Direct evidence for the role of pigment cells in the brain of ascidian larvae by laser ablation
Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Garden City, Kouto 3-2-1, Akoh-gun, Hyogo 678-1297, Japan
* Author for correspondence (e-mail: mtsuda{at}sci.himeji-tech.ac.jp)
Accepted 13 January 2003
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Summary |
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Key words: otolith, ocellus, eye, ascidian, larva, swimming behavior
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Introduction |
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The ascidian larva, composed of only 2600 cells, has a primitive nervous
system that is said to comprise only about 100 neurons (Nicol and
Meinertzhagen,
1988a,b
;
Meinertzhagen and Okamura,
2001
). Not only do ascidian larvae have among the smallest numbers
of neurons in any central nervous system
(Meinertzhagen and Okamura,
2001
), but the central nervous system (CNS) in the tadpole also
extends most of the length of the animal and is divided into three parts, an
anterior sensory vesicle, a visceral ganglion and a caudal nerve cord
(Katz, 1983
;
Nicol and Meinertzhagen,
1991
). The anterior sensory vesicle is a single large vesicle in
which lie two types of pigment cells, the anterior and posterior pigment
cells, called the otolith and the ocellus, respectively. The ultrastructure of
these pigment cells has been described by Dilly
(1969
,
1962
), Eakin and Kuda
(1971
), Torrence
(1986
) and Ohtsuki
(1991
). The otolith is a
spherical mass of pigment granules connected to the floor of the sensory
vesicle by a narrow stalk. The ocellus is cup-shaped and contains many small
pigment granules. In addition to the ocellus and the otolith, a third type of
presumptive sensory organ has been described in some species, consisting of
globular bodies with membranous tubules and a ciliary organ. The function of
this sensory receptor is unknown. Eakin and Kuda
(1971
) proposed that it may
function as a hydrostatic pressure detector, but others
(Dilly, 1969
;
Reverberi, 1979
) have
characterised it as a photoreceptor.
The ascidian larva has a characteristic pattern of swimming, consisting of an initial period when it swims upward followed by a period when it swims or sinks downwards to settle and metamorphose. In nature, the initial phase serves to distribute the larvae, and in the second phase the larvae seek out the undersides of eelgrass blades or other suitable sites for attachment and metamorphosis. The putative sensory organs such as the ocellus, otolith and globular bodies are thought to guide the swimming behaviour of the larvae.
The larvae of Ciona intestinalis, one of the species studied in
most detail, have been reported to pass through photopositive and
photonegative phases (Dybern,
1963). Svane and Young
(1989
), however, suggest that
Ciona intestinalis larvae remain photonegative during their entire
free-swimming period. Many observations have shown that larvae at the
beginning of the free-swimming period swim upward both in the dark and in
light. Our observations showed that within 3 h of hatching, the larvae do not
show any photoresponse (Nakagawa et al.,
1999
; Tsuda et al.,
2001
). These results indicate that a negative response to gravity
or hydrostatic pressure effectively leads the larvae to the water surface. Our
motion analysis results, which are consistent with many other observations,
showed that larvae start swimming when the intensity of light is decreased
(Tsuda et al., 2003
),
suggesting that larvae swim downward due to a negative photoresponse. We also
showed that when stimulated by repeated onset and cessation of light, the
larvae exhibited sensitization and habituation of the swimming response
(Tsuda et al., 2003
).
The functions of three putative sensory organs of ascidian larvae the ocellus, otolith and pressure organs have been suggested from morphological information alone. To date there has been no direct evidence for their function from physiological and behavioural studies. In the present work, the functions of these putative sensory organs were studied using the behaviour of free-swimming larvae. Geotropism (movement in response to gravity) was studied by observing the upward swimming stage (within 3 h of hatching) with or without laser ablation of the anterior and posterior pigment cells. Phototropism (movement toward or away from light) was studied by observing the photic behaviour of the larvae with or without laser ablation of the anterior and posterior pigment cells. Prestropism (response to pressure) was studied by recording the effect of hydrostatic pressure on the swimming behaviour of the larvae.
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Materials and methods |
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Laser microsurgery
Larvae that demonstrated negative geotactic ability were anesthetized with
0.02% MS-222 (Ethyl m-aminobenzoate methanesulfonate, Nakarai, Kyoto, Japan).
Larvae in the anesthetized condition were transferred to a chamber in a glass
slide with a plastic spacer (6 mm in diameter, 150 µm in depth) and covered
with coverslip. They were examined under Normarski DIC optics using an
Axioplan 2 microscope (Carl Zeiss, Jena, Germany) attached, via the
epifluorescence port, to a small nitrogen laser (Micropoint Ablation Laser
System; MP VSL337, Carl Zeiss, Jena, Germany) coupled with a dye laser
[Coumarin dye (440 nm)]. To ablate the anterior pigment cell in the sensory
vesicle (i.e. the otolith), the laser microbeam was focused onto the short
stalk that interconnects the vesicle wall and its distal foot in the lumen of
the vesicle. The posterior pigment cell (i.e. the ocellus) was ablated by
focusing the laser onto the centre of the pigment. Intact control and ablated
larvae were transferred to a thin quartz optical cell (10 mmx40
mmx3 mm) at 18°C in a constant-temperature incubator (AG-HC090X,
Nihon-ika, Osaka, Japan).
Behavioural observation
Upward swimming behaviour of the larvae
Both intact and ablated larvae were transferred to a thin quartz optical
cell (widthxdepthxlength=10 mmx40 mmx3 mm) in order to
observe the two-dimensional (2-D) upward swimming behaviour. The larvae in the
thin optical cell were illuminated by nonactinic far-red illumination
(wavelength 640 nm; 20 far-red photodiodes were placed by the side of the
cell) and visualised as bright larvae on a dark background. In a side-on view,
bright larvae on the dark background were monitored with a CCD camera (30
frame s-1; Photron FASTCAM-Net, Osaka, Japan) which was rigidly
mounted on the side of a constant-temperature incubator (AG-HC090X, Nihon-ika,
Osaka). The 2-D movement of a larva in time was analysed using modular
software, `Image Tracker PTV' (Digimo Corp., Osaka, Japan).
Photic behaviour of the larvae
The photic behaviour of 100 larvae was analysed by the ExpertVision system
as shown in previous papers (Nakagawa et
al., 1999). Intact control and ablated larvae were transferred to
a transparent plastic container (widthxlengthxdepth, 50
mmx60 mmx15 mm). In order to prevent geotaxis of the larvae, the
depth of the seawater was 5 mm. The swimming behaviour of the larvae was
monitored by non-stimulus far-red illumination (wavelength 680-800 nm with the
combination of cut-off filter, O-68, and IR-cut filter, IRA-20A: Toshiba,
Tokyo, Japan) at 18°C in the constant-temperature incubator. The stimulus
was monochromatic light obtained by the combination of an interference filter
(494 nm with a full width of <18 nm at half-maximum, KL-series: Toshiba,
Tokyo), a UV cut-off filter (L39: Toshiba, Tokyo, Japan) and neutral density
filters (Kenko, Tokyo, Japan) in front of a 300 W slide projector. Delivery of
the light stimulus was controlled by means of an electromagnetic shutter
(C-79-1: Chuo Precision Industrial Co. Ltd., Tokyo, Japan). The shutter was
coupled to the digitising unit of an automated tracing system (Motion Analysis
Corp., Santa Rosa, CA, USA) that controlled the delay between an event marker
used to initiate data acquisition and the delivery of the stimulus.
The motion of the free-swimming larvae was recorded by an
infrared-sensitive CCD camera (XC-77: Sony, Tokyo, Japan) that was connected
to a video processor. Analysis was done using modular software `ExpertVision'
(Motion Analysis Corp., Santa Rosa, CA, USA). The video processor detected
areas of high contrast which, in this case, were bright larvae on a dark
background. The centre of each larva in successive frames was connected into
paths representing the 2-D movement of each individual larva in time. The
linear speed, in mm s-1, was defined as the distance between
consecutive centers in a path divided by the time taken to travel this
distance (Sundberg et al.,
1986; Sager et al.,
1988
).
Swimming behaviour under hydrostatic pressure
The effect of hydrostatic pressure on the swimming behaviour of the larvae
was studied. Larvae collected within 3 h after hatching were transferred to
the optical quartz cell (widthxdepthxlength=10 mmx40
mmx10 mm) connected to a gas pressure apparatus (High Select GH-Type;
Chiyoda Seiki, Tokyo, Japan).
The larvae in the optical quartz cell were illuminated by nonactinic far-red illumination (wavelength 640 nm; 20 far-red photodiodes were placed by side of the cell) and visualized as bright larvae on a dark background. In side-on view, bright larvae against a dark background were monitored with a CCD camera (30 frames s-1; Photron FASTCAM-Net, Osaka, Japan), which was rigidly mounted on the side of a constant-temperature incubator (AG-HC090X, Nihon-ika, Osaka, Japan). The 2-D movement of a larva in time was analysed using modular software `Image Tracker PTV' (Digimo Corp., Osaka, Japan).
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Results |
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To determine whether the anterior pigment and/or posterior pigment cells play a role in this tendency to swim upwards, the swimming behaviours of four groups of larvae (intact, preanesthetized, anterior pigment and posterior pigment cell-ablated) were tested. 1-2 animals from each group were placed on the bottom of an optical quartz cell, and were recorded with a CCD camera from a side window (10 mmx40 mm) as they swam horizontally. They were illuminated by 20 far-red photodiodes placed on each side of the optical cell.
Fig. 1 shows several traces from CCD recordings of the paths taken by the intact larvae within 3 h after hatching. The x, y coordinates of the swimming paths were measured every 20 ms. This allowed us to plot the 2-D swimming paths and the x, y coordinates of the initial (xi, yi) and final (xf, yf) positions of the larvae. Most of the intact larvae showed upward swimming behaviour, but produced different patterns of traces such as straight (Fig. 1A), spiral (Fig. 1B), curved (Fig. 1C) and random (Fig. 1D). Some of the larvae swam diagonally or erratically (Fig. 1D). Swimming directions and patterns of the intact larvae in the xy plane are shown in Fig. 2. Each point in Fig. 2 shows the final position (xf, yf) of the larvae after swimming, where the coordinates of the initial position (xi, yi) of each larva were adjusted to the origin of the coordinate axes (0, 0). The different symbols used for the points in Fig. 2 represent the different patterns of swimming behaviour of the larvae, i.e. straight (open circle), spiral (open triangle), curved (solid circle) and random (solid triangle). These results suggest that most of the larvae initially (within 3 h of hatching) swam upward, though some of them swam diagonally or erratically.
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In order to evaluate any statistical difference between the patterns before and after lesion, the results in Figs 2 and 4 were re-examined. The larvae whose final position was within (shaded area) or beyond 45° of the y-axes in Figs 2 and 4 were counted for each swimming pattern and the probability of each swimming pattern was plotted (Fig. 5). The final position of 77% of the 60 control larvae (A in Fig. 5) was located within 45° of the y-axes. 72% of the 69 posterior pigment cell-ablated larvae also finally located within 45°, as for the intact controls, but only 27% of the 56 anterior pigment cell-ablated larvae located finally within 45° of the y-axes. The majority of the control and the posterior pigment cell-ablated larvae showed a straight or spiral swimming pattern, but those of anterior pigment cell-ablated larvae showed a random swimming pattern. Thus, ablation of the anterior pigment cell greatly reduced upward swimming and increased the extent of random swimming behaviour, but ablation of the posterior pigment cell affected neither swimming direction nor pattern.
These results suggest that the posterior pigment cell is not linked to the upward swimming behaviour.
Photobehaviour
In previous papers (Nakagawa et al.,
1999; Tsuda et al.,
2001
), we showed that larvae of Ciona intestinalis
changed their photic behaviour during the course of development. Newly hatched
larvae showed no response to a light stimulus at any intensity. 4 h after
hatching, larvae were induced to start swimming upon cessation of
illumination, and to stop swimming upon onset of illumination
(Tsuda et al., 2003
). The
maximum speed of swimming increased with time up to 8 h after hatching and
then plateaued.
Fig. 6A shows swimming speeds of intact larvae with time in response to repeated stimuli consisting of the onset (for 6 s periods) and cessation (for 1.5 s periods) of light (494 nm; 5.0x10-3 J m-2 s-1) from the slide projector. Intact larvae started swimming when the light was switched off, reaching a maximum speed at 0.6 s, after which the swimming speed decreased gradually. The swimming speed of the larvae slowed abruptly (after 1.5 s) when the light was switched on. These results show that larvae start swimming in response to the cessation of light and stop swimming with its onset. Fig. 6B shows the photoresponse of the pre-anesthetized larvae using the same photo-stimuli as in Fig. 6A, showing that anesthesia had no effect on the photoresponse of the larvae.
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After being anesthetized, either the anterior pigment or posterior pigment cell in the sensory vesicle was ablated by laser as shown in Fig. 3, and Fig. 6 shows the swimming photoresponse of larvae, anterior pigment (Fig. 6C) and posterior pigment (Fig. 6D) cell-ablated 5 h after hatching. Anterior pigment cell-ablated larvae start swimming in response to the cessation of light and stop swimming with its onset (Fig. 6C), like the intact or anesthetized controls (Fig. 6A,B, respectively). By contrast, the posterior pigment cell-ablated larvae did not show any response to light cessation or onset (Fig. 6D). These results clearly show that the posterior pigment cell, but not the anterior pigment cell, is responsible for the photoresponsive component of swimming behaviour.
Effect of pressure on swimming behaviour
Besides the otolith and the ocellus, the larvae of Ciona
intestinalis possess a third type of presumptive sensory organ. It is
situated in the dorsal posterior wall of the neural vesicle and projects into
its lumen, and consists of numerous globular or ovoid bodies. Its function is
still disputed; according to Dilly
(1962,
1969
) it is a second type of
photoreceptor; Eakin and Kuda
(1971
), on the other hand,
consider it to be a pressure-detection organ. In contrast to the otolith and
ocellus, these globular bodies do not contain pigment cells and the diameter
of the bodies are as small as 2 µm. The larvae of Ciona possess
more than ten such bodies and thus it is very difficult to ablate all of them
by laser. Instead, we investigated the effect of pressure on the swimming
behaviour of the larvae to ascertain whether the pressure organs are involved
in controlling the upward and downward swimming behaviours. Ciona
larvae swim actively in more-or-less regular short bursts, which may last for
several minutes, but usually last 1-5 s or less, with interburst intervals of
5-20 s. Pressure was applied to the optical cell containing the larvae 5 s
after beginning to record the behaviour by CCD camera.
Fig. 7A shows the time profiles
of the swimming speeds of the bursts of 12 individual larvae 2 h after
hatching. The larvae swam actively in more-or-less regular bursts, which
lasted for 1-5s. Application of a hydrostatic pressure of 2 atm (1
atm=1.013x105 Pa) was applied to the quartz optical cell
(Fig. 7B) neither caused
swimming larvae to stop, nor induced stationary larvae to start swimming. The
starting times of the individual bursts after the observation in
Fig. 7B were measured at five
different pressures, from 1.1 to 2.0 atm, corresponding to a sea depth of
approximately 1-10 m (Fig. 7C),
but no larvae were induced to stop or to start swimming. Thus swimming
frequency or swimming period are not affected by pressure, which suggests that
the presumed pressure-detection organ is not involved in the control of larval
swimming behaviour.
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Discussion |
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If this is the case, when the statocyte is removed from the sensory vesicle, the larvae should be unable to recognize the direction of gravity or the direction of the horizontal plane. When the otolith was removed from the sensory vesicle by laser ablation, the larvae lost upward swimming behaviour, as shown in Fig. 4A. One explanation for these results is that without the statocyte body, the otolith lacks a way to deform the dendrites and thus the animal can no longer sense the direction of gravity. Since ascidian larvae do not exhibit a photoresponse at this initial swimming stage, the larvae swim upward by using gravity orientation.
As shown in Fig. 5, the
swimming behaviour of the larvae with or without the otolith differs not only
in the upward component but also in the pattern of swimming. Because of their
placement beside the neck of the statocyte in the intact larvae
(Torrence, 1986; Otsuki,
1991), the two dendrites (anterior and posterior), would be differentially
deformed and comparison of the activities of the two sensory neurons by
ganglion cells could provide the animal with directional information.
The initial period of larval life is characterized by upward swimming.
Larvae do not show a photoresponse for the first 4 h after hatching
(Kajiwara and Yoshida, 1985;
Nakagawa et al., 1999
). As
shown in Fig. 7, pressure
changes between 1.1 and 2 atm did not induce larvae either to start or to stop
swimming. Thus it is concluded that the otolith is solely responsible for the
upward migration of ascidian larvae after hatching.
In the latter half of their life, larvae sink downward, which in nature
presumably aids them in selecting a suitable site for attachment and
metamorphosis. In a previous paper, we showed that larvae of Ciona
show a response to shading 4 h after hatching
(Nakagawa et al., 1999). They
were induced to start swimming upon the cessation of illumination, and to stop
swimming upon the onset of illumination
(Tsuda et al., 2003
). The
photopigment responsible for phototaxis in the ascidian larva is still
controversial. It was shown that the action spectrum of larval photic
behaviour was similar to the absorption spectrum of human rhodopsin
(Nakagawa et al., 1999
;
Tsuda et al., 2001
). The
presence of rhodopsin in the ocellus was shown by a retinal protein imaging
method (Ohkuma and Tsuda,
2000
). Three opsins of Ciona intestinalis have been
cloned and expression patterns in larvae detected by whole-mount in
situ hybridization. Ci-opsin1 mRNA was found only in
photoreceptor cells of the ocellus
(Kusakabe et al., 2001
).
Ci-opsin2 is homologous to Ci-opsin1 and is expressed on the
dorsal side of the sensory vesicle, but not in the ocellus
(Kusakabe et al., 2002
). Since
Ci-opsin3 is a retinal G-protein-coupled-receptor (RGR) homologue and
functions as a retinal photoisomerase
(Nakashima et al., in press
),
it is not responsible for phototaxis. Thus, the two opsins Ci-opsin1 and
Ci-opsin2 are candidates for the visual pigment responsible for phototaxis in
the ascidian larvae.
Electron microscopic examination of the Ciona larvae shows that
the ocellus consists of three parts: the pigment cup, the photoreceptor cells
and the lens cells (Dilly,
1964). A single cup-shaped cell filled with membrane-bound pigment
granules lies between the lens and the retinal cells. The whole structure of
the retinal cells was visualized by antibody against Ci-arrestin
(Nakagawa et al., 2002
;
Horie et al., 2002
). The
pigment granules were arranged to prevent stray light from falling on the
photoreceptor endings. The photoreceptor membranes of the retinal cell were
situated inside the concavity of the pigment cup cells, which were stained by
antibody against Ci-opsin1 (Kusakabe et
al., 2002
).
As shown in Fig. 3D, the laser was focused onto the center of the posterior pigment cell to ablate the photoreceptors in the ocellus. The posterior pigment cell-ablated larvae lost all response to a light stimulus, i.e. both the cessation of light and its onset (Fig. 6D). Since the posterior pigment cell contained only Ci-opsin1, Ci-opsin1 is responsible for the photoresponse during larval swimming.
In addition to the ocellus and the otolith, a third type of presumptive
sensory organ has been described in some species, consisting of globular
bodies with membranous tubules and a ciliary organ. The function of this organ
is unknown. Eakin and Kuda
(1971) proposed that it may
function as a hydrostatic pressure detector; however, the present work shows
that the globular bodies are not involved in the swimming behaviour of the
larvae (Fig. 7).
In conclusion, the functions of two distinct pigment cells in the brain vesicle were determined by laser ablation. The larvae with anterior pigment cells ablated lost the upward swimming behaviour. Thus, the anterior pigment acts as the statocyte body in response to gravity or inertia and the deformation of the dendrites of mechanoreceptors may provide the animal with a means of gravity detection. The larvae with posterior pigment cells ablated lost all response to a light stimulus. Ci-opsin1, the photopigment of the ocellus, is responsible for the photoresponse during larval swimming. Since swimming behaviour is not affected by pressure, the reason for the presence of any pressure detector remains uncertain.
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Acknowledgments |
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