the peripheral nervous system of an ascidian, botryllus schlosseri, as revealed by cholinesterase...

Invertebrate Biology 120(2): 185-198. 0 200 1 American Microscopical Society, Inc.

The peripheral nervous system of an ascidian, Botryllus schlosseri, as revealed by cholinesterase activity

Paolo Burighel,'?" Marina Sorrentino,' Giovanna Zaniolo,' Michael C . Thorndyke,2 and Lucia Manni'

'Dipartimento di Biologia, Universith di Padova, via U. Bassi 58/B, 1-35121 Padova, Italy *School of Biological Sciences, Royal Holloway College, University of London, Egham, Surrey TW20 OEX, U.K.

Abstract. In this study we present the first detailed description of the motor component of the peripheral nervous system of an ascidian showing its three-dimensional organization and the spatial relationships between nerves and contiguous organs. Nerves of the oozooid and blastozooid of the colonial ascidian Botryllus schlosseri were analyzed using a histochemical method for detecting cholinesterase activity in whole-mount preparations and in sections for light and electron microscopy. Except for the neural gland and gonads, all tissues are well innervated by cholinesterase-reactive neurites. Each blastozooid of the colony possesses an individual nerve plexus which is not in continuity with that of adjacent zooids. The innervation of the mantle, oral and cloaca1 siphons, branchial basket, heart, and gut are described. Most organs possess a complex network of nerves often with multiple origins from different pathways. A sophisticated pattern is described for the first time in the gut, which receives innervation from the endostyle, the roof of the branchial basket, and the posterior mantle. Dilated axonal regions but no obvious cell bodies were recognized in the peripheral nervous system revealed by cholinesterase activity. The localization of nerves is discussed together with a consideration of their physiological role.

Additional key words: cerebral ganglion, cholinesterase, Tunicata, Urochordata

The nervous system of ascidians demands attention for several reasons. (1) Recent data indicate that the larval nervous system has many characteristics in common with the nervous system of vertebrates and confirm a shared ancestry with other chordates (Wada et al. 1996; Corbo et al. 1997). (2) The nervous system exhibits different modalities of form during the life cycle of a species. In the embryo, it differentiates from a neural plate by a mechanism typical of chordates. At metamorphosis, the larval nervous system regresses and a new one is formed in the oozooid. This comprises an invertebrate-like cerebral ganglion which, in some species, shows an unusual capacity for regeneration (Schultze 1899; Bollner et al. 1995, 1997). In colonial species, moreover, a new nervous system is formed in each blastozooid during budding (Brien 1933). Although the early stages of formation differ in oozooid, blastozooid, and regenerating animals, an identical final organization is achieved (Bollner et al. 1995; Burighel et al. 1998; Manni et al. 1999). ( 3 ) The

Author for correspondence. Tel: 39 049 8276185; Fax: 39 049 8276199; e-mail: [email protected]

relationship between the central and peripheral nervous system (PNS) seems to be different from that seen in other groups of metazoans. This is borne out by the fact that the ciliary feeding mechanism shows relative autonomy from the central nervous system and in some species animals can survive and respond to external stimuli after ablation of the cerebral ganglion (Hoyle 1952; Mackie & Wyeth 2000, for review). (4) The ability to propagate electrical signals is not limited to the nervous system in that some colonial species have excitable epithelia able to transmit electrical signals between zooids and coordinate behavioral responses in the colony (Mackie 1995a).

Most reports concentrate on the larval central nervous system and its development during embryogen- esis. A number of genes involved in the formation of the larval nervous system have been identified and a complete cell lineage reported (Satoh 1994). Recently, the central and PNS of the larva were described showing the extent of both sensory and motor pathways (Takamura 1998; Vorontsova et al. 1997; Sotgia et al. 1998; Sorrentino et al. 2000).

In comparison, knowledge of the nervous system of

186 Burighel, Sorrentino, Zaniolo, Thorndyke, & Manni

adults, in particular the PNS, is rather limited (Good- body 1974; Bone & Mackie 1982; Arkett et al. 1989; Mackie & Wyeth 2000) because of the difficulty of detecting nerves using conventional histological techniques. The adult possesses a neural complex, derived from an anterior remnant of the embryonic neural tube during metamorphosis (Satoh 1994; Manni et al. 1999). It comprises the cerebral ganglion and associated neural gland, which extends posteriorly as the dorsal strand. The PNS derives from nerves emerging from the ganglion and from sensory cell bodies located in the periphery (Mackie & Wyeth 2000). A visceral plexus, showing gonadotropin releasing hormone (GnRH) immunoreactivity and associated with the dorsal strand, has also been recognized (Mackie 199Sb; Powell et al. 1996; Tsutsui et al. 1998).

Millar (1953), in whole-mount preparations of adults of Ciona intestinalis, described the general innervation pattern, including paired anterior and posterior nerves and an unpaired visceral nerve emerging from the ganglion. More detailed observations are rather scanty, especially those on the gut; Millar (1953, p. 42) stated, ‘‘It was rarely possible to follow the nerve fibers far through the connective tissue towards the epithelium of the gut, and nerve endings have not been seen.”

More attention has been given to the innervation of the branchial basket because of its implication in control of ciliary activity. In an elegant series of experi- ments, Mackie and colleagues (1974) showed that ciliary control in Corella was largely mediated by a network of fine nerve fibers originating as branches from the visceral nerve. Staining for acetylcholinesterase (AChE) activity allowed the innervation of the branchial basket to be described in detail (Arkett et al. 1989). Such activity was not restricted to the synaptic sites but was present throughout the neurons. Recently, Mackie & Wyeth (2000), using electrophysiological and histochemical methods in the mantle and branchial basket of normal and deganglionated solitary ascidi- am, found no evidence of a peripheral nerve net, i.e., one with cell bodies lying within the net itself. With AChE histochemistry and immunolabelling against tubulin and GnRH, they distinguished between motor and sensory neurons, the first having cell bodies in the ganglion and the second, in the periphery.

Attention has also been focused on the dorsal strand nerve plexus because of its evolutionary implications for the control of reproduction in vertebrates. The plexus, which is not labeled by AChE reaction product, was investigated using antisera against GnRH (Mackie 1995b; Powell et al. 1996; Tsutsui et al. 1998). The plexus extends to viscera and gonads and

may possibly arise by delamination of epithelial cells from the dorsal strand (Fedele 1938).

Thus, considering the peculiar features of the ascidian nervous system and the scarce information about the general pattern of adult innervation, we analyzed the motor component of the PNS in moids of the colonial ascidian Botryllus schlosseri PALLAS 1766. Old reports (Pizbn 1893) were limited to nerve pathways in the dorsal mantle. In this species, each colony orig- inates from a swimming larva, which metamorphoses into a sessile zooid and grows by cycles of blastoge- netic generations. Its adult zooids resemble individual solitary ascidians in their general body organization. The small size of the zooids facilitates mapping nerve pathways in whole-mount preparations, showing the three-dimensional network and the spatial relationships between contiguous organs. Data were obtained from whole-mount specimens by staining for AChE, and by scanning electron microscopy (SEM) as well as light (LM) and transmission electron microscopy (TEM). Specimens for TEM were also treated to reveal cholinesterase activity.

Methods

The ascidian Botryllus schlosseri (family Styelidae, order Stolidobranchia) forms colonies composed of a great number of small zooids, embedded in a common tunic and arranged in star-shaped systems. Each zooid has its own oral siphon toward the periphery and shares the common cloaca1 siphon at the center of the system (Figs. 1 , 2). Animals used in this study were collected in the lagoon of Venice and cultured on glass in the laboratory (at 18°C) following Sabbadin’s (1960) technique.

Whole-mount preparations. Colonies were anesthetized with MS 222, fixed in Bouin’s fluid, washed in SO% ethyl alcohol, rehydrated and stained with Mayer hematoxylin. After washing in distilled water, they were dehydrated in alcohol and mounted with bal- sam. For AChE reaction, colonies anesthetized and fixed in 4% paraformaldehyde in seawater (pH 7.2) were dissected using a razor blade and treated following the method of Karnovsky & Roots ( 1 964), using 0.1 M sodium maleate buffer (pH 6.0) and acetylthiocholine iodide (Sigma) as substrate. The specificity of the reaction was controlled by omitting the substrate or adding the specific inhibitor 0.03 M neostigmine bromide (Sigma).

Transmission electron microscopy. Selected specimens were fixed in 1 .S% glutaraldehyde buffered with 0.2 M sodium cacodylate, pH 7.4, plus 1.6% NaCl. After washing in buffer and postfixation in 1% OsO, in 0.2 M cacodylate buffer, the specimens were de-

Ascidian peripheral nervous system 187

Fig. 1. Surface view of a colony of Botryllus schlosseri. Hematoxylin. Bud (b); cloaca1 siphon area (cs); endostyle (e); gut (g); neural complex (arrows); oral siphon (0s); tunic (tn). Scale bar, 400 Km.

hydrated and embedded in Araldite. Thick sections (1 pm) were counterstained with toluidine blue; thin sections were given contrast by staining with uranyl ac- etate and lead citrate. Micrographs were taken with a Hitachi H-600 electron microscope operated at 80 kV. To reveal AChE, tissue was treated following the method of Gautron (1982), prefixing the specimens in 4% paraformaldehyde and 0.2% glutaraldehyde buffered with 0.2 M sodium cacodylate, pH 7.4, and using acetylthiocholine perchlorate (Sigma) as substrate. Specimens were then postfixed in 1% OsO, in 0.2 M

pericoronal& sin1 I C

cacodylate buffer, dehydrated, and embedded in Ar- aldite. Ultrathin sections were stained with uranyl ac- etate and lead citsate. Controls were treated as described above, but without the substrate.

Scanning electron microscopy. Zooids were fixed as described for TEM. After dehydration, zooids were dissected in order to eliminate any tissue covering the ganglion. Specimens were then critical-point dried, sputter-coated with gold palladium, and observed using a Cambridge Stereoscan 260.

All figures were acquired with a Duoscan (Agfa), elaborated with Corel Photo-Paint, and composed with Corel Draw 8 (Corel Corp.).

Results Organization of the nerve plexus is described for

both blastozooids and oozooids of Botryllus schlosseri. We report the pattern of innervation of the blastozooid (Figs. 1, 2), considering first the cerebral ganglion and nerve roots, and then the innervation of each organ (oral and atrial siphons, branchial basket, digestive system, and heart). The oozooid exhibits a pattern of innervation similar to that of the blastozooid. The only differences between the two are mentioned at the end of this section.

Cerebral ganglion

The localization of AChE allowed us to describe the organization of the cerebral ganglion and the routes of motor nerves leaving it and to map the detailed inner-

phageal becula end

subendc - - n stomach D sinus A n D

1st

a L Fig. 2. Diagram of a blastoxooid. A: sagittal section; B: transverse section through posterior region of branchial basket and gut. The mantle (gray) is delimited by epidermis and atrial epithelium. The branchial basket is double-walled and its interspace is in continuity with the mantle at the base of the oral siphon (where the pericoronal sinus runs), at the level of the whole subendostyIar region, and via branchial (not shown) and visceral trabeculae.


Figs. 3, 4. Anterior nerve roots (ar) formed by numerous parallel axons running together. SEM. Fig. 3. The cerebral ganglion is dissected to show the fibrous medulla (md). Scale bar, 10 pm. Fig. 4. An enlargement of the area marked by a rectangle in Fig. 3 showing axonal processes (arrows) starting from peripheral neurons of the cortex (cx). Scale bar, 2 pm. Fig. 5. Nerve root with a peripheral neuron sending its axon (arrow) along the nerve. A fibrous sheath (sh) envelops the nerve (n). Blood cells (bc). TEM. Scale bar, 3 pm. Fig. 6. Detail of a nerve showing axonal processes con-

vation pattern of the dorsal mantle. The cerebral ganglion lies close to the oral siphon, adjacent to the neural gland and dorsal organ (Fig. 2a). It comprises a medulla of densely packed neurites surrounded by a cortical ring of cell bodies (Fig. 3). Neuronal cell bodies were also found in the roots of nerve bundles which extend from both the anterior and posterior ends of the ganglion (Figs. 3-5). The axons possess microtubules, vesicles, secretory granules, mitochondria, and occasional large vacuoles. The nerves are not myelinated and lack typical glial cells but are surrounded by a fibrous sheath recognizable in TEM (Figs. 5 , 6). No synapses are apparent between the axons in the nerve bundles. The anterior and posterior roots are paired and far more developed than the lateral roots (Figs. 7, 8). A fifth, large nerve, the visceral nerve, emerges postero-ventrally from the ganglion and is directed towards the viscera running in the roof of the branchial basket. No dorsal strand plexus was observed.

As the nerve bundles pass farther from the ganglion, they split into thinner bundles (Fig. 7) that innervate mantle, oral siphon, and endostyle anteriorly. Lateral nerves innervate the lateral mantle and converge ventrally. Posterior nerves supply the posterior mantle and cloaca1 region, then pass to the viscera through the visceral trabeculae. Generally, nerves were found to run perpendicular to the discrete circular muscle fibers of the body wall musculature. AChE reaction product at the TEM level was seen in the cerebral ganglion and along the nerves (Figs. 9, lo). Concentrations of electron-dense precipitate characterized the intercellular spaces between neurites of the ganglionic medulla; perinuclear cisternae and endoplasmic reticulum cisternae of ganglionic cell bodies were labeled. The precipitate was also visible in the intercellular spaces between muscle and nerve fibers. The neural gland and its posteriormost part, the dorsal organ (Burighel et al. 1998) (see Fig. 2a), appeared not to be innervated.

Innervation of oral and atrial siphons

Anterior roots from the ganglion form the pericoronal nerves, which split repeatedly to innervate the oral siphon and the anterior mantle with thinner branches (Fig. 7). The pericoronal nerves encircle the siphon and meet at the anterior end of the endostyle.

t

taining cross and longitudinal sections of microtubules (nit), granules (arrows), and various kinds of vesicles (arrowheads). A fibrous sheath (sh) envelops the nerve. TEM. Scale bar, 0.5 pm.


sal languet, which forms, together with the languets of other blastozooids, the common cloacal siphon of each star-shaped system of zooids. A sheet of tunic sepa- rates contiguous zooids.

Branchial basket

At the base of the oral siphon, pericoronal nerves from the anterior roots meet at the anterior end of the endostyle. Each nerve comprises a bundle of axons, which approach the opposing axons and partially cross, giving rise to 2 pairs of nerves running in parallel along the endostyle (Figs. 13-15). Nerves flank the endostyle and form the main ventral nerve chains of the blastozooid. Posteriorly, they split repeatedly into numerous small nerves that innervate the viscera and heart. The median nerves, which are the main subendostylar nerves, are thicker than the lateral nerves and run at the level of endostylar Lones 2 and 3, whereas the lateral subendostylar nerves lie adjacent to zones 5 and 6 (Fig. 15). Numerous connections link the main and lateral subendostylar nerves of the same side, whereas no connection was seen between the main subendostylar nerves of opposite sides. Some free endings arising from subendostylar nerves were seen in the wall of the endostyle.

The lateral walls of the branchial basket are perfo- rated by elliptical ciliated stigmata elongated antero- posteriorly (Martinucci et al. 1987). Transverse interstigmatic sinuses run between successive rows of stigmata and connect the dorsal with the subendostylar sinus. They also communicate with the longitudinal interstigmatic sinuses and the sinuses running within

Fig. 7. Diagram of the nerves of tile dorsal mantle as re- the 3 pairs Of longitudinal folds. This entire sinus net- vealed by AChE activity. A: dorsal view; B: lateral view of work is richly innervated. The branchial basket re- the cloacal region. The visceral nerve (not shown) lies in a ceives nerves both from the lateral subendostylar deeper plane. nerves ventrally and from the visceral nerve dorsally.

The visceral nerve splits into 2 large roots, which

The nerves split further into progressively smaller bundles, which eventually reach the siphon apex (Fig. 11) . The siphon is extremely richly innervated, which is in keeping with the numerous circular muscle fibers and presumptive secondary sensory cells present in the tentacles (Burighel & Cloney 1997). Each tentacle of the oral siphon receives a nerve (Fig. 12), which arises from the nerve ring at the siphon base.

divide into numerous dorso-ventrally oriented branches running in the transverse interstigmatic sinuses. Posteriorly, the visceral nerves reach the esophagus, then split into thin nerves directed towards the viscera. Two nerves in each transverse interstigmatic sinus ul- timately send single axons into the longitudinal interstigmatic sinuses (Figs. 16-19) so that each stigma is completely encircled by a nerve ring. The lateral sub- -

Posterior roots from the ganglion divide into 2 or 3 major bundles passing to the dorso-lateral mantle (Fig. 7). Median bundles run parallel on the atrial siphon roof. At the base of the cloacal siphon they form a nerve ring (the pericloacal nerves) running along the pericloacal sinus. Some smaller nerves pass to the dor-

endostylar nerves contribute in the same manner to stigmata1 innervation. In contrast, no nerves coming from the pericoronal nerves or from the branchial trabeculae that connect the branchial basket to the lateral wall of the mantle were seen to reach the branchial walls.


Digestive system

The branchial sac continues posteriorly with the esophagus, which turns ventrally to open into the stomach (Fig. 20). The esophageal wall is longitudi- nally folded and consists of ciliated, columnar mucous cells and scattered endocrine cells containing small granules in the basal cytoplasm (Burighel 1970) (Fig. 21). The sac-like stomach possesses 8 parallel folds extending its entire length. The intestine leaves the stomach ventrally and runs upwards to the left of the branchial sac, ending dorsal to the atrial chamber. Part of its wall is encrusted by the pyloric gland, consisting of numerous blind ampullae connected to a net of anastomosing tubules that join into the collecting duct. This duct opens into the stomach at the base of the pyloric caecum, a digitiform outgrowth of the gastric wall (Burighel 1970) (Fig. 20).

The esophagus, stomach, and intestine are innervated by a complex network of nerves deriving from the left main and lateral subendostylar nerves, the visceral nerves, and the nerves of the posterior mantle, which reach the gut via visceral trabeculae (Fig. 22). The nerves run in the space between the visceral and peri- visceral epithelium comprising the peri-esophageal, perigastric, and peri-intestinal lacunae (Fig. 2b).

The esophagus is richly innervated by fibers derived mainly from the visceral nerves. Several thin axonal processes contain numerous electron-dense granules, rough endoplasmic reticulum profiles, and small Golgi fields. These processes are often associated with endocrine cells (Fig. 21).

The nerves of the stomach are found mainly at the level of the folds (Fig. 23) and derive from the left subendostylar nerves, from the visceral nerves, and from the posterior mantle nerves via left and right gastric trabeculae.

In contrast, innervation of the intestine is sparse, except for the area of the pyloric gland, where a complex network is visible. Thin nerves join the gastric and intestinal networks, running along the connective tissue supporting the collecting duct (Fig. 20).

Heart

The heart, located in the ventral mantle between the endostyle and the stomach, is in form of a curved, double-walled cylinder of external pericardium and in- ternal myocardium, which are in contact along the raphe (Nunzi et al. 1979). It is innervated by posterior branches from the right subendostylar nerves (Fig. 24), which split repeatedly and are directed towards the raphe. It is also innervated by nerves from the latero- posterior mantle directed perpendicularly towards the heart. TEM reveals diffuse AChE reaction product on

the pericardium and scarce product on the myocardium. Visceral innervation is summarized in Fig. 25.

Oozooid

The oozooid has 4 or 5 long protostigmata oriented dorso-ventrally, whereas the blastozooid has parallel rows of small elliptical stigmata elongated antero-posteriorly. Whole-mount oozooids show a basic pattern of innervation similar to that seen in blastozooids. Mi- nor differences, such as at the level of branchial stigmata, reflect the different anatomical organization of oozooid organs, since the same source of the nerves and the same pattern of branching was seen in both the blastozooid and oozooid.

Discussion

Mackie & Wyeth (2000) studied the histology of portions of the mantle and branchial sac in normal and deganglionated specimens of two solitary ascidians using AChE histochemistry and immunolabelling. The use of antibodies against tubulin and GnRH allowed them to distinguish the exclusively sensory units from the motor AChE positive fibers, but so far, similar techniques applied by us to Bott-yllus schlosseri have failed to give comparable results (unpubl. obs.). How- ever, in this ascidian, the use of AChE histochemistry labels a complex nervous network which extends to the periphery and into most of the organs.

Generally, AChE has a pivotal role in neuromuscular synapses but is also found in alternative molecular forms on external surfaces of cells and in various cytoplasmic regions. Moreover, AChE can be involved in non-cholinergic roles and its activity plays a critical role in the development of various tissues (see Mas souliC et al. 1993, for a review). In developing ascid ians, the AChE reaction product labels embryonic muscle cells (Whittaker 1973, 1980) and larval adhe- sive papillae (Coniglio et al. 1998); in adults, acetyl- choline plays its main role in regulating muscle and ciliary activities (Florey 1951, 1967; Arkett et al. 1989; Mackie & Wyeth 2000). The cholinergic response seems to be modulated by various neuropep- tides (Thorndyke & Georges 1988).

In this study, we describe in detail the nerve plexus of a colonial ascidian zooid, as revealed by the AChE histochemical method, which can be considered a general, high-resolution marker for motor nerves. For the small blastozooids of B. schlosseri, this technique allowed us to follow the complete pathways of nerves from the ganglion to the mantle and to visceral organs including the gut, where nerves have not previously been described (see Goodbody 1974).

In agreement with Arkett et al. (1989) we found that


Fig. 8. AChE reaction in nerves of the dorsal mantle. Nerves from the anterior and posterior root5 (arrows) are thicker than lateral nerves (arrowheads). Anterior at top. Whole-mount specimens. Scale bar, 70 pm. Fig. 9. AChE reaction in cerebral ganglion. Intercellular spaces between iienrites of medulla (md) are densely labeled. Cortex (cx). TEM. Scale bar, 2 pni. Fig. 10. AChE reaction in muscle and nerve fibers. Axons (asterisks) between muscle fibers (mf) are labeled by electron-dense product. TEM. Scale bar, 0.5 pm. Figs. 11, 12. AChE reaction in whole-mount specimens. Fig. 11. The oral siphon (0s) possesses numerous axonal endings (arrows). Scale bar, 70 pm. Fig. 12. In each tentacle (t) of the oral siphon (0s) is a process positive to AChE (arrowheads). Scale bar, 40 pm.

the reaction product for AChE is not limited to synaptic sites, but is distributed throughout the entire neuron. Also, we assume that the staining marks the ma- jority of nerves in that many ascidian nerves are mixed, containing axons of both sensory and motor neurons (Bone & Mackie 1982). Thus, as in other ascidians (Mackie & Wyeth 2000), exclusively sensory neurites, before entering nerve bundles heading for the ganglion, cannot be visualized.

SEM and TEM observations show that the nerves

comprise numerous fascicles and are not myelinated, nor are they supported by glial cells, but by a fibrous matrix. Synapses were never detected along the nerves and were restricted to the medulla of the cerebral ganglion, as reported previously (Arkett et al. 1989; Ko- yama & Kusunoki 1993; Burighel et al. 1998; Manni et al. 1999).

In zooids of B. schlosseri, the main nerves branching from the ganglion show the pattern characteristic of all ascidians studied so far: paired anterior nerves


Fig. 13. AChE reaction in a whole-mount specimen. The pericoronal nerves (pn) meet at the anterior end of the endostyle (e) and partially cross to form the main (msn) and lateral (lsn) subendostylar nerves. Scale bar, 30 km. Fig. 14. AChE reaction on an oblique thin section of the endostyle. The electron-dense reaction product marks the position of main (msn) and lateral (Isn) subendostylar nerves. Zones 4, 5, and 6 of the endostyle are indicated. TEM. Scale bar, 5 km. Fig. 15. Diagram of a transverse section of the endostyle to show the position of subendostylar nerves. Numbers 1-8 indicate the 8 zones of the endostyle. Fig. 16. Transverse section of stigmata. Two interstigmatic longitudinal nerves (arrows) lie at the base of two clusters of ciliated cells. TEM. Scale bar, 2.5 bm. Fig. 17. Detail of a stigma in which the presence of a nerve (arrows), similar to those of Fig. 16, is indicated by electron-dense product of the AChE reaction. Cilia of the stigmata1 cells (arrowhead). TEM. Scale bar, I pm. Fig. 18. AChE reaction in a whole-mount specimen. Detail of stigmata near the endostyle (e). Nerves running into the transverse interstigmatic sinuses (arrows) come directly from the lateral subendostylar nerve (lsn) and send branches into each longitudinal interstigmatic sinus forming a nerve ring (arrowheads) around the stigma. Main subendostylar nerve (msn). Scale bar, 40 pm.

giving off branches to the oral siphon and mantle; tween oozooid and blastozooid reflect the different or- paired posterior nerves supplying the atrial siphon and ganization of the branchial basket in the two forms. mantle; an unpaired visceral nerve emerging ventro- Each blastozooid has its own plexus and no nerve posteriorly and directed to the viscera (Goodbody fibers interconnecting zooids or running in the tunic 1974; Burighel & Cloney 1997). Minor differences be- were recognized. Thus, the coordinated behavior of


19 Fig. 19. Diagram of the innervation pattern of the branchial basket. A: dorsal view; B: ventral view.

blastozooids in the same colony could be mediated by the excitable epithelium lining the tunic blood vessels, which transmits action potentials from cell to cell via gap junctions (Mackie & Singla 1983).

All organs, except the neural gland and gonads, show extensive innervation. For the neural gland, no nerve endings were previously reported on the basis of ultrastructural observations (Ruppert 1990; Burigh- el et al. 1998), whereas, for gonads, some visceral nerve branches were described in Ciona in the terminal part of the oviduct, where a muscular sphincter is

present (Millar 1953; Goodbody 1974). Possibly, the absence of innervation in B. schlosseri may reflect the fact that it has very specialized gonads with vestigial male and female gonoducts (Mukai & Watanabe 1976; Zaniolo et al. 1987). However, gonads could be reg- ulated by systems not detectable with AChE. For example, in most species, a dorsal strand plexus of GnRH immunoreactive cells extends from the ganglion to the gonads and participates in control of sexual activity (Mackie 1995b; Powell et al. 1996; Craig et al. 1997; Tsutsui et al. 1998).

Most organs of B. schlnsseri receive a complex network of nerves of different origin. This is particularly evident in the gut, which receives branches from the visceral, subendostylar, and mantle nerves, and in the branchial basket, which receives branches from visceral and subendostylar nerves. The physiological implications of this pattern are not known: it could be related to developmental processes that provide each organ with a dual innervation. Equally, it could reflect the functional (excitatory or inhibitory, sensory or motor) role of axonal endings arising from different areas of the cerebral ganglion. This multiple origin of fibers innervating the same organ is not in agreement with the view that somatic innervation is entirely associated with paired anterior and posterior nerves, and that the visceral innervation is confined to a single unpaired posterior nerve (Millar 1953; Goodbody 1974).

Somatic system Fig. 20. Diagram of the innervation pattern of the gut in a dorsal view. Esophagus and rectum are displaced to show nerves (seen in dorsal view, the rectum lies above the esophagus).

A network of nerves in the mantle radiates from the ganglion and converges ventrally with axons particularly concentrated at the level of oral and atrial si-


Fig. 21. Esophageal epithelium showing, scattered among ciliated mucous cells, an endocrine cell (asterisk) with numerous electron-dense granules. A nerve process (arrowhead) is visible in the connective tissue supporting the epithelium. Perivis- ceral epithelium (pe). TEM. Scale bar, 1.5 pm. Fig. 22. AChE reaction. Arrows indicate a nerve running in an intestinal trabecula. TEM. Scale bar, 3.5 pm. Fig. 23. Transverse section of a gastric fold. A nerve (arrow) at the base of ciliated cells is marked by AChE reaction product. Reaction product is also dispersed in some intercellular spaces. Basal lamina (bl). TEM. Scale bar, 1.5 pm. Fig. 24. Section of endostyle (e) and heart (h). AChE activity labels branch of a subendostylar nerve (arrows) reaching the heart at raphe level (arrowhead). TEM. Scale bar, 2 km.

phons, as would be expected from their established sensitivity and the rich musculature of their sphincters.

The role of AChE in regulation of ascidian muscular activity is well supported by the evidence that muscle excitation is inhibited by curare, a specific cholinergic blocker (Florey 1951; Nevitt & Gilly 1976; Mackie & Wyeth 2000). With TEM we showed that the AChE reaction product diffuses in the nerve/muscle contact region. We did not analyze in detail muscle cell innervation, which shows differences between tunicates (Bone & Mackie 1982). However, the presence of AChE product in nerve endings in contact with more than one muscle cell cannot by itself indicate the formation of multiple neuromuscular junctions on different muscle fibers from the same nerve.

The presence of many free nerve endings in the oral siphon tentacles was reported in Ciona by Markman (1 9x9, whereas Arkett et al. (1989) concluded that in the examined corellids, the tentacles lack nerves. In B.

schlosseri, the tentacles lack intrinsic musculature and diffuse ciliated cells (unpubl. obs.) but possess a row of monociliated cells, interpreted as presumptive secondary sensory cells (Burighel & Cloney 1997). Thus, the meaning of AChE positive nerves penetrating the tentacles is, at the moment, unclear.

Visceral system

Branchial basket. In B. schlosseri the branchial basket is innervated by branches of the visceral and subendostylar nerves, which together form the nerve rings around each stigma. This differs partially from the condition in some species of Corellidae (Mackie et al. 1974; Arkett 1987; Arkett et al. 1989), in which the branchial innervation is mainly supported by the visceral nerve and only secondarily by subendostylar and mantle nerves via branchial trabeculae. Arkett and colleagues (1989) showed the extent of innervation of


Fig. 25. Diagram of Botryllus schlosseri, ventral view. The ventral epidermis, the epithelia of the right peribranchial chamber, and part of the left peribranchial chamber were removed to show the superficial and deep innervation pattern of the body. Exposed nerves are brown; nerves covered by epithelia are yellow.

the ciliated cells of the stigmata in Corella in$ata and Chelyosoinu productum, which have spiral stigmata delimited by clusters of 7 ciliated cells. All the clusters of each spiral are innervated and most of the axons follow the blood sinus around the stigma as it coils to form the spiral. This neuronal organization supports ciliary control in the branchial basket of Corellidae (Mackie et al. 1974). B. schlosseri possesses very simple, elliptical stigmata delimited by the typical patterns of 7 ciliated cells (Martinucci et al. 1987). Stigmata are well innervated, with characteristic AChE axons, rich in small vesicles and close to stigmata1 cells, in- dicating that the branchial basket of B. schlosseri, although simple, also requires accurate nervous control of ciliary activity.

In the stigmata of Corellu in.utu, single axons ter- minate in synaptic boutons at the bases of the clusters of ciliated cells (Arkett et al. 1989). In B. schlosseri, similar terminal swellings were not recognizable in whole-mount specimens, not even in those organs, such as the oral siphon, in which we could follow the whole pathway of single fibers.

The subendostylar nerves form the main ventral nervous tracts of the blastozooid with branches directed to branchial basket, heart, and gut. Free axonal endings extend to the endostyle, possibly for regulation of secretory and/or ciliary activity. Primary sensory cells, reported in the endostyle of Cionu by Markman (1958), were not found in B. schlosseri using TEM.

Digestive system. A dense network of nerves has not previously been reported in association with the alimentary tract. Millar (1953, p. 42) described in Ciona “the presence of some branching and occasion- ally anastomosing nerve fibres but over most of the gut these nerve fibres are scarce.” lntestinal musculature is rarely present and food is transported by cilia along the canal (Burighel & Cloney 1997). Tn Ciona a small sphincter muscle surrounds the anus (Millar 1953), and according to Goodbody ( I 974), “there is little evidence to suggest that any other part of the canal has a special innervation.” In contrast to previ- ous reports on other ascidians, the current method al- lows us to reveal a complex network of nerves in- vesting the gut of B. schlosseri, branching on the


connective tissue supporting the esophagus, stomach, and intestine.

Although no exhaustive data on the neuronal control of intestinal activity are available, numerous studies suggest that the regulation of gut secretory activity is by the typical, basally granulated, endocrine cells found scattered among enterocytes. A variety of endocrine cell types have been described through the use of antisera raised against vertebrate neurohormonal peptides and it seems likely that ascidian peptides function as regulatory agents in digestion (Fritsch et al. 1982; Thorndyke & Georges 1988): for example, it has been shown that cholecystokinin and bombesin have significant stimulatory effects on stomach secretory activity in Styela c law (Thorndyke 1977).

In addition to typical nerve fibers, the esophagus of B. schlosseri possesses fibers particularly rich in electron-dense granules resembling those that accumulate in the basal cytoplasm of esophageal endocrine cells. The ultrastructural characteristics of these fibers, with an abundance of rough endoplasmic reticulum and Golgi membranes, suggest that they are specialized for neurohormonal peptide production and secretion. Probably, they participate, together with the endocrine cells, in the neurohormonal control of esophageal activity. The source of these fibers is not clear and although B. sclzlosseri lacks a typical dorsal strand (Bur- ighel et al. 1998), the possibility remains that these fibers represent a derivative of a dorsal strand plexus.

The nerves of the stomach are mainly localized in the pockets at the base of cap regions which contain ciliated mucous cells and scattered endocrine cells (Burighel & Milanesi 1975); thus a relationship between the latter and the adjacent neurons is plausible. The most heavily innervated region of the intestine is the middle segment, which displays a thick network of nerve fibers running among the tubules and ampullae of the pyloric gland. This segment comprises epithelial cells in various stages of necrosis, while the scantily innervated proximal and distal segments in- clude absorbtive, endocrine, and ciliated mucous cells (Burighel 1970; Burighel & Milanesi 1977).

In general terms, we assume the innervation of the gut epithelium may support various functions: (1) sensory, by means of free axonal endings: (2) control of ciliary activity for food transportation; (3) regulation of pyloric gland cells and associated gut cell metabo- lism; (4) integration with endocrine cells for the regulation of gut activity via neurosecretory fibers, such as is seen in the esophagus.

Heart. The possibility that cardiac activity is reg- ulated by the nervous system has long been debated, but it now seems certain that the heart is controlled by

a myogenic mechanism involving several pacemakers (reviewed by Goodbody 1974).

In B. schlosseri, the heart has a simple organization and its development and ultrastructure have been described (Nunzi et al. 1979). Our observations on whole-mount specimens reveal that numerous nerve fibers invest the heart from the right subendostylar nerves and latero-posterior mantle nerves, forming a sort of cardiac plexus, the details of which were dif- ficult to follow. Nerve bundles have been identified in the pericardium of other ascidians, sometimes running in the raphe but never penetrating the myocardium (Goodbody 1974). Bone & Whitear (1958) suggested that the nerve plexus of the pericardium has a sensory role and that the fibers may respond to pressure chang- es in the heart forming part of a sensory-motor reflex arc to the pericardial smooth muscle and striated myocardial cells. It seems likely that the nerve fibers recognized in B. schlosseri participate in a sensory-motor plexus, as proposed for other ascidians (Goodbody 1974).

The analysis of TEM for AChE reaction product shows the presence of nerves from the endostyle innervating the raphe. We were unable to find unequiv- ocal evidence that nerve fibers enter the raphe, reaching the myocardial cells. However, AChE reaction product was widely dispersed on the lumenal surface of myocardial cells close to the raphe and on the pericardium. We cannot exclude the possibility that the localization of the electron-dense granules represents an artifact due to dispersal or diffusion of reaction products. Equally, however, AChE could be produced locally. The possibility that cardiac cells secrete ace- tylcholine into the heart lumen where it acts as a local humoral modulator was suggested by Kalk (1970). Thorndyke & Georges (1988) pointed out that, with no direct evidence for this hypothesis, there remains the possibility that other chemical regulators such as neurohormonal peptides may be involved in the control of heart beat. Our data on the presence of AChE suggest that there may well be a cholinergic element also in the cardiac regulatory pathway.

Peripheral autonomy

Our observations on B. schlosseri show that a complex network innervates most organs, and axons often exhibit large swellings recalling cell bodies in shape and dimension. However, the ultrastructural analysis failed to reveal the presence of nuclei in these swellings. Arkett et al. (1989) observed nerves with similar swellings in Corellidae. Given the lack of evidence for peripheral nerve cell bodies and synapses along the nerves, one of the most unusual and astonishing fea-


tures of ascidians is the relative autonomy of some functions from the cerebral ganglion and the existence of a peripheral conduction system that survives degan- glionation. This problem was recently investigated by Mackie & Wyeth (2000), who confirmed that motor cell bodies lie entirely within the ganglion. The only neurons with cell bodies in the periphery, except for those of the dorsal strand plexus, are exclusively sensory. Thus, the capacity of deganglionated specimens to respond to stimulation and to maintain spontaneous activity was attributed to the existence of interconnected motor nerve terminals or interconnected sensory neurites or some combination of the two. At present, in the absence of direct morphological evidence, the finding of the very complex network of B. schlosseri gives support to the hypothesis of Mackie & Wy- eth (2000) that ascidians maintained the cerebral ganglion for coordination and for rapid defensive responses, whereas devolved certain functions to the periphery as an adaptation to sessile life.

Acknowledgments. We express our thanks to Drs. G.O. Mackie and Q. Bone for providing valuable criticism of the manuscript. We thank Mr. Miolo for technical assistance and Mr. Friso for drawings. This investigation was supported by grants from M.U.R.S.T. to PB; MCT thanks the University of London Central Research Fund and BBSRC Grant INS 028 I9 for support.

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