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First published online November 17, 2006
Journal of Experimental Biology 209, 4676-4689 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02556
Neural control of the velum in larvae of the gastropod, Ilyanassa obsoleta
Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada
Author for correspondence (e-mail:
Roger.Croll{at}dal.ca)
Accepted 20 September 2006
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Key words: serotonin, dopamine, norepinephrine, monoamines, FMRFamide, Leu-enkephalin, neuropeptides, cilia, veliger, muscle, mollusc
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;
Kandel, 1976), but such work
has focused almost exclusively upon adults with little attention paid to the
larvae. And yet, examination of larval behaviour and its neural control would
provide insight into how these organisms survive this critical life stage and
further our understanding of the development and evolution of the molluscan
nervous system.
Molluscan larvae typically use a velum, which is rimmed by one or two bands
of cilia, to swim and feed (Fretter,
1967; Mackie et al.,
1976). The longer, pre-oral cilia are located on the outer edge of
this organ and generate the propulsive forces necessary for swimming. Shorter,
post-oral cilia, when present, are located on a nearby parallel ridge and
collect food particles downstream from the pre-oral band. Separating the two
bands is a food groove, in which the combined currents of ciliary activity
entrap and transport food particles to the mouth
(Chia and Buckland-Nicks,
1984; Strathmann and Leise,
1979).
The basic anatomy and function of the velum have been well established, but
little is known about neural control over this organ, although velar
innervation has long been known to exist. For example, Carter
(Carter, 1928) first provided
histological evidence for neural innervation of the velum in larval nudibranch
molluscs. More recently, Mackie et al.
(Mackie et al., 1976) and
Marois and Carew (Marois and Carew,
1997b) used electron microscopy to confirm innervation of pre-oral
cells in Mangelia nebula and Aplysia californica, and in the
latter species, demonstrated that serotonergic fibres contributed to such
innervation. In fact, immunocytochemical studies have now demonstrated
serotonergic innervation of the vela of various gastropods
(Dickinson et al., 2000;
Dickinson et al., 1999;
Kempf et al., 1997;
Page and Parries, 2000) and
bivalves (Croll et al., 1997;
Plummer, 2002), although the
cell types of the targets are generally unknown. Finally, physiological
evidence also exists suggesting that momentary arrests of pre-oral cilia on
the velum might be elicited by neural input
(Arkett, 1987;
Leise and Hadfield, 2000).
Serotonergic control of ciliary beating in developing molluscs has been
best studied in the fresh water gastropod Helisoma trivolvis, which,
like other pulmonates, develops in ovo and has a greatly reduced
velum and larval nervous system (Croll,
2000; Croll and Dickinson,
2004). H. trivolvis uses a ciliated foot to rotate within
the egg capsule to enhance aeration of the capsular fluid
(Diefenbach et al., 1991;
Marois and Croll, 1991). A
putative sensorimotor neuron detects intracapsular hypoxia and stimulates
ciliary beating via local release of serotonin
(Kuang et al., 2002). Although
these findings may be generally applicable to closely related species
(Uhler et al., 2000),
questions arise about whether direct-developing pulmonate gastropods are
representative models for understanding the control of ciliary activity on
vela of free-living molluscan larvae. In particular, ciliated cells on the
foot of H. trivolvis are only known to possess serotonergic
innervation whereas serotonergic, catecholaminergic and peptidergic axons
extend into the vela of larval caenogastropods
(Dickinson and Croll, 2003;
Dickinson et al., 1999;
Page and Parries, 2000),
heterobranch gastropods (Dickinson et al.,
2000; Kempf et al.,
1997) and bivalves (Croll et
al., 1997; Plummer,
2002).
Questions also arise regarding the neural control of the larval retractor
muscle, which extends bilaterally into the velum. Large contractions of this
muscle occur either spontaneously or following collisions with the surface or
other objects and result in complete retraction of the velum into the shell
(Fretter, 1967). Subtle
changes in the tone of this muscle could also reposition the velum and affect
the direction of swimming. Neural innervation of the velar musculature has
been described in developing caenogastropods
(Dickinson and Croll, 2003;
Page and Parries, 2000) and
heterobranchs (Kempf et al.,
1997), but it remains unclear how this innervation contributes to
maintenance and control of muscle activity.
In the present study we characterized the neural control of the velum and
ultimately the behaviour of the developing caenogastropod, Ilyanassa
obsoleta. We first used immunocytochemistry to examine neurotransmitters
previously identified in larval I. obsoleta
(Dickinson and Croll, 2003).
We then tested the hypotheses that these putative neurotransmitters affect the
activity of cilia and muscles in the isolated velum and the behaviour of
intact larvae. Finally, we also tested the hypothesis that monoamines are
released endogenously to regulate normal swimming behaviour.
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). Briefly, egg capsules were gathered from aquaria containing
adults collected from the wild and were kept in 1 l containers of aerated,
filtered (0.22 m pore size) seawater (FSW; from the Dalhousie University
Aquatron System). Upon hatching, larvae were transferred to culture systems
similar to those described previously
(Miller and Hadfield, 1986)
and fed daily with Isochrysis galabana (Clone T.ISO; Provasolin
Guillard Center for Culture of Marine Phytoplankton, West Boothbay Harbor, ME,
USA) at a final concentration of 
15 000-20 000 cells ml-1. All
larvae used for experiments described below were 7-10 days post-hatching.
Morphology
Immunocytochemistry
Immunolabelling followed procedures outlined previously
(Dickinson and Croll, 2003).
Larvae were anaesthetized using a 7.0% solution of MgCl2 for
several minutes until fully extended from their shells. Specimens labelled
with polyclonal anti-FMRFamide, anti-serotonin (ImmunoStar, Inc., Hudson, WI,
USA), or anti-Leu-enkephalin (Chemicon International, Inc., Temecula, CA, USA)
antibodies or a monoclonal anti-alpha-tubulin antibody (DM1A clone; Sigma
Chemical Co., Mississauga, ON, Canada) were fixed in 4% paraformaldehyde (PFA)
in phosphate-buffered saline (PBS; 50 mmol l-1
Na2HPO4 and 140 mmol l-1 NaCl, adjusted to pH
7.2) for 10-30 min at room temperature (22-24°C). For immunolabelling with
a monoclonal tyrosine hydroxylase (TH) antibody (ImmunoStar, Inc., Hudson, WI,
USA), larvae were fixed in 100% methanol for 10-30 min at -20°C.
After fixation, shells were decalcified for 12-24 h with a solution of 80% 0.23 mol l-1 ethylenediaminetetraacetic acid (EDTA) and 20% 0.1 mol l-1 sodium acetate and specimens were then permeabilized with 4% Triton X-100 in PBS overnight at 4°C. Tissues were next incubated for 3-7 days (4°C) in one of the primary antibodies listed above, diluted 1:500-1:1000 in PBS with 1.0% goat or sheep serum and 1.0% Triton X-100. (No significant differences were noted in staining over these dilutions and incubation times.) This was followed by 1-2 days incubation in secondary antibodies: goat anti-rabbit (for polyclonal primary antibodies) or sheep anti-mouse (for monoclonal primary antibodies) conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA), FITC or rhodamine (Bio/Can Scientific, Mississauga, ON, Canada) and diluted 1:50 in PBS.
Muscle labelling
Some larvae fixed in 4% PFA and labelled with anti-5-HT, Leu-enkephalin or
FMRFamide were double labelled with phalloidin (which marks F-actin) as
described (Degnan et al.,
1997). Briefly, after incubation in secondary antibodies
conjugated with FITC or Alexa Fluor 488 and subsequent washing, specimens were
incubated in a 1:100 dilution of phalloidin (labelled with
tetramethlyrhodamine B isothiocyanate; TRITC; Sigma Chemical Co.) for 1-4 h.
The alcoholic fixation needed for localization of TH-like immunoreactivity was
incompatible with subsequent F-actin staining.
Mounting and viewing
Specimens were mounted in glycerol (3:1) in 0.1 mol l-1 Tris
buffer (pH 8.0) with 2% n-propyl gallate added to prevent fading
(Longin et al., 1993) and then
viewed with a Zeiss LSM 510 confocal microscope. Images were created by
superimposing stacks of 10-80 images obtained through stepped sequences of
focal planes at 0.10-0.80 µm intervals. Projections were created with Zeiss
LSM 510 software. Images were assembled into plates and labelled using
Photoshop 7.0 (Adobe Systems, Inc., San Jose, CA, USA).
Controls
As negative controls, larvae were processed without incubation in primary
antibodies; such specimens exhibited no detectable fluorescence.
Pre-absorption controls were also performed for anti-FMRFamide and
anti-Leu-enkephalin. Synthetic FMRFamide and Leu-enkephalin (Sigma Chemical
Co.) were added at a concentration of 200 µg ml-1 to the 1:500
dilutions of their respective antibodies. The antibodies were pre-absorbed for
24 h at 4°C and then spun for 10 min at 5000 r.p.m. in a bench-top
centrifuge. Larvae were incubated in the supernatant and processed as
described above. These specimens did not exhibit immunoreactivity. Positive
controls included parallel processing of embryonic and larval Lymnaea
stagnalis and Aplysia californica with known labelling patterns
(Croll and Voronezhskaya,
1995; Marois and Carew,
1997a; Marois and Croll,
1992).
Photodiode recordings of ciliary beating in isolated velar lobes
Preparation of the isolated velum
Larvae were anaesthetized with 7% MgCl2 and fully extended vela
were transected lateral to the eyespots, hence peripheral to the apical organ
and developing cerebral ganglia. This produced single isolated velar lobes on
which the cilia continued to beat. Pieces of velum were next pipetted through
three washes of fresh FSW and then suspended in 1 ml FSW within a silicone
compartment adhered to a glass slide. A glass pipette, pulled to a fine tip
and then broken and polished to yield a lumen of 20-30 µm, was mounted on
polyethylene tubing embedded in the wall of the chamber and connected to a
syringe. Vela were positioned near the tip of the pipette, and application of
light suction immobilized the specimens. Such vela were left to acclimatize
for 2 min during which FSW was continuously replaced (2 ml
min-1).
Drug administration
Drug perfusions of 10 ml were used because preliminary experiments
indicated that this volume completely washed a dye solution from the chamber.
Drugs were introduced in ascending order of concentration from 10-9
mol l-1 to 10-8 mol l-1 at intervals of at
least 2 min after cilia resumed their normal beat patterns following washouts
of approximately 30 ml FSW. For higher drug concentrations different specimens
were used for each trial. Six specimens were tested at each of four
concentrations (10-6-10-9 mol l-1) for each
drug (see Table 1), all at room
temperature (22-24°C).
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Photoelectric recordings
Immobilized vela were observed with a 40x Zeiss
long-working-distance, fluid-immersion objective. Beating cilia could be
viewed through the oculars while images were also projected onto a white
screen via the camera port of a Leitz Aristoplan microscope in a
darkened room. Such images were positioned over a small hole (diameter 1 mm)
in the screen. A photodiode mounted behind the hole recorded changes in light
intensity caused by the shadows of beating cilia. The roughly sinusoidal
output was displayed on an oscilloscope (Tektronix, Beaverton, OR, USA) and
further downloaded to a computer through Digidata 1320A software in
conjunction with Axoscope 8 (both from Axon Instruments, Inc., Union City, CA,
USA). Shadows caused upward deflections on the traces, in which height
corresponded to the intensity, and width to the duration, of the shadowing.
Data acquisition was started approximately 2 min after completion of drug
perfusions and continued for 3 min per trial. Calculations of ciliary beat
frequency (CBF) were based upon time intervals required for eight maximal
downward deflections during a regular beating pattern (i.e. without ciliary
arrests; see below).
Comparisons between experimental groups were performed through standard one-way analyses of variance (ANOVA) and Dunnett's tests for multiple pairwise comparisons (P<0.001). All statistics were performed with SPSS (Chicago, IL, USA) statistical software. Oscilloscope traces selected for presentation were filtered (lowpass filter: 20-30 Hz) with Clampfit 8.0, and assembled using Photoshop 7.0 (Adobe, San Jose, CA, USA). Schematic diagrams were constructed using Corel Draw 11 (Corel Corp. Ltd., Ottawa, ON, Canada).
Photoelectric measurement of post-oral ciliary beating frequency
Activity of post-oral cilia was assessed with similar recording procedures.
However, these cilia could not be monitored when the pre-oral cilia were
beating, since the larger cilia obscured their smaller neighbours. We
therefore measured activity of the post-oral cilia only during drug-induced
arrest of the pre-oral cilia (see below).
Measurements of muscular contractions and ciliary arrests
Temporary cessations of regular ciliary beating were occasionally noted in
the photoelectric recordings. Simultaneous viewing of the velum through
microscope oculars permitted further description and counting of these ciliary
arrests. A computerized event marker was used to record the occurrence of such
events before and during applications of different compounds. Observations
were made of four specimens at each drug concentration for 1-2 min during
measurement of ciliary beating. Differences in frequency were analyzed through
one-way analyses of variance (ANOVA) and Dunnett's tests for multiple pairwise
comparisons (P<0.05).
Behavioural tests on whole larvae
Vertical distribution test
Approximately 50 larvae were placed into vertical polystyrene pipettes (30
cmx1 cm outer diameter), containing 10 ml of seawater and one
neuroactive chemical (Table 1).
After 30 min, each column was drained into two fractions: the bottom
versus the top of the column. (Preliminary experiments indicated that
monoamines were effective at 10-4-10-6 mol
l-1 concentrations in whole larvae.) The larvae in each fraction
were then counted. A total of eight trials were performed for each chemical at
each concentration. Neuropeptides were not tested in this experiment since
preliminary studies indicated that they did not retain their effectiveness for
the 30 min duration of the experiment.
Feeding rates
A matched pair design was used to calculate relative feeding rates of
larvae (equation 1) when exposed to the various chemicals
(Table 1). Tests were performed
in 50 ml plastic tissue culture flasks (VWR, Canlab, Mississauga, ON, Canada)
containing Isochrysis galbana (Clone T.ISO) in seawater at
concentrations generally ranging between 100 000 and 200 000 cells
ml-1 at which larvae ingest at relatively constant rates
(Dickinson, 2002). Each trial
(N=8 per drug), consisted of a pair of flasks with one containing
algae and the chemical tested but no larvae (A in equation) and the other
containing equal concentrations of the algae and chemical plus 25 larvae (B in
equation). After each trial, flasks were sampled (1 ml) and the algae killed
by adding 20 µ1 of 4% PFA. The algal concentrations were determined by
standard counting methods using a Brightline haemocytometer (VWR, Canlab).
Additional controls, serving to normalize the data, consisted of averaged
algal counts for flasks containing algae plus larvae without any chemical (D
in equation), or alternatively algae alone (C in equation). Neuropeptides were
not tested in this experiment since their effectiveness did not last the
length of the experiment.
 | (1) |
where A is the number of cells ml-1 (algae + chemical), B is the number of cells ml-1 (algae + chemical + larvae), C is the average number of cells ml-1 (algae) and D is the average number of cells ml-1 (algae + larvae).
Frequency of locomotory arrests: ciliary arrest and velar contractions
The frequency of locomotory arrests and effects of drugs
(Table 1) on this behaviour
were counted over 5 min periods with intact larvae swimming freely in small
depression slides (2 cm diameter). This arrangement permitted continuous
monitoring of the animals at low magnification (15-30x), but did not
permit discrimination between isolated ciliary arrests and subtle contractile
arrests. We therefore pooled all visible arrests into a single count. A total
of 10 larvae were tested for each chemical.
Statistics
Data were subjected to tests for normality and a one-way analysis of
variance (ANOVA). Alternatively, for those trials that failed to show normal
distribution a Kruskal-Wallis one-way ANOVA on ranks was conducted. Dunnett's
tests for multiple pairwise comparisons were used to identify drug trials that
differed significantly from the controls (P<0.05).
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).
Fibres containing serotonin (Fig.
1C), FMRFamide and Leu-enkephalin
(Fig. 1D) possessed numerous
varicosities along the basal edges of the pre- and post-oral ciliated cells
(also see Fig. 2) and along the
velar musculature (serotonin, Fig.
2A; FMRFamide, Fig.
2B,C; Leu-enkephalin, Fig.
2D), which originated in the main retractor muscles and formed
attachments near the basal edges of the pre-oral cells. Additional bands of
muscle fibres ran parallel to the food groove along the basal edges of
pre-oral cells. TH-immunoreactive cells were located along the posterior edge
of the food groove close to the post-oral ciliated cells
(Fig. 2E). Processes from
TH-immunoreactive cells ran along the basal edges of the post-oral ciliated
cells and also extended across the food groove to the basal edges of the
pre-oral cells (Fig. 2F).
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Serotonin applied to the isolated velum significantly increased CBF in a dose-dependent manner (Fig. 4) for all concentrations tested (P<0.001). Notably, when administered at 10-6 mol l-1, serotonin led to an approximately threefold increase in ciliary beating (Fig. 3B). Analyses of photodiode recordings with increased temporal resolution (Fig. 5) demonstrated that the effects of serotonin on CBF were accompanied by graded changes in the beat pattern. Thus, with increasing concentrations, phase 1 remained clearly recognizable, while phases 2 and 3 became less distinct, thereby suggesting possible changes in the stiffness of the cilia, their stroke angles or their positions.
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By contrast, catecholamines caused significant dose-dependent decreases in CBF (Fig. 4). Application of dopamine at 10-6 mol l-1 caused complete arrest of the pre-oral cilia in five of six specimens (see below). Perfusions of dopamine at lower concentrations (10-7-10-9 mol l-1) did not halt ciliary activity but led to significant dose-dependent decreases of CBF in all groups (P<0.001). Photodiode recordings obtained during dopamine perfusions suggest that the three-phase cycling of ciliary activity was largely preserved (Fig. 3C), although there were several irregular peaks on the traces. Norepinephrine also caused slowing of CBF (Fig. 4), although it was less effective than dopamine in reducing ciliary beating, and did not cause complete, sustained ciliary arrests at the highest concentration administered.
Neuropeptides did not significantly alter the CBF of pre-oral cilia at any of the concentrations tested (Fig. 4). However, application of both FMRFamide (Fig. 3D) and Leu-enkephalin (data not shown) markedly changed the three-phase waveform normally recorded in control conditions; recordings obtained from these experimental groups generally had single, broad, upward deflections that were flanked by irregular smaller peaks.
Post-oral ciliary beat frequency
When pre-oral cilia were beating, they obscured the activity of the smaller
post-oral cilia, thereby preventing the measurement of their CBF. During
dopamine-induced arrests of the pre-oral cilia (10-6 mol
l-1 concentration), however, we observed that the post-oral cilia
continued to beat (Fig. 6) at
an average CBF of 13.34 Hz (±2.11 Hz; N=4), which was
significantly faster than the pooled normal CBF of the pre-oral cilia
(P<0.001). Recordings of post-oral ciliary activity did not yield
the clear patterns of multiphasic cycles of the pre-oral cilia. Rather, each
cycle was generally characterized by a single large upward and a variable (one
to two) number of smaller deflections.
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In control specimens, contractile arrests also occurred at a low mean frequency of 1.50±1.29 contractions m-1 (Fig. 9). Serotonin, dopamine and norepinephrine had no significant effects on the frequency of muscular contractions compared to the control level. A striking effect, however, was observed after application of FMRFamide, which caused much more frequent contractile arrests at all concentrations tested (Fig. 9). At the highest FMRFamide dose administered, the contractions accompanying the arrests were more vigorous and occurred in immediate succession to one another, thus causing the velum to twitch constantly for sustained periods. During such periods, the pre-oral cilia remained inactive and were curled onto the velum. Leu-enkephalin had a similar effect to FMRFamide in enhancing the number of contractile arrests (Fig. 9).
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In contrast to serotonin, both dopamine and norepinephrine significantly decreased the percentage of larvae in the top half of the water column in dose-dependent manners (Fig. 10). Spiperone, conversely, resulted in a significant increase in the percentage of larvae in the top half of the column. However, alprenolol induced a significant decrease in the percentage of larvae in the top half of the water column whereas haloperidol had no significant effect.
Feeding rate
An overall significant difference in feeding rates was observed following
exposure of larvae to transmitters (Fig.
11; P<0.001). When larvae were exposed to
10-6 mol l-1 and 10-5 mol l-1
serotonin, the feeding rates increased significantly. Exposure to
10-4 mol l-1 serotonin resulted in no significant
differences from control levels but again a number of larvae were observed to
be lying on the bottom of the test chamber at this concentration. Applications
of both dopamine and norepinephrine decreased feeding rates in dose-dependent
manners from 10-6 mol l-1 to 10-4 mol
l-1 concentrations.
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Locomotor arrests
Applications of neurotransmitters resulted in an overall significant
difference in the average number of locomotor arrests
(Fig. 12;
P<0.001). The control larvae produced approximately 0.87
(±0.99) arrests m-1. Both serotonin and dopamine
significantly increased the average frequency of locomotor arrests at
concentrations of only 10-5 mol l-1. At 10-4
mol l-1, both serotonin and dopamine immobilized many larvae,
however, in serotonin they were contracted into their shells whereas in
dopamine they appeared to be flaccid and extended. Norepinephrine at a
concentration of 10-4 mol l-1 also significantly
increased the average frequency of locomotor arrests. Both FMRFamide and
Leu-enkephalin caused significant dose-dependent increases in the frequency of
arrests with 40- to 50-fold increases over control values at the highest
concentrations.
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Serotonin and catecholamine regulation of pre-oral cilia
Serotonergic axons were localized throughout the velum and particularly
along the bases of the pre-oral cells, and serotonin caused dose-dependent
increases in CBF on the isolated velum. Serotonin also increased swimming and
feeding in whole larvae, thus reflecting the heightened ciliary activity seen
in vitro. These results are largely consistent with previous
research, which has shown that serotonin stimulated ciliary beating in adult
(Aiello, 1990;
Audesirk et al., 1979;
Cadet, 2004;
Catapane, 1983) and developing
molluscs (Kuang et al., 2002;
Uhler et al., 2000) and in
animals from other phyla (Wada et al.,
1997).
We also provide evidence that catecholamines decrease ciliary activity and thus have opposite effects to serotonin. Catecholamine-containing neurones are located near the post-oral cells, but fibres arising from these cells appear to contact the pre-oral cells. Dopamine and norepinephrine had dose-dependent actions leading to a gradual decline and ultimately arrest of pre-oral ciliary beating on the isolated velum. Similarly, application of these transmitters depressed swimming and feeding in intact larvae (see below).
There is ample anatomical evidence, to suggest that catecholaminergic
innervation of ciliated cells is widespread in adult
(Cadet, 2004;
Cain and Woodward, 2002) and
developing (Croll et al.,
1997; Dickinson et al.,
1999; Plummer,
2002; Voronezhskaya et al.,
1999) molluscs. The identity of endogenous catecholamine(s),
however, generally remains unclear. Here we used immunocytochemistry to detect
tyrosine hydroxylase, an enzyme involved in the synthesis of both dopamine and
norepinephrine. Chemical analyses of adult and larval molluscs suggested that
dopamine is the most abundant catecholamine
(Cann-Moisan et al., 2002;
McCaman, 1984;
Pani and Croll, 1995;
Pires et al., 2000a), in
accordance with descriptions of various actions for dopamine in molluscs
(Ascher, 1972;
Swann et al., 1982a;
Swann et al., 1982b;
Swann et al., 1982c). However,
many studies (see above) have also detected significant concentrations of
norepinephrine (see also Pires et al.,
2000b), while yet other evidence suggests possible roles for
norepinephrine in modulating larval behaviours
(Coon et al., 1985;
Pechenik et al., 2002).
Clearly, further work must determine the specific roles for dopamine and
norepinephrine in the larval nervous systems of molluscs.
Possible mechanisms for aminergic effects
The cellular actions of serotonin and catecholamines are not always clearly
understood in other populations of ciliated cells, although serotonergic
enhancement of ciliary beating generally appears to be mediated by an increase
of intracellular calcium and activation of downstream messengers (e.g. nitric
oxide) in molluscs (Cole et al.,
2002; Doran et al.,
2003) and mammals (Gertsberg
et al., 2004). In fact, nitric oxide synthase has been identified
in I. obsoleta veligers and might therefore play a role in modulation
of larval behaviours (Thavaradhara and
Leise, 2001). The cellular mechanisms that underlie the actions of
catecholamines on ciliated cells are also relatively unknown, but appear to be
more complicated than those underlying serotonergic effects. Both excitation
and inhibition of ciliated cells have been suggested
(Beiras and Widdows, 1995;
Cain and Woodward, 2002;
Malanga, 1975). Furthermore,
both central (Cadet, 2004) and
peripheral (Paparo and Murphy,
1975) mechanisms have been postulated to account for the effects
of catecholamines. For example, centrally acting dopamine depresses beating of
the lateral cilia in M. edulis, possibly through inhibition of
peripheral serotonin release (Cadet,
2004). However, it was shown in this same species that local
catecholamines could also inhibit ciliary beating, possibly by increasing
intracellular calcium within the ciliated cells
(Paparo and Murphy, 1975), and
may resemble calcium dependent ciliary arrests in other molluscs
(Arkett et al., 1987;
Stommel and Stephens, 1985).
Thus, paradoxically, both serotonergic excitation and catecholaminegic
inhibition may initially arise from increases in intracellular calcium but
modulatory pathways diverge thereafter.
Regulation of pre-oral ciliary beat waveform
Although serotonin and catecholamines clearly affect CBF, changes in
waveforms in the photodiode recordings suggest other possible actions as well.
One limitation of photoelectric recordings is that they most clearly represent
the movement of cilia along a single plane. However, cilia normally move in
three dimensions during a single beat cycle
(Blake and Sleigh, 1974).
Moreover, dopamine has previously been shown to affect ciliary beat plane in
sea urchin larvae (Wada et al.,
1997). The changes in waveform that we recorded following
administration of neurotransmitters may thus represent changes in beat plane
or stiffness of the cilia. Such changes in waveform were particularly evident
following administration of FMRFamide and Leu-enkephalin, which had no effect
on CBF. Thus, while various neuropeptides have previously been shown to affect
CBF in adult T. diomedea (Willows
et al., 1997) and also in the lateral gill cilia of the clam
Mercenaria mercenaria (Gainey et
al., 1999), our results suggest that such substances may also
change the plane of ciliary beating. Clearly, high speed video microscopy will
be a necessary component of future studies aimed at understanding the full
spectrum of effects that neurotransmitters may induce upon ciliary
beating.
Neural control over post-oral cilia
Our data suggest that the pre-oral and post-oral cilia may be affected
differently by neural input. For example, whereas both pre-oral and post-oral
cilia stop during muscular contractions, only the pre-oral cilia stop during
spontaneous, isolated arrests or in isolated arrests following applications of
catecholamines. Also, pre-oral cilia were completely immobilized by dopamine
at high concentrations, but post-oral ciliary beat at speeds that exceeded
normal pre-oral ciliary beating. This suggests that pre-oral ciliary cells are
inhibited by catecholamines, but post-oral ciliated cells may be unresponsive
to dopamine or may in fact respond to catecholamines with an increase in
ciliary beating, although this hypothesis must be tested by observations of
post-oral CBF under normal conditions. Similar variations in the
responsiveness of different populations of ciliated cells have been reported
for serotonin in H. trivolvis
(Doran et al., 2004) and
neuropeptides and dopamine in the clam Mercenaria mercenaria
(Gainey et al., 1999),
suggesting that a different catecholamine receptor or even a lack thereof on
the post-oral ciliated cells is plausible.
Neural control over the velar musculature
The cilia on the velum play essential roles in locomotion and feeding. The
velar musculature also serves important functions in the generation of such
behaviours (Fretter, 1967).
Our anatomical data indicated that serotonergic and peptidergic fibres are
abundant along muscles, which both radiate into the velum and form a
circumferential band near its outer rim. Catecholamine-containing axons, in
contrast, are sparse along the radial muscle fibres although they are present
near circumferential muscle.
Consistent with numerous previous reports of peptidergic effects on
molluscan muscles (Brezden et al.,
1999; Hernadi et al.,
1998), we found that both FMRFamide and Leu-enkaphalin induced
vigorous muscular contractions at all concentrations administered. Such
contractions were also often correlated with ciliary arrests suggesting that
these two actions are coupled, possibly via the peptidergic neural
elements. Finally, in addition to eliciting repeated short contractions in the
velar muscles, tonic contractions of muscles may also be commanded by the
larval nervous system to modulate the angle of ciliary beating, which
ultimately may account for both changes in the waveforms of ciliary activity
(see above) and for changes in direction of swimming of the whole larvae (see
below).
The lack of effects of serotonin on musculature in the present study may be
explained in view of known effects of this amine on other molluscan muscles.
For example, in buccal muscles of adult Aplysia californica,
application of serotonin does not cause contractions but rather enhances
subsequent contractions elicited by other inputs
(Fox and Lloyd, 2002;
Hurwitz et al., 2000;
Kupfermann and Weiss, 1982).
It will therefore be interesting to observe interactions between synaptic
inputs to the velar muscles in future research.
Neurotransmitters and analogues affect whole larval behaviour
Administration of monoamines and neuropeptides to whole larvae elicited
changes in behaviour which were largely consistent with those expected from
observed effects on cilia and muscles in isolated vela; however, such effects
were only seen at higher concentrations, presumably because of restricted
access to internal receptors. The pre-oral cilia provide the propulsive force
used by the larvae to swim upward against the pull of gravity, and thus, one
would expect that increased ciliary beating caused by serotonin would result
in net upward movement in a water column as observed in the vertical test.
Conversely, decreased ciliary beating and heightened levels of ciliary arrests
caused by both dopamine and norepinephrine would be predicted to result in net
downward movement of larvae within the column.
Ciliary beating also underlies feeding, and our behavioural observations are again consistent with predictions derived from findings in the isolated velum. Thus as expected, serotonin increased feeding rates whereas catecholamines decreased algal intake. The highest concentrations of serotonin and dopamine, however, also appeared to have non-specific effects, thus confounding the results.
In addition to supporting our observations of the effects of neurotransmitters on cilia on isolated vela, these behavioural studies also permitted tests of the hypothesis that monoamines are released endogenously in whole larvae to tonically regulate ciliary beating. For example, fluoxetine, a selective serotonin re-uptake inhibitor and mianserin, a serotonin antagonist, both known to be effective in molluscs (see Table 1), would only be expected to affect swimming in the specific inverse manners observed if serotonin was being released endogenously. By contrast, the catecholamine antagonist spiperone, suggested to be a nonspecific antagonist of catcholaminergic pathways in molluscs (see Table 1), caused a net upward movement of larvae in the water column, as would be the predicted outcome from inhibiting tonically released catecholamines. However, alprenolol (see Table 1) caused net downward movement in the column, which is opposite to the hypothesized effect, whereas haloperidol (see Table 1) had no significant effect at all. Clearly, future studies would be aided by a more extensive pharmacological characterization of the catecholaminergic receptors involved in controlling ciliary beating in the larvae.
In a final set of behavioural experiments, we demonstrated that locomotor arrests in freely swimming larvae are affected by neuropeptides. As expected from findings in the isolated velum, applications of both FMRFamide and Leu-enkephalin led to large, dose-dependent increases in the number of locomotor arrests displayed by intact larvae. Smaller increases in the frequency of arrests elicited by catecholamines were also expected from our observations of isolated vela, but brief arrests of only the pre-oral cilia may have been overlooked in our low magnification observations of swimming larvae. Flaccid paralysis of larvae exposed to the highest concentration of dopamine also confounded these results. Thus, only the slight increase in the frequency of arrests elicited by serotonin was unexpected from our findings of the effects of transmitters on isolated vela. We suggest that this increased frequency of arrests may have been the result of increased numbers of collisions by more rapidly swimming larvae with the water surface and small suspended objects, and therefore not an accurate indicator of the rate of spontaneous arrests. More detailed observations of either free-swimming larvae in larger containers or tethered larvae to prevent such collisions, should be a goal of future studies.
Conclusions
Data from the present study offer new insight into neural control
mechanisms over the cilia and muscles of the velum, which together mediate
major larval behaviours of free-living veligers, such as I. obsoleta.
The findings suggest that neural control over the velum is more complex than
might have previously been expected for such a simple organism, and in some
regards it is reminiscent of autonomic control in vertebrates, with effectors
often innervated by dual, antagonistic controls and where blocking receptors
of one division reveals its significant, ongoing tonus
(Nilsson and Holmgren,
1994).
We specifically investigated regulation of the CBF and ciliary arrest rates of the pre-oral cilia and provided data concerning the CBF of the post-oral cilia. We also provided evidence of neural regulation of an intricate network of velar muscles. Together these effectors are responsible for generating larval swimming and feeding, which we showed to be influenced by neurotransmitter analogues in whole larvae, thereby confirming the endogenous roles of the identified neural substrates.
Clearly, much more work is required to understand neural control of larval
behaviour in gastropods. Electrophysiological studies are particularly needed
since only a very small number of studies have been published to date using
this approach (Arkett et al.,
1989; Arkett et al.,
1987; Leise and Hadfield,
2000). Furthermore, the larval nervous system of I.
obsoleta probably contains other transmitters, [as suggested by Dickinson
and Croll (Dickinson and Croll,
2003)], which potentially mediate additional controls over velar
effectors than those examined in this initial survey. Nonetheless, the present
paper, together with previous detailed studies of larval neuroanatomy
(Dickinson and Croll, 2003;
Lin and Leise, 1996)
demonstrate that I. obsoleta may prove to be a valuable system for
basic understanding of how neurons regulate behaviours in such planktotropic
larvae. [But see for preliminary results
(Pires and Penniman, 2003)
using another model species, Crepidula fornicata].
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Acknowledgments |
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Footnotes |
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* Present address: Whitehead Institute, 9 Cambridge Center, Cambridge, MA
02142, USA 
Present address: Molecular Medicine Program, Ottawa Health Research
Institute, Ottawa Hospital, Ottawa, Ontario K1H 8L6, Canada
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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