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First published online August 17, 2006
Journal of Experimental Biology 209, 3405-3412 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02421
Development of swimming behaviour in the larva of the ascidian Ciona intestinalis
1 Neurobiology Laboratory Stazione Zoologica `Anton Dohrn', Villa Comunale
I-80121 Naples, Italy
2 Royal Swedish Academy of Sciences Kristineberg Marine Research Station,
SE-45034 Fiskebäckskil, Sweden
* Author for correspondence at present address: Dipartimento di Biologia `L. Gorini', Università degli Studi di Milano, Via Celoria 26, I-20133 Milano, Italy (e-mail: giuliana.zega{at}unimi.it)
Accepted 1 July 2006
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Summary |
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Key words: shadow response, electrophysiology, muscle field potentials, nervous system, locomotion, Ciona intestinalis
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Kats, 1983;
Bone, 1992). Ascidian tadpoles
actively swim by bending their tail with alternating symmetrical contractions.
They also produce asymmetrical contractions, or `tail flicks', which help the
larvae escape from the chorion membrane and, later, to change direction
(Mast, 1921).
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; Meinertzhagen and
Okamura, 2001; Meinertzhagen
et al., 2004). Muscle contractions are driven by five pairs of
motor neurons, found in the visceral ganglion, that project axons to the
muscle fibres of the tail (Fig.
1B) (Bone, 1992;
Cole and Meinertzhagen, 2004;
Brown et al., 2005). The
control of swimming has some similarities to vertebrate spinal networks
suggesting the existence of some `universal' chordate features. For example,
larval motor neurons innervating the tail express cholinergic promoters and
genes (Takamura et al., 2002;
Yoshida et al., 2004),
neuromuscular junctions are cholinergic
(Ohmori and Sasaki, 1977),
while GABA is present in the visceral ganglion and, as in vertebrates, has
been shown to modulate swimming (Brown et al.,
2005).
Impinging on the `lower' motor network of the visceral ganglion are fibres
emanating from the `higher' part of the nervous system, which includes the
sensory vesicle. The sensory vesicle contains the two main sensory organs that
allow the larva to detect light and gravity: the ocellus and the otolith
(Fig. 1A,B). The ocellus is a
pigmented cell associated with 17-30 photoreceptor cells and a lens
(Eakin and Kuda, 1971;
Nicol and Meinertzhagen, 1991;
Horie et al., 2005). The
otolith is a single pigmented cell, connected to neurones via the
floor of the sensory vesicle (Tsuda et
al., 2003a; Nagakawa et al.,
2002; Sakurai et al.,
2004). These two organs are mainly involved in the perception of
environmental cues that drive ascidian tadpole behaviour
(Tsuda et al., 2003a;
Sakurai et al., 2004;
Di Jiang et al., 2005).
The behaviour of larvae changes during the free swimming phase. For
example, larvae have been reported to switch their behaviour from
photopositive to photonegative during the presettlement period
(Grave, 1920;
Millar, 1971). Resting larvae
are stimulated to swim when passing from light to dark conditions and this
reaction is known as the shadow response
(Mast, 1921;
Kajiwara and Yoshida, 1985;
Young and Chia, 1985;
Bone, 1992). Ciona
savignyi larvae were found to develop the shadow response 1.5 h after
hatching (Kajiwara and Yoshida,
1985), while C. intestinalis larvae became sensitive to a
reduction in light around 4 h after hatching and during the dark period they
swim faster (Kawakami et al.,
2002; Tsuda et al.,
2003b).
The objective of this work was to record the muscular activity underlying
larval swimming during the course of larval life and to determine the time of
onset of the shadow response and its influence on larval behaviour. The
recording method did not seem to alter significantly the development of
swimming behaviour, as restrained larvae demonstrated similar behaviour to
that observed with video or by direct observation. This work focuses on the
first 6 h period post-hatching since during this phase larval structures
complete their development and competence for metamorphic change is acquired
(Degnan et al., 1997;
Eri et al., 1999;
Davidson and Swalla, 2001;
Horie et al., 2005;
Nakayama et al., 2005). We
used a high level of precision because video analysis of swimming larvae does
not reflect linearly the output of the nervous system at all stages of larval
development; because some larval muscle activity is not concerned with
swimming (e.g. changes in direction), and because swimming occurs at
intermediate Reynolds numbers in seawater, initial tail movements do not
produce instantaneous velocities (McHenry
et al., 2003).
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Materials and methods |
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Electrophysiological recordings
For electrophysiological recording, glass micropipettes were drawn from
borosilicate glass of 1.5 o.d. on a microelectrode puller (Model P87 Sutter
Instrument Company, Novato, CA, USA). The electrodes were mounted on a
micromanipulator and their tips broken under microscopic control, so that the
internal diameter was about four-fifths the diameter of the larval tail. Using
coarse manipulation of the microscope stage and micromanipulation of the
electrode, the larval tail was placed in close contact with the tip of the
electrode. Then, negative suction was rapidly applied and the larval tail was
drawn into the pipette to about two thirds of its length. Muscle action
potentials were recorded differentially between the inside of the pipette and
the seawater of the bath, and amplified (WPI model DAM 80 World Precision
Instruments Ltd, Aston, UK) 10 000x with reference to a silver chloride
pellet placed in the bath. Signals were AC-coupled and passed between 0.1 Hz
and 10 000 Hz. They were then digitised and stored, using a Digidata 1200 data
acquisition system, and analysed using Clampfit software (version 9.0) (Axon
Instruments Inc, Molecular Devices Corporation, Sunnyvale, CA, USA). A
custom-built shutter was controlled by 5 V control pulses delivered from the
Digidata board, allowing a step-down in the light intensity to be applied for
5 s. In order to determine the exact age of larvae used in the recordings, a
pool of newly hatched larvae was sampled and transferred to a new Petri dish.
Then some larvae were put into a 5 cm Petri dish in FSW and placed under the
microscope. When drawn into the pipette, larvae showed some inhibition of
swimming activity that lasted around 15 min. Therefore all the experimental
runs were started 20 min after the suction electrode was attached to the tail.
All experiments were carried out at 20°C and the Petri dish was perfused
with FSW (8 ml min-1). Larval activity was recorded in a series of
1 min sweeps, every 5 min, under constant light conditions or with 5 s
light-off. The light-off stimulation was always given within the first 30 s of
the sweep so that the after-effects of the response could be studied.
Plots of instantaneous frequencies of potentials vs time and mean frequency of potentials were obtained from raw traces. The duration of each interval of larval swimming activity, also termed `burst', and the quantity of activity for each sweep (sum of all burst durations), both with light-off stimulation and in constant light, was obtained. Recordings were made from newly hatched larvae and from larvae up to 6 h post-hatching (h.p.h.). The activity of each larva was recorded for a maximum period of 3 h.
Photographs
To establish the time of onset of the shadow response independently of the
suction electrode method, larvae were placed in a square tank (3 cm), and were
photographed from above in light conditions. Photographs were taken before and
5, 30 and 60 s after shading (see Kajiwara
and Yoshida, 1985). The ambient temperature was 20°C.
Data analysis and statistics
Analysis of variance (ANOVA) was performed to test whether the mean values
of muscle potential frequencies associated with different larval activities
were significantly different. Mean frequencies of muscle field potentials of
different larval activities were calculated from 2 s of sampled traces, with
the exception of tail flick values, since these often lasted for shorter
periods. For evaluation of the after-effects of the light-off stimulus,
average values were obtained from 2.5 s of the sampled trace. The effect of
larval age, hours post hatching, and presence or absence of dark stimulation
on quantity of activity per sweep (s), were also evaluated. Linear regression
analysis was used to examine if trends observed in the after-effects of the
light-off stimulus were significant. Values are means ± s.d.
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), i.e.
potentials occurred singly or in trains on a flat baseline
(Fig. 2A). To examine possible
time- or activity-dependent changes in swimming rate, the instantaneous
frequency of each tail contraction was plotted vs time
(Fig. 2B). Significantly
different frequencies of potentials were found for three distinctive larval
behaviours: tail flicks, spontaneous swimming bursts and shadow responses
(dark-induced activities). Tail flicks occurred at 9.8±2.2 Hz
(N=7), spontaneous swimming bursts at 22.8±2.4 Hz
(N=24) and shadow responses at 32.1±3.9 Hz (N=24)
(ANOVA test: F=148.9; P
0.00001) (data from
Fig. 2C). For both tail flicks
and spontaneous swimming, the frequencies did not alter significantly with
time (Fig. 2C). Trains of tail
flicks were often recorded in larvae up to 3 h.p.h., both before and after
swimming bursts (Fig. 2A,B).
Later they occurred rarely.
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After-effects of the shadow response.
In Fig. 2B, the scatter plot
of instantaneous frequency of muscle potentials in a 3.5 h.p.h. larva, shows a
gradual decrease after the 5 s dark period. We observed that the frequency of
potentials of the shadow response activity remained higher than the
spontaneous swimming frequency for a period after the light was switched on
again. A linear regression model explained about the 31% of the frequency
decrease in time (N=15, R2=0.310;
F=116.257; P0.00001)
(Fig. 4). The mean frequency of
tail contractions, for each time interval, was significantly higher than the
frequency of spontaneous swimming (recorded before the light off stimulation)
for 25 s after the beginning of shadow response
(Table 2).
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Quantity of swimming activity
To examine if there were changes in the quantity of swimming with time, the
shadow response duration, the duration of spontaneous swimming, mean burst
duration and quantity of total swimming activity during larval aging was
calculated. The duration of the shadow response stimulated by 5 s light-off
was very variable and no significant trend was found during larval aging (data
not shown). The mean duration of the shadow response was 20±10.4 s and
the minimum duration was 5 s, corresponding to the duration of the imposed
dark period. The mean duration of spontaneous swimming bursts changed
significantly during the three periods considered. In larvae from 0-2 h.p.h.,
bursts lasted for 10.2±14.2 s, while in older larvae, from 2-4 h.p.h.
and from 4-6 h.p.h., mean burst duration was 4.1±5.1 s and
5.7±6.0 s, respectively (ANOVA: F=11.304,
P0.0001; Tukey's Post Hoc: 0-2 h.p.h. vs 2-4
h.p.h. P0.0001; 0-2 h.p.h. vs 4-6 h.p.h.:
P0.002; 2-4 h.p.h. vs 4-6 h.p.h.: no significant
difference) (N=20) (Fig.
5).
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0.00001; effect of presence or absence of light-off stimulation
vs total activity F=18.495, P0.00001;
interaction: larval age x presence or absence of light-off stimulation
F=22.803, P<0.00001). From 0 to 2 h.p.h., larvae under
constant light swam for 40.1±15.1 s per sweep, while larvae stimulated
by the 5 s light-off swam for 31.0±17.8 s. In the next two periods,
larvae under constant light conditions swam for shorter times per sweep when
compared to larvae stimulated by the light-off (2-4 h.p.h. and 4-6 h.p.h.
without light-off: 15.2±11.2 s and 20.3±11.1 s; 2-4 h.p.h. and
4-6 h.p.h. with light-off: 33.1±14.4 s and 36.2±12.5 s) (ANOVA:
2-4 h.p.h. F=27.242, P0.00001; 4-6: F=35.269,
P0.00001) (N=40)
(Fig. 6). These results showed
that there was a significant decrease in the quantity of activity during aging
if larvae were not `stimulated' by light-off. On the other hand, in the three
different time intervals considered, there was an increase in the total
activity of the larvae during sweeps with light off stimulation. Moreover in
each of the three periods, there was a significant difference between mean
total activity in sweeps without light-off compared to that in sweeps with
light-off. Under our experimental conditions, larvae from 0 to 2 h.p.h. swam
for longer in the absence of dark stimulation while later on (2-4 and 4-6
h.p.h.) they swam for longer times only if stimulated by a light step down,
when compared to 0-2 h.p.h. larvae.
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). In agreement with
this view, we often observed tail flicks in the period immediately preceding a
`bout' of spontaneous symmetrical swimming and also in the first movements of
the shadow response. However, tail flicks were not an absolute prerequisite
for both types of symmetrical swimming and it has been observed that they
could determine a change in direction in free-swimming larvae
(Mast, 1921;
Bone, 1992). Symmetrical swimming activity, or spontaneous activity, was characterized by rates of around 20 Hz, which is about double the frequency of the single tail twitches. The fact that both tail flicks and symmetrical swimming have such tightly controlled frequencies would seem to suggest that both are subject to control by impulses coming from the main sensory organs in the trunk, which act to coordinate firing rate and, in consequence, muscle contractions. In terms of control, it should be noted that there was no superposition of tail flicks, spontaneous swimming or shadow response. In other words, there is a final common pathway for nervous system output, and cross-inhibition ensures temporal separation of the different behaviours.
The onset of the shadow response in C. intestinalis larvae occurred at 1.5 h.p.h., and this was confirmed by photographs of swarms of larvae taken before and after shading. This showed that even when restrained in the suction electrode, larvae develop shadow responses at the same time as freely moving specimens. The frequency of potentials of the shadow response activity was initially no different from the frequency of spontaneous swimming. About half an hour after its appearance, the frequency of the potentials during the shadow response increased to 30-35 Hz. Under our experimental conditions, even actively swimming specimens reacted to light-off by increasing the frequency of muscle contractions (tail beating).
Our results on the timing of the onset of the shadow response are in
accordance with observations made on C. savignyi
(Kajiwara and Yoshida, 1985).
Kajiwara and Yoshida described how different larval behaviour, from the onset
of the shadow response to the beginning of the photonegative period, was
related to the different developmental stages of the ocellus, which becomes
fully differentiated 3.5 h after hatching. In particular, the authors
described how pigment granules are gradually accumulated while the sensory
structure of the photoreceptor is folded into its definitive form. It is
possible that changes in the ability to respond to dark stimulation are
determined by a particular course of development of the light sensing organ.
In C. intestinalis, Horie et al. described that in 1 h.p.h. C.
intestinalis larvae all the photoreceptor cells are already present,
while their nervous connections expanded remarkably only later, at around 3
h.p.h. (Horie et al., 2005).
Therefore, the observed increase in frequency of the shadow response activity
could be related to the completion of the development of the light sensory
organ and of the neural network connecting the sensory organs to motor area in
the visceral ganglion.
It has been reported that in C. intestinalis larvae the shadow
response did not develop until 4 h.p.h.
(Kawakami et al., 2002;
Tsuda et al., 2003b). Such
different observations could be explained by the different temperatures at
which the experiments were carried out (20°C vs 18°C).
Temperature strongly influences the timing of development in Ciona
embryos and it is possible that larvae kept at a lower temperature develop the
shadow response later. Perhaps the major difference, however, was that the
determination of the onset of this response was obtained by carrying out video
recording and producing a mean linear speed for a large number of swimming
tadpoles. As larval swimming speed does not deviate significantly from that of
spontaneous swimming until 2 h.p.h., it would be difficult to determine the
onset of this response using the video recording method. In any case, and in
accordance with our results, it was established that linear speed of swimming
increased to a maximum value during the dark period and then decreased when
the light was on again (Tsuda et al.,
2003b).
The frequency of potentials of shadow response activity showed
after-effects. Maximum values were recorded during the 5 s dark period, while
after that, when the light was on, the frequency gradually decreased for 25 s
until it was equal to the frequency observed during spontaneous swimming.
Ascidian photoreceptors are of the hyperpolarizing type
(Gorman, 1971) and darkness
should produce a depolarizing response, giving an excitatory stimulus to
nearby neurones. This stimulus could determine a change in muscle tail
contraction frequency, through excitation of interneurone circuits that drive
the firing rate of motor neurones in the visceral ganglion. Such
interneurones, located close to the photoreceptors and forming part of a
retinal territory that sends `descending' neuronal process to the visceral
ganglion, have been detected morphologically
(D'Aniello et al., 2006). When
the light is on, hyperpolarization of photoreceptors occurs and the motor
response frequency begins to wane, following the trend shown in
Fig. 4. Brown et al. localized
GABA immunoreactivity in the nervous system of larvae of C. savigny,
particularly at the level of the sensory vesicle and the visceral ganglion
(Brown et al., 2005). Their
pharmacological results with Ciona intestinalis showed that GABA is
released during swimming and could act as a modulator of swimming frequency.
Another potential inhibitory transmitter system in ascidian larvae is
dopamine. Moret et al. detected tyrosine hydroxylase (dopamine synthesis
rate-limiting enzyme) expression in the hypothalamus-related domain of the
sensory vesicle of C. intestinalis larvae
(Moret et al., 2005) and
suggested that dopamine could be involved in modulating larval locomotion.
These authors showed that dopamine synthesis begins only some hours after
hatching. It could be that one of these inhibitory neurotransmitters cause the
decrease in frequency of muscle contraction after dark stimulation, when the
light is on. Indeed the increase in frequency of the shadow response with time
may reflect a gradual disappearance of inhibitory control over the `normal'
(spontaneous) swimming rate.
The duration of the shadow response was very variable during the course of
larval life. The fact that such a diffuse reaction among ascidian tadpoles has
an unpredictable duration during larval aging, supports the hypothesis that
this response is not involved in locating shaded habitats and does not allow
larvae to encounter a suitable place for settlement with a higher probability
(Young and Chia, 1985;
Svane and Young, 1989). Its
most probable function is to help orientation of swimming larvae in light,
first in photopositive and later in photonegative taxis. A mechanism was
described supporting the hypothesis that the shadow response may help the
larvae to orientate towards or away from the light direction
(Mast, 1921). Mast noted that
while swimming, ascidian tadpoles continuously rotate on their longitudinal
axis clockwise and they twitched the tail in different directions depending on
the orientation of the ocellus to the light source As the unpaired
photoreceptor of the ascidian tadpole is situated on the right posterior wall
of the sensory vesicle, he suggested that the orientation of larvae to light
could be the result of one or more reactions, caused by the alternate shading
and illumination of the optic nerve endings, owing to the rotation of the
tadpoles on the longitudinal axis.
Mean burst duration and mean total activity per sweep were higher in larvae
up to 2 h.p.h. than in older ones. As a consequence, under our experimental
conditions, ascidian larvae swam for longer time intervals and more often
during the first hours after hatching, compared to older ones. It is
reasonable to suppose that, under natural conditions, newly hatched larvae are
more active to improve dispersal, while older larvae swim less and most
probably sink for longer, to increase the chance of finding a suitable place
to settle (Bone, 1992;
McHenry, 2005). Our
observation provides additional evidence that, even if restrained in the
suction electrode, larvae retained an apparently normal behaviour.
To date the neural networks connecting the sensory vesicle to motor neurons and how they might drive ascidian larvae locomotion are still poorly known. The method used here to characterize ascidian larvae behaviour is an essential first step towards describing in detail how single neurone behaviour and networks work together to produce whole-larva behaviour.
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Acknowledgments |
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