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Journal of J Comp Physiol (1982) 149: 179 193 Comparative Physiology • A Springer-Verlag 1982 Common Visual Response Properties of Giant Vertical Cells in the Lobula Plate of the Blowfly Calliphora R. Hengstenberg Max-Planck-Institut für Biologische Kybernetik, Spemannstrasse 38, D-7400Tübingen 1, Federal Republic of Germany Accepted June 21, 1982 Summary. 1. The common response properties to simple visual stimuli (light impulses, light steps, and movement of simple patterns at different speeds) has been investigated by intracellular recording from Giant Vertical Cells (VS) in the lobula plate of the blowfly Calliphora erythtroccephala. 2. The impulse response begins < 10 ms after onset of the photoreceptor signal (Fig. 6), and shows several phases which gradually subside within about 0.5 s. Very late events, which would hint at recurrent or far-reaching sidepaths, were not observed. 3. The step response is highly non-linear in that both, the increase and decrease of brightness elicit transient depolarization. The excitatory transients are followed by inhibitory waves (Figs. 7, 8), similar to those observed in impulse responses. The possible significance of this succession of excitation and inhibition is discussed. 4. Vertical movement of arbitrary patterns (dot, edges, bar, and gratings) elicit, invariably and irrespective of contrast polarity, depolarizing responses with downward movement, and hyperpolarizing responses with upward movement (Fig. 10). Both responses increase nonlinearly with contour length (Fig. 11 ). Possible mechanisms, and the functional significance of such nonlinear summation are discussed. 5. The velocity dependence of movement responses to periodic gratings was investigated at both high and low pattern luminance and contrast. Under these conditions VS-cells respond best at a contrast frequency of ≈2 Hz, which corresponds with that of velocity dependent optomotor reactions. 6. These results confirm earlier findings that Abbreviations: HS horizontal system; VS vertical system giant vertical cells have many response properties in common. They are best suited to perceive widefield motion, which occurs when a fly performs rotatory and translatory movements in a resting environment. VS-cells are therefore most likely involved in the visual control of such movements. 7. The present results are not sufficient to indicate which of the VS-cells contribute to which of the optomotor reactions. A subsequent publication will be addressed to these questions. Introduction The lobula plate of the blowfly Calliphora contains amongst others two classes of remarkably large tangential neurons: 3 ‘Horizontal Cells’ (HS), and 11 ‘Vertical Cells' (VS) (Fig. 1). They were originally termed this way for anatomical reasons (Pierantoni 1976). By subsequent intracellular recording, and dye injection, HS-, and VS-cells were found to respond specifically to movements in the visual field. Their preferred directions correspond roughly with their class names. Extensive studies of the response properties of HS-cells indicate that they probably contribute to the visual control of yaw movements (for review see Hausen 1981). Much less is known about VS-cells. Despite the general similarity of its members this class is quite heterogeneous. A study on the structure of VS-cells (Hengstenberg et al. 1982) has shown that each of the 11 VS-cells is distinguished from the others by its position in the neuropil and its particular dendritic structure. It was further shown that major structural aspects of the different VS-cells are surprisingly constant in different individuals of Calliphora. Similarly, signs of physiological hetero0340-7594:82:014910179 803.00 180 R. Hengstenberg: Common Visual Responses in Giant Vertical Cells of Calliphora geneity of VS-cells have been found (Hausen 1976a; Eckert and Bishop 1978; Hengstenberg 1981 b). According to this dualism of gross similarity, which defines the class of VS-cells, and the distinct individuality of its members, the present work deals with response properties that are common to all VS-cells, and which define physiologically the class of VS-cells. A subsequent article will aim at the discrete response characteristics of its individual members. Materials and Methods Animals All experiments were performed with female Calliphora erythrocephala Meig. (Insecta, Diptera) from laboratory cultures, 4-6 days post emergence. Preparation Flies were anaesthetized ≤ 1 min in nitrogen, wings and legs amputated, wounds sealed with wax, and the body fixed to a metal holder. After at least 4 h of recovery from anaesthesia, the head was tilted ca. 30° ventrally and secured to the thorax with wax. Access to the lobula complex was gained through a window in the occipital cuticle. The overlying fatbody, and M. retractor haustelli were removed. Neck muscles and the pulsatile head organ were transsected. After amputation of the proboscis near its base, the esophagus was pulled caudad out of the brain, closed by wax, and fixed to the thorax. The tracheal system was usually left intact: for some experiments, how ever, it was necessary to remove one branch, which overlies the axons of VS-cells. Control of Viabilty In view of the extensive dissection, necessary for stable intracellular recording, the viability of the preparation was checked by extracellularly recording from a prominent movement sensitive interneuron (H1) in the lobula plate. It responded for > 24 h in the same manner, as in much less extensively dissected specimens. Experiments were usually completed within <6 h after dissection. Penetration and Cell Identification Recording electrodes (3%, aqueous Procion Yellow M4-RAN 200500 MΩ), and reference electrodes were placed under microscopic control. The recording electrode was carried by a hydraulic microdrive (D. Kopf), and cell penetration was aided by a piezoelectric jolter (Hengstenberg 1981 a). All potential differences stated in text and figures refer to the reference electrode placed in the hemocoel. Mostly the axons of VS-cells were impaled at the proximal margin of the lobula plate. Dye was injected by dc-iontophoresis ( ≤ 5 nA , ≥ 1 µC) through the recording electrode by a constant current source, which allowed observation of the electrical activity of the neuron during iontophoresis. Detailed procedures for cell penetration, dye injection, histology, fluorescence microscopy. and cell reconstruction are published elsewhere (Hengstenberg and Hengstenberg 1980: Hengstenberg 1981 a; Hengstenberg et al., in press). Recording and Staining Strategy The lifetime of intracellular penetrations varies from a few seconds in most cases up to 2 h in very few cases. About 10 min R. Hengstenberg: Common Visual Responses in Giant Vertical Cells of Calliphora 181 Data Processing To evaluate the recorded data off-line, the signals were either filmed or processed with a signal analyzer (Intertechnique, Histomat S). Statistical analyses were performed if necessary by electronic integration of single responses during a period of stimulation. Further evaluation involved a data printer (Kienzle, D44c), and a programmable calculator (Hewlett Packard, 97). Results Giant Tangential Neurons are required for dye injection if satisfactory stainings are to be obtained. At least the same time is required if even simple measurements are to be made under stationary recording conditions. Since the end of a penetration is never predictible, a complementary strategy has been applied in successive penetrations: (a) One or only a few stimulus response cycles were recorded, at the risk of statistical uncertainty of the results, and emphasis was placed upon dye injection and cell identification. (b) Most of the time was dedicated to recording, in order to ensure stationarity and statistical significance of the results, at the risk of injecting too little or even no dye for cell identification. (c) In a few cases, dye was injected by low current ( < 1 nA) dciontophoresis while responses were recorded. Data from the latter experiments were only used for control purposes. Stimulus Arrrangement The flies faced two circular backprojection screens of 60° or 90° diameter, whose axes were in the equatorial plane of the fly's head, and displaced by ±45 laterally (Fig. 2). A dull black diaphragm between the screens prevented optical crosstalk between the two stimulus fields. Since the visual field of Calliphora covers almost the entire solid angle around the animal, only 13%-30% of the total visual field could be stimulated this way. Figure 1 shows 12 of the 14 giant tangential cells of the lobula plate, in a frontal view of the right optic lobe from behind, as they are displayed by retrograde transsynaptic cobalt diffusion, and silver intensification. Three Horizontal Cells (HS) and 11 Vertical Cells (VS) are present per lobula plate in different depths of the neuropil. Figure 1 shows HSand VS-cells separately, because each class covers completely the area of the lobula plate, i.e. the retinotopic input array of this neuropil. Only VS-cells will be considered here; their individual structure has been previously described in detail (Hengstenberg et al. 1982). Electrical Activity in the Neuropil The vast majority of neurons in the lobula plate and other visual neuropils are unipolar (Strausfeld 1976) and their initial process, connecting the perikaryon with the rest of the cell, is very thin (≈ 2 µm) and comparatively long (20-50 µm). When somata are impaled, a steady, membrane potential of - 75 mV < U < -40 mV is recorded, but neither spontaneous nor stimulus-induced potential changes could be observed. This indicates that cell somata are electrically uncoupled from the bulk of VS-cells, and all recordings have to be made by blind penetration of fibres in the neuropil. Conventional types of electrical signals are encountered there, depending upon which type or part of neuron has been impaled. The giant tangential cells (HS and VS) have in most penetrations a comparatively low average membrane potential of -40 to -50 mV (Fig. 3). lt appears ‘noisy’ by continuous synaptic activity 182 R. Hengstenberg: Common Visual Responses in Giant Vertical Cells of Calliphora tic potentials. A study of the regenerative processes, associated with the output side of these cells, will be presented later. Impulse Responses (e.g. Fig. 9). Both, the average potential, and the degree of fluctuation depend upon the stationary level of brightness. Figure 3 shows amplitude histograms of a VS-cell, when dark-adapted (I ≤_ 0.1 cd/m2and when bright-adapted (I=100 cd/ m2). lt is on average depolarized by 2 mV in brightness, and the fluctuation around the mean value is about twice of that in darkness. Responses to visual stimulation consist usually of such membrane potential shifts, and concomitant changes of fluctuation (e.g. Fig. 9). So far, overshooting action potentials could not be demonstrated to occur anywhere in these neurons under the prevailing recording conditions. Indications of electrical excitability have however been found in the axons and terminal arborizations of HS- and VS-cells (Hengstenberg 1977; Eckert 1979; Eckert and Bishop 1978; Hausen 1981 , Eckert and Hamdorf 1981). For the present work, the graded potential changes in response to visual stimulation are used to characterize the input organization of VScells, because this activity is probably caused by summation of many excitatory and inhibitory postsynap- Very bright, brief flashes, when diffusely delivered to large areas of the compound eye, present a strong, instantaneous perturbation of the visual system. Several thousand photoreceptors are simultaneously excited. Their signals travel along an equally large number of parallel fibres centripetally, and along particular pathways even through the whole central nervous system (Hengstenberg, unpublished). Several kinds of results can be expected when the response of central interneurons to such an unspecific perturbation is measured: (a) A neuron may not respond at all, and may therefore lack any connection with the visual system. (b) Neurons, with very specific visual stimulus requirements may be ‘surprised’ this way, and induced to reveal their visual input, even if their specific stimulus is not available or not yet known. (c) For apparently visual interneurons, the response delay indicates their position in a chain of neurons, relative to photoreceptors on the input side, and to other neurons in the vicinity. (d) Different pathways between photoreceptors, and an interneuron under investigation may be revealed by impulse responses consisting of several excitatory and/or inhibitory waves. Impulse responses of VS-cells were recorded in 29 penetrations. The responses are very similar in different VS-cells, and will therefore be presented in general terms. Figure 4 shows impulse responses of an unidentified VS-cell at two different flash frequencies. Both were recorded after ≥30 s adaptation to the particular flash frequency. Under both conditions.. the first sign of a response is a small negative deflection, which is due to a field potential that can also be recorded extracellularly (Fig. 6, inset), or in any of the cell somata. At 0.2 Hz, the next event is a singular action potential, whose amplitude depends upon the average resting potential in darkness, upon the degree of dark adaptation.. and possibly other factors. In penetrations of low average membrane potential (U > -45 mV), or in the bright adapted state only a small, positive peak follows the field potential at the time where the action potential is elicited in dark adapted penetrations. The variable amplitude of the action potential indicates that the regenerative mechanism is partially inactivated in these neurons (Hengstenberg 1977). At 0.2 Hz flash frequency, the action R. Hengstenberg: Common Visual Responses in Giant Vertical Cells of Calliphora potential is followed by a series of humps and notches, superimposed upon a transient depolarization of < 10 mV. At 2 Hz flash frequency, the depolarizing transient is smaller, and the potential fluctuations are reduced (Fig. 4). Figure 5 shows averaged impulse responses of the same VS-cell as in Fig. 4 in comparison to impulse responses of a photoreceptor axon and a lamina monopolar cell, which were recorded under the same conditions, but in a different animal. In all records, the transient increase of activity decays with approximately the same time course. That is to say that an instantaneous perturbation does not seem to elicit longlasting reverberations, which might occur if the wave of excitation would travel along very complicated and/or recurrent pathways. The high frequency fluctuations, seen in single responses (Fig. 4) are largely suppressed in the averages, indicating that they are not specifically related to the synchronizing flash. There are, however, two negative going waves (Fig. 5, asterisks). the first at about 60 ms after the flash, and the second after about 120 ms. The first is present in practically all impulse responses recorded so far in VS-cells, the second is often less clearly recognizable. It is currently not known whether these negative going waves, superimposed upon a transient depolarization arise from a temporary inactivity of the depolarizing inputs or from distinct hyperpolarizing inputs. Such delayed inhibition could in principle be an essential part of the elementary process of movement detection (Thorson 1966; Torre and Poggio 1978). The results presented 183 here, are, however, not sufficient to prove or disprove this possibility (see Discussion). Figure 6 shows impulse responses of RA, LM and VS at higher time resolution to reveal how the impulse perturbation travels through the optic lobe. The inset shows records at 2 Hz flash frequency inside and just outside the same VS-cell. The initial negative deflection in intracellular records of VS-cells at 0.2 and 2 Hz flash frequency is equally present in the extracellular recording. 184 R. Hengstenberg: Common Visual Responses in Giant Vertical Cclls of Calliphora lobula complex. This is a very short time compared to the further 5 ms which are required to elicit the action potential in the VS-cell. It is therefore probable that the field potential is not locally generated by the input elements of VS-cells- it may even originate in the lobula or medulla. Step Response The impulse responses (Figs. 4- 6) give no indication whether the transient excitation of VS-cells is caused by the increase or decrease of brightness which occur almost simultaneously in flashes of 20 µs duration. Step responses are appropriate to decide this question, and have been recorded in 21 VSpenetrations. Since impulse responses decline within about 500 ms, square pulses of ≥0.5 s may be considered as a succession of ‘ON’- and ‘OFF’-steps. Figure 7 shows the step response of a VS-cell: (a) in the dark-adapted state, and (b) in a state of bright adaptation, which corresponds roughly to that of movement stimuli, which are used later in this work. The dark adapted response is quite complex: it starts again with a negative going field potential, followed by an abortive action potential, a transient, depolarizing peak and, a considerable increase of fluctuation of the membrane potential about its average dark value (Fig. 7a). Light off is followed by even larger fluctuations, superimposed upon a somewhat slower depolarizing transient. When flashes of 0.5 s duration are repeated at 1 s intervals, the step response changes gradually from the form shown in Fig. 7a to that in Fig. 7b. presumably due to an average bright adaptation. As before, the response shows depolarizing transients both at the onset and offset of light, but the potential fluctuations during brightness are much smaller than in the dark adapted state. The detailed time course of step responses thus varies with adaptation, but also from flash to flash, when a steady state of adaptation is reached. Similar variations are seen in different penetrations from presumably different VS-cells, and have not been studied in detail. lt is therefore rather a fieldpotential of unknown origin than a constituent part of the transmembrane signal of VS-cells. For comparison, the latency of the responses was defined as the intersection of the tangent through the inflexion point of the rising phase with the resting potential level. Figure 6 shows that a large proportion of the total latency of the VS-cell response is caused by the photoreceptor latency (6.5 ms), that the perturbation needs less than 5 ms to travel from the lamina through the external chiasma, through the medulla, and through the internal chiasma into the The general features of step responses of VScells are revealed, when a large set of measurements are pooled and compared with averaged step responses of a photoreceptor and of a lamina monopolar cell. The photoreceptor responds in a tonic manner to the input step, and the monopolar cell gives a phasic signal (Fig. 8). In both cases, the sign of the potential change reverses when the direction of brightness change is reversed. VS-cells, however, respond to increasing as well as to de- R. Hengstenberg: Common Visual Responses in Giant Vertical Cells of Calliphora 185 lowed by inhibitory waves, similar to those observed in impulse responses. Figure 8 shows that only one such wave occurs after each of the respective depolarizing transients in step responses. The inhibitory wave after 'o n ' is comparatively shallow, and attains its minimum about 120 ms after the onset of the depolarizing transient. The inhibitory wave after light 'off' is shorter, and attains its minimum at about 45 ms after the beginning of the depolarizing ‘off’-transient. Both characteristics of the inhibitory waves in step responses (the delay after the depolarizing transient, and the different duration) bear striking similarity with the two inhibitory waves, observed in the impulse response (Fig. 5): the first, short-lived inhibitory wave occurs about 46 ms after the onset of the VS-response, and the second, slower inhibitory wave occurs at about 115 ms. Comparison with the inhibitory waves of step responses suggests that the first inhibitory wave of impulse responses is caused by the 'off-aspect' of the light impulse, whereas the later, and slower inhibitory wave of the impulse response is caused by the 'on-aspect' of the light impulse. The close correspondence of delayed inhibitory waves in step responses, and in impulse responses, is quite surprising in view of the different stimulus conditions (step: Ipeak=100 cd/ m2, dI/d t≈ 2 x 104 cd .s/ m2; impulse: Ipeak=250,000 cd /m2,dI/dt≈2.5 x 1010 cd.s/m2). Similar nonlinear responses of VS-cells, and especially the succession of excitation and inhibition, have also been recorded in VS-cells of Phaenicia (Soohoo and Bishop 1980). creasing brightness with a transient depolarization. Such nonlinear responses are expected in movementsensitive interneurons like VS-cells, because the essential process in directionally specific movement detection is a nonlinear interaction between adjacent input channels (Hassenstein and Reichardt 1951). The nonlinearity seen here, is, however, not necessarily due to this mechanisms. It could equally be caused by an unspecific 'self-interaction' (Poggio and Reichardt 1976). In step responses, the initial depolarizing transients after the onset and offset of light are fol- The succession of transient excitation and inhibition, as demonstrated in the present investigation may be related to the elementary process of directionally specific movement detection (Torre and Poggio 1978). A delayed inhibition could however equally arise from other mechanisms, for example from recurrent inhibition of input channels (Poggio et al. 1981). Specific experiments will he necessary to reveal the significance of the successive excitatory and inhibitory waves in impulse-and step responses of VS-cells. Movement Responses and Directionality VS-neurons have been termed 'Vertical cells' because their dendritic arborizations span the whole dorso-ventral extent of the retinotopic input array of the lobula plate (Pierantoni 1976). Some of them have later been found to respond predominantly to vertical movement in the ipsilateral visual field (Dvorak et al. 1975; Hausen 1976a, b: Hengstenberg 1977. 1981 b; Eckert and Bishop 1978: 186 R. Hengstenberg: Common Visual Responses in Giant Vertical Cells of Calliphora companied by an increase of fluctuation frequency, and a decrease of fluctuation amplitude relative to periods where the pattern does not move. This can be expected if the response is generated by superposition of many asynchronous ipsp's and' or exclusion of epsp's. The movement responses of VS-cells have been tested in 47 penetrations, and all respond in a very similar manner to vertical pattern movement. Some VS-cells (VS1, VS5-VS10) respond additionally to horizontal movements. The functional implications of such responses will be reported separately. Physiological data about VS11 are presently not available. The similarity of responses to vertical movements in the visual field defines the most important common response property of these neurons. Together with their structural similarities (Hengstenberg et al. 1982), the notion of a distinct class of neurons ('Vertical System', Pierantoni 1976) appears justified. VS-cells should however not be regarded as ‘general purpose neurons’ for any vertical movements, because several vertically sensitive neurons with different structural and functional characteristics are known in the lobula plate (cf. Hausen 1981). Responses to Movements of Basic Patterns Soohoo and Bishop 1980). This response behaviour is illustrated in Fig. 9 for VS2. The meaning of the arrow symbols, which specify movement stimuli is illustrated in Fig. 2. This cell does not respond to any kind of horizontal movement. It is, however, depolarized as long as the pattern is moving downwards, and at the same time the frequency and amplitude of membrane potential fluctuation increase. probably because of an increased rate of epsp's impinging upon the cell. Upward movement causes a steady hyperpolarization, ac- In previous investigations, the movement responses of VS-cells were elicited by extended striped patterns, moving at constant angular velocity. In order to characterize the functional organization of the receptive field of VS-cells, a series of 12 experiments was performed, where four local, and one extended pattern were used: (a) A 10° x 10° black square on white background; (b, c) single edges of either positive or negative brightness change, when moved across the receptive field; (d) a black bar of 10° width on white background; and (e) a periodic grating of 20° spatial wavelength. The patterns were moved 60° up and 60° down at 24°:s by a position-controlled servo system. The stimulus fields of 70° x 70° were arranged as in Fig. 2, i.e., their center axes coinciding with the equatorial plane of the eye. Because of the fixed arrangement of the stimulus fields in the anterior part of the visual field, only distal VS-cells (VS2-VS4) could be studied this way. In order to ensure stationarity of the responses, the phases of pattern movement were separated by 3 s rest, where the pattern was present and illuminated, but not moving. Furthermore, the sequence of measurements shown in Fig. 10 was repeated several times, usually until the cell was lost. R. Hengst enberg: Common Visual Responses in Giant Vertical Cells of Calliphora Figure 10 illustrates the essential results of these experiments, which will he discussed sequentially: VS-cells respond to arbitrary patterns in a qualitatively similar manner; all patterns elicit depolarizing responses when moving downwards, and all elicit hyperpolarizing responses when moving upwards. There is neither a unique preference for small discrete objects (Fig. 10a e), nor a recognizable threshold which would indicate a 'least area requirement'. The moving square stimulates at any time no more than 25 out of about 6,000 visual elements of the compound eye, i.e. less than 0.5%. The sign of the responses to moving edges (Fig. 10b, c) is determined by the direction of 187 movement, and not by the sign of the local brightness change. This invariance of the responses against contrast reversal immediately proves the directional selectivity of movement responses in VS-cells. Responses to discrete patterns (Fig. 10a-d) attain maximal amplitudes shortly after the beginning of the downward movement and towards the end of the upward movement. This indicates the presence of a sensitivity maximum for preferred (downward), and reverse (upward) movement about 10° dorsal of the equatorial plane of the head. The limited extent of the stimulus field prohibits, however, definite statements about the spatial sensitivity distribution of VS-cells. The amplitudes of both depolarizing and hyperpolarizing responses increase with contour length of the pattern. This is to say that VS-cells respond most vigorously when sufficiently textured, extended patterns are moved in the appropriate direction and speed across the receptive field. The electrical activity of VS-cells suggests that their graded responses are due to superposition of a multitude of asynchronous synaptic potentials. The vigorous responses to extended, well-structured patterns is therefore likely to arise from widefield summation of local signals as provided by elementary movement detectors with small receptive fields (Buchner 1976: Buchner et al. 1978). In the simplest case, the response amplitude may increase linearly with increasing contour length of the pattern, because an increasing number of movement detectors is activated. To determine the dependence of VS-cell responses from the contour length of the pattern, live cycles of up/down movement were averaged for each of the patterns of Fig. 10. Data on edge movements were pooled to restitute the dorsoventral symmetry as in the other patterns. The peak amplitudes are plotted against the contour length of the respective patterns. Hyperpolarizing responses are plotted with inverse sign to facilitate comparison. Figure 11 shows the result of this procedure: The amplitudes of de- and hyperpolarizing responses increase monotonically with contour length, but by no means linearly. If the slope of the curves is taken to express the response gain (mV/degree of contour length), it is seen that this gain decreases from > 30 mV/deg for small patterns to < 0.01 mV /deg for extended patterns. This kind of saturation nonlinearity is to be expected when the driving potential of synaptic channels is reduced by large changes of the postsynaptic membrane potential (see Discussion). Figure 11 shows further that the relative ampli- 188 R. Hengstenberg: Common Visual Responses in Giant Vertical Cells of Calliphora tudes of de- and hyperpolarizing responses to patterns of low contour length ( < 150°) arc about equal. At high contour length, however, the depolarizing response is significantly smaller than the hyperpolarizing one (Fig. 11, 490° ; 8 penetrations. 4 4 tests). When the luminance of the moving grating is reduced (I ≈ 0.1cd/m2; 4 penetrations, 23 tests), this asymmetry vanishes, as in case of patterns with low contour length. The additional suppression of depolarizing responses at high stimulus strength cannot be explained by synaptic saturation (see Discussion). These results show that VS-cells respond to arbitrary patterns, moved anywhere in their receptive field, if the direction and velocity of movement are appropriate. The sign of the responses depends only upon the direction of movement, and their size increases with brightness, contrast and contour length as long as the pattern texture is transmitted with full contrast by the optics of the eye (Götz 1964, 1965). Apparently, VS-cells collect movement signals which originate at different locations in their receptive field, and integrate these over large parts of the visual field. The positional information associated with local signals is lost by this process. Contrast Frequency Dependence The observation that VS-cells respond best, when stimulated by large field movements, suggests that they contribute to compensatory optomotor reflexes which stabilize a fly's position and orientation in space. When studied with periodic gratings of spatial wavelength λ (deg), moved at different angular velocities w (deg/s), such reflexes depend upon the contrast frequency (CF=w/λ,), rather than upon w. Maximum reactions were observed between 1 < CF < 5 Hz (Hassenstein and Reichardt 1956; Fermi and Reichardt 1963; Götz 1964, 1968, Götz and Wenking 1973; Srinivasan 1977, Wehrhahn 1978a; Blondeau and Heisenberg 1982). Similar values hold for various visual interneurons (cf. Hausen 1981). However, this range is not universal in flies, since other movement dependent behaviour like the landing reaction is best elicited between 6 <CF< 10 Hz (Eckert and Hamdorf 1981; Wehrhahn et al. 1981), and a few interneurons are known which respond best at high contrast frequency (Hengstenberg 1973; cf. Kirschfeld 1979). In order to corroborate the suggestion that VScells contribute to velocity-dependent optomotor reactions, the contrast frequency dependence of R. Hengstenberg: Common Visual Responses in Giant Vertical Cells of Calliphora their movement responses was measured in 34 penetrations. In some of these experiments, response saturation was minimized either by reduction of pattern luminance (I= 10 cd,/m2, n=7) or by reduction of pattern contrast (m=0.06; n=15). Figure 12 shows one of the response curves, obtained with low contrast. Under all conditions and in all penetrations, responses were confined to 0.01 < CF < 10 Hz. Maximal responses were obtained with CF≈2 Hz for both, depolarizing and hyperpolarizing stimuli. By dye injection this has been shown to hold true for the vertical cells VS1-VS4, and VS6VS9. VS5 has not been stained in these experiments, but responds vigorously at CF= 2 Hz. No data are presently available for VS 10 and VS 11. These results show that the majority, and most likely all VS-cells respond best at contrast frequencies which coincide with those of different velocity dependent behaviour. Their responses are not compatible with the higher range of contrast frequencies which optimally elicit the landing response. Discussion Common Properties of Vertical Cells Studies of the structure of Vertical Cells in Calliphora have shown that each of these neurons has a characteristic structure, which is largely invariant in different individuals (Hengstenberg et al. 1982) and even in different species (Eckert and Bishop 1978). On the other hand, VS-cells are quite similar to one another. This similarity of structure was found to correlate with a similarity of basic response properties. The results presented here agree, where comparable, with those of previous investigations (Hausen 1976a, b; Hengstenberg 1977, Eckert and Bishop 1978; Soohoo and Bishop 1980). Apparently, VS-cells respond best, when appropriately textured, extended patterns are moved at appropriate speed, and in appropriate direction across their receptive fields. Their optimal contrast frequency (2 Hz) coincides with that of velocitydependent optomotor reactions, and there is little doubt, that VS-cells are involved in their control. Electrical Activity The electrical activity of the movement sensitive giant tangential neurons (HS, VS, CH) in the lobula plate has been controversial ever since it has become possible to record from these neurons. Under the present recording conditions, VS-cells respond consistently with graded potential chan- 189 ges, occasionally with small spikes superimposed. They are, however, capable to generate overshooting action potentials, at least when hyperpolarized. The regenerative mechanism of the axon membrane seems therefore to be largely inactivated under the prevailing conditions. The current state of the discussion about coding in these neurons is given in Hausen (1981). For the present investigation, the graded potential changes in response to visual stimulation, were used to characterize the synaptic input organization of VS-cells. These signals depend quantitatively upon the average membrane potential level, because the dynamic range of synaptic channels depends upon their driving voltage (V = E - U), and therefore upon U. Under the prevailing recording conditions the average resting potential of VS-cells ranges from -40 mV to -50 mV. Consequently, a variable degree of asymmetry of the graded potential changes in response to visual stimulation was observed: in records with small resting potentials ( U ≈ -40 mV), depolarizing, and hyperpolarizing responses were about symmetrical. In records with larger resting potentials (U≈ -50 mV), depolarizing responses were enhanced, and hyperpolarizing ones reduced. This is to be expected if the reversal potential of ipsp's is, as usual, more negative than that of epsp's. Impulse- and Step Responses At first sight, it may not seem sensible, to investigate quite sophisticated interneurons like VS-cells by simple stimuli like diffuse impulses, and steps of light. It is in fact true that very little about the specific response properties of any interneuron can be revealed this way. The instantaneous, and synchronous perturbation, set up by a light impulse, does however yield information which is not accessible by specific movement stimuli, where the input elements of VS-cells are excited sequentially. Especially the impulse response has proved to be helpful to reveal the time history of an instantaneous perturbation, travelling through the optic lobe. The transition time of 9.7 ms from the beginning of the photoreceptor response to the first event (action potential) in VS-cells leaves plenty of time for several synaptic interactions, and for signal conduction even along very thin fibres. If a minimum of three synaptic stages is assumed (one in the lamina, one in the medulla, and one in the lobula plate), each requiring maximally 0.7 ms at 22° C (Hengstenberg 1971), then there are still 7.5 ms left for signal conduction, and further synaptic interactions. In order to localize more 190 R. Hengstenberg: Common Visual Responses in Giant Vertical Cells o1 Calliphora accurately the time consuming processes during the passage of an impulse perturbation through the optic lobe, comparable measurements at different stages will be necessary. Since the photoreceptor latency contributes a large proportion of the total delay, and since this depends strongly upon different parameters like dark adaptation, flash intensity, and temperature (Payne and Howard 1981), it will be advisable to record the photoreceptor response in such experiments as an internal standard. The nature of the later phases of impulse- and step responses is at present not clear, especially that of the delayed negative going waves. They may either- he due to a temporary interruption of excitatory input or due to delayed inhibitory input. The fact that the peak of the negative wave following the on-transient in step responses is more negative than the resting potential in darkness seems to favour the second possibility. The same is seen in step responses of VScells of Phaenicia (Eckert and Bishop 1978, Fig. 10; Soohoo and Bishop 1980, Fig. 6). The nature, and functional significance of these delayed inhibitory waves is at present obscure. They are, however, of considerable interest because the nature and location of `elementary movement detectors' (cf. Poggio and Reichardt 1976; Götz and Buchner 1978; Buchner et al. 1978; Torre and Poggio 1978; Srinivasan and Dvorak 1980) is still unknown. As proposed previously (Hausen 1976a, 1981) two radically different organizations are possible: (a) local movement detection takes place in the medulla, directionally specific movement signals are conducted along small field fibres into the lobula plate, and are integrated by widefield tangential neurons. (b) the input signals to VS-cells are not movement-specific but provide appropriately filtered flicker signals. Elementary movement detection may then take place by nonlinear interaction between adjacent synapses on small patches (possibly dendritic `spines') of tangential cells (cf. Torre and Poggio 1978). Current knowledge about the response properties, and identity of medullary neurons neither suffices to discriminate these possibilities, nor to suggest further ones. The Torre-Poggio model of directionally specific movement detection requires a fast excitatory, and a delayed inhibitory channel. whose signals interact in close vicinity on a patch of postsynaptic membrane. The optimal contrast frequency of such a movement detector can be estimated from the time delay between excitatory and inhibitory signals by CF ≈ 1/4·∆t (Poggio and Reichardt 1973). The sequence of excitation, and inhibition, as observed in impulse- and step responses is very suggestive in this respect. An optimal contrast frequency of about 2 Hz could be predicted from the 120 ms delay of the slow inhibitory wave, which follows the excitatory on-transient (Fig. 8). This would agree very well with the observed optimal contrast frequency of VS-cells (Fig. 13). Similarly, an optimal contrast frequency of about 5.6 Hz could be predicted from the 45 ms-delay of the fast inhibitory wave, which seems to be superimposed upon the excitatory off-transient (Fig. 8). The present results on the contrast frequency dependence of VS-cells, does not suggest the existence of such movement detectors. However, in previous studies of various other tangential cells of the lobula plate (Hausen 1981) and of velocity-dependent optomotor behaviour (Wehrhahn 1978a), the contrast frequency optimum of responses was found close to 5 Hz. The delayed inhibitory waves could equally arise from entirely different processes. The vertical system of movement detection could, for example, include a gain control mechanism, based upon feedback inhibition of the input channels, as proposed for the horizontal yaw torque system (Poggio et al. 1981). When such a mechanism is synchronously activated by light impulses or -steps, a succession of excitatory, and inhibitory, events can be expected. In this case, the time delay of the inhibitory wave would reflect the time of transmission along the inhibitory feedback loop. The observation of delayed inhibitory waves in impulse- and step responses of VS-cells is certainly not sufficient to prove, or to discard any of these possibilities. Further evidence will be required to reveal their functional significance. The synchronous activation of a multitude of input channels by light flashes reveals, however. functional properties of VScells. which remain obscured, when input channels are asynchronously activated by pattern movement. Movement Responses The movement responses of VS-cells, agree, where comparable, very well with previous findings (Dvorak ei al. 1975, 1976a, b; Hengstenberg 1977, 1981, Eckert and Bishop 1978; Soohoo and Bishop 1980). In particular it was found that the responses of all VS-cells (except VS11) to vertical pattern movement are very similar. From the responses to diffuse brightness changes one should expect that even VS-cells like VS2 show a flicker response when stimulated with horizontal movement. The local depolarizing transients after light- R. Hengstenbcrg: Common Visual Responses in Giant Vertical Cells of Calliphora 191 to explain the asymmetry of de- and hyperpolarizing responses seen in Fig. 11 at high contour length, because the ratio of these responses should remain constant if only the number of input elements is varied. The voltage-dependent potassium conductance of the axon membrane of VS-cells (Hengstenberg 1977) is likely to produce a variable shunt conductance which must be expected to suppress depolarizing membrane potential changes selectively. Although the membrane mechanisms of' nonlinear summation are trivial, their functional significance is not. If flies move through a variegated environment, they are confronted with rapid changes of' overall ‘movement stimulus strength’ and may want to correct their flight maneuvres independent of this quantity. Nonlinear summation, as observed in VScells, is the first, simplest und fastest mechanism which can contribute to such adaptation because it is an inherent property of the summation process itself. This mechanism is also economic when only the energy expenditure of VS-cells is considered because synaptic saturation reduces the transmembrane flow of ions. It is, however, uneconomic with respect to the energy expenditure of input elements. It may therefore well be that the optomotor control network for vertical movements contains also means of feedback inhibition Nonlinear Summation of the input elements as in case of the horizontal The process of widefield integration of a multitude system (Hausen 1981). of local movement signals in VS-cells could in principle be almost linear, if the activation of single input channel would only produce a minute voltage change ( < 1 mV) at the output site of the dendrite. If however single input channels produce a sizeable Contrast Frequency Dependence voltage change ( > 1 nmV), summation of many such It was shown for 8 of the 11 VS-cells in Calliphora signals yields a large change of membrane potential that these neurons respond best to a moving grating at towards the equilibrium potential of the involved about 2 Hz contrast frequency. A similar value was synaptic channels. This in turn reduces the driving previously stated for a vertical cell in Phaenicia voltage acting upon the synaptic conductance, and (Soohoo and Bishop 1980). Responses of opposite the current per synaptic site is smaller than in case polarity which are elicited by movement in of single activation. Nonlinear summation may opposite directions, have the same optimum and therefore be conceived as a means to maintain a high range of contrast frequencies (Fig. 11). This suggests gain for singly activated input elements and to reduce that the underlying processes of movement detection the risk of overloading the output of a neuron when have very similar properties in either direction many input elements are active. despite of the different synaptic mechanisms which Varying the number of simultaneously must underly de- and hyperpolarization. The range activated input elements of VS-cells by choice of patof contrast frequencies to which VS-cells respond terns with different contour length (Fig. 11) has best, coincides very well with that of velocity revealed that widefield integration in VS-cells dependent optomotor behaviour, and not with the probably involves this mechanism because moved higher range of contrast frequencies (CFmax≈ 8Hz) gratings elicit large average potential changes. This which optimally elicits the landing response, and effect must be even more dramatic when instead of' the which was so far only found in one tangential neuron of the lobula plate and in descending fibres of the limited stimulus field (∆θ=60° ) the whole cervical connectives. receptive field of VS-cells (∆θ=180° ) is stimulated. The common response properties of giant vertiSynaptic saturation is however not sufficient on and -off should add to a net depolarizing response even if they arise asynchronously at different sites of the dendritic arborization. The absence of such an average flicker response with horizontal pattern movement (Fig. 9) is most likely due to the transient nature of the intensity responses (Figs. 5- 8) and the much lower rate of local brightness change with the moving pattern (dI/dt <103 cd·s/m2) than with step stimuli (dI/dt ≈ 2 x 104 cd·s/m2). The movement responses to different patterns have proved the previous assumption that VS-cells respond maximally, when extended, well textured patterns are moved at appropriate speed, and in appropriate direction across the receptive field of VS-cells. This behaviour is in sharp contrast to that of the orthopteran LGMD (lobula giant movement detector; Rowell et al. 1977), which responds best, when small objects are moved in arbitrary direction anywhere in the receptive field, and which is inhibited by large field movement of extended patterns. Therefore, there is little doubt that VS-cells are involved in the optomotor control of locomotion, and that VS-cells are used by the fly to perceive its movement in space.