effect was obtained with 100 mM SNAP (n 8). In the experiment shown in Fig. 3B, the basal discharge increased to 42 ^ 2.7%. In contrast, SNAP induced a 25.2 ^ 6.7% increment in the response to mechanical stimuli. A complete recovery of the electrical activity was observed after a 45 min washout of the drug. SIN-1 (1 mM to 1000 mM; n 58) was tested on the discharge rate of the afferent ®bers of the semicircular
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Fig. 3. Actions of SNAP in the electrical activity of the semicircular canal afferent neurons. (A) Concentration±response relationship of SNAP (n 25) does not show a concentration-dependent activity in the range between 0.1 and 10 mM; larger concentrations produced a signi®cant excitatory action (paired Student's t-test, P , 0.05). Each point represents the mean ^ S.E.M. of at least ®ve experiments. The asterisk indicates statistical signi®cance from control value (100%, dotted line) using a paired Student's t-test (P , 0.05). (B) Top, spikes/bin versus time plots of the control recording of the basal discharge (1 min) and of mechanically evoked response of the vestibular afferent ®bers. Middle, perfusion of 100 mM SNAP increased the basal and mechanically evoked activity. Bottom, washing (45 min) with Ringer's solution. Bin 200 ms.
Fig. 4. (A) Concentration±response relationship (n 58) between SIN-1 concentration and electrical activity basal (X), peak (7) and the minimum (A) of the response to mechanical stimuli. Each point represents the mean ^ S.E.M. of at least eight experiments. Asterisk indicates statistical signi®cance from control value (100%, dotted line) using a paired Student's t-test (P , 0.05). (B) Top, spikes/bin versus time plots of the basal and mechanically evoked discharge of vestibular afferent ®bers in control conditions. Middle, after 10 min of bath perfusion with 10 mM SIN-1 the baseline of the mechanical response increased signi®cantly by 61.9 ^ 22.5%, consequently the deacceleratory phase of the mechanical stimulus was no longer able to completely inhibit the afferent ®ber discharge (arrow). Bottom, excitatory action of SIN-1 was partially reversible during 20 min of washing. Bin 200 ms.
canal (Fig. 4A). At low concentrations SIN-1 (1 mM and 10 mM, n 8 and n 12, respectively) had no signi®cant effect on the basal discharge and on the mechanically evoked peak response of the afferent neurons from the semicircular canals (paired Student's t-test, P . 0.05). At
a concentration of 1 mM, bath application of SIN-1 (n 8) consistently elevated the basal discharge of these ®bers to 22.6 ^ 18.4%, and the peak response to mechanical stimuli to 8.1 ^ 5.1% above the control discharge, although this action was not statistically
461
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Fig. 5. (A) Intracellular recording of an afferent ®ber in control conditions. (B) After 10 min of bath administration with l-NOARG 1 mM. Note that l-NOARG produced a decrease in the spike discharge rate and a hyperpolarization (248.4 ^ 2.65 vs 251.4 ^ 2.01 mV) of the mean membrane potential (n 5). The dotted lines indicate 0 mV. (C) Averaged action potentials (10 spikes) in control condition (continuous line), and after 10 min l-NOARG 1 mM perfusion (dotted line). Action potential parameters (amplitude, duration, AHP amplitude) were not signi®cantly affected by l-NOARG, although membrane potential was slightly hyperpolarized (paired Student's t-test, P . 0.05).
signi®cant. It is worth noting that after 10 min of bath perfusion, SIN-1 1 mM signi®cantly modi®ed the baseline (minimum) during the mechanical stimulation, producing a signi®cant increase of 41.7 ^ 16.4% in the mean spike discharge of the afferent ®bers (paired Student's t-test, P , 0.05). With 10 mM SIN-1 (n 12) also signi®cantly increased the baseline (minimum) of the mechanically evoked response to 61.9 ^ 22.5% of the control value (paired Student's t-test, P , 0.05) (Fig. 4B). In contrast, higher concentrations of SIN-1 (100 mM, 300 mM and 1000 mM; n 13, 11 and 14, respectively) diminished the spike discharge rate of the afferent ®bers. Maximal inhibitory effect in this study was attained with 1000 mM SIN-1 (n 14), basal activity signi®cantly decreased in 30 ^ 12.5% (paired Student's t-test, P , 0.05) but not the mechanically evoked peak response (17.2 ^ 13.3%; paired Student's t-test, P . 0.05). The baseline of the mechanical response was not signi®cantly affected. During repeated applications of SIN-1 no obvious hypo- or hypersensitivity was observed. Intracellular recordings To determine whether NO acts at the synaptic level or directly on the afferent ®bers, we studied the capability of NOS inhibition and NO to modulate the action potentials and EPSPs of vestibular nerve ®bers. Spontaneous action
potentials and EPSPs were measured in control conditions and 10 min after bath perfusion with l-NOARG or SNAP. Bath perfusion of l-NOARG 1 mM (n 5) resulted in a 3 mV hyperpolarization from the mean membrane potential (248.4 ^ 2.65 vs 251.4 ^ 2.01 mV) associated with a decrease in the frequency of discharge (Fig. 5A, B). As shown in Fig. 5C, the averaged action potential amplitude (51.4 ^ 3.9 vs 48.6 ^ 6.26 mV), the duration (1.06 ^ 0.08 vs 1.06 ^ 0.09 ms) and the AHP amplitude (4.72 ^ 0.46 vs 4.75 ^ 0.49 mV) were not signi®cantly modi®ed (paired Student's t-test, P . 0.05). Analysis of the frequency and mean amplitude of EPSPs recorded in control conditions and after l-NOARG administration reveals that this NOS inhibitor signi®cantly decreased the frequency of the EPSPs in 32.3 ^ 1.4% (paired Student's t-test, P , 0.05) without changes in their amplitude (1.5 ^ 0.4%). l-NOARG 1 mM did not signi®cantly alter the CV of the neurons recorded (0.72 ^ 0.10 vs 0.77 ^ 0.12) (paired Student's t-test, P . 0.05). SNAP 100 mM (n 4) affected neither the mean amplitude (1.45 ^ 0.55%) nor the frequency of EPSPs (4.55 ^ 2.32%). In contrast, SNAP produced a depolarization of 2.5 mV of the mean membrane potential (259 ^ 2.34 vs 257 ^ 2.48 mV) and decreased the input resistance (88.35 vs 76.41 MV) of afferent neurons. SNAP 100 mM increased the spike discharge rate of all the neurons that were recorded (Fig. 6A, B). The averaged action potential amplitude (59.87 ^ 6.0 vs 56.2 ^ 4.8 mV), duration (1.25 ^ 0.18 vs 1.3 ^ 0.17 ms) and AHP amplitude (3.37 ^ 0.93 vs 3.7 ^ 0.93 mV) were not signi®cantly modi®ed (paired Student's t-test, P . 0.05) (Fig. 6C). Analysis of all the neurons recorded showed that SNAP signi®cantly increased the CV from 0.65 ^ 0.23 to 0.86 ^ 0.53 (paired Student's t-test, P , 0.05). DISCUSSION
These results provide evidence indicating that NOS inhibitors may inhibit, and NO donors may facilitate the electrical activity of afferent neurons. We found that bath application of NOS inhibitors, l-NOARG and l-NAME decreased the basal electrical activity of the afferent neurons of the semicircular canals from the axolotl in a concentration-dependent manner. The preparation showed higher sensitivity to l-NOARG than to lNAME. These results coincide with several reports indicating that l-NOARG is more potent than l-NAME both in vitro and in vivo. 22 In our experiments, the biological effect of l-NOARG was partially reversed by washing with Ringer's solution. In contrast, l-NAME inhibition of the basal electrical activity was completely reversible. The effects of l-NOARG and l-NAME hint at the presence of NOS in the preparation and can be explained by the competitive action with l-arginine for the substrate-binding site. Previous reports described the presence of NOS in the vestibular system. 7,14,26 NADPH diaphorase histochemistry of the axolotl vestibular system showed the afferent ®bers and hair cells predominantly stained. 7 In the chinchilla, NADPH
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Fig. 6. Intracellular recording of an afferent ®ber in control conditions (A), and after 10 min of bath perfusion with SNAP 100 mM (B). Note the increase in the spike discharge rate. SNAP 100 mM produced a small depolarization (259 ^ 2.34 vs 257 ^ 2.48 mV) associated with a decrease in input resistance (88.35 vs 76.41 MV) of the afferent ®bers (n 4). (C) Averaged action potentials (10 spikes) in control condition (continuous line), and after 10 min SNAP 100 mM perfusion (dotted line). Action potential parameters (amplitude, duration, AHP amplitude) were not signi®cantly affected by SNAP, although membrane potential was slightly depolarized (paired Student's t-test, P . 0.05).
diaphorase-positive staining was observed in efferent boutons, rather than in afferent boutons, and in some hair cells predominantly of type I. 26 In the adult rat vestibular system, NOS has been found in ®bers of the vestibular ganglion and boutons throughout the crista ampullaris. 14 Recently, NOS I and III, as well as soluble guanylyl cyclase (sGC), have been detected in the vestibulocochlear system of mice 15 and in the vestibular system of guinea-pigs. 16 The selective expression of the NOS II isoform during mouse vestibulocochlear receptorgenesis has also been described. 1 In the guineapig vestibular system, studies using a ¯uorescent indicator show the production of NO in hair cells. 33 All of these results support the presence of NOS in the vestibular system of several animal models, and are consistent with our present ®ndings indicating that NO signi®cantly contributes to the basal discharge, and to the response of afferent ®bers to mechanical stimuli. In the present study we observed that SNAP produced an excitatory action on the basal and mechanically evoked discharge, but the concentration±reponse curve is ¯at, except for the concentration of 100 mM which produced a signi®cant excitatory action. It should be considered that SNAP is an unstable nitrosothiol; its half-life can vary from seconds to hours at pH 7.4 depending on the experimental conditions. SNAP is also very photosensitive and highly susceptible to many factors, including temperature.6,23 SNAP at concentrations
higher than 100 mM produced an irreversible block of the afferent ®ber discharge. Although NO is an important messenger molecule, when generated in high concentrations it can produce cellular damage, consequently explaining this last effect. 11 SIN-1 is a sydnonimine, highly water-soluble and stable without signi®cant decomposition when stored protected from light at 48C. 6 SIN-1 at concentrations lower than 100 mM increased the basal discharge and the peak response to mechanical stimuli. At higher concentrations SIN-1 decreased the basal and the mechanically evoked activity. In this regard, it has been reported recently that SIN-1 has dual effects: facilitation and suppression on laryngeal motoneurons in the nucleus ambiguous. 35 In our experiments, after 10 min of bath perfusion with SIN-1, it signicantly modi®ed the baseline during the mechanical stimulation, producing an increase in the mean spike discharge of the afferent ®bers. This effect is similar to the previous one reported when glycine was added to the Ringer's solution in which the electrical discharge of the semicircular canal afferent ®bers increased during the deacceleratory phase of sinusoidal mechanical stimulus. 30 This action was dependent on the stimulus duration, and was blocked by NMDA antagonists and also by extracellular magnesium. These results provide support for a signi®cant role of NMDA receptors in mediating the activation of afferent neurons, and also probably participating in some type of adaptative changes in the gain of the afferent synapse mediated by the activation of NOS and NO generation. 29,30 There is evidence that NO leads to an elevation of cyclic guanosine monophosphate (cGMP) levels in the cytoplasm by the activation of the cytosolic enzyme sGC. 18,4,2 cGMP then mediates the physiological effects of the pathway, often by activating a cGMP-dependent protein kinase. Furthermore, it is known that high cGMP levels reduce the inward potassium conductance (IK,1) of the vestibular hair cells in the rat. 3 The reduction in the K 1 current may lead to increased changes in membrane potential due to the mechano-electrical transducer currents, therefore the release of the excitatory neurotransmitter may also increase. Moreover, patch clamp recordings have shown that NO donors, such as sodium nitroprusside and nitroglycerin, inhibit a low voltageactivated potassium current (IK,L) speci®c to mammalian type I vestibular hair cells. 8-Bromo-cGMP, the membrane-permeant analog of cGMP, also inhibited the onset current. 5 Chen and Eatok 5 suggest the possibility that, in vivo, IK,L can be turned off by a retrograde messenger released by the calyx endings from primary afferent neurons. This retrograde messenger could be NO. Considerable data suggest that NO is capable of modifying the electrical activity of neurons. 11 In our preparation the basal ®ring rate and the frequency of EPSPs were decreased by the NOS inhibitor l-NOARG, suggesting that it acts at the presynaptic level, inhibiting neurotransmitter release. It has been argued that NO produced in one cell acts principally on NO targets in other cells. Thus, NO produced in the vestibular hair cells diffuses
Nitric oxide in the vestibular system
to the afferent neurons and acts there. In contrast, perfusion of the NO donor SNAP increased the ®ring rate and decreased the input resistance of the vestibular afferent neurons, thus suggesting a postsynaptic action of SNAP. This compound also increased the CV of the interspike intervals, indicating that NO donors could modify the regularity of discharge of vestibular afferents. The basal and the mechanically evoked electrical activity were differentially affected by l-NOARG and SNAP (the latter at the highest concentration used). This implies that, as has been previously suggested, 12 the mechanisms determining the resting basal discharge and the mechanically evoked response are probably different. Both the release processes 12 and the type of postsynaptic receptors involved 29 can explain the differential effect of NO upon the basal and the mechanically evoked activities.
463
Particular interest has revolved around NO production following NMDA receptor activation, making NO an attractive candidate for a retrograde messenger involved in the correlation of pre- and postsynaptic activity during changes in synaptic ef®cacy. Our results indicate that apart from the role that NO may have in blood ¯ow, immune response and metabolic regulation, NO plays a signi®cant role in the sensory coding in the vestibular system, participating as a neuromodulator of the afferent discharge, thus in¯uencing the overall gain of the system. These results imply that NO-related drugs may have potential use in the clinical treatment of vestibular disorders. AcknowledgementsÐThe authors thank Professor Augustine Udeh for proof reading the English manuscript. This work was partially supported by CONACyT grant J28904-N to A. Flores.
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