J Comp Physiol A (1986) 159: 827-840 Journal of Comparative Sensory, Neural,

and

P h y s i o l o g y A B....lo~, Physiology

�9 Springer-Verlag 1986

Identified target-selective visual interneurons descending from the dragonfly brain

Robert M. Olberg Department of Biological Sciences, Union College, Schenectady, New York 12308, USA

Accepted August 1 i, 1986

Summary. 1. Eight large interneurons descending in the dragonfly (Aeshna umbrosa, Anax junius) ventral nerve cord from the brain to the thoracic ganglia were identified anatomically with intracel- lular dye injection (Fig. 3). All eight were strictly visual and responded only to movements of small patterns, such as black squares, 'targets', moving on a white background.

2. The target interneurons all projected from the protocerebrum at least as far as the metatho- racic ganglion. Within the protocerebrum they ar- borized in the posterodorsal neuropil region, near the base of the circumesophageal connectives (Fig. 3).

3. The receptive fields of six of the cells were large, including most of the forward hemisphere of vision. For five of these, spiking responses were often restricted to a much smaller region within the receptive field, with stimulation of other areas yielding only subthreshold responses (Figs. 4 and 5, Table 1).

4. The pattern of selectivity for target size var- ied, with some neurons responding only to small targets, some showing consistent responses over a wide range of target sizes, and one preferring larger targets (Fig. 6, Table 1).

5. Five of the interneurons were directionally selective. Movement in the antipreferred direction elicited hyperpolarizing responses in two of them. Movements of large patterns, such as a checker- board pattern covering the forward hemisphere, elicited opposite directional responses, i.e., hyper- polarizations in the preferred target direction and subthreshold depolarizations in the antipreferred direction (Fig. 7). A large pattern moving in any

Abbreviations: D I T Dorsal intermediate tract; D M T Dorsal median tract; M D T Median dorsal tract; VNC Ventral nerve cord; D C M D descending contralateral movement detector

direction inhibited the response to target move- ment (Fig. 8).

6. These neurons mediate, in part, the visual control of flight orientation. I propose that they convey turning signals to the wing motor in re- sponse to objects moving relative to the animal.

Introduction

Descending interneurons in insects receive input from the sensory structures of the head and pro- vide input to thoracic motor centers to initiate or guide behavior (Tanouye and Wyman 1980; Blon- deau 1981; Pearson and Robertson 1981; Bacon and M6hl 1983; Bicker and Pearson 1983; Rei- chert and Rowell 1985). From studies of descend- ing interneurons in a variety of insects, such as grasshoppers (Catton and Chakraborty 1969; Rowell 1971; Bacon and Tyrer 1978; Simmons 1980; Rowell and Pearson 1983; Kien and Altman 1984), flies (Strausfeld and Bacon 1983), and moths (Olberg 1983), two generalizations emerge: 1) Many descending interneurons are multimodal and therefore appear relatively nonspecific to the experimenter (but see Olberg 198t b; Reichert et al. 1985) and 2) single descending interneurons usual- ly do not elicit movements by themselves. The only published exceptions to the second generalization are the descending interneurons which elicit song in the cricket (Bentley 1977) and the giant fibers which initiate escape in flies (Tanouye and Wyman 1980; Tanouye and King 1983).

If descending interneurons appear relatively nonspecific in their sensory reponses, how is the precision which characterizes many insect behav- iors (e.g. Collett and Land 1978) achieved? Kien

828 R.M. Olberg: Identified visual target interneurons in the dragonfly

A E l e c t r o d e ~ ~ B

Projection L,/ I / Screen ~ /

Fig. 1 A, B. A, Visual stimulation arrangement. Dragonfly was mounted ventral side up and tilted 15 ~ up from horizontal. The head was centered between the back edges of a translucent, wrap-around projection screen in line with the center of the front 90 ~ x 90 ~ facet of the screen. B, View of screen from the front. Square target 4 ~ visual angle at (a) has following dimen- sions at indicated locations (HorxVer t ) : b) 2.1~ ~ c) 1.4 ~ x 1.4 ~ d) 4 ~ x 2.8 ~ e) 2 ~ x 2 ~ f) 1.4 ~ x 1.4 ~ (Visual angle cal- culated from the center of the dragonfly's head)

and Altman (1984) suggest that most behavior is probably directed by many descending interneu- rons acting in concert. Simultaneous transmission by many parallel channels carries the potential for sharpening the precision of the sensory informa- tion.

A group of dragonfly descending interneurons are of interest because they do not fit the above generalizations. They are purely visual and re- spond only to the movement of small patterns or 'targets' (Olberg 1981a). Most are also selective for target direction. When stimulated intracellu- larly some target interneurons can elicit move- ments of from two to all four wings (Olberg 1978).

In the present study I have used intracellular microelectrodes and Lucifer Yellow injection to identify eight individual target interneurons. This paper presents their anatomy, directional prefer- ences, receptive fields, size selectivity and responses to several standard stimuli.

Materials and methods

Research animals. The animals used in this study were adult males and females of 2 dragonfly species, Aeshna umbrosa and Anaxjunius, both of the family Aeshnidae. They were collected as larvae and reared in the laboratory (23 ~ 16L/8D light cycle) on a diet consisting mainly of mealworms (Tenebrio). After adult eclosion they remained at 23 ~ for 2 days before being moved to a 4 ~ cold room. I usually used the animals within one week after emergence.

Preparation. Using a mixture of beeswax and rosin, I waxed the animal's ventral surface behind the metathoracic legs to a steel rod. I waxed the back of the head to the thorax dorsally and on each side. I fixed the rod to a magnet stand, with the animal ventral side up at a 15 ~ angle to horizontal, anterior end up (Fig. 1). (Extracellular recordings from dragonfly target

interneurons (Olberg 1981 a) showed that their directional pref- erences remained constant relative to the animal whether it was dorsal or ventral side up.)

I cut off the legs and removed the ventral thoracic cuticle anterior to the metathoracic leg sockets. To reduce movement from flight muscles, I cut all of the nerve roots from the meso- and metathoracic ganglia. To further reduce movement and to expose the recording site, I removed the mouthparts and gut from mouth to abdomen.

A manipulator on the magnet stand held a small, stainless steel capillary tube. The tube (1) applied pressure downward and backward on the prothoracic ganglion to stabilize the re- cording site, (2) regulated the saline level around the recording site, and (3) contained a Ag/AgC1 ground electrode.

Electrophysiological recording. I used glass micropipettes filled with Lucifer Yellow CH (3% Lucifer in 1% LiC1 in the tip, 1 M LiC1 in the shaft) to penetrate axons in the cervical connec- tive just posterior to the subesophageal ganglion. After filling the electrodes, I etched their tips slightly by dipping them in a 20 x dilution of hydrofluoric acid and washing them in dis- tilled water. The two movements of a micromanipulator (Zeiss, Jena) were used to hold the microelectrode and the stainless steel spoon which supported the fused connective. This record- ing situation was quite stable; penetrations often lasted for more than an hour.

The signal from the electrode was amplified by a preampli- fier (WPI M707). The signal to be recorded on magnetic tape passed through a high pass filter ( t a u = l l 0 ms) to eliminate the DC component. I used a 4 track cassette recorder (Fostex) with FM adapters (Vetter) to store the signal for later analysis. Signals were analysed with a digitizing waveform analyzer (Data Precision DATA 6000) and printed out with a digital plotter (HP 7470A).

Visual stimuli. I rear-projected moving images on a translucent plastic screen which covered nearly half of the spherical field of vision of the dragonfly. The front face of the screen was a square covering 90 ~ x 90 ~ visual angle. Dorsally and to both sides, the screen angled back to cover the region of space be- tween 45 ~ and 90 ~ back (Fig. 1).

The stimuli were black and white patterns photographed on Kodak Ortho film projected from a 2 m distance with a 35 mm slide projector (Leitz Ortholux). The light intensity (sur- face luminance) of the white areas on the screen, viewed from the animal's side, was 170 cd/ft 2.

With this projection arrangement, both the size and the shape of the projected pattern, measured in visual angle from the surface of the eye, varied over the surface of the screen. Figure 1 B shows examples of the size changes when measured in visual angle at the dragonfly eye. These resulted from both the variation in distance between the eye and screen and the variation in projection and viewing angle. For example, a square 4 ~ on a side when projected on the center of the 90 ~ square screen face was only 1.4 ~ visual angle when projected on the corner. Here I will refer to each target by its size when projected at the center of the screen. Variation was continuous over each of the 4 faces of the screen, but there were discontinu- ities at the borders between faces.

The beam reflected off a front-surface mirror before reach- ing the screen. I produced pattern movements by tilting the mirror horizontally or vertically, using the movement of two loudspeaker cones to produce the deflections. These were driven by a power amplified signal from a trapezoid generator. The beam passed through a window in an opaque screen to restrict its width to approximately the size of the screen, but some projected movement was visible on the wall behind the animal.

R.M. Olberg: Identified visual target interneurons in the dragonfly 829

within the cord and to determine its tract through the ganglion. I photographed good sections within the connective and gangli- on under phase contrast with Polaroid 667 Film and marked the fluorescing axonal cross sections.

To determine the three dimensional arrangement of den- dritic arborizations I sectioned the brain sagittally (30 gm) and reconstructed the neurons from the sections with a drawing tube.

In the adult dragonfly, the brain and optic lobes bend up- ward to lie in a plane that is nearly at right angles to the plane of the rest of the nervous system. Dorsal tracts in the subesophageal and thoracic ganglia are therefore continuous with and homologous to posterior tracts in the brain. For sim- plicity I will refer here to the posterior surface of the brain as its dorsal surface.

Fig. 2. Identification of target interneuron axons in cross sec- tion of prothoracic ganglion. Top : Double exposure, using epi- fluorescent illumination and phase contrast to show individual- ly stained axon (MDT 1, arrow) with background detail. Bot- tom: Enlargement of area outlined above showing the locations of the target interneurons. Two arrows from DIT3 indicate observed variation in the axon location. There was some varia- tion in the order of MDT2, 3, and 4, but order shown here was most typical. (Scale bar 50 gin)

Control experiments showed that these movements did not elicit responses in the identified neurons except for the neuron (DMTI) whose receptive field was in the backwards direction.

One series of experiments (Fig. 8) required simultaneous movements of a small black target and a larger black and white striped grating pattern. The grating pattern consisted of a series of equally spaced black stripes, printed with a Kodak .copying machine on a transparent acetate sheet. I used an XY recorder (MFE) to move the grating back and forth about 5 cm in front of the projection screen. The target was projected through the acetate sheet onto the screen. The black target was visible even on the dark stripes as a darker black square.

Anatomy. After recording from an axon, I injected Lucifer Yel- low (5-10 nA) for 7 to 30 min. After allowing an additional diffusion time of 3045 rain, I dissected out the head and thor- acic ganglia. These I fixed in 4% formalin in a phosphate buffer solution (10min) followed by 4% formalin in methanol (45 min). I dehydrated the ganglia in propanol and cleared them in methyl salicylate. Each filled neuron was drawn using a Nikon drawing tube on a fluorescence microscope (Olympus BHS) using a fluorescent pencil and UV light source to illumi- nate the drawing. The ganglia were then embedded in soft Spurr resin for sectioning. The cervical connectives and prothoracic ganglion were sectioned transversely (20 gin) to locate the axon

Results

M o s t l a rge d i a m e t e r a x o n s in the a d u l t d r a g o n f l y t h o r a c i c ne rve c o r d were v i sua l . T h e e igh t i n t e r - n e u r o n s d e s c r i b e d he re h a v e been g r o u p e d t o g e t h e r b e c a u s e t hey s h a r e d c e r t a i n c h a r a c t e r i s t i c s , b o t h a n a t o m i c a l a n d p h y s i o l o g i c a l . T h e y al l d e s c e n d e d f r o m the b r a i n , a n d the i r a x o n s were a m o n g the l a rges t in the v e n t r a l ne rve c o r d ( V N C ) . T h e y al l r e s p o n d e d se lec t ive ly to s m a l l p a t t e r n ( t a rge t ) m o - t i on a n d n o t to t he m o v e m e n t o f l a rge t e x t u r e d b a c k g r o u n d s . E a c h was r e c o r d e d a n d d y e - i n j e c t e d th ree o r m o r e t imes a n d s h o w e d c o n s i s t e n t s e n s o r y r e s p o n s e s a n d a n a t o m y .

O f the e igh t t a r g e t i n t e r n e u r o n s , five s h o w e d d i r e c t i o n a l p re fe rences . A t l eas t o n e t a r g e t i n t e r - n e u r o n in e a c h c o n n e c t i v e was se lec t ive fo r e a c h d i r e c t i o n , left , r igh t , u p a n d d o w n . ( D i r e c t i o n a l p r e f e r e n c e s wil l be p r e s e n t e d he re o n l y fo r the in- t e r n e u r o n s w h o s e a x o n s e x t e n d d o w n the r i g h t connec t ive . T h e i r s y m m e t r i c a l p a r t n e r s in the lef t c o n n e c t i v e s h o w e d the e x p e c t e d r eve r sa l in p r e f e r - ences to lef t a n d r igh t . ) Seven o f t he i n t e r n e u r o n s h a d r e c e p t i v e f ie ld cen te r s s o m e w h e r e in the fo r - w a r d v i sua l h e m i s p h e r e , a n d the e i g h t h ( D M T 1 ) l o o k e d b a c k w a r d (F ig . 3).

Anatomical identification

I n j e c t i o n o f L u c i f e r Y e l l o w i n t o the a x o n s w i t h i n the ce rv i ca l c o n n e c t i v e b e t w e e n the s u b e s o p h a g e a l a n d p r o t h o r a c i c g a n g l i a u s u a l l y e n a b l e d m e to see a n d d r a w the s o m a a n d m a j o r d e n d r i t i c a r b o r i z a - t ions w i t h i n the b r a in . T h e dye - f i l l ed a x o n p ro f i l e s were a l w a y s v i s ib le in c ross s ec t i ons o f the ce rv ica l c o n n e c t i v e a n d the p r o t h o r a c i c g a n g l i o n (F ig . 2, top) .

I h a v e g r o u p e d a n d n a m e d the t a r g e t i n t e r n e u - r o n s b a s e d o n the l o n g i t u d i n a l t r a c t t h r o u g h w h i c h t hey p a s s in the p r o t h o r a c i c g a n g l i o n . T a r g e t i n t e r - n e u r o n a x o n s a l w a y s t r a v e l e d t h r o u g h one o f t h r e e

Fig. 3. Anatomical identification of the giant descending target interneurons. Camera lucida drawings (center) of Lucifer Yellow stained neuron profiles in the brain, subesophageal ganglion and prothoracic ganglion viewed from dorsal aspect. Reconstruction of serial sagittal sections (upper right) gives view of the same neuron looking from the right side of the brain (dorsal is left, ventral right). Arrows in cross-sections of the cervical connectives (left top) at the anterior margin of the prothoracic ganglion

and cross-sections of the prothoracic ganglion (left bot tom) show positions of the axons, retouched with white ink. (Scale bars 50 ~tm). Diagram of projection screen (lower right) indicates location and direction of greatest response to target movement. (MDT3 and DIT3 were not directionally selective. DMT1 gave no responses to target movement on the screen - see text.) In this and remaining figures, upward and rightward on screen mean upward and rightward with respect to the animal. (H, Horizon; Z, line directly above the animal)

834 R.M. Olberg: Identified visual target interneurons in the dragonfly

Table 1. Anatomical and response characteristics of the target interneurons. Where there were discrepancies, the most commonly observed responses are shown. Soma locations are with respect to axons recorded in the right connective

Cell MDT1 MDT2 MDT3 MDT4 DIT1 DIT2 DIT3 DMT1

Number of recordings 5 3 3 4 5 8 3 6 Soma location in brain LT RV LT RV LV RV LD LD Left (L), Right (R), Top (T), Dorsal (D) Ventral (V) Directional preference U L ND D R L ND - Left (L), Right (R), Up (U), Down (D) Nondirectional (ND) Spontaneous spike rate 0 0 0 0 0 0 0 0 Max. number of spikes to 7 10 4 20 20 8 17 8 target motion Light on response E E E 0 E E E E EPSP(E), No response (0) Light off response E E E S E E S E EPSP(E), Spike (S) Air puff response 0 0 0 0 E 0 0 0 EPSP(E), No response (0) Greater response to = = B B B = B - white (W) or black (B) target? Preferred target size A S L S S A A - Small (S), Large (L), All (A) Range in number of quadrants 8-10 7-10 7-11 4 7 10-12 10-12 12 - where EPSPs were elicited Range in number of quadrants 0- 7 1- 5 2- 5 4 1 11 1- 3 8 I0 - where spikes were elicited

longitudinal tracts through the prothoracic gangli- on, the median dorsal tract (MDT), dorsal inter- mediate tract (DIT) or the dorsal median tract (DMT). The tract chosen by a given interneuron was consistent, and even the position of the axon within the tract showed little variation (Fig. 2, bot- tom).

The eight target interneurons descended from the protocerebrum of the brain and projected at least as far as the metathoracic ganglion. I could discern very little branching below the prothoracic ganglion, due to the distance from the recording site. The soma location and branching pattern within the brain and axon locations within the cer- vical connective and prothoracic ganglion were used to identify the cells anatomically (Fig. 3).

Within the brain the processes of the target interneurons were restricted to the protocerebrum (Fig. 3). The somata of three of them (MDTI, MDT3, and DIT3) were located very near one an- other in an antero-dorsal group of somata just me- dial to the globuli cell cluster. All three of these cells sent their axons down the connective contra- lateral to their somata. The somata of DIT2, MDT2 and MDT4 were located very near one an- other, in a thin cortex of ventral cell bodies even with or slightly medial to the alpha lobe of the

mushroom bodies. All of their axons projected down the ipsilateral connective. The soma of DIT1 was also ventral, but lateral to those above. The soma of DMT1 was located against the mid-dorsal surface of the brain.

All of the target interneurons arborized in the postero-dorsal region of neuropil very near the point where their axons left the brain via the circumesophageal connective (Fig. 3). None of these interneurons made visible associations with the mushroom bodies, central body or protocere- bral bridge.

Within the protocerebrum the branching pat- tern of the neurons was quite consistent from ani- mal to animal, making each neuron easily recog- nizable by its profile alone. Their large size, consis- tent sensory responses, and consistent locations within the longitudinal tracts of the prothoracic ganglia provided further evidence that the neurons were uniquely identified (Fig. 3).

There was no obvious correlation between the profiles of the neurons within the brain and their receptive field centers or directional preferences. Figure 3 includes the target locations and move- ment directions on the projection screen to which each neuron responded maximally. Notice that DMTI , whose soma and axon were not close to

R.M. Olberg: Identified visual target interneurons in the dragonfly 835

Fig. 4. Response of DIT2 to target movement on various screen locations. Outlines indicate the entire screen divided into 45 ~ • 45 ~ quadrants. Target (4 ~ black square) moved right, left, down, and up with movements centered across each quadrant. Bars under traces show 200 ms stimulus in indicated direction. Only one spike (s) was elicited by target movements (spike clipped to show synaptic potentials). Note subthreshold EPSPs on most of screen and IPSPs to antipreferred (rightward) target movement. (Move- ments 45~ ms; Scale bar 5 mV; Cell 6-28-1)

any of the others, was the only cell whose receptive field was not in the forward direction.

Electrophysiological recordings

Upon penetrating an interneuron, I presented a preliminary set of sensory stimuli to determine whether it was a target interneuron. I first moved a white card (7.5 • 12.5 cm) in various locations and directions around the animal ca. 3 cm from the eye. The 3 mm black dot at its center was an effective stimulus, producing spiking in all of the target interneurons. A control card without a dot never elicited spiking from the forward looking tar- get interneurons (the backward looking DMT1 could not be tested in this way). A card covered with black and white stripes also failed to elicit spikes when moved at a variety of velocities.

To test the neuron for mechanosensory re- sponses, I blew air onto the animal from the front and sides, moved each of its wings up and down and stroked its thorax and abdomen with a soft

brush. None of these stimuli elicited spiking in the target interneurons. Table 1 summarizes some ana- tomical and sensory properties of the target inter- neurons.

In most target interneuron recordings com- pound EPSPs and IPSPs were visible even though the recording site in the axon was a long distance (ca. 2 mm) from, and separated by a ganglion from the probable site of spike initiation in the brain. Such long length constants were due to the large diameter of the axons. I did not see PSPs in other, smaller axons descending from the brain recorded during the study. Furthermore, when the largest diameter fibers, M D T I and D M T I , were pene- trated below the prothoracic ganglion, PSPs were no longer visible.

Receptive fields

To determine the receptive field size and location, I moved a 4 ~ or 16 ~ target up, down, left and right in each of the twelve 45 ~ x 45 ~ quadrants compris-

836 R.M. Olberg: Identified visual target interneurons in the dragonfly

Cel l 9 - 0 5 - a 2

�9 P r e f e r r e d

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-H

-Z

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Cel l 1 1 - 1 8 - b l

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Fig. 5. Variation in responsiveness and apparent receptive field size. Recordings of DITI in two different animals (left and right). Top screens show responses to rightward (preferred) target movement across entire 180 ~ screen along horizontal lines indicated. Bottom screens show responses to three leftward target movements. In this and remaining figures, spikes are clipped to show PSP activity. (Scale bars 200 ms, 5 mV)

ing the projection screen (Fig. 4). The receptive field area in which this target motion evoked EPSPs was much greater than the area in which movements elicited spiking (Fig. 4). To quantify this I counted the number of screen quadrants in which I saw PSPs to target motion and the number in which I saw spikes (Table 1). For all but MDT4 and DMT1 movements nearly anywhere on the screen (10 to 12 quadrants) elicited EPSPs.

The receptive field area in which target move- ment elicited spiking varied greatly across prepara- tions for a given cell type. Figure 5 shows traces recorded from DIT1 in two different preparations. Rightward, preferred-direction movements elicited a total of only 4 spikes within a very restricted region in the first DITI, but the same stimuli eli- cited 67 spikes in the second. The underlying pat-

tern of compound EPSPs in the two rcordings, however, was almost identical. The peaks of depo- larization of the left cell in Fig. 5 corresponded to the highest spike densities in the right cell. What appeared to vary was the overall level of excitabili- ty. Even antipreferred motion (lower screens of Fig. 5) elicited 9 spikes in the second DIT1, while it elicited none from the first. Similar variability occurred for MDTI and MDT2; the variability was less extreme in MDT3, DIT2, and DIT3 (Ta- ble 1). For all of these neurons, variability between preparations was much greater than variability over time within a given preparation. The spiking receptive field area of MDT4 was quite consistent among preparations. It was always confined to a ca. 30 ~ wide vertical strip along the midline above the horizon.

R.M. Olberg: Identified visual target interneurons in the dragonfly 837

DIT1 DIT2

3 2 ~

1

Fig. 6. Target size selectivity in DIT1 and DIT2. Square black targets of indicated sizes moved in preferred direction (right for DIT1, left for DIT2) during the period indicated by the stimulus marker below bottom traces. Checkerboard pattern (8 ~ black and 8 ~ white squares) covered entire screen. (All movements 45~ Scale bar 5 mV; DIT1 cell 9-5-2; DIT2 cell 9-2-1)

-=-=-=-=-_-. ,,,,,,,~.~ ~ , ~ , ~ , r ~ ~ -

, ; _ - ; _ - -

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I Fig. 7. Opposing directional preference in responses of target interneuron to target and checkerboard pattern movement. Pre- ferred direction for target movement in this cell (MDT4) was downward (trace 3). Downward movement of large pattern showed hyperpolarizing response (trace 1) and upward pattern movement showed depolarizing response (trace 2). (Movement 45~ ms; Scale bar 5 mV; Cell 7-12-a2)

Selectivity for target size

All of the target interneurons responded to object movement but not to movement of a large textured background. This visual discrimination occurred over a range of velocities from 45~ to 225~

When moving black squares of various sizes were presented to these interneurons, their re- sponses fell into one of three types (Table 1). Some cells (DIT1, MDT2, MDT4) consistently preferred

the smaller targets, 2 ~ or 4 ~ on a side, over the larger targets, 16 ~ or 32 ~ (Fig. 6). Other neurons (DIT2, DIT3, MDTI) responded about equally to targets of all sizes (Fig. 6). One neuron (MDT3) consistently preferred the larger targets, showing little or no response to 2 ~ or 4 ~ squares. Target interneurons rarely showed even a single spike to movements of a checkerboard pattern, which cov- ered the entire projection screen (Fig. 6, bottom traces).

Directional selectivity

Five of the target interneurons showed clear direc- tional selectivity (Fig. 3); movement in their pre- ferred direction elicited spikes, and movement in the antipreferred direction usually did not (Fig. 7). For two neurons (MDT2, DIT2), compound IPSPs indicated that target movement in the anti- preferred direction was inhibitory (Fig. 4).

The directionally selective target interneurons (MDT1, MDT2, MDT4, DIT1, DIT2) also showed direction-dependent subthreshold re- sponses to movement of the large checkerboard pattern. For these cells, the preferred direction for the large pattern movement was opposite to that for the small target (Fig. 7, cf. 2nd and 3rd traces). Checkerboard pattern movements very rarely eli- cited any spikes from target interneurons, but these invariably resulted from pattern movement in the

838 R.M. Olberg: Identified visual target interneurons in the dragonfly

- , , . . , , J , , . , , , , , . . . . . . . . .

- I l l i III I I . . . . . . . . . .

II1'tll

Fig. 8. Responses to target movement with and without a mov- ing background. DITI produced spikes in response to right- ward 4 ~ target movement (trace 3) and was depolarized (trace 2) by leftward movement of a striped pattern (8 ~ black and white stripes covering entire screen), but leftward stripe move- ment completely inhibited response to the rightward target movement (trace 4). Bottom trace shows recovery of target response in the absence of stripe movement. The five traces are shown in the sequence they were obtained (i.s.i.=15 s). (Movements 45~ ms; Scale bar 5 mV; Cell 9-5-2)

antipreferred direction for targets. Checkerboard movement in the direction preferred for the target elicited hyperpolarizing responses (Fig. 7, top).

Since the target interneurons preferred opposite directions of target and background motion, per- haps they would respond maximally to a combina- tion of target and background movements in oppo- site directions. Figure 8 shows the results of an experiment designed to test this hypothesis. Al- though this DIT1 interneuron showed spikes to rightward target movement and a depolarization to leftward background movement, the combina- tion of the two produced no spikes at all. This result was confirmed in two other preparations (DIT1, DIT2).

Discussion

The most important properties of the dragonfly target interneurons are: (1) their axons are very large (and therefore easily recorded and recogniz- able), (2) they are highly selective visual interneu- rons, responding only to the movement of relative- ly small objects and (3) the majority are direction- ally selective.

Receptive field structure

An unexpected finding in this investigation was that receptive field size for a given neuron may vary greatly between preparations if spikes are

used as a criterion (Fig. 5, Table 1). The visual area in which there was some subthreshold response, either depolarizing or hyperpolarizing, to target mot ion was usually much greater than the limited region in which targets elicited spiking responses. When these subthreshold responses are taken into account, the size and ' topography ' of the receptive field, its peaks and valleys of excitability, were quite consistent for a given neuron from prepara- tion to preparation.

It seems unlikely that the spike variability be- tween preparations is an artifact of the recording situation. It is possible that electrode injury to the axon could cause the spread of depolarization to the spike initiating zone, influencing spike produc- tion. However, such injuries would also reduce PSP amplitude at the recording site due to reduced input resistance. Such systematic differences in PSP amplitude were not seen (Fig. 5).

The variable receptive field area in which spik- ing occurred was apparently a function of the over- all excitability of the cell, that is, of the ability of the visually evoked graded potentials to elicit spiking. Three possible mechanisms may underlie the differences in excitability from cells of the same type: 1) postsynaptic responses, al though qualita- tively similar, may vary in their amplitudes, 2) spike threshold may vary or 3) tonic excitation or inhibition may bring the cell membrane closer to or farther from threshold. The available data do not exclude any of these possibilities.

Directional selectivity

Some target interneurons showed opposing direc- tional preference to target and background mot ion (Figs. 7 and 8). One explanation for such direction- ally opposed responses is that the target inter- neuron receives tonic inhibition from a wide field movement detector with the identical directional preference. A large textured pattern moving in the preferred target direction would then hyperpolar- ize the target interneuron. The depolarization seen in response to the same pattern moving in the op- posite direction would result from disinhibition, due to the suppressed activity of the wide field detector.

Wide field movement detectors with the appro- priate directional preferences to mediate the cir- cuitry proposed above are present in the dragonfly CNS (Olberg 1981 b). However, the above explana- tion does not account for the inhibition of target response by the large textured pattern moving in the reverse direction from the target (Fig. 8, trace 4). The implication of this inhibition is that

R.M. Olberg: Identified visual target interneurons in the dragonfly 839

although background motion in the antipreferred direction elicits a depolarizing response, this depo- larization is in fact inhibitory. This suggests the presence of at least two different ionic mechanisms, one hyperpolarizing and one slightly depolarizing, underlying inhibitory inputs to the target inter- neurons. The nature of the inhibition underlying directional and size selectivity will be considered in more detail in a later paper.

Comparison with other descending interneurons in insects

In its size, structure, and location within the central nervous system, MDT1 bears a resemblance to the orthopteran Descending Contralateral Movement Detector (DCMD, for review see Rowell 1971). The soma size and location, the profile within the brain, and the axon size and location within the nerve cord are all quite similar (cf. O'Shea et al. 1974). This suggests that MDT1 and DCMD may be homologous giant neurons. Like MDTI, DCMD responds maximally to small pattern movement and is inhibited by large pattern move- ment (Palka 1967; Pinter 1977, 1979).

There are differences, however, in the visual properties of DCMD and MDT1. Whereas DCMD's visual input is strictly from the contralat- eral eye, MDT1 receives binocular input, strongly weighted for input directly in front of the animal. MDT1 is selective for upward pattern movement. DCMD is not directionally selective.

In general, the large, descending interneurons that have been described in insects have not been as selective as the dragonfly target interneurons in their visual properties. Although directionally selective descending visual interneurons have been found in grasshoppers (Kien 1974, 1975; Reichert et al. 1985) and in the moth Manduca (Rind 1983), these have responded to wide field movement. The target interneurons described here represent an ad- ditional, inhibitory step in the processing of the visual signal. A target moving in the preferred di- rection is only excitatory if it does not exceed a certain dimension. Beyond that point, inhibition outweighs the excitation produced by the target.

Role of the target interneurons in behavior

There are two reasons to assign an important be- havioral role to the target interneurons. First is the size of their axons. The eight axons represent more than 10% of the total cross-sectional area of each connective. Their large size implies a selec- tive advantage to rapid transmission of the infor-

mation they carry down the cord. Second is their direct influence on the flight system. High fre- quency (200 Hz), intracellular stimulation of some individual target interneurons produces move- ments of from two to four wings, even when the animal is not flying (Olberg 1978; Olberg in prepa- ration). Their selective visual responses and their influence on the flight musculature imply that the target interneurons control, in part, oriented flight responses to objects whose images move across the dragonfly's retina.

The moving objects of greatest importance to a flying dragonfly are flying insect prey, flying pre- dators, and other dragonflies. Six of the target in- terneurons are dominated by visual input from a forward direction above the horizon. This is the direction in which prey are viewed during the ap- proach, the dragonfly sweeping up from below its intended prey (Mayer 1957). Potential predators (birds) also usually approach from above. Thus both prey and predators would elicit a response from various combinations of the target interneurons. It seems likely that the activity of the target inter- neurons governs the first turning response of the flying dragonfly to the moving prey or predator.

Acknowledgements. I am indebted to David Wohlers for teach- ing me embedding and sectioning techniques, and to Dawn Tamarkin for her technical help. I thank Robert Pinter for help with some of the experiments and with light level measure- ments, and Andrea Worthington for her critical review of the manuscript. This work was supported by National Science Foundation Grant No. BNS-84-06254, by a Cottrell College Science Grant from Research Corporation, and by grants from the Union College Faculty Research Fund.

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