THE JOURNAL OF COMPARATIVE NEUROLOGY 295~248-267(1990)

Organization of the Auditory Pathway in the Thoracic Ganglia of Noctuid Moths GEORGE BOYAN, LEZ WILLIAMS, AND JAMES FULLARD Molecular Neurobiology Group, Research School of Biological Sciences, Australian National University, Canberra City ACT 2601, Australia

ABSTRACT We describe the neuroarchitecture of the noctuid thoracic nerve cord and use this framework to interpret the organization of the auditory pathway responsible for escape behaviour in noctuid moths. Noctuid moths possess only two auditory receptors (Al, A2), in each ear. The axon of the A1 cell projects initially to a glomerulus located ventrally and medially in the metathoracic ganglion, where it bifurcates. One branch ascends in the ventral intermediate tract to the brain, the other descends in the ventral intermediate tract into abdominal neuromeres of the metathoracic ganglion. Both axons arborize in the median ventral and ring tracts in each neuromere. The central projections of the A2 cell remain largely within the metathoracic ganglion. The axon bifurcates at the midline and directs arborizations dorsally to the dorsal intermediate and median dorsal tracts, and ventrally into the ring tract where the arborizations overlap those of the A1 afferent. The afferent projections remain ipsilateral to the ear of origin. We describe a posterior auditory association area in the metathoracic ganglion in which the major arborizations of several identified interneurones overlap those of the A1 afferent and make monosynaptic connections with it. These interneurones all respond tonically to sound stimuli. We have also identified the projections of the A1 afferent, interneurones, and motor neurones in the segmentally equivalent anterior auditory association area of the mesothoracic ganglion. An interneurone with major arborizations in the same tracts as the A1 afferent, and receiving monosynaptic input from it, is described. The arborizations of higher order interneurones lie mainly in dorsal tracts along with those of flight motor neurones. All the interneurones in this anterior centre respond phasically or phasic/tonically to sound stimuli. The relevance of this anatomical organization for predator avoidance behaviour is considered and the organization of auditory pathways in tympanate insects compared. Key words: insect nervous system, neuroanatomy, audition, avoidance behavior

The avoidance behaviour of noctuoid moths in response to the echolocating calls emitted by a predatory bat is one of the best-documented interactions in ethology (see Roeder, '65; Miller, '82 for reviews). The entire behaviour is mediated by only two receptor cells ( A l , A2) in each ear, and the physiological properties of these cells have been extensively researched (see Miller, '83; Fullard, '87). The afferents, in turn, activate interneurones within the central nervous system (CNS) (Paul, '74), a number of which have recently been identified (Boyan and Fullard, '86, '87, '88; Boyan et al., '88). At present our knowledge of the anatomical organization of central pathways in the thoracic ganglia of noctuoid moths is based largely on observations of stained cells in wholemount preparations (Paul, '73; Kondoh and Obara, '82; Surlykke and Miller, '82; Rind, '83; Boyan and Fullard, '86, '87, '88; Madsen and Miller, '87; Agee and Orona, '88). These data suggest areas in the pterothoracic ganglion o 1990 WILEY-LISS, INC.

where interactions between auditory afferents, interneurones, and flight motor neurones might occur. However, an understanding of how avoidance behaviour is organized at the cellular level requires that physiological interactions be demonstrated between identified cells whose anatomical relationships can be incorporated into the general neuroarchitectural framework of the CNS. We previously proposed a preliminary model of how avoidance behaviour might be organized based on connectivities between afferents and interneurones (Boyan and Fullard, '86, '87, '88). At that time we could not relate the anatomical projection patterns of identified cells to demonstrated physiological interactions because a description of the neuroarchitecture of the

Accepted December 7,1989. Address reprint requests to Dr. G.S. Boyan, Zoologisches Institut, Universitkit Basel, Rheinsprung 9,4051 Basel, Swiizerland.

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Abbreviations AAA Aff Ant Abdl AbdZ aRT ax CNS CT d D dB DC I-VI DIT DMT DT dVCLIl EPSP IN ind int n IT LDT LVT

anterior auditory association area afferent projections anterior abdominal neuromere 1 abdominal neuromere 2 anterior part of Ring tract axon central nervous system C tract direct dorsal decibel dorsal commissures I-VI dorsal intermediate tract dorsal median tract dorsal tract dorsal part of ventral commissure loop I1 excitatory postsynaptic potential interneurone indirect intermediate neuropile I tract lateral dorsal tract lateral ventral tract

ganglionic core was lacking (c.f. locust, Tyrer and Gregory "821 or cricket, Wohlers and Huber "851). In this article we provide a description of the structure of the ganglionic core and use this as a frame of reference to describe the anatomy of auditory afferents, interneurones, and flight motor neurones in four genera of noctuid moths. We then examine the physiological inputs from these afferents to identified interneurones and motor neurones in the light of these new anatomical data. We propose that auditory information processing occurs in so-called auditory association centres that are distributed segmentally and where each association centre carries out particular transformations of the input signal. We consider the relevance of this neuroanatomical organization to avoidance behaviour in the moth, and on the basis of comparisons with hemimetabolous insects, discuss the evidence for a common organization or "Bauplan" for insect auditory pathways. A preliminary account of our findings has been published in the form of an abstract (Boyan et al., '88).

MATERIALS AND METHODS Animals Four species of noctuid moths were used in the experiments. 1. Heliothis uirescens: adult male and female moths were reared from pupae obtained from the University of Guelph and were 1 to 4 days postemergence at the time of experimentation in the Department of Physiology, University of Alberta, Edmonton, Canada. 2. Catocala ceragama: moths were caught as adults in the wild with an ultraviolet collecting light and used within 2 days a t Queen's University Biology Station, Chaffey's Lock, Ontario, Canada. 3. Agrotis infusa: moths were caught as adults in the wild around Canberra with an ultraviolet collecting light and used within 3 days at the Research School of Biological Sciences, Australian National University. 4. Acronicta americana: adults were caught as adults a t Queen's University Biology

MDT meso meta MN mtr MVT PAA PVC RT S

SMC

SOG SPL TR TT V VAC VCI VIT VLT VMT VNC YVCLII

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median dorsal tract mesothoracic ganglion metathoracic ganglion motor neurone midline trachea median ventral tract posterior auditory association area posterior ventral commissure Ring tract section supramedian comrnissure suboesophageal ganglion sound pressure level trachea T tract ventral ventral association centre ventral commissure I ventral intermediate tract ventral lateral tract ventral median tract ventral nerve cord ventral part of ventral commissure loop I1 white noise sound stimulus

Station and used for cobalt backfilling only. Subsequent anatomical analyses were performed in Canberra.

Neuroachitecture of the ganglionic core The neuroarchitecture of the meso- and metathoracic ganglia of the moth is described according to the nomenclature provided in the detailed guides for the same ganglia in the locust (Tyrer and Gregory, '82), cockroach (Gregory, '74), and cricket (Wohlers and Huber, '85). Tracts in the moth were named according to their positions relative to one another, to nerve roots, tracheae, and with respect to the ganglion as a whole; commissures were named according to their order of appearance, from anterior to posterior, in the ganglion. The resulting plan largely corresponds to the description of identified structures in these other insect, species. We have been conservative in interpreting and naming the structures observed. For example, where two or more fibre bundles were consistently found in close proximity to one another and well separated from other groups of bundles, we considered them part of the one tract, but still drew the outline of each bundle within the tract in our summaries of transverse sections. The description of the external features of the ganglion such as nerve roots and tracheae follows the nomenclature of Nuesch ('57).

Histology The neuroarchitecture of the ganglionic core was established on the basis of ganglia stained according to Wigglesworth's osmium tetroxide-ethyl gallate method as described in Williams ('75). Ganglia were then embedded in araldite and transverse sections (25 fim thickness) cut. Cobalt backfills of the tympana1 nerve (ITINlb) were performed by using cobaltic lysine for 12-15 hours a t 8°C. Ganglia were fixed in Bouin's medium, silver intensified (Bacon and Altman, '77), embedded in araldite, and sectioned at 25 pm. Ganglia containing neurones that had been

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250 intracellularly stained with Lucifer Yellow were dehydrated, cleared, and the stained cells photographed in wholemount with an epifluorescence microscope. The ganglia were subsequently embedded in araldite and sectioned at 50 wm for reconstruction of stained cells. The nomenclature for describing the interneurones here is the same as that used by us in previous studies (Boyan and Fullard, '86, '87, '88; Boyan et al., '88).

Dissection,stimulation, recording, and staining Moths were prepared for dissection and presented with auditory stimuli with the aid of the methods, apparatus, and calibration procedures described previously (Boyan and Fullard, '88). Intracellular recording and staining of interneurones, along with extracellular recording and stimulation of afferents in IIINlb, proceeded as previously described in Boyan and Fullard ('88).

RESULTS Neuroarchitecture of the pterothoracic ganglion The basic framework of the ganglion is provided by the bundles of nerve fibres that run anterior/posteriorly (tracts), or transversely (commissures) linking areas of more diffuse fibres (neuropile). In addition there are the peripheral nerve roots, and cell body fibres (neurites) from populations of neurone cell bodies. Nine longitudinal tracts run through each half of the ganglion core and are named according to their position. The dorsal tracts are: the median dorsal tract (MDT), the lateral dorsal tract (LDT), the dorsal median tract (DMT), the dorsal intermediate tract (DIT). The ventral tracts are: the ventral lateral tract (VLT), the ventral intermediate tract (VIT), the lateral ventral tract (LVT), the median ventral tract (MVT), the ventral median tract (VMT). We have been able to identify six dorsal commissures, numbered DCI-VI from anterior to posterior; and four ventral commissures: ventral commissure I (VCI), the dorsal and ventral parts of ventral commissural loop I1 (dVCLII, vVCLII), the supramedian commissure (SMC), and the posterior ventral commissure (PVC). We have identified the following vertical and oblique tracts: the T tract (TT), ring tract (RT), C tract (CT), and I tract (IT). The roots of peripheral nerves generally run obliquely in the ganglion, but apart from IIINl of the metathoracic ganglion, which contains the auditory afferents, we have not concentrated on their course through the ganglia. In noctuid moths the meso- and metathoracic ganglia are fused to form a pterothoracic ganglion, which also includes two fused abdominal neuromeres (Singh and Srivastava, '73; Tsujimura, '83; see below). We found no obvious differences between the pterothoracic ganglia of the four species of moths used in this study, either in gross external appearance, internal neuroarchitecture, or the morphology and physiology of identified afferents and interneurones.

Mesothoracic ganglion The mesothoracic ganglion is considered first because it has remained unisegmental and so best illustrates the basic organization from which the fused ganglia arose.

The mesothoracic ganglion has six paired peripheral nerves (IIN1-6) and a dorsal median nerve. The neuroarchitecture of the mesothoracic ganglion is summarized in the nine transverse sections shown in Figure la-i, each section taken at the plane indicated in the schematic. Many of the features referred to are visible in the photomicrographs shown in Figure 4. Anteriorly, the nine longitudinal fibre tracts are visible symmetrically arranged on each side of the ganglion (Fig. la). The LDT is divisible into several fibre bundles for most of its course through the ganglion, and these bundles vary in their proximity to the fibre bundle we have labelled the MDT. This division is somewhat arbitrary, and all the bundles may be best considered part of a general dorsal tract. The DIT is also divisible into two bundles, separated by a third smaller bundle, which could be traced to the periphery and contains sensory afferents; the MVT consists of two bundles; and the VIT also appears to consist of two bundles more posteriorly in the ganglion. The first transverse commissures to appear are DCI, running between, then below the DMT tracts on each side (Fig. l b ) , DCII running dorsal to DIT and between DIT and LDT (Fig. lc), and DCIII running ventral to DIT at the level of VLT (Fig. lb,c,d). At the extreme ventral border with the cortex is the VCI (Fig. l b ) followed by the vVCLII (Fig. Id), which forms part of a vertical ring in the ganglion and whose dorsal equivalent (dVCLII, Fig. lc) forms the anterior border of a prominent tract known as the ring tract (RT, Fig. ld,e). The R T is fused across the midline anteriorly as the dVCLII and then forms two cylindrical halves, one on each side of the midline between each VIT as it courses posteriorly (Fig. ld,e) and is again fused across the midline posteriorly as the SMC (Fig. If). Anterior in the ganglion lies a very prominent region of fine fibrous neuropile called the ventral association area (VAC). It lies mostly posterior to VCI, ventral to VMT, and dorsal to the vVCLII (Fig. la,b,c). The VAC is fused across the midline anteriorly in the ganglion (Fig. l a ) , and then as it courses posteriorly splits into two cylindrical halves, one on each side of the midline (Fig. la,b). The most anterior vertical tract is the T tract (TT, Fig. lc,d), which runs up the midline between the DMT on each side, posterior to DCI and anterior to DCIII. Ventral to DCII the TT turns laterally passing between LDT and DIT on each side and so giving the tract its T-shape. The I tract (IT, Fig. Id) runs vertically lateral to each VLT. Further posterior is the C tract (CT, Fig. le,f), which forms a crescent-shape vertical tract beginning dorsally and running between DIT and VIT. I t then runs medial to each VIT and, proceeding ventrally, curves between VMT and MVT on each side (Fig. If). Three commissures appear in the sections occupied by the CT in the ganglion. DCIV crosses the ganglion dorsal to DMT and passes laterally between DT (MDT,LDT) and DIT on each side (Fig. If). In the transverse sections shown, DCV appears to cross the ganglion anterior to DCIV (Fig. le,f). This is because a lobe of DCV extends anteriorly over DCIV and is connected with the remainder of DCV by a bridge, which extends posteriorly just dorsal to DCIV. This arrangement is confirmed both in transverse sections taken a t the appropriate levels in the metathoracic ganglion (Fig. 2f,g,h), and in sagittal sections (see Fig. 5). As seen in transverse section, DCV follows the same path in the ganglion as DCIV, passing dorsal to DMT and laterally between DT and DIT on each side (Fig. le,f,g). The third commissure to appear is the SMC, which is found

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Fig. 1. Mesothoracic ganglion of Agrotis infusa, drawings of transverse sections showing chief anatomical features. Sections were taken a t the levels (a-i) indicated on the ganglion shown schematically, and in ventral view, at top left. Longitudinal fibre tracts are abbreviated here as

follow^: 1 = MDT, 2 = LDT, 3 = DMT, 4 = DIT, 5 = VLT, 6 = VIT, 7 = LVT, 8 = MVT, 9 = VMT. See abbreviations list for other abbreviations used. Scale bar: 100 ym.

ventral to DMT and between each VLT (Fig. If). The SMC is the posterior part of the ring tract described earlier (dVCLII in Fig. lc; RT in Fig. ld,e). Posteriorly in the ganglion two commissures become visible: dorsally DCVI forms a coherent bundle crossing just below DIT and DMT and curving dorsally just lateral to each DIT (Fig. lg,h); ventrally, PVC crosses the ganglion below the VMT and MVT, and above the LVT (Fig. lh). Further posterior a prominent midline trachea marks the boundary between the mesothoracic and metathoracic neu-

romeres (Fig. li). Fibres can be seen entering the ganglion from peripheral nerve root IIN6 (dorsally) and IIN5 (ventrally) (Fig. li).

The metathoracic ganglion Transverse sections of the metathoracic ganglion a t the levels indicated in the schematic are shown in Figures 2 and 3. The ganglion consists of a metathoracic neuromere (Fig. 2) fused with the neuromeres of the first two abdominal segments (Fig. 3) a t the levels indicated (Fig. 3j,p). Several

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Fig. 2. Metathoracic ganglion of Agrotis i n f ~ s drawings ~, of transverse sections showing chief anatomical features of the metathoracic neuromere. Sections were taken a t the levels (a-i) indicated on the ganglion shown schematically, and in ventral view, at top left. Longitudi-

nal fibre tracts are abbreviated here as follows: 1= MDT, 2 = LDT, 3 = DMT, 4 = DIT, 5 = VLT, 6 = VIT, 7 = L\T, 8 = MVT, 9 = VMT. Stippled area indicates the projection from peripheral nerve root IIIN1. Scale bar: 100 urn.

of the features referred to are visible in the photomicrographs shown in Figure 4,and the description below should be read in conjunction with the plan of the ganglion as seen sagittally in Figure 5. The pattern of nerve roots entering the metathoracic ganglion is much simpler than for the mesothoracic ganglion, with only two peripheral nerve roots (IIIN1 anteriorly and IIINP posteriorly), and a dorsal median nerve present.

The basic neuroarchitecture of the meso- and metathoracic neuromeres is similar with the exception of the major projection anteriorly into the metathoracic ganglion of fibres via IIIN1, including those from the tympanum (via branch IIINlb) (Fig. 2a,b). The projection of fibres from IIINl enters the ganglion dorsally and symmetrically on each side, and has an anterior and a posterior portion. The anterior portion courses obliquely below DIT towards the

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Fig. 3. Metathoracic ganglion of Agrotis injusa,drawings of transverse sections showing chief anatomical features of the fused first and second abdominal neuromeres, respectively. Sections were taken at the levels Q-s) indicated on the ganglion shown schematically, and in ventral view, a t the top left of Figure 2. The sequence of sections

midline, and at the level of VLT this projection bifurcates, with one bundle turning dorsally to terminate a t the VIT, whereas the other bundle continues on its oblique course and ends anteriorly at the VAC (Fig. 2a,b). The posterior portion of the IIINl projection enters the metathoracic ganglion more dorsally than does the anterior portion, and fibres from this posterior portion terminate in and around DIT and DT (MDT,LDT) (Fig. 2b,c). The sensory fibres in

continues directly from those shown for the metathoracic neuromere. Longitudinal fibre tracts are abbreviated as follows: 1 = MDT, 2 = LDT, 3 = DMT, 4 = DIT, 5 = VLT, 6 = VIT, 7 = LVT. 8 = MVT, 9 = VMT. Scale bar: 100 pm.

DIT are found grouped between the two bundles drawn in the transverse sections. The appearance of commissures and vertical and oblique tracts then follows the same order as in the mesothoracic ganglion. Dorsally, DCI, DCII, and DCIII (Fig. 2a-d) appear in the same order as in the mesothoracic ganglion (Fig. lb-d). Posterior to DCI, between it and DCIII, lies the vertical T tract (TT, Fig. 2b,c), which again bifurcates

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Fig. 4. Photomicrographs showing transverse sections of the pterothoracic ganglion of Agrotis infusa stained according to the Wigglesworth’s osmium tetroxide-ethyl gallate method. Sections were taken at the levels (a-c) indicated on the ganglion shown schematically, and in ventral view, a t top left. Scale bar: 100 pm.

dorsally to run laterally under each LDT (Fig. 2d,e). DCIII, in fact, splits into anterior (slightly more ventral) and posterior (slightly dorsal) fibre bundles (Fig. 2c,d,e). The anterior bundle is found a t the same level as the first ventral tracts to appear (VCI, dVCLII, Fig. 2c), and the posterior bundle at the level of the vVCLII (Fig. 2e). The fibres of dVCLII lie dorsal to VMT and form the fused anterior section of a region of intermediate neuropile also known as the ring tract (Fig. 2c,d,e). The fibre bundle of dVCLII then turns posteriorly and bifurcates into two cylindral halves labelled RT, one on each side of the ganglionic midline medial to VIT (Fig. 2f). Lateral to RT on each side and looping around the VIT is the dorsal part of CT (Fig. 2f), and lateral to VLT is the I T (Fig. 2f). Dorsally, the anterior lobe of DCV crosses the ganglion above DMT and then turns laterally between DIT and LDT (Fig. 2f). Farther posterior, DCIV appears dorsally and crosses the ganglion in the same way as DCV (Fig. 2g), whereas ventrally the cylindrical halves of R T fuse across the midline as the SMC (Fig. 2g). As in the mesothoracic neuromere, DCV has a bridge that passes dorsal to DCIV and links its anterior and posterior lobes; the posterior lobe then extends back to the level of DCVI and the PVC (Figs. 2h,i, 5 ) . At the level of the PVC the midline trachea marks the division between the metathoracic and first abdominal neuromeres (Fig. 2i).

Abdominal neuromeres of the metathoracic ganglion The neuroarchitectural features that characterize the metathoracic neuromere above are essentially preserved in both of the fused abdominal neuromeres (Abdl: Fig. 3j-0; Abd2: Fig. 3p-s). Fusion has been accompanied by a compression of commissures in the anterior-posterior axis but no change in the relative position, or order of appearance, of tracts and commissures (see Fig. 5 ) .The major differences in neuroarchitectural organization are the absence in the abdominal segments of a prominent projection equivalent to IIIN1, the presence of the ventral nerve roots IIIN2, and the compression of tracts into a single fused connective exiting the ganglion posteriorly. Otherwise the abdominal neuromeres largely reflect the anatomical organization of the mesothoracic ganglion and need not be described in detail again. The general appearance of the ganglionic core and some of the neuroarchitectural features described above can be seen in the photomicrographs of transverse sections in Figure 4. A sagittal view of the posterior part of the mesothoracic and the fused metathoracic and abdominal neuromeres is shown in Figure 5 . The distribution of transverse commissures described above can be see with reference to a dorsal (DMT) and ventral (VMT) longitudinal fibre tract located in the

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Fig. 5. Drawing showing the chief anatomical features of the posterior part of the rnesothoracic, and the complete mukisegmented rnetathoracic ganglion of Agrotis infusa, as seen in sagittal view after reconstruc-

tion from sections. Two midline longitudinal fibre tracts (DMT, VMT) are shown for reference (stippled area). Dashed lines indicate the approximate boundaries of the neuromeres. Scale bar: 100 pm.

ganglionic midline. Note that the order and position of tracts and commissures remains despite the compression that has accompanied fusion of the abdominal neuromeres.

comprising the ring tract (aRT, RT, SMC, Fig. 6A,e,f). All terminals are characterized by large numbers of varicosities or “blebs.” The segment of the axon that turns anteriorly projects into the lower glomerular lobe (Fig. 6A,c) and does not arborize in the metathoracic ganglion. Instead it runs farther anterior in VIT to the mesothoracic ganglion where it branches extensively in the VIT, sending branches into dVCLII, intermediate neuropile (aRT), R T and SMC (Fig. 6A,b), which are almost identical in form to those seen in the metathoracic neuromere (c.f. Fig. 6A,e). Again, all these branches bear large numbers of varicosities or “blebs.” Farther anterior in the mesothoracic ganglion, two axon profiles (the A1 and B fibres) can be seen closely apposed in VIT (Fig. 6A,a) and they leave the mesothoracic ganglion medioventrally in the ipsilateral connective en route to the brain. There are therefore segmentally repeating arborizations from the axons of the A1 and B cells in VIT into dVCLII, aRT, RT, and SMC (Fig. 6A,b,e), and the form of these arborizations is also very consistent between preparations (c.f. Fig. 6B,b). The axon of the A2 cell enters the metathoracic ganglion in the same afferent tract as the A1 and B afferents but does not project to the glomerulus in the ventral midline of the ganglion (Fig. 7). Instead, the axon leaves the afferent tract while still dorsal and turns medially, almost a t right angles (Fig. 7,a). Just before reaching the ganglionic midline the axon bifurcates and directs branches dorsally into DIT and MDT, and ventrally into dVCLII, intermediate neuropile (aRT), and the ring tract (RT, SMC) (Fig. 7a). where they overlap the projections of the A7 and B afferents (c.f. Fig. 6A,e). These ventral projections do not extend anteriorly

Central projections of tympana1 afferents Three profiles with a morphology consistent with their being the A l , A2, and B afferents, as described previously from wholemount preparations (Surlykke and Miller, ’82; Agee and Orona ’88) were reconstructed from transverse sections following cobalt backfills of IIINlb (Figs. 6,7). The central projections of the auditory A1 afferent and nonauditory B afferent were so similar as to be indistinguishable in our sections, and the description below therefore applies to both cells. All three afferents enter the CNS via nerve root IIINl anteriorly and dorsally in the metathoracic ganglion. The axons of the A1 and B cells run obliquely and ventrally, following, without branching, the afferent tract towards the ganglionic midline (Fig. 6A,d). As described above (Fig. 2a), the afferent tract bifurcates near its termination into a glomerulus with upper and lower lobes; the two lobes fuse with the respective bundles forming the VIT farther anterior (Fig. lg-i) and posterior (Fig. 2d) in the pterothoracic ganglion. The axons of the A1 and B afferents also bifurcate just prior to reaching the glomerulus, forming a T-shape arborization with anteriorly and posteriorly directed axons (see Figs. 6A inset, 10A). The posteriorly directed axon of each cell projects into the upper glomerular lobe and arborizes extensively throughout the glomerulus and into dVCLII, a R T and MVT (Fig. GA,d,e). I t then continues posteriorly in VIT to the first abdominal neuromere sending branches medially into the parts of intermediate neuropile

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Fig. 6. Morphology of the A1 and B tympanal d e r e n t s as seen in transverse sections of the pterothoracic ganglion of Acronicta americ a m following cobalt backfilling of peripheral nerve IIINlb. Sections shown are from two different preparations (A,B) and represent summaries of sections taken at the levels (a-f) indicated by the stippkd areas on the schematic, viewed ventrally, at top left. The schematic also shows

adrawing of the A1 afferent as seen in wholemount. The morphologies of the A1 and B afferents are too similar for their projections to be separated here. Longitudinal fibre tracts are abbreviated as follows: 1 = MDT, 2 = LDT, 3 = DMT, 4 = DIT, 5 = VLT, 6 = VIT, 7 = LVT, 8 = MVT, 9 = VMT. Scale bar: 100 p m .

into the mesothoracic ganglion, although the dorsal projections do so in some preparations. The dorsal projections also continue posteriorly within DIT and were stained as far as the first abdominal neuromere (Fig. 7b,c).

glion based on the segmentally repeated arborizations of the A1 afferent. The posterior auditory association area (PAA). A number of sound-sensitive interneurones with major arborizations located anteriorly in the metathoracic ganglion, which might therefore receive input directly from the A1 afferent, have previously been identified in wholemount (Boyan and Fullard, '86,'87). Two of these interneurones are

Anatomy of sound-sensitive interneurones We have evidence for a t least two association areas processing auditory information in the pterothoracic gan-

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A2 Meta hfJ

Fig. 7. Morphology of the A2 tympanal afferent as seen in transverse sections of the metathoracic ganglion of Acronicta arnericana following cobalt backfilling of peripheral nerve IIINlh. Sections shown represent summaries of sections taken at the levels (a-c) indicated by the stippled areas on the schematic, viewed ventrally, at top left. The schematic also

shows a drawing of the A2 afferent as seen in wholemount. Longitudinal fibre tracts are abbreviated here as follows: 1 = MIIT, 2 = LDT, 3 = DMT, 4 = DIT, 5 = VLT, 6 = VIT, 7 = LVT, 8 = MVT, 9 = VMT. Scale bar: 100 Fm.

reconstructed below from transverse sections following intracellular staining with Lucifer Yellow (Fig. 8 IN 501; Fig. 9 IN 504). Interneurone 501 has a soma located ventrolaterally in the metathoracic ganglion (Fig. 8e), and an axon that ascends in MVT (Fig. 8a) to the brain via the connective on the contralateral side. The commissural (crossing) segment of IN 501 is located in dVCLII (Fig. 8d), and links the axon in MVT with the major arborizations that project anteriorly (Fig. 8b,c) and posteriorly (Fig. 8e) in VIT on each side. There are also projections from the axon into the cylindical fibre bundle of the ring tract (Fig. 8d). The anteriorly directed projections in VIT on the ipsilateral side are “spiny” in character, whereas those on the contralateral (axon) side bear numerous varicosities or “blebs” (Fig. 8b). A characteristic feature of IN 501 is the prominent branch leaving the commissural segment in VIT on the ipsilateral side and projecting dorso-laterally into the afferent tract (Fig. 8c). The soma of IN 504 lies in the dorsoventral midline, extremely laterally and anteriorly in the metathoracic ganglion (Fig. 9c). The major arborizations of IN 504 are found in dVCLI1 and the various parts of the ring tract (aRT, RT, SMC). A branch that originates in the arborizations on the soma side crosses to the contralateral side in dVCLII, projects anteriorly in VIT, and exits the ganglion as the axon medially and ventrally in the contralateral connective en route to the brain (Fig. 9a,b,c). Branches originating in dVCLII on both sides enter VIT on the contralateral side and arborize (Fig. 9h,c). Prominent branches run from VIT dorsolaterally into the afferent tract and almost to the edge of the neuropile (Fig. 9b,c), parallelling the projection in IN 501 (Fig. 8c). Posterior to dVCLII the major arborizations

run symmetrically in each of the cylindrical fibre bundles of the ring tract (Fig. 9d) and join posteriorly to form a commissural (crossing) segment in SMC. IN 504 therefore has segments crossing in two commissures-anteriorly in dVCLII (Fig. 9c), and posteriorly in SMC (Fig. 9d). Symmetrical branches spread laterally from the posterior conimissural segment into VIT on each side, and project posteriorly in VIT to the abdominal neuromeres of the pterothoracic ganglion (Fig. 9d,e). These posterior branches bear extensive varicosities on their terminals. To provide a three-dimensional view of the relationship between the projections of the A1 afferent and IN 501 in the pterothoracic ganglion, neurone profiles were reconstructed from section as seen parasagittally and with respect to four longitudinal fibre tracts (Fig. 10). The arborizations of A 1 and IN 501 clearly overlap within VIT on the soma side, and both afferent and interneurone arborize in dVCLII, aRT, RT, and SMC in a segmentally repeating fashion. The arborizations of the A1 afferent and IN 501 remain almost completely ventral in the ganglion. T o test for connectivities, intracellular recordings were made from three identified sound-sensitive interneurones (INS501,503,504),as simultaneous extracellular recordings were made from the A1 afferent in the tympanal nerve (Fig. 11A). Spike triggered averaging shows that each spike in the A 1 afferent evoked an EPSP of large amplitude (about 2-3 mV), and short, constant latency (2 ms) in INS 501,503, and 504 (Fig. 11A). Simultaneous intracellular and extracellular recordings of the A 1 afferent (see also Fig. 1 in Boyan and Fullard, ’88)show that the arrival times of A1 spikes in the ganglion allow for a synaptic delay of only 0.5-0.8 ms to the appearance of EPSPs in these neurones, consistent with a monosynaptic connection with the A 1 afferent.

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Fig. 8. Morphology of I N 501 as seen in transverse sections of the pterothoracic ganglion of Agrotis infusa following intracellular staining with Lucifer Yellow. Sections shown represent summaries of sections taken a t the levels (a-e) indicated by the stippled areas on the ganglion outline at the top left of the figure. Ganglion outline shows a drawing of

IN 501 as seen in wholemount and viewed dorsally. Morphology of IN 501 was consistent in 5 preparations. Longitudinal fibre tracts are abbreviated as follows: 1 = MDT, 2 = LDT, 3 = DMT, 4 = DIT, 5 = VLT, 6 = VIT, 7 = LVT, 8 = MVT, 9 = VMT. Scale bars: 100 pm.

The B cell has a very similar gross morphology to that of the A1 and also projects in VIT (Fig. 6). However, the B cell does not respond with excitation to sound stimuli, and simultaneous recordings show that the B cell did not evoke any activity in INS 501,503, or 504 (Fig. 11). The A2 afferent has a different arborization pattern in the pterothoracic ganglion than the A l , although the ventral part of the projection is directed to the region of intermedi-

ate neuropile also occupied by A 1 (Figs. 6A,e, Fig. 7a). The A2 afferent has a higher auditory threshold than the A l , and its activity is often masked by that of the A1 in extracellular recordings. However, fast stimulus repetition rates presented over extended periods and at high sound levels produce adaptation in the A l , but not A2, response. This feature was used to selectively adapt the A 1 afferent so that any postsynaptic events correlated with activity in the A2

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Fig. 9. Morphology of IN 504 as seen in transverse sections of the pterothoracic ganglion of Agrotis injusa following intracellular staining with Lucifer Yellow. Sections shown represent summaries of sections taken a t the levels (a-e) indicated by the stippled areas on the ganglion outline a t the top left of the figure. Ganglion outline shows a drawing of

IN 504 as seen in wholemount and viewed dorsally. Morphology of IN 501 was consistent in four preparations. Longitudinal fibre tracts are abbreviated as follows: 1 = MDT, 2 = LDT, 3 = DMT, 4 = DIT, 5 = VLT, 6 = VIT, 7 = LV’I’, 8 = MVT, 9 = VMT. Scale bars: 100 pm.

fibre could be detected. Simultaneous recordings of interneurone 504 (intracellularly), and the Al,A2, and (nonauditory) R fibres (extracellularly) in the tympana1 nerve, show that in the case of IN 504, adaptation of activity in A1 was accompanied by an almost total loss of response in IN 504 despite the fact that the sound stimuli still elicited up to seven A:! spikes per burst (Fig. 11Bi,ii). No postsynaptic events in IN 504 could be unequivocally correlated with spikes in the A2 afferent.

The anterior auditory association area (AAA). Four sound-sensitive interneurones (INS 701,507,401,102) were intracellularly recorded and stained in a localized region of the mesothoracic ganglion, and their projections form part of an anterior auditory association area (Fig. 12).

Morphological properties The soma of IN 701 is located ventrally in the posterior midline of the mesothoracic ganglion (Fig. 12); the commis-

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Fig. 10. Morphology of the A1 afferent (A,i) and IN 501 (B,i) in the pterothoracic ganglion of Catocala ceragama (Al) and Agrotis infusa (IN 501) as seen in parasagittal view following reconstruction from sections. Several longitudinal fibre tracts (DT, DIT, VIT, MVT) are shown for reference (stippled). The axon of A1 enters the ganglion via IIINl and projects anteriorly and posteriorly in VIT on the ipsilateral side. Most of the branches drawn are in dVCLII and the ring tract (aRT,

RT, SMC) of each neuromere. The dendritic arborizations of IN 501 (B,i) are in VIT on the ipsilateral side to both the soma and to A 1 (A,i), and also project into the ring tract; the axon of IN 501 runs anteriorly in MVT on the contralateral side. Accompanying planar views show the A1 afferent (A,ii) and IN 501 (B,ii) as viewed dorsally in wholemount following intracellular staining with Lucifer Yellow, but in different preparations than the reconstructed cells. Scale bars: 100 pm.

sural segment crosses the ganglion ventrally at the level of the SMC; and the major dendritic processes are located mostly anterior to the commissural segment and on the ipsilateral side. These processes are ventral, mainly in VIT, but with branches into intermediate neuropile (aRT) and the ring tract. The commissural segment bifurcates into ascending and descending axons on the contralateral side. The ascending axon gives off a recurrent branch to the anterior arborizations in VIT on the ipsilateral side, and then exits the ganglion in the contralateral connective and runs probably in VIT to the brain. The descending axon (most likely also in VIT) directs segmentally repeated branches into the metathoracic and abdominal neuromeres and exits the pterothoracic ganglion posteriorly via the single abdominal connective. The arborizations of IN 701 therefore occupy the segmentally equivalent area of neuropile in the mesothoracic ganglion to those of INS 501 and 504 in the metathoracic ganglion (Figs. 8,9). The soma of IN 507 is located posteriorly, laterally, and ventrally of the midline in the mesothoracic ganglion (Figs.

12, 13A,c). Transverse sections show that the commissural segment crosses the ganglion in PVC and projects to LVT contralaterally, and to VLT on both sides (Fig. 13A,b,c). The major dendritic arborizations of IN 507 are directed anteriorly of the commissural segment and originate from the VLT on each side. The arborization on the ipsilateral side projects dorsally to DIT (Fig. 13A,a),whereas those on the contralateral side remain in VLT as they project anteriorly almost into the connective. The axon originates in LVT and projects to the brain in the connective on the contralatera1 side. A prominent branch runs posteriorly in DIT from the cell body fibre and the medial side branches project into VTT on the ipsilateral side. The most prominent anatomical feature of IN 401 is that all its major processes are restricted to the same hemiganglion as the soma (Fig. 12). The soma is located very anteriorly and ventrally in the mesothoracic ganglion. The major cell processes are located dorsally in the ganglion at the level of DCII. A prominent branch runs laterally from

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90dB SPL Fig. 11. A. Intracellular recordings of sound-sensitive interneurones (INS501, 503,504) from their neuropilar segments in the pterothoracic ganglion of Catocala ceragama (upper traces), and simultaneous extracellular recordings of afferents (A1,B) from their axons in the tympanal nerve on the soma side (lower traces). EPSPs were evoked in each interneurone by spikes in the A1 afferent (left panel), but there was no activity associated with spikes in the B afferent of the same preparations (right panel). Traces are averages of 128 sweeps each triggered by the afferent spike. Scale bars: vertical, (intracellular record only) 0.5 mV: horizontal, lms. B. IN 504 receives input from the A1 hut not A2 afferents. (i) Intracellular activity in IN 504 (upper trace) and extracel-

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lular activity of the A1 tympanal afferent (lower trace) recorded simultaneously in response to sound stimuli (not shown) directed at the ear on the soma side. Stimulus tones (10 ms, 16 kHz, 70 dB SPL) evoked bursts of spikes in the A1 afferent and accompanying depolarizations in IN 504. Stimuli at higher sound levels (90 dB SPL) were then presented for several seconds prior to the record shown in (3)and this lead to the partial adaptation of the response in the A1 afferent, but did not affect the response in A2. Depolarizations in IN 504 still accompanied occasional spikes in the A1 afferent, but no postsynaptic potentials could be unequivocally correlated with the many spikes in the A2 cell. Scale bars: vertical, (intracellular record only) 10 mV; horizontal, 25 ms.

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Fig. 12. Interneurones of the anterior auditory association area (AAA) in the mesothoracic ganglion of Catocala ceragama (INS 701, 401), Heliothis uirescens (IN 507) and Agrotis infusa (IN 102). Draw-

ings of interneurones are from wholemount preparations viewed dorsally, and follow intracellular staining with Lucifer Yellow on at least two occasions. Scale bars: 100 pm.

the midline to about halfway across the ganglion before turning and running back on itself almost to the midline again. This process then turns anteriorly a t right angles and forms the axon that exits the ganglion medially in the ipsilateral connective. Major arborizations are also found projecting posteriorly in the ganglion.

The most localized arborizations belong t o IN 102, which is found anteriorly and laterally in the mesothoracic ganglion a t about the anterior/posterior level of commissures

DCII and DCIII (Figs. 12, 13B). The soma lies ventrolaterally in the ganglion, and dendritic processes run dorsally from the major segment near LDT and spread laterally and

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Fig. 13. Morphology of IN 507 (A) and I N 102 (B) as seen in transverse sections of the mesothoraic ganglion following intracellular staining with Lucifer Yellow. Ganglion outlines accompanying each set of sections contain the respective cell viewed in wholemount, and also

indicate the levels a t which transverse sections were taken to produce the summary sections shown. Longitudinal fibre tracts are abbreviated here as follows: 1 = MDT, 2 = LDT, 3 = DMT, 4 = DTT, 5 = VLT, 6 = VIT, 7 = LVT, 8 = MVT, 9 = VMT. Scale bars: 100 fim.

medially to envelope the fibre bundles comprising LDT. Further processes run medially to surround the fibre bundles of DIT.

adapted strongly on maintained stimulation (Fig. 14Ciii). Spike-triggered averaging revealed that spikes in the A1 afferent on the ipsilateral side evoked large amplitude (1-2mV) EPSPs in IN 701 at a short and constant latency of 2.4 ms (Fig. 14D). High frequency (100 Hz) electrical stimulation of the tympana1 nerve showed a 1:l correspondence between stimulus and evoked EPSP, at a constant latency, and with no decrement (Fig. 14E). The latency of the response to 16kHz tones presented to the ear on the ipsilateral side was a minimum of 5.5-6.0 ms at 90dB (SPL) (c.f. 5.1-5.2 ms for INS such as 501, 503, 504 in the P M , Boyan and Fullard, '86). This slightly longer latency is probably because the major processes of IN 701 lie in the mesothoracic ganglion (Fig. 12) rather than the metathoracic ganglion where the afferents enter the CNS. The measured axon diameter of the A1 afferent (5 K r n when stained) is sufficient to support the conduction of action potentials a t the required velocity ( < l m/s) to evoke EPSPs in IN 701 in the time allowed. The data are consistent with the connection between the A1 afferent and IN 701 being direct, and mediated by synapses located in the dendritic arborization in VIT and the ring tract in the mesothoracic ganglion. Spike-triggered averaging showed that there was no input to IN 701 from the B cell (Fig. 14D). Higher order interneurones. The responses to sound of the other interneurones identified in the AAA could be

Physiological properties The four interneurones described above fall into two physiological classes according to whether they receive their input directly (IN 701), or indirectly (INS 507, 401, 102) from the A1 afferent. However, they share one prominent feature: they all respond phasically to auditory stimuli. First-order interneurone. In the most sensitive preparation, IN 701 (Fig. 12) had an auditory threshold of about 40dB SPL at 16kHz (Fig. 14A), which matched that of the A1 afferent. The response to tones of all intensities consisted of an initial rapid depolarization that supported one, at most two, action potentials, and then declined rapidly. A second depolarization, which remained subthreshold to all stimuli, commenced 5-6ms after the initial action potential, and this second depolarization expanded into a plateau potential with longer stimulus durations (Fig. 14B). The initial depolarization and plateau potential were both generated by the summation of individual EPSPs evoked 1:l by the A1 afferent (Fig. 14Ci,ii). A ripple effect consisting of individual EPSPs on a summated response was often evident during the plateau phase (Fig. 14B,Ciii). However, despite little subsequent change in the instantaneous spike rate of the A1 afferent, the initial response of IN 701

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Fig. 14. Simultaneous recordings of IN 701 (intracellularly, upper traces), and the tympanal nerve (extracellularly, lower traces), in response to auditory (A-D) and electrical (E)stimulation of tympanal afferents in Catocala ceragama. A. Stimulus tones (16 kHz, black bars) delivered to the ipsilateral ear evoked spiking activity in afferents, and a depolarization in IN 701, which remained highly phasic at all sound levels. Stimulus bars indicate onset and duration of tones. B. Longer duration (50 ms) tones at high intensity evoked a tonic response in the afferents, but still only the initial phasic depolarization in IN 701, followed by a subthreshold plateau depolarization that was maintained for the duration of the stimulus. C. The A1 afferent was made to spike over a wide range of instantaneous frequencies by presenting white noise stimuli (WN) of varying (uncalibrated) intensities (i,ii,iii). A t low instantaneous frequencies (i,ii) spikes in the A1 afferent could be seen to evoke EPSPs 1:l in IN 701. Stimuli that evoked higher, maintained, instantaneous spike frequencies in the A1 afferent (iii) still only evoked an initial phasic depolarization in IN 701, followed by a gradually declining subthreshold plateau depolarization (see also B above). D. Spike-triggeredaveraging shows that the A1 afferent (upper panel), but not the B afferent (lower panel), evoked EPSPs of short latency in IN 701. Traces are averages of 128 sweeps. E. High frequency (100 Hz) electrical stimulation of the tympanal nerve on the soma side elicited EPSPs 1:1, and at a short and constant latency. Sweeps are shown superimposed and transient deflections are stimulus artefacts. Scale bars: vertical, (intracellular records only) A (10 mV); B,C (8 mV); D,E (1.4 mV); horizontal, A,B,C (50 ms); D (2.8 ms); E (3.6 msl.

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characterized as phasic (IN 102, Fig. 15Bi),phasic/tonic (IN 507, Fig. 15Ai1, or dependent on activity elsewhere in the CNS (IN 401, Fig. 16). In further recordings of these neurones (not shown), their auditory thresholds were high (70 dB SPL), the responses labile and subject to decrement (INS507,401), the response strength independent of stimulus intensity (IN l02), and the response latencies long (IN 507: 9.6ms, Fig. 15Aii; IN 102: 21.4ms, Fig. 15Bii), all of which are consistent with an indirect input from the A1 afferent. The responses of IN 401 to sound were distinguishable from those of other interneurones because they varied in strength depending on the level of the membrane potential a t the time of stimulation. In a quiescent preparation, interneurone 401 responded to high intensity (90 dB SPL) tones with a depolarization that supported a slowly adapting spiking discharge (Fig. 16A). Spike-triggered averaging shows that this response was not directly evoked by the A1 (or the B) afferent (Fig. 16Bi,ii). The membrane potential of IN 401 oscillated spontaneously or could be brought into oscillation by high frequency (200Hz) electrical stimulation of auditory afferents in peripheral nerve IIINlb (Fig. 16C). Such oscillations were not observed in the other neurones described in this study. The rhythmical depolarizations had a mean cycle time of 12Hz (Fig. 16D) equal to approximately half the wingbeat frequency of the intact insect (unpublished observations). Sound presented when the membrane potential was at rest evoked a strong response (Fig. 16A), whereas the same stimulus presented to the preparation while the membrane potential oscillated evoked a much smaller, or no, response (Fig. 16D). By using the very regular spiking of the B afferent (whose rate was unaffected by sound) as a marker, a sound stimulus could also be shown to modulate the period of oscillations in I N 401 (Fig. 16D). Motor neurones. The output stage of the neural circuitry described above is represented by the motor neurones that innervate the flight muscles. Two depressor flight motor neurones innervating the forewing muscles-a basalar (Fig. 17A) and a dorsal longitudinal (Fig. 17B) motor neurone-were intracellularly recorded and stained in the

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Fig. 15. Intracellular recordings of IN 507 in a preparation of Heliothis uirescens (A), and IN 102 in a preparation of Agrotis infusa (B),in response t o tones (stimulus bars) delivered to the ipsilateral ear. Stimulus bars represent 16 kHz, 80 dB SPL in ‘A’, 16 kHz, 90 dB SPL in ‘B’, and indicate onset and duration of tones. Responses to several successive stimuli are shown superimposed in B(i), and lower panels (Aii,Bii) show evoked EPSPs averaged over 64 and 32 stimulus repetitions respectively. Scale bars: vertical, (intracellular records only) Ai,Bi (3 mV); Aii (3.3 mV): Bii (0.8mV); horizontal, Ai,Bi 17 ms): Aii (3.3 ms), Bii (8.3ms).

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16kHz 90dB Fig. 16. A. lntracellular recording of IN 401 (upper trace), and extracellular recording of activity in the tympanal nerve (lower trace), in response to a sound stimulus of 16 kHz, 90 dB SPL (black bar) presented to the ipsilateral ear in a quiescent preparation of Catocala ceragama. Response in the tympanal nerve consisted mainly of spikes in the A1 and A2 afferents; spikes in the B afferent occurred spontaneously before (arrow) and after (labelled B) the sound stimulus. B. Spike-triggered averaging reveals that neither the A1 afferent (B,i) nor the B afferent (B,ii) evoked activity in IN 401; 64 sweeps are shown averaged. C. High frequency (200 Hz) electrical stimulation of the tympanal nerve on one side of a quiescent preparation evoked rhythmical depolarizations in IN 401. Transient deflections are stimulus artefacts. D. Portion of an on going sequence of rhythmical activity in IN 401. Oscillations normally occurred at a mean rate of 12 Hz (approximately half the wingbeat frequency of the intact preparation). Spontaneous firing rate of the B afferent was very regular (arrows) and unaffected by sound stimulation in this preparation. Effect of sound on rhythmical depolarizations IN 401 was demonstrated by measuring the interval from each B spike to the first action potential of the following rhythmical burst in IN 401 (distance from dashed line to solid line). This interval remained very constant except when a sound stimulus (black bar) was presented and shortened (advanced) the interval to the next burst. Scale bars: vertical, (intracellular records only) A (3 mV); B (0.7 mV); C,D ( 5 mV); horizontal, A (17 ms); B (1.4 ms); C,D(23ms).

anterior part of the mesothoracic ganglion. Both flight motor neurones arborize extensively in dorsal regions of the ganglion, around MDT, LDT, and DIT. This pattern is repeated by most motor neurones, for example, those that enter the ganglion via IIINl in the next posterior neuromere and whose arborizations also envelop MDT, LDT, and DIT (Fig. 17C). Both basalar and dorsal longitudinal motor neurones were inhibited by 16kHz, 90dB (SPL) tones presented to the ear contralateral to the soma in quiescent preparations (Fig. 17A,B). Spike-triggered averaging shows no direct input to these motor neurones from either the A1 or B afferents (Fig. 17A,B).

DISCUSSION Neuroarchitecture of the ganglionic core The neuroanatomical system used to analyze the gross structure of the ganglionic core in the locust (Tyrer and Gregory, '82; Pfluger et al., '88) and cricket (Wohlers and

Huber, '85) has also proven applicable to the pterothoracic ganglion of the moth (Figs. 1-5). We were able to recognize the same nine longitudinal fibre bundles (tracts), six transverse commissures, and various association areas in the moth as in these orthopteran insects. The similarity in the projection of fibres entering the metathoracic ganglion via IIINl in the moth (Fig. 2a-c) and locust (Pfluger et al., '88), for example, is striking. It was more difficult, however, to generalize about the grouping of fibre bundles into tracts. Although our groupings largely conform to those reported for the locust (Pfluger et al., '88), tracts such as DIT and LDT are clearly not unitary in the moth (Figs. 1-3), and comparisons between species are complicated by the fact that the number of fibre bundles constituting a given tract can vary as fibres join or leave the tract in each segment along the CNS. Since the organization of fibre bundles in a tract must reflect the array of exteroceptors providing afferent fibres, the arrangement and type of appendages to be moved, and hence the population of interneurones necessary for coordination, it is not surprising to find some interspecific differences at this level. One consequence of the anatomical analysis above is that we can confirm neuroanatomically (Fig. 5) the fusion of the metathoracic and two abdominal neuromeres suggested by studies of the metamorphosis of the CNS (Singh and Srivastava, '73; Tsujimura, '83). The fusion of these neuromeres has not disturbed the arrangement of fibre bundles found in unisegmental ganglia and follows closely the pattern of fusion of the same segments in locust (Tyrer and Gregory, '82).

Neuroanatomy of central circuits The central projections of the A l , A2, and B afferents have a very similar gross morphology to those reported previously for other noctuid species (Surlykke and Miller, '82; Agee and Orona, '88). The data presented here show that the projections of the A1 afferent and sound-sensitive interneurones, such as IN 501 and IN 504, overlap extensively in VIT, MVT, dVCLII, and the various parts of the ring tract (aRT, RT, SMC) (Figs. 6, 8,9). Our physiological results confirm that INS 501,503, and 504 receive monosynaptic input from the A1 afferent (Fig. 11A; Boyan and Fullard, '88; Boyan et al., '88). Although the location of the synapses was not established, the prominent branch that INS 501 and 504 direct into the afferent tract is a strong candidate (Figs. 8c; 9c). There are no dorsal projections from the A1 afferent (Fig. 6), and no overlap with arborizations from premotor (Fig. 13B) or motor neurones (Fig. 17C). Consistent with this, we found no direct input to flight motor neurones from the A1 afferent (Fig. 17A,B). The B cell is not excited by sound in tympanate moths (Lechtenberg, '71), makes no connections with any of the interneurones or motor neurones tested here (Figs. 11A, 14D, 16Bii, 17A,B) despite projecting in the same tracts and commissures as the A1 afferent (Fig. 6), and it plays no known role in avoidance behaviour. The third tympanal afferent to enter the CNS is the A2 cell and its projections lie mainly, but not exclusively, in the metathoracic ganglion (Fig. 7; Surlykke and Miller, '82; Agee and Orona, '88). The arborizations are found dorsally in DIT, with additional branching ventrally to areas of intermediate neuropile (ring tract) occupied by the A1 afferent and INS 501 and 504 (Figs. 6-9). T o date we have found no central connections for the A2 cell, but do have evidence that IN 504 receives direct input from the A l , but

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Fig. 17. Morphologies of basalar (A) and dorsal longitudinal (B) flight motor neurones in the mesothoracic ganglion of Catocala ceragama are shown drawn from wholemount and viewed dorsally, following intracellular staining with Lucifer Yellow on a t least two occasions. Insets show that sound (10 ms tone a t 16 kHz, 90 d B SPL, stimulus not shown) presented to the ear contralateral to the innervated side (arrow) inhibited both the motor neurones (upper traces) while evoking a summed excitatory response in A1 and A2 afferenta in the tympana1 nerve (lower traces). Spike-triggered averaging (middle and lower panels) shows that neither the A1 nor B afferent on the contralateral

side directly evoked any response in these motor neurones. Traces are averages of 64 sweeps. C. Morphology of two unidentified motor neurones as seen in transverse section of the mesothoracic ganglion of Acronicta arnericana following cobalt backfilling of IIIN1. Longitudinal fibre tracts are abbreviated as follows: 1 = MDT, 2 = LDT, 3 = DMT, 4 = DIT, 5 = VLT, 6 = VIT, 7 = LVT, 8 = MVT, 9 = VMT. Scale bars: vertical, (intracellular records only) upper panel, A (60 mV), B (30 mV); middle and lower panels, A,B (4.5 mV); horizontal, upper panel, A (60 ms), R (150 ms); middle and lower panels, A,B 9 ms; anatomy, A,B (260 pm), C (200 gn).

not the A2, afferent (Fig. llA,B). This implies that there must be some distinction in the central circuitry processing information from these afferents. Some of the physiological properties of the A2 cell, such as its higher auditory threshold, suggest a role in the later stages of avoidance behaviour (Roeder, ’64). Although there is no evidence yet for a direct input from the A2 cell to flight circuitry, this is possible given the dorsal projections of A2 (DIT, Fig. 7), which would overlap those of‘flight motor pathways at least in the metathoracic ganglion (Orona and Agee, ’88).

information processing occurring in the auditory association areas of different segments. In the posterior association area (PAA), the interneurones receiving direct input from the A1 afferent (INS 501, 503, 504, Figs. I l A , 18) can now be shown to have ventral arborizations (Figs. 8,9), and respond tonically (“repeater”) to auditory stimuli (Boyan and Fullard, ’86, ’87, ’88; Boyan e t al., ’88). Interneurones responding phasic/tonically or phasically (INS 502,505,506,508,509,511, 101) have higher thresholds and longer latencies consistent with indirect inputs from the A1 cell (Fig. 18; Boyan and Fullard, ’86), but may receive input from the A2 cell. In the anterior association area (AAA) within the mesothoracic ganglion, IN 701 receives direct input from the A1 afferent (Fig. 14C,D,E),and has arborizations in the segmentally equivalent areas of neuropile (VIT, ring tract) to INS 501 and 504 of the PAA. However, IN 701 responds phasically (“pulse marker”) to sound (Fig. 14A), probably because of different synaptic integration properties, as no inhibitory potentials were ever observed. The other interneurones so far identified in the AAA (Figs. 12-16) have largely phasic or phasic/tonic responses, higher auditory thresholds, labile responses, and long latencies. Taken together these properties suggest an indirect input from the A1 afferent, as was confirmed in the case of I N 401 by the appropriate double recordings (Fig. 16). INS 507, 401, and

Segmental organization of the auditory pathway In noctuid moths the A1 afferent and identified interneurones ascend in parallel from the metathoracic ganglion to the brain (Surlykke and Miller, ’82; Boyan and Fullard, ’86), arborizing in VIT, MVT, dVCLII, and the ring tract (aRT, RT, SMC) in each neuromere (Figs. 6,8,9). A serial array of interneurones has been identified in several neuromeres in this pathway (Boyan and Fullard, ’87),forming segmentally repeating auditory association areas, two of which are depicted in summary form in Figure 18. Auditory interneurones in the moth have been traditionally classified as “repeater” (tonic) or “pulse marker” (phasic) according to their temporal response properties (Roeder, ’66). We feel that this classification also has relevance to the type of

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pathways controlling the movement of the forewings, which in the moth provide the primary thrust for flight.

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101

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A common “Bauplan”for auditory pathways?

AAA (meso)

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ear Fig. 18. Summary schematic showing organization of identified sound-sensitive interneurones into anterior (AAA) and posterior (PAA) auditory association areas with respect to input from the A1 afferent only. Interneurones listed have been identified in this or previous studies (Boyan and Fullard, ’86) and shown to make direct (d) or indirect (ind) connections with the A1 afferent. The anterior associationarea also receives projections from interneurones originating posteriorly, and a number of interneurones from both centres project in parallel with the A1 afferent to the brain. Anatomical overlap of projections demonstrated in transverse sections suggests possible outputs from the AAA to flight motor neurones (MN), but to date there are insufficient data to make a prediction for the PAA.

102 have the physiological responses and dorsal projections overlapping those of flight motor neurones (Figs. 13, 17; Kondoh and Obara, ’82; Rind, ’83; Madsen and Miller, ’87; Orona and Agee, ’88), which suggest a putative role as premotor neurones in avoidance behaviour. Indeed, the rhythmical oscillations in membrane potential and sound sensitivity of IN 401 (Fig. 16) place this cell at the interface of the auditory and flight motor pathways. Although the sample size of identified neurones (14) is still small, we believe that some generalizations concerning the organization of auditory information processing in the pterothoracic ganglion can be made (Fig. 18). If the two association centres described so far are representative of those in the VNC, then it appears that a core group of interneurones located ventrally in each centre receives tonic auditory input from the A1 afferent, and this information is then transformed directly (e.g., IN 701, Fig. 16), or indirectly (e.g., INS507, 401, 102, Figs. 15, 16), into a phasic or phasichonic output to the dorsal motor system. Anterior centres also receive a parallel (tonic) input from the A1 afferent (Fig. 10) and more posterior interneurones (e.g., INS 501, 503, 504, Figs. &10; Boyan and Fullard, ’86). Further, a comparison of neurone response types in the two association areas described so far suggests that the proportion of neurones with “pulse marker”-type physiologies, and with arborizations overlapping those of motor pathways in dorsal neuropile, appears to be higher in the more anterior association area (Boyan and Fullard, ’86, ’87). This may represent a segmentally organized change in information processing that directs predominantly phasic information to

A comparison of the organization of the auditory pathway in the various tympanate insects reveals that the VIT, MVT, dVCLII and the various parts of the ring tract (aRT, RT, SMC) contain the projections of auditory afferents and first-order interneurones in Lepidoptera (Figs. 6-9), Orthoptera (locust: Tyrer and Gregory, ’82; Pearson e t al., ’85; Halex et al., ’88; Romer et al., ’88; bushcricket: Romer et al., ’88; cricket: Wohlers and Huber, ’85; Atkins and Pollack, ’87), Dictyoptera (mantid: Yager and Hoy, ’87, ’89), and probably Hemiptera (cicada: Wohlers et al., 1979). In the locust, auditory aeerents entering the metathoracic ganglion ascend to a t least the suboesophageal ganglion (SOG) and send projections into the ring tract and VIT a t each segmental level (Halex et al., ’88). A serially homologous array of auditory interneurones has been described in the thoracic ganglia (Pearson e t al., ’85), along with an auditory association area in the SOG comprising through fibres, ascending, and local interneurones, with arborizations in VIT, MVT, and the ring tract (Boyan and Altman, ’85). This parallels closely the anatomical organization described above for the moth, and changes in auditory information processing have also been described as one moves anteriorly up the VNC of the locust (Kalmring, ’75; Silver et al., ’80). At the gross level, the auditory neuropiles clearly occupy homologous regions of the CNS in different insect groups even when the ears are located on different parts of the body and the f i e r e n t s enter the CNS via different nerve roots. Audition and phonotactic behaviour derive from an anatomical organization or “Bauplan,” which is conserved in holometabolous (Lepidoptera) and hemimetabolous (Orthoptera, Dictyoptera) insects with very different lifestyles.

Acknowledgments This study was funded in part by operating and equipment grants to J.H.F. from the Natural Sciences and Engineering Research Council of Canada and the University of Toronto; to G.S.B. from the Alberta Heritage Foundation; and to K.G. Pearson from the Medical Research Council of Canada. We appreciate the financial support of J.L.D.W. provided by Prof. Dr. K.-E. Kaissling, Max-Planck Institut, Seewiesen. We thank Dr. Raleigh Robertson for the use of facilities at Queen’s University Biology Station, and Dr. P.E. Teal of the University of Guelph for kindly supplying individuals of H. uirescens. Dr. E.E. Ball kindly criticised the manuscript.

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