chemical communication in heliothine moths

J Comp Physiol A (1995) 177:545 557 �9 Springer-Verlag 1995

T. A. Christensen" H. Mustaparta. J. G. Hildebrand

Chemical communication in heliothine moths VI. Parallel pathways for information processing in the macroglomerular complex of the male tobacco budworm moth Heliothis virescens

Accepted: 13 April 1995

Abstract The chemical and temporal features of the sex-pheromone emitted by Heliothis virescens females are encoded by a diverse array of output pathways from the male-specific macroglomerular complex (MGC) in the antennal lobe. Most output neurons (29 out of 32) were activated by antennal stimulation with the principal component of the sex-pheromone blend of this species, (Z)-ll-hexadecenal. Six neurons were excited solely by this component, 8 neurons also responded to the second essential blend component, (Z)- 9-tetradecenal, and 14 neurons displayed equivalent responses to the two. Many neurons also effectively encoded the onset and duration of the stimulus. In one additional neuron, a prolonged excitatory response (synergism) was evoked only by the blend of the two components, indicating that some MGC neurons function as 'blend detectors'.

In contrast to the situation in Helicoverpa zea, none of the MGC neurons in H. virescens responded selectively to (Z)-9-tetradecenal, suggesting that these two noctuid species employ different neural strategies to encode information about their respective pheromone blends.

Three MGC-output neurons responded selectively to (Z)-ll-hexadecenyl acetate, an odorant released by some sympatric species that disrupts normal upwind flight to pheromones. Thus, changes in the attractant and deterrent chemical signals, as well as the physical features of these odor plumes, are encoded in the MGC

T. A. Christensen ( l ~ ) �9 J. G. Hildebrand Arizona Research Laboratories, Division of Neurobiology, 611 Gould-Simpson Building, University of Arizona, Tucson, AZ 85721, USA

H. Mustaparta Department of Zoology, University of Trondheim, AVH, N-7055 Dragvoll, Norway

across a diverse parallel array of output pathways to the protocerebrum.

Key words Interneuron �9 Odor coding �9 Olfaction �9 Pheromone " Tobacco budworm

Abbreviations AL antennal lobe �9 AN antennal nerve �9 16:AL hexadecanal - MGC macroglomerular complex �9 14:AL tetradecanal �9 Zll-16.AL (Z)-I 1-hexadecenal �9 Z11-16:A C (Z)- 11 -hex adecenyl acetate �9 Z9-14:AL (Z)-9-tetradecenal" Z9-14.FO (Z)-9-tetradecenyl formate

Introduction

In many species of moths, the molecular 'image' of a sex-pheromone stimulus released by a conspecific female is encoded by neural circuits that reside within a sexually-dimorphic group of enlarged glomeruli known as the macroglomerular complex (MGC; for recent reviews see Masson and Mustaparta 1990; Boeckh and Tolbert 1993; Christensen 1995). A glomerular organization, as seen in the olfactory systems of widely-divergent species including mam- mals, is also the most prominent feature of the MGC in the antennal lobes (ALs) of many male moths (Koontz and Schneider 1987; Christensen et al. 1991; Hansson et al. 1992, 1995). There is also abundant evidence in several moth species that the anatomically separate glomeruli of the MGC represent functionally distinct processing units (Christensen et al. 1991, 1995; Christensen and Hildebrand 1994; Hansson et al. 1991, 1992, 1995). The striking anatomical, physiological, and neurochemical similarities in the glomerular organization of vertebrate and insect olfactory systems have led several authors to speculate that olfactory circuits in these two groups may be governed by similar

546 T. A. Christensen et al.: Glomerular processing of olfactory information in a moth

mechanisms of synaptic organization (Christensen and Hildebrand 1987a, b; Strausfeld 1989; Boeckh et al. 1990; Boeckh and Tolbert 1993).

As more is learned about the behaviors that are triggered or modulated by pheromones in insects, it becomes increasingly apparent that these chemical signals do not only serve a species-specific function. It is well known that chemicals serving as part of an attractive pheromone in one species can also mediate a different type of communication between sympatric species. The two heliothine moths Heliothis virescens (F.) and Helicoverpa (formerly Heliothis) zea (Boddie), for example, are widely sympatric in North and South America (King and Coleman 1989). Both are strongly polyphagous, feeding on more than 100 different plant species, including agricultural staples such as corn, cotton, soybeans and wheat. The sexual attractants of these two species are well studied and have been identified as a 6- and a 4-component blend in H. virescens and H. zea, respectively. In both species, (Z)-ll-hexadecenal (Zll-16:AL) is produced in the greatest amount, and at least one other pheromone component is necessary to evoke high levels of male mate-seeking behaviors (Vetter and Baker 1983, 1984). Another re- quirement is that the two principal pheromone components must be presented in the correct ratio in order to evoke optimal attraction. In H. virescens, the ratio of the two principal components (Zl l -16:AL and (Z)-9- tetradecenal or Z9-14:AL) is about 15 : 1 (Pope et al. 1982), whereas in H. zea, the ratio of the two principal components (ZI 1-16 : AL and (Z)-9-hexadecenal or Z9- 16:AL) is about 70:1 (Pope et al. 1984). The chemical differences in the attractive blends of these two species certainly contribute to reproductive isolation, but the involvement of chemical deterrents has also been revealed through behavioral assays. For example, one component produced by H. virescens females, Z9- 14 : AL, also inhibits the attraction of sympatric H. zea males (Klun et al. 1980a, b). This phenomenon of interspecific interruption has been observed in a number of lepidopteran species (Hansson et al. 1986; Grant et al. 1988; Lucas and Renou 1989 and references therein). Another interruptant produced by the females of several sympatric species (Arn et al. 1992) and also by H. virescens males (Teal and Tumlinson 1989) is an analog of Zl l -16:AL, (Z)-ll hexadecenyl acetate (Zl l - 16 : AC). A population of receptor neurons selective for Z l l -16 :AC has been found in the antennal sensilla of H. virescens males (Berg et al. 1995), and recent behavioral results show that this chemical signal profoundly interrupts normal pheromonal attraction in H. virescens males (N.J. Vickers, A. Mafra-Neto, T.C. Baker unpublished).

In order to understand the functional organization of the MGC in H. virescens males, it is essential to know what types of information are carried in each MGC pathway, and how that information might ulti- mately be used in behavioral attraction and arrestment.

The results from this study support the notion that different features of the chemosensory environment are represented by a limited number of parallel pathways in the glomeruli that comprise the MGC in H. virescens males, and that the MGC glomeruli are functionally- distinct processing units. We present a hypothetical model of how this parallel array of MGC outputs could relay different messages to the protocerebrum that reflect changes in the chemical and physical features of the insect's olfactory environment. Some of this work has appeared elsewhere in preliminary form (Christen- sen et al. 1994).

Materials and methods

Preparation

Heliothis virescens (F.) were obtained as pupae from the Insect Attractants, Behavior, and Basic Biology Research Laboratory, USDA-ARS in Gainesville, Florida. Males and females were separated into different containers, so that upon emergence, males would be isolated from the females' sex pheromone. For all experiments, males 1-3 days post-emergence were selected. The procedures used in preparation for intracellular recording from MGC neurons have been described in detail elsewhere (Christensen et al. 1991).

Stimulation

The preparation was situated so that the ALs were facing upward and the antenna ipsilateral to the recording site would be stimulated with a variety of airborne odorants. The odor-delivery apparatus has been described in detail (Christensen and Hildebrand 1987b; Christensen et al. 1989,1990). The duration and frequency of stimulus pulses were regulated by a computer-controlled stimulator. When given a command pulse, a solenoid-controlled valve (General Valve Corp., # 1-28-900) diverted the charcoal-filtered air from the antenna to a glass cartridge containing a piece of filter paper bearing the odorant(s). Stimuli were separated by at least 1 min to avoid sensory adaptation and synaptic fatigue.

The identified sex-pheromone components isolated from H. virescens females were obtained as synthetic compounds ( > 99% purity) and were tested individually or in blends. These were Zl l -16: AL, Z9-14: AL, hexadecanal (16 : AL), and tetradecanal (14 : AL) (Roelofs et al. 1974; Tumlinson et al. 1975; Klun et al. 1980a; Pope et al. 1982; Teal et al. 1986). We formulated a blend of synthetic components that was well within the dynamic response range of H. virescens male antennae (Almaas and Mustaparta 1990, 1991; Christensen et al. 1990). The 4 components were combined into a blend in a 15 : 1 : 2 : 1 ratio, and diluted in n-hexane to the final concentrations (0.1-10 ng of Z l l -16 :AL, and the appropriate amounts of the other 3 components in 5 gl of solvent). In some experiments, the two principal components, Z l l - 1 6 : A L and Z9-14:AL, were combined in a 1:1 ratio to test for additive effects. The two minor components (14 :AL and 16:AL) were also combined in a 1:1 blend and presented independently of the principal components. Several pheromone ana- logs were also tested. Z l l - 1 6 : A C has been isolated from the female pheromone glands of some sympatric noctuid species (see Arn et al. 1992), and from the hairpencil glands of H. virescens males (Teal and Tumlinson 1989). We also tested (Z)-9-tetradecenyl formate (Z9- 14:FO), a chemical mimic of Z l l - 1 6 : A L (Tumlinson 1979). All synthetic compounds were kindly provided by Dr. J. Tumlinson, USDA-ARS, Gainesville, Florida, USA.

T. A. Christensen et al.: Glomerular processing of olfactory information in a moth 547

Table 1 Comparison of the physiological profiles and numbers of M G C interneurons found in two sympatric heliothine moths, Hel io th i s

virescens and Hel icoverpa zea ~

responsiveness to each odorant n for each species

Cell type Z11-16 : AL Z9-14 : AL Z9-16: AL blend H. virescens H. zea

Z11-16: AL-type 1 + o o + 6 14 (low sensitivity) Z l l -16 :AL- type 2 + + + + + + 8 5 (high sensitivity) Z11-16: AL/Z9-14 :AL + + o + 14 I b Z9-14:AL/Z9-16:AL o + + + + + + 0 7 Blend Synergist o o NT + + + 1 2 Z l l - 1 6 : A C o o o + + c 3 NT

Mean responses are represented by the following symbols: o, no response; + , tonic response up to 15 spikes/300-ms stimulus; + + , phasic-tonic response with > 15 spikes timeqocked to the stimulus; + + + , synergistic response outlasting stimulus for more than 3 s (see text); NT, not tested. Note that responses in every cell type are primarily excitatory al l . zea data from Christensen et al. (1991) bCell no. 5 of Fig. 6 in Christensen et al. (1991) CThese neurons will only respond if Z11-16:AC is presented, whether separately, or in a blend with other components

Morphology

The procedures for visualizing dye-injected neurons are detailed in Kanzaki et al. (1989). After physiological characterization, Lucifer Yellow was injected by passing negative DC current (10 nA-min). Brains were fixed for 1 h in 2.5% formaldehyde in 0.1 M phosphate buffer containing 3% sucrose (NJ Strausfeld, personal communication). The brains were then dehydrated, cleared in methyl salicylate, and documented as wholemounts by fluorescence photo microgra- phy. Brains were returned to 100% ethanol and embedded in Spurr's resin. Tissues were sectioned at 25 lam, each section was photo- graphed with color slide film, and the neuron was reconstructed from the color slides.

Results

Characterization of odor-information-processing neurons

Intracellular recordings were obtained from a total of 32 presumptive projection neurons (PNs) in the ALs of H. virescens male moths. All impalements were made directly in the neuropil of the MGC where the largest-diameter neurites of PNs (but not local interneurons) are most accessible. As found in other pheromone information-processing PNs in H. zea and H. virescens (Christensen et al. 1989), subthreshold synaptic potentials as well as action potentials were readily observed. Background activity varied from cell to cell, but it rarely exceeded 10 impulses/s. Owing to the instability inherent in these small preparations, we were unable to record the responses in each cell to antennal stimulation with every component and blend. Nevertheless, the responses recorded allowed us to place each neuron into one of several broadly-defined response categories (Table 1). Neurons that responded only to the major pheromone component, Z11-16: AL, were classified as "Zl1-16 :AL-type 1" neurons in ac-

cordance with the nomenclature used by Christensen et al. (1991). A second group of cells that responded strongly to Z l l - 1 6 : A L and also to other stimulants was classified as "Zl l -16: AL-type 2" neurons. A third group of cells responded in a qualitatively and quantitatively similar fashion to both essential pheromone components and was classified as "Zl 1-16 : A1/Z9- 14:AL" neurons. One neuron that responded in a unique fashion to Z l l - 1 6 : A L and Z9-14:AL presented together was classified as a "Zl 1-16 : AL + Z9- 14:AL-synergist" neuron. Finally, 3 neurons did not respond to the pheromonal aldehydes but did respond to the chemical analog, Z11-16 : AC. Multiple represen- tatives from the different response classes allowed us to make qualitative and quantitative comparisons of response properties between neurons. For two cells stained with Lucifer Yellow, the response properties of the neurons could be correlated with their morphology.

Z l l -16 : A L - t y p e 1 : phys iology

About 19% of the neurons examined (6 out of 32) responded to antennal stimulation with Z11-16 : AL, but not to Z9-14 : AL up to 10 ng. One neuron was also tested with Z9-16:AL (1 ng) but showed no response to this odorant, which is a minor pheromone component in this species. An example of the selective response of a Zl l -16:AL-type 1 neuron is shown in Fig. 1. In each type-1 neuron, the stimulus caused a small (1-2 mV) membrane depolarization that resulted in a brief burst of action potentials. In two neurons, the response far outlasted the 300-ms stimulus presentation (Fig. 2; cells 1 and 2 in Fig. 3), and these long-lasting firing patterns were very reproducible (Fig. 2). In the other four cells, responses were closely time-locked to the stimulus (cells 3,4, 5 and 6 in Fig. 3). Four cells were tested for


A ZI 1-16".AL, I0 ng

B zg-14:AL, 10 n g

C

Fig. 1A C Selective response of a Z l l -16 :AL- type 1 neuron to Z l l -16 :AL, one of the principal components of the pheromone blend of H. virescens females. Same neuron as cell no. 6 in Fig. 3. Stimulus marker ( = 300 ms) indicates onset and duration of the stimulus-carrying airflow. This cell responded to Z l l - 1 6 : A L at 10 ng (A), but not to Z9-14: AL (the second principal component), at the same concentration (B). The 4-component pheromone blend evoked a qualitatively and quantitatively similar response as that of Z11-16:AL alone, but with a shorter latency (C). Abbreviations for stimuli are defined in the Methods

A ZlI-16:AL, 0.I ng

Zg-14:AL, 0.I ng

D Repeat C

3 0 0 m s

Fig. 2A D Tonic and prolonged responses of a Z] 1-16:AL-type 1 neuron to Z11-16 : AL. The excitatory response to Z11-16: AL was robust and reproducible (A,B), but no response was elicited by Z9-14: AL (C, D). Downward deflection preceding stimulus marker is a 10 mV calibration pulse. Other responses from the same neuron are shown in Fig. 3 (cell no. 1)

Z l l-16 : A L type-2 : physiology

Eight neurons (25%) responded to Z l l - 1 6 : A L and also to Z9-14: AL, but these neurons were uniformly more sensitive to the principal pheromone component, Z l l - 1 6 : A L (Figs. 4-6). At a dosage of only 0.1 ng, Z l l - 1 6 : A L evoked a membrane depolarization with a phasic-tonic pattern of spike activity that closely followed the stimulus duration (Fig. 4A). The same amount of Z9-14 : AL evoked a smaller depolarization and a reduced and more tonic pattern of firing activity (Fig. 4B). We also tested stimuli of differing quality as a test of the cell's breadth of tuning. As for the type-1 neurons, type-2 neurons were not selectively responsive to mixtures. The responses to two mixtures that con- tained Z l l - 1 6 : A L were similar to the responses to Z11-16:AL presented alone (Fig. 4C-D). A mimic of Zl1-16 : AL (Z9-14 : FO; see Materials and methods) evoked a response that was both reduced and delayed compared to that evoked by the natural pheromone component (Fig. 4E). Two neurons of this type were tested with Zl1-16: AC, and one responded tonically with a depolarization and a train of spikes (Fig. 4F). This same neuron was also tested with Z9-16:AL and tobacco-plant volatiles (Fig. 4G and H, respectively). These olfactory stimuli both evoked only a small membrane depolarization without spiking activity.

As a group, the more broadly responsive type-2 neurons were also the most sensitive neurons encountered, and more sensitive to the presence of Z11-16 : AL than the type-1 neurons (Figs. 4-6). Not all type-2 neurons, however, were equally sensitive, as shown in Fig. 5. Antennal stimulation with 0.1 ng of Z l l -16 :AL evoked a small depolarization and fewer than 10 spikes in one neuron (Fig. 5A), while the same stimulus evoked a 15-mV depolarization and more than 8 times as many spikes in another (Fig. 5C).

Another distinguishing feature was that all type-2 cells also gave unambiguous responses to stimulation with Z9-14:AL as shown above (Fig. 6). Addition of Z9-14 : AL to Z11-16 : AL in either a 1 : 1 or 1 : 15 blend ratio, however, did not alter the response in a system- atic fashion (Fig. 6). In most cases, responses to the blend were similar to the responses evoked by Z l l - 16 :AL alone.

responses to the 4-component H. virescens blend (up to 10ng of Z l l - 1 6 : A L plus the appropriate amount of the other 3 components). The addition of other components (in either of the two blend ratios tested; see Materials and methods) did not affect the excitatory responses to Zl l -16:AL. While all 6 neurons of this type exhibited a selective response to Z11-16 : AL, none were depolarized by more than 1-2 mV by this component, and most responses in these neurons did not exceed 4 spikes per 50-ms bin width in the histograms shown in Fig. 3.

Z l l -16 : AL- type 2:morphology

Two neurons that were characterized as type-2 were subsequently stained with Lucifer Yellow (Fig. 7). The soma was found in the medial cell group (MC), and all dendritic arborizations within the AL were restricted to only one of the four glomeruli recently identified in the MGC (Hansson et al. 1995; Fig. 7A). As the primary neurite crossed the inner margin of the largest glomerulus, designated glomerulus a, it divided into two main branches. One branch arborized in only the


Fig. 3 Response spectra for all of the Z11-16: AL-type 1 neurons found in this study. Peristimulus-time histograms show the temporal characteristics of responses and allow for a direct comparison between responses from different neurons. X-axis = 0-1000 ms, stimulus marker = 300 ms, Y-axis = number of spikes/50-ms bin. BLEND refers to the 4-component blend as described in Methods. B L A N K refers to an odorless, mechanosensory stimulus. A blank space indicates that the stimulus was not tested

CELL # 1 2 3 4

BLANK 12

4

0 J r f I i t i r i | = I i I i i l l l T i I

z,,_,..o., . o L . 1 ng 12 7

8 ~

o .§ L t i t i i i i

10 ng

5

7

ZT" t ~ I I I I 1 - u

Z9-14:AL ~2

0.1 ng ! t , ~ , , , I

1 ng

10 ng

r , ~ j r r ' l ' ~ F i V

BLEND 12

4 I, 0 ~ l ~ l r ~ - F - I .

! r m , f ~ , , r I , l i

(1:1) (1:1) (15:1) 0.1 ng 1 ng 1 ng

6

i

8

4

0

(15:1)

10 ng

lateral portion of the glomerulus (Fig. 7B). The second branch gave rise to dense arborizations that were completely confined to the medial portion of the glomerulus. The remaining glomeruli of the MGC (b, c and d), were completely devoid of any neuronal pro- cesses from this cell (Fig. 7B). A single axon ran through the inner antenno-cerebral tract to the protocerebrum, where axon terminals were clearly visible in both calyces of the ipsilateral mushroom body (Fig. 7C). Partial staining of one additional neuron revealed similar morphological characteristics.

Z11-16: AL/Z9-14 : AL neurons :physiology

Fourteen neurons (44%) responded to both Z l l - 16 : AL and to Z9-14 : AL in a qualitatively and quantitatively similar manner. An example of one of these neurons is shown in Fig. 8. In each neuron, both components evoked a 10 15mV depolarization with a phasic-tonic pattern of spike activity, as compared to

a blank, which elicited only a small (5 mV) depolarization that did not reach spike threshold. A summary of the responses in all 14 of these particular neurons is shown in Fig. 9. As shown for the Z l l - 1 6 : A L type-2 neurons, these cells displayed a wide range of sensitivities to both pheromone components, but the temporal pattern and magnitude of the responses to Z11-16:AL and Z9-14:AL in these neurons were similar (Figs. 8 and 9). In most cases, the time course of each spiking response approximated the stimulus duration. Only in cases in which spontaneous activity was present did the response outlast the stimulus by more than 100 ms.

Addition of Z9-14:AL to Z l l - 1 6 : A L in either a 1 : 1 or 1 : 15 blend ratio did not alter the responses dramati- cally, but in most cases where blends were tested (11 neurons), responses to the blend were noticeably different from the responses evoked by Z l l - 1 6 : A L or Z9-14:AL presented alone. In 7 neurons (cells 1, 2, 7, 8 1 ! in Fig. 9), the blend evoked a greater membrane depolarization and increased spiking activity over the responses to the individual components, and

550 T.A. Christensen et al.: Glomerular processing of olfactory information in a moth

2O mV

Zll-16:AL. 0~1 ng t 300 ms

o., . IIPIIIIIJI/

o , IP ISJJlIII /

zg-14:Formate, 0.I ng

LLltl lt Z11-16:AC. 0.1

Zg-16".J.L, 0.I ng

H tobacco head space

Fig. 4A H Physiological responses of a Z11-16:AL-type 2 neuron to various pheromonal and non-pheromonal stimuli (other responses shown in Fig. 6, cell no. 4). Stimulus marker ( = 300 ms) indicates onset and duration of the stimulus-carrying airflow. Down- ward deflection preceding stimulus marker is a 10 mV calibration pulse. Type 2 neurons showed a higher sensitivity than the Z l l - 16: AL-type 1 neurons in Fig. 3. This neuron responded similarly to any stimulus containing Z l l - 1 6 : A L (A,C and D), but also responded to Z9-14:AL (B), Z9-14:FO (E), and to Z l l -16 :AC (F) The pheromone analog Z9-16: AL evoked only a small depolarization (G), as did a mixture of tobacco (host plant) volatiles (H)

ZII-16:AL, 0.1 ng A

II

Fig. 5A C Responses of three Zl l -16:AL-type 2 neurons to ZI1- 16:AL at 0.1 ng, illustrating the range of sensitivities observed in this neuronal class. Other responses shown in Fig. 6 : A, cell no. 6; B, cell no. 2; C, cell no. 1

In addition to Z11-16:AL and Z9-14:AL, this neuron was tested with 1 ng of Z9-16: AL, a blend of the two minor pheromone components at 1 ng each, and 1 ng of Z9-14: FO, but it did not respond to antennal stimulation with any of these odorants (Fig. 11). Two additional Z11-16:AC neurons are shown in Fig. 12. These neurons illustrate the range of sensitivity observed in this class of neurons. In contrast to the neuron in Fig. 11, the upper neuron of Fig. 12 had a threshold sensitivity below 0.1 ng, and the lower neuron was strongly depolarized, with its spiking temporarily at- tenuated, in response to 0.1 ng. In both neurons of Fig. 12, the responses to Zl l -16: AC were robust and reproducible.

in one case (cell 4), the blend evoked a slightly reduced response.

Z l l -16 : AL + Z 9-14 : AL synergist neuron

Out of the 32 neurons examined, one displayed characteristics of a "blend specialist" (Fig. 10). This neuron responded weakly to 1 ng of Z l l - 1 6 : A L and was only slightly depolarized by 1 ng of Z9-14:AL. If the two stimuli were presented together, however, the neuron responded with a prolonged train of spikes that lasted nearly 7 s beyond the period of stimulation (Fig. 10).

Z 11-16 : A C neurons

Three neurons responded to antennal stimulation with the acetate analog, Z l l -16 :AC, but not to either Z l l - 16:AL or Z9-14 : AL. One example is shown in Fig. 11.

Discussion

It is now well established that pheromone plumes, like any natural odor stimulus, possess both chemical and physical qualities that are affected by changes in environmental conditions (Murlis and Jones 1981; Muftis 1986; Murlis et al. 1990; Moore 1994). From behavioral studies, we also know that changes in the chemical attributes of the pheromonal stimulus (i.e., blend composition and component ratios) result in dramatic changes in male orientation flight (Linn and Roelofs 1983; L i n n e t al. 1984,1986; Witzgall 1990; Vickers et al. 1991). Similarly, alterations in the physical attributes (the spatio-temporal structure of individual filaments of pheromone within the plume) also result in significant behavioral changes (Willis and Baker 1984; Baker et al. 1985; Baker and Haynes 1987; Vickers and Baker 1992,1994; Mafra-Neto and Card6 1994). Conse- quently, we wanted to know how these aspects of the


Fig. 6 Response spectra for all of the Zl1-16 : AL-type 2 neurons found in this study that responded more strongly to Z l l -16 :AL than to Z9-14:AL at the same concentration. X-axis = 0 I000 ms, stimulus marker = 300 ms, Y-axis = number of spikes/50-ms bin. A blank space indicates that the stimulus was not tested

CELL #

1 2 3 4 5 6 7 8

i i T - - r ~ i i J ~ i J ~ I I f f I I J I J s i i ,

Z11-16-AL ~

0.1. ng

1 2

l o ~ o i i t i ~ r r i p i t ~ i

1 2

0 I , i r r i i i J i J

(1:1) e 4

0.1 n 9 o

1 2

1 2

BLEND 8 (15:1) 4

0.1 ng o

1 2 ~ r ~ 1 ng o 8 4

odor quality and temporal features of the stimulus are encoded by neural circuits in the insect's brain.

In the present study, five different physiological classes of pheromone-information-processing PNs in the ALs of H. virescens males were identified according to the selectivity, sensitivity, and time course of their responses to the sex-pheromone components shared by several heliothine moth species. Several of these classes of neurons are strongly analogous to those found in the closely-related sympatric species, Helicoverpa (Helio- this) zea (see Table 1 and Christensen et al. 1991). As found in H. zea males, most MGC neurons in H. virescens were excited by the principal pheromone component of both species, Zl l -16:AL. Of these 29 neurons, Zl l -16:AL-type 1 neurons were the least sensitive, but responded exclusively to Zl l -16:AL. Z l l - 1 6 : A L - type 2 neurons, which were among the most sensitive neurons found, responded best to Z l l - 16 : AL, but responded also to Z9-14 : AL (Figs. 4-6). As in the MGC of H. zea, the different tuning characteristics of type-1 and type-2 PNs may reflect input from

different physiological populations of Zl l -16: AL receptor neurons (Almaas and Mustaparta 1990, 1991). It appears very likely that type-1 neurons receive direct or indirect input exclusively from the least-sensitive Z11- 16:AL receptor neurons of the ipsilateral antenna. Similarly, the increased sensitivity of type-2 neurons, including their responses to Z9-14:AL, may reflect a selective input from the most sensitive Z l l -16 :AL receptor neurons, which also show a secondary responsiveness to Z9-14:AL (Almaas and Mustaparta 1990, 1991; Berg et al. 1995). The morphological features of type-2 neurons are also consistent with their physiological characteristics. In both type-2 PNs that were stained with Lucifer Yellow, the dendritic arborizations were confined to one MGC glomerulus - the largest of the 4 glomeruli, a (Fig. 7). Furthermore, in another recent morphological study, the axons from both Z l l -16 :AL and Z9-14:AL - selective sensory neurons were also shown to converge in the a glomerulus (Hansson et al. 1995). Similarly, we found several type-2 PNs in H. zea that also had extensive


A t

~IGC MC

d

,

B am

i ' ' b

Fig. 7A Diagram of the male H. virescens brain, viewed frontally. Arrow at the top indicates the approximate midline of the brain. The macroglomerular complex (MGC) comprises 4 glomeruli (a d), shown in greater detail in R A N antennal nerve; LC lateral cell group; MC medial cell group; Oe oesophageal foramen; OL optic lobe. Orientation marker: d dorsal; I lateral. Scale marker = 300 lam. B Reconstruction of a Z 11 - 16 : AL-type 2 projection neuron (cell no. 3 in Fig. 6) filled intracellularly with Lucifer Yellow. The soma (s) was in MC. One major branch of the primary neurite innervated the lateral portion of glomerulus a (a~), but a portion of this neuropil was not innervated (*). Another major neuritic branch extended den- drites throughout the medial portion of glomerulus a (a,,). The axon (arrow) enters the protocerebrum through the inner antenno-cerebral tract. Scale marker = 100 lam. C At a depth of 50 gm from the back of the brain, the terminal arborizations in both the medial and lateral calyces of the ipsilateral mushroom body (Ca) were visible in the protocerebrum. Scale marker = 200 gm

- I

Fig. 8A-C Responses of a Zl1-16: AL/Z9-14: AL neuron that responded equally to Zl1-16: AL and to Z9-14: AL. A A mechanosensory stimulus evoked only a small membrane depolarization. In contrast, Z11-16:AL at 1 ng evoked a stronger depolarization and burst of spikes (B), and virtually the same response was evoked by Z9-14:AL at 1 ng (C). The responses of other cells in this category are shown in Figure 9

these neurons, along with the other neuron types we have described, could together encode blend information as an 'across-fiber' pattern of activity from the MGC (see below).

dendritic branching in the largest MGC glomerulus, but dendritic branches were also found in other MGC glomeruli (Christensen et al. 1991). Similar principles of organization therefore seem to govern the processing of Z l l -16 :AL information in H. zea and H. virescens. In both species, the importance of the principal component of the pheromone is clearly reflected in the large proportion of output neurons in both species that respond to this odorant (Table 1). Furthermore, in both species, this information is preserved for higher processing in at least two output channels:one that is activated by very low ambient pheromone concentrations (type-2 PNs), and another that is only activated at higher concentrations (type-1 PNs).

A third output pathway is equally sensitive to Z11- 16:AL and Z9-14:AL information (Table 1). The responses to both pheromonal components in the Z l l - 16:AL / Z9-14 : AL neurons differ distinctly from those found in the Zl l-16:AL-type 2 neurons. The nearly equivalent responses to the 2 pheromonal components suggest that these neurons integrate a combined input from both Z11-16:AL and Z9-14:AL receptor neuron populations (Figs. 8 and 9). Thus the neurons in this pathway respond to both components of the blend, but an individual neuron cannot discriminate the blend from either component presented separately (Table 1). This does not, however, exclude the possibility that

Different neural pathways encode Z9-14:AL information in H. zea and H. virescens

In the case of H. zea, Z9-14: AL operates as an interspecific signal that interrupts the attraction of males (Klun et al. 1980a, b), whereas in H. virescens, it serves as an essential component of the species-specific pheromone blend (Vetter and Baker 1983). Our results with H. virescens now point to some interesting differences in the way information about this essential component is processed in the MGCs of these two closely-related species. In contrast to a significant number of PNs in H. zea that responded selectively to Z9-14:AL and Z9-16:AL (Christensen et al. 1991), none of the neurons encountered in H. virescens exhibited this response profile. In H. zea, there is physiological and behavioral evidence that this single pathway may perform a dual function: it may participate in behavioral attraction if stimulated by Z9-16:AL released by a conspecific female, but because it is more sensitive to Z9-14 : AL, the presence of an H. virescens female producing a large amount of this antagonist will evoke an elevated level of spike activity that signals an inappropriate blend ratio, resulting in the disruption of upwind flight (Christensen et al. 1991; Vickers et al. 1991). The apparent lack of these specialized Z9- 14:AL/Z9-16:AL neurons in H. virescens suggests


1 2 3 4

B L A N K 8

' L . . ~ , ~ o . . . . . . ~ , I , , � 9 4~'~,,'~ , ,...., , .

C E L L #

5 6 7 8 9 10 11 12 13 14

! t

I ng

Z 9 - 1 4 : A L

0.1 ng

I ng

BLEND (1:1)

0.1 ng

I ng

! t,_.,,, i_..

~ | _

(15:1) 8 4

0.1 ng o , , ~ I i I

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Fig. 9 Response spectra for all of the Z 11-16 : AL/Z9-14 : AL neurons that responded in a similar fashion to the 2 principal pheromone components used in H. virescens sexual communication. X-axis = 0 to 1000ms, stimulus marker = 300ms. Y-axis = number of spikes/50 ms bin. A blank space indicates that the stimulus was not tested

important differences in the functional organization of the MGCs in these two species. In H. virescens males, it appears that behavioral antagonism is mediated by an MGC pathway that is independent of the pathways that carry species-specific pheromone information, and this antagonist pathway furthermore arises in one particular MGC glomerulus. Several lines of evidence point in this direction. First, on the antennae of H. virescens males, one population of receptor neurons is selectively responsive to Z l l -16 :AC (Berg et al. 1995), a compound which has now been found to eliminate upwind flight and source location when added to an otherwise attractive blend of Z l l -16 :AL and Z9- 14:AL (Vickers N J, Mafra-Neto A, Baker TC, unpublished). Second, when these neurons are filled and traced into the brain, their axons project to a specific location in the MGC, glomerulus c (Hansson et al. 1995). Third, in addition to the AL neurons that responded to pheromone components, three neurons found in the present study responded exclusively to Z11-16:AC and probably received input from the re-

ceptor neurons tuned to Z11-16:AC (Berg et al. 1995). Z11-16:AC is produced by the hairpencil glands in H. virescens males (Teal and Tumlinson 1989) and also by the female pheromone glands of other sympatric heliothine moth species, as a component of their species- specific pheromone blends (Arn et al. 1992). Thus the Z l l -16 :AC pathway in H. virescens males could be- come activated if the male were approaching a conspecific female being courted by another male, or if he

[3 Z9-14-.AL, I n g

Fig. 10A-C Responses of a Z l l -16 :AL+Z9-14 :AL-synerg i s t neuron. The neuron was depolarized and fired spikes in response to Z l l -16 :AL presented alone (A), responded only with a depolarization to Z9-14:AL alone (B), but showed a much stronger and prolonged response to the mixture of Z11-16 : AL and Z9-14: AL. In C, the depolarization and spiking lasted for more than 3 s beyond the end of the stimulus


A BLANK

B ZII-16:AL, 1 ng

C Z 9 - 1 4 : ~ , I ng

D Zg-16:AL, I ng

14:AL + 16:~.,, 1:1, 1 N

F Z9-14 : l~ rmat~ , 1 ng

Fig. l l A - G Recordings from a Z l l -16 :AC neuron illustrating its extreme olfactory selectivity. Of the 6 odors and odor combinations tested, only Z11-16: AC evoked a phase-locked excitatory burst of spikes

A ZII-16:AL, 0 . I ng

E ~ , Z I I - 1 6 : A C , 0.1 ng 120 mV

Fig. 12A F Responses of two Z11-16:AC neurons illustrating the range of sensitivities to this odorant. A D No responses were registered to Z l l -16 :AL or to Z9-14:AL at 0.1 rig. A dose-depen- dent response was seen, however, with Zl l -16 :AC. In the neuron shown in E-F , a mechanosensory stimulus evoked a small membrane depolarization (not shown), but Z11-16:AC evoked a much stronger membrane depolarization accompanied by spikes. The spike amplitude gradually decreased as the cell depolarized (possibly as a result of sodium inactivation), and then increased again as the cell repolarized. As shown in F, this response was also very reproducible

encountered a female of a sympatric species that released Z11-16 : AC. The Zl1-16 : AC - selective interneurons in the AL (Figs. 11 and 12) may therefore function as part of a labeled line that relays an arrestment signal to higher-order centers that control flight. Future studies will report on the morphologies and axonal projections of these important neurons, as fur- ther evidence for the existence of a separate labeled line for chemical signals that interrupt attraction.

Another important output pathway from the MGC, one with the most complex integrative properties yet encountered in this neuropil, is represented by the Z l l - 1 6 : A L + Z9-14:AL synergist neuron. Only one such neuron was encountered in H. virescens, but its physiological characteristics closely resemble those of 3 other neurons of this type found in H. zea (Christen- sen et al. 1989, 1991). A blend of these two components evoked a much stronger and prolonged excitatory response than either component presented separately (Fig. 10). This type of neuron appears to receive input from both Zl l -16: AL and Z9-14: AL receptor neuron pathways, but through unknown integrative mechanisms, converts "component" information to "blend" information. We need to study more of these neurons in both species to see if maximal activity is evoked by the conspecific blend, or instead by another blend or ratio that is more like that released by sympatric females.

A working model of MGC functional organization

On the basis of our physiological evidence, we conclude that the diversity in the different types of MGC-PNs observed in H. zea, and now H. virescens, ensures that the olfactory systems in these insects are well equipped to respond to the many possible variations in the physical and chemical features of a pheromone plume that a male moth is likely to encounter in nature. Based on the response characteristics we have observed, we would also predict that this functionally-diverse array of a relatively small number of parallel output pathways could relay different messages to the protocerebrum under different environmental conditions (see Fig. 13). At present, this is only a hypothetical working model, but we believe that the observed neuronal diversity that is evident in the MGCs of heliothines and other moth species suggests a wide range of possible stimulus conditions that can be encoded and dis- criminated in the brain. If, for example, a quiescent male is situated far downwind of an approaching pheromone plume, we expect that upon initial contact, the most sensitive Z 11-16 : AL type 2 output pathway would be the first to be activated. Since these neurons cannot encode or discriminate the complete blend on their own, and other pathways are still below activation threshold, we hypothesize that this pathway may be involved in generalized behavioral arousal (Fig. 13). Arousal could lead to random flight activity, which


Fig. 13A, B Summary diagram of the different types of output pathways (depicted as arrows) found in the MGC of H. virescens males, and a hypothetical model that shows how the pattern of activity across this diverse array of outputs might change under different environmental conditions. The shading of each arrow (from white, through two shades of gray, to black) represents the approximate level of activity in each pathway. White arrows reflect inactivity, black arrows reflect maximal activity. A Our physiological results would predict a different pattern of activity across this array under conditions of threshold behavioral arousal and upwind anemotaxis in the plume. When a male is located at such a distance from the source that only the high-sensitivity, component-specialized neurons are activated, this pattern could lead to behavioral arousal (first column of arrows). This situation is contrasted with that during upwind anemotaxis (second column), which would result when pathways sensitive to other features of the pheromone blend are also recruited into the MGC output pattern. The pattern across the array of outputs is not static, but changes with the natural fluctuations in the chemical and physical aspects of the plume (see text). B Similarly, behavioral arrestment could result from excessive pheromonal stimulation of the array (third column), or under conditions where antagonists (such as Z11-16:AC) are present (fourth column). Thus changes in the relative contributions of the different functional pathways in the same output array could potentially lead to different behavioral outcomes

groups working independently with different species have demonstrated that male moths respond to single filaments of pheromone by making an upwind surge (Mafra-Neto and Card6 1994; Vickers and Baker 1994). In H. virescens, it has been proposed that successful upwind flight is composed of reiterations of these 'phasic' surges, followed by a long-lasting 'tonic' casting response to the loss of odor (Baker 1990; Vickers and Baker 1994). While we cannot be sure of their exact roles as yet, it is particularly intriguing that we have found both types of responses among MGC neurons in H. virescens and other species: phasic responses that can encode temporal information, and tonic responses that signal exposure to pheromone with a long-lasting burst of action potentials. In sum, through this small but diverse array of parallel output pathways, the MGCs of H. virescens, H. zea and other species, are well suited to monitor changes in the chemical and temporal features of the insects' environment - features that determine specific behaviors, both toward and away from the source.

Acknowledgements We wish to dedicate this paper to the memory of our good friend and colleague, Ed Arbas. We thank P. Randolph for assistance with histology and C.A. Hedgcock, R.B.P., for photo- graphic assistance. We also thank Drs. E. Arbas, T.C. Baker, S. Hannaford, B.S. Hansson and N.J. Vickers for many helpful dis- cussions, and B.S. Hansson and N.J. Vickers for helpful comments on the manuscript; Dr. P. Teal (USDA-ARS, Gainesville, Florida) for supplying H. virescens pupae; and Dr. J. Tumlinson (USDA- ARS, Gainesville, Florida) for supplying synthetic and purified pheromone components. This research was supported by USDA Competitive Research Grant #92-37302-7634, and NATO Grant for International Collaboration #0149/88.

increases the male's chances of contacting a region of the plume where the blend of components is above threshold, thus triggering upwind anemotaxis. Then, as each filament is encountered, a transient increase in concentration would evoke activity across the entire array of MGC pathways, that together can encode the species-specific blend. This pattern of activity would then be repeated each time a filament is encountered, thus sustaining the behavior in the plume.

As illustrated in Fig. 13, each type of MGC-output pathway is most efficient at monitoring a particular feature of the insect's olfactory environment. In addition to exhibiting chemical specificity, some MGC neurons can encode important temporal features of the stimulus, such as the arrival and duration of the pheromone filament. (Christensen et al. 1989; Figs. 1, 4, 11 in this study). These findings at the level of neural processing are particularly relevant in light of behavioral evidence that spatio-temporal information is important to sustained upwind flight and eventual location of the odor source. Using carefully-controlled plume delivery systems that mimic the intermittent structure of the plume (Vickers and Baker 1992), two

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