With 1 plate and 10 text-figures Printed in Great Britain
LOCUST WIND RECEPTORS
I. TRANSDUCER MECHANICS AND SENSORY RESPONSE*!
BY JEFFREY M. CAMHIf
Biological Laboratories, Harvard University
{Received 17 June 1968)
INTRODUCTION
Wing movements in locust flight derive chiefly from a central neuronal signal generator determining the temporal pattern of flight muscle contractions (Wilson, 1961; Wilson & Weis-Fogh, 1962). However, several sensory inputs can modify this basic pattern in important, though subtle, ways (Weis-Fogh, 1949, 1956a; Goodman, 1959; Gettrup & Wilson, 1964; Wilson, 1963; Gettrup, 1966; Dugard, 1967; Waldron, 1967). One such input derives from the bank of aerodynamic sensory setae on the dorsal surface of the head in the desert locust (Schistocerca gregaria Forsk.). Guthrie (1964) found these to be trichoid sensilla possessing hollow shafts 70-250 fi long, and all curving forward in slightly different directions. He observed a single sensory neurone under each seta, responding to deflexion of the shaft.
Weis-Fogh (1949, 1950) discovered that directing at the setae of a tethered locust a ' head on' wind stream (analogous to the relative wind which the insect itself creates in free flight) results in flapping flight, provided that no tarsi make contact with solid surfaces. Flight continues as long as the wind flows. Weis-Fogh also observed (1949, 1950) that wind directed toward the head from one side, as occurs in free flight during a yaw, induces the locust to turn into the wind, in an apparent yaw correction man-oeuvre. The minimum angle of relative wind needed to produce the turn was about five degrees. However, since these locusts were severely encumbered by the measuring apparatus, the actual accuracy may be even greater.
The ability to respond to wind direction with such accuracy poses the question—by what means does the nervous system accomplish the directional coding implied by the behavioural response? Haskell (1959) briefly reported that wind flowing from the front or side of a seta evokes a slowly adapting train of sensory impulses, while flow from behind produces no response. Sviderskii (1967) extended these observations to the setae of Locusta migratoria. Guthrie (1964) suggested that a shaft's curvature in some way limits its freedom of motion, thereby imposing some directionality on the sensory response.
The experiments reported here were designed to provide a more complete answer to the question of direction coding, and to lay the groundwork for recordings made from interneurones, reported in the succeeding papers of this series (Camhi, 1969a, b).
• A preliminary report of this work has been published (Camhi, 1967).
t This work was supported in part by a predoctoral fellowship from National Institutes of Health. X Present address: Section of Neurobiology & Behavior, Division of Biological Sciences, Cornell
336 J. M. CAMHI
MATERIALS AND METHODS
All experiments were performed on male desert locusts (Schistocercagregaria Forsk.,
phasis gregaria), between 2 and 6 weeks after final moult. Locusts, obtained from the
Anti-Locust Research Centre, London, were maintained at 27 + 30 C. and were fed with fresh grass (fresh lettuce and dried grass during winter), bran middlings and water. Wind stimulation experiments required an open-throat wind tunnel having the following specifications: wind stream at least 8 mm. diameter, laminar over a velocity range of 0^5—4-5 m./sec.; capability to change very rapidly (within about 50 msec.) wind velocity with the range 0-4-5 m./sec.; capability to make equally rapidly changes of wind angle up to 150.
A glass tube of inner diameter 13 mm. met these requirements when connected by a hard rubber hose 3 m. long to the building compressed air supply, equipped with an accurate reduction valve. By fastening the glass tube to the rim of a wheel the wind could be directed from any angle towards the insect's head, located exactly at the wheel's central axis. The rubber hose was supported centrally above the insect in such a way that turning the wheel did not affect wind velocity. The open end of the jet was always 1 cm. from the locust's head, which was entirely within the region of laminar flow. A Flow Corporation (55A1) hot-wire anemometer served to calibrate the wind jet. For electrophysiological experiments, CO2 or cold anaesthesia immobilized the
insect during dissection. The exposed tissues, if kept in Pringle's (1938) or Weis-Fogh's (19566) saline at temperatures of 27 ± 30 C , remained responsive for hours. Saline-filled pipette electrodes, slipped over the cut tip of a seta's shaft, recorded a sensory response when the pipette deflected the shaft. Wind-stimulated sensory responses could be recorded by teasing out a nerve containing sensory axons and either drawing up a tiny bundle into a pipette electrode or laying a bundle across fine-tapered chlorided silver electrodes. Electrical stimulation was by chlorided silver electrodes. Nerve impulses were amplified by a differential input, capacitance-coupled amplifier (Grass P4), displayed on a dual beam oscilloscope (Tektronix 502A) and recorded on moving or stationary film with a Grass C4 camera. Electrical stimulation was by a Grass SD5 stimulator, producing rectangular pulses of less than 1 msec, duration.
RESULTS
(1) The sensory response to wind
Text-figure 3 is a polar plot of this cell's responses, showing the plateau frequency as a function of wind direction at three different flow velocities. As these curves indicate, for any flow velocity, the cell responds maximally to wind flowing in the plane of the shaft's curvature. In each of the five cells studied the optimal direction differed by less than 3° from the observed angle of shaft curvature. This was within the error of observation, about + 5°.
Shaft
Socket
Dendrite
Cuticle
Cell body
A x o n
SO/'
Text-fig. 1. Aerodynamic sensory seta, schematic drawing. All cuticular elements are stippled. Note the alignment of the two asymmetries—shaft curvature and dendritic attachment. Similar to diagram by Guthrie (1964).
^
200 ji V. 0 2 sec.
Text-fig. 2. Sensory responses to wind. Recorded in the left circumoesophageal connective. Wind at 4-5 m./sec. from io° left of centre. Upward arrow indicates the approximate moment of wind onset; downward arrow, wind cessation. Record reads left to right. The initial burst of spikes levels off within J sec. to a plateau frequency.
338 J. M. CAMHI
quantitatively as the angle between the two half-peak values of the curve. The morei acute the half-peak angle, the more accurate is the direction response.
The sensory response plotted in Text-fig. 3 is fairly directional, showing a half-peak angle of 540 ± 40 for the three wind speeds. Two other axons recorded gave values of 48° + 30 and 57° ±6°.
Text-figure 3 also shows that impulse frequency is approximately linearly related to wind velocity, a point which I shall consider in the Discussion section.
180°
Text-fig. 3. Wind direction against plateau spike frequency. Maximal response to wind from 15° left. Arrow indicates angle of seta curvature plane, 130 left. O, 4-5 m./sec.; x , 3-0 m./sec.; • , 1-5 m./sec. The graph shows that the sensory frequency depends sharply on wind direction, and is about linearly related to wind speed.
(2) Transducer mechanics
cross-section the shaft is circular with minute, regular flutings around the entire circum-ference.) There also existed the possibility of some structural asymmetry within the socket itself. I investigated each of these three potential sources of directional information—dendrite, socket, and shaft—under conditions precluding interference from the other two.
To determine the contribution of the eccentric dendrite attachment to direction coding, I recorded sensory responses using a pipette electrode fitted over the shaft tip.
180°
Tert-fig. 4. Deflecting direction against spike frequency. Recorded with pipette electrode over shaft. 50 /* deflexions. Maximal response to bending is from 20° left. Arrow indicates angle of seta curvature plane, 20° left. Note that this response is less directional than the sensory response to wind (Text-fig. 3).
With the pipette very firmly attached to a calibrated micromanipulator, advancing the manipulator could deflect the shaft a known distance in any desired direction. Bending always occurred at the socket, the rigid shaft retaining its shape during all movements. The use of tactile, rather than wind, deflexion precluded any possible aerodynamic effect of the shaft curvature. This method of stimulation probably also negated any influence of the socket's elastic resisting force because, by comparison, the force used here to deflect the seta was enormous.
Sixteen experiments gave complete sets of data. Text-figure 4 shows the polar curve for a typical seta deflected 50 /i in different directions. (Wind deflects a seta
34° J- M. CAMHI
about the same distance.) The optimal direction for each seta was within io° of its curvature plane. The frequency at this optimal direction was about ioo spikes/sec. Half-peak angles for the i6setaewere85° ± 130. This, then, is a less directional response than that of the sensory axon in wind, and therefore the eccentric dendrite attachment cannot by itself account for the directional properties of the receptors.
Determining any preferred direction prescribed by the socket's elastic force called for a method of comparing the magnitudes of the forces required to deflect the shaft in
180°
Text-fig. 5. Deflecting direction against relative deflexion force. Bending seta 50 /* required least force from about 200 right. Arrow indicates seta curvature angle, 27° right. This socket
behaviour is much less directional than the wind-stimulated sensory response (Text-fig. 3).
different directions. Carefully excising a small strip of cephalic cuticle and gently peeling away its loose hypodermis did not visibly deform the cuticle or setae. Such strips could be mounted on a transparent glass dial which was rotatable through 3600, and individual setae could then be observed with transmitted light through a compound microscope. It was crucial, for the avoidance of optical errors, that the tip and base of any seta measured be in perfect vertical alignment.
that the loop must advance to produce a given shaft deflexion is proportional to the socket's elastic resisting force.* I determined the shaft deflexion using an ocular micrometer, and the loop movement by reading the manipulator's calibration. At different angles (relative to the direction of shaft curvature) I imposed on the shaft a 50 [i tip deflexion and recorded the loop movement. Repeating the measurement at different angles (different settings of the transparent dial) allowed determination of the relative force as a function of angle. Complete sets of data were taken for eight setae. Text-figure 5 shows a typical polar plot of relative force for a 50 ji deflexion of a freshly prepared seta. As the graph indicates, the socket does impose upon the shaft a preferred direction. However, the half-peak angles for the eight sockets studied were very broad, 1830 ± 250. The optimal directions again were closely similar to the direc-tions of shaft curvature.
Studies of the socket's morphology indicate a possible structural basis for this mechanical behaviour. Plate 1 show photomicrographs of the outer and inner surfaces of a strip of cuticle just after its excision from the head. Just behind each seta is a clear cuticular specialization, visible from either surface. Because sectioning this material proved difficult, I did not attempt a detailed structural investigation. However, it seems reasonable that the clear area is related to the socket's force asymmetry, since its outline is almost identical in shape, and just opposite in orientation to the polar curve of the deflexion force.
In order to study any possible aerodynamic influence of the shaft curvature upon the seta's direction discrimination, it was necessary to replace the direction-giving socket with an ' ideal', non-directional socket. An individual shaft could be plucked or excised delicately from its socket and remounted in a tiny drop of rubber cement applied to the surface of the transparent dial. Holding the shaft in place with a tiny probe on a micromanipulator allowed the glue to dry with the base and tip in vertical alignment. Perfect alignment was crucial for avoiding optical errors of measurement.
A preliminary test, using the silk fibre deflecting method, indicated whether such a newly synthesized socket was in fact non-directional (polar curve a circle about the zero point). I selected 12 such non-directional mountings for further study.
A Pasteur pipette replaced the wider tube of the wind jet and directed towards the shaft a horizontal stream of wind. Flow was laminar at all velocities used. When the wind flowed, the shaft deflexion, measured by an ocular micrometer, was proportional to the shaft's aerodynamic drag for that particular angle between shaft curvature plane and wind direction. Repeating the measurement at different rotational settings of the dial allowed a determination of drag as a function of angle, f
Text-figure 6 shows the polar curve of a typical shaft's relative aerodynamic drag for different wind velocities. The curve is bimodal, the two maxima corresponding closely to the direction of shaft curvature and the exact opposite direction. The half-peak angles for the front mode (angle of curvature) of the 12 shafts measured were 1030 ± 120. The opposite mode gave more variable readings. However, this latter mode has no
• The elastic characteristics of the silk fibre are unknown. If the elastic modulus were not a constant, the measurements reported here would underrate, rather than exaggerate, any asymmetrical forces of the socket.
342 J. M. CAMHI
obvious biological significance, since deflecting a seta forward elicits no sensory response (Text-fig. 3).
In summary, then, each shaft's asymmetric drag, each socket's asymmetric elastic force, and each dendrite's eccentric attachment contributes in registering that sensory cell's preferred direction. No one of these three factors appears sufficiently sharp directionally to produce by itself the high degree of angular sensitivity observed in wind-stimulated sensory recordings. Presumably the sensory cell sums the three directional effects to produce its more accurate angular discrimination.
180°
Text-fig. 6. Wind direction against shaft relative drag. Drag maximal for wind from 8° right. Additional peak for wind from 175° left Wind velocity: 0-3 m./sec., 0-1-5 m./gec. Arrow indi-cates seta curvature direction, 9° right. This response is less directional than the wind-stimulated sensory response (Text-fig. 3).
(3) Direction optima for the entire sensory organ
343
(4) Functional neuroanatomy
Guthrie's (1964) light-microscopic observations suggested that at least some of the sensory cells have processes which traverse the entire brain, synapsing in either the suboesophageal, or one of the thoracic ganglia. To investigate this question more fully I first recorded the wind responses of single axons in the circumoesophageal connective. Then, after locating any individual seta whose deflexion would evoke a response in this
Antenna
Ocellus
^ Compound eye
Text-fig. 7. Map of shaft curvature plane angle, for largest seta shafts. Smaller shafts, not shown here, curve as their larger neighbours. The shaft angle is almost identical to the seta's optimum wind direction.
axon, I recorded simultaneously from the axon and the cut tip of the seta. Owing to the small axon diameters, only three successful experiments were performed.
As Text-fig. 8 shows, every impulse was recorded by both electrodes, demonstrating that either no synapses or only one-to-one conducting synapses intervene in the brain. The latency separating the two records of any action potential was about 10 msec. Deflecting any other seta neither elicited an axon response nor altered the axon spike frequency evoked by deflecting the first seta. In each case, the circumoesophageal connective was ipsilateral to the seta inducing the response.
344 J- M. CAMHI
(Text-fig. 9, upper trace). In each of the more than 40 setae studied, the latency was 7-14 msec, giving a conduction velocity of about 0-5 m./sec, a value consistent with the small axon diameter. Contralateral setae gave no response. This antidromic through-conduction requires that if any synapse exists on the pathway from set a to suboeso-phageal ganglion it must be non-polarized. Since Guthrie's (1964) observation
sug-1OO/(V.
0 5 sec.
Text-fig. 8. Simultaneous recordings from a sensory seta and an axon of the circumoesophageal connective, as illustrated in the diagram. Each action potential is recorded by both electrodes.
sens, sensory setae; br, brain; to, suboegophageal ganglion; tu t,, t3 resp., 1st, 2nd and 3rd
thoracic ganglia; Rlt Rlt two recording positions.
r~ "
it*-*—•—w
,100
_ J /.V. 20 msec.
100
1 «V. 40 msec.
,100
20 msec.
gested no brain synapses for at least some sensory cells, and since non-polarized synapses are relatively rare, the most likely condition is that all aerodynamic sensory cells have axons which traverse the brain without synapsing.
When recording from a cut seta tip, stimulation of the cervical connective (just posterior to the suboesophageal ganglion) evoked no response in more than 30 setae tested. This finding suggests that, contrary to Guthrie's (1964) observations, the sensory axons synapse in the suboesophageal ganglion, probably none of them continuing through to the thorax.
(5) Accessory neurone
If, when stimulating the circumoesophageal connective, the stimulus voltage was increased to two or three times threshold, a second unit could be recorded by the pipette over any ipsilateral seta. For each of 22 setae studied, this 'accessory neurone' gave a lower amplitude response and was always slower conducting (latency about 25 msec.) than the sensory cell (Text-fig. 9, middle trace).
MOO^V.
20 msec.
100AV.
0-2 msec.
Text-fig. 10. Pipette recordings over a seta, (a) Sensory impulses (large spikes) produced by deflecting shaft. Accessory cell impulses (arrows) evoked by 1-8 V., i msec, stimulation of cervical connective. Accessory cell spike causes no change in sensory inter spike interval. (6) Continuous sensory discharge produced by deflecting seta. Stimulation of anterior prothoracic connective, 2-2 V., i*a msec., repeated at io/sec. Accessory cell responses (arrow) produced no consistent change in sensory interspike interval.
Stimulation of the cervical connective, while not evoking a sensory response, did induce, in 12 setae studied, one accessory cell impulse per shock (Text-fig. 9, lower trace). The latency was some 30 msec. The accessory cell impulses were again of lower amplitude than sensory spikes produced by simultaneously deflecting the seta. Stimulation of the connective between the prothoracic and mesothoracic ganglia never produced a response in the 12 setae studied. Thus the accessory neurons link the sensory setae with the prothoracic ganglion. This pathway presumably either lacks synaptic interruption, or includes one-to-one transmitting synapses.
346 J. M. CAMHI
sensory cell under each seta (Guthrie, 1964, and this paper). Nevertheless, the possi-bility remained that the accessory cells were sensory in function. Careful scrutiny of the oscilloscope records following bending, twisting or otherwise manipulating the shaft with a recording pipette always revealed only the large sensory spikes. Adding grass extracts or various sugars to the saline of the recording pipette also produced no accessory cell response, though deflexion by such a pipette evoked sensory spikes.
Another possibility was that the accessory neurones function as efferent controls of the sensory cells. Histological studies reveal no muscle cells in the area of the setae, but synaptic control upon the sensory cell itself remained to be tested. Guthrie (1964) cauterized the cephalic cuticle of locusts, destroying most or all of the sensory cell bodies. After allowing 3 weeks for nerve degeneration, he found histologically that some axons persisted in the sensory nerve. One possible interpretation is that the remaining axons belong to efferent neurons.
To see whether the accessory neurones exert efferent control on the sensory response, I first deflected a seta with the pipette electrode, evoking a train of impulses. After a few seconds I stimulated, with repeated shocks, the cervical connective. Each stimulus evoked one spike in the accessory cell, but no sensory impulses. The appear-ance of an accessory cell spike does not correlate with any consistent changes in sensory cell inter-spike interval (Text-fig. 10). The accessory cells therefore appear not to function as efferent controls of the sensory response.
All attempts to evoke an accessory cell response by stimulating more posterior or peripheral parts of the nervous system, including the peripheral nerves to the pro-thoracic ganglion, were without result. Thus both the function and the central con-nexions of these cells remain unknown.
DISCUSSION
The experiments reported in this chapter show that each aerodynamic sensory cell responds to wind with a train of impulses whose initial high frequency adapts to a maintained plateau level. Each sensory cell responds maximally to wind flowing in a specific direction. It is important to recall, however, that the frequency of a wind-stimulated sensory response is a function of both wind direction and wind velocity. This means that without some independent velocity measurement the locust could not employ individual sensory cells to achieve an accurate direction reading such as that implied by Weis-Fogh's (1950) behavioural experiments. As the next paper in this series will show (Camhi, 1969 a), this requirement is met in an interesting way by certain intemeurones.
One factor determining the directional nature of the sensory response was the varia-tion with wind angle of a shaft's aerodynamic drag. This was in one sense a surprising result. Aerodynamic theory prescribes that in conditions of low Reynolds number, drag is determined primarily by overall surface area, and not by the physical conformation of the object. Since the Reynolds number of a single shaft at physiological air speeds is very low, one should expect drag to be relatively independent of wind angle. The empirical result is therefore difficult to reconcile with theory.
sensitivity of the sensory response. However, since each of these three factors is measured in different units (drag, elastic force, and spikes per second) it is impossible to sum these quantitatively in any meaningful way. It is therefore also impossible to determine which, if any, of the three factors exerts the greatest effect in determining the directional properties of the sensory response.
Finally, Nicklaus (1965) has shown that responses from wind receptor hairs on the cockroach cercus are directionally sensitive. Those hairs resemble in many ways the ones presently investigated, suggesting that organs of this type may be fairly general features of insect sensory equipment.
SUMMARY
1. The sensory cell innervating each wind-receptor hair on the face of the desert locust responds to wind with a slowly adapting train of impulses.
2. Each sensory cell responds maximally to wind flowing in a specific direction. The optimal direction for any sense cell is the same as the angle of curvature of its hair shaft.
3. The optimal wind direction has been determined for each sensory cell of the organ.
4. Three independently measured factors determine a sense cell's direction re-sponse : drag asymmetry of the curved shaft, elastic force asymmetry of the socket, and the eccentric attachment of the dendrite.
5. The sensory cells probably continue uninterrupted through the brain, synapsing first in the suboesophageal ganglion.
6. An accessory neurone of unknown function and unidentified central connexions links each seta with the prothoracic ganglion.
My thanks to Professors Ian Cooke and Kenneth Roeder whose critical reading of this manuscript was most helpful.
REFERENCES
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WEIS-FOGH, T. (19566). Tetanic force and shortening in locust flight muscle. J. exp. Biol. 33, 668-84. WILSON, D. M. (1961). The central nervous control of flight in a locust. J. exp. Biol. 38, 471-90. WILSON, D. M. (1963). Astretch reflex controlling wingbeat frequency in grasshoppers. J. exp. Biol. 40,
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EXPLANATION OF PLATE
Plate 1
JEFFREY M. CAMHI (I)
