pheromone-modulated optomotor response in male gypsy moths, lymantria dispar l.: directionally...

J Comp Physiol A (1990) 167:707-714 Journal of Sensory,

Comparative . . - , and

l~alndology

�9 Springer-Verlag 1990

Pheromone-modulated optomotor response in male gypsy moths, L ymantria dispar L.: Directionagy selective visual interneurons in the ventral nerve cord Rober t M. Olberg 1 and M a r k A. Willis 2 ,

t Department of Biological Sciences, Union College, Schenectady, NY 12308, USA 2 Department of Entomology, University of Massachusetts, Amherst, MA 01003, USA

Accepted July 4, 1990

Summary. In response female pheromone the male gypsy moth flies a zigzagging path upwind to locate the source of odor. He determines wind direction visually. To learn more about the mechanism underlying this behavior, we studied descending interneurons with dye-filled microelectrodes. We studied the interneuronal responses to combinations of pheromone and visual stimuli.

1. We recorded 5 neurons whose directionally selective visual responses to wide field pattern movement were amplified by pheromone (Figs. 2-6).

2. The activity of the above neurons was more closely correlated with the position of the moving pattern than with its velocity (Fig. 4).

3. One neuron showed no clearly directional visual response and no response to pheromone. Yet in the presence of pheromone it showed directionally selective visual responses (Fig. 6).

4. We recorded 4 neurons whose directionally selective visual responses were not modulated by pheromone (Fig. 7), ruling out the possibility that the effect of the pheromone was simply to raise the activity of all visual neurons.

5. Our results suggest that female pheromone amplifies some neural pathways mediating male optomotor responses, especially the directionally selective responses to the transverse movement of the image, both below and above the animal.

Abbreviations: CNS central nervous system; VNC ventral nerve cord

* Present address (and address for correspondence concerning this manuscript): Arizona Research Laboratories, Division of Neu- robiology, 611 Gould-Simpson Building, University of Arizona, Tucson, AZ 85721, USA

Key words: Insect - Moth - Optomotor - Pheromone - Vision

Introduction

The localization of an upwind (or upstream) odor source is a problem handled by the sensory systems of many animals. Many male moths, for example, are known to orient upwind in response to sex pheromone released by a conspecific female. For a moth or any animal which is walking, upwind locomotion is a relatively simple form of orientation behavior; an animal anchored to the earth needs only simple mechanosensory information to determine the wind direction. For example, the flight- less male silkworm moth, Bombyx mori, walks a zigzagging path upwind to a source of female pheromone. An attractively simple model, proposed by Kramer (1975), accounts nicely for the observed behavior. The model has only two requirements: (1) the moth must sense the direction of the wind and adjust its upwind turning tendency with respect to relative wind direction, and (2) when it detects pheromone, the moth must add to the upwind turning tendency a flip flopping signal to turn constantly, first in one direction and then in the other. Correspondingly there are in the Bombyx ventral nerve cord (VNC) directionally selective, mechanosensory interneurons and pheromone triggered flip-flopping interneurons (Olberg 1983) whose response patterns match the requirements for this simple model of orientation.

Flight upwind to an odor source is a more difficult orientation problem. For a flying animal, it is not possi- ble to use mechanosensory information alone to deter-

708 R.M. Olberg and M.A.

mine wind d i rec t ion . Excep t du r ing r a p i d turns or in t u rbu l en t cond i t ions , re la t ive wind will a lways be f rom d i rec t ly head -on , regard less o f t rue wind di rec t ion . A n ex te rna l f r ame o f reference is needed, therefore , and m o s t f lying an ima l s o b t a i n the needed i n f o r m a t i o n visual ly ( K e n n e d y and M a r s h 1974). In theory , the flying an ima l cou ld l o o k down , see its course over the g r o u n d and , a s suming it k n o w s its a l t i tude , also de te rmine its veloci ty over the g round . I t cou ld c o m p a r e these to its head ing and its a i r speed to ca lcula te the d i rec t ion and m a g n i t u d e o f the wind.

Does the f lying m o t h m a k e the vec tor ca lcu la t ion ou t l ined above or does it use some s impler rule to or ien t its z igzagging f l ight u p w i n d to a source o f sex phe ro - m o n e ? The p rev ious p a p e r (Will is and Card6 1990) re- views some o f the poss ib le s t ra tegies which m o t h s cou ld use and presen ts a b e h a v i o r a l analys is a i m e d at d is t ingu- i shing be tween these hypotheses . In this s tudy we used a n o t h e r a p p r o a c h to address this ques t ion . We reco rded single neu rons in the V N C o f the gypsy m o t h to determine the na tu r e o f thei r visual and p h e r o m o n a l responses and to l ea rn h o w the visual and chemica l signals in te rac t wi th in the ind iv idua l in te rneurons .

A l t h o u g h m u c h excel lent w o r k has been done to un- d e r s t a n d the p rocess ing o f p h e r o m o n e and o the r o l fac to- ry signals in the m o t h a n t e n n a and b ra in (see Chr is ten- sen and H i l d e b r a n d 1987 for review) there is li t t le avai l- able i n f o r m a t i o n a b o u t w h a t p h e r o m o n e i n f o r m a t i o n is t r a n s m i t t e d by descend ing in t e rneurons f rom the b ra in to the l o c o m o t o r y centers wi th in the thorac ic gangl ia . To ou r k n o w l e d g e there have been no o the r studies which e x a m i n e d the in t e rac t ion be tween visual and pher - o m o n e signals in moths . However , behav io ra l s tudies wi th gypsy m o t h s in t e thered fl ight (Preiss and K r a m e r 1983; Preiss a n d F u t s c h e k 1985) showed tha t the presence o f p h e r o m o n e m o d u l a t e s o p t o m o t o r responses . As d iscussed in the p rev ious pape r , one hypo thes i s for the m e c h a n i s m o f u p w i n d o r i en t a t i on in f lying gypsy m o t h s (Preiss and K r a m e r 1986) calls for a s imple enhancemen t o f o p t o m o t o r response in the presence o f p h e r o m o n e . A c c o r d i n g to this hypothes i s , the u p w i n d fl ight is due to the m o t h ' s o p t o m o t o r sys tem min imiz ing the t rans- verse c o m p o n e n t o f visual m o v e m e n t be low the animal .

These behav io r a l resul ts p r o m p t e d us to examine whe the r the female p h e r o m o n e could be shown to mod i - fy the responses o f d i r ec t iona l ly selective visual interneu rons in the ma le gypsy m o t h V N C , neurons with a p p r o p r i a t e response charac te r i s t i cs to med ia te o p t o m o - tor behav io r . O u r resul ts show tha t some, bu t no t all, o f the d i r ec t iona l ly selective visual in t e rneurons descending in the V N C are mod i f i ed in their visual responses by the s imu l t aneous presence o f female p h e r o m o n e in the a i r s t r e a m f lowing over the an tennae .

Material and methods

The preparation. Male gypsy moth pupae, Lymantria dispar L. (Willis and Card6 1990) were kept at 23 ~ until eclosion. The newly emerged adults were then kept in screen cages until they were used in experiments, 2 to 5 days post emergence. They were

Willis: Pheromone-modulated visual interneurons in male gypsy moths

maintained on a long day light cycle (16L/8D). Experiments were begun about 6 h after the onset of light. To assure ourselves that the animals used in electrophysiological experiments were normal, we placed the male gypsy moths in a simple wind tunnel, down- stream from a source containing 100 ng of the female pheromone, disparlure. We used only individuals which exhibited the stereotypi- cal behavioral sequence, which includes upwind flight to the odor s o u r c e .

We glued the animal to a horizontal steel rod with its head extending beyond the end so that its visual field was not occluded either above or below. In some cases the animal was mounted ventral side up and in other cases dorsal side up. The ventral nerve cord (VNC) was exposed between the subesophageal and prothoracic ganglia and kept moist with a moth saline solution (Kaissling and Thorson 1980). For the ventral approach we found it necessary to remove 2 tracheae to expose the VNC. For the dorsal approach, we made a cut down the dorsal thoracic midline. Then we separated the two lateral bundles of flight muscle by gently pulling on the wings. Removal of the gut revealed the underlying VNC.

We lifted the fused cervical connectives of the VNC onto a manipulator-mounted stainless steel platform. To provide added stability, a 26 gauge stainless steel cannula mounted on another manipulator pressed gently backwards on the prothoracic ganglion. This cannula also delivered saline and held the Ag/AgC1 ground electrode.

We used Lucifer yellow filled, thin-walled glass microelectrodes (45 100 MO) to penetrate individual axons in the cervical connec- tive just posterior to the subesophageal ganglion. Negative current injection of 5-10 nA for 10-30 min proved sufficient to fill major branches of the penetrated cells within the brain, subesophageal, and prothoracic ganglia and sometimes also within the fused pterothoracic ganglia.

Before dye injecting each neuron we presented a standard series of pheromone, visual and mechanosensory stimuli to the animal while recording its activity. The signal was amplified (WPI Intra 767) and digitized and stored on video cassette (Vetter PCM Re- cording Adaptor Model 3000) for further analysis.

Stimulus presentation. We designed an apparatus capable of pre- senting combinations of chemical, visual, and wind stimuli (Fig. 1). To convey the pheromone (7R, 8S-cis-epoxy-2-methyloctadecane, (+)-disparlure) to the antennae, continuously flowing air left a fixed plastic tube (10 cm length, 6 mm diameter) aimed at the moth's head from 6.5 cm in front of the head. The pheromone stimulus was applied by quickly replacing a clean plastic screen in the airstream with one containing either 10, 100, or 1000 ng of pheromone. The plastic screens were rectangles (0.75 • 1.3 cm) of cross-stitch backing. Four of these were arranged 7 mm apart along a stainless steel bar so that the airstream hit only one of them edgewise before flowing over the moth's antennae. A servomotor drove a worm-gear which moved the bar rapidly between screen positions. The bar positions were driven by discrete voltage levels with a sliding potentiometer providing the feedback to control positions accurately.

The source of the flowing air was the house compressed air system. The airflow was controlled by a regulator. The air leaving the regulator was filtered and hydrated by bubbling through a water column. The mean velocity of the air at the antennae, mea- sured with a hot wire anemometer (Prosser Scientific Instruments AVM 502), was 0.7 m/s. The mechanical artifact associated with the replacement of one screen by another within the airstream occurred with a latency of about 100 ms from the beginning of the signal to the servomotor and appeared as an initial 10% increase in air velocity lasting about 50 ms followed by a 20% de- crease lasting about 150 ms.

We investigated mechanosensory inputs to the recorded cells by interrupting the flow of clean air over the antennae. Before reaching the delivery tube, the air passed through a solenoid driven valve which could shunt the airstream to an exhaust tube.

A thin (1.5 mm) transparent plexiglass sheet below the animal formed a barrier in the 9 • 14 cm rectangular mouth of an exhaust

R.M. Olberg and M.A. Willis: Pheromone-modulated visual interneurons in male gypsy moths 709

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/ . . . . . . . #'7 /Siabi / Iiziill] " $ ~ ~ S a ~ l t . ,

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Pherornon/escr~een'~s--~ - - t ~ ' ~ ~ ~ Transparent

Lower projection screen Fig. 1. Stimulus arrangement for pheromone, visual and wind stimuli, as described in the text

tube below the animal. With this arrangement, air flowed downward past the stimulus screens into the exhaust system, preventing pheromone from diffusing from one screen to another or from entering the horizontal airstream flowing over the animal unless the screen was directly in the stimulus airstream. The other exhaust opening behind the animal removed the pheromone-contaminated air after it flowed past the animal (Fig. 1). We used TIC14 smoke to confirm visually that all the smoke from each screen passed directly into the exhaust system except when the screen was within the stimulus airstream and that all the smoke in the stimulus airstream passed into the exhaust system after flowing over the animal.

The visual stimuli were black and white slides in a checkerboard pattern, prepared on Kodak fine line reversal film LPD4. They were rear-projected (Leitz Pradolux RT 300 projector with 150 mm Prolux F3.0 lens) from a distance of 2 m onto translucent plastic screens viewed by the moth (Fig. 1). After leaving the projector the beam was reflected by a front silvered mirror which could be tilted either vertically or horizontally to produce corresponding pattern movements on the screen. The mirror movements were produced by two loudspeakers mechanically coupled to the mirror and driven by power-amplified waveforms from a function generator or trapezoid generator. The surface luminance of the white areas of the pattern on the screens, viewed from the animal's side, was 260 cd/m 2. The contrast was 0.9.

The two projection screens were arranged such that one screen was a 90 ~ (wide)x81 ~ (visual angle) rectangle slanting from in front of the moth downward and backward 45 ~ from the vertical. The other screen was a 76 ~ (wide)x 98 ~ rectangle slanting from the horizon in front of the moth upward and backward at 45 ~ to the vertical. Shading one screen or the other allowed us to present the horizontally or vertically moving checkerboard pattern to either the upper screen or the lower screen or to both.

Data analysis and presentation. Because of the long interstimulus intervals (20 s) needed between chemical stimuli and the short recording time usually available before dye injection, we were not often able to repeat stimuli enough times to measure subtle effects and determine average responses. To study the trends in interneuron firing patterns, we played back the recorded signal trace through a window circuit which produced a logic pulse for each spike. Plots of these pulses yielded pseudo-spike traces (Figs. 2, 6). Spike frequency plots were obtained by counting the spike pulses each 100 ms and representing the counts as vertical deflection. We then used a 3-point convolution function (a built-in function of the Data 6000 waveform analyzer) to smooth the frequency plots (Figs. 3, 4).

In some cases the same visual stimuli were presented several times, and we were able to average (unsmoothed) frequency plots to compare visual responses of a cell in the presence and absence of pheromone (Fig. 5a, c). Then, to further study the time course of the response to visual pattern movement, we folded the 3 individual stimulus/response cycles into a single cycle. To do this, we treated each cycle of the 3 cycle stimulus as an individual stimulus and averaged the 3 responses together (Fig. 5b, d).

Results

We recorded int racel lu lar ly 70 times f rom axons in the male cervical connect ives which showed responses to female phe romone . Of these, 17 also showed visual responses. N ine of the visually respond ing cells were sensitive to the direct ion of visual pa t t e rn movement . This repor t deals with these 9 direct ional ly selective axons and reports on the in te rp lay between their responses to p h e r o m o n e and to visual s t imula t ion . All of the visual in t e rneurons projected f rom the p r o t o ce r eb ru m at least as far as the p tero thorac ic gangl ion.

We found 5 in t e rneurons whose di rect ional ly selective visual responses were m o d u l a t e d by the presence of p h e r o m o n e in the airs tream. F igure 2 shows an example of the spiking responses of the mos t t ho rough ly s tud- ied such cell (6224). A l t h o u g h the cell showed little response to p h e r o m o n e - o n a nd only a small, graded response to phe r omone - o f f (Fig. 2a), p h e r o m o n e in the a i rs t ream dist inctly enhanced the direct ional ly selective visual response, increasing the peak spike f requency in response to the lef tward (contra la tera l to the axon) m o v e m e n t of the checkerboard pat tern . Smoothed spike-frequency plots o f this cell (Fig. 3 a) i l lustrate more quant i ta t ive ly the ampl i f ica t ion of the visual response as an approx imate doub l ing of the peak to peak response in the presence of 100 ng of phe romone .

A n o t h e r feature of cell 6224 was that it r esponded best to a ra ther coarse gra ined checkerboard pat tern . No clear visual responses were seen to checkerboards whose spatial per iod were 8 ~ (i.e. squares 4 ~ on a side)

710 R.M. Olberg and M.A. Willis: Pheromone-modulated visual interneurons in male gypsy moths

a. Pheromone responses

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b. Visual responses

Nopheromo.e lllIIlllllill I!!IIII I I I I IUlI I[ I i l

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i s Patlem movement Fig. la , h. Enhancement of directionally selective responses by pheromone, a Descending interneuron (6224> anatomy shown in Fig. 5) in VNC shows graded responses to the removal of pheromone from an airstream flowing continuously over the antennae. Pseudospike traces obtained by plotting logic pulse for each neuron spike. Values refer to weight (in ng) of pheromone applied to a plastic screen which is moved into the airstream for the period indicated by the stimulus trace below, b Same cell shows directionally selective visual responses to a horizontally oscillating, black and white checkerboard pattern (32 ~ spatial period, 0.5 Hz sinusoi- dal movement, 90 ~ movement peak to peak, indicated by stimulus trace below). Leftward (contralateral with respect to the axon) movement is excitatory, and rightward movement is inhibitory. When 100 ng pheromone screen is held constantly in the airstream, the directionally selective visual responses are markedly enhanced

o r 16 ~ b u t a c l ea r ly d i r e c t i o n a l r e s p o n s e was seen to the 32 ~ p a t t e r n (F ig . 3 b).

T h e r e c e p t i v e f ie ld o f cell 6224 was w e i g h t e d heav i l y t o w a r d s the u p w a r d r a t h e r t h a n the d o w n w a r d r e g i o n o f t he v i sua l f ie ld (F ig . 3 c). A l t h o u g h a s m o o t h e d fre- q u e n c y p l o t s h o w s s o m e s u g g e s t i o n o f a d i r e c t i o n a l l y se lec t ive r e s p o n s e to p a t t e r n m o v e m e n t o n the sc reen b e l o w the a n i m a l , i t was m u c h less t h a n the r e s p o n s e to m o v e m e n t o n the sc reen a b o v e the an ima l . T h e re- s p o n s e to m o v e m e n t o n the u p p e r sc reen a l o n e n e a r l y m a t c h e d t h a t to m o v e m e n t o n b o t h screens.

a. Visual responses amplified by pheromone:

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b. Visual responses require coarse grain pattern:

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c. Visual receptive field primarily dorsal:

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Fig. 3 a--c. Spike frequency plots of visual responses of VNC interneuron (6224, same cell as in Fig. 2). Al l traees were obtained by counting spikes during 100 ms time bins and representing the count as a vertical deflection. Resulting curves were smoothed by a 3-point convolution function, a Presence of pheromone (100 ng) in airstream amplifies the directionally selective visual responses. Shown are responses to 32 ~ checkerboard (squares 16 ~ on a side), oscillating horizontally 90 ~ peak to peak, at 0.5 Hz. b Directionally selective visual responses seen to 32 ~ pattern but not to 8 ~ or 16 ~ patterns, e Visual receptive field was primarily dorsal. Stimulus as in a with 100 ng pheromone screen in airstream. Both fields means that the moving pattern was projected onto both the upper and lower screens. For the other traces one of the screens was shaded

Varying Pattern Oscillation Frequency:

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Fig. 4a, b. Spiking response of VNC interneuron shows similar responses over a range of pattern velocities (6224, same cell as in Figs. 2 and 3). a Responses to 90 ~ horizontal oscillation of 32 ~ checkerboard pattern at 3 oscillation frequencies (upward in stimulus traces represents leftward, i.e. contralateral, pattern movement). Spike frequencies plotted as in Fig. 3. h Average (-t-1 SD) of peak spike frequency (highest number of spikes per 100 ms bin) for each cycle of pattern movement at various frequencies. Taken from data obtained as in a


Cell 6224 showed directionally selective visual responses over a wide range of pat tern velocities (Fig. 4). Two points emerge f rom this figure. The first is that, for 0.5 Hz and 1 Hz oscillations, the peaks in spiking response spike frequency are nearly aligned with the peaks in the sine wave stimulus. This was also true for 2 Hz pat tern oscillation (not shown). The second is that over a wide range of pat tern velocities, the peak spike frequency changes very little (Fig. 4b). These results are consistent with the hypothesis that cell 6224 responded to pat tern position rather than to pat tern velocity (see Discussion).

In Fig. 5, the ana tomy and visual response patterns of cell 6224, are compared with those of another, similar- ly responding cell (6212), Cell 6212 also showed directionally selective responses to horizontal pat tern movement towards the side of the animal contralateral to its axon. Like 6224 it showed little response to 8 and 16~ checkerboards but a clear, directionally

selective response to a 32~ pattern. However, un- like 6224, this cell showed graded excitatory responses to pheromone, and its visual receptive field was in the downward rather than in the upward direction.

A comparison of the responses of cells 6224 and 6212 (Fig. 5 a, c) shows that whereas pheromone elicited extra spikes (shaded area in a) only during 6224's excitatory response peaks, pheromone elicited extra spikes in 6212 (shaded area in c) throughout the visual stimulus cycle. However, more 6212 spikes are added during the peaks than during the valleys of the response, implying that the pheromone is amplifying, ra ther than simply adding to, the visual response. The single sine wave cycle average trace (Fig. 5d) shows that the difference between the pheromone and pheromone-free visual response is greater during the preferred visual stimulus phase than during the antipreferred phase. In this graph (Fig. 5 d) the average difference between levels during the first half cycle is 2.83 spikes per 100 ms (SD=0.37 , n = 10) and

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Fig. 5a--d. Pheromone-mediated amplification of directionally selective visual responses of 2 VNC interneurons. Camera lucida drawings show profiles of descending interneurons in brain and subesophageal ganglia, prothoracic ganglia and, for 6224 only, also the pterothoracic ganglia, viewed from above (dorsal). To the right are side views of profiles within the brain and subesophageal ganglia. a Average number of spikes per 100 ms bin (n=4) in response to 3 cycles of oscillating horizontal visual pattern movement with and without 100 ng pheromone source in airstream. Cell 6224 and

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stimuli as in Fig. 3a. Shaded area shows average excess spikes to visual stimulus in the presence of pheromone, b The average response of 6224 to a single cycle of pattern oscillation with (open squares) and without (filled circles) 100 ng pheromone source in airstream, obtained by further averaging the 3 individual cycles of responses shown in a. e Average visual responses (n = 3) of cell 6212 to horizontally oscillating visual pattern with and without 100 ng pheromone source in airstream as in a. d Average 1-cycle visual response, with and without pheromone, as in b


during the second half-cycle the average difference is 5.35 spikes per 100 ms (SD = 1.21, n = 10).

The single cycle average traces (Fig. 5b, d) indicate that for both cells, the peak response was near the time at which the pattern had reached its most contralateral position. For 6224, the peak spike frequency occurred at about 450 ms without pheromone and at about 350 ms with pheromone, whereas the peak of the sine wave used to drive the checkerboard stimulus pattern occurred at 500 ms. Likewise for 6212 the peak response occurred at about 1.45 s without pheromone and 1.35 s with pheromone, with the point of maximal contralateral pattern displacement occurring at 1.5 s (since this cell was recorded on the opposite side of the body). With this sine wave stimulus, the maximum velocity of the pattern (ca. 290~ in the preferred direction occurred at the beginning of the stimulus, i.e. 0 s, and at 1 s, respectively, for the two cells, in both cases far from the peak of response. The timing of the peaks again suggests that the neuron was coding for the position, rather than the velocity, of the pattern.

A final example illustrates an extreme case of the pheromone acting as a modulator of visual responsive-

a. Pheromone

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No pheromone 81lllllllrlt~ll II I Iltlllll [llfl Illli I I II Itltllll IIIII

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Fig. 6a, b. Responses of unidentified descending interneuron to pheromone and visual stimuli alone and in combination. Above each pseudospike trace (see Fig. 2 caption) is plot of number spikes in the 500 ms bin directly below, a Addition of 100 ng pheromone source to airstream (during 5 s period shown in stimulus trace below) elicits no detectable response, b When a (16 ~ checkerboard pat tern is moved horizontally (Left and Right) or vertically (For- ward and Back) on a screen below animal without pheromone, the firing pat tern becomes more bursty but not clearly directionally selective (first and third traces). Cell becomes directionally selective with the addition of 100 ng pheromone source to airstream (second and fourth traces). All pattern motions are 90 ~ during 2 s ramps shown below each spike trace

ness. This cell (1029) whose morphology is unknown showed no detectable response to the female pheromone (Fig. 6a) when it was presented alone. It showed only small visual responses to pattern movement with no clear directional preference. However, in the presence of pheromone it became clearly directionally selective (Fig. 6b).

Does the presence of pheromone in the air surround- ing the antennae simply cause a general arousal of the male moth's nervous system? Our data suggest that this is not the case. We also found 4 directionally selective visual interneurons whose visual responses were not modulated by pheromone. For example, one such cell (527, Fig. 7) showed no distinguishable differences in its visual

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Fig. 7 a l l . Anatomy, visual and auditory responses of a VNC interneuron (527) whose directionally selective visual responses are not modulated by pheromone, a Anatomy of 527, depicted as in Fig. 5. b Average number of spikes per 100 ms bin ( n = 2 ) in response to 3 cycles of oscillating horizontal visual pattern movement with (open squares) and without (filled circles) 100 ng pheromone source in airstream, c The average response of 527 to a single cycle of pattern oscillation with (open squares) and without (filled circles) 100 ng pheromone source in airstream, obtained by further averaging the 3 individual cycles of responses shown in b. d Auditory response of 527 to the click sound made by switching a toggle switch. Lower trace shows a simultaneous microphone recording of the switch's sound. (Calibration 50 ms, 20 mV)


responses to horizontal pattern movement with and without pheromone in the airstream. This cell was not unresponsive to pheromone, however, but gave graded, phasic on- and off-responses (not shown) to the pheromone stimulus with the off-response being slightly greater than the on-response. Like the two cells above, this cell preferred pattern movement toward the contralateral side, and its peak response was about 100 ms before the pattern reached its contralateral end position. The receptive field of this cell included both the upper and lower screens, with responses of equal magnitude to pattern movement on either screen.

In addition to its pheromone and visual inputs, cell 527 also showed auditory responses (Fig. 7d). We noticed this as a burst of 4-8 spikes in response to the click produced as we flipped various toggle switches. The cell responded vigorously to hissing sounds and to the jingling of keys, but not to low frequency vocal sounds.

Discussion

The results presented here indicate that one way that the pheromone signal is used in the CNS of male gypsy moths is in amplifying the signals of certain, directionally selective visual interneurons. The amplification of the visual response is not the result of an overall pheromone- induced increase in neural activity, since the visual responses of some neurons remain unaffected.

Wide receptive field, directionally selective visual neurons have been described in a large number of insect species (see Hausen and Egelhaaf 1989 for review) and have generally been implicated in mediating optomotor behavior. The preceding paper (Willis and Card6 1990) discussed the importance of visual responses to upwind orientation in a pheromone plume. If we assume that the neurons reported here are involved in steering flight, they can provide information about the control of upwind flight in response to female pheromone.

Counterturn generator ?

As discussed in the previous paper (Willis and Card6 1990), one hypothesis for the zigzag pattern in the upwind course followed by male moths in the approach to a source of female pheromone calls for the presence of a counterturn oscillator within the CNS (Kennedy 1983; Baker et al. 1984; Kuenen and Baker 1983; Willis and Baker 1987). Analysis of wind tunnel flights reveals that the longitudinal axis of the moth reverses its direction about 4 times (2 left turns, 2 right turns) each second. If the hypothesized counterturn generator were sending its rhythmic signal via the descending premotor interneurons, then neurons involved in a left or right turn might be expected to show a burst each half second overlaid on their other responses. The responses during the preferred pattern movement of the directionally interneuron shown in Fig. 6 show the predicted bursting pattern of about 2 Hz (Fig. 6, bottom 2 spike traces).

However, since this was our only observation of a pre- sumptive optomotor cell with such a bursting pattern, our data do not provide any strong evidence for the counterturn generator.

Do pheromone-sensitive descending interneurons in the gypsy moth show a flip-flopping pattern of activity in response to repeated pheromone stimuli, similar to the response pattern described in Bombyx (Olberg 1983)? If so, such interneurons might well be expected to carry the sequential left and right counterturning in- structions. Among the 70 interneuron recordings we obtained, none showed flip-flopping responses to pheromone. The pheromone responses of these remaining cells will be the subject of another paper.

Coding for pattern position

What components of the pattern movement are coded by the directionally selective interneurons? Pattern position, velocity, and acceleration are all potentially coded by visual interneurons. If the descending interneurons were coding pattern velocity we would predict (1) the peaks in spike frequency should appear near the mid- point of the pattern sweep, at or shortly after the point of maximal velocity (neuronal activity then reflecting a differentiation of the sine wave used to move the stimulus), (2) the peak firing rate should increase with the higher velocities produced as we oscillate the pattern with higher frequency sine waves, and (3) heightened firing should be limited to the time when the pattern is moving in the preferred direction (or no more than the <100 ms response latency into the antipreferred movement phase). None of these predictions hold for the cells tested in this way. At various pattern movement frequencies, the peak firing frequency occurred near the end of the pattern's sweep in the preferred direction (Figs. 4a, 5). The peak firing rate was similar over a 50-fold range in pattern movement frequency (Fig. 4 b). Finally, in every case, heightened spike rates persisted several hundred (250-850) ms after the pattern reversed direction (Figs. 3-7). We conclude that these cells are not primarily velocity sensitive.

It is also unlikely that these neurons are primarily sensitive to acceleration. If acceleration were the relevant parameter, spike rates should increase with increasing pattern oscillation frequency, which was not the case (Fig. 4b). Peak spike rates remained consistent over a wide range (0.2 to 5 Hz). We conclude that the consistent magnitude of these responses reflects the constant ampli- tude (90 ~ ) of pattern oscillation, that is, that these interneurons primarily indicate pattern position.

For a cell to respond to a change in position in a given direction, and for it to continue to respond until that position is restored (Fig. 5b, d), implies that it has a memory for the previous position. Such visual memory in the optomotor system controlling flight could explain how the male gypsy moth can continue its upwind flight to the odor source even after the wind is terminated (Willis and Card6 1990), a phenomenon also described in the oriental fruit moth (Baker et al. 1984).


An optomotor control system which is responsive largely to position of major contours in the visual field is ideally suited to controlling flight in a consistent overall direction (upwind) while at the same time allowing wide zigzagging turns. Small deviations of flight path would cause only small neuronal responses, allowing wider deviations. As the deviations from upwind become greater, heightened spike rates would turn the animal back across the wind. Although the responses shown here indicate that the neurons sometimes cease responding before the original pattern position is restored, this might be attributable to our use of low frequency pattern oscillations, much lower than the 2 Hz oscillations mea- sured for gypsy moths in flight (Willis and Card6 1990). Along this line, the response to 0.1 Hz oscillation (Fig. 4a), the slowest movement we used, also showed the greatest decrement over time, persisting only about I s into the 5 s period of pattern return.

Behavioral implications

The presence of pheromone-modulated directionally selective interneurons whose visual receptive fields are up- wardly directed (Fig. 3) has implications for the further exploration of the upwind flight behavior. Since previous behavioral studies have focused on the importance of the image flow below the flying moth, it will be impor- tant to test the importance of the visual image above the animal as well. In particular, in both tethered flight and wind tunnel experiments with gypsy moths, beha- viorists have simulated the effect of wind by moving a pattern below the flying animal. Since visual information from above the animal is either lacking (in the case of tethered flight) or contradictory (in the case of wind tunnel flights over a moving floor), the information about op tomotor control obtained using these ap- proaches may not be complete.

For a diurnal, forest dwelling species such as the gypsy moth the highest contrast portion of the visual environment is located above the animal, in the pattern of gaps in the tree cover. Our results suggest that this pattern is used by the animal in orientation. It is interest- ing that our best studied pheromone-modulated visual cell, whose receptive field was directed upward, required a rather coarse-grained (32 ~ pattern for response (Fig. 3 b). Such a cell would presumably respond primarily to relative movement of silhouettes of large branches or major openings of the canopy rather than to the fine texture of the leaves above. Selective response to large visual features is appropriate for a cell which responds to a change in pattern position rather than to flow field velocity. In a speculative scenario, such a cell as 6224

could use the positions of visual landmarks above the moth to maintain a relatively straight overall course through the forest. Its requirement for coarse-grained visual features would result in the animal steering relative to the large invariant characteristics of the visual environment, ignoring more subtle textural properties.

Acknowledgements. We thank R. Card6 for providing the animals and the synthetic pheromone used in this study. A. Worthington provided valuable critical comments on the text. This study was supported by a USDA Competitive Grant USDA-85-CRCR-1- 1686.

References

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