Direct evidence for the role of pigment cells in the brain of ascidian larvae by laser ablation
Motoyuki Tsuda*,
Daisuke Sakurai and
Muneki Goda
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
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Fig. 1. Swimming paths of intact larvae collected within 3 h of hatching as
measured by a CCD camera. The initial position of the larva is marked by a
closed circle and the final position by an open circle. Most of the intact
larvae swam upward, but followed different paths that were characterized as
straight (A), spiral (B), curved (C) and random (D).
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Fig. 2. Swimming direction and pattern of 60 intact larvae in the
x—y plane. Each point in the x—y plane shows the
final position (xf, yf) of the intact
larvae after swimming from the initial position at the origin (0, 0).
Different symbols denote the different patterns of swimming exhibited by the
larvae, i.e. straight (open circle), spiral (open triangle), curved (solid
circle) and random (solid triangle). Most of the intact larvae collected
within 3h of hatching swam upward. The shaded area represents 45° from the
y axis (see Fig.
5).
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Fig. 3. Laser ablation to the anterior and posterior pigment cells in the sensory
vesicle of the larvae. (A) Trunk of the larva, showing the anterior (otolith)
and posterior (ocellus) pigment cells. (B) When the stalk of the otolith
(arrowhead) is ablated by laser, the anterior pigment cell (C; arrow) is
detached from the wall in the lumen of the sensory vesicle and is thus
defocused. (D) The laser is focused onto the center of the posterior pigment
cell (arrowhead), making a hole was made in the center of the posterior
pigment cell (arrowhead) (E). Bars, 50 µm (A); 20 µm (B—D).
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Fig. 4. Swimming directions and patterns of larvae with anterior (A) and posterior
(B) pigment cells ablated in the x-y plane. (A) The ablation of the
anterior pigment cell greatly reduced upward swimming (N=69). (B)
Upward swimming of the larvae with posterior pigment cells ablated was not
affected (N=56). Both pigment cell-ablated larvae followed similar
swimming paths; straight (open circle), spiral (open triangle), curved (solid
circle) and random (solid triangle). The shaded area represents 45° from
the y axis (see Fig.
5).
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Fig. 5. Statistical difference in swimming behaviour of larvae before and after
lesion. The number of the larvae whose final position was located within
45° of the y-axis in Figs
2 and
4 (shaded areas) was counted
for each swimming pattern and the percentage of each swimming pattern was
plotted. (A) The 60 control larvae. (B) The 56 larvae with anterior pigment
cells ablated. (C) The 69 larvae with posterior pigment cells ablated.
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Fig. 6. Swimming speeds of the intact larvae (A), larvae that had recovered from
anesthetia (B), anterior- (C) and posterior- (D) pigment cell-ablated larvae
in response to repeated stimuli consisting of the onset (6 s light period) and
cessation (1.5 s dark period) of light (494 nm; 5.0x10-3 J
m-2 s-1). Intact larvae (A) started swimming when the
light was switched off, and slowed swimming speed when the light was switched
on. Pre-anesthetized (B) and the anterior pigment cell-ablated larvae (C)
showed the same photoresponse as intact larvae. Posterior pigment cell-ablated
larvae (D) showed no photoresponse whether the light was switched on or
off.
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Fig. 7. Effect of hydrostatic pressure on swimming behaviour. (A) Swimming speed of
individual larvae within 3 h of hatching at 2 atm pressure. (B) Time course of
the hydrostatic pressure, applied 5 s after beginning behavioural measurements
(arrow). (C) The time at which larvae began to swim at different pressure
levels (1.1 to 2 atm). Swimming was not correlated with application of
hydrostatic pressure.
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© The Company of Biologists Ltd 2003
