Conduction and Coordination in Deganglionated Ascidians

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Conduction and coordination in deganglionated ascidians

G.O. Mackie and R.C. Wyeth

Abstract: The behaviour of Chelyosoma productum and Corella inflata (Ascidiacea) was studied in normal and deganglionated animals. Chelyosoma productum lived for over a year after deganglionation and the ganglion did not re- generate. Electrophysiological recordings were made from semi-intact preparations. Responses to stimulation and spontaneous activity continued to be transmitted through the body wall and branchial sac after deganglionation. Spread was slow, decremental, and facilitative. Treatment with >10 µg·mL–1 d-tubocurarine abolished all responses, indicating that nerves mediate conduction of excitation after deganglionation. Histological study using cholinesterase histochemistry and immunolabelling with antisera against tubulin and gonadotropin-releasing hormone showed no evidence of a peripheral nerve net in regions showing conduction, contrary to previous claims. The cell bodies of the motor neurones were found to lie entirely within the ganglion or its major roots. Their terminal branches intermingled to form netlike arrays. Sensory neurons were identified with cell bodies in the periphery, in both the body wall and the branchial sac. Their processes also intermingled in netlike arrays before entering nerves going to the ganglion. It is concluded that the “residual” innervation that survives deganglionation is composed of either interconnected motor nerve terminals, interconnected sensory neurites, or some combination of the two. In re-inventing the nerve net, ascidians show conver- gent evolution with sea anemones, possibly as an adaptation to a sessile existence. Résumé : Le comportement de Chelyosoma productum et de Corella inflata (Ascidiacea) a été étudié chez des animaux normaux et des animaux qui ont subi l’ablation de leur ganglion. Les C. productum ont vécu plus de 1 an après l’ablation du ganglion qui n’a pas été régénéré. Des enregistrements électrophysiologiques ont été faits à partir de pré- parations semi-intactes. Les réactions aux stimulations et l’activité spontanée continuent d’être transmises à travers la paroi du corps et le sac branchial après ablation du ganglion. La transmission est lente, décroissante et sujette à la faci- litation. L’administration de plus de 10 µg·mL–1 de d-tubocurarine inhibe toutes les réactions, ce qui indique que les nerfs sont les médiateurs de conduction de l’excitation en l’absence du ganglion. Une étude histologique basée sur l’histochimie des cholinestérases et sur le marquage immunologique au moyen d’antisérums contre la tubuline et l’hormone libératrice de la gonadotrophine n’a pas mis en lumière de réseau de nerfs périphériques dans les régions où il y a conduction, contrairement à des affirmations antérieures. Les corps cellulaires des neurones moteurs sont entière- ment contenus dans le ganglion ou dans ses racines principales. Leurs branches terminales s’entrecroisent selon des ar- rangements en réseau. Les neurones sensoriels ont été identifiés aux corps cellulaires périphériques, aussi bien dans la paroi du corps que dans le sac branchial. Leurs processus forment également des réseaux avant de pénétrer dans les nerfs qui aboutissent au ganglion. Nous concluons que l’innervation résiduelle qui persiste après l’ablation du ganglion se compose de terminaisons nerveuses motrices interreliés ou d’axones sensoriels interreliés ou d’une combinaison quelconque des deux. En réinventant le réseau de nerfs, les ascidies mettent en évidence leur évolution convergente avec des anémones de mer et peut-être devons-nous voir là une adaptation à un mode de vie sessile. [Traduit par la Rédaction] 1639

Introduction Mackie and Wyethlocal contractions, while stronger or more sustained stimulation evokes responses that spread to the other siphon and The behaviour of ascidians shows some remarkable paral- eventually to the body-wall muscles. These contractions re- lels with that of cnidarians. Gentle stimulation of the outer sult in the closure or retraction of both siphons and contrac- surface of a siphon evokes the “protective” or “direct” re- tion of the whole body, according to stimulus intensity. As sponse (Jordan 1907; Hecht 1918), which consists of small in sea anemones (Pantin 1952), the spread of contractions is graded, decremental, and facilitative (Florey 1951; Hoyle Received March 29, 2000. Accepted May 25, 2000. 1952). Moreover, as in cnidarians (Batham and Pantin 1950), G.O. Mackie1 and R.C. Wyeth.2 Biology Department, these responses occur against a background of spontaneous University of Victoria, Victoria, BC V8W 3N5, Canada. activity in the form of rhythmic contractions of the siphons 1 and body wall accompanied by arrests of the branchial cilia. Author to whom all correspondence should be addressed Isolated siphons continue to show rhythmic contractions (Day (e-mail: [email protected]). 2Present address: Department of Zoology, University of 1919; Yamaguchi 1931; Hoyle 1953) and respond to stimu- Washington, Seattle, WA 98195-1800, U.S.A. lation. The isolated branchial sac likewise responds to stimu- 3G. Kingston. 1976. Ciliary arrest in the stigmatal cilia of the lation, giving propagated ciliary arrests, and shows spontaneous solitary ascidians Corella and Ascidia. B.Sc.(Hons.) thesis, rhythmicity (Mackie et al. 1974; Kingston 19763). Thus, University of Victoria, Victoria, B.C. both muscular and ciliary effector systems show a high degree

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of regional autonomy in their responses, combined with dis- effectors. Fedele emphatically rejected the idea of a periph- persed pacemaker capability, much as in cnidarians (Passano eral nerve net and argued that because the motor neurons 1963). have their cell bodies in the ganglion, the motor innervation At the same time, ascidians (unlike cnidarians) have a sin- would degenerate rapidly in deganglionated animals. There- gle, seemingly well developed cerebral ganglion with sub- fore, any “residual” responsivity could not be nervous in origin stantial nerve bundles running out to the effectors, and some but must be due to myoid conduction. The idea of epithelial of their responses are quite unlike those of cnidarians. In the excitability was suggested by ten Cate (1928), who drew a “ejection” or “crossed” reflex, for example, stimulation of parallel with the non-nervous conduction seen in the skin of the inside wall of one siphon evokes contraction of the op- amphibian tadpoles at a stage prior to arrival of the inner- posite siphon, while the stimulated one remains open vation (Wintrebert 1904; Roberts 1971). Our objective in the (Jordan 1907; Hecht 1918). The ganglion has been shown to present work was to clarify the nature of the peripheral con- contain cell bodies of the motor neurons (“ciliary-arrest neu- duction system that survives deganglionation. rons”) that innervate the branchial sac. Intracellular record- Most of the older work was an exploration of the muscu- ings from these cells show that they produce spikes which lar responses of the siphons and body wall (reviewed by ten precede the electrical signals accompanying ciliary arrest in Cate 1931), but subsequent studies of the branchial sac are the branchial sac and appear, therefore, to act as the master relevant to the same questions (Mackie et al. 1974; Kingston pacemakers regulating ciliary activity in the sac (Arkett 1987). 1976, see footnote 3; Arkett 1987; Arkett et al. 1989). Motor Sensory signals from the receptors in the siphons are be- neurons with cell bodies in the ganglion terminate on the cil- lieved to fire the ciliary-arrest motor neurons in a classic iated cells and bring about coordinated ciliary arrests either reflex response (Takahashi et al. 1973; Bone and Mackie in spontaneous rhythmic patterns or as a result of stimula- 1982). Reflexes mediated by the ganglion typically show a tion. When separated from the ganglion, the branchial sac short latency, indicating that the ganglion, whatever its other continues to show coordinated, spontaneous arrests and to functions, plays a role as part of a fast pathway. conduct responses in a diffuse and unpolarized manner. Here, The picture, then, is one of a conventional nervous system too, a peripheral nerve net might be expected to be present, with sensory and motor nerves centred in the cerebral gan- supplementing the innervation from the ganglion, but histo- glion, combined with a semi-independent peripheral con- logical study has provided no support for a conventional ducting “net.” Spontaneity resides both within the central nerve net. A rich motor innervation was revealed by cholin- and peripheral conduction systems. esterase staining (Arkett et al. 1989), but no nucleated nerve The independence of the peripheral net is vividly demon- cells were observed. It was therefore proposed that the pe- strated in animals from which the ganglion has been re- ripheral conduction system that was demonstrated physio- moved. After a period of postoperative recuperation, the animal logically consists of the synaptically interconnected motor- returns to a seemingly normal life. Tonus is restored in the nerve terminals (Mackie et al. 1974; Bone and Mackie 1982). body wall. Spontaneous contractions are exhibited, though If this scenario is true it could serve as a model for the at an altered frequency (Yamaguchi 1931; Bacq 1935). Degan- “peripheral nerve net” innervating the body-wall musculature. glionated animals also respond to tactile and electrical stimu- Chelyosoma productum (Stimpson) (Ascidiacea) was se- lation with responses that spread decrementally and are graded lected for most of the present investigation because it is eas- according to stimulus strength and frequency. Stimulation of ily deganglionated and survives for over a year without the one siphon can still cause contraction of the other (Bacq ganglion regenerating (Hisaw et al. 1966). This is in marked 1934, 1935; Florey 1951), though stronger stimulation is contrast to Ciona intestinalis and Ascidia mentula, which re- needed than in intact animals, and the response is generally generate their ganglia after 3–5 weeks (Schultze 1900; Day slower and less dramatic. Deganglionated animals behave, in 1919; Lender and Bouchard-Madrelle 1964), a process that fact, “like slightly fatigued intact animals” (Hoyle 1952). is explored in several recent articles, most recently by Bollner What is the nature of the peripheral conducting system et al. (1997). We used electrophysiology to verify the earlier that coexists with and survives removal of the central ner- descriptions of peripheral conduction in the body wall both vous system? With the notable exception of M. Fedele, most before and after deganglionation. The earlier accounts all workers in the field (and influential reviewers like von Bud- date from the period before such techniques were available. denbrock 1928) have assumed that there is a peripheral nerve We also used histological and immunocytochemical proce- net of the cnidarian type, i.e., a system of neurons with their dures to try to settle the vexed question of whether ascidians cell bodies in the periphery that provide a pathway for dif- have a conventional peripheral nerve net. Finally, we con- fuse, unpolarized conduction. Florey (1951) concluded that sider alternatives to such a net. A preliminary report of this ascidian muscle receives dual innervation consisting of cen- work has appeared elsewhere (Mackie and Wyeth 1999). tral innervation (the ganglion and motor nerves) on the one hand and largely independent peripheral innervation having Material and methods the character of a nerve net on the other. As histological support for this idea the work of Hunter (1898) is usually cited. Specimens of C. productum and Corella inflata (Huntsman) were However, Hunter himself never claimed to have found cell collected intertidally and from the undersides of floats and boats in and around Friday Harbor, Washington, and Victoria, British Co- bodies in the peripheral nervous system and his Fig. 3, based lumbia, and maintained in the seawater tanks at Friday Harbor on methylene blue preparations, is open to other interpreta- Laboratories and the University of Victoria. Those at Victoria, tions. Other studies (Fedele 1923a, 1937a; Markman 1958; where the seawater system is constantly filtered, were periodically Mackie et al. 1974; Arkett et al. 1989) failed to reveal cell given a particulate food supplement (Fritz Invertebrate Buffet, Fritz bodies in the nerves innervating the muscular and ciliary Chemical Co., Dallas, Texas). Most specimens of both species

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lived for many months in captivity and remained in good condi- fixation in Flemming’s solution (without acetic acid) followed by tion, although there was some mortality in both control animals Heidenhain’s iron haematoxylin, a method used to good effect by and those that had received ganglion surgery. Millar (1953). Methylene blue preparations were also made for supravital study of the nervous system in C. inflata, but penetration Ganglion surgery was often very slow, as noted by previous workers (Hunter 1898; The anatomy of C. productum is well described by Huntsman Markman 1958). (1912) Specimens were anaesthetized in MS 222 (0.02% in seawater). The tunic was then incised to partially free the two large horny Physiology plates that lie between the siphons, and a small fish hook was used The reactions of intact specimens of both C. productum and to retract the right-hand plate (p in Fig. 1A), allowing access to the C. inflata were observed, using tactile and electrical stimulation. ganglion, which was then transected or completely removed (Hisaw Other specimens were anaesthetized and opened out to allow direct et al. 1966). The plate was then returned to its normal position in recording of neuromuscular activity. In the case of C. inflata, the the tunic. The mantle-layer tissues healed rapidly, covering the animal was removed completely from its tunic and a large part of lesioned area. Specimens of C. inflata were deganglionated after the mantle, including the siphons, was pinned out on a Sylgard removal from the their tunics. platform after the viscera were removed. Chelyosoma productum “disk preparations” were made by cutting around the whole animal just Histology below the disk and separating the disk from the lower part. The In the case of C. productum the animal was removed from its tu- disk portion was then pinned out flat, with the tunic down, allow- nic and portions were dissected and pinned out in Sylgard-lined ing access to the nerves and muscles of the mantle from above. petri dishes. In some cases, after the branchial sac was removed, The branchial sac was snipped away and discarded. The mantle the mantle from the whole region around the siphons right out to was pinned out radially to extend the disk-retractor muscles, which the points of insertion of the disk-retractor muscles on the tunic otherwise tended to contract inwards. Stimulating and recording was prepared as a single whole mount. In such preparations the probes were placed at various points and surgical lesions were motor innervation to the entire body-wall musculature could be made using fine-pointed needles or scissors as shown in Fig. 1B. seen. Essentially the same procedure was followed in the case of These preparations needed at least8htorecover from the stress of C. inflata, but more care was needed in removing the mantle from the operation, but after recovery remained responsive for 24–48 h the tunic, especially around the siphons, where the tissues are eas- when kept in running seawater at temperatures below 14°C. A per- ily torn. Pinning of the delicate tissues of C. inflata was best con- fusion system was employed for long-term experiments. This had ducted using cactus spines, stretching the piece out progressively the advantage that after anaesthesia or drug treatment, the prepara- and evenly in all directions. tion could be returned gently to seawater for further observation. Preparations were fixed in 4% paraformaldeyde in 0.1 M Tactile stimuli were delivered by means of a hand-held probe or phosphate-buffered saline at pH 7.3 for about 1 h prior to process- a probe mounted on a loudspeaker activated by current pulses. ing by the “direct colouring” thiocholine method for cholinesterases Electrical stimuli were delivered through concentric bipolar (Karnovsky and Roots 1964), following procedures published pre- stainless-steel electrodes (Model SNE-100, Clarke Electromedical viously (Arkett et al. 1989). This method shows the motor in- Instruments, Reading, U.K.). Extracellular recordings were made nervation well but fails to show the peptidergic neurons of the using polyethylene suction electrodes pulled out to an internal dorsal strand plexus or the sensory neurons with peripheral cell diameter of 50–80 µm. Amplified signals were displayed on an bodies. Permanent mounts were studied using conventional bright- oscillosocpe and stored on an instrumentation tape recorder for field microscopy. later analysis. All electrical recordings were carried out in a Fara- For anti-tubulin fluorescence microscopy, tissues were fixed in day cage, and special pains were taken to eliminate 60-cycle inter- either the same fixative or a picric acid – formaldehyde mixture ference, as the signals recorded were typically in the microvolt (Zamboni and DeMartino 1967). The latter gave better reagent range. penetrability but poorer cytoplasmic fixation. The preparations were incubated in primary antibody followed by standard processing, with controls in which the primary antibody was omitted. Of sev- Results eral antibodies tried, the most useful was a mouse monoclonal raised against alpha tubulin (Amersham N356). Both sensory and Histology motor neurons are well-labelled using this antibody. Treatment was completed with fluorescein isothiocyanate (FITC)-coupled goat anti- Motor innervation mouse secondary antibody. Brief immersion of the preparation in 1 mg·mL–1 propidium iodide prior to mounting allowed the nuclei The large nerve trunks running out from the ganglion in to be observed using the Texas Red filter set. In another set of ex- tunicates are mixed nerves containing both sensory and mo- periments, paraformaldehyde-fixed preparations were treated using tor fibres (Bone and Mackie 1982). In C. productum, fibres antisera raised against Tunicate I gonadotropin-releasing hormone running in the body wall and branchial sac were found to (GnRH) (Powell et al. 1996), which labels sensory neurons as well fluoresce strongly with anti-tubulin antisera. The motor in- as dorsal strand elements in C. inflata (Mackie and Marx 1999). nervation could be visualized selectively by cholinesterase The secondary antibody in this case was coupled to Alexa 488 histochemistry using the procedure of Arkett et al. (1989). (Molecular Probes, Inc., Eugene, Oregon). Labelled preparations As can be seen in Fig. 2A (a control preparation from a were observed with a conventional fluorescence microscope or a freshly dissected animal), the nerves leaving the ganglion laser-scanning confocal microscope (LSM 410, Zeiss). The filter combination used for Fig. 3D allowed both the long- and short- ramify repeatedly and eventually break down into small bun- wave emissions to be visualized simultaneously, avoiding the need dles that are especially abundant in muscular regions. At for an overlay. higher magnifications, small bundles or individual neurites In addition to these special procedures, tissues were studied were traceable to terminations on muscle fibres within the alive with phase-contrast, polarization, and Nomarski optics, and mantle. Nerve-cell bodies were not observed within the some preparations were made by classical staining methods, e.g., peripheral motor innervation.

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Fig. 1. (A) Intact Chelyosoma productum seen from above. Removal of plate p exposes the cerebral ganglion (after Arkett 1987). (B) Chelyosoma productum disk preparation, showing stimulating and recording sites and surgical lesions referred to in the text. The ganglion (G) is shown midway between the two siphons. Muscles (M) surround the disk (disk-retractor muscles) and the siphons are equipped with circular and radial muscles. Nerves radiate from the ganglion, one of which has been cut and sucked into a recording electrode (Rec1). Another recording electrode (Rec2) has been placed on the muscles of a siphon. Tactile or electrical stimuli (Stim) are delivered at another siphon. Cuts were made transversely through the ganglion (Cut1) or through the ganglion and one whole side of the preparation (Cut2). In other preparations the ganglion was removed completely.

Animals from which the ganglion had been removed were cies, as the animals did not survive the operation long enough kept for periods of up to 5 months before being fixed and for useful comparisons with control animals to be made. processed with cholinesterase staining. In no case where we Cholinesterase preparations of the branchial sac of both were sure that the entire ganglion has been removed was the species showed the rich innervation of the stigmatal ciliated ganglion found to have regenerated. While no attempt was cells described by Arkett et al. (1989). Nuclei were absent made to quantify these observations, we found no evidence from these nerves. suggestive of degeneration in the peripheral ramifications of the motor neurons. Not only were the main nerve branches Sensory innervation still strongly reactive (Fig. 2B), but small bundles and fine Using anti-tubulin and anti-GnRH labelling, we confirmed individual neurites were readily distinguishable running in earlier descriptions (Fedele 1923a; Millar 1953) of scattered all areas of the mantle, including tracts running around the unipolar sensory cells whose cell bodies lie just beneath the zone from which the ganglion had been removed (Fig. 2C). mantle epithelia. The cells typically occur in pairs (Fig. 3B), In all regions where the motor innervation was followed out but a few single ones were observed (Fig. 3C, 3D). In our to its terminal ramifications, the fine endings of the motor preparations they were restricted to the siphons, and were neurons intermingled to form a plexus. This could be seen most abundant in the outer mantle epithelium and siphon both in the vicinity of muscle fibres and in muscle-free rims (Fig. 3E). A smaller population of these sensory cells zones (Fig. 2D). Similar observations were made in freshly was also observed in the inner mantle epithelium immedi- prepared control tissues so this is evidently a normal feature ately adjacent to the siphon rim. Each mature sensory cell of the anatomy, not the product of deganglionation. In sum, had a single cilium, 15–20 µm long when fully extended, we found no evidence of degeneration, regeneration, regrowth, projecting externally. In life, the cilia probably lie just below or reorganization of the motor innervation after complete the tunic. As the cilia lie in a different focal plane, they were ganglion removal. not seen in confocal images where the Z series stopped at In C. inflata, cholinesterase and anti-tubulin preparations the epithelial surface (Figs. 4C, 4D). Each sensory cell had a gave a similar picture to that observed in C. productum,in- single neurite. The neurites of the sensory cells intermingled cluding vivid pictures of fine motor nerve endings on mantle- to form plexiform arrays in the periphery (Fig. 3F) before muscle fibres (Fig. 3A), but the effect of ganglion removal on entering nerve bundles heading for the ganglion. Each sen- the motor innervation could not be made studied in this spe- sory cell body contained a single nucleus. Seen as dark

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Fig. 2. Motor nerves and muscles in the mantle of C. productum, shown by cholinesterase histochemistry. (A) Animal with intact ganglion. AS, atrial siphon; G, ganglion, OS, oral siphon. (B) Animal from which the ganglion was removed 158 days previously (to the same scale as A). The ganglion has not regenerated and the nerves have not degenerated. (C) Animal from which the ganglion was removed 85 days previously, showing nerves (arrows) passing around the site of the former ganglion (the empty region at the left). (D) Same animal as in C, showing the reticular pattern of fine motor nerve terminals interconnecting larger nerve bundles.

(nonfluorescing) spaces in Figs. 3B and 3C, the nuclei ap- tubular organizing centres of the epithelial cells as bright peared as a bright objects after propidium iodide staining spots (Figs. 3B, 3C), and their nuclei also stained with pro- (Fig. 3D). Anti-tubulin immunofluorescence showed the micro- pidium iodide (Fig. 3D). The epithelial cells adjacent to

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sensory cells did not appear to be specialized “supporting compound events of up to 500 µV (Fig. 4A). Similar cells.” responses were observed when recording from the atrial si- A special effort was made to determine if sensory cells phon and stimulating the branchial siphon. When a nerve ex- were present in the branchial sac, as such cells have never iting the ganglion was cut and a recording electrode attached been reported in this location and were not seen in studies to its proximal stump, a stimulus to the siphon on the far on C. inflata, using methylene blue staining, phase-contrast side of the ganglion evoked a burst of action potentials microscopy, and cholinesterase histochemistry (Mackie et al. (Fig. 4B) that again summed to produce a larger, irregular 1974; Arkett et al. 1989). Anti-tubulin immunofluorescence deflection. The size and duration of bursts evoked by tactile microscopy on C. productum, however, showed a rich inner- stimulation were found to vary roughly in proportion to the vation stemming from multicellular sensory organs (“sensory strength of stimulation, but the animals rapidly became less papillae”) that are scattered through the epithelium covering responsive with repeated stimulation and needed periods last- the atrial surface of the branchial sac (Figs. 3G, 3H). They ing several minutes to recover responsivity. The smallest were most abundant on the dorsal side of the branchial sac events observed (10–20 µV) probably represent individual and appeared quite distinct from the “cupular organs” re- axon spikes. There was no evidence of consistently large, ported in the epithelium lining the inner side of the atrial si- rapidly conducted units suggestive of giant axons and there phon of Ciona intestinalis (Fedele 1923a; Millar 1953; Bone are no reports of giant axons in the histological literature. As and Ryan 1978), which are remarkable for their resemblance we were primarily concerned with the animal’s ability to to sense organs in the vertebrate acoustico-lateralis system, show propagated responses after ganglion transection or re- having a “flag” or “cupula” of tunic-like material in which moval, we did not explore the peculiar ejection or crossed the sensory cilia are embedded. The sensory pappillae in the reflex, as this response is well known to require an intact branchial sac of C. productum consisted of a cluster of about ganglion (reviewed by Goodbody 1974). 6–8 sensory cells, each with a single neurite. No cupula was Responses to electrical stimulation resembled those to tac- present. The sensory neurites running out from the base of tile stimulation. Low-voltage short-duration shocks evoked the papilla formed a compact bundle. Close to their origin, purely local responses that spread progressively with repeti- the neurites and sensory cell somata could be individually tion, but a single strong shock resulted in immediate con- visualized (Fig. 3H). Propidium iodide showed the nuclei of traction of the other siphon, to the accompaniment of a burst the sensory cells but also brilliantly lit up the nuclei of the of potentials. Conduction velocities in the intersiphonal path- numerous supporting cells and other epithelial cells, making way were hard to estimate accurately because of uncertainty it rather hard to distinguish the primary sensory structures. as to the arrival time of the first (typically small) potentials in the burst, but the fastest reliable values lay in the range Lack of a “conventional” nerve net 20–22 cm·s–1. The ability to detect the finest neurites of both sensory and motor neurons by means of anti-tubulin and anti-GnRH Responses after ganglion transection or removal in immunofluorescence and cholinesterase histochemistry, re- C. productum spectively, and to visualize their nuclei allowed us to scan large areas of the mantle and branchial sac of C. inflata.We Disk preparations that had previously shown normal excit- examined both muscular and nonmuscular regions to deter- ability were unresponsive for several hours after ganglion mine whether or not there is a nerve net, in the sense of a transection or removal but gradually recovered the ability to plexiform array of nucleated bi-or multi-polar neurons, lying respond to mechanical and electrical stimulation. Direct re- in these regions. Our preparations fully support the findings sponses of the siphons could be evoked by gentle stimula- of Fedele (1923a, 1937a) in showing that the only neurons tion and spread decrementally with repeated stimulation. In with cell bodies in the periphery are the sensory cells lying in preparations in which the ganglion had been removed, single the mantle epithelium (and, as we show here, in the branchial moderately strong shocks (which would have evoked con- sac) and that there are no cell bodies in the motor terminal traction of both siphons in a preparation with an intact gan- plexus in either the body wall or the branchial sac. glion) failed to cause a response at the distant siphon (Fig. 4C). When repeated at 10 Hz, however, such stimulation caused responses that propagated right through (Fig. 4D). Single very Physiology strong shocks also produced responses that reached the other Responses of intact animals and dissected preparations siphon. In experiments on animals with transected ganglia, with intact ganglia response latencies varied widely but were always much longer Both C. productum and C. inflata showed the classic protec- than in control preparations with intact ganglia (Figs. 5A– tive (Jordan 1907) or direct (Hecht 1918) responses to external 5C). The fastest conduction velocities obtained in prepara- –1 siphon stimulation. Light stimulation of the outer surfaces of tions with transected ganglia lay in the range 2.5–3.0 cm·s . either siphon caused graded contractions of that siphon, spread- In an attempt to determine the pathway taken by excitation ing, with stronger stimulation, to the other siphon and eventually in animals with transected ganglia, cuts were made through to the body-wall muscles, causing a generalized retraction. the entire mantle tissue at various points. It was found that a A single sharp prod applied to a siphon typically caused strip of intact tissue 3–5mm wide on one side of the ganglion rapid contraction of both siphons. In C. productum disk pre- was sufficient to allow impulses to be conducted through to parations set up with a recording electrode on the branchial the second siphon (Fig. 5C). siphon, a prod to the atrial siphon evoked contraction of both Disk preparations from animals that had been degang- siphons accompanied by a burst of potentials recorded at the lionated over a month previously showed a similar ability to branchial siphon. The potentials summed to produce irregular conduct excitation between the siphons. This finding fits

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Fig. 3. Nerves in Corella inflata (A–F) and C. productum (G, H) shown by immunofluorescence microscopy (A) and confocal microscopy (B–H). (A) Fine nerve processes running along muscle fibres (presumably motor terminals) in the mantle near the atrial siphon (anti-tubulin). (B) A pair of simple sensory cells in the outer mantle epithelium, showing their cilia, one curled and one extended (anti- tubulin). (C and D) Another sensory cell to the same scale as in B. In C the cell is shown by anti-tubulin immunofluoresence alone. In D propidium iodide staining shows nuclei. (E) Sensory cell pairs located around the rim of the atrial siphon (anti-GnRH). (F) Sensory neurites intermingling in plexiform array in the outer mantle epithelium (anti-GnRH). Arrowheads indicate sensory cells. (G) Two sensory papillae and sensory nerves in the branchial wall (anti-tubulin). (H) Sensory papilla enlarged (anti-tubulin).

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Fig. 4. (A) Response of C. productum with an intact ganglion recorded from a siphon (Fig. 1B, Rec2) following a prod with a blunt probe (arrow) on the other siphon (Fig. 1B, Stim). (B) A burst of action potentials recorded from a nerve root leaving the ganglion (Fig. 1B, Rec1) following a similar stimulus. (C and D) In a deganglionated preparation stimuli were delivered at one siphon and recordings made on the other (Fig. 1B, Stim and Rec2). In C, a single shock failed to produce a response in the other siphon, but in D, after a series of three shocks at 10 Hz, excitation got through.

with the lack of evidence of degeneration in the histological Drug experiments preparations. In a series of controlled experiments using C. productum disk preparations, d-tubocurarine was found to block inter- Source of recorded potentials siphonal conduction in both normal and deganglionated In both C. productum and C. inflata it was found to be preparations (Figs. 5D–5F). The lowest effective dose was possible to record the characteristic bursts of electrical events 10 µg·mL–1 and the block was completely reversible at not only with electrodes placed over muscle bundles but also this level, though full recovery took at least 2 h. With stron- from patches of mantle tissue where no muscle fibres were ger concentrations (20–40 µg·mL–1) the preparation some- visible. The “best” (largest amplitude) recordings were made times failed to recover or recovery was incomplete. In a from muscular regions, but the patterns of impulses evoked deganglionated C. inflata,10µg·mL–1 curare blocked all by stimulation were similar in both places, and were also pacemaker activity within 13 min, along with spontaneous similar to the patterns recorded directly from large nerve muscle contractions and ciliary arrests, so the cilia beat con- roots (Fig. 4B). These observations indicate that the bursts tinuously. Treatment with eserine (physostigmine) in con- of potentials are primarily nervous events that are augmented centrations up to 80 µg·mL–1 had no definite effect on the by muscle depolarizations in muscular regions. Ascidian body responses of either C. inflata or C. productum, as Bacq (1935) wall muscles are known from intracellular recordings to give and Florey (1963, 1967) found with C. intestinalis. We were twitch-type responses accompanied by all-or-none action po- unable to demonstrate any effect of α-bungarotoxin on tentials (Nevitt and Gilly 1986). Such events presumably C. productum at 10 µg·mL–1 even after 24 h of treatment. sum with and augment the primary nervous signals. The times taken for curare to take effect and for responses to be restored on return to seawater varied considerably in our Coordinated pacemaker activity in deganglionated C. inflata experiments, probably because of the slowness with which After several hours’ recovery from ganglion removal, some the drug penetrates intact tissues. Tunicate epithelia are well pinned preparations of C. inflata started to show patterns of sealed, with extended tight junctions (Lane et al. 1995; spontaneous activity in the form of regular bursts of small Burighel and Cloney 1997). Preparations of C. productum in (50–70 µV) potentials roughly every 4 min. Recordings made which the inner mantle epithelium was deliberately slit to as- with two electrodes, one on each siphon, showed similar pat- sist penetration generally responded rapidly to the drug. The terns at the two sites despite the absence of the ganglion failure of Scudder et al. (1966) to obtain any blocking action (Fig. 6). In the longer record from which Fig. 6 was taken, with curare on whole, undamaged C. intestinalis may have bursts lasted about 115 s, with 38 pulses per burst and inter- been due to failure of the drug to penetrate, as these authors burst intervals of 127 s (mean values from a sequence of limited their drug tests to 1 h duration. 13 bursts). Because these preparations were pinned out flat, it was not possible to see accompanying muscle contractions except at the edges of the siphons, where small twitches oc- Discussion curring in time with burst potentials were just visible. In one preparation a piece of the branchial sac was left attached to Motor innervation the mantle and it was seen that the cilia lining the branchial Motor nerves leaving the ganglion run out to the periph- stigmata underwent arrest in synchrony with each of the ery, where they associate with muscles and ciliated cells. early potentials in the bursts accompanying muscular con- Cholinesterase histochemistry shows the finest processes, in- tractions in the siphons, confirming observations by Mackie cluding synaptic boutons in the branchial sac of C. inflata et al. (1974). Later in the bursts, the branchial cilia ceased to (Arkett et al. 1989). The fine terminal neurites of the motor show discrete arrests but were maintained continuously in system form plexiform arrays in both the mantle (Fig. 2D) the fully arrested position until the end of the burst, when and the branchial sac, but there appear to be no cell bodies normal beating and metachronal rhythms re-emerged. within this mass of neurites. Nickel backfills (Arkett et al.

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Fig. 5. Electrical stimuli were delivered (*) to one siphon of C. productum and responses were recorded from the other (Fig. 1B, Stim and Rec2). (A–C) Demonstration of conduction via an alternative pathway around the brain after ganglion removal. (A) Control animal with the ganglion intact. (B) The same animal after Cut1 (Fig. 1B). (C) The same animal after Cut2. (D–F) Curare block of the alternative pathway in a deganglionated animal. (D) Preparation in seawater. (E) The same preparation after 20 min in 40 µg·mL–1 d-tubocurarine. (F) The same preparation after5hinseawater.

Fig. 6. Coordinated pacemaker activity in the mantle of C. inflata after ganglion removal. Recordings were made simultaneously from the two siphons (atrial siphon, top line; branchial siphon, bottom line) 5 h after ganglion removal.

1989) showed that in C. productum, all the cell bodies lay C. inflata, like those of Markman (1958) and Aros and within the ganglion except for a small cluster close to the Konok (1969) using C. intestinalis, failed to show cell bod- ganglion in one of the posterior nerve roots. As the nerve ies in the motor innervation outside the brain. It seems cer- root in question (AM3) was the one that gives rise to the vis- tain, therefore, that the great majority, and possibly all, of the ceral nerve, it seems possible that the filled cells (which motor neurons would be rendered anucleate by brain re- were located on the surface of the nerve root rather than moval, and yet C. productum remains responsive to stimula- within it) were part of the dorsal strand plexus (see below) tion and shows spontaneous movements many weeks after and were not motor neurons. Lorleberg (1907) observed a deganglionation. We can only conclude that the distal frag- few supposedly neuronal cell bodies (Ganglienzellen) in the ments of the motor neurons stay alive and functional in the nerve roots of Styelopsis grossularia close to the ganglion, anucleate state. We have not addressed the question of how but these became increasingly rare farther away from the the distal fragments survive, but this is a well-documented ganglion. In Polyandrocarpa misakiensis, Koyama and phenomenon in other invertebrates, particularly crustaceans, Kusunoki (1993) saw “some neuron-like cells” in nerve roots insects, and annelids (reviewed by Bittner 1991). It may be close to the ganglion. Our preparations of C. productum and noted that ascidian nerves run in blood spaces and lack

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Fig. 7. Peripheral conduction pathways, two scenarios: branches of motor axons interconnect synaptically in the periphery (A) and processes of sensory neurons interconnect synaptically and synapse with motor terminals (B). In both scenarios, sensory and motor neurons are additionally linked in reflex arcs via their connections in the ganglion.

cellular sheaths, so uptake of metabolites directly from the the branchial sac, where we describe them for the first time. blood would seem likely. Their afferent neurons form bundles that run to the ganglion via the visceral nerve roots but some bundles also leave the Sensory innervation branchial sac and mingle with nerves in the mantle without Lorleberg (1907) failed to find any evidence of peripheral passing through the ganglion. Such connections might provide sensory cells and it was left to Fedele (1923a) and Millar a pathway for the coordination observed between mantle (1953) to provide the first clear descriptions of them in contractions and ciliary arrests in deganglionated preparations. C. intestinalis. We confirmed and extended these findings using It is unclear if ascidians have sensory neurons with cell anti-tubulin and anti-GnRH immunolabelling, both of which bodies in the ganglion and processes that end freely in the proved suitable for showing a wide variety of neurones, both periphery. Free nerve endings are seen in many places where sensory and motor, in contrast to the cholinesterase method, there are no primary sensory receptor cells and some of which was only effective in showing motor elements. Simple these might be sensory endings. In the oral tentacles, which primary receptor cells lie in both the outer and the inner are highly sensitive to tactile stimuli evoking the crossed re- mantle epithelium of the siphons and have a sensory process sponse (Hecht 1918), Seeliger (1893–1907) described cells or cilium that projects to the exterior. They typically occur with processes projecting to the exterior that might be sen- in pairs or small clusters. Similar cells occur in ascidian lar- sory structures, but his drawings are unconvincing, and sub- vae (Torrence and Cloney 1982) and pelagic tunicates (Bone sequent workers, including ourselves, have been unable to 1959) and are generally regarded as mechanoreceptors (Good- confirm his finding. Burighel and Cloney (1997, Fig. 42) body 1974; Bone and Mackie 1982). Our investigation using figure a presumptive secondary sensory cell in a tentacle of immunolabelling shows the neurites of these cells running Botryllus schlosseri, raising the possibility that some of the below the epithelium, intermingling and forming a netlike “free nerve endings” described by other workers are actually array (Fig. 3F) before entering larger nerve bundles leading postsynaptic or electrically coupled to secondary sensory to the ganglion. Sensory papillae occur in the outer layer of cells lying in the epidermis. Secondary sensory cells occur

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in various larvaceans (Bone and Mackie 1982) but have not own account of a nerve net in the dorsal fold of the branchial been recognized in other tunicates, except for this one report sac (Fedele 1923b, 1927, 1938). The system consists of a on B. schlosseri. plexus of bi- and multi-polar neurons lying within the dorsal blood sinus and closely associated with the dorsal strand Pacemakers (dorsal cord) and visceral nerves. This plexus (“dorsal-strand Intracellular recordings from the ganglion of C. productum plexus”) extends all the way from the cerebral ganglion back demonstrate the presence of motor neurons that generate to the region of the gonads. It can be readily visualized by rhythmic patterns of impulses driving ciliary arrests in the means of methylene blue staining and phase-contrast optics branchial sac (Arkett 1987). However, the branchial sac contin- in living whole mounts (Mackie et al. 1974) and by its ues to show spontaneous rhythmicity after deganglionation immunoreactivity with antisera raised against GnRH (Mackie (Mackie et al. 1974) and this persists for at least 3 weeks 1995; Powell et al. 1996; Tsutsui et al. 1998). The presence (Kingston 1976, see footnote 3). Pacemaker capability must of this nerve net in the dorsal fold made it quite plausible therefore exist at the periphery. This is also clear from stud- that another net existed in the mantle tissue, but, as noted ies on the siphons and body wall of other ascidians (Day above, there is no histological support for such a structure. 1919; Yamaguchi 1931; Bacq 1935; Hoyle 1953). Our present Indeed, the fact that the nerve plexus in the dorsal fold can work on deganglionated C. inflata has shown the emergence be visualized so readily means that if such a plexus were of a rhythmic pattern of muscle contractions coordinated be- present in the mantle, it could hardly have gone undetected. tween the two siphons and blocked by curare. The peripheral The techniques used in the present study allowed us to visual- pacemakers are therefore presumably components of nerve ize both the sensory and the motor innervation in considerable cells but it is impossible to say whether they are located in detail and to detect nuclei where present (i.e., in sensory sensory or motor nerves. cells), and would surely have revealed a nerve net if it were present. Is there a peripheral nerve net? Most students of ascidian behaviour (e.g., Jordan 1907; Alternatives to a conventional nerve net Hecht 1918; ten Cate 1928; von Buddenbrock 1928; Schiller If there is no nerve net, we are left with four possibilities: 1937; Florey 1951) have assumed that there is a peripheral (i) the transmission of excitation by mechanical forces between nerve net in the mantle that survives deganglionation and muscles in the body wall, acting as “independent effectors” provides a basis for continuing responsiveness to stimulation. (proposed by Loeb 1891; Fedele 1923b, 1937b), (ii) conduc- The physiological picture is fairly clear: peripheral conduction in excitable epithelia (suggested by ten Cate 1928), tion pathways showing the characteristics elsewhere associ- (iii) conduction between the interconnected terminals of the ated with nerve nets certainly exist both in intact animals motor nerves (suggested by Mackie et al. 1974), and (iv) con- and after deganglionation. We have confirmed previous re- duction between interconnected branches of the sensory nerves. ports that ascidians respond to stimuli with contractions that Regarding i, myoid transmission is hard to reconcile with spread decrementally for varying distances depending on on the observations of Magnus (1902) and Kinoshita (1910), the strength and frequency of stimulation, and produce graded who found that treatment with cocaine and other drugs that responses in the muscles in a manner reminiscent of sea eliminate all nervous actvity while leaving the muscles ex- anemones. Conduction velocity in the “net” is slower than in citable by electrical stimuli blocked the spread of responses the intact animal by about an order of magnitude. Florey completely. Our own findings with curare, a potent blocker (1951), the most recent physiologist to address this question, of cholinergic synapses, also show that conduction of excita- concluded that there is a twofold innervation of the muscula- tion must depend not on muscles but on nerves, as muscles ture in C. intestinalis, the first consisting of motor nerves are not synaptically linked. This does not mean that the mus- coming from the ganglion and the second consisting of a cles are incapable of independent action when directly stim- peripheral nerve net. After destruction of the ganglion, all ulated, or that they could not act mechanically upon other remaining responses would be mediated by the nerve net. muscles in the locality, but the results of the experiments The term nerve net is generally taken to refer to a plexi- with drugs make it clear that the main peripheral conduction form array of bi- or multi-polar neurons lying outside the pathways surviving deganglionation cannot be muscle-based. central nervous system and constituting a diffusely conduct- Regarding ii, excitable epithelia that conduct behaviourally ing (unpolarized) set of pathways (Mackie 1999). As evi- meaningful signals occur in some ascidian larvae (Mackie dence of such a net in ascidians, many workers have cited and Bone 1976), in the blood vessels linking the zooids in histological observations by Hunter (1898), whose Fig. 3 botryllid colonies (Mackie and Singla 1983), and in various could be interpreted as showing cell bodies within the pe- pelagic tunicates (see Bone and Mackie 1982), raising the ripheral nervous system of Molgula manhattensis. Our find- possibility that the mantle and branchial epithelia in adult ings, however, support Fedele (1923a; 1937a) and Markman solitary ascidians are also excitable and conduct electrical (1958), who found no such nerve net in the mantle, and are signals. Recordings made with electrodes attached to the in agreement with earlier reports (Mackie et al. 1974; Arkett surfaces of the mantle epithelia, however, failed to show the et al. 1989) in showing that there is no nerve net in the large potentials typically associated with epithelial conduc- branchial sac. tion, showing instead much smaller potentials in patterns The assumption by various workers that there is a nerve similar to the bursts of action potentials recorded directly net in the mantle, though emphatically denied by Fedele from cut nerves. Furthermore, curare would not be expected (1923a, 1937a), was (paradoxically) bolstered by Fedele’s to block epithelial conduction, as transmission in all such

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tissues is electrical not chemical (Anderson 1980). The small potheses. Both are highly unconventional and would reflect potentials recorded with suction electrodes attached to the a unique situation among triploblastic animals but, given mantle epithelia almost certainly derive from the plexiform that there is no conventional nerve net, one or other of them, mass of fine nerve terminals running under the epithelium. or some combination of the two, must be true. Regarding iii, if the motor nerve terminals were interconnected synaptically, they could in theory produce the Evolutionary implications equivalent of a nerve net (Fig. 7A). Decremental spread Regardless of the precise nature of the peripheral conduc- would be exhibited if the synapses failed to transmit one-for- tion system that endows ascidians with the functional equiv- one but represented a partial barrier to the spread of excita- alent of a nerve net, it is clear that ascidian evolution has tion. This would occur, for example, if early impulses in a been characterized by decentralization of certain functions burst arriving at a synapse failed to evoke regenerative po- that are more fully centralized in other tunicates. Doliolids tentials on the postsynaptic side but evoked subthreshold and salps deprived of their ganglia show no spontaneous depolarizations (excitatory postsynaptic potentials) that al- swimming or contractions of the body-wall musculature and lowed later ones to produce spikes. The burst would dimin- do not respond to stimulation. However, doliolids show local ish with each synapse crossed and gradually fade out or autonomy in the ciliary control system in the branchial sac, become ineffective in evoking effector responses. A conse- like ascidians (Fedele 1923c; Bone and Mackie 1982). The quence of an arrangement of this sort would be to reduce the ganglion in ascidians is small and simple compared with, for selectivity of muscle excitation. Motor nerves in most animals example, that of salps. It still has a special role to play in function to excite specific effectors, leaving others unexcited maintaining muscle tone, as a fast conduction pathway and (the concept of “labelled lines”), and if motor impulses in- as mediator of certain complex responses, notably the crossed vaded adjacent motor units, it would become hard to obtain response (Day 1919; Bacq 1935; Florey 1951). These func- precise local movements such as directional flexions. In fact, tions, however, are superimposed upon a fundamental neuro- ascidian behaviour lacks directionality. Siphon closures and muscular organization characterized by the decremental, body-wall contractions are more or less symmetrical. In the diffuse, facilitating spread of contractions within and be- case of the isolated branchial sac, impulses generally spread tween the siphons and in the body wall and branchial sac, through the whole preparation, though not necessarily at the often exhibited in rhythmic patterns (Hoyle 1952, 1953), and same velocity in all directions (Mackie et al. 1974). Creation it is this residual innervation that survives deganglionation. of a motor net in which impulses are shared between adja- Evolutionary relationships within the Tunicata remain un- cent motor units might have advantages in “smoothing out” certain (Lacalli and Holland 1998; Lacalli 1999), and it is responses. not clear whether the ancestral urochordate was planktonic In the scenario shown in Fig. 7A, sensory cells do not or bottom-living, but a recent molecular phylogenetic analy- synapse with motor nerves except in the ganglion, and it is sis (Swalla et al. 2000) suggests that the ancestor “probably assumed that the motor plexus itself is directly excitable by retained the tadpole tail, including the dorsal nerve cord, the local stimulation but sensory units could synapse with the notochord and flanking muscle cells through the adult life.” motor plexus in the periphery; some observations support A good case can be made for the origin of ascidians from such a picture. For instance, isolated siphons and siphons in more active, mobile, free-living ancestors (Satoh and Jeffery deganglionated animals remain almost as responsive to vi- 1995; Wada 1998) whose nervous systems would probably brations as those in the intact animal (Magnus 1902), which have been more fully centralized, as in present-day pelagic implies that mechano-receptors are still functional parts of tunicates. The decentralization of functions seen in ascidians the response system with connections to local effectors. would therefore appear to be in some way adaptive in the Regarding iv, sensory cells lying in the mantle and branchial context of their sessile habitat. It may be that processing all sac could send processes not only to the cerebral ganglion sensory information and motor output via a single nerve cen- but also through branches in the periphery to one another tre is not only unnecessary but metabolically wasteful in a (Fig. 7B). Synaptic links would regulate impulse traffic as in situation where many responses can be managed initially by iii above. To excite the muscular or ciliary effectors, the local action systems. Actinian nerve nets have the same po- sensory net would either have to contact the effectors directly tential for organizing actions locally that we see in ascidians or excite them indirectly through the motor innervation. We and their nervous systems show a corresponding lack or low have shown them exciting the effectors indirectly by synap- degree of centralization. Aside from the purely “visceral” sing on motor terminals. However, direct connections be- activities carried out locally in the heart, gut, and gonoducts, tween sensory cells and muscles are known in Hydra littoralis ascidian behaviour consists essentially of opening, closing, (Westfall 1973) and cannot be excluded. A net fashioned out and retracting the siphons, contracting the body-wall mus- of the interconnected branches of sensory neurons would cles, and arresting the cilia in the branchial sac. These com- satisfy the requirement for a set of peripheral conduction ponents are functionally linked and are exhibited in various pathways that could survive deganglionation. A potential dis- degrees depending on the strength of stimulation. There are advantage of this arrangement would be some loss of precision no directional responses and little apparent need for precise in the ability of the animal to localize sources of stimulation, localization of sources of stimulation. Whereas actinians have for sensory signals would tend to invade afferent pathways true nerve nets for organizing local action systems, it would adjacent to those actually stimulated. appear that ascidians have created the equivalent of a periph- At present, the evidence is insufficient for deciding be- eral nerve net by modifying the connectivity patterns of their tween the motor-net (Fig. 7A) and sensory-net (Fig. 7B) hy- motor and (or) sensory nerves. This has allowed them to

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devolve certain functions to the periphery in a way not seen Bone, Q., and Mackie, G.O. 1982. Ch. 11. Urochordata. In Electri- in pelagic tunicates. At the same time they have retained the cal conduction and behaviour in ‘simple’ invertebrates. Edited cerebral ganglion and its major input–output pathways for by G.A.B. Shelton. Clarendon Press, Oxford. pp. 473–535. organizing complex responses and as a fast pathway for Bone, Q., and Ryan, K.P. 1978. Cupular sense organs in Ciona rapid and coordinated defensive responses. (Tunicata: Ascidiacea). J. Zool. (1965–1984), 186: 417–429. Burighel, P., and Cloney, R.A. 1997. Urochordata: Ascidiacea. Chap. 4. In Microscopic anatomy of invertebrates. Vol. 15. Hemichordata, Note added in proof: Chaetognatha, and the invertebrate chordates. Edited by F.W. Harrison and E.E. Ruppert. Wiley–Liss, Inc., New York. pp. 221– A forthcoming paper (Burighel et al. 2000) describes the 347. distribution of nerves in the colonial ascidian Botryllus Burighel, P., Sorrentino, M., Zaniolo, G., Thorndyke, M.C., Manni, schlosseri, as visualized by cholinesterase histochemistry. L. 2000. The peripheral nervous system of an ascidian Botryllus Nerves extend to all organs except for the neural gland and schlosseri. Invert. Biol. In press. gonads. A complex network of nerves is described, but no Day, E.C. 1919. The physiology of the nervous system of the tuni- peripheral cell bodies were observed. The system does not cate 1. The relation of the nerve ganglion to sensory responses. interconnect different zooids within the colony. The picture J. Exp. Zool. 28: 307–335. of this component of the innervation in B. schlosseri thus Fedele, M. 1923a. Sulla organizzazione e le caratteristiche funzionali corresponds in essential respects to what we describe here dell’attività nervosa dei Tunicati. I. Ricerche sul sistema nervosa for the solitary ascidians C. productum and C. inflata. periferico degli Ascidiacea. Atti R. Accad. Lincei Roma Ser. 5, 32: 98–102. Fedele, M. 1923b. Ancora sulla organizzazione e le caratteristiche Acknowledgements funzionali dell’attività nervosa dei Tunicati. II. Attività riflesse ed effetori autonomi negli Ascidiacea. Atti R. Accad. Lincei This work was supported by a grant to G.O.M. from the Roma Ser. 5, 32: 185–188 Natural Sciences and Engineering Research Council of Canada. Fedele, M. 1923c. Attività dinamiche e sistema nervoso nella vita We thank Catherine Pennachietti for collecting some of the dei Dolioli. Pubbl. Stn. Zool. Napoli, 4: 129–240. material used in the experiments in Victoria. Nancy Sher- Fedele, M. 1927. Ancora sulla organizzazione e le caratteristiche wood advised us on aspects of GnRH immunobiology and funzionali dell’attività nervosa dei Tunicati. III. Il sistema nervosoa provided antibodies. Thurston Lacalli and Quentin Bone kindly viscerale. Atti R. Accad. Lincei Roma Ser. 6, 6: 532–537. read and commented on the manuscript. We thank Dennis Fedele, M. 1937a. Ancora sulla inesistenza di una rete nervosa Willows, Director, and the staff of the Friday Harbor Labo- periferica nei Tunicati. Rend. Accad. Naz. Lincei Roma Ser. 6, ratories for space and assistance during phases of the work 25: 656–661. conducted there. Fedele, M. 1937b. Contrattilità ed eccitazione neurogena e miogena negli “Ascidiacea.” Rend. Accad. Naz. Lincei Roma Ser. 6, 26: 31–37. References Fedele, M. 1938. Il sistema nervosa degli ‘Ascidiacea’ nel piano di organizzazione dei Cordati. Rend. Accad. Naz. Lincei Roma Anderson, P.A.V. 1980. Epithelial conduction: its properties and Ser. 6, 27: 370–376. functions. 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