THE PHARMACOLOGY OF EFFERENT SYNAPSES IN THE LATERAL-LINE SYSTEM OF XENOPUS LAEVIS BY I. J. RUSSELL

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1 J. Exp. Biol. (1971) With 1 plate and 13 text-figures Printed in Great Britain THE PHARMACOLOGY OF EFFERENT SYNAPSES IN THE LATERAL-LINE SYSTEM OF XENOPUS LAEVIS BY I. J. RUSSELL Department of Zoology, University of Cambridge (Received 20 October 1970) INTRODUCTION The sensory hair-cells of the acoustico-lateralis system of sense organs have been shown, in the majority of cases, to receive efferent innervation. Studies on the ultrastructure of the sensory epithelium in the inner ear (Wersall, 1956; Engstrom, 1958) gave the first clear demonstration of vesiculated nerve terminals in synaptic contact with the membranes of hair-cells. From their similarity with axon-dendritic synapses in the central nervous system they were thought to be efferent (Engstrom, 1958). Degeneration experiments have confirmed this (Engstrom & Fernandez, 1961). Subsequent ultrastructural studies on the hair-cells of other members of the acousticolateralis system including the lateral-line receptors (Hama, 1962; Flock, 1965, 1967) have also revealed vesiculated nerve terminals in synaptic contact with their membranes. Electrical stimulation of efferent fibres to the cochlea (Galambos, 1956; Fex, 1962) inhibits impulse activity in afferent fibres in the eighth nerve. Recently the inhibitory nature of efferent fibres to the lateral-line organs has also been demonstrated in the clawed toad Xenopus laevis (Russell, 1968, 1970) and in the Japanese sea eel (Katsuki, Haskimoto & Yanagisawa, 1968). In Xenopus, however, stimulation of lateral-line efferent fibres, in the absence of calcium, failed to inhibit afferent impulses. Inhibition was also attenuated or blocked by the presence of D-tubocurarine chloride. These observations have promoted this investigation into the nature of the transmitter released at lateral-line synapses. Its object is to discover what evidence there may be that the transmitter is acetylcholine. Selection of animals METHODS Histochemistry This investigation was carried out on two species of animals: the South African clawed toad, Xenopus laevis, and a freshwater teleost, Acerina cernua. The investigations were not confined to Xenopus for the following reasons: (1) The area of sensory epithelium of individual neuromasts of the lateral-line receptors of Xenopus laevis is small. It occupies an area some 25 ju,m by 10 /tin and containing on average thirty hair-cells. The elongate lateral-line receptors in Xenopus have been called stitches (Harris & Milne, 1966). This term will be used here. (2) Not all lateral-line stitches of Xenopus receive efferent innervation (Gorner, 1968), thus adding a complication to the interpretation of results. Acerina was chosen because of its large lateral-line organs, particularly those in the supra-orbital canal,

2 644 I- J- RUSSELL which have sensory epithelia occupying an area of 1 mm 2. Furthermore, a preliminary neurophysiological investigation of its lateral-line innervation demonstrated tbi presence of considerable efferent nervous activity. Preparation of animals and histological methods Young Xenopus (20-30 g) and Acerina (20 g) were pithed, and appropriate pieces of tissue containing lateral-line organs were removed. In the case of Xenopus these were pieces of skin containing single stitches, and in Acerina the sub-orbital bones containing the sub-orbital canals were removed and cut into short lengths. Each length contained a portion of the canal and a single canal organ. The pieces of tissue were washed for 1 h in isotonic sodium sulphate to displace as much chloride from the tissue as possible, since chloride interferes with the subsequent staining operations (Lewis & Shute, 1961). Frozen sections of the tissue were made by initially freezing the tissue in a mixture of solid CO 2 and methanol at 70 C. Subsequently the tissue was sectioned at 8 fim or 16 /on at 20 C with an International Cryostat. The sections were picked up directly on to slides, thawed in formalin vapour, and fixed in 10% neutral formalin for 1-2 h. They were then washed in distilled water and treated for cholinesterase activity. The cholinesterase method used was the modification of the Koelle & Friedenwald (1949) technique discussed by Lewis & Shute (1961). Serial sections of lateral-line organ were placed singly on consecutive slides numbering 1-5, and one slide was placed into each of five different incubating solutions for periods of 4-6 h. The first incubating solution (Table 1) contained 6 mm acetylthiocholine iodide, 9 mh copper, and 16 mm glycine at a ph of 5-5. The other incubating media (Table 1) contained the same ingredients as the first together with a specific inhibitor. ide Inhibitor Table 1 2 x IO~ 4 M ethapropazine hydrochloride (Lewis, Shute & Silver, 1967) 2 x IO"*M 62C47 (Lewis & Hughes, i960) io~ 4 M neostigmine chloride io~ 4 M versene Cholinesterase visualized Pseudo and acetylcholinesterase Acetylcholinesterase Pseudocholinesterase None None After incubation the slides were removed, washed for 5 min in several changes of distilled water, and placed for 2 min in a solution containing 1 g of sodium sulphide dissolved in 45 ml of N/5 acetic acid at ph 5-5. The sections were removed, rinsed in distilled water, dehydrated and mounted in Canada balsam. Electrophysiology The animals used in these experiments were Xenopus. They were anaesthetized in a o-oi % solution of MS-222 (Tricaine), the brain anterior to the cerebellum was removed and the spinal cord was cut posterior to the second spinal nerve. Method^

3 Efferent synapses in lateral-line system of X. laevis 645 used for recording afferent impulses from single lateral-line receptors and for stimui^ting lateral-line efferent nerve fibres have already been described (Russell, 1970). All pharmacological agents used in these experiments were made up in a saline solution (Russell, 1968) and were pipetted on to the undersides of the receptors and removed by suction. The amount of solution used in each experimental application was 100 ml. The following pharmacological agents were used: acetylcholine chloride, physostigmine salycate, acetyl-/?-methyl choline chloride, propionylcholine chloride, butyrylcholine chloride, carbocholamine chloride, atropine, and D-tubocurarine chloride. In experiments where the calcium concentration of the saline was altered or magnesium was added, the osmotic strength of the saline was maintained constant by appropriate alterations to the sodium chloride concentration. RESULTS Histochemistry Acerina Sections incubated with ethapropazine. Hair-cells and fine fibres of the lateral-line nerve stained intensely (Plate 1, figs. 1,2). Hair-cells in sections incubated for 4 h or more stained diffusely throughout the cell (Plate 1, fig. 1), whereas in sections incubated for only 2 h staining was confined to discrete points within or on the cell surface (Plate 1, fig. 3). No apparent difference existed between sections incubated with and without ethapropazine. Acetylcholinesterase staining was confined to the hair-cells and nerve fibres only, but bone in sections incubated for 3 h or longer stained unspecifically for cholinesterase. This effect was not inhibited by ethapropazine. Sections incubated with 62C47 and neostigmine. Hair-cells and nerve fibres of sections incubated in either 62C47 or neostigmine failed to stain for cholinesterase. The non-specific staining of the bone was, however, not affected by these inhibitors (Plate i, fig. 4). Xenopus A staining reaction for cholinesterase was confined to fine fibres in the trunk and branches of the lateralis vagus at the base of hair-cells in sections incubated in a solution containing ethapropazine (Plate 1, figs. 5, 6). It was observed that staining was absent from nerve fibres in sections incubated in a solution containing 62C47. Pharmacology The action of acetylcholine and related compounds on afferent activity The frequency of spontaneous impulses from afferent lateral-line fibres was unaffected when acetylcholine in concentrations less than IO~ 4 M was pipetted on to the undersides of the stitches which they innervated. But acetylcholine reversibly inhibited afferent impulses if an anticholinesterase, namely physostigmine, was pipetted on to the stitches min before and during the addition of acetylcholine. Total inhibition of afferent impulses by IO~ 8 M acetylcholine in the presence of 5 x IO~ 6 M physostigmine is shown in Text-fig. 1. Physostigmine in the absence of acetylcholine occasionally caused a slight decrease in the frequency of afferent impulses (Text-fig. 1).

4 646 I. J. RUSSELL Text-fig. 2 shows that the same result was obtained when the experiment was repeated with acetyl-/?-methyl choline, which is a cholinergic transmitter substance with a higj affinity for muscarinic receptors. Saline Physostigmine Physostigmine Saline 60 5x1 O-'M/I,5X1 O-*M/I, Acetylcholine j1x10-*m/li Time (min) Text-fig. 1. The influence of acetylcholine on afferent impulses from a single lateral-line stitch. Ordinate: summed impulse frequency from the two afferent fibres innervating a single stitch. Each point is the mean of five readings and the vertical bars represent twice the standard error. Q. Physostigmine Physostigmine 5x10-'M 5X10-*M/ 40 Acetyl-/?-methyl choline! I 5X10-*M/I 30 Saline 20 E SO 60 Time (min) Text-fig. 2. The influence of acetyi-/8-methyl choline on afferent impulses from a single lateralline stitch. Ordinate: summed impulse frequency from the two afferent fibres innervating a single stitch. Each point is the mean of five readings and the vertical bars represent twice the standard error. Butyrylcholine and propionylcholine, which are both cholinergic transmitter substances with affinities for nicotinic receptors, had no apparent influence on afferent impulses when added to lateral-line stitches even in concentrations as high as IO~ 4 M and in the presence of physostigmine (Text-figs. 3, 4). Carbocholamine chloride, a cholinergic compound with a high affinity for nicotinic receptors, which is not inactivated by acetylcholinesterase, was pipetted on to the undersides of lateral-line stitches. At a concentration of 5 x IO~ 4 M it caused a reversibla

5 Efferent synapses in lateral-line system of X. laevis ' 30 inhibition of spontaneous afferent impulses, but at 5 x IO~ 5 M this inhibition was less marked (Text-fig. 5). The action of acetylcholine on the afferent impulses was characterized still further by observing the influence of the eholinergic blocking drugs atropine and D-tuboa. B Time (min) Time (min) Fig. 3 Fig. 4 Text-fig. 3. The influence of butyrylcholine on afferent impulse activity in a single lateral-line stitch. Ordinate: summed impulse frequency from the two afferent fibres innervating a single stitch. Each point is the mean of five readings and the vertical bars represent twice the standard error. Text-fig. 4. The influence of propionylcholine on afferent impulse activity in a single lateralline stitch. Ordinate: summed impulse frequency from the two afferent fibres innervating a single stitch. Each point is the mean offivereadings and the vertical bars represent twice the standard error. Ringer Carbachol Saline 40 Time (min) Text-fig. 5. The influence of carbachol on afferent impulse activity in a single lateral-line stitch. Ordinate: summed impulse frequency from the two afferent fibres innervating a single stitch. 0, 5 x IO~ 4 M carbachol; O, S x IO~ 6 M carbachol. Each point is the mean of five readings and the vertical bars represent twice the standard error. 41 EXB54.

6 648 I. J. RUSSELL curarine on the pharmacologically induced inhibition. Atropine, which specifically blocks the action of acetylcholine at muscarinic synapses, blocked the action of acetyl-. choline on afferent impulses when it was applied to the lateral-line stitches mill beforehand in concentrations of 5 x IO~ 6 M (Text-fig. 6). D-Tubocarine chloride, which tends to block the action of acetylcholine more easily at nicotinic than at muscarinic synapses, blocked the action of acetylcholine on afferent impulses in concentrations exceeding 5 x IO-^M (Text-fig. 7). 60 Q. E 50 ; - 5 v S 40 h 3 cr I" ''5' xt in 2 2 x E <u 00 c Physosti Atropi X X 0 to S i 1 ^ Physostig Acetylch< V > V c i 1 i 1 i i Time (min) Text-fig. 6. The influence of atropine on acetylcholine-induced inhibition of impulse activity from a lateral-line stitch. Ordinate: summed impulse frequency from the two afferent fibres innervating a single stitch. Each point is the mean of five readings and the vertical bars represent twice the standard error. Q. E Q. 40 ( 30VI 20 10»t Physost I5x10 M/l -TC5x Q V T Physo!itigmine - i o 5x1 \ ine Acety Ichol o 5x1 Saline 10 1 I i l l Time (min) Text-fig. 7. The influence of D-tubocurarine chloride on acetylcholine-induced inhibition of impulse activity from a lateral-line stitch. Ordinate: summed impulse frequency from the two afferent fibres innervating a single stitch. Each point is the mean of five readings and the vertical bars represent twice the standard error. Impulse traffic in the lateral-line afferent fibres took some 7-10 min to respond to the action of the cholinergic drugs. This time delay was interpreted as a reflection of the relative inaccessibility of the lateral-line stitches to the drugs. The response time was noticeably shorter in young animals with thin skins.

7 Efferent synapses in lateral-line system of X. laevis 649 The influence of physostigmine on inhibition In order to examine the action of physostigmine on the inhibition of spontaneous activity of single lateral-line afferent fibres caused by the electrical stimulation of efferent lateral-line nerve fibres, it was decided to measure a parameter which would indicate the duration of the inhibitory effect. Physostigmine (an anti-cholinesterase) might be expected to increase the length of inhibition if the transmitter released from the efferent terminal was acetylcholine. The parameter chosen was the period d shown in Text-fig. 8, which is the time between the last of the efferent compound action potentials producing inhibition and the first of the spontaneous afferent impulses to reappear after the cessation of efferent inhibition. Saline Saline Time (mln) Text-fig. 8. The influence of physostigmine on efferent inhibition. Each point is the mean of ten observations on the same stitch and the vertical bars are equal to twice the standard error. Spontaneous afferent activity in nerve fibres from individual stitches was inhibited by trains of efferent impulses, each i s long, separated by 3 s, and containing impulses at a frequency of 120 s -1. A succession of ten consecutive trains of efferent impulses was delivered to each stitch once every 2 min. The period d was measured after each period of inhibition. The addition of physostigmine to lateral-line stitches in concentrations above 1 x 1 o~ 5 M increased the period d by factors up to 80%. For example, in the experiment 41-2

8 650 I. J. RUSSELL illustrated in Text-fig. 8 the period d increased from 45 ms in saline solution to above 70 ms when physostigmine in concentrations of 5 x io~ 5 M was added. Throughout each experiment the resting frequency of afferent impulses remained constant and apparently unaltered by the addition of physostigmine. Atropine and the inhibitory action of lateral-line efferent fibres Spontaneous afferent impulses from single stitches were inhibited by trains of efferent impulses having the same parameters described in the previous section. To assess the influence of atropine on efferent inhibition, the numbers of afferent impulses returning during each period of inhibition were counted before, during and after the addition of atropine in the saline solution bathing the underside of each Saline Atropine 5x10-5 M/l Saline SO 60 Time (min) 70 Text-fig. 9. The influence of atropine on efferent inhibition of impulses in an afferent fibre to a stitch. O. Resting frequency of the afferent fibre. #, Frequency of afferent impulses returning during the inhibitory period (mean of ten readings). Ordinate: frequency of impulses in the afferent fibre expressed as impulses per second. The vertical bars are equal to twice the standard error. stitch. Again a succession of ten consecutive trains of efferent impulses was delivered to each stitch once every 2 min for the duration of the experiment. The application of atropine in concentrations between 5 x IO~ 6 M and 5 x io~ 5 M to the underside of the stitch caused an increase in the number of afferent impulses to appear during the inhibitory period, until eventually stimulation of the efferent lateral-line nerve fibres ceased to exert an inhibitory influence on the spontaneous afferent activity (Textfig. 9). It was noticed that the block exerted by atropine was usually reversible, although recovery was slow and rarely complete (Text-fig. 9). Atropine also caused a slight increase in the frequency of spontaneous afferent activity. Calcium - magnesium interaction Calcium is an important co-factor in the release by nerve impulses of acetylcholine at the amphibian neuromuscular junction (del Castillo & Stark, 1952). Furthermore,

9 Efferent synapses in lateral-line system of X. laevis 651 there is competitive interaction between calcium and magnesium for some carrier jnolecule X (Katz & Miledi, 1964): Ca + X-^CaX; Mg+X->MgX. (1) Dodge & Rahamimoff (1967) have devised a relationship for the interaction between calcium and magnesium in regulating the quantal content (m) of the excitatory postsynaptic potentials (e.p.s.p.) at the amphibian neuromuscular junction. When the concentrations of calcium and magnesium ions at the neuromuscular junction are (Ca^ and (Mg)i, respectively, the quantal content (m) of the e.p.s.p. is given by = K 1 + (CaWMgJJ (2) where K = constant, W = proportionality constant. m 1 can still be achieved by substituting different, but appropriate, values for (Ca^ and (Mg)!, namely (Ca) 2 and (Mg) 2 : ' t, (Ca) 2 (Mg), (3) 1s Text-fig. 10. Spontaneous afferent impulses from a stitch are not inhibited by a burst of efferent impulses when the calcium concentration of the saline bathing the stitch is reduced to o-g mm. (a) Saline solution containing i-8mm-cacl 2. (b) Saline solution containing 0-9 mm-cacl a. Upper trace afferent impulses (a) and efferent impulses (e). Lower trace stimulus of 70 s" x delivered to efferent fibres. If the concentration of calcium is changed from (Ca) x to (Ca) 2, then the concentration of magnesium (Mg) 2 which will maintain unchanged the quantal content of the released acetylcholine is derived from equations (2) and (3): ill (4) K 2 has been calculated for the amphibian neuromuscular junction as 3-o + o-ymm (Dodge & Rahamimoff, 1967). As a starting-point this figure has been used in the present study. It was found that afferent impulses from lateral-line stitches were inhibited when the efferent fibres were electrically stimulated providing the stitches were bathed in a saline containing no magnesium but i-8mm calcium (Text-fig. 10a). Inhibition failed to occur when saline solutions containing o^mm were used (Text-fig. 10b).

10 652 I. J. RUSSELL These conditions were used in equation (4) to plot concentrations of magnesium and calcium in which stimulation of efferent fibres may be predicted to inhibit and nofc inhibit respectively (Text-fig. u). The two curves were tested at points a, b, c, d, an* a i> b lt c lf d 1 and were found to be in agreement with the prediction. In comparison with the efferent synapses, the afferent synapses of lateral-line haircells in Xenopus are insensitive to the presence of calcium or magnesium. In the experiments described above, spontaneous afferent impulses continued under conditions in which electrical stimulation of efferent fibres failed to inhibit afferent impulses (Text-fig. 11). Furthermore, under these conditions, the stitches were mechanically sensitive. 10 r 1s iiiiwinmilliiii' 1s Concentration of magnesium (mm) 12 Text-fig. 11. A plot of the ratios of magnesium and calcium calculated from equation (4). The upper line gives values of calcium and magnesium concentrations which, if used in a saline solution to bathe a lateral-line stitch, are predicted to cause afferent impulses to be inhibited when the efferentfibresare electrically stimulated. The lower line gives values of calcium and magnesium concentrations which are predicted not to permit such inhibition to occur. Points a, b, c, d, a lt b u c lt andrf,are concentrations of magnesium and calcium at which the predictions were tested. The observations which were made, as illustrated by traces (b), (d) and (c.) are in agreement with the predictions. ' Impulses were recorded from afferent fibres to a stitch, and efferent fibres to the same stitch were electrically stimulated with trains of pulses in the usual way. The trains were delivered once every 2 s and caused inhibition. The stitch was then bathed in a saline solution containing no calcium or magnesium but 1 mm EDTA. Inhibition was soon reversibly blocked (Text-fig. 12) and the frequency of spontaneous afferent impulses was increased. Eventually the afferent nerve fibre itself began to produce bursts of impulses spontaneously at frequencies up to 600 Hz; but between bursts, spontaneous impulses of synaptic origin still occurred. Furthermore, the fibres remained sensitive to mechanical stimulation of the hair-cells. Both high calcium (10 HIM) or high magnesium (iomm) concentrations (Text-fig. 13) reduced spontaneous afferent activity, but they did not abolish the responses of the afferent fibres to mechanical stimulation of the hair-cells.

11 Efferent synapses in lateral-line system of X. laevis 653 Saline Calcium-free saline-)-1 mm EDTA Saline 90 r Time (min) Text-fig. 12. The influence of calcium-free saline containing i mm EDTA on spontaneous afferent activity and efferent inhibition. Ordinate: frequency of spontaneous afferent impulses from a single fibre per second. Lower bar represents the period for which efferent inhibition was blocked. Upper bar is the period during which the afferent fibre showed spontaneous bursts of impulses at frequencies up to 600 Hz. (a) 1 ill 1 *JL-J«IIII 1 II I (b) Text-fig. 13. Prolonged exposure to saline solutions containing high CaCl 2 concentrations causes the disappearance of spontaneous afferent impulses from a lateral-line stitch. Upper trace: impulses from a single afferent fibre (a) inhibited by a train of efferent impulses (e); (J) stimulus artifact. Lower trace: train of stimulating pulses delivered to efferent fibres, (a) Recorded 10 min after the stitch had been exposed to a saline solution containing 10 mm-cacl 2. (6) Recorded from same stitch after 30 min exposure to the saline solution containing 10 mm- CaCl a. 1s DISCUSSION Several criteria must be satisfied before a substance can be accepted as a synaptic transmitter. These criteria have been extensively reviewed (Paton, 1958; Curtis, 1961; McLennan, 1963). It is essential that the substance presumed to be the transmitter is released on nerve stimulation and that its pharmacology and its post-synaptic action are the same as those of the natural transmitter. Other criteria include evidence for the presence at the synapse of enzymes capable of the synthesis and inactivation of the presumed transmitter.

12 I n identifying the transmitter released at the lateral-line efferent synapse several important criteria remain to be tested, but features of the lateral-line efferent synaps which have so far been observed are characteristic of cholinergic synapses. Acetylcholinesterase has been located at the base of hair-cells and in the fine nerve fibres of the lateral-line nerve. Although the presence of acetylcholinesterase is a requisite of cholinergic synapses, its presence at a synapse is not irrefutable evidence that the synapse is cholinergic (Lewis, Shute & Silver, 1967). Acetylcholinesterase has, however, been located on the pre-synaptic membrane of efferent synapses on cochlear hair-cells (Hilding & Werdll, 1962) where acetylcholine is the presumed transmitter of the olivo-cochlear bundle (Fex, 1968). Localization of acetylcholinesterase in the cochlea was achieved using electron-microscope techniques. This degree of localization was not achieved with the light-microscopical techniques used here, but the localization of acetylcholinesterase in fine nerve fibres is in'agreement with the known physiology, namely that efferent fibres conduct more slowly than lateral-line afferent fibres. T h e inhibition of spontaneous afferent impulses by the addition of low concentrations of acetylcholine to the lateral-line receptors may be due to one of three causes. T h e excitability of the hair-cell membrane may be reduced, resulting in a drop in spontaneous afferent activity. T h e threshold for impulse generation at the afferent nerve terminal may be increased. Alternatively acetylcholine may be acting on specific receptor sites at the efferent synapse. High concentrations of acetylcholine are needed before any influence can be exerted on membranes without receptor sites specific for acetylcholine. Acetylcholine and carbachol in concentrations of 3 x 10" to 3 x I O - ~ g ml-1 slightly decrease the amplitude of compound action potentials from mammalian myelinated nerve fibres (Armett & Ritchie, 1961). Complete inhibition of spontaneous afferent impulses is achieved in lateral-line nerves by bathing the undersides of the receptors in solutions containing I O - ~ M (2 x 10-7 g ml-l) acetylcholine or 10-OM acetyl-b-methyl choline. These concentrations are about three orders of magnitude less than those needed by Armett and Ritchie to produce a very small effect in conduction in unmyelinated nerve fibre. I t is therefore proposed that acetylcholine is acting at specific sites on the hair-cell membrane. Furthermore, the specific sites have a higher affinity for muscarinic substances such as acetyl-,&methyl choline than for nicotinic substances such as butyrylcholine or carbachol. The specificity of action of acetylcholine is reflected in the actions of D-tubocurarine chloride and atropine on the receptor. T h e inhibition of spontaneous afferent impulses, caused either by tetanic electrical stimulation of efferent fibres or by bathing the receptor in low concentrations of acetylcholine, is blocked by lower concentrations of atropine (5 x ~ )than of D-tubocurarine (5 x ~ ).Thus the actions of atropine and curare on the synaptic inhibition and on inhibition by applied acetylcholine are similar. T h e argument may again be put forward that atropine and D-tubocurarine chloride reduce the excitability of the efferent nerve terminal or, in the experiments where acetylcholine was applied to receptors, that they interact non-specifically with the acetylcholine. Atropine in concentrations of I O - ~ g ml-l, but not I O - ~ g ml-l reduced the effect of acetylcholine on the compound action potentials elicited from mammalian unmyelinated fibres (Armett & Ritchie, 1961), whereas D-tubocurarine even in concentrations of 10-2 g ml-1 had no influence at all. It is unlikely that in the concentrations used, atropine and acetvl- 1

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14 656 I. J. RUSSELL It may be that the calcium-free and high-magnesium salines did not reach the afferent synapses. This is most unlikely because the closely adjacent efferent synapses wera blocked by the same salines (Text-fig. 12). Thus it appears that the afferent trans' mitter is released under conditions unfavourable for the release of transmitter at the lateral-line efferent synapse or neuromuscular junction. The small size of the hair-cells in lateral-line receptors of Xenopus and the small numbers of the efferent synapses have prevented the examination of two important questions. These are: (1) Is acetylcholine released by efferent nerve stimulation? (2) Are the post-synaptic events which accompany electrical stimulation of the efferent fibres and application of acetylcholine to the hair-cell the same? In order to examine these two questions animals must be selected which have large numbers of efferent synapses and hair-cells capable of penetration by micro-electrode. Several characteristics of the efferent synapses of the related cochlear hair-cells have raised the problem of whether one or more transmitter substances are involved. Fex (1968) has evidence that the synapses are cholinergic, but earlier work (Fex, 1962; Desmedt and Monaco, 1962) shows that strychnine and brucine, but not a related drug picrotoxin, block synaptic inhibition of the olivo-cochlear fibres. A theory to reconcile these two findings has been presented (McKinstry & Koelle, 1967), namely that more than one transmitter is released pre-synaptically. The influence of strychnine and related drugs on lateral-line efferent synapses has yet to be described. SUMMARY 1. The nature of the transmitter substance released at the lateral-line efferent synapses was investigated by histochemical techniques in Xenopus and Acerina and by pharmacological methods in Xenopus. 2. The bases of lateral-line hair-cells and fine fibres in the lateral-line nerves reacted positively for acetylcholinesterase in Acerina and to a lesser extent in Xenopus. 3. Acetylcholine (io~ 6 M, and acetyl-/ff-methyl choline (5 x IO~ 6 M), which has a muscarinic action, caused strong reversible inhibition of afferent impulses when pipetted on to the undersides of lateral-line stitches. Carbachol (5 x io~ 4 to 5 x IO~ 5 M) caused a smaller reversible inhibition of spontaneous afferent impulses, but other nicotinic substances (propionylcholine and buterylcholine) had no effect. 4. Physostigmine (5 x IO~ 5 M) prolonged inhibition of afferent impulses by electrical stimulation of efferent fibres, but atropine (5 x IO~ 6 M) blocked it. 5. Calcium and magnesium interact at the efferent synapses in a way similar to that found at the amphibian neuromuscular junction. 6. Arguments are put forward to support the hypothesis that acetylcholine is released at lateral-line efferent synapses. I am most grateful to Dr H. W. Lissmann for his encouragement and interest in this work and to Dr S. H. P. Maddrell and Dr R. W. Meech for advice and comments on the manuscript. This work was supported by a Research Studentship from the Science Research Council, and a Research Studentship from Trinity Hall, Cambridge.

15 Efferent synapses in lateral-line system of X. laevis 657 REFERENCES ft.rmett, C. J. & RITCHIE, J. M. (1961). The action of acetylcholine and some related substances on conduction in mammalian non-myelinated nerve fibres. J. Physiol., Lond. 155, BERANEK, R. & VYSKOCIL, F. (1968). The effect of atropine on the frog sartorius neuromuscular junction. J. Physiol., Lond. 195, CURTIS, D. R. (1961). The identification of mammalian inhibitory transmitters. In Nervous Inhibition (ed. E. Florey), pp Oxford: Pergamon Press. DEL CASTILLO, J. & STARK, L. (1952). The effect of calcium ions on the motor end plate potential. J. Physiol. 116, DEL CASTILLO, J. & ENGBACK, L. (1954). The nature of the neuromuscular block produced by magnesium. J. Physiol., Lond. 124, DEL CASTILLO, J. & KATZ, B. (1954). The effect of magnesium on the activity of motor nerve endings. J. Physiol., Lond. 124, DESMEDT, J. E. & MONACO, P. (1962). The pharmacology of centrifugal inhibitory pathways in the cat acoustico lateralis system. Proc. 1st Int. pharmac. Meet. 8, DODGE, F. A. & RAHAMIMOFF, R. (1967). Co-operative action of calcium ions in transmitter release at the neuromuscular junction. J'. Physiol., Lond. 193, ENGSTROM, H. (1958). On the double innervation of the sensory epithelia of the inner ear. Ada oto-lar. 49. i 9- ENGSTROM, H. & FERNANDEZ, C. (1961). Discussion to C. Smith. Trans. Am. otol. Soc. 49, 58. FEX, J. (1962). Auditory activity in centrifugal and centripetal cochlear fibres in the cat. Ada physiol. Scand. 55, suppl FEX, J. (1968). Efferent inhibition in the cochlea by the olivo-cochlear bundle. Ciba Foundation Symposium on Hearing Mechanisms in Vertebrates (ed. A. V. A. de Reuk and Julie Knight), pp FLOCK, A. (1965). Electronmicroscopic and electro-physiological studies on the lateral-line canal organ. Ada oto-lar. suppl FLOCK, A. (1967). Infrastructure and function in the lateral-line organs. In Lateral-Line Detectors (ed. P. H. Cahn), pp Indiana University Press. FURUKAWA, T. & ISHII, Y. (1967). Neurophysiological studies on hearing in goldfish. J. Neurophysiol. 30, GALAMBOS, R. (1956). Suppression of auditory nerve activity by stimulation of efferent fibres to the cochlea. J. Neurophysiol. 19, G6RNER, P. (1968). Personal Communication. HAMA, K. (1962). Fine structure of the lateral-line organ of the Japanese sea eel. In Electron Microscopy, vol. 11 (ed. S. Breese). New York: Academic Press. HARRIS, G. G. & MILNE, D. C. Input-output characteristics of the lateral-line sense organs of Xenopus laevis. J. Acoust. Soc. Am. 40, HILDING, D. & WERSALL, J. (1962). Cholinesterase and its relation to the nerve endings in the inner ear. Ada oto-lar. 55, JENKINSON, D. H. (1958). The nature of antagonism between calcium and magnesium ions at the neuromuscular junction. J. Physiol., Lond. 138, KATSUKI, Y., HASHIMOTO, T. & YANAGISAWA, K. (1968). Information processing in fish lateral-line sense organs. Science, N. Y. 160, 439. KATZ, B. & MILEDI, R. (1964). The effect of calcium on acetylcholine release from motor nerve terminals. Proc. Roy. Soc. Lond. B 161, KOELLE, G. B. & FRIEDENWALD, J. S. (1949). A histochemical method for localising cholinesterase activity. Proc. Soc. exp. Biol. N.Y. 70, LEWIS, P. R. & SHUTE, C. C. D. (1961). The use of cholinesterase techniques combines with operative procedures to follow nervous pathways in the brain. Bibliotheca anat. 2, LEWIS, P. R., SHUTE, C. C. D. & SILVER, A. (1967). Comfirmation from cholinacetylase analysis of massive cholinergic innervation of the rat hippocampus. J. Physiol., Lond. 191, MCLENNAN, H. (1963). Synoptic Transmission. Philadelphia: W. B. Saunders Co. MCKINSTRY, D. N. & KOELLE, G. B. (1967). Inhibition of release of acetylcholine by strychnine and its implications regarding transmission by the olivo-cochlear bundle. Nature, Lond. 213, PATON, W. P. M. (1958). Central and synaptic transmission in the nervous system (pharmacological aspects). A. Rev. Physiol. 20, RUSSELL, I. J. (1968). Influence of efferent fibres on a receptor. Nature, Lond. 219, RUSSELL, I. J. (1971). The role of the lateral-line efferent system in Xenopus laevis. Jf. exp. Biol. 54, WERSALL, J. (1956). Studies on the structure and innervation of the sensory epithelium of the cristae ampullares in the guinea pig. Ada oto-lar. (suppl.), 126.

16 658 I. J. RUSSELL EXPLANATION OF PLATE The histochemical localization of acetylcholinesterase. Fig. 1. Hair-cells (h) of Acerina staining intensely for acetylcholinesterase (AChE). Fig. 2. Nerve fibres (n) of Acerina at base of hair cells staining for cholinesterase. Blood vessel (bv). Fig. 3. Hair-cells (h) showing a granular staining for AChE after 2 h incubation in acetyl thiocholine. Fig. 4. Hair-cells (h) incubated for 4 h in acetyl thiocholine after pre-treatment with 62C47. Bone (b) remains stained. Fig. 5. Nerve trunk of Xenopus showing small-diameter nerve fibres («) staining for AChE. Fig. 6. Neuromast of Xenopus showing localization of AChE (arrows) at base of hair-cells (A). All sections cut at 16 /an and incubated with ethapropazine. Photographed under phase-contrast. The bars = 40 fan.

17 :.. '. '» < Journal of Experimental Biology, Vol. 54, No. 3 Plate 1 *.t. * - " ' ',» 2 * ; 4 & \rvr^ 6 \ I.J. RUSSELL (Facing p. 658)

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