Single unit activity in the visual pathway of the butterfly Heliconius erato

J. Insect Physiol., 1968, Vol. 14,pp. 1589 to 1601.Pergmnon Press. Printed in Great Britain

SINGLE UNIT ACTIVITY IN THE VISUAL PATHWAY THE BUTTERFLY HELICOIVIUS ERATO*

OF

S. L. SWIHART Department of Biology, State University College, Fredonia, New York 14063 (Received 20 May 1968) Abstract-Extracellular potentials were recorded from the optic lobes, protocerebrum, and ventral nerve cord of the neotropical butterfly He&mius erato adanis. Except for pure ‘on’ and ‘off’ fibres, all neurons demonstrated a dark discharge. Neurons demonstrating a special sensitivity to unidirectional movement, jittery movement, dimming, unilateral stimulation, illumination level, etc., were studied. It is questionable whether neurons responding to such stimuli are homologous in all insects. A hypothesis concerning the neural processing of colour information is presented. INTRODUCTION

A NUMBERof recent events occurring

works have contributed

in the visual pathway

to the understanding

of insects.

of the neural

Most closely studied

have been

(BURTT and CATTON, 1954, 1956, 1959, 1960, 1966; HORFUDGE et al., 1965) and several nocturnal moths (BLESTand COLLETT,1965a, b). Each of these works emphasized a different portion of the visual pathway. Thus, for example, whereas Burtt and Catton concentrated on the medulla externa, intema, and ventral nerve cord, Blest and Collett emphasized the medulla intema and protocerebrum. It is, therefore, difficult to assemble a total picture of the mechanisms involved in the integration of visual information. This work reports a series of experiments which were conducted to determine the nature of the neural activity characteristic of the entire visual pathway of the neotropical butterfly Heliconim erato adank Several reasons suggested the choice of this organism. Firstly, was the background of behavioural information on this form (CRANE, 1955, 1957) which provided data concerning its visual orientation. Secondly, a considerable amount of information has been accumulated concerning the visual mechanisms of this species as revealed by analysis of summated potentials (SWIHART, 1963, 1964, 1965). Finally, the consideration of a butterfly would provide an interesting comparison with the diurnal locust and the nocturnal moths which, while closely related phylogenetically, are quite different behaviourally. Comparisons have been complicated by an unfortunate multiplicity of designations applied to specific neuron types. While both Burtt and Catton, as the diurnal

locust

* Supported by a grant (GB7097X) 101

from the National Science Foundation.

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well as Blest and Collett, utilized descriptive designations (e.g. on, fast-dark, etc.), Horridge et al. chose to introduce an ‘A, B, C’-type series. Both logic and precedent argue in favour of continuing with the descriptive-type designations.

METHODS

AND MATERIALS

These studies were conducted in Trinidad, West Indies. The insects were collected in the wild and maintained in large outdoor cages. Experiments were conducted in a darkened, air-conditioned laboratory. Intact organisms were rigidly mounted with Placticine. KCl-filled capillary electrodes (cu. 10 mQ) were inserted in a dorso-ventral axis through a hole in the head capsule drilled with a minuten pin. Occasionally, a small portion of the capsule, together with the underlying trachea, would be removed so as to assist in placing the electrode. The optic lobes of these insects are large and well developed (Fig. 1). After removal of the superficial trachea, both internal and external chiasma are readily apparent, thereby clearly delineating the major synaptic regions. It is therefore possible to assess with reasonable certainty in which layer the electrode is placed. On some occasions, electrodes were broken off in situ, and dissections were made to determine the path of electrode penetration. An earthed, indifferent, Pt electrode was inserted through the cuticle of the head capsule. Potentials were amplified with a Grass P-6-12 amplifier. Lowfrequency components and amplifier noise were removed with a Krohn-Hite filter. Potentials were displayed on a Tektronix 502 oscilloscope and recorded with either a Grass camera or Ampex tape recorder. As HORRIDGEet aE.(1965) point out, the properties assigned to a neuron in large measure reflect the nature of the stimuli that are utilized. Since it was frequently possible to record from a single neuron for several hours, a variety of stimuli could be employed. These included: (1) A mechanism for providing simple flashes. This consisted of a 100 W incandescent lamp, lens system, Compur-type shutter, and A.O. flexible light guide. One end of the latter was placed very close to an eye with a micromanipulator. This equipment would illuminate nearly the entire eye. A maximum stimulus energy of 15 mW could be delivered with this equipment; however, neutral density filters were usually employed to reduce this. Flashes with an energy of about 1 mW were most frequently employed. The responses to various colours were studied by introducing narrow band interference filters (Blazars) into this optical system. Measurement of the ERG response to these monochromatic flashes provided information concerning an appropriate neutral density filter that could be combined with each interference filter, so as to provide stimuli of equal physiological intensity at each wavelength. (2) In order to assess the response to movement, patterns, and the size of visual fields, a translucent projection screen was erected about 2 ft from the organism, in a plane parallel to the axis of the body. A small 100 W slide projector was mounted about 6 ft behind the screen on a rigid camera tripod. Since the tripod was

FI~. 1. Cross-section of eye, optic lobe, and brain of Heliconius erato. Composite photograph of unstained section, photographed with phase-contrast equipment. I,.G., lamina gangularis; E.C., external chiasma; M.E., medulla externa; I.C., internal chiasma; M.I., medulla interna; G.C., globuli cells; O.N., optic nerve; M.B., mushroom bodies.

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equipped with a pan-head, it was possible to move projected images about on the screen. Various images, including bright squares, disks, concentric circles, ellipses, etc., as well as reciprocal patterns (e.g., dark disks, bright surround) were employed. RESULTS BURTT and CATTON (1966) reported that they recorded spike activity most satisfactorily from the periphery of the synaptic regions of the locust optic lobe, perhaps from the somata of neurons sending fibres into the synaptic regions. These experiments tended to confirm this viewpoint. However, unlike Burtt and Catton, it was found that potentials could also be easily recorded from the butter-By protocerebrum.

Lam&a gangularis In this region, only small amplitude activity was detected. It is not clear whether this activity originates in first- or second-order neurons. Recording from these proved to be extremely difficult. Only on a couple of occasions were the electrodes small enough to clearly reveal the activity pattern characteristic of a single such cell. It was found that these fibres demonstrated a high level of spontaneous activity (dark discharge). The response to a simple flash of white light consists of a transitory increase in discharge frequency, at ‘on’, with very rapid adaptation to a level close to the dark discharge frequency (Fig. 2). Such adaptation is nearly complete within 20 to 50 msec after the onset of increased activity, depending upon the intensity of stimulation. There is no apparent change in the discharge frequency at ‘off’. If, however, the stimulus consists of a spot of light, larger than the visual field, which is moved slowly across the visual field, a burst of activity will occur at both ‘on’ and ‘off’ (Fig. 3). The discharge patterns of such fibres could be explained by assuming strong lateral inhibition (REICHARDT, 1961). Such interaction would account for the very rapid adaptation and would explain how, as the light moved across the eye and adjacent ommitidia were darkened, a cessation of inhibition would produce an ‘off’

effect. Medulla externa At this level, a wide variety of neuron types can be detected. Many of these are quite easy to record and to hold for long periods of time. Unlike locusts (HOFWDGE et al., 1965), however, the position of the electrode is most critical. Experiments to determine the speed of conduction by means of two electrodes recording from the same neuron would be quite impossible in butterflies. All the neurons that were found in this region had at least one property in common, a high level of spontaneous activity. These experiments have, therefore, necessitated a revision of the viewpoint (SWIHART, 1964)thatthe medulla extema contained phasic ‘on' neurons (see descriptionunder

Protocerehm).

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Fig. 6 FIG. 2. Micro-electrode recording from lamina gangularis. Stimulation with simple flash of white light, ca. 0.07 mW. Downward deflection of lower trace indicates period of stimulation (0.5 set). FIG. 3. Recording from lamina gangularis, demonstrating response to a moving spot of light (see text for details of technique). Lower trace records output of photocell placed near centre of visual field. Upward deflection indicates period of stimulation. Time-base is one-half that of Fig. 2. FIG. 4. Response of sustaining fibre of protocerebrum to stimulation with simple one-second flash. White light ca. 1-O mW. Thickened portion of lower trace indicates period of stimulation. Similar responses can be recorded from the medulla intema. FIG. 5. Response of medulla extema sustaining fibre. Stimulation with 1 set simple flash of white light (ca. 1-O mw), cf. Fig. 4. FIG. 6. Rhythmic spontaneously active neuron from medulla extema. Such fibres did not respond to photostimulation. Duration of recording, 2.5 sec.

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The types of neurons characteristically found in this region are as follows: (1) Sustaining jibes. Such spontaneously active neurons demonstrate both tonic and phasic responses to changes in light intensity. They respond strongly to simple flashes, with both ‘on’ and ‘off’ components. However, after a brief period of adaptation, the discharge frequency stabilizes at a level related to the level of ambient illumination. Such fibres have very large, poorly defined visual fields (cu. 47”). Two sub-categories of such neurons have been identified: (a) Those with a higher frequency discharge in the light than in the dark. Such fibres demonstrate pronounced ‘post-off’ inhibition. If stimulated with a flickering light, it can be demonstrated that re-illumination suppresses the appearance of ‘off’ responses. (b) The other type of fibre demonstrates a higher level of activity in the dark than in the light (Fig. 5). With flicker stimuli, the ‘off’ activity inhibits the appearance of ‘on’ responses. (2) Movement-sensitive neurm. Typically, such fibres would demonstrate rates of spontaneous activity as great aa 80 to 90 discharges/set. Such activity was relatively independent of the level of ambient illumination. While such neurons increased their rate slightly with an increase in light intensity, they adapted completely in about 15 sec. While responding only weakly to simple flashes, greatly increased discharge frequencies were produced by any image moving through their visual field, in a preferred direction of movement. Conversely, activity was strongly inhibited by movement in the reciprocal direction. Little or no response could be detected to movements perpendicular to the preferred axis. Fibres responding to movement along an anterior-posterior axis were found much more commonly than those preferring a dorso-ventrally orientated axis. Not uncommonly, preparations were encountered where it was possible to simultaneously monitor the activity of two neurons with reciprocal preferred directions, thus suggesting a ‘paired arrangement of such fibres (Fig. 7). It is possible that sub-categories of such fibres are justified. Thus, some of the movement fibres appeared to give considerably stronger responses to dark objects against a bright surround, while others ‘preferred’ a bright object. Such observations are, however, difficult to quantify, since the differences were in threshold values and not the nature of the response. Complex moving patterns, such as concentric circles, produced responses identical with that elicited by a simple disk pattern. While the visual field of such fibres is quite large (cu. 30”), very small movements are sufficient to produce excitation. With one preparation, strong responses were elicited in normal room lighting when the experimenter stood at a distance of about 10 ft and moved a 3 x 5 in. black card an inch or two. (3) Jittery movement j&-es. Such neurons have a high level of spontaneous activity under all levels of illumination (50-70/set). Simple flashes, slow movement, etc., produce negligible modulation of this activity. The only stimulus parameter found to be effective in producing an excitatory response was fairly rapid movement in any direction. The visual field is very large with diffuse boundaries. The response

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is extremely phasic and is followed by a period of inhibition of approximately equal duration. Fibres responding to similar stimuli have been described in the crayfish (WIERSMAand YAMAGUCHI,1967); however, the crayfish neurons do not demonstrate a dark discharge.

Fig. 7

656 m)r

$96 m)r Fig,

579m)c

52% my

8

FIG. 7. Recording demonstrating activity of two movement-sensitive neurons with opposite preferred directions (anterior and posterior axis). Stimulation effected with a moving projected pattern consisting of a bright ring with dark surround and centre. Lower trace indicates output of photocell mounted on rear projection screen, near centre of visual fields, Upward deflection indicates bright circle moving across photocell. Arrows indicate reversal of direction of movement. Note that fibres are inhibited by movement reciprocal to preferred direction. Duration of recording is 3.5 sec. FIG. 8. Recordings from two sustaining type fibres of medulla intema, demonstrating the responses to various monochromatic stimuli (100 msec duration) of matched physiological intensity. In the deep red (656 mp), the response is virtually entirely due to a single fibre with large amplitude spikes. At 616 m,u, the response of this fibre remains essentially identical, indicating that the intensities were matched; however, there is evidence of increased activity in a second neuron with smaller amplitude spikes. At 596 ml*, only 20 rnp shorter, the activity of the larger neuron is only slightly greater than the dark discharge frequency. At shorter wavelengths, only the smaller amplitude fibre demonstrates any response. If one assumes two retinene-based visual pigments, with absorption maxima separated by as much as 100 rnp (e.g. 520 and 620 mp), the region of effective overlap in absorption spectra would be four to five times greater than that demonstrated by these fibres. It is suggested that there exists a mutually inhibitory interaction between such elements.

(4) Rapidly adapting fibres. These neurons respond strongly to simple flashes with short bursts at ‘on’ and ‘off’. Periods of ‘post-on’ and ‘post-off’ inhibition, of variable duration, are characteristic. Visual fields are usually circular (25-30”). However, several fibres belonging to this category demonstrated elliptical fields with a dorso-ventrally orientated axis. These fibres are probably comparable to

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and CATTON’S(1960) D-units. Two sub-categories of these neurons were detected : (a) Fibres demonstrating Sensitivity to both ipsilateral and contralateral stimulation. Threshold values for both types of stimulation are approximately equal. No consistent differences in the nature of the response to the two types of stimulation can be observed. (b) Neurons responding only to ipsilateral stimulation. Such fibres characteristically demonstrate slight differences in the nature of the response elicited by stimulation with different wavelengths. Four distinctly different patterns have been recorded (Figs. 9, 10). Normally the differences, as indicated by various BURTT

FIG. 9. Diagramma tic representation of various responses of rapidly adapting medulla extema fibres, to monochromatic stimulation. Four kinds of variations in discharge patterns were detected. These were apparent in the degree of ‘post-off’ inhibition (Type A), ‘post-on’ inhibition (Type B), the magnitude of the ‘on’ response (Type C), and the ratio of the magnitude of the ‘on’ to the ‘off’ effects (Type D). simple monochromatic flashes, are slight. It was, however, found that these distinctions can be magnified by observing the response to a flicker consisting of alternate red and blue stimulation. This was provided by a pendulum-like device,

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which carried two interference filters adjacent to each other and swung back and forth in the optical path. Even though the physiological intensity of these colours was matched, some fibres would discharge only during ‘red’ stimulation, whereas others demonstrated the reverse pattern.

FIG. 10. Response of medulla externa rapidly adapting neuron to two wavelengths of monochromatic stimulation (matched intensities). Stimuli of 1 set duration (lower trace). Top trace indicates response to 420 rnp; centre trace demonstrates response to subsequent stimulation with 656 rnp. FIG. 11. Recording of activity of pure ‘off’ fibre from the medulla interna. Lower trace indicates 0.5 set stimulation with ccl. l-0 mW of white light. FIG. 12. Response of protocerebral spontaneously active ‘on’-type fibre to flashes of 20 and 100 msec duration (ca. I.0 mw).

(5) Non-responding neuron. Such fibres demonstrated high levels of spontaneous activity which did not appear to be modulated by photostimulation. Their activity was either a nearly uniform sustained discharge frequency or a rhythmic pattern consisting of five to twenty-five action potentials, followed by a quiet period of approximately equal duration (Fig. 6). Such activity patterns were monitored for extended periods and did not seem to be due to trauma. A discussion of the possible function of such neurons in Calliphora is presented by LEUTSCHERHAZELHOFF and KUIPER (1966). Medulla

interna

The medulla interna is very much smaller than the extema. Much of the tissue proximal to the internal chiasma is in the form of major tracts, passing through the region without apparent synaptic interaction. Only two types of fibres were found to be characteristic of this region. One of these was a spontaneously active neuron which produced a simple, tonic excitatory response when stimulated with a simple flash. Little or no ‘off’ response is produced. The second type of neuron represents the most peripheral element in the visual pathway which did not demonstrate a dark discharge. This was a pure ‘off’ fibre (Fig. 11). Three or four spikes represented the maximum response that could be elicited. Potentials recorded from such fibres were of unusually large amplitude, suggesting a neuron of very large diameter. Pure ‘off’ fibres are apparently absent from the locust visual pathway (BURTT and CATTON, 1960).

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Protocerebrum The protocerebral regions of H. erato are large and well developed with welldefined mushroom bodies. A variety of responses was recorded from this general region. Three of these appeared to have only excitatory inputs.

Fig. 13

A

Fig. 16

B

FIGS. 13 and 14. Response of protocerebral fibre demonstrating binocular interaction. Fig. 13(A) Response to 0.5 set ipsilateral flash. (B) Contralateral stimulation. Fig. 14. Binocular stimulation. FIG. 15. Protocerebral pure ‘on’ fibre. Stimulation with 0.5 set flash of white light, CQ.1.0 mW. FIG. 16. Recording from cervical connectives of the satyrid Taygetis oirgillia. (A) Response to one-second stimulation with ca. 1 *OmW. Note activity of descending ‘on’ and ‘off’ fibres, and ascending giant fibre. (B) Response to the same stimulus, after habituation of giant fibres.

One of these was a phasic ‘on’ response which otherwise resembled the ‘off’ fibre characteristic of the medulla interna (Fig. 15). The number of spikes produced in response to a simple flash was proportional to the stimulus intensity, up to a maximum of about twenty discharges, A second type of neuron which responded in a purely excitatory manner was also detected (Fig. 12). This type of fibre differed from the former in producing a somewhat more tonic response, with spikes of considerably smaller amplitude, and in possessing a low frequency dark discharge. Discharge frequency as well as duration of the response seems to be determined solely by the stimulus intensity. The

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duration of a simple flash (exceeding ca. 10 msec) has little or no effect upon the nature of the response. The third type of fibre can be termed sustaining (Fig. 4). The discharge frequently of such fibres rapidly adapts to a level which is proportional to the level of illumination. The response is very tonic and remains constant for an indefinite period of time. A pronounced ‘post-off’ quiet period is very evident. These neurons appear identical to those described under medulla interna however, they differ from the externa sustaining fibres in that they fail to demonstrate a burst of activity at ‘off ‘. A more complete discussion of such fibres may be found in SWIHART(1965). Two types of neurons demonstrating inhibitory synaptic inputs and a sustained dark discharge were detected. One of these responded with pure inhibition in response to stimulation with simple flashes (fast-dark fibres) (SWIHART, 1964). Small differences between such neurons were noted. Particularly apparent were variations in the extent of the post-inhibitory rebound at ‘off’. Such fibres lack the burst of activity at ‘on’ demonstrated by the medulla externa, sustaining neurons. The second type of neuron was one of the most interesting to be observed (Figs. 13, 14). This spontaneously active fibre responded with pure inhibition to ipsilateral stimulation and excitation in response to contralateral flashes. Stimulation of both eyes produced weak responses which are intermediate in character. Threshold and latencies for both eyes are approximately equal. Similar fibres have been found in moths (BLEST and COLLETT, 1965a). Ventral wve

cord

Since it is well known that photostimulation elicits activity in the connectives of the locust, recordings with silver hook electrodes were made in the cervical region of H. erato. With this technique, it is possible to detect bursts of activity at ‘on’ and ‘off’. However, such activity is of only very slightly greater amplitude than that of the spontaneous activity. Detection of the activity patterns was facilitated by employing a Schmidt trigger and monostable multivibrator. With such techniques, however, it is impossible to determine whether the potentials are due to the activity of one, a few, or many neurons. In the process of conducting comparative studies on other butterflies, it was found that the nerve cord fibres, responding to photostimulation, were unusually large in satyrids, such as Taygetti virgilla. Recordings from this species demonstrated that the potentials were probably due to the activity of only two large neurons in each connective, one discharging at ‘on’, the other at ‘off’ (Fig. 16). In moths (BLESTand COLLETT, 1965a), a greater number of neurons is involved in the ventral nerve cord response. Because of similarity of characteristics, it is suggested that the ‘on’ unit originates in the protocerbrum while the ‘off’ fibre is from the medulla interna. This situation is different than the locust where all nerve cord fibres appear to originate in the optic lobes (BURTT and CATTON, 1959). It may be noted in passing that in Taygetis a well-developed giant fibre system is excited by photostimulation as well as mechanical stimuli applied to body hairs. Repetitive stimulation rapidly produces habituation of this system.

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DISCUSSION

The basic motivation for any study such as this is to delineate the nature of some of the integrative mechanisms, and thereby learn something of the nature of the information input into higher ‘decision’-making centres. Any conclusions concerning the nature of the animal’s ‘umwelt’ based upon techniques such as those employed in thii study must be rendered with caution. Present methods can probably monitor the activity in only a few per cent of the larger neurons. Electron microscope studies of the arthropod optic nerve (NUNNEMACHER, 1966; M&LEAR, 1967) suggest that probably two-thirds of the fibres have a diameter of 0.5 ~1or less. Equally important is the realization that insects tend to utilize large diameter fibres for information more relevant to startle responses than complex social behaviour (KENNEDY,1966). This is particularly well illustrated by the excitation of the giant fibre system of satyrids by photostimulation and to a lesser extent by the descending ‘on’ and ‘off’ fibres which appear to be characteristic of virtually all insect nerve cords. Viewed in this light, the similarities in the responses of locusts, moths, butterflies, etc., is not surprising. While the various forms may possess very different eyes and neural apparatus, evolution has seemingly placed a uniform premium on the development of large-diameter fibres, with large visual fields, responding to sudden flashes, shadows, movement, unilateral stimulation, etc. One cannot help but wonder why endogenously active neurons are so frequently employed to transmit such vital information. The high noise level would seem to be most unsatisfactory for such purposes, especially when the economy of neurons in insect systems keeps redundancy to a minimum. In this connexion, it is interesting to note that virtually every time our micro-electrodes were in a position to simultaneously monitor the activity of two neurons, it was found that their activity patterns were ‘reciprocal’ functions of each other (e.g. Fig. 7). Thus, it was common to simultaneously encounter neurons with opposite preferred directions of movement, inhibitory and excitatory responses to flashes, etc. These observations suggest that such ‘reciprocal’ channels may be used in the same manner that one employs a differential amplifier to produce a high level of common mode noise rejection. Such a technique may account for the high signal-to-noise ratio demonstrated by higher order neurons (e.g. ‘on’ and ‘off’ fibres). A not dissimilar mechanism has been described in the vertebrate visual pathway (JUNG, 1961). Despite the fact that virtually all the neuron types described were tested with monochromatic stimulation, the precise nature of the method of transmission of colour information in the visual pathway remains largely a mystery. However, on the basis of these and other experiments it is possible to formulate a very tentative hypothesis concerning this problem. VONFRISCH(1950) concludes that it is possible to train bees to distinguish four, and only four, different colours. If one assumes four types of receptors (AUTRUM, 1968), such behaviour could be accounted for by the rather general phenomenon of peripheral lateral inhibition. Thus, monochromatic stimulation would produce activity only in the processes of the receptors, of the type responding maximally to

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that wavelength. Colour discrimination would, therefore, be limited to a determination of which one of four possible channels was stimulated. It should be pointed out that utilizing essentially the same data, AUTRUM (1968) constructed a rather different model of colour vision based upon a comparison of activity in receptors, presumably executed at a higher neural level. Our own experience, however, indicates that the action spectra of certain neurons (Fig. 8) demonstrate very little overlap. While a few of the larger neurons tested revealed action spectra considerably narrower than that of the eye, it is likely that most such fibres would be of small diameter, not readily accessible to the usual recording techniques. Higher order neurons which respond to spatial-temporal patterns of special biological significance would necessarily be forced to accept, interchangeably, inputs from any of the four types of processes from a single ommatidium. Small differences in these synaptic connexions result in slight variations in the activity patterns produced by various wavelengths of monochromatic stimulation. SUMMARY (1) The neotropical butterfly H. erato demonstrated the presence of neuron types which generally resembled other insects in the variety of stimuli which were maximally effective. The only unique category would appear to be the ‘pure-off’type fibre. (2) In spite of such similarities, a number of differences exist between H. erato and previously studied forms. These include : (a) a somewhat greater utilization of fibres with high dark-discharge frequencies in H. erato, and (b) considerable variety appears to exist between insects as to where in the visual pathway particular neuron types are located. These differences are especially evident when one compares the role of the protocerebrum in the visual pathway of Lepidoptera and Orthoptera. (3) The observed differences would seem to suggest that a sensitivity to movement, dimming, monocular stimulation, etc., has evolved independently in a number of insect groups. Acknowledgements-The author wishes to gratefully acknowledge the invaluable assistance of Miss CHRISTINEF~INBURG,and most particularly express his appreciation for the untiring efforts of Mr. BARRYANDERSON.

REFERENCES AUTRUMH. (1968) Colour vision in man and animals. Naturwissmschuftm 55, 10-18. BLESTA, D. and COLLETTT. S. (1965a) Micro-electrode studies of the medial protocerebrum of some Lepidoptera-I. Responses to simple, binocular visual stimulation, J. Insect Physid. 11, 1079-1103. BLESTA. D. and COLLETTT. S. (196513) Micro-electrode studies of the media1 protocerebrum of some Lepidoptera-II. Responses to visual flicker. J. Insect PhysioE. 11, 1289-1306. BURTT E. T. and CATTONW. T. (1954) Visual perception of movement in the locust. J. Physiol. 125, 566-580.

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BURTTE. T. and CARTONW. T. (1956) Electrical responses to visual stimulation in the optic lobes of the locust and certain other insects. g. Physiol. 133, 68-88. BURTT E. T. and CAITON W. T, (1959) Transmission of visual responses in the nervous system of the locust. y. Physiol. 146, 492-515. BURTTE. T. and CATTONW. T. (1960) The properties of single-unit discharges in the optic lobe of the locust. J. Physiol. W&479-490. BIJRTT E. T. and CATTONW. T. (1966) Image formation and sensory transmission in the compound eye. Adv. Insect Physiol. 3, 2-52. CRANEJ. (1955) Imaginal behavior of a Trinidad butterfly, HeZiconius eruto hydkru Hewitson, with special reference to the social use of color. Zoologica, N. Y. 40, 167-196. CRANE J. (1957) Imaginal behavior in butterflies of the family Heliconiidae: changing social patterns and irrelevant actions. Zoologicu, N. Y. 42, 135-145. HORRIDGEG., SCHOLFFJ., SHAW S., and TUNSTALLJ. (1965) Extracellular recording from single neurons in the optic lobe and brain of the locust. In The PhysioZogy of the Insect Central Nervous System (Ed. by TREHERNEJ. and BEAMFNTJ.), pp. 165-202. Academic Press, New York. JUNG R. (1961) Neuronal integration in the visual cortex and its significance for visual information. In Sensory Communication (Ed. by ROSENBLITHW.), pp. 627-674. Massachusetts Institute of Technology Press, Cambridge. KENNEDY D. (1966) The comparative physiology of invertebrate central neurons. Adv. camp. Physiol. Biochem. 2, 117-184. LEUTSCHER-HAZELHOFF J. and KUIPER J. (1966) Clock-spikes in the Calliphora optic lobe and a hypothesis for their function in object location. In The Functional Organization of the Compound Eye (Ed. by BERNHARDC.), pp. 483-492. Pergamon Press, Oxford. MCALFAF J. (1967) Comparison of neuron maps of the optic tracts of mouse and crayfish. In Invertebrate Nervous Systems (Ed. by WIFFSMA C.), pp. 263-268. University of Chicago Press, Chicago. NUNNEMACHER R. (1966) The fine structure of optic tracts of decapoda. In The Functional Organization of the Compound Eye (Ed. by BERNARDC.), pp. 363-376. Pergamon Press, Oxford. RFICHARDTW. (1961) Autocorrelation, a principle for the evaluation of sensory information by the central nervous system. In Sensory Communication (Ed. by ROSENBLITHW.), pp. 303-318. Massachusetts Institue of Technology Press, Cambridge. SWIHAFT S. (1963) The electroretinogram of Heliconius erato (Lepidoptera) and its possible relation to established behavior patterns. Zoologica, N. Y. 48, 155-165. SWIHAFT S. (1964) The nature of the electroretinogram of a tropical butterfly. J. Insect Physiol. 10,547-562. SWIHARTS. (1965) Evoked potentials in the visual pathway of HeZiconius eruto (Lepidoptera). Zoologica, N. Y. 50, 55-62. VON ERISCHK. (1950) Bees, Their Vision, Chemical Senses and Language. Cornell University Press, Ithaca. WIERSMAC. and YAMAGUCHI T. (1967) Integration of visual stimuli by the crayfish central nervous system. J. exp. Biol. 47, 409-431.