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Action potentials in the antennae of the blowfly (Calliphora erythrocephala) during mechanical stimulation
y. Ins. Physiol., 1960, Vol. 4, pp. 138 to 145. Pergamon Press Ltd., London. Printed in Great Britain
ACTION POTENTIALS IN THE ANTENNAE BLOWFLY (CALLIPHORA ERYTHROCEPHALA) MECHANICAL STIMULATION* DIETRICH Zoologisches
BURKHARDT
Institut, (Received
OF THE DURING
Universitlt 1 July
Miinchen
1959)
Abstract-The excitation patterns in Johnston’s Organ during mechanical stimulation were studied by means of microelectrodes in Calliphora erythrocephala. An electromechanical transducer was used to deliver stimuli with a rectangular time course, whereby the funiculus of the antenna was rotated about its long axis. By variation of stimulus intensity, polarity, duration, and frequency, the build-up of excitation patterns may be investigated while the antenna is subjected to a variety of stimulus patterns. The recorded action potentials lead one to the conclusion that the majority of nervous elements are responding only to movements, each phase of movement causing a single spike. Some of the elements observed respond to torsional movements independent of direction; others are sensitive only to movements in one of the two possible directions. During stimuli of short duration, strong interactions between the action potentials released at the beginning and end of stimulation may be observed. These interactions are responsible for the observation that slight changes in stimulus pattern cause major changes in excitation pattern during high frequency repeated stimulation (up to 500 c/s). Because of the numerical preponderance of phasic receptors in the funiculuspedicellus joint, the antenna of the blowfly is a sense organ specialized for sensitive time resolution of air flow stimuli. The antenna therefore exhibits some properties of a hearing organ: it responds to airborne sound of frequencies up to 500 c/s. Especially in the case of single stimulation with short rectangular impulses (click), the potentials are very similar to the nervous component of the cochlear potential of birds released by the same sort of stimuli.
IN a previous investigation (BURKHARDT and SCHNEIDER, 1957) it was shown by behavioural experiments and electrophysiological measurements that the antennae of the blowfly are sensitive, extraordinarily fast-reacting current sense organs. Antenna1 excitation is involved in regulation of the animal’s speed in free flight. A constant stream of air forces the arista to act like a lever arm which turns the funiculus outwards around its long axis. A series of action potentials is thereby released in Johnston’s Organ. In the cited investigations, action potentials were also detected when the animal was subjected to airborne sound at frequencies below 500 c/s (cf. WESTECKER, 1957). However, all attempts to establish behavioural reactions to airborne sound failed. In many animals, there is a close relationship between current sense and hearing, and there is reason to believe that in the course of phylogeny many hearing organs have developed from organs originally used * These investigations were supported by a grant from the Deutschen Forschungsgemeinschaft. 138
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for current detection in vertebrates and insects (SCHWARTZKOPFF, 1959). The properties of the Calliphora antennae lead one to suppose that they represent a transitional stage between organs for current sense and hearing. A thorough seems to be particularly interesting here, electrophysiological investigation especially if the build-up of excitation by static and dynamic deflexions (imitating stimuli of current flow and airborne sound) can be followed with microelectrodes. The experimental animals were taken from cultures of the Zoological Institute in Mtinchen. Males and females were used equally often, as no differences could be observed in their responses. Steel needle or tungsten microelectrodes (BUEEHARDT, 1954; HUBEL, 1957) were used to lead off potentials from the pedicellus, in the medial part of Johnston’s Organ; the action potential was amplified, observed, and recorded synchronously with the stimulus on a double-beam oscilloscope (Tectronix Type 502). For stimulation, the arista or the funiculus was moved by the thinly drawn out, bent tip of a glass rod. The glass rod was glued to a small loudspeaker which was fed from a Grass Stimulator S4. The stimulator delivers rectangular electrical impulses of any desired strength, polarity, and duration, singly or repeated at selected intervals. As in natural stimuli, the funiculus was rotated about its long axis by the artificial stimulus. It was rotated abruptly, maintained its position for the length of the stimulus, Stimuli of current flow can thus be and then returned abruptly to its original position. By increasing stimulus frequency imitated in a simple way by single, longer deflexions. and shortening stimulus duration the build-up of excitation during rhythmic stimulation (in imitation of natural sound stimulation) can be followed up to high frequencies. The loudspeaker employed had a resonance frequency of 350 c/s, therefore a low-amplitude damped oscillation of this frequency was superimposed on the beginning and end of each rectangular stimulation.
In Fig. 1 a typical action potential is shown which arises from a single rotation of the antenna. After a short latency (O-5-2 msec at 20°C) the preparation responds to stimulus onset with a large, di- (or tri-) phasic action potential. Latency and amplitude of this ‘on-wave’ are dependent on stimulus strength (angle-of rotation) (see Fig. 2); the amplitude may be greater than 5 mV. A series of smaller, usually mono- or diphasic action potentials follows the on-wave; their amplitude decreases A comparison with the movements of the during the course of stimulation. stimulating needle shows that this series of action potentials is released by the superimposed oscillations. Towards the end of such a series, single action potentials may drop out of this rigidly stimulus-synchronized succession. During lowfrequency repetitive stimulation, the amplitude of the large on-wave often fluctuates stepwise, in a random manner, within a considerable fraction of its maximum range (Fig. 3). Both facts may. be explained as follows: the individual action potentials, under microelectrode leadoffs, are composed of a small number of rigidly synchronized discharges of single nerve elements. With increasing stimulus intensity, the number of synchronously discharging elements increases (and thereby also the amplitude of the registered potentials). Towards the end of the damped oscillations, only a few elements are still subjected to a stimulation strength above threshold. If threshold happens to vary a little, or stimulus strength changes slightly, part of these potentials may drop out. The action potentials of the
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single elements obey the all-or-none law. Until now, however, it has not been possible to decide if these single discharges originate from the soma or the axons of the sensory nerve cells.
,,D____--o------
I’
ampliiude
,/’
>
om
10
0.1
ltimuld3r
voltage [V]
FIG. 2. Dependence of amplitude and latency of the on-wave upon stimulus intensity. As a measure of stimulus intensity, the voltage on the stimulator is plotted on the abscissa (log scale); the deflection is proportional to the voltage. Latent period is measured from stimulus onset to beginning of on-wave; amplitude is the difference in height between the highest positive and negative deflections (cf. Fig. 1).
During continued deflexion of the antenna no change in activity, compared with that before the stimulus, can usually be observed. Since, in addition, the action potential and the course of movement of the stimulating needle are strictly synchronous, it can be concluded that the elements described here respond only to movement, but not to constant deflexion (phasic receptors). Each phase of movement releases a single action potential with the rapid stimuli employed. At the end of the stimulus, an action potential is obtained which is very similar to that obtained when the stimulus begins. If the traces of on- and off-waves are superimposed on the oscilloscope, one can see that in certain phases the action potentials are congruent, whereas in others they differ from one another (cf. Fig. 3). If the direction of rotation of the antenna is reversed, the action potential at the onset of the stimulus‘now looks like the .potential at the end of the stimulus for the original direction of antenna1 rotation, and vice versa: at the end of the stimulus it appears similar to the previous trace for the onset of the stimulus. In general, the amplitudes of the on-waves for a given rotation direction are smaller, and their latency is somewhat larger, than for the opposite direction of rotation. The partial similarities of the action potentials for various directions of rotation may be hypothetically explained as follows: some of the elements respond only to turning in one direction, whereas others respond to every movement in any direction.
FIG. 1. Phasic action potential in response to abrupt rotation of the antennae. Calibration: horizontal 5 msec, vertical 1 mV. Lower trace: time course of the stimulus. Upper trace: left, the on-wave (diphasic in this case), followed by smaller action potentials which have doubled the frequency of the damped oscillations superimposed on the stimulus (cf. lower trace). For the photograph action potentials of several stimuli were registered superimposed on one another. FIG. 3. Upper trace: superimposed on-waves from stimuli of opposite polarities. Lower Calibration: horizontal 2 msec, vertical trace: time course of the corresponding stimuli. 1 mV. The action potentials of the two oppositely directed stimuli are congruent in certain phases. Similar pictures are observed if on- and off-waves are superimposed. Falling phase of the on-wave retraced for purposes of reproduction.
FIG. 4. Action trace.
Stimulus
current picture with rhythmic stimulation (upper trace). frequency 50 c/s, duration 9 msec. Calibration: 2 mV.
Stimulus:
lower
FIG. 6. Action potentials during constant stimulus frequency and slightly varied stimulus duration. Upper trace: action potential. Lower trace: stimulus. Frequency for both records 80 c/s. Stimulus duration for upper record ca. 4 msec, Iower record ca. 3 msec. Calibration : 2 mV. In the lower record, the off-waves are suppressed and the on-waves fully developed. FIG. 7. Tonic discharge of position receptors. The two records show the action potentials during stimuli of opposite polarities. Stimulus duration always 0.8 sec. Calibration: 2 mV. At the beginning and the end of stimulus, the very high phasic action potentials are visible. Tonic elements are visible betweerrthem in the upper record; before and after them in the lower record. Peaks retraced for better reproduction.
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This is also supported by the interaction between on- and off-waves during short stimulation (to be described later). This response pattern of the nervous elements can be explained by the geometrical arrangement: in general a mechanical stress in a given direction (for example stretching) serves as a stimulus. The receptors, however, have different orientations with respect to the axis of rotation. Thus some will be stimulated only by a certain direction of rotation, some by both possible directions of rotation. In order to investigate the build-up of excitation by rhythmic stimulation, the stimulus frequency is increased during constant short stimulus duration. Up to intervals of 20-30 msec, the action potentials for single stimuli are hardly changed. The investigated movement receptors do not seem to show adaptation. With further shortening of the intervals between stimuli, the on-wave of each stimulus approaches more and more closely the off-wave of the preceding stimulus. When successive intervals are exactly twice as long as stimulus duration, one observes regular rows of alternating on- and off-waves. These action potentials then look exactly like those released by airborne sound; the action potential frequency is equal to the doubled stimulus frequency (WESTECKER, 1957). The potentials described in the investigations of BURKHARDTand SCHNEIDERas arising from airborne sound, thus seem to arise from the rigidly synchronized discharges at the beginning and ending of each torsional vibration stimulus. It is safe to assume, however, that these potentials are not an effect comparable with the microphonic component of the cochlear potential of birds and mammals. These potentials of the insect antenna are much more similar to the nervous component of the cochlear potential, the discharge of the first acoustic neurones. Especially in single stimulation with short rectangular impulses (‘click’) of alternating polarities, the potentials show a striking similarity to those registered when the bird cochlea is subjected to click stimuli of alternating polarities (SCHWARTZKOPFF,1958). In birds also the superimposed potentials of opposite polarities are only partially congruent; the amplitude is greater and the latency shorter for one of the two polarities. PUMPHREY (1940) has already pointed out the similarities between the action potentials in certain insect hearing organs and those of the higher vertebrates. If the stimulus duration is shortened, while the frequency is held constant and not too high, interactions between the on- and off-waves can be observed. With increasing short stimulus duration, the off-wave approaches more closely the series of small action potentials released by the damped oscillations of the stimulator, and finally the on-wave. It does not simply superimpose on these potentials; it is suppressed at certain stimulus durations, but develops fully at others. The latency between stimulus end and off-wave also changes during this shortening as follows: with small off-wave amplitudes a long latency is registered; with larger amplitude latency is shorter (Fig. 5). The height of the discharge-and thus probably the number of synchronously discharging elements-is thus determined for the offwave by the preceding discharges. These shifts in latency are such that the off-wave tends to occupy the position of one of the previous discharges. For example when stimulus duration is shortened, the latency is at first longer, so that it remains
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approximately at the same position where a discharge occurred with longer stimulus duration. When the stimulus duration is further shortened, the latency decreases abruptly, so that now the off-wave occupies the previous position of an earlier-occurring discharge. One must therefore assume strong interactions between the nervous elements participating in the on- and off-effects.
0
2
L
6
8 stimulus duration
10 [msec]
FIG. 5. Dependence of amplitude and latency of the off-wave upon stimulus duration (with short stimuli). The first arrow on the abscissa indicates the time of occurrence of the peak of the on-wave; the following arrows those of the later smaller action potentials. The momentary location of the off-effect on the, abscissa can be calculated as follows: the latent period (the ordinate value) must be added to stimulus duration. When this is done, the abscissa must be viewed as the time co-ordinate. Because of the shifts in latency, the offwaves tend to occupy the position of preceding discharges ; their amplitude thereby changes in the opposite direction to latency.
Two possibilities might be considered here: (1) the elements participating in the on- and off-effects inhibit each other reciprocally; or (2) the same elements participate in the on- and off-effects ; therefore these elements are refractory after the on-wave for a certain period and cannot contribute to the off-wave. By means of periodic stimulation with small variations in stimulus duration or frequency, one can obtain either an action potential synchronous with the stimulus (by suppressing the off-wave) or one with a frequency twice that of stimulation (when the off-wave is fully developed), because of the described interaction (Fig. 6). By means of a certain shortening of the stimulus, the off-wave is suppressed as it approaches the on-wave. In a similar way, by appropriate prolongation of the stimulus, the on-wave of the succeeding stimulus can be suppressed as the preceding By small opposite variations in stimulus off-wave approaches more closely. duration, the on- and off-effects can thus be alternately suppressed; by this means the phase of the action potential can be shifted almost 180” with respect to the stimulus while maintaining the same frequency.
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Minute changes in the stimulus pattern thus cause extreme alterations in the spatio-temporal excitation pattern in Johnston’s Organ. Such nervous effects can only be analysed by isolated investigations of single or few active elements with microelectrodes, and by a careful study of the excitation pattern. A summary starting from the total organ and an evaluation of the effect from a formal physical viewpoint (‘Klirrfactor’ of TISCHNER,1953 and KEPPLER,1958) is not suitable for the biological function of such a complexly-built organ. During rectangular stimuli of long duration, it was sometimes possible to record spike discharges of single nervous elements throughout the duration of the stimulus, in addition to the described on- and off-waves. When the direction of rotation was reversed, this discharge was visible before and after the stimulus, but during the stimulus it was obliterated (Fig. 7). In addition to the phasic receptors responding only to movements therefore, the antennae of Calliphora also possess tonic receptors which react to the deflexion itself (BURKHARDTand SCHNEIDER, 1957; in Locusta migratoria UCHIYAMA and KATSUKI, 1956). However, these receptors seem to be very rare. On the basis of present experiments it cannot yet ‘be decided whether we are dealing with elements of Johnston’s Organ. or with other position receptors. STADTM~~LLER (1955) describes innervated hair tufts between funiculus and pedicellus, which might conceivably participate in the indication of position. By stronger single deflexions of the antenna, one frequently observes a dense, irregular series of relatively large action potentials about 200 msec after the stimulus onset. Observations with the binocular microscope show that the antennae are thereby actively moved. Probably these action potentials are muscular action currents. Apparently the position of the antennae is actively changed by the animal under strong stimulation (cf. HERAN, 1957). In a previous work (BURKHARDTand SCHNEIDER,1957) it could be shown that when the antennae are subjected to a steady air current, irregular spike-like potentials arise. The antennae are made to tremble by the air-flow (by air eddies ?). This result could be confirmed by leading off with microelectrodes. The oscillograms give the impression that during the stimulus by air current, the antenna reacts with an irregular pattern of on- and off-waves (phasic action potentials), similar to the waves which respond regularly to airborne sound. The majority of receptors in the antenna are phasic receptors which respond only to movement. The question arises therefore, whether it is possible that these phasic receptors are also capable of registering mean deflexion of the antennae. It is thought that this could be made possible by the following mechanism: by continuous vibration of the antennae the phasic receptors are continuously re-stimulated. With increasing angle of rotation the number of elements excited also increases. In addition the peak of excitation in the spatial pattern of the sensory cells will shift with various angles of rotation. Thus, through the spatio-temporal pattern of excitation of Johnston’s Organ, the CNS can receive exact information about the strength and time course of the antenna1 deflexion.
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A very exact resolution of the time course of air flow stimuli is ensured by the preponderance of phasic receptors in the antenna1 joint between funiculus and pedicellus. Thus it is not surprising that this kind of current sense organ possesses a great many of the properties of a hearing organ, as shown by the investigations of the build-up of excitation with rhythmic stimulation and comparisons with the bird ear. ZUSAMMENFASSUNG
Die Erregungsmuster im Johnston’schen Organ von Calliphora erythrocephala werden mit Mikroelektroden bei mechanischer Reizung der Antenne untersucht : Ein elektromechanischer Reizgeber dreht den Funiculus wie bei natiirlichen Reizen urn seine LHngsachse ; der Zeitverlauf des Reizes ist rechteckfiirmig. Durch Ver&rderung von Intensitat, Polaritat, Dauer und Folgefrequenz der Reize kann der Aufbau der Erregungsmuster bei verschiedenen Reizmustern verfolgt werden. Aus den abgeleiteten Aktionspotentialen muss geschlossen werden, dass die iiberwiegende Mehrzahl der nervijsen Elemente nur auf Bewegungen anspricht und zwar auf jede Bewegungsphase mit einem einzigen Spike: phasische Receptoren. Ein Teil der Elemente scheint auf jede Bewegung unabhangig von der Drehrichtung anzusprechen, ein Teil nur auf Bewegungen mit einen jeweils bestimmten Drehsinn. Bei kurzer Reizdauer kijnnen starke Wechselwirkungen zwischen den durch Reizbeginn und Reizende ausgelijsten Aktionspotentialen beobachtet werden. Diese Wechselwirkungen bedingen, dass bei periodischer hochfrequenter Reizung (bis 500 Hz) geringfiigige Anderungen des Verhaltnisses zwischen Reizdauer und Pausendauer einschneidende Veranderungen des Erregungmusters zur Folge haben. Durch das ijberwiegen phasischer Receptoren im Funiculus-PedicellusGelenk ist die Fliegenantenne ein Sinnesorgan, welches auf eine besonders genaue zeitliche Aufliisung von StrGmungsreizen spezialisiert ist. Diese Spezialisierung bedingt, dass die Antenne bereits Eigenschaften eines Geharorgans besitzt: Sie spricht auf Luftschall bis zu Frequenzen von 500 Hz an, und die Erregungsvorgange bei einmaligen kurzen Rechteckreizen (Klicks) zeigen auff%llige Parallelitaten zu den nervcsen Komponenten des Cochleapotentials der Vogel unter gleichen Reizbedingungen. REFERENCES BURKHARDTD. (1954) Rhythmische Erregungen in den optischen Zentren von Calliphora erythrocephala. Z. vergl. Physiol. 36, 595-630. BURKHARDT D. und SCHNEIDERG. (1957) Die Antennen von Calliphora als Anzeiger der Fluggeschwindigkeit. Z. Naturf. 12b, 139-143. HERAN H. (1957) Die Bienenantenne als Messorgan der Fluggeschwindigkeit. Naturwissenschaften 44,475. HUBEL D. H. (1957) A tungsten microelectrode for recording from single units. Science 125, 549-550. KEPPLERE. (1958) Uber das Richtungshijren von StechmGcken. Z. Naturf. 13b, 286-284. PUMPHREY R. J. (1940) Hearing in insects. Biol. Rev. 15, 107-132.
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SCHWAETZKOPFF J. (1958) Uber den Einfluss der Bewegungsrichtung der Basilarmembran auf die Ausbildung der Cochleapotentiale von St&x variu (Barton) und Melopsittucus undulatus (Shaw). Z. vergl. Physiol. 41, 35-48. SCHWAETZKOPFF J. (1959) Der akustische Reiz und die GehSrserrregung. Verb. dtsch. zool. Ges, Miinster 1959, in press. STADTM~LLERR. (1955) Untersuchungen zur Anatomie und Histologic der Antennen von Calliphora erythrocephala. Zulassungsarbeit ftir das Staatsexamen, Wiirzburg. TISCHNERH. (1953) Uber den Gehiirsinn der Stechmticken. Acustica 3, 335-343. UCHIYAMAH. and KATSUKIY. (1956) Recording of action potentials from the antenneal nerve of locusts by means of microelectrodes. Physiol. camp. 4, 154-163. WESTECKEE M. (1957) Elektfophysiologische Untewuchungen der Antennenreaktion von Calliphora erythrocephala bei Luftschall. Inauguraldissertation, Wtirzburg.
ACTION POTENTIALS IN THE ANTENNAE BLOWFLY (CALLIPHORA ERYTHROCEPHALA) MECHANICAL STIMULATION* DIETRICH Zoologisches
BURKHARDT
Institut, (Received
OF THE DURING
Universitlt 1 July
Miinchen
1959)
Abstract-The excitation patterns in Johnston’s Organ during mechanical stimulation were studied by means of microelectrodes in Calliphora erythrocephala. An electromechanical transducer was used to deliver stimuli with a rectangular time course, whereby the funiculus of the antenna was rotated about its long axis. By variation of stimulus intensity, polarity, duration, and frequency, the build-up of excitation patterns may be investigated while the antenna is subjected to a variety of stimulus patterns. The recorded action potentials lead one to the conclusion that the majority of nervous elements are responding only to movements, each phase of movement causing a single spike. Some of the elements observed respond to torsional movements independent of direction; others are sensitive only to movements in one of the two possible directions. During stimuli of short duration, strong interactions between the action potentials released at the beginning and end of stimulation may be observed. These interactions are responsible for the observation that slight changes in stimulus pattern cause major changes in excitation pattern during high frequency repeated stimulation (up to 500 c/s). Because of the numerical preponderance of phasic receptors in the funiculuspedicellus joint, the antenna of the blowfly is a sense organ specialized for sensitive time resolution of air flow stimuli. The antenna therefore exhibits some properties of a hearing organ: it responds to airborne sound of frequencies up to 500 c/s. Especially in the case of single stimulation with short rectangular impulses (click), the potentials are very similar to the nervous component of the cochlear potential of birds released by the same sort of stimuli.
IN a previous investigation (BURKHARDT and SCHNEIDER, 1957) it was shown by behavioural experiments and electrophysiological measurements that the antennae of the blowfly are sensitive, extraordinarily fast-reacting current sense organs. Antenna1 excitation is involved in regulation of the animal’s speed in free flight. A constant stream of air forces the arista to act like a lever arm which turns the funiculus outwards around its long axis. A series of action potentials is thereby released in Johnston’s Organ. In the cited investigations, action potentials were also detected when the animal was subjected to airborne sound at frequencies below 500 c/s (cf. WESTECKER, 1957). However, all attempts to establish behavioural reactions to airborne sound failed. In many animals, there is a close relationship between current sense and hearing, and there is reason to believe that in the course of phylogeny many hearing organs have developed from organs originally used * These investigations were supported by a grant from the Deutschen Forschungsgemeinschaft. 138
ACTION POTENTIALS IN THE ANTENNAE OF THE BLOWFLY
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for current detection in vertebrates and insects (SCHWARTZKOPFF, 1959). The properties of the Calliphora antennae lead one to suppose that they represent a transitional stage between organs for current sense and hearing. A thorough seems to be particularly interesting here, electrophysiological investigation especially if the build-up of excitation by static and dynamic deflexions (imitating stimuli of current flow and airborne sound) can be followed with microelectrodes. The experimental animals were taken from cultures of the Zoological Institute in Mtinchen. Males and females were used equally often, as no differences could be observed in their responses. Steel needle or tungsten microelectrodes (BUEEHARDT, 1954; HUBEL, 1957) were used to lead off potentials from the pedicellus, in the medial part of Johnston’s Organ; the action potential was amplified, observed, and recorded synchronously with the stimulus on a double-beam oscilloscope (Tectronix Type 502). For stimulation, the arista or the funiculus was moved by the thinly drawn out, bent tip of a glass rod. The glass rod was glued to a small loudspeaker which was fed from a Grass Stimulator S4. The stimulator delivers rectangular electrical impulses of any desired strength, polarity, and duration, singly or repeated at selected intervals. As in natural stimuli, the funiculus was rotated about its long axis by the artificial stimulus. It was rotated abruptly, maintained its position for the length of the stimulus, Stimuli of current flow can thus be and then returned abruptly to its original position. By increasing stimulus frequency imitated in a simple way by single, longer deflexions. and shortening stimulus duration the build-up of excitation during rhythmic stimulation (in imitation of natural sound stimulation) can be followed up to high frequencies. The loudspeaker employed had a resonance frequency of 350 c/s, therefore a low-amplitude damped oscillation of this frequency was superimposed on the beginning and end of each rectangular stimulation.
In Fig. 1 a typical action potential is shown which arises from a single rotation of the antenna. After a short latency (O-5-2 msec at 20°C) the preparation responds to stimulus onset with a large, di- (or tri-) phasic action potential. Latency and amplitude of this ‘on-wave’ are dependent on stimulus strength (angle-of rotation) (see Fig. 2); the amplitude may be greater than 5 mV. A series of smaller, usually mono- or diphasic action potentials follows the on-wave; their amplitude decreases A comparison with the movements of the during the course of stimulation. stimulating needle shows that this series of action potentials is released by the superimposed oscillations. Towards the end of such a series, single action potentials may drop out of this rigidly stimulus-synchronized succession. During lowfrequency repetitive stimulation, the amplitude of the large on-wave often fluctuates stepwise, in a random manner, within a considerable fraction of its maximum range (Fig. 3). Both facts may. be explained as follows: the individual action potentials, under microelectrode leadoffs, are composed of a small number of rigidly synchronized discharges of single nerve elements. With increasing stimulus intensity, the number of synchronously discharging elements increases (and thereby also the amplitude of the registered potentials). Towards the end of the damped oscillations, only a few elements are still subjected to a stimulation strength above threshold. If threshold happens to vary a little, or stimulus strength changes slightly, part of these potentials may drop out. The action potentials of the
140
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single elements obey the all-or-none law. Until now, however, it has not been possible to decide if these single discharges originate from the soma or the axons of the sensory nerve cells.
,,D____--o------
I’
ampliiude
,/’
>
om
10
0.1
ltimuld3r
voltage [V]
FIG. 2. Dependence of amplitude and latency of the on-wave upon stimulus intensity. As a measure of stimulus intensity, the voltage on the stimulator is plotted on the abscissa (log scale); the deflection is proportional to the voltage. Latent period is measured from stimulus onset to beginning of on-wave; amplitude is the difference in height between the highest positive and negative deflections (cf. Fig. 1).
During continued deflexion of the antenna no change in activity, compared with that before the stimulus, can usually be observed. Since, in addition, the action potential and the course of movement of the stimulating needle are strictly synchronous, it can be concluded that the elements described here respond only to movement, but not to constant deflexion (phasic receptors). Each phase of movement releases a single action potential with the rapid stimuli employed. At the end of the stimulus, an action potential is obtained which is very similar to that obtained when the stimulus begins. If the traces of on- and off-waves are superimposed on the oscilloscope, one can see that in certain phases the action potentials are congruent, whereas in others they differ from one another (cf. Fig. 3). If the direction of rotation of the antenna is reversed, the action potential at the onset of the stimulus‘now looks like the .potential at the end of the stimulus for the original direction of antenna1 rotation, and vice versa: at the end of the stimulus it appears similar to the previous trace for the onset of the stimulus. In general, the amplitudes of the on-waves for a given rotation direction are smaller, and their latency is somewhat larger, than for the opposite direction of rotation. The partial similarities of the action potentials for various directions of rotation may be hypothetically explained as follows: some of the elements respond only to turning in one direction, whereas others respond to every movement in any direction.
FIG. 1. Phasic action potential in response to abrupt rotation of the antennae. Calibration: horizontal 5 msec, vertical 1 mV. Lower trace: time course of the stimulus. Upper trace: left, the on-wave (diphasic in this case), followed by smaller action potentials which have doubled the frequency of the damped oscillations superimposed on the stimulus (cf. lower trace). For the photograph action potentials of several stimuli were registered superimposed on one another. FIG. 3. Upper trace: superimposed on-waves from stimuli of opposite polarities. Lower Calibration: horizontal 2 msec, vertical trace: time course of the corresponding stimuli. 1 mV. The action potentials of the two oppositely directed stimuli are congruent in certain phases. Similar pictures are observed if on- and off-waves are superimposed. Falling phase of the on-wave retraced for purposes of reproduction.
FIG. 4. Action trace.
Stimulus
current picture with rhythmic stimulation (upper trace). frequency 50 c/s, duration 9 msec. Calibration: 2 mV.
Stimulus:
lower
FIG. 6. Action potentials during constant stimulus frequency and slightly varied stimulus duration. Upper trace: action potential. Lower trace: stimulus. Frequency for both records 80 c/s. Stimulus duration for upper record ca. 4 msec, Iower record ca. 3 msec. Calibration : 2 mV. In the lower record, the off-waves are suppressed and the on-waves fully developed. FIG. 7. Tonic discharge of position receptors. The two records show the action potentials during stimuli of opposite polarities. Stimulus duration always 0.8 sec. Calibration: 2 mV. At the beginning and the end of stimulus, the very high phasic action potentials are visible. Tonic elements are visible betweerrthem in the upper record; before and after them in the lower record. Peaks retraced for better reproduction.
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This is also supported by the interaction between on- and off-waves during short stimulation (to be described later). This response pattern of the nervous elements can be explained by the geometrical arrangement: in general a mechanical stress in a given direction (for example stretching) serves as a stimulus. The receptors, however, have different orientations with respect to the axis of rotation. Thus some will be stimulated only by a certain direction of rotation, some by both possible directions of rotation. In order to investigate the build-up of excitation by rhythmic stimulation, the stimulus frequency is increased during constant short stimulus duration. Up to intervals of 20-30 msec, the action potentials for single stimuli are hardly changed. The investigated movement receptors do not seem to show adaptation. With further shortening of the intervals between stimuli, the on-wave of each stimulus approaches more and more closely the off-wave of the preceding stimulus. When successive intervals are exactly twice as long as stimulus duration, one observes regular rows of alternating on- and off-waves. These action potentials then look exactly like those released by airborne sound; the action potential frequency is equal to the doubled stimulus frequency (WESTECKER, 1957). The potentials described in the investigations of BURKHARDTand SCHNEIDERas arising from airborne sound, thus seem to arise from the rigidly synchronized discharges at the beginning and ending of each torsional vibration stimulus. It is safe to assume, however, that these potentials are not an effect comparable with the microphonic component of the cochlear potential of birds and mammals. These potentials of the insect antenna are much more similar to the nervous component of the cochlear potential, the discharge of the first acoustic neurones. Especially in single stimulation with short rectangular impulses (‘click’) of alternating polarities, the potentials show a striking similarity to those registered when the bird cochlea is subjected to click stimuli of alternating polarities (SCHWARTZKOPFF,1958). In birds also the superimposed potentials of opposite polarities are only partially congruent; the amplitude is greater and the latency shorter for one of the two polarities. PUMPHREY (1940) has already pointed out the similarities between the action potentials in certain insect hearing organs and those of the higher vertebrates. If the stimulus duration is shortened, while the frequency is held constant and not too high, interactions between the on- and off-waves can be observed. With increasing short stimulus duration, the off-wave approaches more closely the series of small action potentials released by the damped oscillations of the stimulator, and finally the on-wave. It does not simply superimpose on these potentials; it is suppressed at certain stimulus durations, but develops fully at others. The latency between stimulus end and off-wave also changes during this shortening as follows: with small off-wave amplitudes a long latency is registered; with larger amplitude latency is shorter (Fig. 5). The height of the discharge-and thus probably the number of synchronously discharging elements-is thus determined for the offwave by the preceding discharges. These shifts in latency are such that the off-wave tends to occupy the position of one of the previous discharges. For example when stimulus duration is shortened, the latency is at first longer, so that it remains
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approximately at the same position where a discharge occurred with longer stimulus duration. When the stimulus duration is further shortened, the latency decreases abruptly, so that now the off-wave occupies the previous position of an earlier-occurring discharge. One must therefore assume strong interactions between the nervous elements participating in the on- and off-effects.
0
2
L
6
8 stimulus duration
10 [msec]
FIG. 5. Dependence of amplitude and latency of the off-wave upon stimulus duration (with short stimuli). The first arrow on the abscissa indicates the time of occurrence of the peak of the on-wave; the following arrows those of the later smaller action potentials. The momentary location of the off-effect on the, abscissa can be calculated as follows: the latent period (the ordinate value) must be added to stimulus duration. When this is done, the abscissa must be viewed as the time co-ordinate. Because of the shifts in latency, the offwaves tend to occupy the position of preceding discharges ; their amplitude thereby changes in the opposite direction to latency.
Two possibilities might be considered here: (1) the elements participating in the on- and off-effects inhibit each other reciprocally; or (2) the same elements participate in the on- and off-effects ; therefore these elements are refractory after the on-wave for a certain period and cannot contribute to the off-wave. By means of periodic stimulation with small variations in stimulus duration or frequency, one can obtain either an action potential synchronous with the stimulus (by suppressing the off-wave) or one with a frequency twice that of stimulation (when the off-wave is fully developed), because of the described interaction (Fig. 6). By means of a certain shortening of the stimulus, the off-wave is suppressed as it approaches the on-wave. In a similar way, by appropriate prolongation of the stimulus, the on-wave of the succeeding stimulus can be suppressed as the preceding By small opposite variations in stimulus off-wave approaches more closely. duration, the on- and off-effects can thus be alternately suppressed; by this means the phase of the action potential can be shifted almost 180” with respect to the stimulus while maintaining the same frequency.
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Minute changes in the stimulus pattern thus cause extreme alterations in the spatio-temporal excitation pattern in Johnston’s Organ. Such nervous effects can only be analysed by isolated investigations of single or few active elements with microelectrodes, and by a careful study of the excitation pattern. A summary starting from the total organ and an evaluation of the effect from a formal physical viewpoint (‘Klirrfactor’ of TISCHNER,1953 and KEPPLER,1958) is not suitable for the biological function of such a complexly-built organ. During rectangular stimuli of long duration, it was sometimes possible to record spike discharges of single nervous elements throughout the duration of the stimulus, in addition to the described on- and off-waves. When the direction of rotation was reversed, this discharge was visible before and after the stimulus, but during the stimulus it was obliterated (Fig. 7). In addition to the phasic receptors responding only to movements therefore, the antennae of Calliphora also possess tonic receptors which react to the deflexion itself (BURKHARDTand SCHNEIDER, 1957; in Locusta migratoria UCHIYAMA and KATSUKI, 1956). However, these receptors seem to be very rare. On the basis of present experiments it cannot yet ‘be decided whether we are dealing with elements of Johnston’s Organ. or with other position receptors. STADTM~~LLER (1955) describes innervated hair tufts between funiculus and pedicellus, which might conceivably participate in the indication of position. By stronger single deflexions of the antenna, one frequently observes a dense, irregular series of relatively large action potentials about 200 msec after the stimulus onset. Observations with the binocular microscope show that the antennae are thereby actively moved. Probably these action potentials are muscular action currents. Apparently the position of the antennae is actively changed by the animal under strong stimulation (cf. HERAN, 1957). In a previous work (BURKHARDTand SCHNEIDER,1957) it could be shown that when the antennae are subjected to a steady air current, irregular spike-like potentials arise. The antennae are made to tremble by the air-flow (by air eddies ?). This result could be confirmed by leading off with microelectrodes. The oscillograms give the impression that during the stimulus by air current, the antenna reacts with an irregular pattern of on- and off-waves (phasic action potentials), similar to the waves which respond regularly to airborne sound. The majority of receptors in the antenna are phasic receptors which respond only to movement. The question arises therefore, whether it is possible that these phasic receptors are also capable of registering mean deflexion of the antennae. It is thought that this could be made possible by the following mechanism: by continuous vibration of the antennae the phasic receptors are continuously re-stimulated. With increasing angle of rotation the number of elements excited also increases. In addition the peak of excitation in the spatial pattern of the sensory cells will shift with various angles of rotation. Thus, through the spatio-temporal pattern of excitation of Johnston’s Organ, the CNS can receive exact information about the strength and time course of the antenna1 deflexion.
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A very exact resolution of the time course of air flow stimuli is ensured by the preponderance of phasic receptors in the antenna1 joint between funiculus and pedicellus. Thus it is not surprising that this kind of current sense organ possesses a great many of the properties of a hearing organ, as shown by the investigations of the build-up of excitation with rhythmic stimulation and comparisons with the bird ear. ZUSAMMENFASSUNG
Die Erregungsmuster im Johnston’schen Organ von Calliphora erythrocephala werden mit Mikroelektroden bei mechanischer Reizung der Antenne untersucht : Ein elektromechanischer Reizgeber dreht den Funiculus wie bei natiirlichen Reizen urn seine LHngsachse ; der Zeitverlauf des Reizes ist rechteckfiirmig. Durch Ver&rderung von Intensitat, Polaritat, Dauer und Folgefrequenz der Reize kann der Aufbau der Erregungsmuster bei verschiedenen Reizmustern verfolgt werden. Aus den abgeleiteten Aktionspotentialen muss geschlossen werden, dass die iiberwiegende Mehrzahl der nervijsen Elemente nur auf Bewegungen anspricht und zwar auf jede Bewegungsphase mit einem einzigen Spike: phasische Receptoren. Ein Teil der Elemente scheint auf jede Bewegung unabhangig von der Drehrichtung anzusprechen, ein Teil nur auf Bewegungen mit einen jeweils bestimmten Drehsinn. Bei kurzer Reizdauer kijnnen starke Wechselwirkungen zwischen den durch Reizbeginn und Reizende ausgelijsten Aktionspotentialen beobachtet werden. Diese Wechselwirkungen bedingen, dass bei periodischer hochfrequenter Reizung (bis 500 Hz) geringfiigige Anderungen des Verhaltnisses zwischen Reizdauer und Pausendauer einschneidende Veranderungen des Erregungmusters zur Folge haben. Durch das ijberwiegen phasischer Receptoren im Funiculus-PedicellusGelenk ist die Fliegenantenne ein Sinnesorgan, welches auf eine besonders genaue zeitliche Aufliisung von StrGmungsreizen spezialisiert ist. Diese Spezialisierung bedingt, dass die Antenne bereits Eigenschaften eines Geharorgans besitzt: Sie spricht auf Luftschall bis zu Frequenzen von 500 Hz an, und die Erregungsvorgange bei einmaligen kurzen Rechteckreizen (Klicks) zeigen auff%llige Parallelitaten zu den nervcsen Komponenten des Cochleapotentials der Vogel unter gleichen Reizbedingungen. REFERENCES BURKHARDTD. (1954) Rhythmische Erregungen in den optischen Zentren von Calliphora erythrocephala. Z. vergl. Physiol. 36, 595-630. BURKHARDT D. und SCHNEIDERG. (1957) Die Antennen von Calliphora als Anzeiger der Fluggeschwindigkeit. Z. Naturf. 12b, 139-143. HERAN H. (1957) Die Bienenantenne als Messorgan der Fluggeschwindigkeit. Naturwissenschaften 44,475. HUBEL D. H. (1957) A tungsten microelectrode for recording from single units. Science 125, 549-550. KEPPLERE. (1958) Uber das Richtungshijren von StechmGcken. Z. Naturf. 13b, 286-284. PUMPHREY R. J. (1940) Hearing in insects. Biol. Rev. 15, 107-132.
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SCHWAETZKOPFF J. (1958) Uber den Einfluss der Bewegungsrichtung der Basilarmembran auf die Ausbildung der Cochleapotentiale von St&x variu (Barton) und Melopsittucus undulatus (Shaw). Z. vergl. Physiol. 41, 35-48. SCHWAETZKOPFF J. (1959) Der akustische Reiz und die GehSrserrregung. Verb. dtsch. zool. Ges, Miinster 1959, in press. STADTM~LLERR. (1955) Untersuchungen zur Anatomie und Histologic der Antennen von Calliphora erythrocephala. Zulassungsarbeit ftir das Staatsexamen, Wiirzburg. TISCHNERH. (1953) Uber den Gehiirsinn der Stechmticken. Acustica 3, 335-343. UCHIYAMAH. and KATSUKIY. (1956) Recording of action potentials from the antenneal nerve of locusts by means of microelectrodes. Physiol. camp. 4, 154-163. WESTECKEE M. (1957) Elektfophysiologische Untewuchungen der Antennenreaktion von Calliphora erythrocephala bei Luftschall. Inauguraldissertation, Wtirzburg.