(Received 19 June 1975)

Transcription

1 J. Phy8iol. (1976), 257, pp With 1 plate and 8 text-figure8 Printed in Great Britain INHIBITION BY EFFERENT NERVE FIBRES: ACTION ON HAIR CELLS AND AFFERENT SYNAPTIC TRANSMISSION IN THE LATERAL LINE CANAL ORGAN OF THE BURBOT LOTA LOTA BY AKE FLOCK AND IAN RUSSELL* From the Department of Physiology II, Karolinska Institute, S Stockholm 60, Sweden (Received 19 June 1975) SUMMARY 1. Intracellular recordings were made from morphologically identified hair cells in the lateral line canal organs of the burbot Lota Iota. 2. I.p.s.p.s were recorded from hair cells when the efferent fibres were excited by electrical stimulation of the lateral line nerve. The i.p.s.p.s were abolished when the fish was injected with immobilizing concentration of Flaxedil which is known to block the efferent synapses. 3. The i.p.s.p.s are accompanied by a decrease in the resistance of the hair cell membrane and an increase in the intracellular receptor potential. 4. Spontaneous and mechanically evoked e.p.s.p.s which were recorded intracellularly from the post-synaptic afferent nerve terminals were reduced in amplitude for the duration of the i.p.s.p. INTRODUCTION The central nervous system exerts a centrifugal control over sensory input at different stages in its neural processing. In the cochlea, vestibular system and related lateral line organs of lower vertebrates, this control extends to the receptor cell itself where efferent fibres form axo-somatic synapses with the sensory hair cells (Engstr6m & Wersall, 1958; Wersall, 1956, Hama; 1965; Flock, 1965; Spoendlin, 1973). Stimulation of the efferent fibres modulates electrical activity in the sense organs causing inhibition of spontaneous and evoked afferent impulses, a slow d.c. change in the sensory epithelium and an augmentation of the receptor potential (Galambos, 1956; Fex, 1962, 1967; Sala, 1965; Russell, 1971a; Llinas & Precht, 1969; Klinke & Schmidt, 1970; Flock & Russell, 1973b). The * Neurobiology and Ethology Group, School of Biology, University of Sussex, Falmer, Brighton, Sussex BN1 9QG, England.

2 46 AKE FLOCK AND IAN RUSSELL potential changes brought about by efferent stimulation in the cochlea, vestibular system and lateral line organs are all so similar that it is very probable that efferent fibres act in a similar way on all hair cells of the acoustico-lateralis system. The present paper describes an investigation of the intracellular potential changes which occur in hair cells during stimulation of the efferent fibres. The sense organs chosen for this study were the lateral line canal organs of Lota Iota, the burbot, because they are accessible to experimentation and the hair cells can be penetrated by intracellular microelectrodes (Harris, Frischkopf & Flock, 1970; Flock, Jorgensen & Russell, 1973). Moreover, the efferent fibres are easily excited by electrically stimulating the whole lateral line nerve trunk (Flock & Russell, 1973b). Although this also causes an antidromic conduction block of afferent fibres, this effect is brief compared with the inhibition produced by the efferent fibres. Furthermore the conduction block caused by the antidromic invasion of the afferent nerve fibres is separable from the inhibition because efferent synapses are selectively blocked by Flaxedil whereas afferent synapses remain unaffected (Russell, 1971b; Flock & Russell, 1973a). The results described in this paper indicate that the efferent synapses on hair cells behave very similarly to inhibitory synapses which transmit through the release of a chemical transmitter. Stimulation of the efferent fibres causes an i.p.s.p. which can be recorded intracellularly in the hair cells, and the i.p.s.p. accompanies a reduction in the transmitter release from the afferent synapses of the hair cells. A preliminary report of this work has been published elsewhere (Flock & Russell, 1973a). METHODS Preparation. The experiments were carried out on forty specimens of Lota Iota (burbot) weighing g which were first anaesthetized in a 0-025% solution of MS-222 (tricaine methanesulphonate). The brain was exposed and the forebrain and the spinal cord posterior to the obex were destroyed by cautery. The fish were then removed to a recording tank where they were clamped firmly by a head holder, and respired by water at 120 C perfused over their gills. The cranium was further dissected to expose the supraophthalmic branch of the facial nerve which supplies lateral line organs to the frontal canal on one side of the head. After severing the nerve from its proximal connexion with the medulla it was carefully displaced anteriorly in the brain case. A lateral line organ in the frontal canal was dissected free by removing the over-lying skin to reveal the cupula and sensory epithelium. Providing the flow of water over the gills was sufficient (1.5 l./min) the circulation of blood in the capillary bed beneath the lateral line organ was maintained in good condition for several hours. Stimulation and recording. A glass pipette with a tip diameter of 100 jm was brought in contact with the cupula. It could be sinusoidally displaced, with a frequency of 70 Hz, in the direction of maximum sensitivity of the organ. Stimulus intensity is expressed in db units relative to an empirically determined threshold for

3 EFFERENT INHIBITION OF HAIR CELLS single afferent nerve fibres. A stimulus of 20 db corresponds to a peak-to-peak displacement of 1-2 #am of the glass pipette. A silver-silver chloride electrode was placed close to the base of the cupula to record the external microphonic potential produced in response to the tone and also d.c. potential changes caused by the efferent fibres. The supraophthalmic branch of the facial nerve was stimulated via paired platinum electrodes. Glass micro-electrodes were filled by capillary action (Tasaki, Tsukuhara, Ito, Wagner & Yu 1968) either with 3 M-KCl or with a 6 % solution of Procion Navy Blue H3RS in distilled water (Stretton & Kravitz, 1968). KCl filled electrodes had resistances of MD whereas those filled with Procion dye had resistances of MQ. The electrodes were connected to a d.c. amplifier (MEN- TOR) with a high impedance input (1012 l) which had facilities for current injection and continuous impedance monitoring with a bridge balance circuit. A silver-silver chloride indifferent electrode was inserted into musculature behind the head. The micro-electrode was advanced into the sensory epithelium by a stepping motor micro-drive (Transvertex). In some experiments cells were marked by iontophoretic injection of dye (5 x 10-8 A for see) and the organs were subsequently fixed with glutaraldehyde and embedded in Epon (Kaneko & Hashimoto, 1967). Sections 3 jm thick were cut on an LKB Ultrotome and were examined unstained in a microscope with Nomarski interference-contrast (Zeiss). Data was stored on d.c. and a.c. tape recorders and this was later analysed by a Didac 4000 special purpose computer (Intertechnique). Electron microscopy. The lateral line organ in the frontal canal, which was used in this physiological study, was examined by electron microscopy in order to determine the presence and location of the different types of synapses on the lateral line hair cells. The methods were the same as those described in a previous study on the physiology and ultrastructure of lateral line organs in the supratemporal canal (Flock, 1965). RESULTS Inhibitory post-synaptic potentials from hair cells Large, long lasting hyperpolarizing potentials were recorded intracellularly from some cells in the canal organs of Lota when the lateral line nerve was electrically excited (Text-fig. 1). The time course and shape of these potentials are similar to the extracellularly recorded 'efferent' potentials (Flock & Russell, 1973b), and their amplitude and duration depend on the frequency and duration of the nerve stimulus. With the stimulus parameters used in these experiments, namely 200 shocks/sec for msec the hyperpolarizing potentials were maximally 10 mv in amplitude and outlasted the period of stimulation by msec. In five cases the cells from which hyperpolarizing potentials had been recorded were marked by iontophoretic injection of Procion Navy Blue dye, and they were all identified as hair cells in histological sections (P1. 1). Electron microscopy showed that nerve endings of the efferent type as well as afferent ones innervate the base of hair cells (P1. 1). No lateral synapses between efferent and afferent nerve endings were seen. Hyperpolarizing potentials were not recorded from supporting cells, or from hair cells in fishes which had been immobilized with Flaxedil (gallamine 47

4 48 AXE FLOCK AND IAN RUSSELL triethiodide 3-4 mg/kg body wt.) which is known to pharmacologically block the hair cell efferent synapses (Flock & Russell, 1973a, b). Thus the hyperpolarizing potentials, which have characteristics similar to inhibitory post-synaptic potentials (i.p.s.p.s) recorded elsewhere in the nervous system (Eccles, 1964) are due to the post-synaptic action of the efferent fibres on the hair cells. A B -15 ~~' -200^ -301 mv Text-fig. 1. I.p.s.p.s evoked by electrical stimulation of the lateral line nerve and recorded intracellularly in a hair cell. A, the four traces from the same hair cell show variation in the shape of the i.p.s.p.s even though the stimulus to the nerve is kept constant. B, three successive traces from another hair cell show that the i.p.s.p. is increased when the membrane is depolarized. The period of nerve stimulation is indicated by shock artifacts in A and by the bar beneath the lower trace in B. The horizontal and vertical bars below A apply to all records: 10 msec and 5 mv respectively. Successive i.p.s.p.s were not always similar in shape (Text-fig. 1A) and this may be because hair cells receive several efferent synapses (PI. 1) ot which only a variable number are excited each time the efferent system is excited. It was difficult to maintain stable recording conditions intracellularly in hair cells for more than a few tens of seconds and no attempts were made to measure the reversal potentials of the i.p.s.p.s, but it is evident from fluctuations in the membrane potentials of impaled hair cells that it is probably close to the resting potential. Fluctuations in the depolarizing direction increase the i.p.s.p. while hyperpolarizations cause a decrease in the amplitude of the i.p.s.p. (Text-fig. 1 B). Reai8tanrce change during the i.p.s.p. The resistance of penetrated hair cells was measured in eleven cases by injecting small (0.4 na) current pulses through non-polarizing pipettes filled with KCl and by balancing out the resulting voltage pulses with a bridge circuit. The resistances of the hair cells varied between 10 and 100 MO (mean 40 MQ) and decreased during the i.p.s.p. This was in-

5 EFFERENT INHIBITION OF HAIR CELLS 49 dicated by a decline in the amplitude of the voltage pulse by %. Text-fig. 2 illustrates a resistance change of 5 MQ during an i.p.s.p. in a cell with a resting resistance of 40 MQ. This corresponds to a conductance change due to efferent stimulation of AG 0-2 x 10-6 mho. WL. Text-fig. 2. Impedance change in a hair cell during the i.p.s.p. Constant current pulses of 0-4 na are injected through the recording electrode to produce voltage pulses across the hair cell membrane. When the lateral line nerve is stimulated the amplitude of the voltage pulses decreases indicating a decrease in membrane resistance. Right hand vertical and horizontal bar: 5 mv and 10 msec respectively. Left hand vertical bar 10 MQ, and horizontal bar beneath the trace indicates the time when the lateral line nerve was stimulated at 200 sec-1. Interaction between the i.p.s.p. and the receptor potential Sinusoidal displacement of the cupula produces a microphonic potential which may be recorded by an electrode placed close to the sensory epithelium (Jierlof, Spoor & de Vries, 1952). The lateral line microphonic differs from the cochlear microphonic in that it is twice the frequency of the applied mechanical stimulation, but it resembles the cochlea microphonic in that it is augmented by stimulation of the efferent system (Fex, 1959, 1962; Flock & Russell, 1973b). Intracellular receptor potentials were recorded from cells in the sensory epithelia of lateral line organs. The cells were marked by iontophoretic injection of Procion blue and later identified as hair cells (P1. 1). The receptor potentials are small with maximum amplitudes of 1 mv, 50 db above the threshold for phase locking in the afferent nerve fibres. Unlike the microphonic potential they are the same frequency as the stimulus. This has been discussed elsewhere (see Flock, 1971). During the i.p.s.p. the amplitude of the receptor potential in response to mechanical stimulation 40 db above threshold is increased by about 40 % (Text-fig. 3A). This is equivalent to a 14 db change in the sensitivity of the hair cell (Flock & Russell, 1973 b). Although the amplitude of the receptor potential is increased, the amplitude of the hyperpolarization during the i.p.s.p. is considerably larger (Text-fig. 3B). Intracellular e.p.s.p.s from afferent nerve terminals Occasionally when the micro-electrode passed through the sensory epithelium of the lateral line organs, it penetrated an afferent nerve

6 50 AKE FLOCK AND IAN RUSSELL terminal and recorded small spontaneous excitatory post-synaptic potentials of brief duration (Text-fig. 4A). Nerve impulses were not always recorded and it was presumed that under these circumstances the electrode had entered the unmyelinated portion of the fibre very close to the A of Wo a B.. W 8{ * ww ma~~* - I Il Text-fig. 3. The post-synaptic action of efferent fibres on mechanically evoked receptor potentials in lateral line hair cells. A, a computer averaged trace of ten successive sweeps shows hair cell receptor potentials evoked by two 8 cycle periods of mechanical stimulation at 70 Hz. The increase in the amplitude of the second group of receptor potentials is caused by preceding electrical stimulation of the lateral line nerve with seven shocks at 200 sec-1 which is indicated by the stimulus artifacts. The trace is a.c. coupled between 30 and 600 Hz. Time bar: 100 msec. B, an i.p.s.p. recorded intracellularly from a mechanically stimulated hair cell. The magnitude of the i.p.s.p. is much greater than the receptor potential. Middle trace is the external double microphonic receptor potential which is augmented during the i.p.s.p. Lower trace is the sinusoidal mechanical stimulus at 70 Hz. The bar beneath the lower trace represents the period of electrical stimulation of the lateral line nerve at 200 sec-1. Vertical bar: 1 mv for upper trace and 0 1 mv for middle. Horizontal bar: 50 msec.

7 EFFERENT INHIBITION OF HAIR CELLS 51 synapse, or else that the mechanism which causes e.p.s.p.s to give rise to action potentials in the myelinated portion of the axon had been damaged by the electrode. When dye was injected very fine strands, which sometimes branched, were seen to travel for a distance in the lower half of the sensory epithelium. The e.p.s.p.s were very similar in appearance to those which have been recorded intracellularly from fibres of the goldfish eighth nerve A as_ I B Text-fig. 4. A, four successive traces of spontaneous e.p.s.p.s recorded intracellularly from a lateral line nerve terminal. Vertical bar: 2 mv. Time bar: 5 msec. B, the time delay between the externally recorded microphonic potential and the intracellularly recorded e.p.s.p.s in an afferent nerve terminal of a lateral line organ which was mechanically stimulated at 100 H8z and at 40 db above the threshold for excitation. Upper trace: externally recorded double microphonic potential. Lower trace: intracellularly recorded e.p.s.p.s. The traces are computer averages of 20 consecutive sweeps. The time delay between the peak of the excitatory phase of the microphonic and the peak of the e.p.s.p. is indicated by d. Time bar: 5 msec. (Furukawa & Ishii, 1967; Ishii, Matsuura & Furukawa; 1971) and their amplitudes were mv. The time to the peak of the potential was msec (mean 1-08 msec) and the time to decay was 1-2 msec (mean 1-69 msec). These times are somewhat longer than those measured from

8 52 AKE FLOCK AND IAN RUSSELL e.p.s.p.s recorded in the goldfish eighth nerve, which are 0-64 and 0-8 msec respectively (Furukawa, Ishii & Matsuura, 1972). When the cupulae of the lateral line organs were sinusoidally displaced the e.p.s.p.s followed only one phase of the stimulus (Text-figs. 4B and 5) and the synaptic delay between the peak of the depolarizing phase of the so ~~~~~~S0~~~~~~~~ 4.1 w_ Stimulus amplitude (db) Text-fig. 5. The relationship between the intensity ofmechanical stimulation and the amplitude of the 2nd, 3rd, 6th and 60th e.p.s.p. in response to prolonged mechanical stimulation of the lateral line organ: 64 cycles at 70 Hz. Each point is the computer average of eighty-five consecutive measurements. Ordinate: the intensity of mechanical stimulation is expressed with reference to an empirically determined threshold of excitation for single nerve fibres in the lateral line organ: 0 db. Abscissa: amplitude in arbitrary units. extracellularly recorded microphonic potential and the peak of the e.p.s.p. was msec at 140 C (Text-fig. 4B). In response to low amplitudes of sinusoidal displacement, the e.p.s.p.s did not follow every excitatory phase, but they followed every excitatory phase when the displacement amplitude was large. Furthermore, the size of e.p.s.p.s decline progressively

9 EFFERENT INHIBITION OF HAIR CELLS 53 with time in response to prolonged periods of mechanical stimulation, e.g. 64 cycles at 70 Hz (Text-fig. 5). The rate of decline is rapid for the first 3-4 e.p.s.p.s, successive e.p.s.p.s declining with a ratio of with respect to the preceding e.p.s.p. However, the decline eventually becomes more gradual in the later e.p.s.p.s. This apparent adaptation in the size of the e.p.s.p.s is not long lasting, and their recovery is full if successive periods of stimulation are presented with an interval of at least 1-5 sec. The progressive decline in the amplitude of the e.p.s.p.s with prolonged mechanical stimulation is a property of the afferent synapse and not of the receptor mechanism of the hair cell because the microphonic potentials do not adapt to prolonged stimulation. The adaptation represents a change in the gain of the afferent synapse which is especially noticeable during large amplitudes of mechanical stimulation. In Text-fig. 5 is shown the relationship between the amplitudes of the e.p.s.p.s and the cupula displacement for the successive 2nd, 3rd, 6th and 60th e.p.s.p.s in response to periods of prolonged sinusoidal displacement of the cupula (64 cycles at 70 Hz). The steepest part of these curves lies between 16 and 28 db above threshold. In this region a 6 db change in the amplitude of the displacement causes a 16 unit change in the response of the 2nd e.p.s.p., but only an 8 unit change in the 60th e.p.s.p. Inhibition of the excitatory podt-8ynaptic potentials Electrical stimulation of the lateral line nerve causes a reduction in the amplitude of the mechanically evoked e.p.s.p.s and suppression of the spontaneous e.p.s.p.s recorded intracellularly from afferent nerve terminals for msec following the stimulus (200 shocks/sec for 80 msec) (Text-fig. 6A, B). The time course and envelope of the inhibition of the mechanically evoked e.p.s.p.s is an almost mirror image of the augmentation of the microphonic potential (Text-fig. 7). The inhibition is due to the post-synaptic action of the efferent fibres on the hair cells, and is not due to the antidromic invasion of the afferent nerve terminal because intramuscular injections of Flaxedil in low concentrations (3 mg/kg body wt.) blocks the inhibition. The time course for this effect is rapid (1-3 min) and is the same as that required to block the augmentation of the microphonic potential and the neuromuscular junction (Flock & Russell, 1973b). Text-fig. 6B and C shows the influence of efferent stimulation on mechanically evoked e.p.s.p.s before and shortly after Flaxedil administration. It will be noticed that the 'reset' period caused by antidromic invasion of the afferent fibres is insignificant compared with the time course of inhibition. In Text-fig. 8 the relationship is shown between the amplitude of displacement and the amplitude of the e.p.s.p.s with, and without inhibition. The e.p.s.p.s and the microphonic potential are changed by

10 54 AKE FLOCK AND IAN RUSSELL B L I' 11t1.ii~~~~~~~~~~~~~1111 'Ii" n l liiaaaiaiaaiaaaaaaaaiaa1m11j1%iieieuiiu- Text-fig. 6. For legend see facing page. I -j

11 - 60 C 0 s ow EFFERENT INHIBITION OF HAIR CELLS I I I E < m I 90 I Time (msec) Text-fig. 7. The time course of the recovery from efferent inhibition in a lateral line organ which was mechanically stimulated for 64 cycle at 70 Hz and 30 db above threshold for excitation. 0-0 plots the amplitude of e.p.s.p.s recorded intracellularly from an afferent nerve terminal. *-* plots the amplitude of the extracellular microphonic potential. Each point is the computer average of eighty-five measurements such as illustrated in the sample trace. The period over which the measurements were made is indicated by the time beneath the sample record. Abscissa: the amplitudes of the computer averaged traces of the microphonics and e.p.s.p. were normalized and expressed as arbitrary units. Ordinate: time in msec. Text-fig. 6. E.p.s.p.s recorded intracellularly from afferent nerve terminals in mechanically stimulated lateral line organs are reduced in amplitude following electrical stimulation of lateral line efferent fibres at 200 sec-1 for 80 msec. A, first three traces: the amplitude of the e.p.s.p.s is reduced following electrical stimulation of the efferent fibres during the horizontal bar. Fourth trace: extracellularly recorded microphonic potential is augmented during the inhibition. Fifth trace: the extracellular hyperpolarizing potential recorded in the absence of mechanical stimulation. Bottom trace: mechanical stimulus 70 Hz and 30 db above threshold for excitation. Horizontal bar indicates efferent stimulation for trace 4 and 5. B, upper trace: efferent inhibition of e.p.s.p.s. Lower trace: control record in which the efferent fibres were not stimulated. C, the same fibre as in B, but the inhibitory effect of the efferent fibres on the e.p.s.p.s has been blocked by an I.M. injection of Flaxedil administered 3 min beforehand. Middle trace: control record without efferent stimulation. Lower trace: mechanical stimulus, 70 Hz and 30 db above threshold. The electrical stimulation of the efferent fibres is indicated by stimulus artifacts and also by the line beneath the middle trace in C. Time bar: 100 msec for all traces. Vertical bar: 4 mv for A; 2 mv for B; and 1 mv for C.

12 56 5A E FLOCK AND IAN RUSSELL similar amounts during the post-synaptic action of the efferent fibres (compare Flock & Russell, 1973b). This is about 14 db when the displacement amplitude of the mechanical stimulus is large (40 db above threshold) but only 3 db when the amplitude of the mechanical stimulus is small (10 db above threshold). so _40 L30 20 E Stimulus amplitude (db) Text-fig. 8. Input-output curve of intracellular e.p.s.p.s recorded from an afferent nerve terminal in a lateral line organ which was mechanically stimulated for 64 cycles at 70 Hz. *-@ plots the amplitudes of the e.p.s.p. illustrated in the sample record. *-U plots the amplitudes of the 3rd e.p.s.p. after the stimulation of the efferent fibres at 200 sec-1 for 80 msec. 0-0 plots the amplitudes of the 34th e.p.s.p. Each point is the mean of eightyfive computer averaged traces. Ordinate: amplitude of e.p.s.p.s in arbitrary units. Abscissa: stimulus intensity with reference to an empirically determined threshold for excitation of the lateral line organ. DISCUSSION The hyperpolarizing potentials which have been recorded from lateral line hair cells during stimulation of the efferent fibres are very similar to inhibitory post-synaptic potentials (i.p.s.p.s.) which have been recorded elsewhere in the central nervous system and periphery (Eccles, 1964). The recording of i.p.s.p.s from hair cells also supports previous ultrastructural

13 EFFERENT INHIBITION OF HAIR CELLS 57 findings (Hama, 1965; Flock, 1965) that efferent fibres in the lateral line system act post-synaptically on hair cells. The extracellularly recorded events which occur during inhibition clearly reflect the intracellularly recorded potential changes. The efferent potential (Flock & Russell, 1973b) has the same time course and shape as the intracellularly recorded i.p.s.p., and the intracellular receptor potential, like the microphonic potential, is augmented during inhibition. The efferent fibres act on the lateral line hair cells through the release of a chemical transmitter which is probably acetylcholine (Russell, 1971b). The release of the transmitter causes a decrease of about 12 % in the resistance of the post-synaptic hair cell membrane (Text-fig. 2). This presumably accompanies a change in the ionic permeability of the subsynaptic hair cell membrane; however, the nature of permeability change has not been investigated. This is due to difficulty in maintaining stable intracellular recordings for any length of time. When the membrane potential fluctuates in a depolarizing direction the amplitude of the i.p.s.p. increases, but it decreases when the membrane potential fluctuates in a hyperpolarizing direction. Thus it appears that the action of the efferent synapse is similar to that of many other inhibitory synapses in that it tends to drive the membrane potential to a level close to the resting potential of the cell (Eccles, 1964). The i.p.s.p. is associated with an augmentation of the intracellularly recorded receptor potential of the hair cell (Text-fig. 3), and with inhibition of impulse activity in the afferent nerve fibre (Flock & Russell, 1973b). These relationships have also been observed in the cochlea (see Fex, 1974; Klinke & Galley, 1974) where they have been described as ambiguous (Davis, 1965). However, they can be explained as follows: at each moment the probability of transmitter being released at the afferent synapse is determined by the potential level in the hair cell, it is increased during depolarization and decreased during hyperpolarization. If we assume that the receptor potential is due to the flow of some ion along an electrochemical gradient, then a hyperpolarization of the cell would lead to an increased receptor potential if the equilibrium potential for that particular ion is more positive than the resting membrane potential of the hair cell. Hyperpolarization of the cell, as during the i.p.s.p., would therefore cause at the same time an increased receptor potential and a decreased firing of afferent nerve fibres. The two effects could also be explained in terms of current flow if it is assumed that during inhibition the efferent synapses provide a low resistance pathway which shunts current away from the afferent synapse (Wiederhold, 1967). The decrease in resistance of the efferent postsynaptic membrane during inhibition would permit more current to flow

14 58 5KE FLOCK AND IAN RUSSELL across the hair cell membrane and result in an increase in the amplitude of the receptor potential. The increased receptor potential would not cause an increase in the release of transmitter at the afferent synapse, because of the shunting action of the efferent synapse. It is interesting to observe that the afferent synapses of outer hair cells in the cochlea are often totally enveloped by efferent nerve terminals (Spoendlin, 1973) which could very efficiently shunt current away from the afferent synapse. The efferent innervation of hair cells in the lateral line has not developed to the same extent as in the cochlea although each lateral line hair cell may receive several efferent synapses. The finding that the receptor potential can be influenced by the action of a neurochemical synapse can be taken as evidence that the receptor potential is truly a biological potential and not a mechanical motion artifact. The widely held view that transmission between the hair cells and afferent nerves is mediated by a chemical transmitter is further supported by the recording of spontaneous and mechanically evoked e.p.s.p.s from the afferent nerve terminals in the lateral line system of Lota. The evoked e.p.s.p.s, like those recorded in the goldfish auditory nerve fibres (Furukawa et al. 1972), are time-locked to a mechanical stimulation of the hair cells and they occur in synchrony with one phase of sinusoidal mechanical stimulation. The presence of a delay between the peak of the microphonic potential and the peak of the accompanying e.p.s.p. is a further argument for neurochemical transmission at this synapse. The synaptic delay described here in Lota (0.9 msec) is longer than that recorded at the afferent synapses in hair cells of the goldfish sacculus (0.5 msec). However, it is probable that this difference is only apparent and may be attributed to the different temperatures at which the two experiments were performed, 14 and 22 C respectively. Attempts were not made to raise the temperature of the lateral line preparation because Lota do not tolerate temperatures as high as 220 C. Flaxedil blocks the i.p.s.p. in hair cells, and it releases the afferent e.p.s.p.s from inhibition and leaves them unaffected. This selective action indicates that the efferent and afferent synapses use different transmitters. The efferent synapse in the lateral line appears to be cholinergic (Russell, 1971 b). The nature of the afferent transmitter has not been elucidated but catecholamines (Osborne & Thornhill, 1972), glutamate (Steinbach & Bennett, 1971) and y-amino butyric acid (Flock & Lam, 1974) have all been suggested as possible candidates. The time courses of e.p.s.p.s recorded from the lateral line afferent nerves of Lota are somewhat longer than those recorded from the auditory nerve fibres of goldfish, but even so they are faster than e.p.s.p.s recorded

15 EFFERENT INHIBITION OF HAIR CELLS elsewhere in the nervous systems of lower vertebrates and mammals (Furukawa et al. 1972). Thus the afferent synapse of lateral line hair cells has the potential to transmit temporal information at high frequencies. In this respect the afferent synapse is preadapted to transmit information at frequencies approaching those transmitted in the mammalian cochlea, even though the frequency response of the lateral line organ itself is limited to a maximum of about 200 cycles by the mechanical properties of the cupula (Kuiper, 1956). The e.p.s.p.s are suppressed for the duration of the i.p.s.p. when the efferent fibres are stimulated. The inhibition of the e.p.s.p.s is most probably due entirely to the post-synaptic action of the efferent fibres on the hair cells and is not brought about by inhibition at some other site, such as on the afferent dendrites. This is because the receptor potentials are augmented when the e.p.s.p.s are suppressed, both with the same time course and by similar amounts, namely about 14 db (during mechanical stimulation 40 db above threshold). Furthermore, the inhibition of the e.p.s.p.s, and the efferent augmentation of the microphonic potentials (Flock & Russell, 1973b) are dependent in a similar way on the level of mechanical stimulation of the lateral line organ. Thus the physiological results tend to support ultrastructural findings that axo-dendritic synapses do not exist in the lateral line canal organs of Lota (Flock, 1965), although they are present on the afferent nerve terminals of inner hair cells in the cochlea (Spoendlin, 1973) and on the afferent nerve chalice of type I hair cells in the vestibular system (Wersdll, 1956). It is clear that there is a progressive decline in the amplitudes of successive e.p.s.p.s during prolonged mechanical stimulation of the lateral line organ which is especially marked during large amplitude stimuli (Text-fig. 4B). Ishii et al. (1971) observed a similar phenomenon in the auditory nerves of goldfish in response to loud or continuous sounds which they attributed to the depletion of a readily available store of afferent transmitter. Whatever the cause of the decline in the e.p.s.p.s, it clearly represents a change in the gain of the hair cells. Moreover, the efferent neurones may prevent the decline in response of the afferent synapses during excessive stimulation. The lateral line efferent neurones are active just before and during swimming movements (Russell, 1971 a; Roberts & Russell, 1972). It has been suggested that by inhibiting the lateral line system during the long and excessive stimulation to which it is subjected during swimming movements (Roberts, 1972) the efferent system preserves the sensitivity of the lateral line receptors so that they will be optimally functional immediately swimming ceases. This suggestion remains to be tested, but inhibition is maximal during particularly violent movements (Russell & 59

16 60 AXKE FLOCK AND IAN RUSSELL Roberts, 1974; Russell, 1974) because the efferent neurones are most active at this time (Roberts & Russell, 1972) and their post-synaptic action is most potent during maximal mechanical stimulation of the lateral line organs (Text-fig. 8). We are grateful to Dr P. J. Benjamin and Dr P. Sellick for their helpful comments on the manuscript. This work has been supported by grants from the Swedish Medical Research Council (04X-2461), the King Gustaf V Memorial Fund and Funds of the Karolinska Institute. I. J. Russell was supported by a European Programme Research Fellowship from the Royal Society. This work was carried out at the King Gustaf V Research Institute, Stockholm. REFERENCES DAVIS, H. (1965). A model for transducer action in the cochlea. Sensory receptors. Cold Spring Harb. Symp. quant. Biol. 30, ECCLES, J. C. (1964). The Physiology of Synapses. Berlin: Springer-Verlag. ENGsTROM, H. & WERSXLL, J. (1958). Structure and innervation of the inner ear sensory epithelia. Intern. Rev. Cytol. 7, FEx, J. (1959). Augmentation of the cochlea microphonics by stimulation of efferent fibres to cochlea. Acta oto-lar. 50, FEx, J. (1962). Auditory activity and centripetal cochlear fibres of the cat. Acta physiol. scand. suppl FEX, J. (1967). Efferent inhibition in the cochlea related to hair cell D.C. activity: study of postsynaptic activity of the crossed olivocochlear fibres in the cat. J. acoust. Soc. Am. 41, FEx, J. (1974). Neural excitatory processes of the inner ear. In Handbook of Sensory Physiology, vol. vi. Auditory system, ed. KEIDEL, W. D. & NEFF, W. D., pp Berlin: Springer-Verlag. FLOCK, A. (1965). Electronmicroscopical and electrophysiological studies on the lateral line canal organ. Acta oto-lar. suppl. 199, FLOCK, A. (1971). The lateral line organ mechanoreceptors. In Fish Physiology, vol. v, ed. HoAR, W. S. & RANDALL, D. J., pp New York: Academic Press. FLOCK, A. & LAM, D. M. K. (1974). Neurotransmitter synthesis in inner ear and lateral line sense organs. Nature, Lond. 249, FLOCK, A. & RUSSELL, I. J. (1973a). Efferent nerve fibres: Postsynaptic action on hair cells. Nature, New Biol. 243, FLOCK, A. & RUSSELL, I. J. (1973 b). The postsynaptic action of efferent fibres in the lateral line organ of the burbot Lota lota. J. Physiol. 235, FLOCK, A., JORGENSEN, M. & RUSSELL, I. (1973). The physiology of individual hair cells and their synapses. In Basic Mechanism in Hearing, ed. MOLLER, A. R., pp New York: Academic Press. FURUKAWA, T. & ISHII, Y. (1967). Neurophysiological studies on hearing in gold fish. J. Neurophysiol. 30, FURuKAWA, T., ISHII, Y. & MATSUURA, S. (1972). Synaptic delay and time course of post-synaptic potentials at the junction between hair-cells and eighth nerve fibres in the gold fish. Jap. J. Physiol. 22, GALAMBOS, R. (1956). Suppression of auditory nerve activity by stimulation of efferent fibres to cochlea. J. Neurophysiol. 19, HAMA, K. (1965). Some observations on the fine structure of the lateral line organ of the Japanese sea eel, Lyncozymba nystromi. J. cell Biol. 24,

17 EFFERENT INHIBITION OF HAIR CELLS 61 H& BuS, G. G., FRISCHKOPF, L. S. & FLOCK, A. (1970). Receptor potentials from hair cells of the lateral line. Science, N.Y. 167, , Y., MATSuuRA, S., FURuKAWA, T. (1971). An input-output relation at the synapse between hair cells and eighth nerve fibres in goldfish. Jap. J. Phy8iol. 21, JIERLOF, R., SPOOR, A. & DE VRIEs, H. (1952). The microphonic activity of the lateral line. J. Phyeiol. 116, KANEKO, A. & HAsHImOTO, H. (1967). Recording site of the single cone response determined by an electrode marking technique. Vi-ion Re8. 7, KI NKE, R. & GALiY, N. (1974). Efferent innervation of vestibular and auditory receptors. Phyeiol. Rev. 54, KNIT E, R. & SCHMIDT, C. L. (1970). Efferent influence on the vestibular organ during active movements of the body. Pflugere Arch. gee. Phyeiol. 318, Kuipem, J. W. (1956). The microphonic effect of the lateral line organ. Thesis, Gronigen. LmNNs, R. & PREcHT, W. (1969). The inhibitory vestibular efferent system and its relation to the cerebellum in the frog. Expi Brain Ree. 9, OSBORNE, M. P. & THORNHILL, R. A. (1972). The effect of monoamine depleting drugs upon the synaptic bars in the Inner Ear of the Bullfrog (Rana cateebeiana). Z. ZeUforech. mikroek. Anat. 127, ROBERTS, B. L. (1972). Activity of lateral-line sense organs in swimming dogfish. J. exp. Biol. 56, ROBERTS, B. L. & RuSSELL, I. J. (1972). The activity of lateral line efferent neurones in stationary and swimming dogfish. J. exp. Biol. 57, RuSSEmL, I. J. (1971a). The role of the lateral line efferent system in Xenopus laevie. J. exp. Biol. 54, RussELL, I. J. (1971 b). The pharmacology of efferent synapses in the lateral line system of Xenopu8 laevi. J. exp. Biol. 54, RUSSELL, I. J. (1974). Central and peripheral inhibition of lateral line input during the startle response in goldfish. Brain Ree. 80, RUSSELL, I. J. & ROBERTS,-B. L. (1974). Active reduction of lateral line sensitivity in swimming dogfish. J. comp. Phyeiol. 94, SALA, 0. (1965). The efferent vestibular system. Acta oto-lar. supply. 197, SPoENDLIN, H. (1973). The innervation of the cochlear receptor. In Baeic Mechaniems in Hearing. ed. MoLLER, A. R., pp New York: Academic Press. STEINBACH, A. B. & BENNEmr, M. V. L. (1971). Effects of divalent ions and drugs on synaptic transmission in phasic electroreceptors in a mormyrid fish. J. gen. Phy8iol. 58, STRETTON, A. 0. W. & KRAvrrz, E. A. (1968). Neuronal geometry: determination with a technique of intracellular dye injection. Science, N.Y. 162, T.sAKI, K., TsuiUHARA, Y., ITO, S., WAYNER, M. J. & YU, W. Y. (1968). A simple direct and rapid method for filling microelectrodes. Phyeiol. & Behav. 3, WERAsLL, J. (1956). Studies on the structure and innervation of the sensory epithelium of the crista ampullares in the guinea pig. A light and electronmicroscopic investigation. Acta oto-lar. suppl. 126, WIEDERHOLD, M. L. (1967). A study of efferent inhibition of auditory nerve activity. Ph.D. Thesis, Massachusetts Institute of Technology.

18 62 AXKE FLOCK AND IAN RUSSELL EXPLANATION OF PLATE A, a cell from which an i.p.s.p. was recorded was marked with dye by electrophoretic injection. In this 3 jum section it is viewed with Nomarski interferencecontrast and is identified as a hair cell. It reaches half-way down the sensory epithelium (se). B, the receptor potential recorded from a cell was augmented by firing the efferent nerve fibres. It was marked and is identified here as a hair cell. cu, cupula. C, efferent nerve endings (NE) innervate a hair cell (HC) at its base. The nerve endings contain an abundance of synaptic vesicles and a subsynaptic sac is present along the post-synaptic membrane.

19 The Journal of Physiology, Vol. 257, No. I Plate 1 L'~ions it. ; rX. 1-1 '03*e..0i A"... " I AKE FLOCK AND IAN RUSSELL (Facing p. 62)