evaluation of the behavioral roles of ascending auditory interneurons in calling song phonotaxis by...

J Comp Physiol A (1992) 170:363-372 Journa l of

Neural, and

P h y s i o l o g y A ,~o~, �9 Springer-Vedag 1992

Evaluation of the behavioral roles of ascending auditory interneurons in calling song phonotaxis by the female cricket (Achet a domesticus) Gordon Atkins*, John Henley, Rob Handysides, and John Stout

Department of Biology, Andrews University, Berrien Springs, Michigan 49104, USA

Accepted November 22, 1991

Summary. 1. Inactivating one L 1 results in angular errors and circling during orientation toward the side having the intact L1 in response to calling songs (CSs) whose intensities are below the threshold for L3 (Figs. 2, 3A). When song intensities are increased above the threshold of L3, circling decreases (Fig. 3B).

2. Following inactivation of one L1 and occlusion of the ear providing input to the intact L1, no phonotaxis occurs in response to CSs at 60 dB (below the threshold of L3; Fig. 4A) demonstrating the necessity of L1 for phonotaxis. Orientation, at large and consistent angular errors (Fig. 5A) and circling (Fig. 5B) to the unoccluded side resumes when song intensities are increased above the threshold of L3 (Fig. 4B) suggesting that a single L3 can induce phonotactic responses.

3. Inactivation of o n e L3 causes angular errors in orientation when CSs are above its threshold (Figs. 6A, 7), which are not apparent when CSs are below L3's threshold (Figs. 6B, 7).

4. Inactivation of one L3 and occlusion of the ear providing input to the contralateral L1 and L3, leave only one L1 functioning. This results in turning and circling toward the unoccluded side containing the o n e

functioning L1 (Figs. 6C, 8), thus confirming the suf- ficiency of L 1 for phonotaxis.

K e y w o r d s : P h o t o i n a c t i v a t i o n - R e c o g n i t i o n - Localiza- tion - Audition - Identified interneuron

I n t r o d u c t i o n

Of the relatively large number of auditory neurons described in the prothoracic ganglion of crickets (e.g. Woh- lers and Huber 1982, 1985; Atkins et al. 1984; Stout et al. 1985; Atkins and Pollack 1987a, b) only 2 or 3 pairs

Abbreviation." CS calling song

* To whom offprint requests should be sent

of interneurons have response properties that suggest their involvement with the female cricket's phonotactic response to the calling song (CS) of males, and send axons to the brain where further processing of acoustic stimuli occurs (Schildberger 1984). These ascending auditory interneurons have been intensely studied and their CS encoding properties carefully described in several species (e.g. Wohlers and Huber 1982; Moiseff and Hoy 1983; Schildberger and Hrrner 1988). In Aeheta domestieus females (Stout et al. 1985, 1988; Atkins et al. 1989b, c), the L1 neuron was most responsive to the 5 kHz CS, encoded its temporal parameters, and had a threshold at this carrier frequency of 45-50 dB. The L2 neuron had lowest thresholds (55-65 dB) around 16 kHz, with high thresholds (> 85 dB) at 5 kHz and thus did n o t

encode the properties of 5 kHz CSs. The L3 neuron was most responsive to 16 kHz (thresholds of 55-65 dB), but was also sensitive at 5 kHz (thresholds 65-75 dB) and effectively encoded the temporal structure of the CS.

Behavioral studies of female cricket phonotaxis to CSs (e.g. Pollack and Hoy 1981; Pollack and Plourde 1982; Weber et al. 1981 ; Thorson et al. 1982; Stout et al. 1983; Doherty 1985; Stout and McGhee 1988) have yielded a great deal of information about the features of a male cricket's CS that were attractive to conspecific females. This provides additional basis for suggesting which encoding and response properties of auditory interneurons might underlie their roles in CS phonotaxis.

A few studies have attempted to manipulate the responses of auditory interneurons found in crickets and demonstrate changes in the females' phonotactic behavior resulting from these manipulations. Atkins et al. (1984) and Stout et al. (1985) demonstrated that following the unilateral killing (by photoinactivation) of the L 1, L3, ON1, and ON2 auditory interneurons of female A. domesticus, the orientation behavior of the female to a model CS changed so that the ear providing input to the killed neuron was kept closer to the sound source than the ear on the intact side, sometimes resulting in circling to that side. Inactivation of L2 did not cause changes in orientation, which was not surprising since L2's thresh-

364 G. Atkins et al. : Behavioral role of L1 and L3 in phonotaxis

old at 5 k H z is > 8 5 d B and thus is too high to be func t iona l ly involved in CS phonotaxis . Schildberger and H 6 r n e r (1988) demons t r a t ed essentially the same roles in s t imulus local iza t ion for the AN1, A N 2 and ON1 neurons of female Gryllus bimaculatus by hyperpolar iz ing these neu rons du r ing phonotaxis . No len and H oy (1984), by hyperpolar iz ing and depolar iz ing the I N T - 1 n e u r o n of female Teleogryllus oceanicus dur ing presen ta t ion of s imula ted ba t calls to a flying cricket, showed that this n e u r o n ' s activity was essential for avoidance phonotaxis . Fur ther , depola r iza t ion of I N T - 1 was sufficient to induce steering responses in the absence of sound.

Phonotax is by female G. bimaculatus and G. campe~ stris, which were uni la tera l ly deafened jus t before the test, was clearly demons t r a t ed by H u b e r et al. (1984). This behavior inc luded bouts of circling with the deafened ear to the outs ide of the circle, and or ien ta t ion at an e r roneous angle to the sound source resul t ing from tu rn ing toward the in tac t ear. K o h n e et al. (1992) demonstrated that uni la tera l ly deafened female A. domesticus also responded phonotac t icaUy to CSs using these behav- iors. They fur ther showed that these females discrimina ted between the syllable periods of the model calls by phonotac t ica l ly circl ing at the highest rate in response to syllable per iods tha t were shown by Stout et al. (1983) to be mos t a t t ract ive for females of this species.

This s tudy fur ther elucidates the roles of ascending pro thorac ic aud i to ry in t e rneu rons (which respond to biological ly re levant 5 kHz songs e.g. L1 and L3, Stout et al. 1985) in local izat ion, recogni t ion and response to the male ' s CS by female A. domesticus, using photo inac- t iva t ion as described by Atk ins et al. (1984), and behavioral procedures involv ing one-eared phonotax is de- veloped by K o h n e et al. (1992).

Materials and methods

Animal care. Crickets (Acheta domesticus) were purchased as 4-week- old nymphs from Fluker's Cricket Farm, Baton Rouge, LA, USA and raised to the imaginal molt in large plastic containers at 22 ~ on a 12 : 12 light/dark cycle. All crickets were provided with shelter (egg cartons), cricket chow (Purina) and moisture (water or fresh potatoes). Adult females were separated daily from the main colony and kept in separate 1 1 containers under the conditions described for the nymphs.

Recording and photoinactivation. The stimulus setup for intracellular recording consisted of an open field paradigm which was achieved by playing sound signals through one of a pair of loud speakers (matched pairs of Realistic #40-1379 piezo high frequency speakers) located 82 cm apart on either side of a fiberglass- lined Faraday cage. Sound intensities were calibrated (_+2 dB) midway between the two speakers (the location of the specimen) by a Heath real time spectrum analyzer (AD-1308). Fiberglass insula- tion was used to cover reflective surfaces in the recording area so that signals did not vary more than + 2 dB in the region occupied by the specimen. Synthetic CSs were generated by a custom-made signal generator, amplified and controlled manually or by a computer (DEC PDP 11~03). Syllables (sinusoidal-shaped pulses of sound) having durations ranging from 22-25 ms were grouped into chirps (3 syllables/chirp with a syllable period of 50 ms) and repeated 1.5/s. Carrier frequency (2-18 kHz) and intensity (45-95 dB SPL) could be varied.

Crickets (10-35 days following the imaginal molt) were mounted intact and ventral side up on a wax block. U-shaped pins, which did not penetrate the cricket, where placed over the head, thorax, abdomen and 6 legs (2 pins for each leg) and pushed into the wax block so that most movement was inhibited and so that the posterior tympana were facing the speakers used for stimulation. An additional 2 pins, when positioned in the wax block, held the coxa of the prothoracic legs as far back laterally as possible without damaging them or the pronotum. The prothoracic ganglion was exposed by cutting a small piece of exoskeleton from between the forelegs and folding it back under the U-shaped pin over the thorax. A stabilizing platform was positioned under the ganglion, and a silver wire inserted into the abdomen was used as the reference electrode. The acoustic tracheae were not severed. A small dam was built around the ganglion using coagulated hemolymph and exposed tissues were kept moist with saline (Fielden 1960).

The ventral portion of the prothoracic ganglion was penetrated with thick walled (1.0/0.5) borosilicate electrodes filled with 3% Lucifer Yellow CH (Stewart 1978, 1981) in 0.5 M LiC1 (resis- tance = 100-200 M~). Intracellular recordings were monitored on an oscilloscope and stored on FM tape (Racal Store 4). In each case, the threshold (the minimum intensity in 5 dB increments, required to induce an average of at least 3 spikes/chirp) for 5 kHz songs was determined. Recordings were later digitized and analyzed on a computer (Apple Macintosh).

Individual neurons were stained by iontophoretic injection ( - 2 to - 4 nA DC) for I-3 min. Following staining, the ganglion was illuminated by a 35 mW Helium Cadmium laser (Omnichrome, model 456XM) for 5-20 s during which time the neurons response to auditory stimuli (5 kHz, 85 dB) ceased and the membrane potential approached 0 mV. Photoinactivation followed the description by Atkins et al. (1984) except that it occurred much faster in these experiments due to the more intense light source.

Pre and post inactivation tests of phonotaxis. Phonotaxis was evaluated on a non-compensating treadmill located in a fiberglass-lined chamber (Walikonis et al. 1991). A cricket was attached at the pronotum by a small perpendicular pin that inserted into a small tube of slightly larger internal diameter (within which it could rotate) and thus tethered on the North pole of a styrofoam ball (12 cm diameter) which was suspended by an air stream. In this manner the cricket could turn and walk in any horizontal direction. Walking was detected by two metered wheels (X an Y axis) which lightly touched the equator of the sphere. The angle between the wheels was such that no matter which direction the female moved, the friction between the two wheels and the ball was constant (Walikonis et al. 1991). The data from these wheels were fed into and stored on an Apple Macintosh computer, which mathemati- cally corrected the angle between the X and Y wheels to 90 ~ and plotted the track of the cricket as if it were walking freely. The computer also calculated the average angle of orientation of each track with respect to the speaker. This angle was determined as the angle of the centroid (center of gravity) of a polar orientation diagram which was calculated for each track (see Stout et al. 1983 and Atkins et al. 1984 for complete description of PODs and centroids).

Phonotaxis to model CSs (5 kHz carrier frequency) was tested over a range of syllable periods (30~70 in 10 ms increments, and 90 ms) presented in a standard non-sequential manner at 60 dB and 85 dB. Sound intensities were calibrated (+ 1 dB) at the position occupied by the tethered cricket on the treadmill by a Heath real time spectrum analyzer (AD-1308). Each test lasted 3 min followed by at least 1 min with no sound between each test. Songs were played from a Motorola speaker positioned 75 cm from and facing the treadmill. All tests were completed in the dark. Temperature ranged from 22-24 ~ Only those crickets that exhibited clear phonotaxis to more than 2-3 different SPs were used further. Fol- lowing pretesting of phonotactic orientation the female was prepared for recording from and killing the L1 or L3 neuron as described above.

Following photoinactivation, the electrode, coagulated

CONDITIONS LEFT RIGHT

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CIRCLES Fig. 2. Effect of killing one L1. Tracks and average orientation angles for a female in response to CSs having various syllable periods at 60 dB (below the threshold of L3). Inset shows the typical morphology of L1 (wholemount , ventral side up)

366 G. Atkins et al.: Behavioral role of L1 and L3 in phonotaxis

hemolymph and saline was removed from around the prothorax. The flap of exoskeleton was repositioned over the prothoracic ganglion and the cricket removed from the mounting block. Only those crickets whose legs were functioning (clearly used in a coor- dinated way during walking) were tested further. Phonotaxis was tested in the same manner as in pretests. In addition, these phonotaxis tests were repeated following occlusion of one ear (the ear opposite the side providing the major excitatory input to the photoinactivated neuron) with wax according to Kohne et al. (1992). Postkilling orientation was frequently measured more than once in response to the same stimuli. However, since the females consistently responded with very similar changes in their postkilling phonotactic orientation, as compared to their prekilling orientation, the results of these repeated tests added no information and are not reported.

Morphological identification. The prothoracic ganglion was removed and fixed in 4% formalin in phosphate buffer at pH 7.2 (10 min) followed by 4% formalin in methanol (1 h), dehydrated in absolute ethanol (20 min), and cleared in methyl salicylate (10 min). Whole mounts were visualized, photographed and drawn using epifluorescence microscopy (Leitz Laborlux D). Neurons were identified according to the description of L-shaped neurons by Atkins et al. (1989b, c).

Resul ts

Controls

50 ms syllable period and dropped to almost none or no circling at 90 and 30 ms, respectively (Fig. 3B). Increas- ing the CS intensity to 85 dB (above the threshold of L3) reduced the degree of circling (Fig. 3B).

Unilateral occlusion of the ear on the side providing input to the intact L1 prevented sensory input to auditory neurons on that side of the ganglion (Kohne et al. 1992). Under these conditions (n = 5), (none of the L- shaped neurons would have been responding to sound) there was no orientation to 60 dB CSs (Fig. 4A). For the experiment shown in Fig. 4A, movement resulted during just the presentation of a 30 ms syllable period CS with no clear trend in direction. For each of the 5 animals, when the intensity of the CS was raised to 85 dB (above the threshold of L3), the female walked with large and (especially for 50-70 ms SPs) consistent average angular errors to the expected side (Figs. 4B, 5A) and/or circling (toward the side with the unoccluded ear Figs. 4B, 5B). Since this was the side with a killed LI , and the 5 kHz stimulus was below the threshold typical for L2 neurons, the only one of the 3 identified, ascending, L-shaped auditory interneurons (Atkins et al. 1989b, c) that could have responded was the L3 neuron. Circling, al though more variable and less than shown for L1 (Fig. 3), was syllable period dependent (Figs. 4B, 5B).

Placing the tip of an electrode in the anterior Ring Tract (acoustic neuropil, Wohlers and Huber 1985) without penetrating an audi tory neuron, and illuminating the prothoracic ganglion with the beam of a HeCd laser, caused no obvious or consistent changes in the subsequent orientat ion of a female A. domesticus (n = 5) to model 5 kHz, 85 dB CSs (Fig. 1 shows a representative example). This allows interpretation of the changes in orientation subsequent to photoinactivat ing auditory interneurons as resulting f rom the loss of the neuron killed, rather than as a result o f the procedures used.

Photoinactivation of L1

In the 5 females tested with unoccluded ears, photoinac- tivating a single L 1 consistently resulted in angular errors in orientation to the sound source (as compared to the female's orientation before killing) and/or circling whose direction was always dependent on which side of the ganglion the inactivated neuron was located in, for all of the syllable periods and intensities presented (during which the female walked - occasionally the female did not walk in response to CSs with 30 or 90 ms syllable periods that are out o f the species range). I f the L 1 which received excitatory input f rom the left ear was killed, the female's orientation deviated or circled to the right toward the side providing input to the intact L1 (Fig. 2), in an apparent a t tempt to equalize input to acoustic pathways on both sides of the female's central nervous system. The average angular errors of the females were rather consistent f rom female to female, especially for CSs with the most attractive syllable periods (50, 60 ms, Fig. 3A).

In response to 60 dB CSs, the degree of circling toward the intact side was maximal in response to a CS with a

Photoinactivation of L3

Unilateral photoinactivation of an L3 in 4 females caused their orientation to deviate (in response to all 5 kHz, 85 dB CSs) in every case away f rom the side with a killed L3 (Fig. 6A). The angular deviation was more variable and, on average, smaller (Fig. 7) than that shown for L1 (Fig. 3A). Two of the 4 females showed rather large angular errors, while the other 2 females oriented with smaller error angles. When the CS intensity was reduced to 60 dB (below the threshold of L3, but still above the threshold of L1), the females continued to orient to the CS, but with no consistent angular errors in orientation (Figs. 6B, 7, in contrast to the response of a female with a unilaterally inactivated L1 to this same stimulus, Fig. 2).

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Fig. 3A, B. Behavior following unilateral photoinactivation of L1. A Average angular errors (resulting from turning toward the side with an intact L1) for 5 females orienting to 60 dB CSs with the indicated syllable periods. B The mean number of circles to the side providing input to the intact L1, exhibited by females (n = 5) during orientation trials to CSs having various syllable periods. Error bars: standard error of the mean �9 60 dB, �9 85 dB

G. Atkins et al. : Behavioral role of L1 and L3 in phonotaxis 367

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Occlusion of the ear contralateral to the side with a killed L3 (of the 3 ascending L cells, only L1 on the unoccluded side could have responded in this situation) resulted in circling orientation toward the unoccluded side (Fig. 6C). Under these conditions the number of circles in the female 's orientation was syllable period dependent, with the greatest average, number of circles occurring during the female 's response to a CS with a syllable period of 50 ms (Fig. 8).

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Fig. 5A, B. Behavior following unilateral inactivation of L1 and occluding one ear (same conditions as in Fig. 4B). A Average angular errors (resulting from turning toward the side with an intact L3) for 4 females orienting to 85 dB CSs with the indicated syllable periods. B Circling behavior at 85 dB with a single L3 functioning. Graph showing the mean number of circles to the side providing input to the functional L3, exhibited by females (n= 5) during orientation trials to CSs having various syllable periods. Error bars: standard error of the mean

D i s c u s s i o n

Controls

Killing a cricket auditory neuron by intense illumination of the ganglion with blue light, after iontophoret ic injection of Lucifer yellow, resulted in elimination of spiking, postsynaptic potentials and membrane potential (Atkins et al. 1984). In the present study and in Atkins et al. (1984), such neurons gave no further responses to the stimulus, regardless of its intensity. The controls used in this (penetrating the acoustic neuropil with a microelectrode without killing a neuron) and our previous studies (killing single sensory neurons, or the high frequency L2 neuron, in the acoustic neuropil, Atkins et al. 1984; Stout et al. 1985; Atkins et al. 1989a) assure that changes in angular orientation by the female following unilateral photoinact ivat ion of either the L1 or L3 auditory interneurons are the result of eliminating the killed neuron f rom the central auditory pathways, rather than an arti- fact o f the procedures involved in photoinact ivat ion (Fig. 1). However, perhaps the best control resulted f rom unilaterally inactivating an L3 neuron and demonstra t - ing that the 60 dB stimulus, which was above the threshold of the bilaterally intact L1 neurons, but below the threshold of the L3 neuron, caused no consistent angular


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Fig. 6A-D. Effect of killing one L3. Tracks and average orientation angles for a female in response to CSs having various syllable periods A at 85 dB (above the threshold of L3) and B at 60 dB (below the threshold of L3). Mean angular errors are 122 ~ to the

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conditions as in Fig. 6C). Graph showing the mean number of circles to the side providing input to the functional LI, exhibited by females (n =4) during orientation trials to CSs having various syllable periods. Error bars: standard error of the mean

errors (Figs. 6B, 7). Increasing the stimulus intensity to 85 dB (above the threshold of the single L3) consistently created angular errors to the expected side (Figs. 6A, 7).

Criteria for concluding that a neuron is an essential element in stimulus recognition and response

The ideal procedures for demonstrating whether or not an identified prothoracic auditory neuron pair was an essential element in the nervous system pathways which lead to CS localization and/or recognition, would be to bilaterally eliminate them. However, since there are three L-shaped, ascending auditory interneurons that are inti- mately intertwined with each other, and with other auditory interneurons (Atkins et al. 1989b) there is very low probability of successfully penetrating, filling with Luci- fer yellow and inactivating the same L-shaped auditory neuron on both sides of the prothoracic ganglion. Therefore we have approached the evaluation of whether an auditory neuron is an essential element in the pathways leading to call recognition and response in another way.

Covering both anterior and posterior tympana with wax eliminates spiking in the prothoracic auditory neurons on the occluded side for female G. campestris and G. bimaculatus (Huber et al. 1984) and female A. domesticus at the intensities utilized in this study (Kohne et al. 1992). CSs presented to females on a treadmill or in an

arena with an occluded ear caused angular errors in orientation (compared to preocclusion tracks) resulting from the female turning toward the side with the unoccluded ear. These error angles resulted from an attempt by the female to balance the sensory input into the central auditory neurons on either side of the prothoracic ganglion (Huber et al. 1984; Kohne et al. 1992). Fre- quently the female with a unilaterally occluded ear (orienting on either treadmill or arena) continuously turned toward the unoccluded side, resulting in a circular path, with the occluded ear to the outside of the circle. Kohne et al. (1992) demonstrated that the rate of circling by female A. domesticus was, on average, greatest in response to CSs with the most attractive syllable period (50 ms, Stout et al. 1983; Stout and McGhee 1988). Thus circling in response to a CS by one eared females is an indication that the female is phonotactically orienting and the rate of circling distinguishes between the different syllable periods, indicating recognition of the CS.

If the activity of a single, unilaterally isolated prothoracic auditory interneuron leads to circling in response to the CS, whose rate is syllable period dependent, it can be concluded that the neuron is in the pathway leading into the female's CS recognition mechanisms.

If, in a one-eared cricket, killing an auditory neuron receiving input from the unoccluded ear, eliminates all phonotactic orientation that occurs under these conditions, that neuron is an essential element in the pathways leading to call recognition and response by these one-eared crickets, and by extension, in the normal, two- eared condition.

Behavioral criteria for demonstrating that a neuron is involved in calliny song localization

Unilateral inactivation of the L1 or L3 auditory interneurons should create an imbalance between the input of sound induced neuronal activity through the two ears into central auditory neurons. Using the principle of turning toward the side most stimulated (Huber et al. 1984) in an at tempt to balance the activity of bilaterally paired auditory neurons, the female cricket should walk at an angle with respect to the sound source, which results f rom turning toward the side with the intact member of the neuron pair, whose magnitude is related to the degree of bilateral asymmetry. Phonotactic circling, which results f rom continuously turning toward the side with an intact member of a bilateral pair of auditory neurons indicates that the imbalance created was too large to be resolved by orienting at a constant error angle. Thus, these two criteria - turning toward (with respect to the female's prekilling orientation) and/or circling toward the intact side - constitute evidence that the inactivated auditory neuron is involved in the cir- cuitry involved with localization of the source of the calling song.


Effects of unilaterally &activating the L-shaped, ascending auditory interneurons

The auditory interneurons that ascend from the prothoracic ganglion are usually considered to be first order interneurons (Hennig 1988) that conduct encoded information about acoustic stimuli up to higher auditory processing pathways in the brain of crickets (Schildber- ger 1984). While most studies have described two pairs of ascending auditory interneurons (Wohlers and Huber 1982, 1985; Hennig 1988), Stout et al. (1985) described 3 functionally distinct classes of ascending auditory interneurons (L1, L2, L3) in female A. dornesticus and sug- gested that they might be coresident in the same female. This suggestion has been recently confirmed by Atkins et al. (1989a, b) by demonstration of 3 morphologically and physiologically distinct types of L-shaped, ascending auditory interneurons that are coresident in the same hemiganglion.

Photo&activation of L2

In a previous study, unilaterally killing an L2 had no effect on phonotaxis in response to 5 kHz CSs (Atkins et al. 1984). Since L2 has a 5 kHz threshold that is above the intensities used in orientation tests (Stout et al. 1985; Atkins et al. 1989a, b), no effect of killing this neuron was expected. Thus L2 plays no demonstrable role in the CS phonotaxis of female A. domesticus.

The syllable period dependent phonotactic orientation and circling, in response to 85 dB CSs, by females with an inactivated L 1 and the contralateral ear occluded (Fig. 4B), suggests that a higher threshold, ascending auditory neuron was brought into play that could also induce phonotaxis. The reduction in syllable period dependent circling by females with L1 killed and with both ears functioning further suggests that when this higher threshold class of auditory neurons was bilaterally active, the imbalance created by unilateral killing of L1 was reduced (Fig. 3B). Since the L3 neuron has already been demonstrated to play a role in CS phonotaxis (Stout et al. 1985 and Fig. 6A), it is possible that L3 might be the higher threshold neuron described above and thus might lead into the localization and recognition networks, and is probably sufficient to induce phonotaxis. However, the involvement of other higher threshold neurons, such as TN1, although apparently not involved in phonotaxis of female G. bimaculatus (Schildberger and Hrrner 1988), cannot be ruled out for A. domesticus. Unilaterally inactivating both L1 and L3 on the same side and testing if phonotaxis could still occur, would be necessary to an- swer this ambiguity in our data.

Schildberger and Hrrner (1988) demonstrated that AN I's role (in G. bimaculatus females) in stimulus localization was similar to that demonstrated for L1 by Stout et al. (1985) and confirmed in this study. However, their experimental paradigm did not allow for evaluation of ANI's role in CS recognition.



The relatively large angular errors and circling in- troduced by unilaterally inactivating an L1, resulting from turning toward the side providing input to the intact L1 by a female on the treadmill (Fig. 2) demonstrate that the L1 is involved in the neural pathways that lead to localization of the stimulus. This confirms the same conclusion, resulting from similar experiments done in an arena by Stout et al. (1985).

The elimination of phonotaxis in response to 5 kHz, 60 dB CSs by a female with a unilaterally inactivated L1, following waxing of the ear on the side providing input to the intact L1 (Fig. 4A), indicates that the functional L1 of a one-eared female is necessary for phonotaxis in response to CSs produced at this intensity.

Since L1 is necessary for phonotaxis in response to 5 kHz CSs below 80 dB, syllable period specific rates of circling by females with a single L1 (Fig. 3) indicates that input to the call recognition and response centers, from a single L1, is sufficient for the occurrence of CS recognition and phonotaxis by the female.

These results strongly suggest that for females with both ears functional, transfer of the response of the paired Lls to an attractive CS, to higher recognition (Schildberger 1984) and response control networks is both necessary and sufficient for inducing the female's phonotactic response.

As demonstrated by Stout et al. (1985) and in this paper, unilaterally inactivating an L3 neuron introduces angular errors into the orientation of female A. domesticus in response to more intense 5 kHz CSs (85 dB in this study) which results from turning toward the side providing input to the intact L3 (Figs. 6A, 7). Thus the L3 is involved in the pathways that lead to stimulus localization. The more direct orientation, without consistent error angles that resulted (Figs. 6B, 7) when the stimulus intensity was reduced to 60 dB (below the threshold of L3) was expected because of the bilateral pair of Lls present. The absence of circling in response to an 85 dB stimulus was not surprising because of the paired L1 neurons that would respond to the CSs in addition to the single L3 neuron present only on one side.

The continued phonotactic orientation and syllable period dependent rate of circling that resulted following occlusion of the ear on the side with an intact L3 (Figs. 6C, 8) was expected due to the presence of an L1 neuron on the unoccluded side (with the killed L3). This is consistent with the suggestion that the response of a single L1 neuron to a CS leads to CS recognition, and is sufficient to initiate and sustain phonotaxis. Since the degree of circling in this experiment, using 85 dB CSs, was very similar to the rate of circling when a single L1 was stimulated at 60 dB (Fig. 3), it is less likely that neurons other than L3 significantly effect phonotaxis when brought into play by the increased stimulus intensity.


Comparison to other cricket species

Numerous studies have characterized cricket auditory neurons, and based on their response properties, proposed functions that these neurons might play in phonotaxis. Only a few investigations evaluated the behavioral roles of cricket auditory neurons by inactivating them and measuring the resulting changes in behavior (Atkins et al. 1984; Nolen and Hoy 1984; Stout et al. 1985; Schildberger and H6rner 1988). Reversible hyperpolarization was used to reduce or eliminate the response of auditory neurons to sound by Nolen and Hoy (1984) and Schildberger and H6rner (1988), while Atkins and Stout (Atkins et al. 1984; Stout et al. 1985; and the present investigation) employed intense illumination of Lucifer yellow-filled neurons to completely stop their responses to sound. Each of these procedures, while quite different in application, results in reduction or elimination of an auditory neuron's response to sound, and thus should yield comparable changes in behavior.

Nolen and Hoy (1984) evaluated the role of the high frequency neuron, INT-1 in predator avoidance, and clearly demonstrated that negative flight phonotaxis ceased when this neuron was hyperpolarized during presentation of model bat calls. While the results we present on the roles of L1 and L3 in positive walking phonotaxis in response to model CSs are not comparable, it is possible that the high frequency neuron L2, which we have shown does not play a role in CS phonotaxis (Atkins et al. 1984), might be involved in negative flight phonotaxis in A. domesticus.

Schildberger and H6rner (1988) chose not to compare our earlier work (Atkins et al. 1984; Stout et al. 1985) with theirs, based on the differences between the inactivation procedures described above, and on their concern that behavioral measures of phonotaxis in an arena, such as we used (Atkins et al. 1984), were less reliable than measurements made on a treadmill and might invalidate the conclusions reached in these papers. The behavioral testing procedures we used in the present study involved a noncompensating treadmill of similar design to the one used by Schildberger and H6rner (1988). Not only do the results we report in this study on the roles of L1 and L3 in stimulus localization, using a treadmill, fully cor- roborate our earlier findings using an arena (Stout et al. 1985), but they are also in agreement with the roles of the AN1 and AN2 neurons of G. bimaculatus in stimulus localization proposed by Schildberger and Hrrner (1988). AN1 and L1 apparently play similar roles in stimulus localization as inactivation by killing or hyperpolarization caused large errors in the angular orientation of either species in response to model CSs. AN2 and L3 are also similar in that inactivation causes smaller errors in angular orientation than does L1 or AN1. However, in the absence of the lower threshold Lls, stimulation of a single L3 causes very large error angles (Figs. 4B, 5A) or circling (Figs. 4B, 5B). It is possible that had Schildberger and Hrrner used an experimental paradigm that allowed them to stimulate AN2 in the absence of the lower threshold AN1, they would have found it to be more important in stimulus localization. In the experi-

ments with AN2 (Schildberger and H6rner 1988), only half of the neurons evaluated resulted in a change in orientation during hyperpolarization. Possibly, this was due to the continued but reduced response they showed for AN2 during hyperpolarization while presenting the sound ipsilaterally. Conversely, all of our experiments in which L3 was unilaterally killed resulted in the expected change in angular orientation. Killing the L3 neuron completely eliminates its activity under any conditions, and would thus yield larger side to side differences between the L3s during sound stimulation. It is also possible that since the effect of hyperpolarization on turning velocity was observed chiefly in animals in which AN2 had a low threshold in the 5 kHz region (Schildberger and H6rner 1988), some of their animals had AN2s with high 5 kHz thresholds that were more L2-1ike than L3- like. If this were the case, the effect of hyperpolarization on a neuron that was giving little response to the stimulus would be less likely. Since Schildberger and H6rner's experimental paradigm was limited to evaluating the role of AN1 and AN2 in stimulus localization, their possible involvement in pathways leading to stimulus recognition cannot be compared with our results for L1 and L3.

The results of this study confirm the findings of Stout et al. (1985) that the L1 and L3 neurons are involved in localization of the source of an attractive CS. More importantly, however, this study extends our under- standing of the roles that the L1 and L3 neurons play in cricket phonotaxis by demonstrating that for CSs below the intensity threshold of L3, activity of the L 1 neuron in response to a CS is necessary and sufficient for phonotaxis by the female. While activity of a single L3 neuron in response to a CS can probably also induce phonotaxis, this situation would not occur naturally since the Lls would already be responding due to their 25-30 dB lower threshold for 5 kHz CSs. Stout et al. (1988) demonstrated that the response of the L1 to CSs of increasing intensity typically saturated between 75 and 80 dB, at or just above the threshold of L3. Thus, we suggest that the paired L1 and L3 neurons both lead into the CS localization and recognition networks most likely found in the brain (Schildberger 1984) and that they fractionate the range of CS intensities encountered by the female, with the L1 neuron pair encoding intensities below approximately 75 dB, and the L3 neurons encoding intensities above that value.

The importance of the roles of the L 1 and L3 auditory neurons in phonotaxis is further emphasized by the par- tial regulation of the phonotactic behavior of female A. domesticus through juvenile hormone III control of the threshold of the L1 neuron (Stout et al. 1991) and syllable period specific response properties of the L3 neuron (Henley et al. 1992).

Acknowledgements. This research was funded by a National Science Foundation research grant BNS 8510251 and BNS 8819817. We thank Dr. A. Stumpner for critical review of this paper.


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