A wonderful network unraveled - Detailed description of capillaries in the prosomal ganglion of scorpions
© Klußmann-Fricke et al.; licensee BioMed Central Ltd. 2014
Received: 21 November 2013
Accepted: 27 February 2014
Published: 1 May 2014
Though it has long been known that the prosomal ganglion of scorpions is supplied by a dense system of arteries, the pattern of this network has never been described and analyzed in detail. Using MicroCT in combination with computer aided 3D-reconstruction we provide the first detailed description of the pattern of arteries in the prosomal ganglion of Brotheas granulatus (Scorpiones, Chactidae) and other scorpion species.
The entire prosomal ganglion in scorpions is supplied by a network of arteries that branch off the major arteries of the anterior aorta system. The most prominent of these are the nine transganglionic arteries which run through the nerve mass along the midline of the body and branch terminally, i.e. below the neuropils, into smaller arteries. These arteries reticulate into a dense network between the surrounding somata and the centrally located neuropil structures of the ganglion.
We demonstrate the presence in the prosomal ganglion of scorpions of a capillary system made up of afferent arteries which deliver hemolymph into the ganglion and efferent arteries which transport the hemolymph out of the ganglion. Adopting the structural definition used for vertebrate circulatory systems, this capillary network can also be termed a bipolar rete mirabile (located as it is between afferent and efferent arteries) analogous to those found in vertebrates and some echinoderms.
Within the rete mirabile of the scorpion prosomal ganglion, some regions (i.e. neuropils) are better supplied than others. The structural information provided here can now be used in functional neuronal studies to determine the physiological and computational significance of the various neuropils in the complex scorpion nervous system.
The central nervous system (CNS) is one of the most important organ systems in animals, so ensuring that it is supplied with nutrients and oxygen is a crucial matter. In arthropods, the central nervous system consists of a 'syncerebrum' and a number of free segmental ganglia which are situated ventrally in the body and therefore known as the ventral nerve cord . In arachnids, the syncerebrum is fused with a taxon-specific number of ventral nerve cord ganglia to form the condensed prosomal ganglion (Pg; ). A prosomal ganglion combines a dorsal supra- and a ventral subesophageal ganglia. Classically, supra- and subesophageal ganglia are connected by circumesophageal connectives , but these are not distinguishable in arachnids , where the supraesophageal ganglion sits broadly on the anterior part of the subesophageal ganglion. Only in lateral view can the rather thin esophagus be taken as a fictive border between the two major ganglionic masses.
It has long been known that the prosomal ganglion in scorpions is supplied by a number of vessels [4, 7–9], and even that these vessels form a dense network [2, 6]. However, the structure and pattern of the network has never been studied in detail. At first glance, the fine arteries in question could be interpreted as capillaries, which in vertebrates are defined as networks of small-diameter vessels (5 – 10 μm) that connect arteries and veins. Capillaries are characterized by the presence of exclusively endothelial layers and connective tissue, and are where the exchange of oxygen and nutrients takes place . As veins are not present in arthropods , the older literature on arthropods (or more precisely on crustaceans) defines capillaries either as small-caliber vessels continuous with the efferent arteries and without visible intima , or as vessel networks that link afferent and efferent vessels .
This paper provides the first detailed description of the morphology of the cerebral vasculature in scorpions. It also identifies parts of the prosomal ganglion that are better supplied than others and discusses possible reasons for this pattern. We discuss the terminology commonly used to describe capillaries and retia mirabilia and the possible adoption of these terms to arthropod circulatory systems in general. Furthermore, we hypothesize on how the pattern of arteries described might be established during ontogeny.
Gross morphology of the prosomal ganglion and the vascular system which supplies it
From its origin at the heart, the anterior aorta (Figure 1; Ao) runs in an anteroventral direction to the posterodorsal part of the supraesophageal ganglion (Figure 3A-C; Ao) where the cheliceral arteries branch off and run around the supraesophageal ganglion laterally (Figure 3B, C; Ch). Anterior to the supraesophageal ganglion these arteries bend upwards and run into the chelicerae (Figure 3C). Shortly after this bend, a supracerebral artery (Figure 3B; Sc) emanates either from the left or the right cheliceral artery (see  for details on variation).
After the cheliceral arteries have branched off dorsally to the thin esophagus, the anterior aorta bifurcates into two lateral trunks from which subsequently the arteries of the pedipalps and the four pairs of walking legs branch off (Figure 3C; Pp, I-IV). On top of the subesophageal ganglion, the lateral trunks curve backwards, reunite, and give rise to the ventral vessel (Figure 3C; Vv). This artery runs atop the ventral nerve cord in a posterior direction through the entire opisthosoma, supplying the free ganglia of the nerve cord.
Afferent systems of the prosomal ganglion
The prosomal ganglion is exclusively supplied by arteries which emanate from the anterior aorta system. The most prominent of these are the nervous midline arteries, which emanate from the aortic trunks. The midline arteries can be broken down into the protocerebral artery (Pa) and the paired esophageal arteries (Ea) in the supraesophageal ganglion, and the transganglionic arteries (Tga) in the subesophageal ganglion (Figure 3D, E). Wherever they come into direct contact with the prosomal ganglion, the arterial subsystems of the anterior aorta (e.g. the cheliceral arteries and the leg arteries) give rise to many other small arteries too (Figure 5C, D).
The first midline artery, i.e. the protocerebral artery, runs in an anterior direction through the prosomal ganglion while the last, i.e. the 8th transganglionic artery (Figure 3D; 8th asterisk), emanates in a ventral direction. The 9th transganglionic artery is a special case since it does not run through the subesophageal ganglion but emanates from the ventral vessel at the posterior border of the prosomal ganglion (not shown). This relatively thick artery runs in a ventral direction and branches terminally into one anterior and one posterior artery. The former anastomoses with the 8th transganglionic artery and the latter splits after a short distance and gives rise to the pectinate arteries.
Supraesophageal ganglion supply
The arcuate body is mainly supplied by a number of ventrally oriented arterial loops (Figure 4D; asterisks) which emanate from the network of arteries within and around the protocerebrum. In addition, there are two small arteries which emanate from the supracerebral artery a short distance after its spilt (see above, Figure 3B). These two arteries run ventrally, then also form loops and anastomoses with the other arteries that supply the arcuate body (Figure 4D; arrowheads).
The cheliceral neuromere is mainly supplied by the esophageal arteries, the only paired midline arteries. They emanate from the lateral trunks of the anterior aorta (Figure 4F; Ea) laterally to the esophagus (Figure 4F; dashed green lines), at the point at which the latter passes through the prosomal ganglion. Each of the esophageal arteries runs in an anterior direction alongside the esophagus, ramifying distally into smaller arteries which anastomose with the surrounding cerebral arteries. Additionally, small arteries run ventrally to supply the pedipalpal neuropil (see below). As described for the protocerebrum, the arteries reticulate directly at the border between the neuropil and the surrounding somata, also forming loops that protrude into the neuropil.
At the anterior border of the neuropils, the arteries supplying the corresponding neuromeres unite to form two laterally positioned frontal collecting arteries (Figure 4A, B; Fca) which run in a ventral direction before finally discharging into the pedipalpocoxal arteries (see below).
Subesophageal ganglion supply
The subesophageal ganglion is mainly supplied by the transganglionic arteries and the small arteries which emanate from the pedipalpal arteries and leg arteries.
The posterior neuropil is mainly supplied by the last three transganglionic arteries, which emanate from the ventral vessel. These arteries also branch terminally and reticulate on the ventral surface of the posterior neuropil (Figure 6C; dotted yellow line). Two additional rows of arteries that emanate from the ventral vessel laterally to the transganglionic arteries surround the borders of the posterior neuropil. There are fewer loops in the posterior neuropil than in the anterior neuromeres (Figure 6C). Furthermore, the pectinate neuropils were not observed to have their own supply.
The ventral collection arteries (Figure 7C; white dashed lines) are situated between the border of the subesophageal neuropils and the ventral cluster of somata, forming a network with numerous interconnecting smaller arteries (Figure 7D; Vca). The main collection arteries run in an undulating course the whole length of the subesophageal ganglion, giving rise to four pairs of coxal arteries which leave the ganglion ventrally of each hemi-neuromere of the corresponding leg neuromere (Figure 7B, C; CaI + IV). The first two pairs of coxal arteries are the coxapophysal arteries, which supply the maxillary glands (see  for details). The maxillary glands are located in the coxapophyses of the first two pairs of walking legs.
Comparative morphological aspects within scorpions
The protocerebral arteries, the paired esophageal arteries and the nine transganglionic arteries which branch terminally into smaller arteries were found in all studied taxa (Figure 8B; Pa, Ea; Tga). Neighboring transganglionic arteries are interconnected. The arteries that supply the neuromeres of the prosomal ganglion also reticulate around the borders of the neuropil structures, sending twisted loops between the nerve fibers (Figure 8B, C, G; arrowheads). The postneuropilar arteries of the limb neuromeres are present in all studied scorpion species (Figure 8A, C, D; arrowheads). Aside from the transganglionic arteries, the most prominent arteries to run into the prosomal ganglion are those emanating posterolaterally from the leg arteries. We also observed that the degree of vascularization of certain parts of the ganglion (e.g. the central ganglion, see above) is higher than in others (Figure 8E; dashed yellow line).
Comparative morphological aspects
The arterial patterns described above with a capillarization in the prosomal ganglion, protocerebral arteries, the paired esophageal arteries and the nine transganglionic arteries and the described efferent arteries can be hypothesized to belong to the ground pattern in scorpions. They may even represent apomorphies for scorpions, but this would need to be tested further using out-groups with complex arterial systems, such as tetrapulmonates (i.e. spiders and their close relatives; see below).
We did, however, also find some taxon-specific differences in the pattern of arteries which supply the prosomal ganglion. In Buthidae, for example, the cerebral vasculature is not as dense as in B. granulatus, and the arteries within the prosomal ganglion are not as highly organized (Figure 8A). There are some other differences in the cerebral vasculature of the different scorpion taxa which may provide further morphological characters for phylogenetic analysis (see ).
Within ‘ pulmonate’ arachnids, Araneae possess similarly complex arterial systems . E.g. in the spider Cupiennius salei (Ctenidae), the supraesophageal neuromeres are supplied bilaterally by ventral branches of the cheliceral artery, arteries which split off from the two branches of the anterior aorta, and by arteries which branch off the first leg artery . The arcuate body is supplied anteriorly by arteries emanating from the cheliceral arteries which enclose the arcuate body ventrally and dorsally. Posteriorly, the arcuate body is supplied by small arteries emanating from the anterior aorta . The subesophageal neuromeres are supplied medially by twelve unpaired transganglionic arteries and laterally by six pairs of arteries which branch off from the leg and pedipalpal arteries. The center of the neuromeres remains largely free of arteries but Huckstorf et al.  observed that the neuropils are often supplied by fine arteries which run in tight loops through the subesophageal ganglion.
Similar patterns of intraganglionic arteries have also been observed in Uropygi and Amblypygi (Klußmann-Fricke B.-J., Pomrehn S.W., Wirkner C.S. unpublished data). It can be suggested, then, that extensive supply to the prosomal ganglion is part of the ground pattern of Tetrapulmonata (i.e. Araneae and Pedipalpi), a group that is clearly monophyletic e.g. [19–22]. However, Scorpiones seem to be more closely related to Opiliones [21, 22] than to Tetrapulmonata. Considering the similarities between the pattern of intraganglionic arteries in Tetrapulmonata and that in scorpions, it seems likely that a similar system of arterial supply to the nervous system was already present in the arachnid ground pattern.
Capillaries and retia mirabilia in arthropods?
In vertebrates, capillaries are defined as networks of small-diameter vessels (5 – 10 μm) that connect arteries and veins. Other definitions include further structural criteria such as the presence of exclusively endothelial layers and the presence of connective tissue. Functionally speaking, capillaries are defined as the location where the exchange of oxygen and nutrients takes place . Due to the lack of veins in arthropods, the relevant literature defines capillaries in this taxon as a network of arteries connecting afferent and efferent arteries [12, 13, 18, 23]. Since the afferent arteries of the anterior aorta and the small arteries in the prosomal ganglion are directly connected to efferent arteries (e.g. coxal arteries), the vasculature in the prosomal ganglion does represent a form of capillarization. Moreover, Lane et al.  state that in scorpions, at least, these channels seem to be lined by endothelial-like layers, making it likely that they are true arteries which are continuous with the rest of the HVS (hemolymph vascular system). In scorpions, then, it does seem possible to talk about capillary networks without flouting the above definitions. Capillary beds connecting afferent and efferent arteries have also, in vertebrates  and some echinoderms , been termed retia mirabilia, though the definitions of this phenomenon vary. In vertebrates a rete mirabile is an artery or a vein that branches immediately into a network of small vessels  forming numerous anastomoses. If the branches of the network reunite to form an artery again, the phenomenon is known as a bipolar rete mirabile[26, 27]. In the medical literature, a rete mirabile is also defined as the branching of an artery or vein that then reforms into an artery or vein . Examples are the glomeruli malpighii in the human kidney and the portal system in the liver. In other vertebrates, such networks are found in the head arteries of mammals and in the feet of Anseriformes (ducks, geese, swans and their allies) and Spheniscidae (penguins) [29, 30], for example. In most of the Anglo-American literature, including the McGraw-Hill “Dictionary of Scientific and Technical Terms” , a rete mirabile is defined as “a network of small blood vessels that are formed by the branching of a large vessel and that usually reunite into a single trunk; believed to have an oxygen-storing function in certain aquatic fauna”. The definition provided in UBERON , an integrative multi-species anatomy ontology, also includes functional aspects, as does that in Wikipedia on which it is based. As we prefer structural definitions of morphological terms (see [1, 33, 34]), we use the above definitions [26, 27] to define the vasculature in the prosomal ganglion of scorpions as a bipolar rete mirabile on the grounds that the main arteries branch into capillaries which form numerous anastomoses and, after passing the prosomal ganglion, reunite first into the collection arteries and then into the efferent coxal arteries.
The vasculature in the prosomal ganglion of Brotheas granulatus corresponds to a high degree with the neuronal organization of the ganglion. The neuropils of the incorporated ganglia are amply supplied by the surrounding arteries, which reticulate on the surface of the neuropils and send loops into them. This is probably to be viewed in the context of the energy requirements of these synapsis-rich structures. Both the somata and the synaptic terminals of nerve cells display a high density of mitochondria and can also be assumed to be involved in a high level of metabolic activity . Thus, the anteroventral regions along the subesophageal midline (i.e. the central ganglion) which exhibit a large number of synaptic contacts and a high level of neuronal activity requiring oxygen and nutrients  are also particularly well-supplied. The same applies to the arcuate body . On the other hand, the pectinate neuropils do not seem to be particularly well-supplied, despite the fact that according to Wolf & Harzsch , numerous synaptic contacts are found in this area. The degree of capillarization in the CNS of spiders  and decapod crustaceans  is comparable to that in scorpions and, as in scorpions, certain distinct areas are better-supplied than others. Sandemann  reported that the capillary system in the neuropil areas of the optic lobe in Carcinus maenas is most elaborate in known synaptic areas. The next best-supplied structures are the somata, while the axons, main nerve bundles and axon chiasmata receive little supply via capillary systems. The same is true of B. granulatus, where the synaptic areas and peripherally arranged somata are better supplied than the rest of the nervous tissue, as the arteries reticulate at the border of the neuropils. No distinct supply to the tracts and neurite bundles was observed.
Another suggestion by Sandemann  is that dense vascularization could be related to the possible synthetization of transmitter substances in the synaptic terminals. However, as immunohistochemical studies of invertebrate nervous systems show that the somata of immunoreactive neurons also emit a clear signal when transmitter substances are labeled , we suggest it is more probable that the numerous mitochondria located in the synaptic contacts need to be supplied intensively with oxygen and nutrients.
In summary, the areas of greatest complexity in capillary networks seems to indicate those areas of the neuropil which require the greatest turnover of fresh hemolymph. This insight and the fact that scorpions have a moderate size and are easy to obtain could be used as the basis for a study into the physiological correlations between oxygen supply and consumption and variations in the neuronal and computational significance of distinct neuropil areas.
Causes for arterial patterns
As described above, the ganglionic arteries are positioned relatively exactly on the border between the centrally located neuropils and the surrounding somata. This raises the question of why and how this particular pattern becomes established during ontogeny. Unfortunately, little is known about the pattern formation and development of arteries in arthropods, so we can only make suggestions as to the reasons behind the patterns observed in the ganglionic vasculature.
One possible explanation could be that nervous system development actually plays a role in arterial pattern formation. McClendon  describes free spaces remaining along the midline of the paired anlagen of adjacent neuromeres in scorpions, into which it is conceivable that the transganglionic arteries grow. The smaller supplying arteries then emanate from the transganglionic arteries and surround the hemi-neuromeres of the ganglia. This, however, is based on the assumption that arteries develop subsequently to the central nervous system anlagen, though it is certainly known from malacostracan crustaceans that the heart only starts to develop once the nervous system anlagen are present (G. Jirikowski, pers. comm.).
Another explanation could be a physiological constraint. Like the tracheae of insects , the arteries may grow along an oxygen gradient, forming networks around structures with a high oxygen and/or nutrient demand.
We could demonstrate that the vascularization in the prosomal ganglion of scorpions is a closed system of capillaries between afferent and efferent arteries. Thus, this capillary network can also be termed a bipolar rete mirabile, adopting the structural definition used for vertebrate circulatory systems. However, similar structures have so far only been termed in vertebrates and some echinoderms.
Similar patterns of intraganglionic arteries have also been observed in Uropygi, Amblypygi, and Araneae. Considering these structural correspondences between the pattern of intraganglionic arteries in Tetrapulmonata and that in scorpions, it seems likely that a similar system of arterial supply to the nervous system was already present in the arachnid ground pattern.
Within the rete mirabile of the scorpion prosomal ganglion, some regions (i.e. neuropils) are better supplied than others. The areas of greatest complexity in capillary networks seem to indicate those areas of the neuropil which require the greatest turnover of fresh hemolymph. The structural information provided here can now be used in functional neuronal studies to determine the physiological and computational significance of the various neuropils in the complex scorpion nervous system. The fact that scorpions have a moderate size and are easy to obtain could lead to new model organisms for physiological studies on correlations between oxygen supply and consumption and variations in the neuronal and computational significance of distinct neuropil areas.
Materials & methods
Data were gathered from two individuals of the chactid Brotheas granulatus in which the injection technique (see below) worked best and provided a complete image of the cerebral vascular system. The taxon sampling also included fourteen buthids, nine vaejovids, four scorpionids, four liochelids, three iurids, two diplocentrids, two chactids, a bothriurid, a chaerilid, a euscorpiid, a pseudochactid and a superstitioniid (see Table 1 in ). Scorpion higher classification follows Prendini and Wheeler .
In order to prepare injections of the circulatory system, the acrylic casting resin Mercox CL-2R/2B (Vilene Comp. Ltd, Tokyo, Japan) was injected into the heart of ether-anaesthetized specimens using micropipettes (Hilsberg pipettes, diameter 1.0 mm, thickness 0.2 mm; pulled with a KOPF Puller 720). The resin was mixed with approximately 0.05 mg MA initiator prior to injection and placed in a 5 ml syringe which was used to fill the pipettes. The pipettes were placed on an adjustable instrument holder in a mechanical micromanipulator and the tips inserted through the intersegmental membrane between the mesosomal tergites directly into the heart. The specimens were left for several minutes after injecting to allow the resin to polymerize and temper. Specimens destined for MicroCT were fixed in Bouin’s fixative, contrasted with iodine  and then critical-point dried (BAL-TEC CPD 030; EMITECH K850). Alternatively, specimens were macerated for 1–2 days by repeated immersion in 10% potassium hydroxide at ambient temperature, followed by washing in a solution of 2 g Pepsin in 10 ml of 2% HCl .
Scanning Electron Microscopy (SEM)
Casts for SEM were air-dried, coated with gold or palladium (BAL-TEC SCD 005; Leica SCD500), and studied using a SEM (LEO 1430; Zeiss DSM 960A), as described by Wirkner and Richter . Corrosion casts were mounted on a specimen holder  to achieve a homogeneous black background.
X-ray imaging was performed with a Phoenix nanotom® (Phoenix|x-ray, GE Sensing & Inspection Technologies) high-resolution MicroCT system in high-resolution mode, using the program datos|x acquisition (target: molybdenum, mode: 0–1; performance: ca. 8–13 W; number of projections: 1080–1440; detector timing: 1000–1500 ms; voxel size ca. 2–10 μm). A volume file was generated using the software datos|x reconstruction and a stack of virtual sections exported with the software VGStudio max. The original μCT image stack of B. granulatus on which most results are based is stored in a public repository (MorphDBase: https://www.morphdbase.de/?B_Klussmann-Fricke_20140304-M-0.1; https://www.morphdbase.de?B_Klussmann-Fricke_20140305-M-1.1)
The 3D-reconstruction of the MicroCT-generated virtual sections was carried out using the software Imaris 7.0.0 (Bitplane©). A scene was created in the program module “Surpass”, and the volume-rendering function chosen to visualize the entire data set. The contours of the organs studied were marked with polygons on each virtual cross-section using the “Surfaces” function. Different functions (“Isoline” and “Distance”) were used for segmentation. Stacks of polygons were visualized via surface rendering.
Figures were arranged into plates using Corel® Graphics Suite X3. Bitmap images were embedded into Corel Draw X3 files and digitally edited using Coral Photo Paint X3.
The authors thank Stefan Richter for fruitful discussions on neuroanatomical terms, Günther Jirikowski for providing interesting details about malacostracan development, and Katarina Huckstorf for discussions on the cerebral vasculature of spiders. Lorenzo Prendini (AMNH) is thanked for providing lab space and several specimens and species identification during CSWs research stay in 2004 at the AMNH. Lucy Cathrow improved the English of this paper.
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