The distribution of neurones immunoreactive for ?-tyrosine hydroxylase, dopamine and serotonin in the ventral nerve cord of the cricket, Gryllus bimaculatus

Cell Tissue Res (1995) 280:583-604 Cell&Tissue

Research �9 Springer-Verlag 1995

The distribution of neurones immunoreactive for -tyrosine hydroxylase, dopamine and serotonin in the ventral nerve cord of the cricket, Gryllus bimaculatus Michael H6rner, Ulrike Sp6rhase-Eichmann, Johannes Helle, Briine Venus, Friedrich-Wilhelm Schiirmann

I. Zoologisches Institut, Abteilung ftir Zellbiologie, Universit~t G6ttingen, Berlinerstrasse 28, D-37073 G6ttingen, Germany

Received: 23 August 1994 / Accepted: 4 December 1994

Abstract. The cellular localization of the biogenic amines dopamine and serotonin was investigated in the ventral nerve cord of the cricket, Gryllus bimaculatus, using antisera raised against dopamine, [3-tyrosine hydroxylase and serotonin. Dopamine- (n<70) and serotonin-immunoreactive (n_<120) neurones showed a segmental arrangement in the ventral nerve cord. Some neuromeres, however, did not contain dopamine-immunoreactive cell bodies. The small number of stained cells allowed complete identification of brain and thoracic cells, including intersegmentally projecting axons and terminal arborizations. Dopamine-like immunostaining was found primarily in plurisegmental interneurones with axons descending to the soma-ipsilateral hemispheres of the thoracic and abdominal ganglia. In contrast, serotonin-immunostaining occurred predominantly in interneu- tones projecting via soma-contralaterally ascending axons to the thorax and brain. In addition, serotonin-immunoreactivity was also present in efferent cells and afferent elements. Serotonin-immunoreactive, but no dopamine-immunoreactive, varicose fibres were observed on the surface of some peripheral nerves. Varicose endings of both dopamine- and serotonin-immunoreactive neurones occurred in each neuromere and showed overlapping neuropilar projections in dorsal and medial regions of the thoracic ganglia. Ventral associative neuropiles lacked dopamine-like immunostaining but were innervated by serotonin-immunoreactive elements. A colocalization of the two amines was not observed. The topo- graphic representation of neurone types immunoreactive for serotonin and dopamine is discussed with respect to possible modulatory functions of these biogenic amines in the central nervous system of the cricket.

Key words: Dopamine - Serotonin - Tyrosine hydroxylase - Immunocytochemistry - Nervous system, insect - Gryllus bimaculatus (Insecta)

Correspondence to: M. H6rner

Introduction

Biogenic amines, such as the catecholamines dopamine (DA) and octopamine or the indolamine serotonin (5- HT), are widely distributed in nervous systems through- out the animal kingdom. It has been shown that amines acting as neuroactive substances influence the perfor- mance of specific behavioural tasks in both vertebrates and invertebrates (Grillner et al. 1986; Kravitz 1989). For studies focusing on the modulatory effects of amines in the central nervous system (CNS), several arthropod preparations have proved to be advantageous because identified neurones can be analysed in the functional context of a well-defined behavioural paradigm (Bicker and Menzel 1989; Harris-Warrick et al. 1989). Whereas the structure and function of putative octopamine-containing neurones (Agricola et al. 1988; Orchard et al. 1993; Stevenson and Sp6rhase-Eichmann 1995) have been thoroughly investigated, the organization of the dopaminergic and serotonergic system is less well understood. The effects of 5-HT on different behavioural patterns have been studied in some insect species (Brook- hart et al. 1987; N~issel 1987; Bicker and Menzel 1989). Both 5-HT and DA have been shown to act as peripheral transmitters of identified neurones innervating locust and cockroach salivary glands (Gifford et al. 1991). Al- though some of the cellular actions of DA have been characterized (Davis and Pitman 1991), the functional role of DA in the CNS is less well understood and has only been investigated in a few invertebrate preparations (Michelsen 1988; Menzel et al. 1989; Goldstein and Camhi 1991).

The presence of DA and 5-HT was originally demonstrated in the nervous tissue and body fluids of insects by histochemical and biochemical methods (Klemm 1974, 1976; Evans 1980; Mercer et al. 1983; Budnik and White 1988; Nagao and Tanimura 1988). Putative serotonergic and dopaminergic neurones in the insect CNS have subsequently been revealed by more specific immunocytochemical techniques for several insect species (5-HT: Klemm 1983; Scht~rmann and Klemm 1984; Tyr-

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er et al. 1984; N~ssel 1987; DA: Viellemaringe et al. 1981; N~issel et al. 1988; Sch~ifer and Rehder 1989; Schtirmann et al. 1989; Distler 1990; Orchard et al. 1992; Watson 1992; Wendt and Homberg 1992).

As yet, most studies cited have mainly focused on the distribution o f putative DA- or 5-HT-containing neurones in the brain or are restricted to particular immunoreactive (it) cells (Distler 1990; Milton et al. 1991) or neuropiles (Elekes et al. 1987). In addition, only a few DA- ir or 5-HT-ir elements have been identified in sufficient detail to allow tracing of terminal arborizations or intersegmental projections. In locusts, 5-HT-ir (Tyrer et al. 1984) and DA-ir (Wendt and Homberg 1992) brain cells project to the ventral ganglia but little is known about their connections and targets in the ventral nerve cord (VNC; Watson 1992). Therefore, al though much of the neural circuitry generating overt behaviour is localized in the ventral ganglia, details o f the chemoneuroana tomy of aminergic pathways in the ventral nervous system re- main undescribed. This is remarkable since possible amine actions are not restricted to the brain itself and selective partially antagonistic effects of DA and 5-HT administration have previously been demonstrated in ventral ganglia (Claassen and Kammer 1986; Goldstein and Camhi 1991; Casagrand and Ri tzmann 1992).

We have used well-characterized antisera against dopamine, ~3-tyrosine hydroxylase (TH, required for dopamine synthesis and thus a marker for DA-containing neurones) and 5-HT in the present investigation to demonstrate the distribution of identifiable DA/TH-i r and 5- HT-ir somata in the V N C of the cricket, GryIlus bimaculatus. Preliminary accounts of parts o f this work have been published in abstract form (Sp6rhase-Eichmann and Schtirmann 1988; Venus et al. 1992; H6rner et al. 1994).

Materials and methods

Animals

Male and female crickets (Gryllus bimaculatus de Geer) freshly moulted or up to 2 weeks after imaginal ecdysis were collected from a breeding colony (28~ 12h-12 h light-dark cycle, feeding ad libitum). The animals were anaesthetized before preparation by cooling (4~ for 20 rain) and were then pinned down ventral side up in a wax dish. During preparation, the nervous system was per- manently kept under fixative (see below). In order to demonstrate the morpholgy of intersegmental fibres and their connections within the VNC, single ganglia and complete ganglia chains with intact connectives from the brain to the terminal ganglion were dissected.

Immunocytochemistry

Dopamine. The location and morphology of putative dopaminergic neurones was analysed using two different antisera against (1) TH (monoclonal mouse antibody, code KTHM 788, purchased from Incstar, Stillwater, Mo., USA), the rate limiting enzyme cata- lyzing the o-hydroxylation from tyrosine to L-DOPA, which is de- carboxylated to dopamine and (2) DA coupled to thyroglobulin with glutaraldehyde (polyclonal rabbit antibody; Steinbusch and

Tilders 1987). The following protocol was used for both antisera. Fixation took place in paraformaldehyde 4% (v/v) in Taghert buffer (Taghert et al. 1982; pH 7.2, 12-16 h, 4-8~ In addition, fixation in 5% glutaraldehyde (v/v) in Taghert buffer containing 0.2% ascorbic acid (v/v) was used, which improved the staining intensity of material treated with DA antiserum, in some cases. To facili- tate the penetration of the antisera, whole-mounts were rinsed in buffer (pH 7.2, 1-3 days, 4-8~ containing 0.25% (w/v) Triton X-100 (Sigma, Deisenhofen, Germany). Prior to incubation with the primary antibody, the tissue was pre-incubated in normal goat serum (10% v/v; Dakopatts, Hamburg, Germany; 1 h, 4-8~ An- tisera were diluted in 0.01 M Taghert buffer with 0.25% Triton X- 100 (1:1000 v/v for TH; 1:500 for DA) and washed in the same buffer thoroughly between successive antiserum incubations (1-2 h).

The peroxidase-antiperoxidase technique (PAP technique; Sternberger 1986) was used as a marker for immunoreactions to both TH and DA in whole-mounts and sections. After incubation of the primary antibody against TH, we sequentially employed a second polyclonal rabbit anti-mouse serum (Dakopatts, Denmark; 1:40 v/v in 0.25% Triton X-100, 3-5 h, 4-8~ and a mouse anti- rabbit-peroxidase complex as the third antiserum (Dakopatts, Denmark; 1:30-1:160 v/v in 0.25% Triton X-100, 24-48 h, 4-8~ For the demonstration of DA, a second goat anti-rabbit antibody (1:40 v/v; Nordic Immunology, No. 3203) and a rabbit PAP-complex (Dakopatts; further details see above) were used. All other steps were the same as described for TH. The staining process for both TH and DA antisera was monitored during treat- ment of the preparations with 0.02-0.05% 3,3'-diaminobenzidine tetrahydrochloride (v/v; DAB, Aldrich, Steinheim, Germany) in TRIS-HC1 buffer (final pH 8.4) containing 0.005-0.01% (v/v) H202. The staining reaction was stopped by rinsing the preparations in TRIS-HC1 buffer containing 1% sodium azide (NaN 3, v/v). Whole-mounts were dehydrated and mounted in Durcupan (Fluka, Neu-Ulm, Germany).

Serotonin. For the immunocytochemical demonstration of 5-HT- containing neurones a well-characterized monoclonal rat antiserum (Seralab MAS 055, YC5/45, Sussex, UK; Consolazione et al. 1981) and a polyclonal rabbit antibody (Ges. f. Immunchemie und -biologic, Hamburg, Germany, No. JBL43HZT), both conjugated to bovine serum albumin with paraformaldehyde, were employed. Two different fixation protocols were used for these antisera: (1) paraformaldehyde fixative 1.5-4% (v/v) in Taghert buffer (pH 7.6, 6-20 h, 4-8~ with an additional fixation period in the same fixative (pH 11.6, 1-3 h, 4-8~ (2) a two step fixation with pre-fix- ing (10 min, at room temperature) in Bouin fixative (15 ml satu- rated aqueous picric acid, 4 ml 37% v/v paraformaldehyde solution, 1 ml 100% acetic acid) and subsequent fixation as described in (1). The primary monoclonal antiserum was used in a 1:250 dilution (v/v, in 0.01 M Taghert buffer, pH 7.2 containing 0.25% Triton X-100, 1% normal goat serum, 0.25% bovine serum albumin; 12-24 h, 4-8~ For the demonstration of 5-HT in the tissue, we employed a biotinylated rabbit anti-rat antibody as a second antiserum (Vector No. BA 4000, Wiesbaden, Germany, 1:40 v/v, in 0.01 M Taghert buffer with 0.25% Triton X-100, 3-4 h, room temperature; ABC technique; Hsu et al. 1981). After repeat- ed washes in buffer (see above), the preparations were incubated in a 1:1 solution containing avidin and biotinylated peroxidase (diluted 1:40 v/v from the stock solution, Vectastain ABC-kit No PK 4000 in Taghert buffer 12-16 h, 4-8~

We used the staining protocol as described by Sp/Srhase-Eich- mann et al. (1987) with the polyclonal rabbit antiserum (1:1000 dilution in the same buffer system as mentioned above without bovine serum albumin) employing the PAP technique. The DAB reaction for both antisera was identical with that used for the immunocytochemical detection of DA.

In addition to the whole-mount preparations described above, Vibratome sections (thickness:40-70 gin) were made from head, thoracic and abdominal ganglia. After fixation (protocols: see above for each of the different antisera used), horizontal, sagittal

and frontal sections were made from agarose-embedded ganglia following the procedures described in detail by SpOrhase-Eich- mann et al. (1992).

Controls and specificity tests

Possible unspecific labelling was tested for all antisera used. In negative control experiments, sections and whole-mounts of ventral ganglia were examined after omission of the primary, secondary or third antiserum used. No staining could be observed after this procedure. Positive controls were performed by diluting the primary antisera against DA, TH or 5-HT in steps between 1:100 and 1:5000. This led to a gradual decrease of staining intensity. Ir- respective of the chosen dilution of the antiserum, the same set of neurones was always stained. In tissue tests, omission of the employed goat or rabbit sera slightly increased the level of back- ground staining but revealed no additional immunopositive elements for either of the antisera used (Sternberger 1986). The specificity of the polyclonal anti-DA serum has been thoroughly tested in vitro and in tissue tests (Steinbusch and Tilders 1987; Stein- busch et al. 1991). Possible cross-reactivity of the anti-DA serum with 5-HT, histamine and adrenaline has been tested in gelatin diffusion tests by Steinbusch et al. (1991). In tissue control experiments, no DA-ir elements were detected in whole-mounts or Vi- bratome sections after preabsorption of the anti-DA serum with DA (100-500 gg/ml DA-HC1) according to Steinbusch et al. (1991).

According to the specifications of the manufacturer, the anti- TH serum is specifically directed against the mid-portion of the enzyme molecule, where regions of species homology have been described. The same antiserum has previously been employed in a number of studies in insects (N~issel and Elekes 1992; Orchard et al. 1992; Ali et al. 1993). Pre-absorption tests could not be per-

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formed for the anti-TH serum since no original antigen was avail- able. Immunostaining for DA and TH yielded identical results in the cricket CNS.

The specificity of the polyclonal antiserum against 5-HT was tested according to Klemm (1983) by pre-absorbing the primary antiserum (500 ~tg/ml 5-HT-creatinine-phosphate). After standard immunohistological processing, no labelling was observed in the controls. Pre-absorption tests were omitted for the monoclonal antibody against 5-HT, since its specificity has been convincingly demonstrated I Consolazione et al. 1981).

Data analysts

Stained material was analysed in the light microscope with a drawing tube (Olympus BH2). With respect to classifcation of soma clusters or single cells, the diameters of somata were measured from photographs or camera lucida drawings. For the identification of immunoreactive structures and a comparison with defined tracts and neuropile regions (Wohlers and Huber 1985) within the VNC, serial sections of osmium-ethyl-gallate-treated tissue (Wig- glesworth 1957) were used. In addition, single salivary gland cells were filled iontophoretically with Lucifer-Yellow to allow comparison with antibody-stained somata. To estimate possible segmental homology between single cells or cell groups, the number, location, segmental and intersegmental projections, symmetries of arborizations and the variation of staining intensity of immunola- belled structures were evaluated. Immunoreactive somata or cell groups were classified according to their ganglionic location and numbered along the anterio-posterior axis (SOG=suboesophageal, PTG=prothoracic, MSG-mesothoracic, MTG=metathoracic, AG=abdominal and TG=terminal ganglion). A pre-fix indicating the antigen detected by immunolabelling was added (e.g., DA- SOGl=most anterior cell or cell group in the SOG showing DA- like immunoreactivity).

Table 1. Characteristic features of DA/TH-ir and 5-HT-ir elements in the CNS of Gryllus bimaculatus

Cerebral Suboesophageal Prothoracic Mesothoracic ganglion a ganglion ganglion ganglion

DA/TH 5-HT DA/TH 5-HT DA/TH 5-HT DA/TH 5-HT

No. of cells (mean) -200 -70 13.3 26.8 6.8 17.5 3.5 14.5 Miminum-maximum 180-210 60-80 10-28 22-32 4-10 5-34 2-8 6-22

Fibre number in one 6-8 14-20 8-10 6-10 8-12 9-13 8-12 12-16 connective posterior to the relevant ganglion

Metathoracic Abdominal Terminal ganglion ganglion (1-4) ganglion

DA/TH 5-HT DA/TH 5-HT DA/TH 5-HT

No. of cells (mean) 3.3 17.5 Miminum-maximum 2-4 8-37

Fibre number in one 10-12 10-12 connective posterior to the relevant ganglion

1.2 5.3 6.6 28.9 b 0-2 1 - 12 2-8 2 2 4 3 b

10-12 10-12 - -

a Cerebral ganglion without optic lobes b Numbers for males and females

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Table 2. Comparison between DA/TH-ir and 5-HT-ir neurones in the CNS of Gryllus bimaculatus

DA/TH 5-HT

No. of brain cells

No. of ventral cord cells

Segmental arrangement

Variation of cell number

Sexual dimorphism

Neurone types

Morphology of identified single cells

Axon profiles in fibre tracts

Central fibre projections

Peripheral projection/fibres

Location and morphology of varicose projections

180-210 cells (without optic lobe) No ascending elements identified in the brain

-60-70 cells Mostly bilateral symmetric single cells

Some neuromeres without cell bodies Cell number decreasing along the

anterior/posterior axis (most cells in the SOG)

No variation of lateral and large medial neurones High variation of small medial cells

Not observed

Plurisegmental interneurones with wide-field projections

Primarily descending thoracic interneurones One pair of efferent axons in SOG (SOG2 neurones) No sensory fibres observed

Ventro-lateral or -medial soma position most common

Descending cells show soma-ipsilateral projections Plurisegmental projections through the whole CNS

Descending fibres from cells of SOG, PTG and MTG in LDT

Descending fibres from tritocerebral ceils in VMT Small axon calibres of intersegmental fibres

All neuromeres with DA/TH-ir projections Sensory neuropile without specific labelling Common projection of brain/thorax cells in the TG

Efferent: exclusively salivary glands (SN1 via N. 7b)

Mainly dorso-median from descending brain cells Mainly dorso-lateral from descending SOG

and thoracic neurones No innervation of associative neuropiles,

such as the VAC Only central varicosities; no varicosities on nerves

60-80 cells (without optic lobes) Terminals of ascending fibres in the brain

120 cells Mainly bilateral symmetric cell groups

All neurones with labelled cell bodies Cell number _+ comparable along

anterior/posterior axis (most cells in the TG)

Variations primarily in small medial cells

Females with more efferent cells in the TG

Intersegmental wide-field interneurones Mostly ascending or intrasegmental interneurones Some efferent fibres in the SOG and TG Sensory projections from peripheral thoracic nerves

Dorso-lateral soma position most common Ascending cells show soma-contralateral projection Intersegmental projections over 2-3 ganglia Prominent intrasegmental auditory cell (ONI)

Ascending fibres from the abdomen in the VIT

Descending fibres from brain cells in DMT Axon calibres decreasing in anterior direction

All neurones with 5-HT-ir-projections Dense innervation of ventral sensory neuropile Common projection of ascending cells in the brain

Efferent: salivary glands (SN2 via N. 7b) Efferent: genital musculature from the TG Sensory: unidentified and mechanoreceptors in MSG

Mainly dorso-medial from brain fibres Mainly dorso-lateral from ascending cells

Numerous afferent projections in the VAC

Central varicosities and peripheral varicosities on nerves (SOG and TG)

Results

Distr ibut ion o f immunoreact ive neurones f o r DA and TH

The present ana tomica l descr ip t ion is based on the l ight- mic roscop ica l ana lys is of who le -moun t prepara t ions (n=54 for TH; n=15 for DA) and serial Vibra tome sections. With respect to the different ant isera used, the re- cons t ruc t ion of in te rsegmenta l f ibres was more eas i ly achieved in an t i -TH- t rea ted mater ia l , whereas s taining o f central var icosi t ies appea red to be more intense with an- t isera agains t D A itself. However , nei ther the inspect ion o f who le -moun t prepara t ions nor the h i s to logy of ser ia l sect ions revea led any s ignif icant d i f ferences as far as ( I ) the number and segmenta l dis t r ibut ion of ident i f ied somata, (2) the loca t ion and number of in te rsegmenta l f ibres in the connect ives or (3) the m o r p h o l o g y and pro jec t ions o f the main D A / T H - i r var icos i t ies in the central neuro- pi les were concerned. Therefore , all data for D A and TH immunocy tochemis t ry are presented together. A total number of up to 260 cel ls showed D A / T H immunoreac - t ivi ty in the bra in and V N C (Table 1). A l though cell

Fig. 1A-E TH-ir neurones in the cerebral, suboesophageal (SOG), prothoracic (PTG) and mesothoracic ganglia (MSG); whole-mount preparations, anterior to the top. A Cerebral ganglion with a bilateral symmetric group of three neurones (white asterisks) in the tritocerebral lobe with descending axons, rostral view. x115. B Montage of two planes of focus of the SOG (inset showing a Lucifer-Yellow single-cell staining of one SOG2 neurone); black arrows, axon from SOG2 neurone extending through N. 7b; white arrows, descending axons from neurones marked in A; asterisks, lateral paired somata found only in some preparations; black arrowheads, descending axons from SOG4 neurones. x95. C Detailed view of the anterior region of the SOG with rami- fications of SOG1 cells (black arrowheads, somata not shown) and SOG2 neurones, x170. D Large ventro-median PTG3 neurones (white star) and small PTG2 and PTG4 cells in the PTG. x130. E Montage of two planes of focus demonstrating the ventro-medial through-fibres and the main neurites and dorso-lateral axons of the large PTG2 somata (out of focus); star, position of somata shown in D; white arrows, medially descending axons from neurones marked in A; black arrowheads, axons from SOG4 neurones shown in B; black arrows, descending axons from PTG3 neurones shown in D. x130. F MSG2 somata (asterisks) with ascending axons; white arrows, descending axons from neurones shown in A. x130. Scale bars: 100 Bm

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clusters appeared to be common in the brain, bilateral symmetric single cells were most often present in the VNC. Segmental homology between DA/TH-ir neurones could be asserted only for some cells.

Since whole-mount preparations allowed the tracing of complete intra- and intersegmental projections in the VNC, the morphology of some DA/TH-ir cells was com- pletely investigated. With the exception of two suboesophageal neurones with efferent projections (salivary gland neurones, see below), all identified DA/TH-ir cells in the cricket CNS could be classified as interneurones. No evidence for either afferent or efferent fibres was found. Among intersegmental neurones those with ipsilaterally descending axons were most frequently found in the VNC (Table 2). As shown in Table 1, the number of labelled cells varied between preparations. We found that cell groups consisting of small, faintly stained somata showed more variations concerning their number and location than intensely labelled cells.

Cerebral ganglion. Since we found no major differences between the distribution of DA/TH-ir cells in our preparations and those in other orthopterans (Nyhof-Young and Orchard 1990; Wendt and Homberg 1992), only a brief description of DA/TH-ir neurones is given here for the cricket brain. In comparison with the ventral cord ganglia, the brain (without optic lobes) contained the largest number of DA/TH-ir elements (180-210). DA/TH-ir somata were arranged in bilateral clusters and were concentrated in the protocerebral lobes (about 140-170 cells) compared with the deuto- and tritocerebral lobes (30-50 cells). Most of the DA/TH-ir brain cells were interneurones restricted to the brain. In the anterior portion of the tritocerebrum, one cell group of three somata (soma diameter: 20-25 gm) with ipsilaterally descending axons was identified in each hemisphere (Fig. 1A). Their intersegmental projections were traced through the whole VNC to the TG (Figs. t A-F, 2C, F). In the thoracic ganglia, these descending axons passed medially in close vicinity to each other within the ventro-median tract (VMT; Fig. 3A-C, D). The course and the morphology of fibre projections will be described below in more detail.

SOG. A total number of 28 bilaterally symmetric DA/TH-ir cells was labelled in all three neuromeres of the SOG (Figs. 1B, 4A). Single paired anterior (TH- SOG2) and posterior (TH-SOG4) neurones could be discerned from grouped anterior (TH-SOG1), medial (TH- SOG3) and posterior (TH-SOG5) cells. In a few preparations, an additional lateral cell pair (Figs. 1B, 4A) was present. The two largest and most intensely stained dorsal DA/TH-SOG2 cells (diameter: 60 gin) were the only labelled neurones in the whole ventral cord with efferent soma-contralateral projections crossing via the mandibular commissure. The efferent fibres left the SOG via peripheral nerve (N) 7b (Fig. 1B) to extend to the salivary glands, where multi-terminal varicose projections were seen. TH-SOG2 cells were intracellularly injected with Lucifer-Yellow (see inset Fig. 1B) and could thus be un- equivocally identified as SN1 neurones similarly described by Gifford et al. (1991) in locusts.

Anterior to the prominent TH-SOG2 cells, a group of three ventro-lateral somata (TH-SOG1, n---6; diameter: 20-25 gm) was reproducibly labelled in each hemisphere. The fasciculated axons projected dorsally to the mandibular neuropile forming parallel collaterals that crossed the ganglion midline (Fig. 1C). A single strongly labelled cell pair was present in a posterio-medial position near the neck connectives (TH-SOG4, n=2; diameter: 40 gm) forming ipsilateral axons descending laterally in the connectives (Figs. 1B, E, 2E-G, 3F). These fibres (diameter: 3-5 gm) were traced to the TG, where their varicose endings were observed (Fig. 2C, G). Addi- tional DA/TH-ir somata (TH-SOG5; n=0-4; diameter: 30 gin) occurred at the posterior ganglion border in a more lateral position (Fig. 1B). Their primary neurites projected anterio-ventrally but could not be traced further.

In contrast to the strongly stained lateral DA/TH-ir elements, medial cell groups (TH-SOG3, n=0-10; diameter: 15-20 gin) appeared only faintly labelled and axonal arborizations were never discernible. Moreover, the position and number of medial cells varied considerably and labelled somata could often not be detected.

The axons descending from the tritocerebral neurones (diameter: 5 ~tm; Fig. 1A) were traced through the SOG and whole VNC (Figs. 1E, F, 2E-G, 4). They run in a dorso-lateral position in the brain connectives (Fig. 1B) and curved ventro-medially in the SOG to pass in close vicinity to each other within the VMT. Medial collaterals were visible at the border between the labial and mandibular neuromeres.

PTG. Only 10 DA/TH-ir interneurones were present in this ganglion. Three bilateral symmetric single cells (TH-PTG1,3,4) and one medial soma group (TH-PTG2) were labelled (Figs. 1D, E, 4B). The largest and most intensely stained somata were found in the posterior part of the PTG in a ventro-median position (TH-PTG3, n=2,

)

Fig. 2A-G. Comparison of DA-ir (A-C, left) and TH-ir (D-G, right) neurones in the metathoracic (MTG), free abdominal (AG) and terminal ganglia (TG); whole-mount preparations, dorsal views, anterior to the top. A Fourth free AG with pattern of varicose collaterals originating from intersegmental DA-ir fibres running in a lateral position (note the absence of continously stained intersegmental axons), x130. B Anterior part of the TG showing the arrangement of DA-ir collaterals and the most anterior paired DA-TGI somata (asterisks; compare with C, G); black arrows, descending axons from thoracic nemones, x160. C Paired dorso- lateral DA-TGt-3 somata in the TG (compare with G); black arrow, axon from lateral intersegmental fibre, x90. D Third free AG with posterior TH-AGI somata; black arrow, lateral intersegmental fibre, x180. E Fourth free AG with a pattern of varicose collaterals emerging from lateral intersegmental TH-ir fibres (compare with A; black arrow, stained intersegmental fibres), x130. F Ar- rangement of TH-ir fibres in the fused abdominal segments of the MTG; white arrows, median axons from neurones descending from the brain; black arrows, axons from cells descending from the SOG and PTG. xlS0. G Montage of two planes of focus of the TG showing four pairs of dorsal TH-TG1-4 somata and the terminal arborizations of lateral intersegmental TH-ir fibres (compare with C; black and white arrows as in F; asterisks additional TH- TG4 somata not found in DA-ir preparations), x90. Scale bars: 100 ~xm

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diameter: 30-35 gin). From the ventral somata (Fig. 1D), the neurites curved dorsally to form ipsi- and contralateral projections giving rise to descending axons (diameter: 3-5 gin; Fig. 1E) that were traced down to the TG. The axons in the soma-ipsilateral lateral dorsal tract (LDT) gave off mainly dorso-lateral collaterals in the thoracic ganglia and AG (Figs. 1E, 2E-G, 3A, C, 4B-G). Another ventral pair of TH-PTG1 somata (n=0-2; diameter: 10-15 gm) was found in an anterior position near the entrance of the neck connectives. Their axons crossed the ganglion midline via the dorsal commissure VCI (Wohlers and Huber 1985) and divided contralaterally in an ascending axon giving off medial collaterals and a lateral branch with projections near the entrance of N. 5 (Fig. 4B). The ascending axon formed varicose terminals in the dorsal labial and maxillar neuropiles of the SOG. Additional cells were visible in a ventro-median location (TH-PTG2, n--0-4, diameter: 15-20 gin, Fig. 1D), but were only faintly labelled.

Descending axons from tritocerebral brain cells were constantly found near the midline of the ganglion within the VMT (Figs. 1E, F, 2E-G, 3A-C, E, F), where small collaterals formed varicose projections. In contrast, the axons of the SOG- and PTG-descending elements occu- pied more lateral regions and were located within the LDT (Fig. 3A).

Dorsal and ventral neuropiles were connected by collaterals emerging from the LDT (Fig. 3C). DA/TH-ir varicose terminals were observed in the neuropilar regions with similar density (Fig. 3A, D). Only the ventral association centre (VAC) showed no DA/TH-ir innervation (Fig. 3A). Intersegmental fibre tracts were often sur- rounded by varicose DA/TH-ir endings (Fig. 3A, C).

MSG. A maximum of eight DA/TH-ir bilateral symmetric somata was stained in this ganglion (Figs. 1F, 4C). As observed in other ganglia, medial somata (TH- MSG1, n=0-6, diameter: 15-20 gin) varied in number and could be discerned from two regularly stained elements (TH-MSG2, diameter: 20 gin) lying in a posterio- lateral position near the roots of the connectives. Axons of the ventro-median TH-MSG1 cells were traced only within the MSG. Posterior TH-MSG2 cells showed ipsilaterally ascending axons that projected anteriorly in parallel with the VMTs and were thus different from DA/TH-ir neurones in the SOG or PTG in a posterio- ventral position.

Lateral and medial intersegmental fibre profiles could be identified in the MSG. Descending axons from brain cells were found exclusively in medial regions where collaterals were discernible (Figs. IF, 3C). Descending axons of somata of the SOG and PTG were located in the LDT giving off collaterals innervating mostly lateral neuropilar regions.

MTG. Only two bilaterally paired cells were regularly stained (TH-MTGI,2, diameter: 20-30 gm, Figs. 2F, 4D) in the MTG neuromere. Medial somata (TH-MTG1) appeared only weakly labelled in a ventral position with no coherent staining of fibres. These cells were found only in about half of the preparations. Ventral TH- MTG2 somata in a posterio-lateral position were located

in the first fused abdominal neuromere of the MTG. Ip- silateral arborizations with fine varicosities emerged from the neurite that projected anterio-dorsad. The descending axons (diameter: 3-5 gm) entered the LDT and could be traced to the TG. The same pattern of axonal collaterals as described for other thoracic ganglia was observed in the MTG. Descending neurones of the brain showed medial arborizations, whereas descending neurones of the SOG and PTG exhibited lateral arborizations. This "ladder-like" arrangement of collaterals (Fig. 2B-G) possibly represented segmentally homologous arborizations.

AG. In the well-stained abdominal preparations (Fig. 2A, D, E), DA/TH-ir cell bodies (TH-AG1, n=0-2, diameter: 10 gin) were detected exclusively in the third unfused abdominal ganglion (Fig. 2D). The projections of these neurones appeared to be restricted to the ganglionic neuropile and no indications for intersegmental fibres originating from these abdominal cells were observed. On the other hand, up to six intersegmental fibres were seen in each ganglion hemisphere (Figs. 2A, E, 4E, F). The most prominent projections emerged from lateral intersegmental fibres originating from the descending thoracic elements. Anterior and posterior collaterals from these axon profiles extended to medial regions forming a similar "ladder-like" projection pattern to that found in the MTG. Medial intersegmental fibres from descending brain cells gave off only a few collaterals that were restricted to medial regions of the ganglion.

Terminal ganglion. The TG consisted of five fused neuromeres (Murphy 1981). The most posterior neuromere was reduced and contained only a few cells (Panov 1966). A set of three bilaterally paired cells (DA/TH- TG1-3) was constantly found in the TG with both antise-

)

Fig. 3A-F, DA-ir and TH-ir elements in tracts and neuropiles of the prothoracic ganglion (PTG); 50-~tm Vibratome sections. A Cross-section through the medial region of the PTG showing dense DA-ir innervation with varicose arborizations in associative neuropile areas, whereas most ventral sensory neuropiles lack DA-ir innervation; axon profiles are present in the LDT (black arrowheads) and VMT (white arrows); encircled area, VAC region free of DA-ir structures, x160. B Cross-section through the anterio-ventral region of the PTG; white arrows, three TH-ir axon profiles in each VMT; white arrowheads, TH-ir collaterals within the intrasegmental LVT. x250. C Cross-section through a dorso-medial region showing stained profiles and collaterals in different tracts; asterisks, neurites of medial TH-ir somata marked in Fig, 1D; white arrows, 2-3 axon profiles regularly found in the VMT; black arrows, neurites from the large medial somata marked in Fig. ID. xlT0. D Micromorphology of DA-ir varicosities in the medio-lateral neuropile of the PTG. x300. E Horizontal section through the ventral part of the PTG; white arrows, medially descending fibres within the VMT (compare with B); black arrows, neurites emerging from the somata marked in Fig. 1D; encircled area, position of the VAC lacking TH-ir innervation (compare with A). x130. F Cross-section through the connectives posterior from the PTG showing small intersegmental TH-ir fibres; each connective contains two dorsal profiles from neurones descending from the SOG and PTG (black arrows) and 2-3 ventral fibres from brain neurones (white arrows), x150. Scale bars: A, C, E, F: 100 ~tm; B, D: 50 ~tm

t_,h

SOG

S(

D o p a m i n e - ' l m u n o s t a i - - - - -

MT

M'

A E

\

PTG 2

PTG3

!

B

P-- ! -

MSG1

C

Fig. 4A-G. Complete mapping of TH-ir neurones in the suboesophageal (SOG), thoracic, abdominal (AG) and terminal ganglia (TG). The ganglionic distribution of all DA/TH-ir somata, their main arborizations, the course through intersegmental fibre tracts

G

and their terminal projections are drawn from a whole-mount preparation. A SOG; B PTG; C MSG; D MTG; E third free AG; F fourth free AG; G TG. Scale bar: 100 gm

593

ra used (Figs. 2C, G, 4G). An additional cell pair (TH- TG4) was stained in in the 10th neuromere in some preparations. Except for the TH-TG4 ceils (diameter: 10-15 gin), which were observed in a medial position at the posterior border of the ganglion, the other three somata pairs (DA/TH-TG1-3, diameter: 20-25 gin) were located dorso-Iaterally close to the lateral margin of the ganglion. Neurone reconstruction revealed that all projections of DA/TH-ir cells remained within the TG.

In addition, a dense plexus of varicose fibres was seen emerging from the plurisegmental brain and thoracic interneurones described above. In dorso-medial neuropile regions, lateral DA/TH-ir collaterals (Fig. 2B, C, F) from thoracic cells showed the typical symmetrical pattern that was also observed in more anterior abdominal segments. Accumulated varicosities from these fibres were observed in the cercal glomeruli in the region of the giant fibre dendrites (Fig. 2G). Terminals originating from medial fibres could be attributed to cells descending from the brain.

Distribution of 5-HT-immunoreactive neurones

A total number of 200 neurones showed 5-HT-ir labelling in the CNS including the brain (without optic lobes; Tables 1, 2; n=105 preparations). Of these somata, 120 were found in the VNC. Each neuromere contained 5- HT-ir neurones that were mainly arranged in bilateral symmetric cell clusters. The majority of 5-HT-ir cells were interneurones. In addition, efferent neurones in the SOG and TG and sensory fibre projections in the thoracic ganglia were regularly observed. The two antisera revealed the same 5-HT-ir neurones and arborizations. As a general rule, each neuromere contained a posterior bilateral symmetric 5-HT-ir cell group with up to three large neurones per hemiganglion. They showed either intrasegmental or ascending intersegmental projections in the soma-contralateral ganglion hemisphere, as described for serial homologous cells derived from a common neuroblast in the grasshopper embryo (Taghert and Goodman 1984). Serial homology could reasonably be assumed for the large posterior cells described in the present study (referred to a s 5-HTseg neurones in the following paragraphs). In contrast, smaller medially arranged cell clusters were less intensely stained and showed more variable numbers.

Varicosities showing immunoreactivity for 5-HT were present in all neuropile areas and appeared especially concentrated in ventral regions. A plexus of 5-HT-ir varicose fibers was also stained on the surface of peripheral nerves of the SOG and TG.

Cerebral ganglion. The distribution of 5-HT-ir somata in the insect brain has previously been described (e.g. Bish- op and O'Shea 1983; Schtirmann and Klemm 1984; Tyr- er et al. 1984; Schtirmann and Woermann 1986; N/~ssel 1987; Homberg and Hildebrand 1989). Therefore, only a brief survey is given here. In the brain (without optic lobes) of GrylIus bimaculatus, about 60-80 5-HT-ir cells were distributed in bilateral symmetric cell clusters; ad-

ditionally four single 5-HT-ir cells could be identified in each hemisphere. Descending fibres, originating from a prominent soma cluster located on either side of the midline of the frontal protocerebrum and also identified in the locust brain (Williams 1975; Tyrer et al. 1984), were traced to the ventral ganglia. Their axons running in the dorso-median tract (DMT) were characterized by medial tufts in distinct ganglionic regions of the VNC. The descending axons were traced until the 1 st unfused AG. In the deuto- and tritocerebrum, many terminals from ascending serial homologous suboesophageal and thoracic 5-HT~g cells were present.

SOG. All 5-HT-ir cells in this ganglion (n=20-32; Figs. 5A-E, 8A) were regularly arranged in four bilateral symmetric cell clusters and two unpaired median groups, one in an anterior and one in a posterior position. Be- cause of their characteristic morphological features, three of the four lateral groups of 5-HT-ir somata occur- ring in each of the three SOG neuromeres were regarded as serial homologues (5-HT-SOGseol-3; n=2-6, diameter: 15-20 ~tm; Figs. 5A, B, 8A). ~'he most anterior unpaired dorso-median cell cluster (5-HT-SOG1; n=0-6; diameter:30 gin) varied in staining intensity. However, in some preparations, their efferent axons were identified in the N. mandibularis (similar efferent 5-HT-ir neu- tones supplying the muscle adductor mandibularis have been described in the locust SOG; Baines et al. 1990).

Compared with all other 5-HT-ir somata, the bilateral symmetric cluster in the ventral region of the first SOG neuromere (5-HT-SOG2; n=2-t2; diameter: 50-80 gin; Fig. 5C) with large 5-HT-ir somata did not appear uni- formly stained. Instead, granular particles were seen in the cytoplasm, indicating the neurosecretory character of these cells (Panov 1980; Raabe 1982). The varicose fibre projections regularly present on the surface of peripheral SOG nerves (Fig. 5D, branch of the N. mandibularis) probably belonged to these neurones.

In animals starved before preparation, one additional ventral 5-HT-ir cell pair (5-HT-SOG3; n=0-2; diameter: 30-40 gin) with efferent axons in N. 7b was observed in a posterio-medial location (Fig. 5E). Dense 5-HT-ir varicose terminals from these fibres were found in salivary gland tissue [Fig. 5F; cf. a 5-HT-ir cell with the same efferent innervation pattern of the salivary glands as described in the locust by Tyrer et al. (1984)]. The large 5-HT-SOG3 cells were accompanied by a group of small medial somata (5-HT-SOG4; n=0-8; diameter: 10 ~tm). Intersegmental fibres (Table 1) were found both in dorsal and ventral tracts; medial tufts near the ganglionic midline were typical for axons descending from the brain (axon diameter: 5-7 gin; Fig. 5G). The most intense 5- HT-ir labelling of the neuropile was observed in the anterio-ventral region (equivalent to the ventral thoracic association centres, which also contain terminals from sensory fibres; Kien et al. 1990).

PTG and MSG. In the PTG and MSG, the number and fibre projections of 5-HT-ir neurones were similar and therefore their arrangement is described together. Immu- noreactive somata could be classified into five bilateral

594

symmetric groups of interneurones, the most posterior of which represented the serial homologous 5-HTse ~ cells mentioned above (Figs. 5H, I, 6A, 8B, C). In addition, sensory fibres were present in N. 2 (Fig. 7D, E). The bilaterally paired 5-HTseg cells (n=4, diameter: 30 gm) were located in the posterio-lateral ganglion region (Figs. 5H, I, 6A). Each group comprised one intra- and one intersegmentally projecting element. The ascending axons (diameter: 5-7 gm) of the latter in the ventro-mediate tract (VIT) were traced to the SOG. Both neurone types projected to the soma-contralateral ganglion hemisphere via a ventral commissure located posterior from the posterio-ventral commissure (Fig. 7B, F, H). Bilater- ally arranged 5-HT-PTG/MSG4 cell clusters (n=0-20, diameter: 10-15 gm) were associated with 5-HT~e~ neurones but were located further anteriorly (Figs. 5H, 6A, 8B, C). Dense fibre projections were found in all associative neuropile areas. Median collaterals formed prominent anterio-dorsally projecting processes (Fig. 7G, F, H) in close vicinity to fibre tufts originating from plurisegmental 5-HT-ir cells descending from the brain (Fig. 76).

Anterio-lateral 5-HT-PTG2 cells (n=2, diameter: 20 gin) allowed further identification of their arborizations. Histological analysis of Vibratome-sectioned ganglia (n=22 preparations) revealed a characteristic ~2- shaped morphology of 5-HT-PTG2 cells, similar to that of identified auditory neurones (ON1, Wohlers and Hu- ber 1982; Fig. 7B). Possible homologues of 5-HT-PTG2 neurones were detected in the anterior region of the MSG (Fig. 6A; 5-HT-MSG2; n=2, diameter 20 ~tm) but their fine branches were not investigated in further detail. Compared with the 5-HT-ir neurones described so far, the remaining ventral 5-HT-PTG/MSG1,3 somata were smaller and varied considerably in staining intensity and position.

Immunoreactivity for 5-HT was also found in some sensory elements�9 In the MSG, sensory fibres entered via N. 2 and projected into the ipsilateral portion of the VAC (Fig. 7D, g).

Intersegmental fibres descending from 5-HT-ir cells in the brain gave off characteristic fibre tufts at the level of the peripheral nerve roots. Ascending fibres from 5- HTseg neurones located in the MSG or MTG were observed in the VIT and lateral-ventral tract (LVT; Fig. 7A, C).

MTG. The MTG comprised three fused neuromeres, each of which contained at least two bilateral groups of the serial homologous 5-HTs~g neurones (5-I-IT- MTGsegl-3, Figs�9 6B, 8D). However, differences in number and morphology were observed in the MTG compared with thoracic 5-HTseg cells�9 The metathoracic neuromere only contained one single 5-HT s cell pair eg (diameter: 30-40 gin). As in the thoracic ganglia, small 5-HT-MTG4 somata (n=4-12, diameter: 10 gin) were associated with them�9 Whereas the cell morphology of 5-HT-MTGseA cells in the metathoracic segment was similar to that observed in other thoracic segments, the axons from the 5-HT-MTGse_2 neurones (n=2, diameter: 20-30 gm) m the first abdominal neuromere did not pro-

ject into the VIT but into the LVT. The second fused abdominal neuromere contained two laterally positioned 5- HT-MTGse 3 somata (n=4, diameter �9 30-40 gin) with

g . . �9

contralaterally ascending axons in the VIT or LVT, respectively. These fibres were traced to the anterior region of the PTG. Anterior 5-HT-MTG1/2 (n=0-4, diameter: 10-15 ~tm) and medial 5-HT-MTG3 (n=0-9, diameter: 10-20 gin) showed variable staining and could not be traced further.

AG. Bilateral symmetric 5-HT-AGse z neurones (n=2-6, diameter: 30 gin; Figs. 6C, 8E, F) and associated ventro- median cells (5-HT-AG1, n=0-6, diameter: 10-15 ~tm) were observed in the 1st unfused AG. The 5-HT-AGse ~ elements were always intensely labelled and showed, un- like homologous cells in thoracic ganglia, intersegmentally ascending axons in the LVT and VIT. In addition, a fine ipsilaterally descending projection was observed in some cases. Ventro-median 5-HT-AG1 neurones often appeared less intensely stained and were only present in the 1 st unfused AG. Fine collaterals orginating from intersegmental 5-HT-ir fibres were observed in all ganglionic regions. Medial fibres (Figs. 5G, 7G) could be as- cribed to neurones descending from the brain.

TG. In this ganglion, we classified three dorso-lateral (5- HT-TGsegl-3) and four ventro-lateral soma groups (5- HT-TG1-4) in each hemiganglion. In contrast to the thoracic ganglia and AG three further groups of efferent cells (5-HT-TG5-7) occurred in the TG. Their number showed sex-specific variations with more 5-HT-ir elements being regularly stained in females (Figs. 6D, 7G). Dorso-lateral cell groups (5-HT-TGsegl-3, n=l-5, diameter: 15-30 gm) were found in neuromeres 7-9 (nomen- clature according to Murphy 1981). As in the thorax, these cells had intersegmentally ascending axons (diameter: 5-7 gm) and reached the soma-contralateral ganglion hemisphere via ventral commissures (Fig. 6D).

Fig. 5A-I. Neurones immunopositive for 5-HT in whole-mount preparations and Vibratome sections of the suboesophageal (SOG) and prothoracic ganglia (PTG); anterior or dorsal to the top. A Anterio-dorsal aspect of the SOG with lateral 5-HT-SOGs~.I neurones, dorso-median 5-HT-SOG1 cells and mtersegmental fibres. x170. B Posterio-ventral aspect of the SOG with lateral 5-HT- SOG,eg3 neurones forming a ventral commissure (asterisk) that gives rise to ascending axons (black arrows), x170. C Cross-section through the anterior region of the SOG with ventral 5-HT- SOG2 somata and intersegmental fibre profiles (black arrows) in the dorsal connectives, x90. D Plexuses of varicose fibres on the N. mandibularis of the SOG. x200. E Large efferent ventro-medial 5-HT-SOG3 neurones innervating the salivary glands. Note the small median 5-HT-SOG4 cells, x200. F Varicose fibres in the salivary gland tissue emerging from efferent 5-HT-SOG3 cells shown in E. x360. G Dorso-medially descending axons (black arrowheads) forming fibre tufts in the PTG. x340. H Sagittal Vibratome section of the PTG with 5-HT-PTG,eg neurones and small 5-HT- PTG4 cells; black arrows, intersegmentally ascending fibres from other ganglia, x140. I Whole-mount preparation of the PTG (one hemiganglion shown) with 5-HT-PTGse cells and their intraganglionic (white arrowhead) and mtersegmentally ascending (black arrow) fibre projections, xlS0. Scale bars: A-C, H, I: 100 gin; D- G: 50 ~xm

595

596

597

Ventro-lateral 5-HT-TG1-4 somata (n=0-4, diameter: 15-30 btm) were observed in close medial vicinity to each dorso-lateral 5-HT-TGseg cell group. The former neurones projected to the so-ma-contralateral ganglion region. However, no ascending axons could be identified. The axons crossed the ganglion midline via ventral commissures (Fig. 6D), separate from those described for the 5-HT-TGseg neurones.

Furthermore, two single neurones and one posterio- median group of efferent cells could be discerned in each ganglion hemisphere (Fig. 6D). The two single ventro-lateral 5-HT-TG5/6 neurones (diameter: 40-50 gin) sent axons into the ventral ipsilateral N. 8 (5- HT-TG5) and N9 (5-HT-TG6), respectively. These 5-HT- ir cells were regarded as being homologous to motoneu- roues identified in Acheta (Hustert and Topel 1986) supplying specific genital muscles in crickets. The anterior 5-HT-TG5 cells were never observed in male crickets. Sex-specific differences were also detected in the ventro-median 5-HT-TG7 neurones that lay near the posterior ganglion border and that sent efferent axons to the hindgut nerve. Whereas cells were clustered (n--4-11, diameter: 35-50 gm) in female crickets, the equivalent neurones (n=2-13) were scattered in the posterior region of the ganglion in male crickets. Peripheral varicose fibres on the surface of N. 8 (Fig. 6E), N. 9 and the hindgut nerve (Fig. 6F) originated from 5-HT-TG5, -6 and -7 cells, respectively. Central varicose projections of 5-HT- ir fibres were concentrated in the anterio-ventral neuropile and in dorso-medial regions of the posterior neuromeres (Fig. 6D).

Discussion

Our results achieved with different antisera (poly- and monoclonal antibodies) directed against different anti-

Fig. 6a-E Neurones immunopositive for 5-HT in the meso- (MSG), metathoracic (MTG), abdominal (AG) and terminal ganglia (TG); whole-mount preparations; anterior to the top). A Gen- eral arrangement of somata and projections in the MSG; black arrows, ascending axons of 5-HT-MSGseg cells; white arrowheads, intraganglionic projections from 5-HT-MSGseg neurones; black arrowhead ascending fibres from 5-HT-ir cells in the MTG are accompanied by small 5-HT-MSG4 cells, x70. B Pattern of 5-HT- MTGseg cells and their major fibre projections in the MTG; ascending axons (black arrows) and ganglionic projections (white arrowheads) from 5-HT-MTG e neurones (somata partially out of sg focus), x110. C Second free abdominal ganglion with 5-HT-AGseg neurones with intersegmentally ascending axons (medial neurones partially out of focus), x170. D Medio-ventral aspect of the TG of a female cricket with efferent 5-HT-TG5-7 neurones (asterisks); one pair of segmental homologous 5-HT-TGsegl neurones is visible in the anterior ganglion region; the others are out of focus (ventral commissures are partially out of focus). Note the prominent varicosities in anterior and posterior neuropiles (white arrows), x190. E Varicose fibres on distal branches of N. 8 of the TG. Projections emerge from an efferent 5-HT-TG5 neurone observed only in female crickets (compare with D). x190. F Vari- cose fibres on the hindgut nerve originating from efferent 5-HT- TG7 neurones (compare with D) in the TG of a female, x145. Scale bars: 100 btm

gens (dopamine itself, TH and 5-HT) together with specificity tests and controls, reliably indicate the cellular detection of the two amines in question. The investigation of the whole VNC confirms and extends the assumption of similar labelling of DA-ir and TH-ir somata in insects (N~issel and Elekes 1992). In the TG, which contains six DA-ir neurones (Elekes et al. 1987; see also Fig. 2C), two additional TH-ir elements have been stained in some preparations (Fig. 2G). A similar distribution of TH-ir and DA-ir neurones has also been found in the CNS of vertebrates (Gonzales and Smeets 1991). Tracing of stained fibres in whole-mount preparations and subsequent histological analysis have resolved novel features concerning the segmental arrangement, single cell morphology and neuropile architecture of DA/TH-ir and 5- HT-ir elements. Comprehensive cell mapping has allowed a description of the topography and interrelation- ship of major DA and 5-HT pathways showing that DA/TH-ir and 5-HT-ir neurones contribute to the VNC layout in different ways.

Distribution of DA/TH-ir and 5-HT-ir neurones and projections

As summarized in Tables 1 and 2, both DA/TH-ir and 5- HT-ir neurones are limited in number. The quantity of ir neurones corresponds well with data from locusts where only 1% of the total cell number per segment shows 5- HT immunoreactivity (Taghert and Goodman 1984). Comparable numbers of cells containing other biogenic amines have been detected by immunocytochemistry in the cricket VNC (octopamine: Sp6rhase-Eichmann et al. 1992; histamine: H6rner et al. 1994). Other transmitters, such as gamma-aminobutyric acid are typically more abundant and present in up to 20% of the ganglionic neurones (SpOrhase-Eichmann et al. 1989). A small number of distinct DA-ir and 5-HT-ir wide-field neurones with intersegmental projections innervates all segments from the brain to the TG. Aminergic terminals with typical varicose endings appear co-distributed in dorso-median neuropiles of the whole VNC. Although the diffuse aminergic projections often show considerable overlap, the axons can be assigned to specific cell bodies. A co-localization of DA and 5-HT can be excluded. In addition, a co-localization of 5-HT or DA with either octopamine (Sp6rhase-Eichmann et al. 1992) or histamine (H6rner et al. 1994) seems unlikely. Wide- field projection neurones with varicose terminals have also been found in aminergic cells of the vertebrate CNS (Consolazione and Cuello 1982). However, despite these morphological similarities, a detailed histological analysis has also revealed substantial differences between DA-ir and 5-HT-ir cells in the cricket CNS.

The majority of DA/TH-ir somata are bilateral symmetric single cell pairs different from the clustered 5- HT-ir neurones. DA-ir cells have been demonstrated to be clonally related and therefore segmentally arranged in Drosophila (Huff et al. 1989). In the cricket, however, segmental repetition of DA/TH-ir somata has only rarely been observed. Evidence for a serial homologous ar-

598

Fig. 7A-H. Elements immunopositive for 5-HT in tracts and neuropiles of the pro- (PTG; A-C, H) and mesothoracic ganglia (MSG, D-G); 50-gm Vibratome sections, dorsal or anterior to the top). A Cross-section through the PTG in the region of the anteri- or root of N. 5 with 5-HT-ir innervation in all neuropile areas. Note that ventral neuropiles, namely the VAC (encircled areas),

show the highest density of varicosities; fibre tufts of descending brain neurones (asterisk), and intersegmentally ascending (white arrowheads) and intrasegmental projections (black arrows) of 5- HT-PTGs~g neurones, x130. B Horizontal section through the ventro-median region of the PTG showing the ~-shaped morphology of the 5-HT-PTG2 neurone in the anterior and the typical pos-

599

rangement of DA/TH-ir somata is seen only for prominent medial DA/TH-ir cells in the SOG (DA/TH-SOG4 Fig. 1B) and PTG (DA/TH-PTG3 Fig. 1D, E). Both show a similar soma position, axonal branching and descending projections. The similar pattern of axonal collaterals from descending cells in the abdominal portion of the VNC and the regular arrangement of DA/TH-ir neurones in the TG (Fig. 2A-G) point to a segmental supply of the VNC by DA/TH-ir elements. A segmental arrangement of DA/TH-ir somata may, therefore, be re- placed by segmental repetition of fibre elements in the AG.

As opposed to DA/TH-ir neurones, the 5-HT-ir cells demonstrated in this study are segmentally arranged cor- responding to the serial homologous distribution of 5- HT-ir neurones demonstrated in other insect species (Taghert and Goodman 1984; Tyrer et al. 1984; Elekes et al. 1987; N~issel 1987; van Haeften and Schooneveld 1992). Homologues of the large posterior 5-HTse_ neurones demonstrated here have been shown to be derived from a common neuroblast in grasshoppers (Taghert and Goodman 1984).

Most DA/TH-ir neurones identified in the cricket are plurisegmental interneurones with descending axons that converge in the thoracic ganglia and AG. All DA/TH-ir brain and thoracic neurones identified so far show their main arborizations and descending axons on the soma- ipsilateral side. Parts of this descending DA-ir pathway have also been identified in the locust brain, thorax and abdomen (Orchard et al. 1992; Watson 1992; Wendt and Homberg 1992). Compared with locusts (Watson 1992; n=14 DA-ir cells) the larger number of DA-ir cells found in crickets (Tables 1, 2) might represent species-specific differences, although discrepancies caused by the use of different antisera cannot be excluded. Most of the neurones identified by Watson (1992), however, correspond to DA/TH-ir elements found in the cricket VNC.

In contrast to the mainly descending DA/TH-ir projections, the most prominent 5-HT-ir varicose plexuses

terior commissure (asterisk) of the 5-HT-PTGseg neurones. The f~- shaped 5-HT-PTG2 neurones have prominent projections in the auditory neuropile (white arrows), x120. C Cross-section near the anterior margin of the PTG; ascending fibres (black arrows) in the LVT and VIT, and descending brain fibres (black arrowheads) in the DMT. Note imrnunopositive intersegmental sensory elements (asterisks). x150. D Horizontal section through the anterio-ventral region of the MSG showing bundles of parallel 5-HT-ir sensory fibres (black asterisks) entering the MSG through N. 2; intersegmental 5-HT-ir fibres (black arrowheads) are visible in the connectives, x130. E Horizontal section through the MSG showing terminals of 5-HT-ir sensory fibres in the anterior VAC (encircled area), xll0. F Cross-section through the posterior commissure formed by 5-HT-MSGse 8 neurones invading large areas of the MSG. x340. G Horizontal section through the posterio-median region of the MSG showing a close arrangement of axon-collaterals (white arrowheads) from ascending thoracic neurones with descending axons (black arrowheads) originating from brain neurones. x230. H Sagittal section through the median region of the PTG; collaterals of commissural fibres (white arrowheads) curve anterio-dorsad. Note that the ventral 5-HT-ir commissure (asterisk) is separated from the posterior ventral commissure (black arrow). x90. Scale bars: A-E, H: 100 btm; F,G: 50 btm

belong to interneurones with axons ascending contralaterally to the soma position. They do not span the whole CNS, as do DA-ir fibres, but innervate the neuromere of the soma and the neighbouring 1-2 ganglia. This also applies to brain neurones whose descending axons have been followed until the 1st unfused abdominal neuromere and do not reach the TG, as has been observed for DA/TH-ir elements in the brain.

In addition, local 5-HT-ir interneurones have been identified in the VNC. All morphological details of one prominent local neurone (5-HT-PTG2; Fig. 7B) such as (1) the anterio-lateral position of the soma, (2) the course of the neurite in the ventral commissure I and (3) the shape and micromorphology of the dendrites not crossing the ganglion midline are similar, if not identical, with those of the acoustic ON1 interneurone described by Wohlers and Huber (1982, 1985). Immuno- cytochemical data from Hardt and Agricola (1991) also suggest the presence of 5-HT in the ON1 neurone. In addition, in staining of histamine-ir neurones in the cricket VNC, we have not detected any labelled cell bodies in the PTG, although intersegmental fibres appear heavily stained (H6rner et al. 1994). We, therefore, suggest that 5-HT is present in the ON1 cell, although Skiebe et al. (1990) have shown that histamine mimicks some of the effects of ONI activity.

Furthermore, efferent DA/TH-ir and 5-HT-ir fibres are observed in the SOG. Based on our selective immunostaining and additional single-cell Lucifer-Yellow injections, we suggest that the DA/TH-SOG2 neurones (Fig. 1B, C) projecting to the salivary glands of GrylIus are homologous to DA-ir SNl-cells in the SOG of other orthopterans (Gifford et al. 1991). The remarkably intense and invariant immunostaining of these cells corre- lates well with the high DA content determined bio- chemically (Gifford et al. 1991).

The medial 5-HT-SOG3 cells with efferent axons in N. 7b observed in crickets probably correspond to the SN2-cells innervating the salivary glands in locusts and cockroaches described by Gifford et al. (1991). The fact that these neurones show immunostaining only in starv- ing animals indicates a variable cellular 5-HT content depending on the physiological state. Biochemical studies have shown that the content of 5-HT in the SN2 cell is only about three times higher than in controls (Gifford et al. 1991), a result that might explain variable immunostaining.

Whereas efferent DA-ir cells exclusively occur in the SOG, efferent 5-HT-ir neurones are also present in the TG. In female crickets, two additional efferent 5-HT-ir cells compared with males are found that have been shown to innervate the genital chambre (Hustert and To- pel 1986).

Afferent DA-ir projections have never been observed in the CNS. Instead, a histological inspection shows that the ventral neuropiles, which have been described as sensory projection centres (Tyrer and Gregory 1982; Wohlers and Huber 1985; Kien et al. 1990), are the only central neuropiles devoid of DA/TH immunostaining.

In contrast, terminals of sensory 5-HT-ir projections entering the CNS via the peripheral nerves are found in

600

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MTG 3

MTGseg 1

MTGs,

A D

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601

the thoracic ganglia in the ventral neuropile, exactly in those regions that are devoid of DA/TH-ir projections (compare Figs. 3A, 7A). The ventral sensory centres show the most intense 5-HT immunolabelling in the whole VNC. Afferent 5-HT-ir projections in the thorax are similar to the projections from exteroceptive bristle hair afferents described by Johnson and Murphey in Acheta (1985). Sensory cells from the proprioceptive chordotonal organ have been found in the same location in locusts (Lutz and Tyrer 1988).

Distribution of DA/TH-ir and 5-HT-ir varicosities

As opposed to the occurrence of peripheral nerve 5-HT- ir varicosities, we have found no DA/TH-ir varicosities outside the CNS neurilemma (except salivary gland innervation) or associated with neurohemal organs. DA/TH-ir projections appear to be restricted to the central neuropiles indicating that DA-ir neurones are typically interneurones.Varicose terminals showing immunoreactivity for 5-HT are observed on the surface of peripheral nerves that can be regarded as possible sites for neurohemal release. Furthermore, 5-HT-ir varicose fibres present on SOG and TG nerves are arranged in two different ways: in the SOG, varicose fibres extend from proximal to distal regions of the N. mandibularis. In fa- vourable preparations, thin connections of the paired anterior 5-HT-SOG2 cells and peripheral varicosities are visible. The cell bodies show a granular matrix, typically found in neurosecretory cells (Panov 1980; Raabe 1982). Possible homologues of 5-HT-SOG2 cells in crickets have also been described in a number of other insects (Davis 1987; N~issel and Elekes 1985; Br~iunig 1987; Homberg and Hildebrand 1989; van Haeften and Schoo- neveld 1992). In the TG, varicose fibres are found in distinct distal regions of peripheral nerves. They can be attributed to efferent cells (5-HT-TG5-7, Figs. 6D, 7) and have, therefore, been called secretomotor neurones (Knowles and Bern 1966).

Functional aspects

The two biogenic amines investigated here by an immunocytochemical approach have been detected biochemi- cally in considerable amounts in all parts of the cricket CNS (Nagao and Tanimura 1988). The content of DA and 5-HT is higher in the brain than in the VNC, corre- sponding to the larger total number of DA/TH-ir and 5- HT-ir neurones in the head ganglia. The fact that the presence of synthesizing enzymes and degradation prod- ucts has been determined in the CNS indicates the po- tential role of DA and 5-HT as neuroactive substances. Ultrastructural investigations of DA-ir (Elekes et al. 1987; Nfissel et al. 1988) and 5-HT-ir (N~issel and Elekes 1984, 1985) neurones in the insect CNS indicate the putative transmitter character of the two amines. However, accumulation of 5-HT-immunolabelling in dense-core vesicles has also been observed at non-synaptic sites, possibly indicating a paracrine release and a neuromodu- latory function (see N~issel 1987). A possible neuromod-

ulatory action of DA has been postulated by Davis and Pitman (1991) who have investigated the physiology of the CI-neurone in the cockroach. Moreover, in vertebrates, ultrastructural and physiological investigations point to a paracrine release of biogenic amines (Bach-y Rita 1993).

The cellular effects of both amines have been most extensively studied in peripheral targets such as the salivary glands, which are innervated both by DA-ir and 5- HT-ir fibres (Klemm 1972). Evans and Green (1990) have shown that DA has both a direct electrical effect on the salivary gland cells and that it induces secretion via a separate DA-sensitive G-protein-coupled adenylate-cy- clase mechanism. Futhermore, 5-HT has been shown to act via G-protein-coupled enzyme systems (Evans and Green 1990; Ali et al. 1993) similar to those of many amine receptors involved in modulatory circuits (see Roeder 1994). The DA-ir (SN1) and 5-HT-ir (SN2) salivary effector cells identified in the SOG seem to control secretion collectively in the salivary gland, since they are co-activated during feeding behaviour (Schachtner and Br~iunig 1993).

Compared with the studies dealing with the peripheral effects of DA and 5-HT cited above, our knowledge of physiological amine actions in the insect CNS is much more limited (Menzel et al. 1989; Erber et al. 1993). In the thoracic ganglia, DA administration influences the flight motor pattern (Claassen and Kammer 1986). This effect can be antagonized by 5-HT. DA furthermore af- fects the well-described cockroach escape behaviour, as DA injections into the MTG potentiate synaptic transmission between ventral giant fibres and leg motor neurones (Goldstein and Camhi 1991; Casagrand and Ritz- mann 1992). Again, 5-HT has the opposite effect, decreasing the response of the leg motor neurones. Given the intersegmental DA/TH-ir and 5-HT-ir projections in the TG, together with the arborizations of local interneu- tones demonstrated here and in Acheta (Elekes et al. 1987), one would expect a most pronounced effect of DA and 5-HT in this ganglion. However, experiments in cockroaches with bath-applied DA or 5-HT have not revealed significant effects in the TG of cockroaches (Goldstein and Camhi 1991). Interestingly, modulation of sensory responsiveness has been observed in many interneurones involved in the control of cricket escape behaviour (H6rner et al. 1989; H6rner 1992; Gras and H6rner 1992) or phonotactic walking (Schildberger and H6rner 1988) and could partially rely on amine effects.

Despite the lack of unequivocal ultrastructural and physiological data on the aminergic cells presented here, morphological features such as the wide-field DA/TH-ir and 5-HT-ir neurones with their multi-terminal varicosities and highly divergent plurisegmental projections indicate a modulatory function as suggested by Kravitz (1989). The limited number Of aminergic neurones invading most neuropiles of the VNC seems to be well suited to mediate collective spatial and temporal coordi- nation of ensembles of neurones in different parts of the CNS involved in behaviour.

On the other hand, the substantial overlap of DA/TH- ir and 5-HT-ir varicosities in the thoracic ganglia indi-

602

cates a two-fold control of identical functional networks in one and the same ganglion, pointing to a concerted action of the two amines. Al though areas o f superim- posed aminergic arborizations do exist, we can show that they belong to different cell types representing function- ally distinct neurone systems. This might explain the antagonistic interactions of 5-HT and DA in the cricket escape system (Goldstein and Camhi 1991; Casagrand and Ritzmann 1992). Interactions between 5-HT and other biogenic amines have also been observed in other invertebrates (e.g. 5-HT/octopamine; Kravitz 1989) and vertebrates (e.g. 5-HT/norepinephrine; B loom 1981).

In conclusion, DA appears merely as a central neuroactive compound acting predominant ly in integrative and motor centres in dorso-medial neuropile regions where terminals of plurisegmental descending DA/TH-i r interneurones show converging projections in the thorax and TG. In contrast, 5-HT is found in other cell types and is mainly represented in segmentally arranged ascending and local interneurones connecting neighboring ganglia. In addition, 5-HT immunoreact ivi ty occurs in afferent cells and in a number o f efferent neurones with peripheral varicose fibre projections. Therefore, 5-HT may be in- dependently released in different compartments, pointing to a multifunctional role including the control o f afferent, central and efferent circuits.

Acknowledgements. We appreciate the valuable comments of Dick N~issel and Heribert Gras concerning our manuscript. Further- more, we thank Klaus Schildberger for helpful discussions. We should also like to thank Marion Knierim-Grenzebach and Marg- ret Klages for technical assistance and their patience in helping us to assemble the figures. This work was supported by the Deutsche Forschungsgemeinschaft, grants Ho 1507/2-1 and Schu 374/9-2.

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The distribution of neurones immunoreactive for ?-tyrosine hydroxylase, dopamine and serotonin in the ventral nerve cord of the cricket, Gryllus bimaculatus