J. exp. Bid. (1981), 9». i43-"S3 143With 7 figures

Printed in Great Britain

ELECTRICALLY EVOKED COURSE CONTROL IN THE FLYCALLIPHORA ERYTHROCEPHALA

BY JEAN BLONDEAU*

Max-Planck-Institut fiir biologische Kybernetik, Spemannstrasse 38, D-7400 Tubingen

(Received 28 July 1980)

SUMMARY

Neurones in the optic ganglia of free-moving and fixed CalHphora wereelectrically stimulated with extracellular microelectrodes. The maximumdiameter of the stimulating focus was 20 /im in the antero-posterior axis.Course control and landing manoeuvres could be elicited by stimulating inthe lobula plate but not in the lobula or medulla. With stimulation in thevicinity of the H neurones, yaw responses were evoked. Direction of theresponse was dependent on stimulation polarity. This supports thehypothesis that H neurones mediate optomotor responses. Stimulation inthe posterior part of the lobula plate, close to the V neurones, elicited pitch,lift and thrust responses as well as landing reactions.

INTRODUCTION

The neuronal basis of optomotor responses in insects has been the subject of manyinvestigations (for a partial review see Hausen, 1977). A set of giant neurones in thelobula plate of the fly (Braitenberg, 1972; Pierantoni, 1973; Pierantoni, 1976), havebeen shown to be sensitive to movement in a large part of the visual field (Bishop &Keehn, 1967; Bishop, Keehn & McCann, 1968; Dvorak, Bishop & Eckert, 1975;Hausen, 1976; Eckert & Bishop, 1978). They make synaptic contact with a smallnumber of large interneurones which descend through the cervical connective to theventral ganglion. The lobula plate giant neurones thus form part of a link between thechain of visual processing neurones and the motor control centres in the thorax.

The present paper describes the behaviour of free-moving and fixed Calliphora inresponse to electrical stimulation within the optic ganglia.

Animals MATERIALS AND METHODS

Experiments were carried out with females of Calliphora erythrocephala, 6-10 daysold, from the Institute's stock. A few control experiments were performed withmales within the same range of age and weight. Only flies which showed flightactivity in the breeding cages were selected for the experiments. Care was taken touse animals that looked completely healthy, as many flies have damaged wings afterieing in the breeding cages for a few days.

•* Present address: Rflntgenring 11, UniversitBt WUrzburg, Institut fiir Genetik und Mikrobiologie.

144 JEAN BLONDEAU

Room conditions

All experiments were carried out at room temperatures of 19-21 °C and 50-70%relative humidity. Ambient illumination in the room was normally 60-150 lx, andoccasionally 5 lx.

Preparation of animals

Flies were briefly anaesthetized with carbon dioxide (for about 5 s) and glued toa cardboard triangle by means of a molten mixture of beeswax and resin or, asa control for the effect of heating with wax, with dental epimine plastic (Scutan).The cemented point was located frontally on the thorax so as to leave the flightmovements undisturbed. The fly was fixed to a rack which allowed the head to bepushed down under microscopic control, without damaging the cervical connective.A paper barrier was put around the neck of the fly to prevent the animal from puttingits feet in the prepared part of its head. The head was pushed down to an angle ofabout 700 from the normal position. A small hole (0-5 x 0-5 mm) was made in theback wall of the head just behind the lobula plate, using a broken razor blade. Theair sac, which then became visible, was gently pushed to the side, but not removed.The proboscis muscles had to be sectioned, as their contraction induce largemovements of the brain. Fat tissue, which sometimes covered the ganglion, wassucked out with a fine tube connected to a vacuum pump. The lobula plate was theneasily accessible. A tungsten reference electrode was glued with the wax mixtureto a heating wire driven by a micromanipulator and was gently pushed against thelobula plate surface, as close as possible to the position projected for the stimulatingelectrode, but without penetrating the ganglion. This pressure produced very goodmechanical stabilization of the tissue in the vicinity of the stimulated point.Subsequently, the electrode's shaft was cemented to the head's wall by a minute dropof Scutan cement and freed from the manipulator after the wax joint has been meltedby the heating filament. The tungsten stimulating electrode was pushed just farenough into the tissue to bring the shoulder at the tip (see Fig. 1) into contact withthe surface of the ganglion, and was then cemented to the wall of the head. Theremaining hole in the head was covered with dental cement. The head was broughtback into its normal position and fixed to the thorax with the wax-resin mixture. Thewires leading to the electrodes were cemented to the cardboard support. The fly wasthen fed. After it had rested for one hour, its behaviour was tested. An animal wasconsidered normal if it tried to escape and fly away when attempts were made tocatch it from any direction and if no obvious signs of pathological behaviour weredetected. The fly was then left undisturbed for a further two hours before anexperiment.

Electrodes

Insulated tungsten electrodes (Fig. 1) were used for most experiments. Just beforeuse, the insulation was removed from the electrode tip by sending a short currentpulse (1 /iA for 10 to 20 ms) through the electrode as its tip was brought into contactwith the surface of a bath of either etching fluid or mercury. Mercury was used whe^the exposed area was to be very small (1 fim or less).

Electrically evoked course control in C. erythrocephala

5-8

2-4 (im-

4-Ea.

Fig. i. Tungsten electrode for chronic implantation. The shoulder in the electrode, at20-200 /*m from the tip, was formed by electrolytic etching. The electrode is inserted into thenervous tissue until the shoulder prevents further penetration. This ensures better precisionand stability of the electrode tip's depth than would be possible by simple stereotacticpositioning. The connecting wire was made of copper, io fim in diameter.

Glass microelectrodes were used for some experiments with fixed flies. Theelectrodes were filled with physiological saline (modified from Case, 1957) or withioomM cobalt chloride solution in water ( + 5% gelatine to avoid leakage if theelectrode tip diameter exceeded 2 /im). The electrode tip was finally ground to therequired diameter (1 to 10 /im) and bevelling angle (about 450) on a grinding wheel.

StimulationCurrent stimulation was applied from a constant current source (made in the

institute's workshop) controlled by a pulse generator (T. Sachs). The intensity ofpne stimulating current was chosen to be a function of the surface area of the exposedtip. A value of 10 nA/(/im)z was never exceeded. Generally, stimulation was made

146 JEAN BLONDEAU

with negative impulses of 0-5 to o-6 ms duration that were spaced by 5-200Stimulation was also attempted with positive impulses and DC current injection ofeither polarity. To ascertain that the stimulus location coincided with the tip of theelectrode, the reference electrode was placed at different positions in the body ofthe fly. This was repeated for different positions of the stimulating electrode. Thereaction of the fly always depended on the postion of the stimulating electrode andnot on the position of the reference electrode as long as the total current and currentdensity was kept low (current below i/iA; current density below 10 nA/(/mi)2).Total currents of more than 5 /tA elicited traumatic reactions such as convulsionsand coma; current density above 10 nA/(/*m)2 produced tissue coagulation in thevicinity of the electrode tip.

Stimulus location

The stimulating electrode was stereo-tactically brought into place. To know theposition of the stimulated zone with respect to anatomical landmarks, it was necessaryto generate a histologically visible trace of the tip of the electrode. When tungstenneedles were used, marking was achieved at the end of the experiment by means ofa high frequency current (1 MHz) from a signal generator, which coagulated thetissue in the area of the electrode tip and made the site of stimulation visible inhistological sections (Fig. za). When glass microcapillaries were used, the stimulatingelectrode was filled with cobalt chloride solution. After the stimulation, positivesquare impulses of 50 ms duration were sent 10 times a second through the electrodefor 10 to 20 seconds with an intensity of about 10 nA/(/im)a of electrode tip area.The brain was rinsed for 10 min with a 1 % ammonium sulphide solution in salineprior to fixation. Fig. 26 shows an example of such a marking experiment.

Histology

The brain was fixed overnight with a 2% glutaraldehyde solution in saline(at pH 7-2), dehydrated in alcohol, embedded in paraffin and cut at 15 fim thickness.

Recording of motor responses

Most of the reported results were obtained from freely moving flies. In thissituation, the animal has solely to carry the electrodes and part of the weight of theconnecting wires, which altogether and in the worst case amounted to a maximumof 10 mg. The two wires used to connect the stimulating apparatus to the fly were10 fim in diameter and about 1 m long. The effect of their stiffness on the fly'sbehaviour in most cases could be neglected but the fly's movements were limited bythe length of the wires. Responses were recorded with movie cameras (at 18 pictures/s).Event markers in the field of the camera indicated stimulation periods. The positionand orientation of the fly was measured frame by frame from the films.

Similar observations were obtained from the stimulation of fixed walking flies ina device kindly provided by E. Buchner (see Buchner, 1976). The fly was fixed ina clamp by the cardboard piece glued to its thorax and its head was tilted down asdescribed earlier. The head was kept in this position for the experiment, andhole in it was left open to allow electrodes to be inserted repeateadly. The fly

Journal of Experimental Biology, Vol. 92 Fig. 2(a)

50 fim

Fig. 2. Histological localisation of the tip of the electrode, (a) Frontal section through the fly'sbrain. The arrow points to the mark left by high frequency burning of the tissue at theelectrode tip (silver staining after Bodian). (6) Horizontal section through the brain. Thearrow points to the cobalt sulphide mark left by a glass microelectrode (unstained section - darkfield illumination).

BLONDEAU (Facing p. 146)

Electrically evoked course control in C. erythrocephala 147

^ a small polystyrene ball floating on an air cushion. In this way, electrically stimu-lated behavioural sequences were largely free of visual feedback. In these experimentsno motion pictures were taken and rotations of the ball were not recordedautomatically.

RESULTS

Electrical stimulation of the lobula plate was carried out successfully on a total of48 flies. Twenty-two of them were stimulated with chronic implanted tungstenelectrodes; the remaining 26 were stimulated with glass capillaries. A further 8 trialsended unsuccessfully (i.e. no reaction could be elicited at the indicated current levels).No reaction could be evoked that would not fit with one of the types described below-

The effects of electrical stimulation of the lobula plate depended upon positionof the stimulation. Three functional layers were found, roughly parallel to the frontof the lobula plate.

Stimulation of the frontal third of the ganglion produced very pure yaw manoeuvres,in both flying and walking flies. Direction of the manoeuvres depended on the polarityof the stimulus. Negative current pulses (i.e. the stimulating electrode was the cathode)caused the fly to turn in the direction opposite to the stimulated side. Reversing thestimulus polarity also reversed the direction of the evoked yaw (Figs. 3 and 4). Thisexperiment was extremely repeatable. Similar results were obtained over a widerange of stimulation frequencies (10 to 250 pulses/s with a pulse duration of 0-5 ms);even DC currents evoked yaw. With a fixed fly, the frontal layer of the lobula platewas probed dorso-ventrally and medio-laterally to find the region of highestsensitivity to electrical stimulation. Keeping the stimulation current constant, theintensity of the response appeared to be strongest when the lateral equatorial zonewas stimulated, getting weaker the more the stimulus was moved mediad, and noresponses or very weak ones could be obtained from the polar regions of the ganglion(Neural projection of zenith and nadir in the visual field). Transient responses ofopposite polarity to the reactions described happened in about 10% of stimulationsin the frontal layer of the lobula plate. These transients appeared at the beginningand/or at the end of the stimulation and were of short duration (Fig. 5).

Stimulation of the middle third of the lobula plate with negative impulses causedfixed and freely walking flies to walk sideways away from the stimulated side. Nosimilar effect was observed during flight. The walking reaction could be evoked overa wide field in the frontal plane (i.e. medio-laterally and dorso-ventrally). The mosteffective region for stimulation was found to be in the middle of the lobula plate.The response could only be obtained with trains of negative impulses (0-5 msduration and at rates of 50 to 200 impulses/s). D.C. currents and positive impulsesfailed to elicit the reaction.

Stimulation of the caudal third of the lobula plate evoked several reactions whichall involved manoeuvres in the sagittal plane (stimuli were negative impulses witha rate of 50 to 200 impulses/s).

Stimulation of the ventral middle third of this layer induced a landing reaction inflight (defined as a spreading of all legs without interruption of the wing beat)...ThisRiding reaction was paired with the return of the antennae to a more or less verticalposition (in flight, the antennae are thrust forward). A somewhat similar reaction

148 JEAN BLONDEAU

Fig. 3. Yaw reactions induced in a free-flying Calliphora by electrical stimulation of theanterior layer of the lobula plate. The diagram was obtained by the frame by frame analysisof films (constant motor speed of 18 frames per second). The plotted angles refer to thedirection of the fly at the beginning of the sequence.

Fig. 4. Effect of stimulation polarity upon the yaw induced in a freely walking fly. Stimulationwith negative impulses caused the fly to turn away from the stimulated side, positive impulsesinduced a yaw towards the stimulated side. The diagram illustrates a stimulation sequence inwhich the stimulus applied through a chronic electrode in the anterior layer of the lobulaplate was inverted after a short pause of about 10 s duration. The starting position has beenarbitrarily chosen o°. Stimulation does not account for the fine structure of yaw.

Electrically evoked course control in C. erythrocephala 149

Fig. 5. Typical transient antagonistic yaw response observed in a free-flying animal at thebeginning of a stimulation period. After a long period of fairly straight flight (unstimulated),at the onset of the stimulus, the fly first turns towards the stimulated side before swiftlytaking a sustained course away from the stimulated side.

was elicited when the electrode was inserted into the lateral border of the ganglion,but in this situation only the front legs were spread forward (see Eckert & Bishop,1978). This last reaction could, surprisingly, also be evoked in the walking fly, whichseemed to try to catch something in front of itself and above its head.

Stimulation of the lateral half of the caudal layer evoked upward pitch in flight.Stimulation of the medial half of the layer caused the walking fly to lower its body

onto the ground. In free-flying individuals, stimulation in this area caused an increasein lift and thrust together with upward pitch. This reaction started relatively lateafter the beginning of the stimulation. Fig. 6 is a rough summary of all the results.

DISCUSSION

The elicited behavioural responses had a co-ordinated appearance. It should beemphasized that at low current densities confused motor activity like tumbling,convulsion, leg shaking or rivalry between behavioural activities was never observed.When the stimuli were applied during flight, the forces exerted by the animal didnot decrease. When walking animals were stimulated, the evoked movements werefluent sequences, even when the behaviour evoked appeared but rarely in theunstimulated fly. All evoked behaviour was, however, observed in free-moving flies£ the absence of any artificial stimulation. It thus seems that the electrically evokedbehaviour patterns closely mimicked those existing naturally.

JEAN BLONDEAU

Horizontal section

Anterior

LobulaMedulla

Lobula platePosterior

Frontal section

Lobula plaitDorsal

u

Medulla

YawVentral

Sideslip

Pitch'(Lift/thrust)

Landing(front legs only

Landingreaction

(complete)

Fig. 6. Summary of the approximate delimitations of the areas in the lobula plate in whichresponses could be elicited by electrical stimulation. The hatched areas in the left row (a)represent these zones in a schematic horizontal section through the fly's brain; the right row (6)depicts the same domains in a frontal section. Marking of the stimulation points was not madein a way systematic enough to allow a precise mapping of the ganglion.

In the present study, care was taken to keep the focus of stimulation as small aspossible. Electrode tips were made small to concentrate the stimulating current, andcurrents were made small to reduce the probability that low resistive channelssomewhere in the neuropile could concentrate the current lines sufficiently to generatea 'ghost* stimulation point (Huber, i960). No precise measurement of the range ofthe stimulation could be made, but after a response was elicited, a rough indicationwas provided by the distance the electrode had to be pushed forwards before theresponse disappeared. This distance was 5 to 7 /tm with electrodes of less than 2 fimof exposed tip. A smaller value would be unlikely to be obtained since the range ofmechanical instability of the preparation was 5 /tin.

In comparing responses observed in this study with the available data for thjilobula plate neurones, due consideration should be made of the experiment™

Electrically evoked course control in C. erythrocephala IS 1

Fig. 7. General sketch of the distribution of the giant interneurones in the lobula plate ofCalliphora. This figure was kindly provided by K. Hausen (see Hausen, 1977). A, anterior;P, posterior.

situation. In the present experiments, the artificial commands were superimposed onthe flies endogenous movements. Furthermore, the evoked manoeuvres caused visualfeedback which was probably not suppressed by efference copy. The latter problemcould be eliminated using the apparatus described in the next paper (Blondeau, 1981).The problem was partly overcome in this study with fixed flies walking on a styrofoamball. In future experiments, white-eyed flies with 'neutralized' corneas (Francescini& Kirschfeld, 1971) might be used to achieve satisfactory open loop conditions, whileavoiding complete darkness.

Production of yaw by stimulation of the anterior region of the lobula plate (seeFig. 7 for a general sketch of the lobula plate's architecture) is in accord with thepresence of H neurones in this region (Pierantoni, 1973). However, the direction ofthe yaw response indicates a more complicated effect than just stimulation of theH neurones. Turns away from the stimulated side were made when the extracellularstimulation was made with negative pulses, which should depolarize nearby cells,Depolarization of H neurones should produce turns towards the stimulated sideiHausen, 1976). It should be noted, however, that transient responses of oppositepolarity were occasionally observed at the beginning and end of a stimulation period.

152 JEAN BLONDEAU

Current applied to the middle layer of the lobula plate can be expectedstimulate the V(2> neurones (Hausen, 1976). These neurones are depolarized byupwards as well as back to front movements in the ipsilateral visual field (Hausen,1976). The exact connection of V(2) cells are still unknown, but it seems probablethat they, too, make direct contact to descending neurones (Hausen, 1976). Thenature of the sideways walk that could be elicited in this medial layer is too littleunderstood to make a comparison with the properties of the V(2) cells meaningful(see also Eckert & Bishop, 1978). If this walking was caused by a transient rollreaction (counteracted by the autonomous balance mechanism of the thorax) itspolarity would be compatible with a direct effect upon the V(2) neurone (Hausen,1976).

Pitch, lift and thrust were elicited by stimulation of the posterior layer of thelobula plate. V and V(1) neurones which cover the posterior surface of the lobulaplate are depolarized by ipsilateral downward movement (Hausen, 1976; Eckert &Bishop, 1978). As in the case of the electrically stimulated yaw response, the polarityof the pitch response is the opposite of what would be expected if the negativestimulus were to depolarize V neurones. Recent behavioural studies on a mutant ofDrosophila (J. Blondeau, in preparation) indicate that V cells are involved in visuacontrol of pitch and roll. Optomotor lift and thrust responses seem to be independentof V neurones in Drosophila (Heisenberg, Wonneberger & Wolf, 1978). Theexcitability of these responses in the posterior layer of the lobula plate in Calliphoramight suggest the existence of other neurones in this layer which are involved inaltitude control, but the following explanation is at least as plausible. If one keepsin mind that in walking flies forward movement could never be elicited by electricalstimulation, the late appearance of the increase in lift and thrust after the onset of thestimulus in flight may indicate that these forces are a secondary effect of the pitchresponse in order to avoid stall and loss of altitude.

In summary, the agreement between the properties of neurones in the lobulaplate and the behavioural responses elicited by weak electrical stimulation in theirvicinity indicates the key role that individual neurones seem to play in invertebratebehaviour.

REFERENCES

BISHOP, L. G. & KEKHN, D. G. (1967). Neural correlates of the optomotor response in the fly.Kybernetik 3, 288-295.

BISHOP, L. G., KEKHN, D. G. & MCCANN, G. D. (1968). Motion detection by interneurona of opticlobes and brain of the flies Calliphora, Phoenicia and Musca domatica. J. Neurophytiol. 31, 509-525.

BLONDEAU, J. (1981). Aerodynamic capabilities of the fly Calliphora as revealed by a new technique.J. exp. Biol. 93, 155-163.

BRAITENBKRO, V. (1972). Periodic structures and structural gradients in the visual ganglia of the fly.In Information processing in the visual system of arthropods (ed. R. Wehner), pp. 3-15. Berlin,Heidelberg, New York: Springer.

BUCHNEH, E. (1976). Elementary movement detectors in an input visual system. Biol. Cybernetics 24,85-101.

CASE, R. (1957). Differentiation of the effects of pH and COi on the spiracular function of insect*.J. cell. comp. Physiol. 49, 103-113.

DVORAK, D. R., BISHOP, L. G. & ECKERT, H. (1975). Intracellular recordings and staining of direction-ally selective motion detecting neurones in the fly optic lobe. J. comp. Physiol. 100, 5-23.

ECKERT, H. & BISHOP, L. G. (1976). Anatomical and physiological properties of the vertical cellsMthe third optic ganglion of Phoenicia sericata (Diptera, Calliphoridae). J. comp. Physiol. ia6,

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ANCESCINI, N. & KIRSCHFELD, K. (1971). Etude optique in vivo des elements photorfcepteure daml'oeil compost de Drotophila. Kyberbnetik 8, 1-13.

HAUSEN, K. (1976). Functional characterisation and anatomical identification of motion sensitiveneurones in the lobula plate of the blowfly Calliphora erythrocephala. Z. Naturforsch. 31, 629-633.

HAUSEN, K. (1977). Struktur, Funktion und Konnektivitflt bewegungsempfindlicher Interneurone imdritter optischen Neuropil der Schmeissfliege Calliphora erythrocephala. Doctoral Thesis universityof Tubingen (W. Germany).

HEISENBERG, M., WONNEBERGER, R. & WOLF, R. (1978). Optomotor blind H11 a Drosophila mutantof the lobula plate giant neurones. J. comp. Phytiol. 124, 287-206.

HUBER, F. (i960). Unterauchungen Uber die Funktion des Zentralnerven-systems und insbesonderedes Gehirns bei der Fortbewegung und bei der Lauterzeugung der Grillen. Z. vergl. Phytiol. 44,60-13*.

PIBRANTONI, R. (1973). Su un strato nervoso nel cervello della Mosca. In Atti delta prima riuniarescientifica plenaria (Camogli, dicembre 1973). Soc. Ital. Biofis. Pura e Applicata, pp. 231-249.

PiERANTONi, R. (1976). A look into the cockpit of the fly. The architecture of the lobula plate. Cell Titt.Ret. 171, 101-122.