Serotonin-immunoreactive neurons in scorpion pectine neuropils: similarities to insect and crustacean primary olfactory centres?

Zoology 115 (2012) 151–159

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Serotonin-immunoreactive neurons in scorpion pectine neuropils: similarities to insect and crustacean primary olfactory centres? Harald Wolf a,b,∗ , Steffen Harzsch c a b c

Institute for Advanced Study Berlin, Wallotstraße 19, D-14193 Berlin, Germany Institute of Neurobiology, University of Ulm, Albert-Einstein-Allee 11, D-89081 Ulm, Germany Zoological Institute and Museum, Department of Cytology and Evolutionary Biology, University of Greifswald, Soldmannstraße 23, D-17487 Greifswald, Germany

a r t i c l e

i n f o

Article history: Received 10 March 2011 Received in revised form 17 October 2011 Accepted 20 October 2011 Keywords: Arthropoda Mandibulata Chemosensory glomeruli Serotonergic neurons Scorpion pectine neuropils

a b s t r a c t The pectines of scorpions are a single pair of mechano- and chemosensory appendages located ventrally behind the most posterior pair of walking legs. They are used for probing the substrate in behaviours such as prey tracking and courtship. The sensory afferents on the pectines supply large segmental neuropils with a conspicuous glomerular structure. The pectine neuropils thus bear similarities to insect and crustacean deutocerebral chemosensory centres associated with the antennae, but they also possess idiosyncratic features. One characteristic property of many insect and decapod crustacean olfactory neuropils is their innervation by single, or very few, large serotonergic (inter-) neurons. This feature, among others, has been proposed to support homology of the olfactory lobes in the two arthropod groups. A possible serotonergic innervation of the scorpion pectine neuropils has not yet been studied, despite its apparent diagnostic and functional importance. We thus examined serotonin-immunoreactivity in the pectine neuropils of Androctonus australis and Pandinus imperator. Both scorpion species yielded similar results. The periphery of the neuropil and the matrix between the glomeruli are supplied by a dense network of serotonin-immunoreactive (5-HT-ir) arborisations and varicosities, while the glomeruli themselves are mostly free of 5-HT-ir fibres. The 5-HT-ir supply of the pectine neuropils has two origins. The first is a pair of neurons on each body side, up to 30 ␮m in diameter and thus slightly larger than the surrounding somata. These cell bodies are and associated with the neuromeres of the genital and pectine segments. The situation is reminiscent of the 5-HT supply of insect and crustacean olfactory and antennal neuropils. The second 5-HT innervation of the pectine neuropils is from a group of some 10–20 ipsilateral neuronal somata of slightly smaller size (15–20 ␮m). These are part of a much larger 5-HTir group comprising 70–90 somata. The whole group is located more anteriorly than the single soma mentioned above, and associated with the neuromere of the last (4th) walking leg. When compared to data from other arthropods, our findings may suggest that glomerular organisation is an ancestral feature of primary chemosensory centres innervated by arthropod appendages. This idea needs further scrutiny, although supporting evidence may have been overlooked previously, due to the small size of chemosensory neuropils in walking legs and in reduced segmental appendages. © 2012 Elsevier GmbH. All rights reserved.

1. Introduction The striking similarities between the chemosensory, and especially olfactory, primary neuropils in animals have long intrigued both neuroanatomists and physiologists (see reviews by Strausfeld and Hildebrand, 1999; Eisthen, 2002). In groups related as distantly as higher vertebrates and arthropods, the afferent chemosensory receptor neurons terminate in structures called olfactory glomeruli. Glomeruli are more or less spherical, dense synaptic neuropils that

∗ Corresponding author at: Institute of Neurobiology, University of Ulm, AlbertEinstein-Allee 11, D-89081 Ulm, Germany. E-mail address: [email protected] (H. Wolf). 0944-2006/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2011.10.002

are well delineated from the surrounding tissue and usually occur in large groups. Their boundaries are formed by glia and the surrounding tissue consists of tracts formed by axons of the chemosensory afferents and of interneuron processes involved in further signal processing (Anton and Homberg, 1999; Strausfeld and Hildebrand, 1999; Eisthen, 2002; Homberg, 2005; Schmidt and Mellon, 2010). Two major factors have been implicated in this ubiquitous glomerular organization. The first is functional necessity, as suggested by the fact that glomeruli allow spatial segregation of inputs from chemoreceptor types with different response profiles. Indeed, the different types of receptor neurons project to particular glomeruli, often just to one glomerulus (e.g., Hansson and Christensen, 1999; Hansson and Anton, 2000; Vosshall and Stocker, 2007). This segregation by receptor neuron populations, and thus

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by groups of chemicals perceived, should facilitate further processing and extraction of odour features. Thus, these conformities in architecture across widely separated phyla could be the result of common selective pressures to perform the same tasks (Strausfeld and Hildebrand, 1999; Eisthen, 2002). In the second place, and not to the exclusion of the first argument, glomerular organisation may be an ancient feature of primary chemosensory neuropils in arthropod animals. The similarities noted above might thus represent a plesiomorphic character set and reflect a common origin of arthropod chemosensory neuropil structure (review in Schachtner et al., 2005). While this latter assumption may appear unlikely for more distantly related animal groups such as vertebrates and arthropods, a common origin would appear quite possible within either of these groups. It has been argued convincingly that the olfactory neuropils associated with the antennae of hexapods (insects) and decapod crustaceans share a number of specific features which suggest homology (Schachtner et al., 2005; but see also the discussion in Strausfeld, 2009). Among these shared features are (i) cholinergic olfactory receptor neurons that each project to just a single glomerulus (uniglomerular neurons), (ii) GABAergic local interneurons that connect several glomeruli (multiglomerular neurons), (iii) projection interneurons of similar morphology that connect the primary neuropil to higher brain centres, and – of particular importance in the present context – (iv) supply of the olfactory neuropil by a single or very few (Johansson, 1991; Dacks et al., 2006) large serotonin(5-HT)-immunoreactive neurons, i.e., a specific innervation of the olfactory neuropil by these neurons that do not supply other areas. A recent study in scorpions (Chelicerata) has provided evidence for a similar glomerular organisation of these animals’ primary chemosensory neuropils, although distinct differences in detail are obvious (Wolf, 2008). Intriguingly, though, scorpions’ chemosensory appendages are not antennae located on the animals’ heads, but rather a single pair of comb-shaped appendages on the ventral side of the posterior body region, just behind the posterior pair of walking legs (Fig. 1). These appendages, the pectines, are thus well suited to probe the substrate both chemically and mechanically (Brownell, 1988; Gaffin and Brownell, 1992, 1997). The pectines are innervated by conspicuous, large neuropils located posteriorly in the fused suboesophageal ganglion mass (Fig. 2) (Wolf, 2008). These neuropils have a glomerular organisation reminiscent of the olfactory/antennal lobes in insects and malacostracan crustaceans. The fact that the pectine neuropils are associated with a different set of arthropod appendages, as regards segmental location, use in behaviour and structural detail, raises the question posed above under a more specific perspective. Is this glomerular organisation due to functional necessity, or is it a basic property of the chemosensory neuropils of the arthropod appendage – be it an antenna, a pectine, or even a walking or swimming leg? While this question will be impossible to answer in the near future, the serotonergic supply of the pectine neuropils may provide important arguments for the discussion. In this context, the present study sets out to elucidate one particular common characteristic of the chemosensory neuropils in insects and decapod crustaceans, namely, the supply by a single or very few large serotonin-immunoreactive (5-HT-ir) interneurons (Johansson, 1991; Schachtner et al., 2005). The possible presence of such a neuron has so far neither been examined for the scorpion pectine neuropil, nor indeed for any part of the scorpion central nervous system. At the same time, this feature and the properties of such a neuron, if present, should provide valuable answers to the above question. A single large 5-HT-ir neuron supplying the pectine neuropil with dense and homogeneous arborisations that occupy most of the neuropil matrix would correspond closely to the situation in malacostracan crustaceans and insects. It would thus

support the above hypothesis that such a single 5-HT interneuron supplying the chemosensory module of the appendicular neuropil is a basic – plesiomorphic – character of the arthropod appendage neuropil. 2. Materials and methods 2.1. Animals Pandinus imperator (Koch 1841) was obtained from a local supplier (b.t.b.e. Insektenzucht GmbH, Schnürpflingen, Germany). Androctonus australis (Linnaeus 1758) was captured in the Egyptian desert and imported by Alaa El-Din Sallam (Zoology Department, Suez Canal University, Ismailia, Egypt); no import permits were required. The animals were kept for up to four weeks and fed on locust or cricket larvae before they were used for experiments. 2.2. Immunohistochemistry The animals (n = 5 for each species) were anaesthetised for at least 1 h on crushed ice prior to dissection. The body was opened from the ventral side and the central nervous system dissected in phosphate buffered saline (0.1 M PBS, pH 7.4). The fused supra- and suboesophageal ganglion mass was fixed overnight in 4% paraformaldehyde (PFA) in 0.1 M PBS, ph 7.4, at 4 ◦ C. After fixation, the tissue was washed for 4 h in several changes of PBS and subsequently sectioned (80 ␮m) with a HM 650V vibratome (Microm International GmbH, Walldorf, Germany). Sections were pre-incubated overnight in PBTX (0.3% TritonX-100, 2% bovine serum albumine in 0.1 M PBS, pH 7.4) at 4 ◦ C. Specimens were then incubated in a solution with the primary antibodies overnight at 4 ◦ C. We used polyclonal rabbit anti-serotonin antiserum (diluted 1:2000 in PBTX; ImmunoStar Inc., Hudson, WI, USA, Cat. No. 20080, Lot No. 541016). Alternatively, the specimens were labelled with a monoclonal mouse anti-Drosophila synapsin “SYNORF1” antibody (1:10 in PBTX; provided by E. Buchner, Universität Würzburg, Germany; Klagges et al., 1996). After incubation in a solution with the primary antibody, tissues were washed in several changes of PBS for 4 h at room temperature and incubated in secondary Alexa Fluor 488 or Alexa Fluor 546 IgGs (1:50; Invitrogen Inc., Eugene, OR, USA) overnight at 4 ◦ C. Nuclei in synapsin-labelled preparations were stained with DAPI (4 ,6diamidino-2-phenylindoldihydrochloride) according to standard procedures (3 × 10−5 M in PBS; Merck KGaA, Darmstadt, Germany; cf. Harzsch and Hansson, 2008). The antiserum against serotonin used in this investigation is a polyclonal rabbit antiserum raised against serotonin coupled to bovine serum albumin (BSA) with paraformaldehyde. The antiserum was tested by the manufacturer using standard immunohistochemical methods. According to the manufacturer, staining with the antiserum was completely eliminated by pre-treatment of the diluted antibody with 25 ␮g of serotonin coupled to BSA per ml of the diluted antibody. We repeated this control with the serotonin–BSA conjugate that was used for generating the antiserum as provided by ImmunoStar (ImmunoStar Inc., Hudson, WI, USA; Cat. No. 20081, Lot No. 750256; 50 ␮g of lyophilised serotonin creatinine sulfate coupled to BSA with paraformaldehyde). Pre-adsorption of the antibody in working dilution with the serotonin–BSA conjugate at a final conjugate concentration of 10 ␮g/ml at 4 ◦ C for 24 h completely blocked all immunolabelling. We performed an additional control and pre-adsorbed the diluted antiserum with 10 mg/ml BSA for 4 h at room temperature. This pre-adsorption did not affect the staining, thus providing evidence that the antiserum does not recognise the carrier molecule on its own. The manufacturer

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Fig. 1. (A) Semi-schematic drawing of a scorpion (Pandinus imperator) in ventral view. The fused suboesophageal ganglion mass is indicated in black (see detailed view in Fig. 2), partly hidden by the sutures of the prosoma cuticle, which is otherwise drawn as if transparent. (B) The pectines are located ventrally, just behind the walking legs, shown in more detail here. The frame indicates the magnified area from (A). Note the comb-like pectine structure; the individual pegs bear dense arrays of mechano- and chemosensors (Foelix and Müller-Vorholt, 1983; Brownell, 1988).

also examined the cross-reactivity of the antiserum. According to the data sheet, the following substances did not react with the antiserum diluted to 1:20,000 using the horse radish peroxidase (HRP) labelling method: 5-hydroxytryptophan, 5-hydroxyindole3-acetic acid, and dopamine, each tested with 5 ␮g, 10 ␮g, and 25 ␮g amounts. The monoclonal mouse anti-Drosophila synapsin “SYNORF1” antibody (provided by E. Buchner, Universität Würzburg, Germany) was raised against a Drosophila GST–synapsin fusion protein and recognises at least four synapsin isoforms (ca. 70, 74, 80, and 143 kDa) in Western blots of Drosophila head homogenates (Klagges et al., 1996). In an analysis of crayfish homogenates, this antibody

stained a single band at ca. 75 kDa (Sullivan et al., 2007). In a Western blot analysis comparing brain tissues of Drosophila melanogaster and Coenobita clypeatus, the antibody provided identical results for both species, staining one strong band around 80–90 kDa and a second weaker band slightly above 148 kDa (Harzsch and Hansson, 2008), suggesting that the epitope which SYNORF 1 recognises is strongly conserved between the fruit fly and the hermit crab. Similar to the situation in D. melanogaster, the antibody consistently labels brain structures in representatives of all major subgroups of the malacostracan crustaceans (Harzsch et al., 1997; Beltz et al., 2003; Harzsch and Hansson, 2008) in a pattern that is consistent with the assumption that this antibody does in fact label

Fig. 2. The pectine neuropils in the fused suboesophageal ganglion mass. (A) Horizontal sections (80 ␮m) through the ventral portion of the ganglion. Colours illustrate different tissue elements: red, neuropil regions (anti-synapsin immunohistochemistry); blue, cell bodies (DAPI stain for cell nuclei); green, connective tissue (background fluorescence). Section (i) is ventrally adjacent to section (ii), both showing corresponding neuropil portions since the ganglion bends dorsally towards the posterior connectives. (B) Semi-schematic drawing of the suboesophageal ganglion mass, ventral view (cf. Wolf, 2008). The supraesophageal ganglion (brain) is indicated as dotted outline, the pectine neuropils and tracts as dotted areas. The main nerve roots are indicated, supplying the pedipalps, the four pairs of walking legs (1–4), the genital segment, and the pectines; the connectives extend toward the unfused ganglia of the opisthosomal nerve cord. Anterior is at the top.

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synaptic neuropils in Crustacea. The antibody also labels neuromuscular synapses both in Drosophila and in Crustacea (Harzsch et al., 1997) and synaptic neuropils in ancestral clades of protostomes, Chaetognatha (Harzsch and Müller, 2007) and platyhelminths (Cebria, 2008), suggesting that the recognised epitope is conserved over wide evolutionary distances. In additional control experiments for possible nonspecific binding of the secondary antisera, we omitted the primary antiserum, replaced it with blocking solution, and followed the labelling protocol as above. In these control experiments, no staining occurred. 2.3. Microscopy, image processing and nomenclature Our analysis is based on at least 5 successfully processed brains per species, with the labelling patterns consistent between specimens. The specimens were mounted in Mowiol embedding medium (Calbiochem; Merck KGaA, Darmstadt, Germany) on standard slides for microscopy and viewed with a Zeiss Axio Imager equipped with the Zeiss ApoTome structured illumination device for optical sectioning (“grid projection”). Confocal scans were performed with the laser scanning microscope Zeiss LSM 510 Meta, and digital images processed with the Zeiss AxioVision software package (Carl Zeiss MicroImaging GmbH, Jena, Germany). This allowed the reconstruction of thick sections from stacks of optical scans or confocal scans. In some cases, reconstruction of thick sections was effected not just from image stacks from a single vibratome section but from neighbouring vibratome sections as well, if these neighbouring sections were not distorted and allowed such reconstruction. Double-labelled specimens were generally analysed in the multi-track mode, in which the two lasers operate sequentially, and narrow band-pass filters were used to assure a clean separation of the labels and to avoid any crosstalk between the channels. All images were black/white inverted and processed with Photoshop (Adobe Systems, San Jose, CA, USA) using global picture enhancement features (brightness/contrast/inversion). The neuroanatomical nomenclature used in the present study follows the standard recently proposed by Richter et al. (2010). 3. Results 3.1. Pattern of serotonin immunoreactivity in the suboesophageal ganglion mass The fused suboesophageal ganglion of the scorpion occupies a considerable portion of the scorpion prosoma (Fig. 1). It consists of the neuromeres belonging to the chelicere and pedipalp segments, the segments of the four pairs of walking legs, and the genital and pectine segments (Fig. 2). The pattern of 5-HT-ir is repetitive across these segments, despite some quantitative variation in detail (Fig. 3). Groups of 5-HT-ir somata occur bilaterally in the ventral layer of cell bodies. Depending on the body segment, these groups contain about 60 to almost 100 neuronal somata. The number of immunolabelled neurons appears to be related to the size of neuropils supplied, i.e., the large pectine neuropil is associated with about 100 serotonergic somata and the pedipalp neuromere with the second largest number, while the leg neuromeres have some 60–80 somata. The small genital neuromere is difficult to distinguish from the pectine neuromere but appears to possess fewer than 60 somata. However, it is impossible to determine exact numbers in the densely packed groups. Soma diameter ranges from just above 15 ␮m to almost 30 ␮m. By comparison, the largest motoneuron somata just exceed 50 ␮m. The 5-HT-ir soma groups are located medially, some 150–250 ␮m from the ganglion midline, and behind the nerve root supplying the respective appendage, almost halfway towards the more posterior nerve root.

From these soma groups, the primary neurites ascend in a bundle dorsally towards the neuropil (Fig. 4A and B). There they form numerous arborisations that supply much of the neuropil in a sparse though fairly homogeneous pattern (Fig. 4A, B and D). Especially the neuropils associated with the nerve roots of the appendages, and the nerve roots themselves (Fig. 3A; 1 and 2 indicate nerve roots of walking legs), are supplied by these homogeneously arranged branchings of 5-HT-ir neurons. Also, 5-HT-ir axons form tracts that extend along the length of the ganglion and connect the two body sides by extending across commissures (Figs. 3B and 4A, arrows). One major tract extends dorsally and medially, about 150–200 ␮m from the ganglion midline, along each hemiganglion (Fig. 3B). It is accompanied by a more scattered tract with immunolabelled neurites that appears to connect mainly the neighbouring segments in a slightly more lateral position (Fig. 4A; labelled areas ventral and lateral of the arrows). A pair of prominent immunolabelled commissures connects the two body sides in each segment (Fig. 3B). 3.2. Pattern of serotonin immunoreactivity in the pectine neuropils In contrast to the sparse and rather homogeneously scattered 5-HT-ir neurite supply of most neuropil regions in the suboesophageal ganglion, the pectine neuropils receive profuse 5-HT-ir neurite arborisations and varicosities. These form dense networks in some regions whereas others are completely spared (Fig. 5). The supply to the outer portions of the pectine neuropils is particularly dense, being reminiscent of a rind surrounding the glomerular structures. There is also a dense supply to the fibre tracts leaving the neuropils anteriorly. The central portions of the glomeruli are completely devoid of immunolabelled neurites (asterisks in Fig. 5B, D and E). The pectine neuropils are quite isolated from the remaining ganglion mass all around their perimeters. They are innervated only from two anterior tracts (Wolf and Harzsch, 2002; Wolf, 2008) and, of course, posteriorly through the afferent pectine nerves (Fig. 2). Accordingly, 5-HT-ir innervation also originates from the anterior, from a paired bundle of primary neurites that originate in the 5-HT-ir soma groups noted above (Fig. 4A–C; marked by double arrowheads in Fig. 4B). There are about 10–20 ipsilateral serotonergic somata with axons targeting the pectine neuropil on each side of the body. These cell bodies are part of a much larger group, comprising approx. 70–90 (or even up to 100) somata of similar size as the surrounding unlabelled neurons (ca. 15–20 ␮m). This soma group is associated with the neuromere of the fourth walking leg. However, the small neuromeres of the genital and pectine segments are skewed and distorted to an extent that often makes the attribution of their neuronal elements impossible and may lead to confusion with elements from the fourth walking leg neuromere (Wolf and Harzsch, 2002; Wolf, 2008). Therefore, it has to remain unclear at present whether the small subgroup of 5-HT-ir cell bodies supplying the pectine neuropils actually belongs to the fourth walking leg neuromere or if it is of more posterior origin. The 5-HT-ir fibres leading into the pectine neuropil are connected to the longitudinal tracts of immunoreactive fibres that extend along the length of the suboesophageal ganglion and receive contributions from all segmental groups of 5-HT-ir neurons (see above and Fig. 3B). There is also a commissure just anterior of the pectine neuropil that contains many 5-HT-ir axons mingling with the supply of the pectine neuropils (Figs. 4D and E, 5B and C; indicated by double arrowheads in Fig. 5C). There is a second source of 5-HT-ir innervation to the pectine neuropils on each body side. This source consists of a pair of ipsilateral immunolabelled neurons (Fig. 5C and F; arrow in Fig. 5F) or sometimes just a single cell body (arrows in Fig. 4B) slightly larger

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Fig. 3. Horizontal sections through the suboesophageal ganglion, 5-HT immunocytochemistry. (A) Pandinus imperator; (A (i)) longitudinal section through the ventral-most part of the ganglion showing the segmental groups of somata labelled with serotonin-like immunoreactivity (5-HT-ir; indicated by arrows on the right side). The anteriormost pair of large soma groups corresponds to the pedipalp neuromere. (A (ii)) illustrates the pair of 5-HT-ir soma groups associated with the pectine neuropils. Structures are shown in their proper position with respect to the section in (A (i)), but are combined from sections located 120–200 ␮m further dorsally to account for the upward bend of the posterior ganglion part (cf. Fig. 2). (B) Androctonus australis; longitudinal section through the dorsal-most part of the ganglion showing the longitudinal tracts and the commissures which exhibit 5-HT-ir. Due to the bent shape of the ganglion, only the anterior commissures are visible, which correspond to the pedipalp and the anterior two walking leg neuromeres. In (A) and (B), scans of two 80 ␮m vibratome sections were combined; total thickness of suboesophageal ganglion mass is ca. 1200 ␮m. Labelling as in Fig. 2; SEG, ventral portion of supraoesophageal ganglion (brain); asterisk indicates oesophageal opening in the ganglion. Ganglion midline indicated by dashed line in this and the following figures; anterior is at the top.

(diameter up to 30 ␮m) than the surrounding cell bodies and associated with the neuromeres of the genital and pectine segments. These 5-HT-ir somata are thus slightly more posterior than the group of cell bodies mentioned above, located just lateral of the pectine neuropils. Nonetheless, in Fig. 4 both sources of 5-HT-ir innervation to the pectine neuropil are visible in a frontal section across the anterior end of the neuropils. The neurites supplying the pectine neuropil cannot be disentangled; it is thus not possible to determine any differences in the innervation patterns of the single 5-HT-ir soma and the more anterior soma group. Judging by neurite diameters it appears that the neuron pair, as well as the occasional single cell, makes a significant contribution to 5-HT-ir innervation of the pectine neuropils, probably in excess of the roughly 5% that would be expected by sheer numbers. The 5-HT-ir somata on one side of the body appear to supply the pectine neuropils on both sides with immunoreactive neurites crossing through a commissure just anterior of the pectine neuropils (Figs. 4D and E, 5B and C).

4. Discussion 4.1. Pattern of serotonin immunoreactivity in the suboesophageal ganglion The pattern of immunoreactivity in the fused portion of the scorpion prosoma ventral nerve cord is reminiscent of the situation in hexapods (insects), decapod crustaceans, and the few chelicerates studied so far (see review in Harzsch, 2004), regarding more general features of the serotonin-immunoreactive neurons, as described in previous studies for insects (Bishop and O’Shea, 1983; Tyrer et al., 1984; Nässel and Cantera, 1985; Longley and Longley, 1986;

Bräunig, 1987; Breidbach, 1987; Cantera and Nässel, 1987; Vallés and White, 1988; Griss, 1989; Radwan et al., 1989; van Haeften and Schooneveld, 1992; Lundell and Hirsh, 1994; Boleli et al., 1995; Hörner et al., 1995), malacostracan crustaceans (Beltz and Kravitz, 1983; Real and Czternasty, 1990; Johansson, 1991; Harrison et al., 1995; Antonsen and Paul, 2001; Harzsch, 2003), and chelicerates (Breidbach and Wegerhoff, 1993; Harzsch et al., 2005b). These common features are: (i) the presence of a pair of bilaterally symmetric 5-HT-ir soma groups per neuromere in the ventral cell cortex; the number of somata per group may range from 1 in representatives of Homarida and Caridea to 2–3 in Brachyura and Astacida and 4 in the harvestman, an opilionid; likewise, 2–3 cells are present in various representatives of the insects; (ii) the connection of these neuron groups via longitudinal tracts that run bilaterally along the dorsal part of the ventral nerve cord and up to the brain; (iii) the connection through a pair of commissures in each segment; (iv) scattered 5-HT-ir arborisations that supply much of the segmental neuropil; (v) 5-HT-ir fibres surrounding the nerve roots, sometimes even outside the ganglion itself, in some arthropods (lobster: Beltz and Kravitz, 1983; insects: e.g., Nässel and Elekes, 1985; Davis, 1985, 1987; Bräunig, 1987; Griss, 1989; Schachtner and Bräunig, 1995). These similarities of 5-HT-ir structures in the scorpion nervous system suggest a similar function of serotonergic innervation in scorpions and the other arthropod groups (cf. arguments in

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Fig. 4. Frontal cross-sections through the suboesophageal ganglion, illustrating patterns of serotonin-like immunoreactivity (5-HT-ir) in Pandinus imperator. (A) Section through the anterior half of the pectine neuropil (cf. Fig. 2B) giving an overview of the ganglion structure. Arrows indicate longitudinal tracts with 5-HT-ir (cf. Fig. 3B). (B) Magnified ventral area (= boxed area in A) showing the pectine neuropils and their surroundings in more detail. Arrows indicate two single neuronal somata that innervate the pectine neuropils; double arrowhead indicates a large segmental soma group that sends a few neurites into the pectine neuropils, with many more branches ascending further to join tracts and commissures and supply other neuropil areas. The neurites branching off into the pectine neuropils are indicated by three arrowheads. (C) Magnification of boxed area in (B); ventral-most neurites branching off towards pectine neuropils indicated by light arrowheads. (D) Section from a slightly more anterior area, showing the commissure between left and right pectine neuropils (cf. Fig. 5B and C for horizontal sections). (E) Magnification of boxed area in (D) showing many 5-HT-ir commissural neurites, indicated by the arrow. Dorsal is at the top. (A), scan of 80 ␮m section; (B)–(E) projections of 5 optical sections spanning 23 ␮m.

Harzsch, 2004). Notable differences between scorpions and insects or crustaceans are displayed in the number of neuronal cell bodies in the segmental groups. In the scorpion, there are approx. 60–100 5-HT-ir somata in each neuromere half. Assuming that these large groups of 5-HT-ir somata correspond to the groups of just 1–4 somata in the other arthropods, the situation is reminiscent of that in the motoneurons and inhibitory interneurons. For these types of neurons, there are five to ten times more cells in the scorpion than in corresponding soma groups of the other arthropods (Bowerman and Burrows, 1980; Wolf and Harzsch, 2002; Harzsch et al., 2005a). For instance, a scorpion muscle is innervated by about 12–20 motoneurons, while an insect muscle typically receives 3–4 motoneurons, although there may be 14 in exceptional cases (Burrows and Hoyle, 1973; Burrows, 1996). Mechanosensors, too, are usually supplied by a larger number of sensory neurons in scorpions than in mandibulate arthropods (Foelix and Schabronath, 1983). Functional significance and evolutionary aspects of this comparatively large number of cell bodies remain enigmatic, particularly the question of whether or not the scorpion reflects a plesiomorphic character state. In this context it is noteworthy that in the horseshoe crab (Limulus ployphemus), a member of the Chelicerata just like the scorpions, segmental groups of 5-HT-ir neurons also possess up to 10 neurons (Harzsch et al., 2005b).

4.2. Pattern of serotonin immunoreactivity in the pectine neuropils The most intriguing result of the present study is the finding that pectine neuropils are supplied by two relatively large 5-HT-ir neurons per side, which probably belong to the associated neuromere, and additionally by another 10–20 axons per side, with the corresponding somata associated with the neuromere of the 4th walking leg, or perhaps the genital or pectine segments. The pectine neuropils’ supply by two relatively large 5-HT-ir neurons per side is reminiscent of a particularity of insect and malacostracan crustaceans insofar as their antennal/olfactory lobes, which are associated with a pair of predominantly chemosensory antennae, are supplied by one or very few large serotonergic neurons, called dorsal giant neurons, in the deuterocerebrum (Sandeman and Sandeman, 1987; Schachtner et al., 2005). This similarity in serotonergic innervation has, among other features (see Section 1), been proposed to support homology of the antennal/olfactory lobes in the two arthropod groups (Schachtner et al., 2005; but see also the discussion in Strausfeld, 2009 and general aspects in Kutsch and Breidbach, 1994). Other distinguishing features of the insect and crustacean olfactory systems, such as (i) the uniglomerular projections of cholinergic olfactory receptor neurons, (ii) the GABAergic local inhibitory interneurons that connect

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Fig. 5. Supply of the (posterior) pectine neuropils by neurites with serotonin-like immunoreactivity (5-HT-ir). (A) Overview of the pectine neuropils in the horizontal plane. Roots of the 4th walking leg nerves are just visible (marked 4). Horizontal sections of pectine neuropils in (B) Androctonus and (C) Pandinus at higher magnification illustrate the glomerular structure; a central glomerulus is indicated by * in (B). Note the dense 5-HT-ir arborisations and varicosities, except in the cores of the glomeruli. The anterior commissure, indicated by double arrowheads, contains several 5-HT-ir neurites (cf. Fig. 4D and E for frontal sections). Arrow in (C) indicates a pair of (bilaterally arranged) 5-HT-ir somata which supply the pectine neuropil. (D) and (E) Cross-sections of the pectine neuropil again illustrate the glomerular structure; the glomeruli (central ones marked with *) are mostly devoid of 5-HT-ir neurites, but are surrounded by a network of 5-HT-ir arborisations; dense 5-HT-ir supply also to the cortex of the pectine neuropil that contains the afferents. Double arrowheads mark supply of the pectine neuropils by fibres from the lateral 5-HT-ir soma group (cf. Fig. 4B and C). Higher magnification in (F) illustrates that these are indeed two somata, discernible by two primary neurites that originate from the cell bodies, marked by rows of arrowheads. Anterior is at the top in (A–C), (F) and dorsal is at the top in (D) and (E). Ganglion midlines are indicated by dashed lines. (A) projection of 9 optical sections spanning 40 ␮m in total; (B–D) 5 optical sections, spanning 20 ␮m; (E) 3 optical sections, spanning 3 ␮m; (F) 9 optical sections, spanning 34 ␮m.

different glomeruli, and (iii) the connection of the primary olfactory neuropil to higher brain centres by sets of projection interneurons of similar morphology, have not yet been analysed. And indeed, even the homology of the serotonergic supply to the olfactory lobes in insects and crustaceans is controversial. Nonetheless, the above similarity to the situation in insects and crustaceans was unexpected, considering the fact that, on the one hand, pectines are segmental appendages just like the antennae of insects and crustaceans, but on the other hand, they are located in a quite different and less specialised body segment. The antennae of insects and crustaceans are associated with the deutocerebrum and have been specialised in chemosensory and tactile exploration of the environment for a long time during evolution. Pectines, in contrast, are part of the tagma bearing the walking legs and are close to the unfused opisthosomal segments. It appears that they have also specialised in the chemosensory and tactile exploration of the environment for a long time, and they may have already been present in the fossil marine group of eurypterids (Dunlop and Webster, 1999), although their function in these animals is not yet clear. The observation of just two 5-HTir neurons per side in the presumed pectine neuromere was also unexpected considering the usually much larger number of neurons in a given soma group, as indicated above. Thus, this in itself is a

significant similarity to the situation in insect and crustacean olfactory lobes.

4.3. Speculations on possible homologies The present finding that 5-HT-ir innervation is supplied by two large neurons in the scorpion pectine neuropils would seem to suggest a set of interconnected hypotheses: (i) In the scorpion, serial homology (homonomy) of 5-HT-ir neurons associated with the pectine neuropils and the segmental ganglia appears rather straightforward. Observations suggesting such homonomy are: similarities in the position of the 5-HT-ir somata and soma clusters in the neuropil, as well as in their primary neurite courses and in their connections to the longitudinal tracts and the commissures. (ii) The insect and malacostracan deutocerebral giant serotonergic neurons may be regarded as deutocerebral homologues of the serotonergic neurons in the trunk neuromeres (cf. Schachtner et al., 2005). (iii) (Serial) homology of the 5-HT-ir neurons supplying the pectine neuropil with the prominent deutocerebral serotonergic

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neurons innervating the mandibulate olfactory lobes would appear to follow from the two above hypotheses. (iv) 5-HT-ir innervation of chemosensory neuropils associated with an appendage might be a plesiomorphic character within the arthropods and a fundamental component of the neuronal circuitry in the ground pattern of all arthropod trunk ganglia.

These hypotheses are primarily intended to stimulate further research and we recognise that several aspects are speculative or controversial. One such controversy is the possible homology of the serotonergic supply to the olfactory lobes of malacostracan crustaceans and insects (Schachtner et al., 2005) noted above. The arguments listed in Section 1 support homology, while different lateralisations of the giant serotonergic cells innervating the antennal lobes contradict this assumption. In insects, the giant serotonergic cells cross the midline of the brain, while in malacostracans they stay ipsilateral within their brain hemisphere. In arthropod appendages that are not primarily chemosensory, innervation by one of the 5-HT-ir neurons in the respective trunk ganglion may not have been recognised as being associated with chemosensory input to this ganglion. Obvious reasons for such possible ignorance are the minor importance of chemosensory input in many trunk ganglia, and the small size of the associated neuropil areas (e.g., Newland et al., 2000). This line of argument may also apply to a possible glomerular neuropil organisation of the appendicular chemosensory neuropil, which may be quite inconspicuous considering the small neuropil size. Alternatively, chemoreceptors on walking legs may primarily alert the animal to aversive chemicals, rather than functioning in analysing chemical composition (e.g., Newland et al., 2000). This function may not require glomerular neuropil organisation. As outlined in Section 1, glomerular organisation of primary chemosensory neuropils is certainly advantageous, and probably even essential, for chemosensory signal processing in the central nervous system. This line of argument has been used to explain the glomerular structure of chemosensory neuropils in distantly related animal taxa, such as mammals and insects (Strausfeld and Hildebrand, 1999; Eisthen, 2002). Of course, this argument also applies to chemosensory neuropils in the various body segments of an animal species. If glomerular organisation is a prerequisite for chemosensory signal processing, this should hold for all body segments. One may thus also expect glomerular organisation in the segmental chemosensory neuropil areas associated with walking legs or mouthparts. In fact, such organisation should then represent the basic architecture of the arthropod appendicular neuropil. Interesting insights into this issue should result from an examination of serotonin-like immunoreactivity in the neuropils associated with other arthropod appendages that have a major chemosensory role. Insect maxillae and their derivates, whip spider pedipalps, or crayfish maxillipeds would be suitable objects for further scrutiny (Strausfeld, 1998). It has to be considered, however, that glomerulus-like organisation can serve the subdivision and functional segregation of primary projection neuropils in any sensory modality (see Wolf, 2008). Neuropil compartments reminiscent of glomeruli are observed, for instance, in the insect auditory system, obviously supporting frequency representation (Römer, 1983), and in several insect mechanosensory neuropil areas (Murphey et al., 1980, 1985; Burrows, 1996). For more detailed comparisons, the definition of what constitutes a glomerulus may require elaboration. Important features to consider include afferent innervation towards the centre of the glomerulus and efferent innervation from its periphery, the presence of a glial sheath, and perhaps a more or less spheroidal shape (Schachtner et al., 2005; Wolf, 2008).

Acknowledgments Experiments were performed at the Max-Planck-Institute in Jena, in the Department of Bill Hansson; H.W. is particularly grateful for his hospitality, and S.H. for continuing support, use of MPI infrastructure, and fruitful discussions. We gratefully acknowledge Alaa El-Din Sallam for providing the Androctonus scorpions, and Erich Buchner (Würzburg) for providing the SYNORF1 antiserum. The Institute for Advanced Study Berlin hosted H.W. during data evaluation and writing, and it provided a stimulating environment for discussion with several fellow biologists. This study was supported by DFG grant HA 25408-1.

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