AN ACRIDID AUDITORY INTERNEURONE

Transcription

1 J. Exp. Biol. (1969), 51, With 9 text-figures Printed in Great Britain AN ACRIDID AUDITORY INTERNEURONE I. FUNCTIONAL CONNEXIONS AND RESPONSE TO SINGLE SOUNDS BY C. H. FRASER ROWELL* AND J. M. McKAYf Zoology Department, Makerere College, Kampala, Uganda, University of East Africa {Received 3 January 1969) INTRODUCTION An abdominal tympanum and associated array of more than 8 sensilla (Grey, i96) is present in many of the Acridoidea, and many, though not all, of the species with such an ear also stridulate (see, for example, Dirsch, 1966). The complexity of the ear, and its occurrence in groups which do not stridulate, suggests that it is a generalpurpose hearing organ which can be expected to provide a variety of environmental information to the C.N.S.: in this it resembles the other orthopteran ears and contrasts with the lepidopteran ears of the noctuid type (Roeder & Treat, 1957) which seems to be a simple mechanism serving only very specialized functions. Grey (i96), Popov (1965), Michelsen (1966) and Murray (1968) have recently investigated the anatomy and physiology of the acridid ear and have demonstrated heterogeneity among the sensilla. There have been a number of accounts of recordings from the tympanic nerves and the central nervous connectives, but relatively few individual auditory neurones have been individually characterized (see Table 4; Discussion). The tympanic nerve enters the metathoracic ganglion and at least some primary sensory axons run to the mesothoracic ganglion (Yanagisawa, Hashimoto & Katsuki, 1967). A majority of authors describe the first undoubted auditory interneurones as arising in the mesothoracic ganglion, though there are exceptions (see Table 4); whether or not these mesothoracic neurones are supplied by primary receptor axons, or by small interneurones synapsing with the receptor axons in the metathoracic ganglion, or by both these inputs, is not yet clear. There are a considerable number of auditory interneurones in the pro-mesothoracic connectives (Horridge, 1961), differing in size of recorded impulse and in response characteristics. At least some of them receive input from both ipsilateral and contralateral tympanic nerves. In this paper we give a more detailed description of one of these ascending auditory interneurones. This particular unit was selected because it was easily activated by white-noise clicks made with the fingernails, and because its action potentials record as large and unambiguous signals from the intact connectives. We will refer to this unit as the alpha neurone (alpha, and not a, to avoid confusion with the noctuid ear terminology). This first paper deals with the connexions and response of this unit to the parameters of a single stimulus; we also present some data on another auditory interneurone (the beta neurone), which has properties which contrast interestingly Present addresses: *Zoology Department, University of California, Berkeley, California, U.S.A. t Anatomy School, Cambridge University, England.

2 232 C. H. FRASER ROWELL AND J. M. MCKAY with those of the alpha neurone. A second paper (Rowell & McKay, 1969) concerns the response of the alpha unit to repeated stimuli, including its habituation characteristics and its long-term variation in response level, and also concerns the central factors influencing its response. MATERIALS AND METHODS The insect used was the locally common Gastrimargus africanus Saussure, a grasshopper closely related and very similar to the more familiar Locusta migratoria. Adult males and females were obtained from laboratory culture. The legs were removed and the nervous system was exposed ventrally. Silver wire hook electrodes were used, and, as indifferent electrode, a stainless steel pin in the head. The sound stimuli used were white-noise clicks and pure sine-wave tones generated by an oscillator via an envelope-shaping circuit and an ionic high-frequency loudspeaker. The upper and lower limits of this circuitry were about 2 and 45 kcyc./sec. The shaping circuit ensured a tapering start and finish to sine-wave signals, devoid of transients (Figs. 1, 5, 7). When sound was directed at alternatively the contralateral and ipsilateral ear relative to the recording electrode, the animal was rotated relative to the loudspeaker, and not vice versa. This maintains the same pattern of echoes from the environment, a pattern which is often important with high-frequency sound. Stimuli were monitored by a calibrated Bruel and Kjaer in. microphone placed in the position of the insect ear, connected either to an oscilloscope or to a Bruel and Kjaer sound-level meter Type 223 with octave filter set Type The amplified nervous activity, microphone output and a sweep-triggering signal were recorded on a multi-channel AM/FM magnetic tape recorder and subsequently analysed by photography or long-persistence oscilloscope display, or with a stimulusgated electronic counter. Sound intensities are given in text in db relative to a reference intensity of -2 dynes/cm 2. The results here presented are based on an analysis of experiments on 67 different animals. RESULTS No differences between the sexes of the grasshoppers were noticed in any of our experiments. A. Anatomy of the alpha neurone and its input connexions An appropriate auditory stimulus produces activity in the tympanic nerve which is recorded with an external electrode as a compound potential (Fig. ia). Activity is also recorded c. 5 msec, later in the meta-mesothoracic connective (Fig. ib). This may be the same compound potential seen in the tympanic nerve modified by the passive electrical properties of the connective, or it may include small spikes of interneuronal origin, as implied in the accounts of Horridge (1961), Suga (1963) and Popov (1965); the matter was not investigated further. No element of this signal has a fixed time-relationship with the alpha neurone. The alpha unit is first recorded in the meso-prothoracic connective, and is delayed some 1 msec, relative to the activity recorded from the meta-mesothoracic connective. It transmits 1:1 through the prothoracic ganglion with a delay of less than 2 msec. (Fig. id) and similarly through the suboesophageal ganglion, entering the brain. There is one of these

3 Acridid auditory interneurone 233 urn 1 Fig. 1. Records of the response of the alpha neurone and antecedent connexions to a pulse of 3 kcyc./sec. sound. The lower trace shows the response of the alpha neurone, recorded in the pro-mesothoracic connective, in all the photographs. A. Tympanic nerve and alpha neurone. B. Meso-metathoracic connective and alpha neurone. C. Lack of activity in the abdominal-metathoracic connective during alpha neurone response. D. Alpha neurone in both neck connective and in pro-mesothoracic connective. E. Stimulus. Duration of pulse, 3 msec, amplitude + 8 db. The different records derive from different animals, with varying responsiveness to a standard sound stimulus.

4 234 1 H. FRASER ROWELL AND J. M. MCKAY Prothoracic ganglion Sound- Ipsi lateral ear " Contralateral ear / Mesothoracic Metathoracic ganglion Ipsilateral ear -Sound Brain L Suboesophageal I ganglion Prothoracic ganglion Mesothoracic Metathoracic Ipsilateral ear Contralateral ear Fig 2 ^Tympanic' nerves Fig. 3 Fig. 2. Diagram showing the sequence of lesions, and stimulating and recording arrangements used in experiments to test input connexions and directionality. Recording electrodes are placed on the pro-mesothoracic connectives; sound is presented from alternately right and left-hand sides, before and after cutting one of the tympanal nerves. The neck connectives and the abdominal-metathoracic connectives are cut. Fig. 3. Diagram showing the apparent anatomy and input connexions to the alpha neurone. There is such a unit in each of the paired connectives, and only one is shown in this diagram. The connexion between the primary sensory axons of the tympanic nerve and the alpha neurone may or may not involve a synapse in the metathoracic ganglion; this is symbolized by a query mark. The contralateral input to the alpha neurone is less effective than the ipsilateral; this is symbolized by the broken line and smaller + symbol on the contralateral side. The position of the alpha neurone cell body is not known.

5 Acridid auditory interneurone 235 units on each side of the animal, and except in so far as they share inputs (see below) they appear to be independent of each other. In contrast to Horridge (1961), we found no activity in response to sound in the metathoracic-abdominal connective (Fig. ic). Presumably the units he described in Locusta are not activated by the same stimuli as the alpha neurone. Lesion Sound Animal J68 J69 J7 Table 1. Response of ipsilateral and contrdialeral alpha neurones to unilateral sound, before and after cutting one or both tympanic nerves J68 J69 J7 (Figures are the mean number of action potentials per response, derived from a train of 1 pulses at i/sec.) None both TN intact Ipsilateral 27 4"i Contralateral Ipsilateral Contralateral A. Right-hand units Taking the intact ipsilateral response as 1 %, the above: responses as mean percentages are as follows: A. Right-hand units Nil B. Left-hand units Nil Lesion Ipsilateral (a) Both tympanic nerves intact ioo (b) Contralateral tympanic nerve only cut 43 to Ipsilateral tympanic nerve only cut 14 (d) Ratio of (&) + (<:) to (a) 57 (e) Both tvmoanic nerves cut: Nil B Right-hand TN cut o-i o. Left-hand units o Summarizing: Both TNs cut Ipsilateral Response of alpha neurone to isound (%) Contralateral Nil Contralateral The contribution of each tympanic nerve to one alpha unit can be ascertained by the experiment summarized in Fig. 2, in which recordings are made of the activity of both units to both ipsilateral and contralateral sound, first with both ears connected, then with only one, and lastly with none. The results are summarized in Table 1, and partly illustrated in Figs. 9, 1. Cutting the tympanic nerve of the ear ipsilateral to the sound reduces on the average to 14% the response in the alpha neurone on the same side as the lesion, and reduces less markedly but considerably (to 23 %) the response in the alpha neurone on the opposite side. Cutting the contralateral tympanic nerve approximately halves (43 %) the response of the alpha neurone ipsilateral to the sound, and reduces still more (to 2%) the contralateral response. These results show that each unit receives excitatory inputs from both ears, the Nil Nil

6 236 C. H. FRASER ROWELL AND J. M. MCKAY ipsilateral ear contributing more, and that there is no inhibitory connexion between the ipsilateral and contralateral systems. In this it contrasts with the "I" fibre of the tettigoniids Gampsocleis (Suga & Katsuki, 1961) and Homorocoryphus (McKay, 1968), and with the beta neurone of Gastrimargus ( C, below). Table 1 also shows that the combined excitatory input of the two tympanic nerves to the alpha neurone is greater than would be expected on the basis of the effect of each summed, suggesting that spatial summation acts in a nonlinear fashion at the synapse. These findings on the connexions of the alpha neurone are summarized in Fig. 3. Table 2. Frequency response of the alpha neurone (Response to trains of pulses of sine waves of different frequency; each train consists of 1 pulses, each 4 msec, long, with a rest of 25 min. separating each train. Stimulus amplitude + 75 db throughout.) 5 kcyc./sec. iokcyc./sec. 3okcyc./sec. First Mean of 1 First Mean of 1 First Mean of 1 response responses response responses response responses Animal J25 Nil Nil 3 i J27 Nil Nil J28 Nil Nil B. Response of the alpha neurone to stimulus parameters (i) Frequency. The unit is very sensitive to white-noise clicks, giving a good response to a fingernail click more than 2 ft. away. It gives no response at all to low sine-wave frequencies, and at a sound intensity of + 9 db it first responds to 4 msec, pulses of sine wave of increasing frequency at between 5 and 8 kcyc./sec. The response gets larger with increasing frequency, and is largest at the highest test frequency used, 4 kcyc./sec. (Table 2). The upper limit was not ascertained, nor the frequency response in terms of amplitude of stimulus required for a constant response, because of the limitations of the apparatus. (ii) Amplitude. Absolute threshold intensity could not be measured reliably, as the auditory system was more sensitive than the monitoring apparatus. Threshold for 4 msec. pulses at 3 kcyc./sec. is between +5 and +6odB and the number of nerve impulses elicited by such a pulse increases with increasing intensity up to approximately +9 db. Above this value the system saturates and there is no regular increase at least up to +11 db (Fig. 4). A louder stimulus produces on average not only more action potentials per response, but also a shorter latency of the first action potential. The negative correlation between number of action potentials and latency is low (r = o*6 to *2) though significant; the correlation is lower when the response level is low. The constants of regression equations describing response and latency vary greatly even between replicates of the same experiment on the same animal; the source of the variation appears to be the latency. This variation probably invalidates any attempt to correlate response level and latency over a range of experimental conditions or between different animals, and the most that can be said is that stimulus conditions or lesions to the c.n.s. which tend to increase responsiveness also shorten the average latency.

7 Acridid auditory interneurone 237 (iii) Duration. The response to a sustained tone is more or less tonic, slowly adapting, but there is great individual variation (Fig. 5). Some animals cease to respond after 5 msec, of stimulation, others continue for many seconds to the same stimulus. It is possible that this variation is due to an as yet uninvestigated central factor influencing responsiveness. Adaptation is more apparent in the response to short pulses of sound of increasing duration, in which mean impulse frequency declines exponentially with increasing pulse length (Fig. 6). c o o, I 6 i J38 A J37 3 a, Z Sound intensity at ear (db) Fig. 4. Response of alpha neurone to increasing stimulus amplitude. The stimulus was a 3 msec, pulse of 3 kcyc./sec. sine wave. This was presented at intervals of 1 min., the intensity being varied randomly over 6 db steps. The results from two separate animals are plotted. The thresholds for both are similar, and animal J37 gives a larger response at high amplitudes than J 38. Both response curves level off at between 8 and 9 db. J42 Sound Sound 1 msec. 1 J43 Fig. 5. Response of the alpha neurone to a continuous 3 kcyc./sec. tone, recorded in the promesothoracic connective. Intensity of stimulus, +8 db. Time scale, 1 msec. Records from 2 separate animals, J42 and J43, are shown. J43 responds to the 3 kcyc./sec. tone in the presence of considerable background noise up to 1 kcyc./sec, which can be seen on the upper trace and which evokes no response. (iv) Directionality. Acridid ears, like those of noctuid moths, are known to have considerable directional sensitivity, especially at high frequencies (Katsuki & Suga, i96; Autrum, Schwartzkopf & Swoboda, 1961; Payne, Roeder & Wallman, 1966). The characteristics of the alpha neurone are adequate to preserve at least some of this directional information. Differences in amplitude at the two ears could be

8 238 C. H. FRASER ROWELL AND J. M. MCKAY represented by differences in either or both frequency of impulses or latency in the response of the two alpha units. The directional properties of the system were investigated by recording simultaneously from both connectives with a sound source placed laterally, normal to the animal's long axis, and then rotating the animal through 18 so that the sound impinged on the opposite ear. This 18 transposition is the extreme directional shift, and any directional capability should show most clearly under these conditions. 3 J39 o 2 o. 2 n_ Duration of stimulus (msec.) Fig. 6. Adaptation in the response to short pulses of sound. The ordinate shows the mean frequency of impulses in the response to pulses of 3 kcyc./sec. sound of constant amplitude and increasing duration. Each point represents the mean of three responses by the same animal. Regression equation: log 1 impulse frequency = 2-41 o-2t, where/ = pulse length in msec. Coefficient of correlation (r) = -99. The line through the points is drawn by eye. Table 3. Directionality of the alpha neurone. Comparison of the response to a unilateral sound of the two units on the basis of (a) number of action potentials, (b) latency of first action potential, (c) combination of (a) and (b) (Each figure represents the mean of 1 animals. N in table represents total number of responses heard by these 1 animals. Further explanation in the text.) Ipsilateral Ipsilateral signal Ipsilateral signal exceeds less than signal equal to contralateral contralateral contralateral 4 Criterion (a) Spike no. (b) Latency (c) Spike no. + latency discrimination (%) discrimination (%) IO discrimination (%) N (trii The response to a standard series of 2 pulses of 3 kcyc./sec. sound, each 4 msec, long and of amplitude +82 db, pulse repetition rate i/sec, was recorded in both alpha neurones; 5 animals were tested with both sound directions, receiving a

9 Acridid auditory interneurone 239 total of 213 sound pulses. A specimen response is shown in Fig. 7. The responses were analysed with respect to (a) the number of impulses in ipsilateral and contralateral units; (b) the latency of the first impulse in ipsilateral and contralateral units. As both these measures were shown to vary with intensity, they are expected to be related. A. Intact RHS, ipsilateral LHS, contralateral Expt. 1 Ipsilateral Contralateral B T. nerve cut LHS, ipsilateral' RHS, contralateral Expt. 4 Sound Sound Fig. 7 Fig. 8 Fig. 7. Response of ipsilateral (upper) and contralateral (lower) alpha neurones to a lateral sound source emitting a 3okcyc./sec. pulse. Note that the contralateral unit gives a weaker response and a longer latency; both are typical but not invariable features. Further explanation in the text. Length of stimulus, 6 msec. Fig. 8. Response of alpha and beta neurones to a pulse of 3 kcyc./sec. sound before and after section of one tympanic nerve. See experimental design in Fig. 2. A. Experiment 1. Intact animal. Upper: RHS connective, ipsilateral to sound. Lower: LHS connective, contralateral to sound. Note beta unit activity in ipsilateral connective only, alpha unit activity in both ipsilateral and contralateral connectives; compare with Fig. 7. B. Experiment 4. RH tympanic nerve cut, animal rotated relative to sound. Upper: LHS connective, ipsilateral to sound. Lower: RHS connective, contralateral to sound. Ipsilateral beta activity doubled, no response contralaterally. Ipsilateral alpha unit activity slightly decreased by loss of contralateral input, contralateral response totally lost. Duration of stimulus, 5 msec. Further explanation in the text. Table 3 shows that on criterion (a) alone the animals on average discriminated direction correctly in 57% of all trials, discriminated wrongly in 13% and achieved no discrimination in 3%. On criterion (b) alone the corresponding figures were 54, 21 and 25%. When both criteria are considered simultaneously, on the basis that compatible or single indications of direction are accepted, but contrary indications or no indications rejected as indiscriminable, the performance improves perceptibly; 68% of trials are correctly discriminated, only 1% incorrectly discriminated and

10 24 C. H. FRASER ROWELL AND J. M. MCKAY (1) Intact 1 J69 a-neurone /?-neurone 3 i-cr \i-a-a (2) S- 3' 3 I (3) RH tympanic nerve cut 3 5,- (4) 5 1- V-*-i^ck^a-ii-B-i-ii-rf^-Ai 3 Stimulus presentation number 1 15 Fig. 9. Activity of alpha and beta neurones of an individual animal (J69) during the experiment shown in Fig. 2. Ordinate, number of action potentials per response, abscissa, number of stimulus presentation. There are 15 presentations at 1 /sec. in each experiment. LHS neurones are represented by squares, RHS neurones by triangles; ipsilateral units (with respect to the sound source) are indicated by solid symbols, contralateral units by open symbols respectively. Alpha units. The intact animal gives symmetrical responses when rotated relative to the sound source. Both units fire, the ipsilateral one giving a greater response. After section of one tympanic nerve, both fibres fire at a reduced level, but more when the intact ear is ipsilateral to sound. The side with the intact ear is the more responsive. Beta units. The intact animal gives symmetrical responses when rotated relative to the sound source. Only the ipsilateral unit fires, the contralateral unit is totally inhibited. After cutting the ipsilateral tympanic nerve, there is no activity in the ipsilateral beta unit, but a good response from the formerly inhibited contralateral unit. When the cut tympanic nerve is contralateral to the sound, the ipsilateral beta unit gives a very large response. When both tympanic nerves are cut, neither unit responds to sound at all.

11 Acridid auditory interneurone % are not discriminated. Thus the animal gains in performance by the lack of complete correlation between latency and impulse number. C. Properties of the beta neurone In some though not all preparations a second auditory interneurone is seen when recording from the thoracic connectives, which responds with a high-frequency burst to at least some of the same stimuli as the alpha unit. Its action potentials record considerably smaller than those of the alpha unit, and presumably they are usually lost when recording from the intact connective. Its properties can best be illustrated by the following experiment, originally designed to test directionality and input connexions of the alpha unit, and which was shown in Fig. 2. The abdominal and neck connectives were cut in these animals, and in each experiment the animal was subjected to a train of sine-wave pulses, 3 kcyc./sec, p.r.r. i/sec. When both tympanic nerves are intact, only the ipsilateral beta unit fires. After section of one tympanic nerve, sound presented on the same side as the lesion elicits no response from the ipsilateral beta unit, but a large response from the contralateral one, which was previously silent. When the sound is presented to the side with the one remaining intact nerve, it elicits a very large response in the ipsilateral unit, and nothing in the contralateral one (Figs. 8, 9). These results indicate that there is mutual inhibition between the left-hand and right-hand beta-unit systems. The sound stimulus activates the receptors of both ipsilateral (Expts. 1, 2 and 4) and contralateral (Expt. 3) ears, but in the intact animal inhibition of the contralateral system by the ipsilateral is sufficient totally to suppress its response, while the inhibition of the ipsilateral system by the contralateral is only adequate to reduce the evoked response. This difference presumably reflects the directional sensitivity of the two ears ( B(iv)) probably caused by the acoustic shielding of the body. When the ipsilateral tympanic nerve is cut, the contralateral system is disinhibited and gives a response comparable to the ipsilateral unit of an intact animal. When the contralateral tympanic nerve is cut, the disinhibited ipsilateral system gives a very large response. No attempt has been made to locate the site of this mutual inhibition; it could take place between the primary tympanic inputs, or between the two interneurones, or between the interneurone and the contralateral tympanic nerve; and in any of these it could be direct or mediated by an intermediate interneurone. The functional significance of this arrangement is to increase greatly the directionality of the signal. Whereas the alpha unit correctly discriminated only 68%, at best, of all presentations, the beta unit discriminates under the same stimulus conditions with 1% accuracy the contralateral unit never fires. If the alpha and beta neurones were both fed by the same sensilla, as might be implied by the at least partial overlap in their frequency range, then one would expect the feedback applied to the beta system to result in a relatively lower sensitivity than that of the alpha unit. Experiments to test this proposition were performed on relatively few animals, but showed that, on the contrary, in at least some individuals the beta unit fires at sound intensities less than required to activate the alpha unit. This suggests that the two units receive inputs from populations of sensilla which differ in either their number or their sensitivity or their frequency response. 16 Exp. Biol. 51, 1

12 242 C. H. FRASER ROWELL AND J. M. MCKAY DISCUSSION What conclusions about the function of the two auditory interneurones can be drawn from the measurements of their performance? The alpha unit is clearly specialized for the reception of high-frequency sound. This has obvious uses. The genus stridulates by rubbing the hind femora on the elytra, but lacks the well-developed tooth or peg structure, as for example in the truxaline grasshoppers, which results in a peak of emission in the audible range. The sound produced is a high-frequency hiss, and oscilloscope examination confirms that most of the emission is ultrasonic. The animal lives in grass, and grass moved by the wind or other animals emits much high-frequency sound. Insectivorous predators such as shrews and bats emit ultrasound at high intensity; large-scale nocturnal flights are characteristic of many species of solitary grasshopper, and we have caught Gastrimargus at light at night. Haskell (1957) describes a take-off response by Schistocerca gregaria to the noise generated by aflyinglocust, and S. vaga responds similarly to the noise made by a vacuum cleaner. Haskell's recordings did not extend much beyond 1 kcyc./sec, but it seems probable that high frequencies are important in both cases. Popov (1965) performed ablations on the three morphological groups of sensilla in the ear of Locusta, and found that only those with a predominantly low-frequency (less than 1 kcyc./sec.) response gave rise to patterned summated potential, mirroring the characteristic amplitude modulation of the species song, in the recording from the tympanic nerve. The Group I sensilla, responding preferentially to higher frequencies (greater than 1 kcyc./sec), did not; the author accepts that amplitude modulation is the most important parameter of song, and therefore suggests that the high-frequency sensilla are not important in song reception. The alpha neurone, which is driven mainly by high-frequency receptors, might by this argument be adapted for some function other than song reception. However, this argument contains too many assumptions about both song reception and the as yet unrecorded ear sensilla to be particularly attractive. The input arrangements to the alpha neurone seem to favour high sensitivity at the price of directionality. The unit is certainly very sensitive to at least some elements of a white-noise click, and we suspect that its most sensitive frequency is higher than our highest test frequency of 4 kcyc./sec. Amplitude changes are reasonably well encoded over some 3 db of the response range. The unit's rather tonic response to medium length pulses would make it very suitable for signalling the length of such sounds. In a second paper (Rowell & McKay, 1969), we show that there is marked habituation to repetitive stimuli, but that at practical repetition frequencies the response never fails completely. We have much less information about the beta unit, but it is of great interest to see that it is clearly specialized for accurate directional discrimination, and that the feed-back mechanism by which this is achieved is functionally identical to that used by the tettigoniid grasshoppers Gampsocleis and Homorocoryphus (Suga & Katsuki, 1961; McKay, 1968). In other ways the tettigoniid interneurone differs considerably from the beta unit; for example, it is highly phasic in response, shows little habituation in the intact animal, gives a good low-frequency response and is very large in size (McKay, 1968); but directionality is achieved in the same way.

13 Table 4. Summary of interneurones described from the acridid auditory system, abstracted from various authors Author, name of unit, genus I. Rowell & McKay ; alpha neurone, Gastrimargus 2. Rowell & McKay; beta neurone; Gastrimargus 3. Horridge (1961) ; Fig. 3 ; SchistocercalLocusta 4. Yanagisawa et al. (1967), Fig. 3 A ; Locusta 5. Yanagisawa et 1. (1967), Fig. 3 B ; Locusta 6. Horridge (1961), p. 226; Locusta/Schistocerca 8. Popov (1965), Fig. 2; Locusta Minimum Location latency Mesothoracic g. 15 msec. to brain Mesothoracic g. I 5 msec. to brain Mesothoracic g.? to brain Mesothoracic g.? to brain Mesothoracic g.? to brain Metathoracic g.? to abdomen Metathoracic g.? to brain Metathoracic g.? to mesothoracic g. Frequency response, kcyc./sec. I, Range Optimum Comments or above Almost identical response curves, 3-4 probably the same unit.17 input, probably from subgenual b organs Yanagisawa et al. (1967) or by present authors a 2 Extra-tympana1 mesothoracic? Not found by Suga (1963),? Not clearly demonstrated by any other author 3 Accepted by most authors on 9 negative grounds, e.g. failure to 3- record mesothoracic units below mesothoracic ganglion, latency, $ etc. ; but at least some tympanic fibres run to mesothoracic ganglion 3 Receives no input from contra- $ lateral tympanum Receives no input from contralateral tympanum. Sustained tones elicit wider range response Same range as alpha neurone of Rowell and McKay? Same as nos. 3 and 4, ascending to brain? Mixed audiovisual units ; complex habituation ; some modality N interaction - t2 9. Adam & Schwartzkopf (1967); 'broadband neurone' ; Locusta 1. Adam & Schwartzkopf (1967); 'deeptone neurone ' : Locusta I I. Schwartzkopf & Adam (1967) ; ' hightone neurone ' ; Locusta I 2. Rowel1 & Horn unpublished ; Schistocerca Horridge et al. (1965); Class CD, CE neurones; N Locusta 14. Horridge et al. (1965); Class D neurones; Locusta Protocerebrum Protocerebrum Protocerebrum Tritocerebrum Optic lobes, lateral protocerebrum Optic lobes, lateral protocerebrum 27 msec. on only picture? ? Audio fre-? quencies at least 5-7 msec.?? 57 msec. 'No evidence of any frequency discrimination, ' range testedinot given

14 244 C. H. FRASER ROWELL AND J. M. MCKAY Neither the alpha nor beta units seem to have been described before individually, though there is no doubt that they must be included in the sample described by Horridge (1961). The small size of the beta unit action potentials in most recordings and the absence of response of the alpha unit to most of the human audible range suffice to explain this. Table 4 summarizes those auditory interneurones which have been more or less characterized in the acridid system. Even allowing for spurious diversity created by different experimental techniques or interspecific differences, it is clear that the auditory system is most complex. So far no recordings have been made from the split connective, a technique which is almost certain to reveal more auditory units (see McKay, (1968) with the tettigoniid Homorocoryphus). A corresponding complexity of receptors is also being demonstrated. Recordings of the summated potential of the tympanic nerve indicate a response from kcyc./sec. and possibly beyond; although several different classes of receptor have already been demonstrated (see Introduction), they do not yet cover this frequency range, and it may be presumed that further types await description. The auditory system is clearly capable of most sophisticated handling of acoustic information, and is quite unlike the picture commonly presented until recently, sensitive only to amplitude modulation and possibly consisting of only a single type of primary receptor and one ascending interneurone. SUMMARY 1. An auditory interneurone (' alpha neurone') of Gastrimargus africanus is described. It runs from the mesothoracic ganglion to the brain, and receives summating excitatory inputs from both tympanic nerves. There is no inhibition between the two units or their inputs. 2. The lowest effective frequency is 5-8 kcyc./sec. and response is greatest at the highest frequency tested, 4 kcyc./sec. Threshold at 3 kcyc./sec. is approximately + 5 db above -2 dynes/cm. 2, and response increases with amplitude from 6-9 db, after which there is no further increase. Increase of amplitude also shortens response latency; the correlation of latency and response strength is low and very variable. The response is slowly adapting, and mean impulse frequency declines exponentially with increasing length of short sound pulses. 3. Directionality of unilateral sound is signalled by the alpha neurones in only 7% or less of trials, and at least 1% are discriminated 'wrongly'. The low correlation between response strength and latency improves the signalling of directionality. 4. A second 'beta' neurone is described briefly. It signals directionality of a unilateral sound with 1% accuracy, due to mutual inhibition between ipsilateral and contralateral systems, as in some tettigoniid interneurones. 5. Ten or more apparently separate auditory interneurones have now been described in acridids; this parallels the diversity of receptors described in the ear. This work was supported in part by the United States Government under Contract C16 and by Makerere College Research Fund, Grant no We are most grateful to Dr M. C. Pike for advice on statistical treatments, and to Dr R. G. Whitehead for computer facilities. One of us (J.M.M.) was supported by an S.R.C. Studentship.

15 Acridid auditory interneurone 245 REFERENCES ADAM, L-J. & SCHWARTZKOPF, J. (1967). Getrennte nervose Representation fiir verschiedene Tonbereich im Protocerebrum von Locusta migratoria. Z. vergl. Physiol. 54, AUTRUM, H., SCHWARTZKOPF, J. & SWOBODA, H. (1961). Der Einfluss der Schallrichtung auf die Tympanal-Potentiale von Locusta migratoria L. Biol. Zbl. 8, DIRSCH, V. M, (1966). The African Genera of Acridoidea. Cambridge University Press. GREY, E. G. (i96). The fine structure of the insect ear. Phil. Trans. R. Soc. B 343, HASKELL, P. T. (1957). The influence offlightnoise on behaviour in the desert locust Schistocerca gregaria (Forsk.) J. Insect Physiol. 1, HORRIDGE, G. A. (1961). Pitch discrimination in locusts. Proc. R. Soc. B 155, HORRIDGE, G. A., SCHOLES, J. H., SHAW, S. & TUNSTALL, J. (1965). Extracellular recordings from single neurones in the optic lobe and brain of the locust. In The Physiology of the Insect Central Nervous System. London: Academic Press. KATSUKI, Y. & SUGA, N. (i96). Neural mechanism of hearing in insects. J. exp. Biol. 37, MCKAY, J. M. (1968). Some aspects of the physiology of the tettigoniid ear. Ph.D. thesis, University of East Africa. MICHELSEN, A. (1966). Pitch discrimination in the locust ear: observations on single sense cells. J. Insect Physiol. 12, MURRAY, M. J. (1968). Fibre groups in the auditory nerve of the locust. Nature, Lond. 218, PAYNE, R. S., ROEDER, K. D. & WALLMAN, J. (1966). Directional sensitivity of the ears of noctuid moths. J. exp. Biol. 44, POPOV, A. C. (1965). Electrophysiological studies of peripheral auditory neurones in the locust. (In Russian.) J. evol. Biochem. Physiol. 1, ROEDER, K. D. & TREAT, A. E. (1957). Ultrasonic reception by the tympanic organ of noctuid moths. J. exp. Zool. 134, ROWELL, C. H. F. & HORN, G. (1968). Dishabituation and arousal in the response of single nerve cells in an insect brain. J. Exp. Biol. 49, ROWELL, C. H. F. & MCKAY, J. M. (1969). An acridid auditory interneurone. II. Habituation, variation in response level and central control, y. exp. Biol. 51, SUGA, N. (1963). Central mechanism of hearing and sound localisation in insects, y. Insect Physiol. 9, SUGA, N. & KATSUKI, Y. (1961). Central mechanism of hearing in insects, y. exp. Biol. 38, YANAGISAWA, K., HASHIMOTO, T. & KATSUKI, Y. (1967). Frequency discrimination in the central nerve cords of locusts, y. Insect Physiol 13,

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