Regulation of early embryonic behavior by nitric oxide in the pond snail Helisoma trivolvis
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9
* Author for correspondence (e-mail: jeff.goldberg{at}ualberta.ca)
Accepted 22 July 2002
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Summary |
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Key words: Embryonic behavior, nitric oxide, pond snail, Helisoma trivolvis, serotonin, ciliary beating, gastropods, NADPH diaphorase
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Introduction |
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Gastropod molluscs, with their simple behaviors, well-characterized neural
circuits and giant identifiable neurons, are ideal for investigating NO
function at cellular, circuit and behavioral levels. In adult Lymnaea
stagnalis, for example, sensory cells of the lips, as well as a number of
neurons within the CNS, have been shown to contain nitric oxide synthase
(NOS), the enzyme that catalyzes NO production
(Elphick et al., 1995;
Moroz et al., 1994
).
Functional studies on this system indicate that NO is necessary for the
transmission of sensory information to the central pattern generator for
feeding behavior (Elphick et al.,
1995
) and activation of buccal motor patterns
(Moroz et al., 1993
).
Furthermore, cellular experiments on L. stagnalis neurons have
clearly demonstrated neurotransmitter-like actions of NO, including its
release from presynaptic cells in an activity-dependent manner and production
of slow excitatory postsynaptic potentials on a target neuron
(Park et al., 1998
;
Philippides et al., 2000
).
In contrast to studies on adult systems, there is less opportunity to gain
an integrated understanding of the physiological and behavioral roles of NO
during embryonic development. However, recent studies on embryos of the pond
snail, Helisoma trivolvis, suggest that this system is well suited to
examine in depth the function of a simple neural circuit and its associated
behavior during early embryonic development. H. trivolvis embryos are
individually compartmentalized in egg capsules within egg masses that contain
5-50 sibling embryos (Goldberg,
1995). As they develop from zygote to juvenile stages inside these
transparent structures, they display a well-characterized rotational behavior
(Diefenbach et al., 1991
) that
serves to mix the capsular fluid and enhance oxygen diffusion to the embryo
(Kuang et al., 2002
). The
embryonic rotation is driven by three bands of constitutively beating cilia
that are innervated by a bilateral pair of early developing serotonergic
neurons (ENC1s). These embryonic neurons periodically release serotonin onto
their target ciliary cells, producing increases in ciliary beat frequency and
surges in the rate of embryonic rotation
(Diefenbach et al., 1991
;
Kuang and Goldberg, 2001
).
Anatomical studies revealed that each ENC1 has a single apical dendrite that
extends to the embryo surface and is tipped with a putative chemosensory
specialization (Diefenbach et al.,
1998
). Thus, ENC1 appears to be a sensorimotor neuron that
interacts with postsynaptic ciliary cells to regulate the embryonic rotation
behavior.
Since NO has been demonstrated in embryonic and larval gastropods
(Lin and Leise, 1996;
Serfösö et al., 1998), and is present and has physiological action
in both sensory and ciliary systems (Li et
al., 2000
; Sisson,
1995
; Uzlaner and Priel,
1999
), we hypothesized that NO is present and plays an important
role in the ENC1-ciliary neural circuit. Lin and Leise
(1996
) looked at the
expression of NOS using NADPH diaphorase (NADPH-d) histochemistry through
metamorphosis in the marine prosobranch Ilyanassa obsoleta.
Expression was restricted to the ganglionic neuropils, with the most intense
staining occurring in the apical ganglion. Serfözö et al.
(1998
) analyzed NADPH-d
activity during embryogenesis and in early juveniles in L. stagnalis,
and showed that the first expression is within the developing protonephridia,
and later it occurs in cells of the developing pedal ganglia, developing eye
and peripheral sensory neurons of the lips. To test for the expression of NO
in H. trivolvis embryos in the present study, we used NADPH-d
histochemistry on whole embryos. In addition, rotation behavior was analyzed
in embryos that were exposed to pharmacological treatments that either
increase or decrease levels of NO. We now report that ENC1 and dorsolateral
ciliary cells are among a variety of tissues that express NO during early
stages of H. trivolvis embryogenesis. Furthermore, pharmacological
experiments indicated that NO has physiological actions in both ENC1 and
ciliary cells. In light of previous in vivo and cell culture studies
on the ENC1-ciliary neural circuit (Christopher et al.,
1996
,
1999
;
Diefenbach et al., 1991
;
Goldberg et al., 1994
;
Kuang and Goldberg, 2001
),
these results suggest that H. trivolvis provides a unique model
system for investigating NO function at the cellular, circuit and behavioral
levels during embryonic development.
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Materials and methods |
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NADPH diaphorase staining
Whole-mount embryos were removed from egg masses and fixed in 4%
paraformaldehyde dissolved in 0.01 mol l-1 phosphate-buffered
saline for 1-3 h. Embryos were then washed 2x 20 min with Tris-buffered
saline (TBS; 1.0 mol l-1 Tris in HCl, pH 8.4). NADPH-d reaction
product was achieved through incubation of embryos for 3-5 h in 1 mmol
l-1 NADPH, 0.1 mmol l-1 Dicoumarol, 0.5 mmol
l-1 Nitroblue Tetrazolium salts and 0.4% Triton X-100 (Sigma, St
Louis, MO, USA), all diluted in 0.05 mol l-1 TBS. At the end of the
incubation time, the reaction was stopped with ice-cold TBS. In control
experiments, NADPH was omitted from the staining solution. To best preserve
cellular integrity, whole embryos were mounted on glass slides in TBS and
viewed immediately using Nomarski differential interference contrast (DIC)
optics on a Nikon Diaphot TMD inverted microscope with a 40x
objective.
Rotational behavior
All rotational experiments were performed on stage E25 embryos. The outer
membranes on the basal surface of the egg mass were cut open without
disturbing the egg capsules in order to minimize diffusion barriers during
drug treatments. Egg masses were allowed to recover for 60 min in filtered APW
and then transferred to 35 mm Petri dishes (Falcon 1008) containing 2 ml of
either APW or drug-containing APW. Embryos within egg capsules were monitored
with a CCD video camera (JVC model TK-860U) mounted on a dissection microscope
(Zeiss model SR). After 10 min of incubation, the embryonic rotational
behavior was recorded at 2.5 frames s-1 for 10 min using a
time-lapse video cassette recorder (Panasonic model AG-6720). This procedure
was modified when treating embryos with the NO donor sodium nitroprusside
(SNP) because of its high light sensitivity. When using SNP, data was
collected for only 2 min. Given the similarity between the results of the SNP
and S-nitroso-N-acetylpenicillamine (SNAP) treatments, this
change did not appear to affect the results. In every experiment, all drug
treatments and controls were done in parallel dishes on the same day. A
maximum of five embryos were recorded from a single egg mass, and each
treatment group contains data from at least three different egg masses over
two separate experiments.
The rotational behavior was analyzed off-line by playing back the
recordings at 30 frames s-1, 12 times faster than the recording
rate. This faster playback speed facilitated the observation of very slow
rotational movements. Rotations were counted over each recording period and
the data was expressed in rotations min-1 (r.p.m.). To assess the
inter-surge rotation rate and the frequency and amplitude of rotational
surges, rotation rate was quantified every 5 s by measuring the degrees of
rotation achieved in sequential 5 s intervals for 1 min
(Diefenbach et al., 1991).
Surges were operationally defined as sequential periods of increasing rotation
rate that result in a total rise of at least 0.5 r.p.m., followed by a falling
phase in which the rotation rate drops by at least 0.5 r.p.m.
Statistics
All values are means ± S.E.M. In experiments involving multiple
treatment groups (Figs 2,
3), statistical significance
was determined using an analysis of variance (ANOVA) followed by a Fisher's
protected least-significant difference (PLSD) test. In experiments where a
single treatment group was compared to a control group (Figs
5,6,7),
the unpaired Student's t-test was used. Differences were considered
statistically significant at P<0.05.
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Chemicals
Serotonin (creatine sulphate complex; Sigma), sodium nitroprusside
dihydrate (SNP; Sigma), NG-nitro-L-arginine methyl ester
hydrochloride (L-NAME; Sigma) and NG-nitro-D-arginine methyl ester
hydrochloride (D-NAME; Sigma) were dissolved in Helisoma saline (HS;
51.3 mmol l-1 NaCl, 1.7 mmol l-1 KCl, 4.1 mmol
l-1 CaCl2, 1.5 mmol l-1 MgCl2, 5.0
mmol l-1 Hepes, pH 7.35), and then diluted in APW to the final
working concentration. S-Nitroso-N-acetylpenicillamine
(SNAP; Tocris) and 7-nitroindazole (7-NI; Tocris) were dissolved in dimethyl
sulfoxide (DMSO), and then diluted with APW so that the level of DMSO did not
exceed 0.1%. This concentration of DMSO has previously been shown to have no
effect on ciliary beat frequency
(Christopher et al., 1999) or
embryo rotation rate (Goldberg at al.,
1994
). All drug solutions were prepared on the same day that they
were used.
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Results |
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As ENC1s were easily visualized in unstained stage E25-E30 embryos using
DIC optics (Diefenbach et al.,
1998), they could be specifically examined for the expression of
NOS activity. Surprisingly, NADPH-d reaction product usually occurred only in
the distal portion of the apical dendrite, including the chemosensory-like
dendritic knob that penetrates the outside surface of the embryo
(Fig. 1A). Out of 113 stage E25
and E30 embryos mounted in the appropriate orientation to assess the soma and
dendritic knob for NADPH-d staining, 97 (86%) had staining that was restricted
to the dendritic knob. Eight embryos (7%) displayed staining in the ENC1 soma,
apical dendrite and dendritic knob, whereas in another eight embryos (7%),
ENC1 appeared unstained. By stage E35, reaction product was observed in the
soma, throughout the apical dendrite and in the proximal descending neurite of
ENC1 (Fig. 1B) in most embryos.
In contrast, reaction product was never resolved in the distal portions of the
descending primary neurite and its branches. The pedal ciliary cells also
exhibited latent expression of NOS activity, as reaction product was often
observed in these cells by stage E40.
The results of the NOS activity localization experiments are summarized diagrammatically in H. trivolvis embryos in front and side view (Fig. 1C,D). Since ENC1 neurons and ciliated cells that drive the rotation behavior were some of the structures that showed consistent and robust staining, these results prompt the hypothesis that NO plays an important role in mediating the chemosensory and ciliary activities that occur in stage E25-E40 embryos.
Regulation of embryo rotation behavior by NO
To assess whether NO may be involved in mediating or regulating embryonic
rotation behavior, time-lapse videomicroscopy was used to record stage E25
embryos after exposure to NO donors, NO inhibitors, serotonin or vehicle
solutions. In most experiments, a serotonin (100 µmol l-1)
treatment group was included as a positive control to indicate the expected
maximal rotation response (Diefenbach et
al., 1991; Goldberg et al.,
1994
).
Addition of the NO donors SNAP (Fig.
2A) and SNP (Fig.
2B) for 10 min induced a twofold increase in rotation rate, with
the maximal response occurring at a concentration of approximately 10 µmol
l-1. In both cases, the increases produced by higher concentrations
of NO donor (100 µmol l-1 SNAP and 250 µmol l-1
SNP) were slightly lower than that produced by the optimal concentrations,
with the difference being statistically significant only for SNAP
(P<0.05, 10 µmol l-1 SNAP versus 100
µmol l-1 SNAP, ANOVA). In contrast to the twofold maximal
response to NO donors, 100 µmol l-1 serotonin (5-HT) induced a
fourfold increase in rotation rate (Fig.
2A,B), similar to the three- to fivefold increase seen in previous
studies (Diefenbach et al.,
1991; Goldberg et al.,
1994
). To control for the possibility that the responses to the NO
donors were induced by the NO donors themselves or NO by-products, rather than
NO, 10 µmol l-1 SNAP and 25 µmol l-1 SNP solutions
were incubated at 20°C for 24 h in the light before being exposed to
embryos. After this treatment, which would have completely exhausted the
NO-producing activity of these drugs (Van
Wagenen and Rehder, 1999
), they were no longer able to induce an
increase in rotation rate (Fig.
2C). These results suggest that NO may play a stimulatory role in
the rotational behavior of H. trivolvis embryos.
The effects of two different NOS inhibitors were assessed to determine more directly whether NO is involved in regulating rotational behavior under normal conditions. Exposure of embryos to the arginine analog L-NAME (10 mmol l-1) induced a nearly 50% reduction in rotation rate (P<0.01, L-NAME versus APW; ANOVA), whereas the same concentration of D-NAME, a less active enantiomer, had no effect (Fig. 3A). Likewise, the NOS inhibitor 7-NI (100 µmol l-1) caused a similar reduction in rotation rate, from 0.97±0.05 r.p.m. to 0.43±0.02 r.p.m. (P<0.001; Fig. 3B). In both these experiments, serotonin produced the expected three-to fivefold increase in rotation rate. To rule out the possibility that the effects of the NOS inhibitors were due to a non-specific action that disables the rotation behavior, we tested whether addition of a NO donor could bypass the NOS inhibition, and produce a normal stimulatory response (Fig. 3C). Addition of 10 mmol l-1 L-NAME alone produced the expected decrease in rotation rate. In contrast, when either 1 µmol l-1 or 10 µmol l-1 SNAP was combined with 10 mmol l-1 L-NAME, the rotation rate increased in a dose-dependent manner. Taken together, these results indicate that in stage E25 H. trivolvis embryos, normal rotation behavior depends in part on the presence and stimulatory action of NO.
Effects of NO on subcomponents of rotation behavior
Previous studies elucidated two subcomponents of the rotation behavior
displayed by stage E25 embryos (Diefenbach
et al., 1991). Superimposed upon a tonic slow rate of rotation are
phasic bursts of rapid rotation, called surges. Pharmacological and
laser-ablation studies suggested that surges result from the periodic release
of serotonin from ENC1, causing cilio-excitation and accelerated rotation
(Diefenbach et al., 1991
;
Kuang and Goldberg, 2001
).
Basal rotation, on the other hand, is probably due to the constitutive
activity of dorsolateral and pedal ciliary cells
(Goldberg et al., 1994
). To
determine whether NO manipulations affect the basal rotation rate or
rotational surges, embryo rotation rate was measured every 5 s to reveal the
temporal pattern of rotation.
Fig. 4 displays the results from two control embryos exposed to APW (Fig. 4A) and two experimental embryos exposed to 10 mmol l-1 L-NAME (Fig. 4B). Surges were operationally defined as sequential periods of increasing rotation rate that result in a total rise of at least 0.5 r.p.m., followed by a falling phase in which the rotation rate drops by at least 0.5 r.p.m. From this type of data, the rate of slow tonic rotation, amplitude of rotational surges and frequency of rotational surges were quantified to indicate the likely cellular sites of NO action (Fig. 5,6,7). For example, effects restricted to the mean rate of slow tonic rotation would indicate NO action directly on the ciliary cells. In contrast, changes in frequency of surges would suggest that ENC1 is the site of NO action, whereas changes in the amplitude of surges would indicate that either cell type may be involved.
|
In embryos exposed to 10 µm SNAP, the mean rate of slow tonic rotation was significantly elevated in comparison to those exposed to the control vehicle (SNAP, 1.70±0.05 r.p.m. versus APW, 0.89±0.05 r.p.m.; P<0.001; Fig. 5A). Similar results were obtained with a second NO donor, 25 µmol l-1 SNP (Fig. 5B). In contrast, exposure of embryos to the NOS inhibitors L-NAME (10 mmol l-1) or 7-NI (100 µmol l-1) had inhibitory effects, with the mean rate of slow tonic rotation dropping to 0.08±0.01 r.p.m. (P<0.001) and 0.25±0.02 r.p.m. (P<0.0001), respectively (Fig. 5C,D). These results suggest that at least one main site of NO action is on ciliated cells, whereby tonic ciliary beating depends in part on constitutive NO activity.
Examination of the frequency and amplitude of rotational surges may reveal
whether ENC1 is also a main target of NO activity. Whereas the NO donors SNAP
(Fig. 6A) and SNP
(Fig. 6B) did not significantly
affect the frequency of rotational surges, the NOS inhibitors L-NAME
(P<0.001; Fig. 6C)
and 7-NI (P<0.001; Fig.
6D) did significantly reduce surge frequency. In the presence of
these drugs, surges only occurred rarely during 1 min observation periods. In
contrast to the results on surge frequency, NO donors were more effective than
NOS inhibitors in regulating surge amplitude. SNAP (P<0.05;
Fig. 7A) and SNP
(P<0.05; Fig. 7B) both significantly enhanced the amplitude of surges, whereas L-NAME
(Fig. 7C) and 7-NI
(Fig. 7D) had no significant
effects. However, since surges were only rarely expressed in the presence of
NOS inhibitors (Fig. 7A,B),
this conclusion is based on a relatively low sample size (N=3 for
L-NAME; N=3 for 7-NI). Since surges are generated by the activity of
the cilioexcitatory motor neurons, ENC1 s
(Kuang and Goldberg, 2001),
the reduction in surge frequency observed in the presence of NOS inhibitors
suggests that ENC1s are also a main site of NO action in H. trivolvis
embryos.
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Discussion |
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Localized expression of NOS-like activity in H. trivolvis
embryos
The pattern of expression of NOS-like activity was examined in stage
E25-E40 H. trivolvis embryos using NADPH-d histochemistry, a
technique with potential limitations in specificity
(Beesley, 1995;
Blottner et al., 1995
). Whereas
a parallel analysis using NOS immunoreactivity could help confirm this
pattern, NOS isoforms expressed in H. trivolvis embryos are not
reliably recognized by the antibodies that we have tested to date. However,
since pharmacological effects of NOS inhibitors have been observed exclusively
in cells and tissues that are NADPH-d positive
(Goldberg et al., 1999
) (see
below), the NADPH-d technique appears to be a reliable indicator of NOS
activity in H. trivolvis embryos. Non-neuronal reaction product was
observed initially in the paired protonephridia, dorsolateral ciliary bands,
ciliary band lining the dorsal buccal cavity and assorted unidentified cells
scattered throughout the dorsal body wall and primordial mantle. By contrast,
reaction product within neurons was limited to the dendritic knob of each
ENC1, chemosensory-like structures situated on the dorsolateral surface of the
embryo (Diefenbach et al.,
1998
). By stage E40, reaction product was also observed in the
pedal ciliary band, as well as the apical dendrite, soma and proximal
descending neurite of ENC1.
In an NADPH-d analysis of Lymnaea stagnalis embryos, the staining pattern observed at corresponding developmental stages was very different from that described above (Serfösö et al., 1998). Whereas the protonephridia were stained in both species, L. stagnalis embryos had no staining in any of the other structures found to be positive in H. trivolvis. Detection of NADPH-d reaction product first occurred in peripheral and central neurons of L. stagnalis at much later stages of development. Although both H. trivolvis and L. stagnalis are pulmonate basommatophoran pond snails, their respective position within different families suggests that significant differences in embryonic development, structure and function are to be expected.
ENC1s have been proposed to be derived from the apical sensory organ
described in a variety of marine gastropod larvae
(Diefenbach et al., 1998;
Kempf et al., 1997
;
Page and Parries, 2000
). The
expression of NOS-like activity in ENC1s is further evidence of this
relationship, as NADPH-d reaction product has been found in the apical sensory
organ of Ilyanassa obsoleta (Lin
and Leise, 1996
) and Phestilla sibogae
(Meleshkevitch et al., 1997
),
the only larval gastropods tested so far. Pharmacological experiments
suggested that NO inhibits metamorphosis in I. obsoleta
(Froggett and Leise, 1999
;
Leise et al., 2001
), yet
promotes metamorphosis in P. sibogae
(Meleshkevitch et al., 1997
).
Whereas direct-developing gastropods such as H. trivolvis do not have
a true larval stage terminated by an environmentally induced metamorphosis,
our preliminary studies suggest that NO does regulate developmental processes
during H. trivolvis embryogenesis
(Goldberg et al., 1999
).
A surprising outcome of the present study was that NADPH-d staining in ENC1
first occurred in the distal apical dendrite and dendritic knob at stage E25,
whereas staining in the soma and proximal axon was not consistently expressed
until stage E35. Since the dendritic knob is located on the embryo surface and
has structural features typical of chemosensory receptors
(Diefenbach et al., 1998;
Koss et al., 2002
), the
presence of NOS-like activity suggests that NO is involved in chemoreception.
Recent experiments have suggested that ENC1 is an oxygen sensor that excites
postsynaptic ciliary cells in response to hypoxia
(Kuang et al., 2002
). Thus, NO
may participate in oxygen sensing in ENC1. Alternatively, since NO has been
implicated in olfactory signaling in a variety of systems
(Bicker, 2001
;
Breer and Shepherd, 1993
;
Gelperin et al., 2000
;
Schmachtenberg and Bacigalupo,
1999
), ENC1 may be a multimodal sensory neuron, with NO involved
in the detection of unidentified olfactory cues.
The expression of NOS-like activity in ciliated epithelial cells of H.
trivolvis embryos is consistent with reports of NOS expression
(Asano et al., 1994;
Xue et al., 1996
) and NO
production (Li et al., 2000
)
in mammalian ciliary cells. The differential distribution of NADPH-d reaction
product in dorsolateral and pedal ciliary cells at stage E25 correlates well
with our earlier finding that serotonin plays a greater role in ENC1-mediated
cilioexcitation in pedal versus dorsolateral ciliary cells
(Kuang and Goldberg, 2001
).
Furthermore, since ENC1 only directly innervates the most medial cell of each
dorsolateral band (Koss et al.,
2002
), NO signaling may be required to pass the excitation to
adjacent lateral cells.
Physiological effects of NO on ENC1s and ciliary cells
The behavioral effects of pharmacological treatments that either raise or
lower levels of NO in embryos indicated that NO is tonically active, with
sites of action in both ENC1s and their target ciliary cells. The stimulation
of embryo rotation rate produced by two different NO donors, and the
inhibition produced by two different NOS inhibitors, provided strong evidence
that NO plays an excitatory role in generating the normal, ongoing rotation
behavior. Previous studies have established that the rotation behavior has two
basic components: (i) slow tonic rotation mediated by the constitutive
activity of the dorsolateral and pedal ciliary bands; and (ii) periodic
episodes of rapid rotation, called surges, that result from the phasic release
of serotonin from both ENC1s onto their postsynaptic ciliary cells
(Diefenbach et al., 1991;
Goldberg et al., 1994
;
Kuang and Goldberg, 2001
).
Thus, NO may mediate its excitatory effect by acting on various processes,
including ENC1 sensory transduction and integration, ENC1-ciliary synaptic
transmission and tonic ciliary beating. These possibilities were assessed by
quantitative analysis of the subcomponents of the embryo rotation behavior
during responses to pharmacological treatments.
The reduction in frequency of surges caused by NOS inhibitors is the most
direct evidence that ENC1 is a site of NO action. Since NO donors did not
cause an opposite increase in surge frequency, NO may act constitutively
rather than periodically to promote excitatory bursts in ENC1. Whether NO
affects ENC1 activity through a modulation of ion channel activity, as
described in identified Aplysia neurons
(Koh and Jacklet, 2001), or by
acting on other elements in the ENC1 sensory transduction pathway, is
unknown.
The effect of NO donors on surge amplitude indicates either an ENC1 or
ciliary site of NO action. Of these possibilities, presynaptic facilitation of
ENC1-ciliary synaptic transmission is most likely because in cell culture
experiments on isolated ciliary cells, NO donors did not facilitate the
ciliary response to serotonin (Goldberg et
al., 2000). However, the possibility of NO acting postsynaptically
as a neurotransmitter (Park et al.,
1998
) or modulator of the serotonin response pathway cannot be
ruled out.
The effects of NOS inhibitors and NO donors on the nonsurge component of
embryo rotation are the easiest to interpret. They strongly suggest that NO is
a constitutive regulatory element of ciliary beating, providing a tonic
excitatory influence under basal conditions. Furthermore, since these results
were consistent with the effects of NOS inhibitors and NO donors on isolated
ciliary cells (Goldberg et al.,
2000), where there was no opportunity for ENC1-ciliary
interactions, NO must be produced within ciliary cells to act as an endogenous
regulator of ciliary beating. These results do not rule out the possibility
that NO may also function in a neurotransmitter-like manner, diffusing from
ENC1 into ciliary cells to stimulate ciliary beating. In addition, since only
some ciliary cells are innervated by ENC1
(Koss et al., 2002
), the
remaining ciliary cells may depend on the paracrine actions of NO to produce
excitatory responses. These transcellular actions of NO could be tested by
exposing embryos to extracellular NO scavengers.
Studies on mammalian ciliary cells have not yet revealed a complete
understanding of the role of NO in regulating ciliary beating. Stimulation of
ciliary beating by NO was reported to occur in tracheal cells of rats
(Li et al., 2000) and rabbits
(Uzlaner and Priel, 1999
), and
in bovine bronchial cells (Sisson,
1995
). Taken together with the stimulatory action of NO on the
non-surge component of embryonic rotation
(Fig. 5) and isolated H.
trivolvis ciliary cells (Goldberg et
al., 2000
), it appears that ciliary stimulation may be a common
action of NO. However, a study on ovine tracheal cells reported that NO donors
did not affect the ciliary beat frequency
(Salathe et al., 2000
).
Furthermore, only the present study on H. trivolvis embryos indicated
that NO is a constitutive regulator of ciliary activity or cilia-driven
behavior. Application of a NOS inhibitor on its own, which should indicate the
presence of tonic NO activity, had no effect on the ciliary beat frequency of
rat tracheal cells (Li et al.,
2000
). A more extensive investigation of this question in other
ciliary systems is required before concluding whether H. trivolvis
embryonic ciliary cells are typical or unique with respect to NO exerting
tonic control of ciliary beating.
In conclusion, both the histochemical experiments on NOS distribution and
the pharmacological and behavioral experiments on NO function point to a
central physiological role for NO in regulating the ENC1-ciliary neural
circuit at multiple sites of action. NO thus joins serotonin as a
neurotransmitter system expressed early in H. trivolvis embryonic
development that participates in controlling the first behavioral response of
the animal, a hypoxia-induced stimulation of embryonic rotation
(Kuang et al., 2002). Although
not directly addressed by this study, it is highly likely that the
physiological regulation of this behavior involves intimate interactions
between these two neurotransmitters.
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Acknowledgments |
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References |
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