Detection of an auditory nerve--activating substance.

A substance or substances capable of increasing the firing rate of primary auditory fibers is detectable in the perilymph of frogs and guinea pigs subjected to sound stimulation. The increase in firing rate occurs in single units of the frog auditory nerve after perilymph obtained from frogs or guinea pigs during sound stimulation is infused into the frog perilymphatic sac. Perilymph collected from animals maintained in silence failed to cause an increase in firing rate of primary auditory fibers of the frog.

The vestibular hair cells: post-transductional signal processing.

Hair cells in mechanosensory systems transduce mechanical stimuli into biological signals to be presented to and analyzed by the brain. Vestibular hair cells transduce stimuli primarily associated with the organism's orientation and motion in space. When examined superficially it may appear that the hair cells act as passive transducers whereby mechanical stimulation of their hair bundle results in transmitter release at their afferent synapses. In fact, hair cell functions are more complicated, and the mechanical signals are heavily processed even before being encoded in afferent nerve activity. Hair cells are different from one another in morphology, biophysics, transmitter and transmitter receptor complements, not only across different organs (as one might expect), but even in the same organ. This review focuses on hair cell morpho-physiological properties, ionic conductances, neurotransmitters/modulators and their receptors, second messengers and effectors. Special features of hair cell neurotransmission, as the synaptic body and the presence of autoreceptors and local circuits, are also discussed, as is the possibility of a differential modulation of hair cell transmitter release in the resting and mechanically-stimulated states.

Audiogenic seizures and cochlear damage in rats after perinatal antithyroid treatment.

The feeding of goitrogens during pregnancy and lactation causes the offspring of rats to be partially deaf and persistently sensitive to audiogenic seizures. The most potent goitrogen, propylthiouracil, caused severe dysfunction and disorganization of the organ of Corti. Adult seizure-susceptible rats showed increased sensitivity to audiogenic seizures when they were fed propylthiouracil.

Influence of ATP and ATP agonists on the physiology of the isolated semicircular canal of the frog (Rana pipiens).

In the present study, the influence of extracellular ATP and ATP agonists in the physiology of the vestibular organs was examined, using the in vitro model of the isolated semicircular canal of the frog (Rana pipiens). The firing activity of the afferent nerve, the d.c. nerve potential and the transepithelial potential were measured in the absence and presence of mechanical stimulation of the sensory epithelium. Administration of ATP into the perilymphatic compartment, from 10(-12) to 10(-3) M, increased the firing rate of the afferent fibers recorded in the absence of mechanical stimulation. Recordings of the d.c. nerve potential indicated that the afferent fibers were hyperpolarized. The presence of the purine also modified the transepithelial potential. During mechanical stimulation of the sensory epithelium, both the evoked afferent firing and the evoked variation of the d.c. nerve potential were reduced in the presence of ATP. However, ATP did not effect the evoked modulation of the transepithelial potential, evoked by the mechanical stimulation. Administration of the P2x purinoceptor agonists, alpha, beta-methylene-ATP and beta, gamma-methylene-ATP, at concentrations between 10(-12) and 10(-3) M, did not significantly modify the different bioelectrical activities investigated. In contrast, 2-methylthio-ATP, a P2y purinoceptor agonist, more potent and efficacious than ATP in its effect on the spontaneous firing. Concurrently, no modification of the d.c. nerve potential, the transepithelial potential and their variation during mechanical stimulation was observed. In opposition to the ATP effect, the total amplitude of the evoked firing was increased in the presence of 2-methylthio-ATP. These data suggest that extracellular ATP, present in the perilymphatic compartment, may act as a neuromodulator in the vestibular physiology. The effects of the purine appear to be mediated by the activation of a P2y subtype of purinoceptor. The absence of an effect of ATP and 2-methylthio-ATP on the evoked variation of the transepithelial potential suggest that the purine did not affect the processes responsible for the generation of the receptor potential but more likely modified the mechanisms involved in the release of the neurotransmitter from the hair cells and/or acted on the afferent endings.

Indirect evidence for the presence and physiological role of endogenous extracellular ATP in the semicircular canal.

Previous studies have shown that low concentrations of exogenous ATP, added to the perilymphatic fluid, could modify the bioelectrical activity of the isolated semicircular canal of the frog (Rana pipiens). To test the hypothesis that ATP is endogenously present and active in the perilymphatic fluid, the influence of two ATP-purinoceptor antagonists, Reactive Blue 2 and suramin, and of the enzyme, nucleotide pyrophosphatase, were examined. When applied by perilymphatic bath substitution, the three compounds reduced, in a dose-dependent manner, the firing of the afferent fibers monitored in the absence of mechanically-applied stimulation. The response of the afferent fibers, recorded when the sensory cells were mechanically inhibited, was also reduced. No modification of the response of the excitatory phase of the mechanical stimulus was observed in the presence of the two antagonists. In contrast, the signal was significantly reduced by the enzyme. None of the three compounds exhibited an influence on the transepithelial potential, or its variation in response to mechanical stimulation. The ATP-induced modification of the firing rate of the afferent fibers, monitored in the absence of mechanical stimulation, was reduced in the presence of the three drugs. No influence of Reactive Blue 2 and suramin was observed on the increase of the spontaneous firing induced by carbachol. In contrast, the effect of carbachol was decreased by nucleotide pyrophosphatase. The excitatory influence of glutamate on the spontaneous firing was not modified by Reactive Blue 2, while it was slightly increased by suramin and nucleotide pyrophosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)

A synthetic peptide corresponding to the carboxy terminus of human immunodeficiency virus type 1 transmembrane glycoprotein induces alterations in the ionic permeability of Xenopus laevis oocytes.

The carboxy-terminal 29 amino acids of the human immunodeficiency virus type 1 transmembrane glycoprotein (HIV-1 TM) are referred to as lentivirus lytic peptide 1 (LLP-1). Synthetic peptides corresponding to LLP-1 have been shown to induce cytolysis and to alter the permeability of cultured cells to various small molecules. To address the mechanisms by which LLP-1 induces cytolysis and membrane permeability changes, various concentrations of LLP-1 were incubated with Xenopus laevis oocytes, and two-electrode, voltage-clamp recording measurements were performed. LLP-1 at concentrations of 75 nM and above induced dramatic alterations in the resting membrane potential and ionic permeability of Xenopus oocytes. These concentrations of LLP-1 appeared to induce a major disruption of plasma membrane electrophysiological integrity. In contrast, concentrations of LLP-1 of 20-50 nM induced changes in membrane ionic permeability that mimic changes induced by compounds, such as the bee venom peptide melittin, that are known to form channel-like structures in biological membranes at sublytic concentrations. An analog of LLP-1 with greatly reduced cytolytic activity failed to alter the electrophysiological properties of Xenopus oocytes. Thus, by altering plasma membrane ionic permeability, the carboxy terminus of TM may contribute to cytolysis of HIV-1-infected CD4+ cells.

Drugs affecting the inner ear. A review of their clinical efficacy, mechanisms of action, toxicity, and place in therapy.

Many drugs have a site of action within the inner ear. The list includes therapeutic, diagnostic and ototoxic agents. Therapeutic agents are most useful in cases of infections, endolymphatic hydrops, vascular insufficiency, vertigo of peripheral origin, autoimmune disease, otosclerosis (otospongiosis), sudden hearing loss and tinnitus. For infections, the most widely used anti-microbial agents are the penicillins and cephalosporins. There are no antiviral agents that have been proven useful for inner ear viral infections. However, steroids have been of some value for controlling some of the sequelae. Steroids have also been useful in conjunction with ampicillin in cases of syphilitic hearing loss. In cases of endolymphatic hydrops, the diuretics chlorthalidone, hydrochlorothiazide and acetazolamide have been useful. When diuretic and diet therapy cannot control endolymphatic hydrops, ototoxic drugs such as streptomycin have been used. In cases of vascular insufficiency within the inner ear, vasodilators such as carbon dioxide, papaverine, buphenine (nylidrin), naftidrofuryl (nafronyl) and thymoxamine have been recommended, but their true efficacy is questionable. Some success with betahistine has been achieved but the mechanism of this drug's action may be other than vasodilatation. Vertigo is best controlled with antihistamines and anticholinergics and with certain calcium channel blockers. Autoimmune inner ear disease appears to respond to a combination of steroids and cyclophosphamide. Although controversial, current pharmacotherapy for otosclerosis includes sodium fluoride. Sudden hearing loss is treated with a 'shotgun' combination of drugs and/or bed rest. There are as yet no drugs which can be used to routinely reduce tinnitus although some medications may help the patient tolerate the problem. Lignocaine (lidocaine) is useful in diagnosing, and very evanescently reducing, tinnitus. Glycerin (glycerol) is useful in diagnosing endolymphatic hydrops and may at times transiently reduce tinnitus. The drugs most noted for their ototoxicity are the aminoglycoside antibiotics, certain diuretics, non-steroidal anti-inflammatory agents, certain anticancer agents and some miscellaneous chemicals. Some new research drugs are in clinical trials for tinnitus, hearing loss and vertigo, and the rational search for new otopharmacotherapeutic agents is increasing.

Concentration-dependent differential induction of necrosis or apoptosis by HIV-1 lytic peptide 1.

The mechanism by which human immunodeficiency virus type 1 induces depletion of CD4+ T-lymphocytes remains controversial, but may involve cytotoxic viral proteins. Synthetic peptides (lentivirus lytic peptide type 1) corresponding to the carboxyl terminus of the human immunodeficiency virus type 1 transmembrane glycoprotein induce cytopathology at concentrations of 100 nM and above. At these concentrations lentivirus lytic peptide type 1 disrupts mitochondrial integrity of CD4+ T-lymphoblastoid cells and induces other changes characteristic of necrosis. In contrast, at concentrations of 20 nM, lentivirus lytic peptide type 1 potently induces apoptosis. Thus, the mechanism by which human immunodeficiency virus type 1 mediates cell death, necrosis or apoptosis, may depend, in part, on the tissue concentration of transmembrane glycoprotein.

Computer analysis of vestibular responses to putative neurotransmitters.

An inexpensive microcomputer (Commodore 64K) based system was developed for the analysis of neural spike trains. The trains were recorded from single ampullary units in response to mechanical stimulation of the isolated semicircular canal of the bullfrog (Rana catesbeiana). A BASIC program provided a number of options while machine language subroutines generated interstimulus interval (ISI) and peristimulus time (PST) histograms. Up to thirty 5-s spike trains could be combined for analysis (0.1 ms resolution ISI, 100 ms bin width PST). Histograms and summary statistics were saved on floppy disks. The cost of adding this computer system to an existing neurophysiology laboratory is less than US $600 (printed, tape, and disk versions of these programs are available). The system was used to measure vestibular responses to putative vestibular neurotransmitters such as carbachol (an acetylcholine mimic) (Rossi et al., 1980) and gamma-aminobutyric acid (GABA) (Flock and Lam, 1974).

Measurement of the combination tone 2f1-f2 using two-tone forward masking and compound action potential responses.

The presence of the 2f1-f2 combination distortion tone generated when two tones are presented to the cochlea, can be detected using a two-tone complex to forward mask a probe tone. The combination tone is the effective masking agent and the amplitude of the compound action potential response to the probe tone reflects the degree of masking. Excitation patterns, frequency relationships and intensity functions that are obtained with this procedure are consistent with previously published data.

The hair cell acetylcholine receptors: a synthesis.

In this article the evidence concerning the nature of the acetylcholine (ACh) receptors on hair cells is reviewed. A schematic organization of these receptors is offered, based on the evidence as follows. (1) There are two kinds of ACh receptors on hair cells: muscarinic-like and nicotinic-like. (2) The nicotinic-like receptor mediates a hyperpolarizing response to ACh and a consequent reduction in afferent firing. (3) The muscarinic-like receptors mediate both a depolarization and a hyperpolarization of hair cells. (4) The hyperpolarization results in a reduction in afferent firing and (5) the depolarization results in an increase in afferent firing.

In vitro pharmacologic characterization of a cholinergic receptor on outer hair cells.

Acetylcholine (ACh) is the major neurotransmitter released from the efferent fibers in the cochlea onto the outer hair cells (OHCs). The type of ACh receptor on OHCs and the events subsequent to receptor activation are unclear. Therefore we studied the effect of agonists and antagonists of the ACh receptor on isolated OHCs from the guinea pig. OHCs were recorded from in whole cell voltage and current clamp configuration. ACh induced an increase in outward K+ current (IACh) which hyperpolarized the OHCs. No desensitization to ACh application was observed. Cs+ replaced K+ in carrying the IACh. The IACh is Ca(2+)-dependent, time and voltage sensitive, and different from the IKCa induced by depolarization of the membrane potential. When tested at 100 microM, several agonists also induced outward current responses (acetylcholine > suberyldicholine > or = carbachol > DMPP) whereas nicotine, cytisine and muscarine did not. The IACh response to 10 microM ACh was blocked by low concentrations of traditional and non-traditional-nicotinic antagonists (strychnine > curare > bicuculline > alpha-bungarotoxin > thimethaphan) and by higher concentrations of muscarinic antagonists (atropine > 4-DAMP > AF-DX 116 > pirenzepine). Pharmacologically, the ACh receptor on OHCs is nicotinic.

Acetylcholine activates a K+ conductance permeable to Cs+ in guinea pig outer hair cells.

Acetylcholine (ACh), the major neurotransmitter released by efferent nerve fibers in the cochlea, has been shown to activate a Ca(2+)-dependent K+ conductance in outer hair cells (OHCs). Previously we reported that this ACh operated conductance is permeable to Cs+. The purpose of the present study was to characterize further this Cs(+)-permeable channel and its dependency on Ca2+ using isolated OHCs and the patch clamp technique in the whole cell configuration. The changes in the ACh response were examined when Cs+, Ba2+, Cd2+, N-methyl-D-glucamine (NMG+) and tetraethylammonium (TEA+) were placed in the external or internal solutions. Cs+ substituted for K+ in carrying the ACh-evoked Ca(2+)-dependent K+ current. When NMG+/TEA+ was substituted for internal K+ ACh-evoked an inward and an outward current, and Cs+ substituted for external K+ blocked the outward but not the inward current evoked by ACh suggesting it was carried by K+. In the NMG+/TEA+ condition, when the cell was held at different Vh values for an extended period of time, the ACh-induced K+ current rectified. In Ba2+ (3 mM) with zero Ca2+ ACh failed to induce any detectable current and the ACh response slowly recovered from the Ba2+ block, suggesting a block at an intracellular site. Cd2+ (1 mM) readily and reversibly blocked ACh-induced currents even when carried by Cs+. This data suggests that ACh opens a channel selective for K+, conductive to Cs+ and dependent on Ca2+.

Three tests of the hypothesis that glutamate is the sensory hair cell transmitter in the frog semicircular canal.

Three series of experiments were devised to test the hypothesis that glutamate is the transmitter released by sensory hair cells of the frog semicircular canal. These three tests were: 1 - The Tolerance experiment (i.e. making the preparation tolerant to injected Glu yet still capable of responding to endogenous transmitter). 2 - The Glu Decarboxylase experiment (i.e. bathing the preparation in sufficient enzyme to prevent the effects of exogenous Glu by degrading it without affecting the response to endogenous transmitter) and; 3 - The Diltiazem experiment (i.e. using the calcium channel antagonist, diltiazem, to prevent the effect of exogenous Glu and yet not to interfere with endogenous transmitter release and action). The Tolerance and Diltiazem experiments produced results indicative of a clear dissociation between exogenous Glu and natural transmitter. The Glu decarboxylase experiment results were not so clear, producing both evidence for and against the hypothesis.

Histamine and related substances influence neurotransmission in the semicircular canal.

Histamine and other imidazole-containing substances were found to increase ampullar nerve afferent firing rate while both H1 and H2 histamine antagonists effectively inhibited ampullar nerve activity. A specific inhibitor of histidine decarboxylase, the enzyme which catalyses the synthesis of histamine, reduced ampullar nerve firing in a dose-dependent manner. These observations suggest a physiological role for histamine in the inner ear. Maintenance of a response to histamine after de-efferentation of the crista ampullaris supports the hypothesis that the site of action is the hair cell; antagonism of the histamine response by a cholinergic antagonist, atropine, and antagonism of a cholinergically mediated facilitation by the histaminergic antagonist pyrilamine, indicate that the site of action may involve the acetylcholine receptor complex on the crista ampullaris hair cells. The observation that imidazole-containing compounds cause significant effects on semicircular canal neurotransmission provides an important finding with regard to the site of action of antihistamines used for the treatment of vertigo and motion sickness.

Electrophysiological properties and morphology of hair cells isolated from the semicircular canal of the frog.

Hair cells isolated from the crista ampullaris of the frog (Rana pipiens) remained viable for up to 5 h and were studied using whole cell voltage- and current clamp recordings. Morphological characteristics of isolated crista hair cells were compared with hair cells studied in situ using light- and electron microscopy. While other labyrinthine hair cells such as mammalian inner and outer hair cells of the cochlea, saccular hair cells of the frog, and cochlear hair cells of the turtle typically have a cylindrical shape, the crista hair cells in the frog are predominantly bulbous, having a thin elongated trunk projecting from a spherical base just large enough to enclose the nucleus. This shape correlates well with the compressed packing configuration of hair cells of the crista ampullaris observed in situ in the histological material. The support cells often failed to separate adjacent hair cells, particularly the apical ends of the hair cells. Maximal cell density on the sensory epithelial ridge appears to be achieved by this arrangement. The mean resting membrane potential (Vz) of isolated crista hair cells was -44.8 mV. Cells with smooth surfaces and apparent opacity had the most negative Vz potentials. As the cells appeared to deteriorate, there was development of transparency and cell surface granulation. Such cells had more positive initial Vz values. Cells with Vz values more positive than -15 mV exhibited a distinct, contoured nucleus. Cells lacking these indicators of deterioration were characterized by input resistances of 1.9 +/- 0.31 G omega and membrane time constants of 13 +/- 2.5 ms. A large complex outwardly rectifying current was identified which was abolished by substituting Cs+ for K+ in the internal solution. The outward K+ current had two major components: a fast tetraethylammonium (TEA)-insensitive, voltage dependent I(A)-type current which showed voltage dependent inactivation; and a TEA sensitive current which had characteristics of a calcium dependent IK(Ca)-type current. Transient changes (20 ms duration) in membrane potential mimicking that which could be produced by the transduction current during cilial displacement potently modulated the I(A) current. Depolarizing current pulses of greater than 63800 pA were required to elicit membrane voltage oscillations. The resulting membrane potential offset of at least 40 mV is well beyond the magnitude of hair cell receptor potentials making it unlikely that these oscillations would play a role in enhancing frequency selectivity.(ABSTRACT TRUNCATED AT 400 WORDS)

The cholinergic pharmacology of the frog saccule.

Stimulation of the efferent nerves to the vestibular organs of the frog's inner ear produces either facilitation or inhibition of afferent firing. Similarly, application of acetylcholine (ACH), the major transmitter of the efferents, can produce both facilitation and/or inhibition as previously reported [Guth et al. (1986) Acta Otolaryngol. 102, 194-204; Norris et al. (1988) Hear. Res. 32, 197-206]. The firing rates of afferent neurons of the semicircular canal (SCC) using multiunit recordings are generally facilitated by ACH. Conversely, the firing rates of afferent units innervating the saccule are generally inhibited by ACH. This latter inhibition is antagonized by strychnine more potently than by curare, which is more potent than atropine. When inhibition is antagonized by strychnine or curare an underlying facilitation is revealed. The inhibition of saccular afferents by ACH shows desensitization requiring about 20 min to recover. The ACH-induced inhibition is mimicked by nicotine at very high concentrations but not by dimethyl phenylpiperazinium or cytisine. The fact that multiunit afferent firing from the SCC is generally facilitated while that from the saccule is generally inhibited by ACH suggests a different distribution of ACH receptors and receptor types (i.e. muscarinic or nicotinic and their subtypes) in the two organs and demonstrates the usefulness of recording from multiple units simultaneously. The difference in distribution of ACH receptors may be important for understanding the physiology of vestibular efferents.

Cholinergically-induced changes in outward currents in hair cells isolated from the semicircular canal of the frog.

Two cholinergically-induced modulations of membrane conductances have been identified in hair cells isolated from the crista ampullaris of the leopard frog (Rana pipiens), using the whole cell recording configuration of the patch clamp technique. Of 56 crista hair cells tested, 28 showed drug-induced changes in membrane current or membrane potential which were repeatable and could be reversed with washout of drug. The predominant effect (observed in 20 hair cells) of acetylcholine (Ach, 100 microM) to 1mM) or carbachol (1 microM to 50 microM) applied to these hair cells was the reduction of an outward current corresponding to a change in conductance of approximately -0.22 nS. This action by Ach on hair cells has been inferred from previous studies of afferent fiber discharge which reported an increase in firing rate with stimulation of efferent fibers or exogenous application of cholinomimetics (Bernard et al., 1985; Valli et al., 1986; Guth et al., 1986; Norris et al., 1988a). The Ach-induced reduction in outward current was associated with a depolarization of the zero-current membrane potential by approximately +2.5 mV. In a total of 8 hair cells, an Ach-induced reversible increase in outward current was recorded. Changes in conductance were approximately +0.13 nS and were associated with a hyperpolarization of the zero-current membrane potential by approximately -2.2 mV. This current increase is likely to be responsible for the inhibitory post-synaptic potentials (IPSPs) which have previously been recorded intracellularly from acoustico-lateralis hair cells during stimulation of the efferent innervation (Flock and Russell, 1976; Ashmore and Russell, 1982; Art et al., 1984, 1985). Of the remaining 28 hair cells, six cells failed to exhibit any change in membrane conductance or membrane potential in the presence of cholinomimetics while an additional 15 cells exhibited decreases, and 7 cells exhibited increases in outward conductance, during application of Ach or carbachol, which were neither reversible with washout nor repeatable. The Ach-induced decrease in outward current could be reversible blocked by removal of Ca2+ from the external solution. The antagonism of the Ach-induced decrease in outward current by atropine (10(-5) M) suggests that this current may correspond to a facilitatory, 'atropine-preferring' Ach receptor mediated response previously identified in the isolated semicircular canal (Norris et al., 1988a).(ABSTRACT TRUNCATED AT 400 WORDS)

Differential modulation of spontaneous and evoked neurotransmitter release from hair cells: some novel hypotheses.

It has been generally accepted that even in the absence of mechanical stimulation of the transductional elements, a resting depolarizing current exists which is ultimately responsible for the spontaneous release of neurotransmitter. Movement of the transductional elements modulates this resting current and thereby the evoked release of neurotransmitter occurs. Recent data from our laboratory and others have led us to question whether the relationship between spontaneous and evoked neurotransmitter release is as simple as stated. Indeed, a variety of experimental manipulations appear to influence the two modes of release differently. Examination of our results and the results of others has led us to four hypotheses: 1. the two modes of neurotransmitter release are processed differently by the hair cells; 2. cyclic AMP is involved in spontaneous but not evoked neurotransmitter release; 3. there is a positive feedback step involving an excitatory amino acid and its receptor on the hair cell in evoked neurotransmitter release and; 4. different pools of calcium are involved according to the mode of release. Accordingly, there may be several biochemical steps between the transductional movement of the stereocilia at the apex of the hair cells and the ultimate release of the neurotransmitter at the base of these cells. Some of these biochemical steps are different depending on whether the mode of release is spontaneous or evoked. These biochemical steps may amplify or at least interact with the biophysical processes previously described in the hair cells.

ATP modulation of L-type calcium channel currents in guinea pig outer hair cells.

Ca2+ channel currents and their modulation by adenosine 5'-triphosphate (ATP) in acutely isolated guinea pig outer hair cells (OHCs) were investigated using the whole-cell patch-clamp technique. The current-voltage (I-V) relation of OHCs indicated that the Ca2+ channel opened near -30 mV, and the current reached a maximum at +10 and 0 mV in 20 mM Ca2+ and Ba2+ external solutions, respectively. BayK 8644 (BayK, 2 microM) caused a 3.5-fold increase in peak Ca2+ currents and shifted the I-V curves toward more negative potentials. These results suggest that the majority of Ca2+ channels in OHCs have L-type characteristics. The effects of ATP on Ca2+ channels of OHCs were heterogenous. ATP (100 microM) decreased Ca2+ channel currents by 31.7 +/- 5.6% at 0 mV and shifted Ca2+ tail activation curves toward more depolarized potentials in some cells (N = 6). By contrast, in others, ATP enhanced the currents by 43.5 +/- 12.5% at +10 mV (N = 6). In the presence of BayK, however, ATP-induced inhibition or enhancement of Ca2+ channel currents was attenuated. In addition, 100 microM ATP produced little effect on Ca2+ channel currents in another subpopulation of cells (N = 12). This heterogenous neuromodulation of Ca2+ channel currents by ATP may reflect a functional diversity among OHCs.