discrimination of visual motion from flicker by identified neurons in the medulla of the fleshfly...

J Comp Physiol A (1991) 168:653-673 Journal of ~ =,..or,,

Neural, and

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�9 Springer-Verlag 1991

Discrimination of visual motion from flicker by identified neurons in the medulla of the fleshfly Sarcophaga bullata Cole Gilbert 1, Douglas K. Penisten 2, and Robert D. DeVoe*

Department of Visual Sciences, School of Optometry, Indiana University, Bloomington, IN 47405 USA

Accepted April 10, 1991

Summary. 1. Responses to moving contrast gratings and to flicker have been studied in cells in the medulla of the fleshfly Sarcophaga bullata using intracellular recordings and stainings. Medullary neurons responded periodically to flicker. Those which primarily discrimin- ated motion had periodic responses or DC shifts in membrane potentials or increased noise. Intrinsic neurons included a T1 a cell which was directionally selective (DS) and specific non-DS amacrine cells (6 types) arborizing either distal or proximal to the serpentine layer. Among the 12 types of output neurons recorded, 1 projected to the lobula plate, 6 to the lobula (Tm and T2 cells), 3 to both the lobula and lobula plate (Y cells), and 2 to the central brain.

2. Irrespective of their projection, medulla neurons which arborize in the stratum of the L2 terminals respond to flicker as does L2 and have the simplest, primarily periodic, responses to motion. The responses have significant power at the second harmonic of the stimulus temporal frequency suggesting that a non-linear operation, such as multiplication, may occur in the L2 stratum. Cells with arbors coinciding with either of the two levels of L1 terminals have much more complex responses to motion. All cells projecting to the lobula plate responded periodically to movement in some direction(s).

Key words: Insect vision Motion detection Medulla - Intracellular electrophysiology - Identified neurons

Introduction

The neuroanatomy of the fly optic lobe is known in some detail, but proximal to the lamina there are few solid data about specific synaptic connections among

Abbreviat ions: DS directionally selective; LMC lamina monopolar cell; EMD elementary motion detector

1 Present address: ARL - Division of Neurobiology, University of Arizona, Tucson, AZ 85721 USA 2 Present address: College of Optometry, Northeastern State Uni- versity, Tahlequah, OK 74464 USA * To whom offprint requests should be sent

cells. There is also a large gap in our knowledge about the neurophysiological properties of neurons relaying information from the lamina to the lobula and lobula plate. For example, image motion simulated by sequen- tial stimulation of just two neighboring photoreceptors is sufficient to excite the giant horizontal cell, H1, that originates in the lobula plate (Franceschini 1985; Fran- ceschini et al. 1986, 1989; Riehle and Franceschini 1982, 1984), and to release optomotor turning behavior (Kirschfeld 1972). However, when the same two photoreceptors are stimulated simultaneously, simulating image flicker, the resting spike rate of H1 is little affected. The identity and physiology of neurons that make up the pathways between photoreceptors and H1 can only be speculated on, however (Strausfeld 1984). It is not known which cells first detect motion or what their responses to motion look like. One criterion for motion detection is that cells be directionally selective (DS). An- other, as indicated above for HI, is that cells which detect motion can distinguish motion from flicker.

There is no evidence so far that motion can be distin- guished from flicker either by photoreceptors (Hardie 1985; Shaw 1981) or by means of interactions among the 12 other neuron types comprising the lamina cartridges (Laughlin 1984). Indeed, Coombe et al. (1989) have argued that the largest LMCs, L1 and/or L2, are perhaps not involved in motion detection at all. All the above suggest that the medulla is the first optic ganglion where motion detection takes place (DeVoe and Ockle- ford 1976; Mimura 1971). The medulla has been less well studied than the retina or lamina and appears to be considerably more complex. It consists of at least 120 morphologically defined neuronal cell types, with about 40 types per column (Campos-Ortega and Straus- feld 1972; Strausfeld 1976). Some physiological information is beginning to accumulate about this neuropil in orthopterous insects (Osorio 1986; Kelly and Mote 1990) and in flies (review: DeVoe 1985). Activity of cells show- ing DS properties has been recorded in flies extracellu- larly (Mimura 1971, 1972) and intracellularly (DeVoe and Ockleford 1976). Cells in the distal regions of the medulla respond to flicker and motion with the same sorts of ON/OFF responses as are recorded in the more peripheral laminar cells (DeVoe 1980; Penisten 1988).

654 C. Gilbert et al.: Discrimination of visual motion

However , medul lary cells recorded in the proximal medulla respond differently to mot ion compared to flicker. Responses to flicker are concatenat ions o f phasic O N and O F F responses. Dur ing st imulat ion with moving gratings, however, the m e m b r a n e potential is depolar- ized with spikes or ripple riding on top o f the depolarization. The ripple is often asymmetrical , depending u p o n the direction o f mo t ion (DeVoe 1980, 1985). These re- suits indicate that a cellular mechanism responsible for mot ion processing m a y occur at this level in the medulla.

Fur ther suppor t for the above comes f rom experi- ments using a radioactive 2-deoxyglucose technique to label neuronal activity (Buchner e ta l . 1984; Bfilthoff 1986; BiJlthoff and Buchner 1985). There is little difference in the incorpora t ion o f the label in distal regions o f medullas which ' v i ewed ' image mot ion and those presented with image flicker. However , a s t rong difference in neuronal activity, as indicated by label uptake, is seen in the medial and more proximal medulla, and in the lobula plate. Image mo t ion excites cells in these proximal areas much more than does flicker, suggesting again that a mo t ion detecting mechanism occurs in the proximal medulla.

In the lobula plate, the label is differentially taken up depending u p o n the direction o f image mot ion . The layers which label mos t s trongly in response to hor izontal or vertical mo t ion are the layers in which the hor izontal or vertical giant cells have their greatest pos tsynapt ic densities, respectively (Hausen et al. 1980). This pat tern o f labelling occurs even in mutan t s lacking the giants (Bfilthoff and Buchner 1985) and so may imply that the lobular plate giants are DS because they receive exci- ta t ion f rom DS co lumnar input fibers to the lobula plate. Two such groups o f input fibers are k n o w n anatomically: the ret inotopical ly organized T4 cells, at least 4 o f which project f rom each medul lary co lumn (Straus- feld, pers. comm.) , and the T5 cells, at least a pair o f which projects f rom each co lumn in the lobula in Calli- phora (Strausfeld 1984). Members o f each o f these groups terminate in either the hor izonta l layer or in the vertical layer o f the lobula plate. Whe the r or no t DS responses actually occur in such input cells can no t be resolved by the 2-deoxyglucose approach , however : if each medul lary co lumn or pair o f co lumns has separate popula t ions o f small-field DS cells sensitive to oppos ing directions o f hor izontal or vertical mot ion , then the neuropil will appear equally labelled for mot ions in the different directions. Clearly, more definite correlat ions o f a n a t o m y and phys io logy are needed.

To this end, we present here the first extensive report o f m o v e m e n t and flicker responses f rom identified cells o f the fly medulla. All medul la neurons that were stained responded differentially to flicker and mot ion . We recorded a variety o f mo t ion responses in neurons which project f rom the medulla to at least 3 different neuropils, suggesting that mo t ion in format ion processed in the medulla diverges to several targets. However , only a few o f the mot ion responses were themselves directionally selective, and only one DS cell (a T l a neuron) was stained. The dye-coupl ing found a m o n g certain groups o f cells ma y be indicative o f electrical coupl ing between these medul lary neurons.

Materials and methods

Preparation. The methods used were essentially similar to those of DeVoe (1980). Under light CO2 anesthesia the legs of fleshflies, Sarcophaga bullata, were amputated and the stumps sealed with a 2:1 mixture of bees' wax and rosin. The fly was then waxed to a toothpick, and the wings, antennae, proboscis, and head were immobilized with the same mixture. A small hole was cut in the back of the head capsule over the right optic lobe. The tracheal air sacs were drawn away, and the neural sheath behind the medulla was delicately teased away with sharpened tungsten needles. A Ringer's solution (DeVoe and Ockleford 1976) thickened with 0.5 % polyvinyl alcohol (to keep the solution in place) was added to the head capsule as needed. The fly was aligned in a holder using the symmetry of the deep pseudopupils (Franceschini 1975) about the equatorial and mid-sagittal planes as viewed through a small alignment telescope.

Stimulation. Most features of the optical stimulator have been described previously (DeVoe 1980). White light from a 100 W quartz- iodide bulb was projected through a shutter, neutral density wedges, a field-stop iris, various movable patterns on a wheel, a Pechan (rotating) prism (to change the angular orientations of the patterns), X-Y mirror galvanometers, and onto the rear of a frosted glass dome which was viewed by the fly. Luminance of the central portion of the dome with no density in the beam was about 100 Cd/m z. The patterns used were 15 ~ (and sometimes as noted 2.5 ~ ) spatial wavelength square-wave gratings of 91% contrast modulation for motion, and a clear area for flash and flicker. As viewed by the fly, 0 ~ denotes movement of a vertical grating to the right, 90 ~ denotes upward movement of a horizontal grating, 180 ~ is to the left, and 270 ~ is downward. Spatial coordi- nates are reported in degrees as (azimuth, elevation). Intersection of the fly's midsagittal and horizontal planes is straight ahead at (0~176 and each coordinate has maxima at __+ 90 ~ Increasing posi- tive values are to the right and upward. Full-field stimuli, filling the entire dome, subtended 180 ~ at the fly's eyes. Unless otherwise noted, the contrast frequencies used were typically 5.16 Hz with a 15 ~ spatial wavelength grating for motion, and full-field stimuli. An Apple lie computer controlled the stimuli through the keyboard or a joystick.

Recording and staining. Intracellular glass electrodes were formed with a Brown-Flaming micropipette puller to give resistances of 300-1000 MO when backfilled with 4% Lucifer Yellow CH at the tip followed by 10 mM LiC1. The indifferent electrode was a broken micropipette filled with Ringer's solution and placed at the medial rim of the hole in the head capsule. The active electrode was ad- vanced in 1 ~tm steps with a Kopf Hydraulic Microdrive. The responses of impaled cells were led through a WPI M707, high input- impedance amplifier with negative capacitance compensation and an active bridge, displayed on an oscilloscope and stored on FM tape for later analysis. Before each stimulus, the value of the membrane potential was digitized and displayed, and a 10 mV calibration pulse was triggered onto the recorded membrane potential. Signals from the shutter, a potentiometer attached to the patterned wheel, a photoelectric grating scanner and a Datum time code generator were also stored on tape. Before each experiment the time code generator was synchronized with the computer's clock. In this way the experimental protocol written to disk and the responses recorded on tape were indexed by date and time.

After recording a cell's physiology, Lucifer Yellow was ionto- phoretically injected by passing < 1.0 nA hyperpolarizing current pulses (1 Hz, 50% duty cycle) through the active electrode. The cell's membrane potential was monitored between pulses. Injection was stopped immediately if the membrane potential did not return to its previous value. Typically, the amount of staining had to exceed -7 .5 nA.s (e.g. -0 .5 nA x 30 s • 0.5 duty cycle) to justify further histological preparation. If the cell was still impaled after staining, further physiology was recorded. After staining, the brain was fixed with 4% paraformaldehyde in the head capsule for 30 min, then dissected out and fixed in fresh paraformaldehyde

C. Gilbert et al. : Discrimination of visual motion 655

minimally for another 60 min. The brain was dehydrated quickly through an ascending ethanol series and infiltrated overnight with soft Spurr's resin (Spurr 1969). It was then block embedded, cut in 20 p~m thick horizontal sections, and viewed with a Leitz Dialux epifluorescence microscope. Profiles of filled cells were reconstructed from photographic slides of the serial sections. The profiles are presented as their projection in the horizontal plane in superior view against the outline of the pertinent ganglia of the right optic lobe, often just the posterior portion of the medulla. The nomencla- ture of Strausfeld (1970, 1976) is followed when possible.

Data analysis. Cellular responses are presented primarily as records played off magnetic tape onto a Gould 220 pen recorder. Responses which required periodic analysis were played off magnetic tape into an Ortec 4620/4623 Signal Averager for digitization and averaging. Motion waveforms displayed in the figures are averages of 2 responses, whereas flicker waveforms are averages of 3 responses. A custom Fourier analysis program calculated the amplitude and phase relationships of the first 12 harmonics of the stimulus contrast frequency. Harmonics that were significantly different from noise (P<0.05) were determined by the method of Hartley (1949). From these harmonics, the sums of harmonics which best fit the averaged data were determined by eye.

Results

The responses and reconstructions of medulla neurons in 20 brains are presented. Cell bodies in the rind around the medulla are electrically silent, requiring that recordings be made from fibers in the neuropil. The small size of medullary fibers, 1-5 Ilm, and the mechanical instability of the brain allowed only brief penetrations, typically for less than 6 min. Nonetheless, the automated appara- tus described in Methods permitted analyzable data from over 100 cells, from which the examples here were chosen. In general, most of the recorded neurons in the medulla of Sarcophaga are non-spiking, and those cells

which do propagate action potentials are located proximally in the medulla. Further, most medullary neurons respond differently to motion than they do to flicker.

Inputs to the medulla

Only in the distal medulla did we record similar responses to motion and to flicker, and staining of such cells always revealed lamina or retina-derived afferents to the medulla rather than neurons originating in the medulla itself (Penisten 1988). Three types of lamina monopolar cells (LMC), L1, L2, and L3, were recorded from their axon terminals and stained (Figs. 1-3). The monopolar cells were penetrated at relatively shallow depths in the distal medulla and their responses to light ON and OFF recorded at this location are similar to those recorded in the lamina (Laughlin 1984): light ON elicits a transient hyperpolarization, followed by a noisy sustained membrane potential which returns nearly to the resting potential; light OFF elicits a transient depolarization. Fourier analysis (Penisten 1988) of the waveforms of the responses to flicker and motion stimuli demonstrated that the two responses have almost identi- cal power spectra for the odd harmonics for L1 and L2. In the examples shown (Figs. 1 C, 2C) the amplitudes of the ON and OFF transient fortuitously varied according to the orientation of the grating, presumably because of differences in initial position of the 15 ~ grating within a cell's receptive field. (In Figs. 1 and 2 compare the responses to the shutter opening at the beginning of full-field flicker versus at the beginning of motion in different directions.) When the grating is stopped the response shows different potentials, probably because a 7.5 ~ dark stripe happened to lie across the cell's recep-

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Fig. 1. A Reconstruction of a medulla terminal of an LI cell. The conventions of the physiological records are similar throughout this paper and are explained in detail here. B Re- sponse to flicker. Top trace: 1 s time marks. Middle trace: intracellular recording indicating the cell's initial resting potential (in mV) followed by a 10 mV calibration pulse before stimulation begins. Bottom trace is from the shutter with up indicating open. C Response to motion. Top trace again a 1 s timing signal. In- tracellular trace (or traces) as in B with addi- tion of the first direction (in degrees) of the 'forward-stop-back' motion of the striped grating. The response of L1 is similar for oblique orientations (not shown). Bottom three traces stimulus signals: Bottom is the shutter, as in B. The second from bottom is the motion signal from a potentiometer attached to the patterned wheel. When the trace ramps up (down) the grating moves in the first (second) direction. Upward ticks indicate changes in the grating from moving to stopped and vice versa. Third trace from bottom from a photocell which monitors motion of the grating

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C. Gilbert et al. : Discrimination of visual motion

Fig. 2. A Reconstruct ion o f the medulla terminal o f an L2 cell. Response to B flicker and C motion. The response was similar for mot ion along the 130 ~ 310 ~ axis

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Fig. 3. A Reconstruct ion for the medulla terminal o f an L3 neuron and its response to B flicker and C motion. For mot ion along the 00180 ~ axis the response waveforms are different in the two directions

tive field in one stop position, and a 7.5 ~ clear stripe in another. This is important because it indicates that the cell's receptive field is smaller than 7.5 ~ and agrees well with the established small receptive fields of L1 and L2, which are well known from recordings in the lamina (Zettler and Jfirvilehto 1972; Laughlin and Osorio 1989). Furthermore, the stop time potentials, when different from each other (see also Figs. 23 and 24, below), pro- vide valuable information about the spatial properties of less well known cell types that were not penetrated long enough to fully measure their receptive field properties.

In a third LMC, L3, the flicker response (Fig. 3) is very similar to the flicker responses recorded from L1 and L2. However, in L3 the amplitudes of the 1 st and

3rd harmonics drop markedly during motion compared to flicker and in one recording a directionally asymmet- ric response was evident as a 50% decrease in the amplitude of the fundamental frequency of the response from 1.4 to 0.7 mV for 0 ~ and 180 ~ motion, respectively (Pen- isten 1988).

Neurons intrinsic to the medulla

The one strictly columnar cell we recorded and stained is T l a (Figs. 4, 5). Its structure would indicate a small receptive field. Consistent with this are small membrane potential differences between stop times of motion, especially on the 90~ ~ axis. The motion response


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is not only distinctly different from flicker, but the amplitude on the fundamental harmonic is also directionally selective for motion on the 900-270 ~ axis (Fig. 5). This is the first small-field, directionally selective, medulla cell identified in the fly.

Another medulla neuron also shows directional selectivity by means of differences in the amplitude of the fundamental harmonic ripple in the membrane potential (Fig. 6). The response to flicker (Fig. 6A) is similar to that of LMCs but is not as fast. Most of the power is in the fundamental harmonic. The response to motion is only a depolarizing oscillation which varies with direction (Fig. 6B) and which also has most of its power at the fundamental harmonic of the contrast frequency (Fig. 6C). We have recorded DS responses similar to this from another unit, but unfortunately neither was stained.

Few other cell types intrinsic to the medulla confine their arborizations to a single column (Strausfeld 1970, 1976). More commonly, the intrinsic neurons we filled were amacrine cells whose neurites extend into just a few columns or into every column in the ventral half of the medulla (Figs. 7-14). Some types occur in a pattern repeated throughout the entire medulla (Fig. 7),

while other types may occur only once or twice in each medulla (Fig. 14).

A stratum that extends throughout the entire medulla at the level of the deeper L1 endings comprises similarly shaped, probably homologous, dye-coupled amacrine cells (Fig. 7). Around the distal rind 146 somata are stained (Fig. 7). The recorded cell strongly depolarizes to light, with additional small depolarizing transients at both ON and OFF. During motion of the grating the membrane further depolarizes by several millivolts, but no periodicities in the response are seen to motion in any direction (Fig. 7 C). In contrast, the cell was very capable of responding periodically to 5.16 Hz flicker (Fig. 7 B). Thus, the tested contrast frequency (5.16 Hz) was not greater than the flicker fusion rate. The absence of periodicities during motion is therefore most likely a result of phasic input information being lost through averaging over a large receptive field. In a second preparation (not shown; Penisten 1988), the penetrated amacrine cell stained brighter than the other cells of the stratum allowing measurement of the dendritic boundaries of a single amacrine as about 80 ~tm, or 13 columns in diameter.

In other cells periodic components are sometimes seen

658 C. Gilbert et al. : Discrimination of visual motion

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on top of depolarizing mot ion responses. Small amplitude periodicity was recorded during mot ion f rom an amacrine cell similar to Strausfeld's (1970) M: tan 13 (Fig. 8), which arborizes proximally between the serpentine layer and the most proximal stratum, in which dendrites to the T4 cells arborize. The larger branches run perpendicular to the columns and send finer collateral fibers parallel to the columns, where each fiber is serially studded with several swellings, like beads on a string. This neuron demonstrates an orientation-sensitive motion response. In response to mot ion along the 0~ ~

axis, ripple at the fundamental frequency is present, but in response to mot ion along the 900-270 ~ axis, ripple is absent and depolarizations are smaller. The flicker response shows the expected concatenation of the light O N and O F F responses (Fig. 8 B).

Another (unnamed) amacrine cell also has a periodic response to mot ion (Figs. 9, 10). This cell arborizes throughout the proximal medulla in a more irregular pattern than that of M: tan 13, and the fibers lack swellings. The shapes of response waveforms are distinctly different for directions along a given axis of mot ion


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Fig. 9. A Reconstruction of another proximal amacrine cell. Response to (B) flicker, recorded later in the penetration when the resting potential had declined, and (C) motion. Note the shape and amplitude of ripple differs with directions of motion

(compare 0~ ~ and 40~ ~ motion), and the response to motion on the 90~ ~ axis is virtually aperiodic (Fig. 10). From the cell's location in the medulla, it can be estimated that the receptive field is centered on the vertical and horizontal midlines, with an asym- metric dendritic spread. The cell arborizes approximately 300 pm along the dorsal-ventral axis and 150 ktm along the rostral-caudal axis. I f the cell's receptive field corresponds directly to its dendritic spread, it subtends an estimated 40~ ~ vertically and 20~ ~ horizontally. Thus, the motion response is strongly periodic when the 15 ~ grating stripes are parallel to the long (vertical) axis of the oval receptive field, and aperiodic when the stripes are perpendicular to that axis.

A second unnamed amacrine cell, which arborizes in the middle third of the medulla proximal to the serpentine layer, also responded periodically to some but not all directions of motion (Fig. 11). The cell arborizes about 260 pm along the dorso-ventral axis and about 125 pm along the rostro-caudal axis. Given the column spacing indicated in Fig. 11 A, the cell's receptive field should be about 40 columns tall by 20 columns wide, or roughly 60 ~ by 30 ~ which agrees roughly with the receptive field determined physiologically (Fig. 12). The alignment of the more dorsally arborizing branches (lower part of the reconstruction and lower inset box) with medullary columns is more obvious than for the more ventral branches. Further, the more dorsal


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Fig. 11. A Reconstruction of a proximal amacrine cell which was sectioned obliquely, as indicated by the outlines of the lobula plate and the corner of the medulla for each section. The inset boxes indicate that in each section the cell arborized in a discrete stratum in the proximal medulla just above the T4 layer. The hatching around the lower box inset box indicates the spacing and orientation of the medullary columns projected from the distal medulla, where they are more evident. Responses to (B) flicker and (C) motion. Compare the response to motion along the 130~ ~ axis with other directions using the same 15 ~ grating in the middle traces and along the same axis using a 2.5 ~ grating in the bottom trace


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branches innervate columns which are retinotopicalty more posterior than the columns innervated by the cell's ventral branches (note the distance of the branches from the outline of the lateral medulla). That is, the cell's branches are oriented obliquely. The cell's response to flicker is simply a combination of the sustained ON response with depolarizing transients at ON and OFF. The motion response is a sustained depolarization, with ripple primarily at the contrast frequency for motion of a 15 ~ grating along the 130~ ~ axis. In this orientation the pattern stripes are oriented at 40 ~ and are more or less parallel to the cell's branches. Unfortunately, the cell was not tested with motion along the 40o-220 ~ axis, but vertical and horizontal motion produced little or no ripple in the sustained depolarization of the motion

response. Thus, this amacrine cell responds similarly to the previous cell (Figs. 9, 10): motion oriented perpendicular to the long axis of the cell's dendritic field produces a periodic modulation at the contrast frequency, while motion oblique to the long axis produces little or no modulation. Further, the modulation but not the depolarization is abolished with the 2.5 ~ grating (bottom trace in Fig. 11 C), although the contrast frequency is the same.

A third unnamed amacrine cell was recorded coupled to a T2 neuron (Fig. 13). The amacrine cell arborized in a roughly circular pattern of columns, 380 ~tm along the dorso-ventral axis, or about 60 columns, and 300 ~tm along the rostro-caudat axis, or about 47 columns. The cell's response to motion is similar to that of the other proximal amacrine cells, i.e. a vigorous depolarization with small amplitude periodic components at the contrast frequency.

The largest amacrine cell stained in this study arborizes throughout the entire ventral half of the medulla (Fig. 14). The cell has large fibers in the serpentine layer and sends a fine fiber, which terminates in a swelling, into each column just distal to the serpentine layer. The receptive field of the cell, as measured with discrete flashes of a 20 ~ spot (not shown), corresponds directly with the distribution of the swellings. Flicker and motion evoke different responses in this spiking cell: Bursting occurs at the contrast frequency to flicker, whereas to motion only a single burst is evoked at the start of motion in any direction.

Medullary outputs

Two different classes of cells projecting from the medulla were stained; cells projecting to the lobula and lobula plate (Figs. 15-24) and those projecting to the central brain (Figs. 25, 26). Cells projecting to the lobula and lobula plate may be further grouped as those projecting to the lobula plate (Fig. 15), to the lobula (Figs. 16-21), or to both neuropils (Figs. 22-24).

Only one cell which projects solely to the lobula plate

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Fig. 13. A Reconstruction of a recorded amacrine cell dye-coupled to a T2 cell. Response to (B) flicker and (C) motion

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Fig. 14. A Recons t ruc t ion of an amacr ine cell which arbor ized t h r o u g h o u t the ventra l ha l f of the medulla. Response to (B) flicker and (C) mot ion . The response to m o t i o n in the obl ique or ienta t ions was similar (not shown)

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- 3 2 ~ t J ~ d ~ ~ ] l ~ , [ [ ~ I I [ , ~ . . ~ ~ , I ~ , [ i i

~, ~d 45 ~

24J 90 ~

Fig. 15. A Recons t ruc t ion of a co lumnar cell. Response to (B) flicker and (C) mot ion . Com- pare the response to m o t i o n a long the 90o-270 ~ axis with o ther direct ions s t imulated with the same 15 ~ grat ing, and with the same direct ion s t imulated with a 2.5 ~ gra t ing (bo t tom trace)

was recorded and stained (Fig. 15). This was recorded in a male fly and is a previously undescriped cell type, as only T4 cells have been known to project from the medulla exclusively to the lobula plate (Strausfeld 1976). The recorded cell is columnar in the medulla and arborizes in the horizontal layer of the lobula plate. The response to flicker is a concatenation of the ON and OFF depolarizations and fluctuates at the contrast frequency. The response to motion, on the other hand, is a sustained depolarization. With vertical motion there is a superimposed ripple at the contrast frequency (Fig. 15 C). The sustained DC, but not the ripple, is still

present when the 5.1 Hz contrast frequency is produced by motion of a 2.5 ~ grating (bottom trace in Fig. 15C). Although the reconstructed cell profile is projected onto the horizontal plane in Fig. 15A, the spatial position projecting retinotopically to this medullary column is actually at a high elevation (40o-60 ~ in the mid-sagittal plane. Thus, the cell's receptive field would be in the male-specific 'acute zone' (Land and Eckert 1985). At that spatial position in both sexes the horizontal rows of ommatidia are rotated vertically and are inclined 20 ~ 30 ~ from vertical (Hausen 1982). Thus, vertically moving stripes sequentially stimulate neighboring columns in the


-16 ~1 I . . ~..~l . . . . . . . . i ,..,

-22 ~ [

663

Fig. 16. A Reconstruction of a Tm cell and its response to (B) flicker and (C) motion. Fourier analysis (not shown) yielded flicker harmonic amplitudes (mV p-p): 1 = 4.8, 2 = 2.2, 3 = 1.6, 5 = 1.0 and motion harmonic amplitudes (mY p-p) at 75~ 1=2.0, 2=3.3 and at 345~ 1=1.2, 2=3.1

A

B

C -38

L

2 90 ~

Fig. 17. A Reconstruction of a Tm cell similar to Tm13 and several other regularly spaced, but incompletely stained cells. Response to (B) flicker and (C) motion

horizontal array. Informat ion about such periodic stimuli is passed to the horizontal layer of the lobula plate by this type of medulla neuron.

The recorded and stained neurons projecting to the lobula, so-called T m cells (Strausfeld 1970, 1976), all have their dendrites restricted to one or a few columns. The first of these cells (Fig. 16) has dendrites in the outer medulla at the level o f the L2 endings. The response to flicker is a periodic hyperpolarizing modulat ion of the membrane potential at the contrast frequency, similar to L2 but lacking the sharp O F F transients of the flicker response of L2. The response to mot ion is quite

different. There is a non-DS sustained hyperpolarizat ion with depolarizing periodic ripple, primarily at the second harmonic of the contrast frequency. It is hard to see how this could arise f rom simple combinations of flicker- like responses of adjacent input channels. Presumably, some nonlinear, mot ion detecting mechanism is involved.

In another preparat ion (Fig. 17), the recorded T m cell (possibly Tml3 ) was dye-coupled to cells which may be columnar homologues. The non-recorded profiles, though regularly spaced, are incompletely filled. Thus their determination as homologues of the impaled cell


/// B

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~ ~ ...... -Tw-.1 ~-- ,v , ,rw r~v-- ,, -'

o o

, A / - ~ - - - - ~ - ' - - a ~ - - - - ~ - - ~ . ~ L . . _ - - ~ -'-'~ " .~-----..._~ Fig. 18. A Reconstruction of a Tm cell similar to Tm2. Response to (B) flicker and (C) motion

is tentative. The penetrated cell has a more complex response than that of the previous Tm cell (Fig. 16). The flicker response shows the distinct periodicity creat- ed by each OFF response. In contrast, the response to motion is a non-DS plateau depolarization with increased numbers of action potentials. In cells with similar responses, spike frequencies during motion are approximately proportional to log contrast frequency (De- Voe 1980, 1985).

Several variants of Tm2 have been recognized from Golgi studies of Musea (Strausfeld 1970, 1976), and a comparable situation exists in Drosophila among similar looking cells, Tml, Tm2, and Tm9 (Fischbach and Ditt- rich 1989). We have recorded from two Tm2-1ike cells which differ in their response properties, differ slightly in the depth of their axon terminals in the lobula, and differ as to whether or not they arborize in the layer of the L1 terminals in the medulla, just distal to the serpentine layer (Figs. 18-19). One cell, which ends shal- lowly in the lobula and lacks a dendritic arbor just distal to the serpentine layer, has a relatively simple response (Fig. 18). To a clear flash the cell has a rapid biphasic response to ON which declines to a sustained plateau and has a slight undershoot of the resting potential at OFF. Full field flicker evokes a concatenation of the flash responses. The response to motion is a periodic modulation at the contrast frequency and is similar for movement on the 0 ~ 180 ~ axis (Fig. 18 C) and the orthogonal, 900-270 ~ axis (not shown). The amplitudes of the ON responses and the depths of modulation are greater for flicker than for motion. This implies that the receptive field of this Tm2 is probably larger than the stripe widths of 7.5 ~ of the grating used during motion and that the cell thus receives lateral input from neighboring columns.

The second type of Tm2 is accompanied by dye-coupled elements (Fig. 19). The response to a flash is a monophasic transient to ON which decays to a sustained depolarization that is dependent upon the intensity. The response to OFF is a smaller depolarizing transient

which decays more quickly to the resting potential. The response to flicker at lower frequencies is simply a concatenation of the flash response (Fig. 19 B, left). At higher frequencies, the response continues to track the contrast frequency of the stimulus, but the ON and OFF peaks are no longer distinguishable. Although the magnitudes of the periodic responses are reduced, there is still a mean depolarization during flicker, even at 22.7 Hz. The response to motion is different from flicker: the response is generally a slow phasic transient at the onset of motion which decays to a sustained plateau during motion in any orientation (Fig. 19 B, right; orthogonal and oblique orientations were tested at all frequencies with similar results). At higher contrast frequencies, the transient at onset of motion becomes larger and faster, some ripple at the contrast frequency becomes evident in the plateau, and there is a depolarizing rebound at the cessation of motion. In short, the response looks more like that evoked by flicker; i.e. the cell is less able to differentiate motion from flicker at contrast frequencies above 5- 13 Hz. We have seen such decays and rebounds in responses to motion stimulation at high contrast frequencies in other medullary cells. Borst and Egelhaaf (1989) have recently reported a similar phenomenon in the spike rate of H1. The response (not shown) to 500 ms clear flashes of different sizes centered at (42 ~ , 32 ~ ) indicates that the impaled cell integrates over a field about 40 ~ in diameter. The ON mechanism has a steeper areal response function than do the OFF and plateau mechanisms, but all reach an asymptote with spots about 40 ~ in diameter.

Another Tm cell recorded from and stained is a previously undescribed type which is dye-coupled with a Y18 cell (Fig. 20). Because the response is different from that of Y18 stained alone (see Fig. 22 below), the impaled cell was likely the Tm cell. This Tm cell extends dendrites laterally in the stratum of the amacrine plexus (see Fig. 7), sends its axon through the basket of Y18, and terminates in the second layer of the lobula, just medial to the T5 layer. The horizontal retinotopic pro-

A


B FUCKER MOTION

__f Lrt~2yLnyt.n~j L___ 3.16 Hz

5.16 Hz , _ _ F U ~ k__ , -

I I

J I

-62 13.5 Hz

22.7 Hz

Fig. 19. A Reconstruction of a coupled preparation containing an L1, a cell similar to Tm2, and proximal amacrine cell. B Response to various contrast frequencies of flicker (left panel) and motion (right panel) along the 0~ ~ axis. Motion responses were to 15 ~ gratings except the trace with an asterisk, which indicates a 2.5 ~ grating

C - -

Fig. 20. A Reconstruction of a recorded Tm cell and a dye-coupled Y18. Response to (B) flicker and (C) motion

j ec t ion o f the axon t e rmina l is rough ly as b r o a d as the r e t i no top ic p ro j ec t i on o f the dendr i tes , bu t the t e rmina l has a b o u t a 4-fold ver t ica l magn i f i ca t ion . The f l icker and m o t i o n responses are s imi lar to those r eco rded f rom a m a c r i n e cells o f the p lexus (Fig. 7).

The f inal type o f t r a n s m e d u l l a r y n e u r o n r eco rded was a T2 cell (Fig. 21) d y e - c o u p l e d to an amac r ine cell. Cou -

p i ing be tween a T2 and an amac r ine cell was also f o u n d in a r eco rd ing f rom a d i f ferent type o f amac r ine cell (Fig. 13). The T2 a rbor izes in the s t r a t u m o f the m o r e p r o x i m a l L1 te rmina l s a n d m o r e d is ta l ly be tween the s t r a ta o f the L2 t e rmina l s a n d the superf ic ia l L1 te rmi- nals. The responses o f T2 (Fig. 21), bo th to f l icker and to mo t ion , are very s imi lar to those r eco rded f rom neu-


A

B

C

3 4 ~ I . . . . . . ~ .............. ' . . . . . ~ " ' " ' r ' ' ~ v ~ . . . . . . . . . . . . . . . . . ~ - ~ " " . . . . . . . . " " ~ ~,.~ ~ 90 ~ Fig. 21. A Reconstruction of two cells, a T2

and an amacrine cell. The T2 cell was likely the recorded cell as it was slightly brighter than the amacrine cell. Response to (B) flicker and (C) motion

C

B

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Fig. 22. A Reconstruction of a Y18 cell. Re- sponse to (B) flicker and (C) motion

rons in the amacrine plexus at the level of the proximal L1 terminals (Fig. 7). The principal difference is that the T2 response to mot ion shows much less sustained depolarization to 5.1 Hz contrast frequency motion (cf. Figs. 21 C and 7C), perhaps due to a smaller resting potential in the T2 recording.

The third principal class of medullary output cells consists of the Y cells. They are so named because after leaving the medulla the axon bifurcates, usually closer to the lobula plate, and sends processes to both the lobula and lobula plate (Strausfeld 1970). Two preparat ions contain Y18 cells, the dye-coupled preparat ion men-

tioned above (Fig. 20) and a singly filled cell (Fig. 22). The Y18 cell has a basket of dendrites about three columns wide below the serpentine layer, and the axons extend across almost all layers of the lobula and lobula plate. The response to flicker (Fig. 22) is similar to that of the Tm cell shown in Fig. 16, which also has a process at the level of the YI8 basket. Like that Tm cell, the Y18 mot ion response is a sustained hyperpolarization to mot ion in any direction, but with only some slight periodic modulations. These modulat ions might be a remnant of out-of-phase periodic signals f rom multiple inputs, such as a number of T m cells. The Y18 cell,


C

B m _ _

-20

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900 Fig. 23. A Reconstruction of a Y17 cell. Re- sponse to (B) flicker and (C) motion, The motion responses were similar in oblique directions (not shown)

C

B ~

4O ~

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Fig. 24. A Reconstruction of two equally bright Y cells with dendritic arbors at the level of L2. Response to (B) flicker and (C) motion. Four- ier analysis (not shown) yields flicker harmonic amplitudes (mV p-p): 1--7.6, 3 = 1.8 and motion harmonic amplitudes (mV p-p) at 180~ 1=4.5, 2=2.4, 3=1.4

which lacks large modulations during movement, could hardly be presynaptic to the Tm cell, which has large modulations.

The Y17 cell (Fig. 23) arborizes in the stratum of the L2 terminals and responds to flicker with periodic ripple at the contrast frequency. The potential hyperpo- larizes to ON and depolarizes to OFF in a manner similar to that of the LMCs. The response to motion is a sustained depolarization with ripple primarily at the second harmonic of the contrast frequency. It would be expected from the extended dendrite and lobula plate terminal that this cell would have a wide horizontal receptive field. However, particularly along the 90~ ~ axis of motion, but also along the 0~ ~ axis, there are large differences in noise between the responses at the two stop times. This implies that the receptive field of this cell is as small as a 7.5 ~ stripe of the grating.

In another preparation, two equally bright, dye-cou-

pied Y cells in the same medullary column were stained (Fig. 24). Both have their dendrites in the stratum where L2 terminates, both send axons to the horizontal layer of the lobula plate, but each terminates in a separate layer in the lobula. The response to flicker is periodic about the resting potential, primarily at the contrast frequency (Fig. 24), and appears similar to LMC responses. The motion response is also primarily at the contrast frequency, but contains only a depolarizing ripple and has significant power at the second harmonic as well (Fig. 24). Responses to all orientations of motion are similar, but the differences in membrane potential during stops of motion on the 40~ ~ axis indicate a relatively small receptive field for whichever of these two Y cells was recorded.

The last class of medullary output cells to be considered here includes those which project to neuropils of the brain proper. Many fibers leave the medulla anterior-

668

A

,/

C

B

-32


Fig. 25. A Reconstruction of a large cell in the serpentine layer which projects to the central brain. The axon was weakly stained and could not be followed beyond the lobula. Response to (B) flicker and (C) motion

[

r

C_ -3O

Fig. 26. A Reconstruction of a proximal amacrine cell which projects to the central brain. Response to (B) flicker and (C) motion. Note the instability of the hyperpolarization plateau during some, but not other, directions of motion (arrows)

ly t h rough the serpent ine layer in a g r o u p k n o w n as Cuca t t i ' s bundle , which suppl ies the pos t e r io r opt ic t ract . One such cell was r eco rded and filled, bu t no t so comple t e ly as to enable us to fo l low its axon b e y o n d the lobu la (Fig. 25). In the medu l l a the cell ex tends ap-

p r o x i m a t e l y 300 ~tm ver t ica l ly and has two discrete a reas in which fibers course for very shor t d is tances in to the neuropi l . F ibers in the ros t ra l medul la , near the soma, pene t r a t e d is ta l to the serpent ine layer and t e rmina te in t iny bulbs , whereas f ibers in the cauda l medu l l a are


exclusively proximal to the serpentine layer and taper at their ends. The motion responses of this cell consist of large (15 mV) sustained depolarizations with superimposed spikes. During the periodic flicker response (recorded later), the spikes appear to have been reduced or lost (Fig. 25).

The axon of a second cell projecting to the central brain leaves the medulla through the inner chiasm before joining the posterior optic tract (Fig. 26). The dendritic field comprises a dense array of columnar fibers extend- ing through most of the caudal medulla proximal to the serpentine layer. The flash response is a hyperpolarizing transient at both ON and OFF, a type of response rarely seen in medullary cells [cf. DeVoe and Ockleford 1976]. Flicker produces a simple concatenation of the flash responses. The cell is inhibited (hyperpolarized) by motion, but the stability of the membrane potential during motion varies according to the direction of motion (Fig. 26, arrows).

Discussion

In this paper we have presented recordings from and stainings of neurons penetrated in the fly medulla. One finding is that dye may appear in more than the one cell that has been injected with dye. Another is that the cells identified by dye staining have included both columnar and tangential medullary intrinsic cells, and both columnar and tangential medullary output neurons. We have compared responses of neurons to flashes, to flicker, and to motion of gratings. For analyses of periodic components in responses to flicker and motion, we have compared Fourier harmonics for flicker versus motion and as functions of directions of motion. One result of this is that directional selectivity in fly visual cells can be manifested as changes in amplitudes of periodic components, just as has been found in DS cells of the vertebrate retina (DeVoe et al. 1989).

For the remainder of this discussion, we will focus on the phenomenon of dye-doupling among medullary cells, on the physiology of the inputs to the medulla, on motion processing in the medulla, and on further steps in motion processing beyond the medulla.

Dye-coupling

The dye-coupling seen in some preparations is of dubi- ous advantage. It provides information about possible functional connectivity between cells, but it often makes ambiguous the determination of the recorded cell. Before discussing the functional aspects of dye-coupled cells, the real versus the artifactual nature of the coupling must be addressed. Neurons may take up Lucifer Yellow from extracellular space under some conditions. In some vertebrates, bathing the retina for tens of minutes with a Ringer solution containing 0.4% Lucifer and a Ca 2 § chelator results in the uptake of Lucifer by specific types of neurons (Detwiler and Sarthy 1981; Sarthy and Hil- bush 1983). In flies, extracellular Lucifer is selectively

taken up by photoreceptors during tens of minutes of relatively intense blue illumination (Wilcox and Frances~ chini 1984), and neurons of the lamina and medulla take up Lucifer from broken microelectrodes inserted into the neuropil for several hours (Strausfeld, pers. comm.). For these extracellular techniques to be successful, neurons have to be exposed to many more molecules of Lucifer for much longer times than with our intracellular technique.

In our preparations we assume that the Lucifer must be injected into cells. In brains of 3 separate flies, while the electrode was in extracellular space (as indicated by a resting potential of 0 mV), dye was ejected by passing several nanoamperes of current for approximately 4 min (cf. 7.5 nA.s for successful intracellular staining as reported in Methods). No dye staining was found. Further evidence that our technique does not result in Lucifer pooling or entering cells from extracellular space comes from a few preparations in which portions of many cells were stained. In each of these preparations (none shown), a linear pattern of fine fluorescing dots could be traced leading from one cell portion to the next. Typi- cally, the stained portion of a cell ended abruptly and coincided with the linear pattern. Presumably, electrodes with damaged tips produced these patterns by extruding dye into cells penetrated along a track, though no hyperpolarizing current was passed. In each case, the linear pattern of fine fluorescing dots matched the electrode track entrance and depth. In some preparations presented here, some cells fluoresced that were deeper than the impaled cell. We assume that Lucifer injected into the impaled cell passed intracellularly into the others, even in preparations in which no direct fluorescing con- nection could be found, e.g. Fig. 17. A cell soma is obviously connected, hence dye-coupled, to the cell's arborizations, but in many cases the connecting neurite was so fine that we were unable to trace it. The small diameter, hence high electrical resistance, of such fine neurites is probably responsible for the electrical silence of the cell's soma. Such may also be the case with the sometimes untraceable connections between dye-coupled cells. Therefore, dye-coupling among cells through such fine neurites may be more indicative of common lineage than functional connectivity.

Two patterns of functional dye-coupling have been resolved: coupling among 'homologous' cells (Fig. 7) and coupling among unlike cells (Figs. 13, 19-21, 24). If functional dye-coupling indicates connectivity through gap junctions (Stewart 1978), then the two patterns seen in the medulla may serve different functions. The coupling among similar cells (Fig. 7) could serve to increase sensitivity, with a concomitant reduction in spatial resolution. This may explain the lack of periodic components in motion responses. The function of coupling among unlike cells is more difficult to understand. Curiously, such preparations usually involved a small- field columnar cell projecting, most often centrally, through the multicolumnar dendritic arbor of a subser- pentine, wider-field cell, the coupled Y cells of Fig. 24 being an exception. The columnar elements known from previous Golgi studies (Strausfeld 1976), e.g. the Tm2


cell of Fig. 19 and the T2 cells of Figs. 13 and 21, are present in every column. Thus, the dendrites of the amacrines could be coupled to many similar columnar cells, but apparently are not. No wide-field medullary amacrine cell type is known to occur as frequently as medullary columns, though the terminals of many medullary amacrines may be 'synperiodic' with the columns (Campos-Ortega and Strausfeld 1972). Thus, there is a coarsening of the functional units of the retinotopic array mediated by lateral interactions of the subserpen- tine amacrine cells. The polarity of their 'synperiodic' terminals is unknown, but the lack of continuous increase in the response to clear flashes of increasing size (Fig. 19) indicates that the terminals may be outputs rather than inputs.

Since cellular transmission via electrical gap junctions is faster than chemical transmission, coupled units may serve to propagate signals quickly to the lobula plate and/or lobula, and to quickly influence signals in medullary columns surrounding the central through-channel. The finding of dye-coupling, and therefore presumably gap junctions, does not mean that chemical transmission is absent. Some neurons in the optic lobes of flies contribute to both gap junctions and to chemical synapses with the same post-synaptic neuron (Strausfeld and Bas- semir 1983).

Inputs to the medulla

Lamina monopolar cells have been considered motion insensitive (DeVoe 1985). However, previous tests of unstained LMCs with moving stimuli (Dubs et al. 1981; Dubs 1982) measured response modulation only for one direction along a given orientation and did not compare the response with the modulation evoked by motion in the opposite direction or by flicker. In the present study, unambiguously identified L1, L2, and L3 neurons respond similarly to flicker and to motion in various orientations (Figs. 1-3). Recently it has been argued that at high light levels LMCs are optimized to detect moving edges due to the spatially antagonistic center-surround organization recorded for the receptive field of an LMC (Srinivasan et al. 1990). At the same time, it has been argued from several kinds of evidence that phasically responding L1 and/or L2 neurons may not participate in the computation of motion by the optomotor system (Coombe et al. 1989). For example, by evoking a directional response in the HI neuron of the lobula plate in response to small (0.16 ~ steps of a ' random' constant grating they argue that at least one of the inputs to an elementary movement detector (EMD) presynaptic to H1 must respond tonically and with 'memory' of the pattern position. It seems risky, however, to base any conclusions about motion detection upon such' ano- malous resolution' without controls for stimulus arti- facts (Palka and Pinter 1975) and aliasing (Thibos et al. 1987). Moreover, the results of Coombe et al. (1989) and of this paper are consistent with synaptic noise itself being a tonic response. Only when there is synaptic noise in an LMC are there spikes in an Hi neuron that is

simultaneously recorded (Coombe et al. 1989). Likewise, in movement responses of L1 and L2 in Figs. 1 and 2, different synaptic noise and DC potentials at one stopped position of the grating vs. the other imply that phasic LMCs can indeed have a memory of grating position.

Also contrary to the conclusions of Coombe et al. (1989), a) phasic signals in the input channels are indeed sufficient to stimulate the current best model of a motion detector (Egelhaaf et al. 1989), to drive the H1 neuron (Franceschini et al. 1989), as well as elicit optomotor behavior (Kirschfeld 1972). b) Anatomically, it is difficult to identify a tonically responding second order neuron type which could comprise one or both small-field retinotopic input channels. Based on the well known synaptology of the lamina (Strausfeld and N/issel 1981; Shaw 1989) five cell types are postsynaptic to R1-6: the wide-field lamina amacrine (the Am cell), L1-L3, and TI (though Shaw (1984) interprets T1 as postsynaptic to a fibers and therefore a third order neuron). In each cartridge a fibers from at least 2 Am cells are postsynaptic to R1-6, and each Am cell has fibers in 6-15 cartridges (Campos-Ortega and Strausfeld 1973). Such broad spatial connectivity is incompatible with motion processing (Buchner 1976), and it is thus likely that L1- L3 and T1 are among the second order neurons in the optomotor pathway. These 3 LMCs respond phasically to changes in illumination (Figs. 1-3) as does T1 (Jfirv- ilehto and Zettler 1973). c) Coombe et al. (1989) show that the contrast frequency response of lamina monopolar cells in Drosophila and Eristalis extends to higher frequencies than does the optomotor response or the contrast frequency response of wide-field directionally selective neurons of the lobula plate. In Calliphora however, contrast frequency responses that are typical of directionally-selective lobula plate neurons (Hausen 1984), optomotor reactions (Wehrhahn 1985), and lamina monopolar cells (French and J~irvihleto 1978) all have maxima around 5-10 Hz. Thus LMCs are not pre- cluded from being involved in the optomotor pathway. Photoreceptors, which are obviously part of the optomotor pathway, also have higher frequency response characteristics, which are filtered by non-linear interactions among higher-order neurons intervening between R1-6 and the lobula plate.

On balance, a variety of evidence does not preclude any second order lamina neuron from involvement in motion processing. The lamina monopolar cells L1 and L2 are thus viable candidates as distal components of the elementary motion detector. In particular, the medulla stratum comprised of the L2 terminals may be the site of the non-linear lateral interaction in the EMD as will be discussed below.

Motion processing in the medulla

It has been known that a variety of motion responses exists in cells of the medulla (see Introduction) and also that a variety of morphological types has been associated with these responses. All the identified ceils responded


differently to motion than to flicker. For many neurons, such as some amacrine cells (Figs. 8, 9, 11) this difference merely reflects the spatial asymmetry of a neuron's receptive field with respect to the small-field inputs being integrated. However, for other, typically columnar, neurons (Figs. 6, 16, 23, 24) the response to motion can not arise by asymmetrical spatial integration or temporal smearing, but must represent a processing step such as frequency doubling or rectification, which may ultimate- ly contribute to perception of directional motion. None of the identified neurons is as directionally selective as any of the neurons recorded in the lobula plate. But, as will be discussed below, some of the non-directional medulla cells may contribute significantly to the DS responses of the lobula plate giants. In the identified medulla cells, motion information is found to be coded in 3 forms: primarily periodic oscillations (ripple), DC shifts in membrane potentials (sometimes with superimposed ripple and/or spikes), or increased noise.

Identified cells which have their dendrites in those strata of the distal medulla which receive L2 axon terminals respond periodically to motion. The simplest types of periodic responses are seen in cells which respond to flashes and to flicker as does L2 (Figs. 16, 23, 24). These cells respond to full-field motion with slight DC shifts but primarily with periodic fluctuations which are almost as large as those evoked by full-field flicker. This indicates that the cells have relatively small receptive fields. Motion responses may also contain significant second harmonics of the contrast frequency. In one Y cell (Fig. 24), though the motion response is dominated by the fundamental at the contrast frequency, the significant power in the second harmonic acts to rectifiy the modulation during motion compared to flicker.

In two other cells, a Y cell and a Tm, the second harmonic dominates the responses (Figs. 16, 23). The motion responses of these cells and many other unstained medullary cells which have similar flicker responses are, with only two exceptions, not orientational- ly or directionally selective. Thus, the simplest type of motion response in the medulla is a small-field, orienta- tionally isotropic, periodic modulation with components at the fundamental and second harmonic of the contrast frequency. This type of response has been recorded in several columnar cell types which have dendritic arbors primarily in the L2 stratum. That the responses of some medullary neurons have power at the second harmonic of the motion stimulus contrast frequency is significant in terms of modelling the elementary motion detector. Motion detection requires a non-linear operation on neighboring channels which the current best computational model satisfies by a multiplication stage (Egelhaaf etal. 1989). This stage introduces second harmonics which subsequently disappear at a second stage in which output from preferred and null direction half-detectors are subtracted. Depending upon the balance of the subtraction the Second harmonic may disappear completely. The strong second harmonic component in medulla columnar neurons with arborizations in the stratum of the L2 terminals may indicate that the non-linear operation, such as multiplication, occurs at that level. From Golgi

studies, Strausfeld (1984) also has suggested that the medulla columnar neurons which arborize at the level of L2, cell types SUB and Tml, could be small-field components of adjacent channels of the elementary motion detector leading via T4 and T5 neurons to the lobula plate giants. Unfortunately, we did not stain SUB or Tml in this study.

More complex are the responses of those cell whose dendritic arbors overlap one of the L1 strata (Figs. 4, 7, 15, 17-21). The responses to a clear flash are sustained depolarizations with fast depolarizing transients at ON and OFF. The responses to flicker are not like those of LMCs. Compared with the previous group, these neurons are more heterogeneous, both morphologically and with respect to their motion responses. Generally, during motion, the membrane potential is substantially depolar- ized and usually has a periodic ripple which can vary with orientation (Fig. 15) or direction (Fig. 4). Thus, though these cells could be directly postsynaptic to L1, their signals reflect complex synaptic interactions among other cell types of the distal medulla.

In the proximal medulla, we have recorded only from cells whose dendritic arbors innervate several-to-many columns. We did not stain (unambiguously) the columnar output cells T3 or T4. Twice we have stained T4 cells, but both preparations were too ambiguously dye- coupled to determine the impaled cell. Many of the proximal amacrine cells may be involved in functional units which sample the retinotopic array more coarsely, as was discussed previously in reference to dye-coupling. Lastly, the proximal medulla contains dendritic arbors of cells which are inhibited by motion (Figs. 22, 26). This also agrees with the estimated depths of unstained units that were previously found to be inhibited by motion (DeVoe 1980).

Motion processing beyond the medulla

One of the most persistent questions about motion processing in the fly is: What are the identities and response properties of cells presynaptic to the directionally-selective giant neurons of the lobula plate (e.g. HS and VS neurons)? From neuroanatomical studies (Strausfeld 1970, 1976) medulla or lobula neurons which project to the lobula plate are columnar and have dendritic arborizations restricted to one or a few columns. The only identified synaptic inputs to the lobula plate giants are reported from serial reconstruction of transmission electron micrographs of selectively stained small-field neurons: a single T4 to VS7 (h) contact reported by Straus- feld (1984) and several T4 to HS (probably HSE) and T4 to VS contacts shown by Lee and Strausfeld (1989). Physiologically the non-spiking giant neurons of the lobula plate, HS and VS neurons (rev. Hausen 1984), respond directionally to full-field moving grating stimuli with either a sustained depolarization or hyperpolarization. In response to a small-field stimulus (<20 ~ aper- ture: Gilbert et al. 1989), or a stimulus which is relatively narrow in the direction of stripe motion (8.5 ~ by 81 ~ slit: Egelhaaf and Borst 1989; Egelhaaf et al. 1989; cf.


stimuli which are small-field, but elongated in the direction of stripe motion: Hausen 1982), both VS and HS neurons have superimposed on their DC polarizations a ripple which is periodic at the fundamental or second harmonic of the contrast frequency. With stimulation of still smaller fields (< I0~ the DC polarizations are almost abolished, but the directional periodic ripple re- mains (Gilbert et al. 1989). Theoretical and pharmaco- logical studies (Egelhaaf et al. 1990) have shown that blocking the subtraction stage of the model increases the power of the second harmonic of the stimulus contrast frequency in the output of the model. This increase is also seen physiologically in a lobula plate giant upon administration of the GABA antagonist picrotoxin. Fur- ther, measurement of ionic conductances (Gilbert 1990) has demonstrated that the subtraction stage takes place on the membrane of the lobula plate giants. Thus the neuroanatomical and physiological evidence is con- sistant with the cells pre-synaptic to the lobula plate giants being small-field units which respond to motion with periodic fluctuation of their membrane potential. In the present study, all 4 small-field cells identified as projecting to the lobula plate respond periodically to motion in certain directions (Figs. 15, 22-24). The response modulation at the second harmonic of the stimulus contrast frequency could be provided directly by the Y cells (Figs. 23, 24) or indirectly, as suggested by Strausfeld (1984), from a Tm (Fig. 16) through lobula T5 neurons, whose response also shows frequency doubling (Gilbert, unpubl.). However, it is not yet known whether any of these small-field neurons is pre-synaptic to the lobula plate giants or how the small-field lobula plate inputs which do not show frequency doubling (Figs. 15, 22) functionally contribute to motion processing. Thus more needs to be learned not only about the specific inputs to the lobula plate giants, but also what their input signals are during motion. The present results are a first step towards that goal.

Acknowledgements. We thank Drs. Martin Egelhaaf, Wulfila Gron- enberg and Nicholas Strausfeld for their comments on previous versions of the manuscript, Dr. Peter Carras for many helpful dis- cussions of this work, and Joanne DeLone for technical assistance. This research was supported by NIH grant EY05163 to RDD and NIH training grant EY05903 to CG.

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