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1626
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 regenerate. 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 convergent 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 préparations 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 facilitation. 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 entièrement contenus dans le ganglion ou dans ses racines principales. Leurs branches terminales s’entrecroisent selon des arrangements 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]
Introduction
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local contractions, while stronger or more sustained stimulaMackie and Wyeth
The behaviour of ascidians shows some remarkable parallels with that of cnidarians. Gentle stimulation of the outer
surface of a siphon evokes the “protective” or “direct” response (Jordan 1907; Hecht 1918), which consists of small
Received March 29, 2000. Accepted May 25, 2000.
G.O. Mackie1 and R.C. Wyeth.2 Biology Department,
University of Victoria, Victoria, BC V8W 3N5, Canada.
1
Author to whom all correspondence should be addressed
(e-mail: [email protected]).
2
Present address: Department of Zoology, University of
Washington, Seattle, WA 98195-1800, U.S.A.
3
G. Kingston. 1976. Ciliary arrest in the stigmatal cilia of the
solitary ascidians Corella and Ascidia. B.Sc.(Hons.) thesis,
University of Victoria, Victoria, B.C.
Can. J. Zool. 78: 1626–1639 (2000)
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tion evokes responses that spread to the other siphon and
eventually to the body-wall muscles. These contractions result in the closure or retraction of both siphons and contraction of the whole body, according to stimulus intensity. As
in sea anemones (Pantin 1952), the spread of contractions is
graded, decremental, and facilitative (Florey 1951; Hoyle
1952). Moreover, as in cnidarians (Batham and Pantin 1950),
these responses occur against a background of spontaneous
activity in the form of rhythmic contractions of the siphons
and body wall accompanied by arrests of the branchial cilia.
Isolated siphons continue to show rhythmic contractions (Day
1919; Yamaguchi 1931; Hoyle 1953) and respond to stimulation. The isolated branchial sac likewise responds to stimulation, giving propagated ciliary arrests, and shows spontaneous
rhythmicity (Mackie et al. 1974; Kingston 19763). Thus,
both muscular and ciliary effector systems show a high degree
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of regional autonomy in their responses, combined with dispersed pacemaker capability, much as in cnidarians (Passano
1963).
At the same time, ascidians (unlike cnidarians) have a single, seemingly well developed cerebral ganglion with substantial nerve bundles running out to the effectors, and some
of their responses are quite unlike those of cnidarians. In the
“ejection” or “crossed” reflex, for example, stimulation of
the inside wall of one siphon evokes contraction of the opposite siphon, while the stimulated one remains open
(Jordan 1907; Hecht 1918). The ganglion has been shown to
contain cell bodies of the motor neurons (“ciliary-arrest neurons”) that innervate the branchial sac. Intracellular recordings from these cells show that they produce spikes which
precede the electrical signals accompanying ciliary arrest in
the branchial sac and appear, therefore, to act as the master
pacemakers regulating ciliary activity in the sac (Arkett 1987).
Sensory signals from the receptors in the siphons are believed to fire the ciliary-arrest motor neurons in a classic
reflex response (Takahashi et al. 1973; Bone and Mackie
1982). Reflexes mediated by the ganglion typically show a
short latency, indicating that the ganglion, whatever its other
functions, plays a role as part of a fast pathway.
The picture, then, is one of a conventional nervous system
with sensory and motor nerves centred in the cerebral ganglion, combined with a semi-independent peripheral conducting “net.” Spontaneity resides both within the central
and peripheral conduction systems.
The independence of the peripheral net is vividly demonstrated in animals from which the ganglion has been removed. After a period of postoperative recuperation, the animal
returns to a seemingly normal life. Tonus is restored in the
body wall. Spontaneous contractions are exhibited, though
at an altered frequency (Yamaguchi 1931; Bacq 1935). Deganglionated animals also respond to tactile and electrical stimulation with responses that spread decrementally and are graded
according to stimulus strength and frequency. Stimulation of
one siphon can still cause contraction of the other (Bacq
1934, 1935; Florey 1951), though stronger stimulation is
needed than in intact animals, and the response is generally
slower and less dramatic. Deganglionated animals behave, in
fact, “like slightly fatigued intact animals” (Hoyle 1952).
What is the nature of the peripheral conducting system
that coexists with and survives removal of the central nervous system? With the notable exception of M. Fedele, most
workers in the field (and influential reviewers like von Buddenbrock 1928) have assumed that there is a peripheral nerve
net of the cnidarian type, i.e., a system of neurons with their
cell bodies in the periphery that provide a pathway for diffuse, unpolarized conduction. Florey (1951) concluded that
ascidian muscle receives dual innervation consisting of central innervation (the ganglion and motor nerves) on the one
hand and largely independent peripheral innervation having
the character of a nerve net on the other. As histological support for this idea the work of Hunter (1898) is usually cited.
However, Hunter himself never claimed to have found cell
bodies in the peripheral nervous system and his Fig. 3, based
on methylene blue preparations, is open to other interpretations. Other studies (Fedele 1923a, 1937a; Markman 1958;
Mackie et al. 1974; Arkett et al. 1989) failed to reveal cell
bodies in the nerves innervating the muscular and ciliary
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effectors. Fedele emphatically rejected the idea of a peripheral nerve net and argued that because the motor neurons
have their cell bodies in the ganglion, the motor innervation
would degenerate rapidly in deganglionated animals. Therefore, any “residual” responsivity could not be nervous in origin
but must be due to myoid conduction. The idea of epithelial
excitability was suggested by ten Cate (1928), who drew a
parallel with the non-nervous conduction seen in the skin of
amphibian tadpoles at a stage prior to arrival of the innervation (Wintrebert 1904; Roberts 1971). Our objective in the
present work was to clarify the nature of the peripheral conduction system that survives deganglionation.
Most of the older work was an exploration of the muscular responses of the siphons and body wall (reviewed by ten
Cate 1931), but subsequent studies of the branchial sac are
relevant to the same questions (Mackie et al. 1974; Kingston
1976, see footnote 3; Arkett 1987; Arkett et al. 1989). Motor
neurons with cell bodies in the ganglion terminate on the ciliated cells and bring about coordinated ciliary arrests either
in spontaneous rhythmic patterns or as a result of stimulation. When separated from the ganglion, the branchial sac
continues to show coordinated, spontaneous arrests and to
conduct responses in a diffuse and unpolarized manner. Here,
too, a peripheral nerve net might be expected to be present,
supplementing the innervation from the ganglion, but histological study has provided no support for a conventional
nerve net. A rich motor innervation was revealed by cholinesterase staining (Arkett et al. 1989), but no nucleated nerve
cells were observed. It was therefore proposed that the peripheral conduction system that was demonstrated physiologically consists of the synaptically interconnected motornerve terminals (Mackie et al. 1974; Bone and Mackie 1982).
If this scenario is true it could serve as a model for the
“peripheral nerve net” innervating the body-wall musculature.
Chelyosoma productum (Stimpson) (Ascidiacea) was selected for most of the present investigation because it is easily deganglionated and survives for over a year without the
ganglion regenerating (Hisaw et al. 1966). This is in marked
contrast to Ciona intestinalis and Ascidia mentula, which regenerate their ganglia after 3–5 weeks (Schultze 1900; Day
1919; Lender and Bouchard-Madrelle 1964), a process that
is explored in several recent articles, most recently by Bollner
et al. (1997). We used electrophysiology to verify the earlier
descriptions of peripheral conduction in the body wall both
before and after deganglionation. The earlier accounts all
date from the period before such techniques were available.
We also used histological and immunocytochemical procedures to try to settle the vexed question of whether ascidians
have a conventional peripheral nerve net. Finally, we consider alternatives to such a net. A preliminary report of this
work has appeared elsewhere (Mackie and Wyeth 1999).
Material and methods
Specimens of C. productum and Corella inflata (Huntsman) were
collected intertidally and from the undersides of floats and boats in
and around Friday Harbor, Washington, and Victoria, British Columbia, and maintained in the seawater tanks at Friday Harbor
Laboratories and the University of Victoria. Those at Victoria,
where the seawater system is constantly filtered, were periodically
given a particulate food supplement (Fritz Invertebrate Buffet, Fritz
Chemical Co., Dallas, Texas). Most specimens of both species
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lived for many months in captivity and remained in good condition, although there was some mortality in both control animals
and those that had received ganglion surgery.
Ganglion surgery
The anatomy of C. productum is well described by Huntsman
(1912) Specimens were anaesthetized in MS 222 (0.02% in seawater).
The tunic was then incised to partially free the two large horny
plates that lie between the siphons, and a small fish hook was used
to retract the right-hand plate (p in Fig. 1A), allowing access to the
ganglion, which was then transected or completely removed (Hisaw
et al. 1966). The plate was then returned to its normal position in
the tunic. The mantle-layer tissues healed rapidly, covering the
lesioned area. Specimens of C. inflata were deganglionated after
removal from the their tunics.
Histology
In the case of C. productum the animal was removed from its tunic and portions were dissected and pinned out in Sylgard-lined
petri dishes. In some cases, after the branchial sac was removed,
the mantle from the whole region around the siphons right out to
the points of insertion of the disk-retractor muscles on the tunic
was prepared as a single whole mount. In such preparations the
motor innervation to the entire body-wall musculature could be
seen. Essentially the same procedure was followed in the case of
C. inflata, but more care was needed in removing the mantle from
the tunic, especially around the siphons, where the tissues are easily torn. Pinning of the delicate tissues of C. inflata was best conducted using cactus spines, stretching the piece out progressively
and evenly in all directions.
Preparations were fixed in 4% paraformaldeyde in 0.1 M
phosphate-buffered saline at pH 7.3 for about 1 h prior to processing by the “direct colouring” thiocholine method for cholinesterases
(Karnovsky and Roots 1964), following procedures published previously (Arkett et al. 1989). This method shows the motor innervation well but fails to show the peptidergic neurons of the
dorsal strand plexus or the sensory neurons with peripheral cell
bodies. Permanent mounts were studied using conventional brightfield microscopy.
For anti-tubulin fluorescence microscopy, tissues were fixed in
either the same fixative or a picric acid – formaldehyde mixture
(Zamboni and DeMartino 1967). The latter gave better reagent
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 several antibodies tried, the most useful was a mouse monoclonal
raised against alpha tubulin (Amersham N356). Both sensory and
motor neurons are well-labelled using this antibody. Treatment was
completed with fluorescein isothiocyanate (FITC)-coupled goat antimouse secondary antibody. Brief immersion of the preparation in
1 mg·mL–1 propidium iodide prior to mounting allowed the nuclei
to be observed using the Texas Red filter set. In another set of experiments, paraformaldehyde-fixed preparations were treated using
antisera raised against Tunicate I gonadotropin-releasing hormone
(GnRH) (Powell et al. 1996), which labels sensory neurons as well
as dorsal strand elements in C. inflata (Mackie and Marx 1999).
The secondary antibody in this case was coupled to Alexa 488
(Molecular Probes, Inc., Eugene, Oregon). Labelled preparations
were observed with a conventional fluorescence microscope or a
laser-scanning confocal microscope (LSM 410, Zeiss). The filter
combination used for Fig. 3D allowed both the long- and shortwave emissions to be visualized simultaneously, avoiding the need
for an overlay.
In addition to these special procedures, tissues were studied
alive with phase-contrast, polarization, and Nomarski optics, and
some preparations were made by classical staining methods, e.g.,
Can. J. Zool. Vol. 78, 2000
fixation in Flemming’s solution (without acetic acid) followed by
Heidenhain’s iron haematoxylin, a method used to good effect by
Millar (1953). Methylene blue preparations were also made for
supravital study of the nervous system in C. inflata, but penetration
was often very slow, as noted by previous workers (Hunter 1898;
Markman 1958).
Physiology
The reactions of intact specimens of both C. productum and
C. inflata were observed, using tactile and electrical stimulation.
Other specimens were anaesthetized and opened out to allow direct
recording of neuromuscular activity. In the case of C. inflata, the
animal was removed completely from its tunic and a large part of
the mantle, including the siphons, was pinned out on a Sylgard
platform after the viscera were removed. Chelyosoma productum “disk
preparations” were made by cutting around the whole animal just
below the disk and separating the disk from the lower part. The
disk portion was then pinned out flat, with the tunic down, allowing access to the nerves and muscles of the mantle from above.
The branchial sac was snipped away and discarded. The mantle
was pinned out radially to extend the disk-retractor muscles, which
otherwise tended to contract inwards. Stimulating and recording
probes were placed at various points and surgical lesions were
made using fine-pointed needles or scissors as shown in Fig. 1B.
These preparations needed at least 8 h to recover from the stress of
the operation, but after recovery remained responsive for 24–48 h
when kept in running seawater at temperatures below 14°C. A perfusion system was employed for long-term experiments. This had
the advantage that after anaesthesia or drug treatment, the preparation could be returned gently to seawater for further observation.
Tactile stimuli were delivered by means of a hand-held probe or
a probe mounted on a loudspeaker activated by current pulses.
Electrical stimuli were delivered through concentric bipolar
stainless-steel electrodes (Model SNE-100, Clarke Electromedical
Instruments, Reading, U.K.). Extracellular recordings were made
using polyethylene suction electrodes pulled out to an internal
diameter of 50–80 µm. Amplified signals were displayed on an
oscillosocpe and stored on an instrumentation tape recorder for
later analysis. All electrical recordings were carried out in a Faraday cage, and special pains were taken to eliminate 60-cycle interference, as the signals recorded were typically in the microvolt
range.
Results
Histology
Motor innervation
The large nerve trunks running out from the ganglion in
tunicates are mixed nerves containing both sensory and motor fibres (Bone and Mackie 1982). In C. productum, fibres
running in the body wall and branchial sac were found to
fluoresce strongly with anti-tubulin antisera. The motor innervation could be visualized selectively by cholinesterase
histochemistry using the procedure of Arkett et al. (1989).
As can be seen in Fig. 2A (a control preparation from a
freshly dissected animal), the nerves leaving the ganglion
ramify repeatedly and eventually break down into small bundles that are especially abundant in muscular regions. At
higher magnifications, small bundles or individual neurites
were traceable to terminations on muscle fibres within the
mantle. Nerve-cell bodies were not observed within the
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
kept for periods of up to 5 months before being fixed and
processed with cholinesterase staining. In no case where we
were sure that the entire ganglion has been removed was the
ganglion found to have regenerated. While no attempt was
made to quantify these observations, we found no evidence
suggestive of degeneration in the peripheral ramifications of
the motor neurons. Not only were the main nerve branches
still strongly reactive (Fig. 2B), but small bundles and fine
individual neurites were readily distinguishable running in
all areas of the mantle, including tracts running around the
zone from which the ganglion had been removed (Fig. 2C).
In all regions where the motor innervation was followed out
to its terminal ramifications, the fine endings of the motor
neurons intermingled to form a plexus. This could be seen
both in the vicinity of muscle fibres and in muscle-free
zones (Fig. 2D). Similar observations were made in freshly
prepared control tissues so this is evidently a normal feature
of the anatomy, not the product of deganglionation. In sum,
we found no evidence of degeneration, regeneration, regrowth,
or reorganization of the motor innervation after complete
ganglion removal.
In C. inflata, cholinesterase and anti-tubulin preparations
gave a similar picture to that observed in C. productum, including vivid pictures of fine motor nerve endings on mantlemuscle fibres (Fig. 3A), but the effect of ganglion removal on
the motor innervation could not be made studied in this spe-
cies, as the animals did not survive the operation long enough
for useful comparisons with control animals to be made.
Cholinesterase preparations of the branchial sac of both
species showed the rich innervation of the stigmatal ciliated
cells described by Arkett et al. (1989). Nuclei were absent
from these nerves.
Sensory innervation
Using anti-tubulin and anti-GnRH labelling, we confirmed
earlier descriptions (Fedele 1923a; Millar 1953) of scattered
unipolar sensory cells whose cell bodies lie just beneath the
mantle epithelia. The cells typically occur in pairs (Fig. 3B),
but a few single ones were observed (Fig. 3C, 3D). In our
preparations they were restricted to the siphons, and were
most abundant in the outer mantle epithelium and siphon
rims (Fig. 3E). A smaller population of these sensory cells
was also observed in the inner mantle epithelium immediately adjacent to the siphon rim. Each mature sensory cell
had a single cilium, 15–20 µm long when fully extended,
projecting externally. In life, the cilia probably lie just below
the tunic. As the cilia lie in a different focal plane, they were
not seen in confocal images where the Z series stopped at
the epithelial surface (Figs. 4C, 4D). Each sensory cell had a
single neurite. The neurites of the sensory cells intermingled
to form plexiform arrays in the periphery (Fig. 3F) before
entering nerve bundles heading for the ganglion. Each sensory cell body contained a single nucleus. Seen as dark
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Can. J. Zool. Vol. 78, 2000
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 appeared as a bright objects after propidium iodide staining
(Fig. 3D). Anti-tubulin immunofluorescence showed the micro-
tubular organizing centres of the epithelial cells as bright
spots (Figs. 3B, 3C), and their nuclei also stained with propidium iodide (Fig. 3D). The epithelial cells adjacent to
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sensory cells did not appear to be specialized “supporting
cells.”
A special effort was made to determine if sensory cells
were present in the branchial sac, as such cells have never
been reported in this location and were not seen in studies
on C. inflata, using methylene blue staining, phase-contrast
microscopy, and cholinesterase histochemistry (Mackie et al.
1974; Arkett et al. 1989). Anti-tubulin immunofluorescence
microscopy on C. productum, however, showed a rich innervation stemming from multicellular sensory organs (“sensory
papillae”) that are scattered through the epithelium covering
the atrial surface of the branchial sac (Figs. 3G, 3H). They
were most abundant on the dorsal side of the branchial sac
and appeared quite distinct from the “cupular organs” reported in the epithelium lining the inner side of the atrial siphon of Ciona intestinalis (Fedele 1923a; Millar 1953; Bone
and Ryan 1978), which are remarkable for their resemblance
to sense organs in the vertebrate acoustico-lateralis system,
having a “flag” or “cupula” of tunic-like material in which
the sensory cilia are embedded. The sensory pappillae in the
branchial sac of C. productum consisted of a cluster of about
6–8 sensory cells, each with a single neurite. No cupula was
present. The sensory neurites running out from the base of
the papilla formed a compact bundle. Close to their origin,
the neurites and sensory cell somata could be individually
visualized (Fig. 3H). Propidium iodide showed the nuclei of
the sensory cells but also brilliantly lit up the nuclei of the
numerous supporting cells and other epithelial cells, making
it rather hard to distinguish the primary sensory structures.
Lack of a “conventional” nerve net
The ability to detect the finest neurites of both sensory
and motor neurons by means of anti-tubulin and anti-GnRH
immunofluorescence and cholinesterase histochemistry, respectively, and to visualize their nuclei allowed us to scan
large areas of the mantle and branchial sac of C. inflata. We
examined both muscular and nonmuscular regions to determine whether or not there is a nerve net, in the sense of a
plexiform array of nucleated bi-or multi-polar neurons, lying
in these regions. Our preparations fully support the findings
of Fedele (1923a, 1937a) in showing that the only neurons
with cell bodies in the periphery are the sensory cells lying in
the mantle epithelium (and, as we show here, in the branchial
sac) and that there are no cell bodies in the motor terminal
plexus in either the body wall or the branchial sac.
Physiology
Responses of intact animals and dissected preparations
with intact ganglia
Both C. productum and C. inflata showed the classic protective (Jordan 1907) or direct (Hecht 1918) responses to external
siphon stimulation. Light stimulation of the outer surfaces of
either siphon caused graded contractions of that siphon, spreading, with stronger stimulation, to the other siphon and eventually
to the body-wall muscles, causing a generalized retraction.
A single sharp prod applied to a siphon typically caused
rapid contraction of both siphons. In C. productum disk preparations set up with a recording electrode on the branchial
siphon, a prod to the atrial siphon evoked contraction of both
siphons accompanied by a burst of potentials recorded at the
branchial siphon. The potentials summed to produce irregular
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compound events of up to 500 µV (Fig. 4A). Similar
responses were observed when recording from the atrial siphon and stimulating the branchial siphon. When a nerve exiting the ganglion was cut and a recording electrode attached
to its proximal stump, a stimulus to the siphon on the far
side of the ganglion evoked a burst of action potentials
(Fig. 4B) that again summed to produce a larger, irregular
deflection. The size and duration of bursts evoked by tactile
stimulation were found to vary roughly in proportion to the
strength of stimulation, but the animals rapidly became less
responsive with repeated stimulation and needed periods lasting several minutes to recover responsivity. The smallest
events observed (10–20 µV) probably represent individual
axon spikes. There was no evidence of consistently large,
rapidly conducted units suggestive of giant axons and there
are no reports of giant axons in the histological literature. As
we were primarily concerned with the animal’s ability to
show propagated responses after ganglion transection or removal, we did not explore the peculiar ejection or crossed
reflex, as this response is well known to require an intact
ganglion (reviewed by Goodbody 1974).
Responses to electrical stimulation resembled those to tactile stimulation. Low-voltage short-duration shocks evoked
purely local responses that spread progressively with repetition, but a single strong shock resulted in immediate contraction of the other siphon, to the accompaniment of a burst
of potentials. Conduction velocities in the intersiphonal pathway were hard to estimate accurately because of uncertainty
as to the arrival time of the first (typically small) potentials
in the burst, but the fastest reliable values lay in the range
20–22 cm·s–1.
Responses after ganglion transection or removal in
C. productum
Disk preparations that had previously shown normal excitability were unresponsive for several hours after ganglion
transection or removal but gradually recovered the ability to
respond to mechanical and electrical stimulation. Direct responses of the siphons could be evoked by gentle stimulation and spread decrementally with repeated stimulation. In
preparations in which the ganglion had been removed, single
moderately strong shocks (which would have evoked contraction of both siphons in a preparation with an intact ganglion) 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
strong shocks also produced responses that reached the other
siphon. In experiments on animals with transected ganglia,
response latencies varied widely but were always much longer
than in control preparations with intact ganglia (Figs. 5A–
5C). The fastest conduction velocities obtained in preparations with transected ganglia lay in the range 2.5–3.0 cm·s–1.
In an attempt to determine the pathway taken by excitation
in animals with transected ganglia, cuts were made through
the entire mantle tissue at various points. It was found that a
strip of intact tissue 3–5mm wide on one side of the ganglion
was sufficient to allow impulses to be conducted through to
the second siphon (Fig. 5C).
Disk preparations from animals that had been deganglionated over a month previously showed a similar ability to
conduct excitation between the siphons. This finding fits
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Can. J. Zool. Vol. 78, 2000
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 (antitubulin). (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
preparations.
Source of recorded potentials
In both C. productum and C. inflata it was found to be
possible to record the characteristic bursts of electrical events
not only with electrodes placed over muscle bundles but also
from patches of mantle tissue where no muscle fibres were
visible. The “best” (largest amplitude) recordings were made
from muscular regions, but the patterns of impulses evoked
by stimulation were similar in both places, and were also
similar to the patterns recorded directly from large nerve
roots (Fig. 4B). These observations indicate that the bursts
of potentials are primarily nervous events that are augmented
by muscle depolarizations in muscular regions. Ascidian body
wall muscles are known from intracellular recordings to give
twitch-type responses accompanied by all-or-none action potentials (Nevitt and Gilly 1986). Such events presumably
sum with and augment the primary nervous signals.
Coordinated pacemaker activity in deganglionated C. inflata
After several hours’ recovery from ganglion removal, some
pinned preparations of C. inflata started to show patterns of
spontaneous activity in the form of regular bursts of small
(50–70 µV) potentials roughly every 4 min. Recordings made
with two electrodes, one on each siphon, showed similar patterns at the two sites despite the absence of the ganglion
(Fig. 6). In the longer record from which Fig. 6 was taken,
bursts lasted about 115 s, with 38 pulses per burst and interburst intervals of 127 s (mean values from a sequence of
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 occurring in time with burst potentials were just visible. In one
preparation a piece of the branchial sac was left attached to
the mantle and it was seen that the cilia lining the branchial
stigmata underwent arrest in synchrony with each of the
early potentials in the bursts accompanying muscular contractions in the siphons, confirming observations by Mackie
et al. (1974). Later in the bursts, the branchial cilia ceased to
show discrete arrests but were maintained continuously in
the fully arrested position until the end of the burst, when
normal beating and metachronal rhythms re-emerged.
Drug experiments
In a series of controlled experiments using C. productum
disk preparations, d-tubocurarine was found to block intersiphonal conduction in both normal and deganglionated
preparations (Figs. 5D–5F). The lowest effective dose was
10 µg·mL–1 and the block was completely reversible at
this level, though full recovery took at least 2 h. With stronger concentrations (20–40 µg·mL–1) the preparation sometimes failed to recover or recovery was incomplete. In a
deganglionated C. inflata, 10 µg·mL–1 curare blocked all
pacemaker activity within 13 min, along with spontaneous
muscle contractions and ciliary arrests, so the cilia beat continuously. Treatment with eserine (physostigmine) in concentrations up to 80 µg·mL–1 had no definite effect on the
responses of either C. inflata or C. productum, as Bacq (1935)
and Florey (1963, 1967) found with C. intestinalis. We were
unable to demonstrate any effect of α-bungarotoxin on
C. productum at 10 µg·mL–1 even after 24 h of treatment.
The times taken for curare to take effect and for responses to
be restored on return to seawater varied considerably in our
experiments, probably because of the slowness with which
the drug penetrates intact tissues. Tunicate epithelia are well
sealed, with extended tight junctions (Lane et al. 1995;
Burighel and Cloney 1997). Preparations of C. productum in
which the inner mantle epithelium was deliberately slit to assist penetration generally responded rapidly to the drug. The
failure of Scudder et al. (1966) to obtain any blocking action
with curare on whole, undamaged C. intestinalis may have
been due to failure of the drug to penetrate, as these authors
limited their drug tests to 1 h duration.
Discussion
Motor innervation
Motor nerves leaving the ganglion run out to the periphery, where they associate with muscles and ciliated cells.
Cholinesterase histochemistry shows the finest processes, including synaptic boutons in the branchial sac of C. inflata
(Arkett et al. 1989). The fine terminal neurites of the motor
system form plexiform arrays in both the mantle (Fig. 2D)
and the branchial sac, but there appear to be no cell bodies
within this mass of neurites. Nickel backfills (Arkett et al.
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Can. J. Zool. Vol. 78, 2000
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 after 5 h in seawater.
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
within the ganglion except for a small cluster close to the
ganglion in one of the posterior nerve roots. As the nerve
root in question (AM3) was the one that gives rise to the visceral nerve, it seems possible that the filled cells (which
were located on the surface of the nerve root rather than
within it) were part of the dorsal strand plexus (see below)
and were not motor neurons. Lorleberg (1907) observed a
few supposedly neuronal cell bodies (Ganglienzellen) in the
nerve roots of Styelopsis grossularia close to the ganglion,
but these became increasingly rare farther away from the
ganglion. In Polyandrocarpa misakiensis, Koyama and
Kusunoki (1993) saw “some neuron-like cells” in nerve roots
close to the ganglion. Our preparations of C. productum and
C. inflata, like those of Markman (1958) and Aros and
Konok (1969) using C. intestinalis, failed to show cell bodies in the motor innervation outside the brain. It seems certain, therefore, that the great majority, and possibly all, of the
motor neurons would be rendered anucleate by brain removal, and yet C. productum remains responsive to stimulation and shows spontaneous movements many weeks after
deganglionation. We can only conclude that the distal fragments of the motor neurons stay alive and functional in the
anucleate state. We have not addressed the question of how
the distal fragments survive, but this is a well-documented
phenomenon in other invertebrates, particularly crustaceans,
insects, and annelids (reviewed by Bittner 1991). It may be
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
blood would seem likely.
Sensory innervation
Lorleberg (1907) failed to find any evidence of peripheral
sensory cells and it was left to Fedele (1923a) and Millar
(1953) to provide the first clear descriptions of them in
C. intestinalis. We confirmed and extended these findings using
anti-tubulin and anti-GnRH immunolabelling, both of which
proved suitable for showing a wide variety of neurones, both
sensory and motor, in contrast to the cholinesterase method,
which was only effective in showing motor elements. Simple
primary receptor cells lie in both the outer and the inner
mantle epithelium of the siphons and have a sensory process
or cilium that projects to the exterior. They typically occur
in pairs or small clusters. Similar cells occur in ascidian larvae (Torrence and Cloney 1982) and pelagic tunicates (Bone
1959) and are generally regarded as mechanoreceptors (Goodbody 1974; Bone and Mackie 1982). Our investigation using
immunolabelling shows the neurites of these cells running
below the epithelium, intermingling and forming a netlike
array (Fig. 3F) before entering larger nerve bundles leading
to the ganglion. Sensory papillae occur in the outer layer of
the branchial sac, where we describe them for the first time.
Their afferent neurons form bundles that run to the ganglion
via the visceral nerve roots but some bundles also leave the
branchial sac and mingle with nerves in the mantle without
passing through the ganglion. Such connections might provide
a pathway for the coordination observed between mantle
contractions and ciliary arrests in deganglionated preparations.
It is unclear if ascidians have sensory neurons with cell
bodies in the ganglion and processes that end freely in the
periphery. Free nerve endings are seen in many places where
there are no primary sensory receptor cells and some of
these might be sensory endings. In the oral tentacles, which
are highly sensitive to tactile stimuli evoking the crossed response (Hecht 1918), Seeliger (1893–1907) described cells
with processes projecting to the exterior that might be sensory structures, but his drawings are unconvincing, and subsequent workers, including ourselves, have been unable to
confirm his finding. Burighel and Cloney (1997, Fig. 42)
figure a presumptive secondary sensory cell in a tentacle of
Botryllus schlosseri, raising the possibility that some of the
“free nerve endings” described by other workers are actually
postsynaptic or electrically coupled to secondary sensory
cells lying in the epidermis. Secondary sensory cells occur
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in various larvaceans (Bone and Mackie 1982) but have not
been recognized in other tunicates, except for this one report
on B. schlosseri.
Pacemakers
Intracellular recordings from the ganglion of C. productum
demonstrate the presence of motor neurons that generate
rhythmic patterns of impulses driving ciliary arrests in the
branchial sac (Arkett 1987). However, the branchial sac continues to show spontaneous rhythmicity after deganglionation
(Mackie et al. 1974) and this persists for at least 3 weeks
(Kingston 1976, see footnote 3). Pacemaker capability must
therefore exist at the periphery. This is also clear from studies on the siphons and body wall of other ascidians (Day
1919; Yamaguchi 1931; Bacq 1935; Hoyle 1953). Our present
work on deganglionated C. inflata has shown the emergence
of a rhythmic pattern of muscle contractions coordinated between the two siphons and blocked by curare. The peripheral
pacemakers are therefore presumably components of nerve
cells but it is impossible to say whether they are located in
sensory or motor nerves.
Is there a peripheral nerve net?
Most students of ascidian behaviour (e.g., Jordan 1907;
Hecht 1918; ten Cate 1928; von Buddenbrock 1928; Schiller
1937; Florey 1951) have assumed that there is a peripheral
nerve net in the mantle that survives deganglionation and
provides a basis for continuing responsiveness to stimulation.
The physiological picture is fairly clear: peripheral conduction pathways showing the characteristics elsewhere associated with nerve nets certainly exist both in intact animals
and after deganglionation. We have confirmed previous reports that ascidians respond to stimuli with contractions that
spread decrementally for varying distances depending on on
the strength and frequency of stimulation, and produce graded
responses in the muscles in a manner reminiscent of sea
anemones. Conduction velocity in the “net” is slower than in
the intact animal by about an order of magnitude. Florey
(1951), the most recent physiologist to address this question,
concluded that there is a twofold innervation of the musculature in C. intestinalis, the first consisting of motor nerves
coming from the ganglion and the second consisting of a
peripheral nerve net. After destruction of the ganglion, all
remaining responses would be mediated by the nerve net.
The term nerve net is generally taken to refer to a plexiform array of bi- or multi-polar neurons lying outside the
central nervous system and constituting a diffusely conducting (unpolarized) set of pathways (Mackie 1999). As evidence of such a net in ascidians, many workers have cited
histological observations by Hunter (1898), whose Fig. 3
could be interpreted as showing cell bodies within the peripheral nervous system of Molgula manhattensis. Our findings, however, support Fedele (1923a; 1937a) and Markman
(1958), who found no such nerve net in the mantle, and are
in agreement with earlier reports (Mackie et al. 1974; Arkett
et al. 1989) in showing that there is no nerve net in the
branchial sac.
The assumption by various workers that there is a nerve
net in the mantle, though emphatically denied by Fedele
(1923a, 1937a), was (paradoxically) bolstered by Fedele’s
Can. J. Zool. Vol. 78, 2000
own account of a nerve net in the dorsal fold of the branchial
sac (Fedele 1923b, 1927, 1938). The system consists of a
plexus of bi- and multi-polar neurons lying within the dorsal
blood sinus and closely associated with the dorsal strand
(dorsal cord) and visceral nerves. This plexus (“dorsal-strand
plexus”) extends all the way from the cerebral ganglion back
to the region of the gonads. It can be readily visualized by
means of methylene blue staining and phase-contrast optics
in living whole mounts (Mackie et al. 1974) and by its
immunoreactivity with antisera raised against GnRH (Mackie
1995; Powell et al. 1996; Tsutsui et al. 1998). The presence
of this nerve net in the dorsal fold made it quite plausible
that another net existed in the mantle tissue, but, as noted
above, there is no histological support for such a structure.
Indeed, the fact that the nerve plexus in the dorsal fold can
be visualized so readily means that if such a plexus were
present in the mantle, it could hardly have gone undetected.
The techniques used in the present study allowed us to visualize both the sensory and the motor innervation in considerable
detail and to detect nuclei where present (i.e., in sensory
cells), and would surely have revealed a nerve net if it were
present.
Alternatives to a conventional nerve net
If there is no nerve net, we are left with four possibilities:
(i) the transmission of excitation by mechanical forces between
muscles in the body wall, acting as “independent effectors”
(proposed by Loeb 1891; Fedele 1923b, 1937b), (ii) conduction in excitable epithelia (suggested by ten Cate 1928),
(iii) conduction between the interconnected terminals of the
motor nerves (suggested by Mackie et al. 1974), and (iv) conduction between interconnected branches of the sensory nerves.
Regarding i, myoid transmission is hard to reconcile with
the observations of Magnus (1902) and Kinoshita (1910),
who found that treatment with cocaine and other drugs that
eliminate all nervous actvity while leaving the muscles excitable by electrical stimuli blocked the spread of responses
completely. Our own findings with curare, a potent blocker
of cholinergic synapses, also show that conduction of excitation must depend not on muscles but on nerves, as muscles
are not synaptically linked. This does not mean that the muscles are incapable of independent action when directly stimulated, or that they could not act mechanically upon other
muscles in the locality, but the results of the experiments
with drugs make it clear that the main peripheral conduction
pathways surviving deganglionation cannot be muscle-based.
Regarding ii, excitable epithelia that conduct behaviourally
meaningful signals occur in some ascidian larvae (Mackie
and Bone 1976), in the blood vessels linking the zooids in
botryllid colonies (Mackie and Singla 1983), and in various
pelagic tunicates (see Bone and Mackie 1982), raising the
possibility that the mantle and branchial epithelia in adult
solitary ascidians are also excitable and conduct electrical
signals. Recordings made with electrodes attached to the
surfaces of the mantle epithelia, however, failed to show the
large potentials typically associated with epithelial conduction, showing instead much smaller potentials in patterns
similar to the bursts of action potentials recorded directly
from cut nerves. Furthermore, curare would not be expected
to block epithelial conduction, as transmission in all such
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tissues is electrical not chemical (Anderson 1980). The small
potentials recorded with suction electrodes attached to the
mantle epithelia almost certainly derive from the plexiform
mass of fine nerve terminals running under the epithelium.
Regarding iii, if the motor nerve terminals were interconnected synaptically, they could in theory produce the
equivalent of a nerve net (Fig. 7A). Decremental spread
would be exhibited if the synapses failed to transmit one-forone but represented a partial barrier to the spread of excitation. This would occur, for example, if early impulses in a
burst arriving at a synapse failed to evoke regenerative potentials on the postsynaptic side but evoked subthreshold
depolarizations (excitatory postsynaptic potentials) that allowed later ones to produce spikes. The burst would diminish with each synapse crossed and gradually fade out or
become ineffective in evoking effector responses. A consequence of an arrangement of this sort would be to reduce the
selectivity of muscle excitation. Motor nerves in most animals
function to excite specific effectors, leaving others unexcited
(the concept of “labelled lines”), and if motor impulses invaded adjacent motor units, it would become hard to obtain
precise local movements such as directional flexions. In fact,
ascidian behaviour lacks directionality. Siphon closures and
body-wall contractions are more or less symmetrical. In the
case of the isolated branchial sac, impulses generally spread
through the whole preparation, though not necessarily at the
same velocity in all directions (Mackie et al. 1974). Creation
of a motor net in which impulses are shared between adjacent motor units might have advantages in “smoothing out”
responses.
In the scenario shown in Fig. 7A, sensory cells do not
synapse with motor nerves except in the ganglion, and it is
assumed that the motor plexus itself is directly excitable by
local stimulation but sensory units could synapse with the
motor plexus in the periphery; some observations support
such a picture. For instance, isolated siphons and siphons in
deganglionated animals remain almost as responsive to vibrations as those in the intact animal (Magnus 1902), which
implies that mechano-receptors are still functional parts of
the response system with connections to local effectors.
Regarding iv, sensory cells lying in the mantle and branchial
sac could send processes not only to the cerebral ganglion
but also through branches in the periphery to one another
(Fig. 7B). Synaptic links would regulate impulse traffic as in
iii above. To excite the muscular or ciliary effectors, the
sensory net would either have to contact the effectors directly
or excite them indirectly through the motor innervation. We
have shown them exciting the effectors indirectly by synapsing on motor terminals. However, direct connections between sensory cells and muscles are known in Hydra littoralis
(Westfall 1973) and cannot be excluded. A net fashioned out
of the interconnected branches of sensory neurons would
satisfy the requirement for a set of peripheral conduction
pathways that could survive deganglionation. A potential disadvantage of this arrangement would be some loss of precision
in the ability of the animal to localize sources of stimulation,
for sensory signals would tend to invade afferent pathways
adjacent to those actually stimulated.
At present, the evidence is insufficient for deciding between the motor-net (Fig. 7A) and sensory-net (Fig. 7B) hy-
1637
potheses. Both are highly unconventional and would reflect
a unique situation among triploblastic animals but, given
that there is no conventional nerve net, one or other of them,
or some combination of the two, must be true.
Evolutionary implications
Regardless of the precise nature of the peripheral conduction system that endows ascidians with the functional equivalent of a nerve net, it is clear that ascidian evolution has
been characterized by decentralization of certain functions
that are more fully centralized in other tunicates. Doliolids
and salps deprived of their ganglia show no spontaneous
swimming or contractions of the body-wall musculature and
do not respond to stimulation. However, doliolids show local
autonomy in the ciliary control system in the branchial sac,
like ascidians (Fedele 1923c; Bone and Mackie 1982). The
ganglion in ascidians is small and simple compared with, for
example, that of salps. It still has a special role to play in
maintaining muscle tone, as a fast conduction pathway and
as mediator of certain complex responses, notably the crossed
response (Day 1919; Bacq 1935; Florey 1951). These functions, however, are superimposed upon a fundamental neuromuscular organization characterized by the decremental,
diffuse, facilitating spread of contractions within and between the siphons and in the body wall and branchial sac,
often exhibited in rhythmic patterns (Hoyle 1952, 1953), and
it is this residual innervation that survives deganglionation.
Evolutionary relationships within the Tunicata remain uncertain (Lacalli and Holland 1998; Lacalli 1999), and it is
not clear whether the ancestral urochordate was planktonic
or bottom-living, but a recent molecular phylogenetic analysis (Swalla et al. 2000) suggests that the ancestor “probably
retained the tadpole tail, including the dorsal nerve cord, the
notochord and flanking muscle cells through the adult life.”
A good case can be made for the origin of ascidians from
more active, mobile, free-living ancestors (Satoh and Jeffery
1995; Wada 1998) whose nervous systems would probably
have been more fully centralized, as in present-day pelagic
tunicates. The decentralization of functions seen in ascidians
would therefore appear to be in some way adaptive in the
context of their sessile habitat. It may be that processing all
sensory information and motor output via a single nerve centre is not only unnecessary but metabolically wasteful in a
situation where many responses can be managed initially by
local action systems. Actinian nerve nets have the same potential for organizing actions locally that we see in ascidians
and their nervous systems show a corresponding lack or low
degree of centralization. Aside from the purely “visceral”
activities carried out locally in the heart, gut, and gonoducts,
ascidian behaviour consists essentially of opening, closing,
and retracting the siphons, contracting the body-wall muscles, and arresting the cilia in the branchial sac. These components are functionally linked and are exhibited in various
degrees depending on the strength of stimulation. There are
no directional responses and little apparent need for precise
localization of sources of stimulation. Whereas actinians have
true nerve nets for organizing local action systems, it would
appear that ascidians have created the equivalent of a peripheral nerve net by modifying the connectivity patterns of their
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
in pelagic tunicates. At the same time they have retained the
cerebral ganglion and its major input–output pathways for
organizing complex responses and as a fast pathway for
rapid and coordinated defensive responses.
Note added in proof:
A forthcoming paper (Burighel et al. 2000) describes the
distribution of nerves in the colonial ascidian Botryllus
schlosseri, as visualized by cholinesterase histochemistry.
Nerves extend to all organs except for the neural gland and
gonads. A complex network of nerves is described, but no
peripheral cell bodies were observed. The system does not
interconnect different zooids within the colony. The picture
of this component of the innervation in B. schlosseri thus
corresponds in essential respects to what we describe here
for the solitary ascidians C. productum and C. inflata.
Acknowledgements
This work was supported by a grant to G.O.M. from the
Natural Sciences and Engineering Research Council of Canada.
We thank Catherine Pennachietti for collecting some of the
material used in the experiments in Victoria. Nancy Sherwood advised us on aspects of GnRH immunobiology and
provided antibodies. Thurston Lacalli and Quentin Bone kindly
read and commented on the manuscript. We thank Dennis
Willows, Director, and the staff of the Friday Harbor Laboratories for space and assistance during phases of the work
conducted there.
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