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First published online October 17, 2008
Journal of Experimental Biology 211, 3467-3477 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.018952
Control of swimming in the hydrozoan jellyfish Aequorea victoria: subumbrellar organization and local inhibition
Department of Biology and Marine Biology and Center for Marine Science, University of North Carolina, Wilmington, NC 28409, USA
e-mail: satterlier{at}uncw.edu
Accepted 4 September 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: cnidaria, hydrozoa, jellyfish, locomotion, motor control, neurobiology
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). The
tentacles are typically contracted prior to extended swimming bouts, or during
escape swimming. The oblate forms, by contrast, tend to swim with the marginal
tentacles extended and flowing in the contraction wakes. Colin and Costello
(Colin and Costello, 2002)
examined the biomechanics of swimming in several hydromedusan species and
found the prolate forms primarily used a jet propulsive form of locomotion
during a swim contraction. The oblate species derived only a small proportion
of thrust from jet propulsion, rather using a drag-based rowing of the margin
for thrust generation. This latter mechanism produced a series of vortex rings
(toroids) that were well-suited for carrying food particles through the
trailing tentacles, suggesting a more active form of feeding in the oblate
medusae (Colin and Costello,
2002). Thus, the mechanics of bell contraction seem to be related
to feeding strategy, at least for the species investigated.
Despite this apparent dichotomy in swim mechanics and feeding strategies,
all hydromedusae, including those with both bell shapes, have what can be
considered a common, basic organization for neuromuscular control
(Satterlie and Spencer, 1983;
Satterlie, 2002). This
consists of an electrically coupled network of large neurons, found in the
inner nerve ring, which acts as a distributed swim pacemaker system for swim
contractions, and which communicates with overlying epithelial cells
(post-synaptic cells) via chemical synapses
(Anderson and Mackie, 1977;
Anderson, 1979;
Spencer and Satterlie, 1980;
Satterlie and Spencer, 1983;
Spencer, 1981;
Spencer, 1982;
Satterlie, 1985a;
Satterlie, 1985b;
Satterlie, 2002;
Mackie and Meech, 2000;
Lin et al., 2001;
Mackie, 2004). As a second
common feature found in all investigated hydromedusae, long duration action
potentials in the post-synaptic epithelial cells are conducted to subumbrellar
circular muscle cells via gap junctions, to produce (or help produce)
swim contractions (Singla,
1978a; Spencer,
1978; Spencer,
1979; Spencer,
1981; Spencer and Satterlie,
1981; Satterlie,
1985b; Satterlie,
2002; Kerfoot et al.,
1985; Satterlie and Spencer,
1983; Mackie,
2004; Mackie,
1975). Superimposed on these two basic features are
species-specific neuronal and muscular organizations that allow unique
behavioural repertoires for each species. For example, although the circular
muscle sheets of the anthomedusa Polyorchis penicillatus are aneural,
the motor network of coupled neurons extends from the inner nerve ring, up
(orally) within each of the four radial nerves (which lie over the radial
canals), and across the top of each quadrant, so excitation of the muscle
sheet does not occur solely at the margin, but from all four sides of the
quadrant (Lin et al., 2001).
The combination of junctional coupling and action potential threshold is such
that action potentials are conducted throughout the muscle sheet without
significant decrement when generated from the motor network
(Spencer, 1978;
Spencer, 1982;
Spencer and Satterlie,
1981).
In the trachymedusa Aglantha digitale, slow swimming is controlled
by a similar network of inner nerve ring neurons, although activation of the
swim musculature is quite different (see
Mackie, 2004). The marginal
motoneuron network synaptically activates eight radially oriented motor giant
neurons that overlie the radial canals
(Weber et al., 1982;
Kerfoot et al., 1985;
Mackie and Meech, 2000;
Mackie, 2004). The motor
giants produce two types of action potentials, a calcium spike during slow
swimming and a sodium-based spike during escape swimming
(Mackie and Meech, 1985;
Meech and Mackie, 1993). The
motor giants, in turn, activate lateral neurons that help transmit excitation
part of the way across the muscle sheets
(Singla, 1978b,
Weber et al., 1982;
Mackie, 2004). Again, muscle
electrical events are transmitted from muscle cell to muscle cell via
gap junctions, however, the conduction is decremental since contractions in
the slow swimming mode are relatively weak, and restricted to the margins of
each subumbrellar octant.
Polyorchis is a prolate medusa, and produces symmetrical,
relatively uniform contractions of the subumbrella. Superimposed on this
swimming activity is another seemingly common type of behavioural response of
hydromedusae that involves contraction of subumbrellar radial muscles, pulling
the margin of the bell inward and upward toward the manubrium. These radial
responses can be local, as in a feeding response in which a single tentacle or
localized group of tentacles can be turned inward into the bell, ultimately to
contact the manubrium (for prey transfer). A widespread radial response rolls
the entire margin into the bell cavity, as a protective "crumple"
response (Spencer, 1975;
Spencer, 1978). Swimming is
inhibited during a crumple, and sometimes during a feeding response. In either
case, the radial responses are produced by contraction of smooth muscle that
is either restricted to bands overlying with the radial canals
(Polyorchis and several other species) or as more widespread
subumbrellar sheets that overlie the striated swim muscle [as in the
leptomedusae Aequorea, Phialidium and Eutonina
(Satterlie and Spencer,
1983)]. The subject of this study, Aequorea victoria
Murbach and Shearer (previously classified as Aequorea aequorea),
which exhibits this latter organization of radial musculature, provides
another excellent example of the superimposition of unique species-specific
behavioural control on the basic hydromedusan neuromuscular plan.
As a relatively large oblate medusa, Aequorea spends a significant
amount of time swimming, and does so with its tentacles extended. It shows
both localized (feeding) and widespread (crumple) radial responses, but during
the former, it can `turn off' swimming in restricted sections of the bell
while the rest of the subumbrella produces seemingly normal swim contractions
(Satterlie, 1985a). This
localized inhibition is unusual, and requires a mechanistic explanation since
the `basic' hydromedusan swim system includes the through-conducting,
electrically coupled network of large motoneurons in the inner nerve ring
(Satterlie and Spencer, 1983;
Satterlie, 2002). This network
has been studied in Aequorea
(Satterlie, 1985a;
Satterlie, 1985b), where it
was found to function as both the pacemaker network for swimming activity and
the primary motor network for synaptic activation of the overlying epithelial
cells (Satterlie, 1985a). The
neurons produce a rapid action potential burst that activates a single, broad
action potential in the post-synaptic epithelial cells
(Satterlie, 1985a;
Satterlie, 1985b). The
epithelial cells are in electrical contact with the subumbrellar swim muscle
via gap junctions. The `muscle' action potentials are variable in
amplitude, and after a period of rest, they show a progressive increase in
amplitude with each successive swim that is suggestive of a facilitatory
mechanism (Satterlie, 1985b).
Local inhibition caused by mechanical or electrical stimulation of the margin
results in regional hyperpolarization in the swim motor network, and either a
suppression of synaptic transmission to the epithelial cells, or a delayed
burst in the motor network (Satterlie,
1985b).
Here, the method of transmission of muscle action potentials through the subumbrellar circular muscle sheet is investigated, as is the activity of the radial muscle during a radial response. These data are used to provide a physiological explanation of Aequorea's ability to `turn off' swimming in localized parts of the bell during restricted radial responses, and demonstrates yet another species-specific method of neuromuscular control of swimming that is superimposed on the `basic' hydromedusan plan.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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. For
dye injections, the electrode tips were filled with 4% Lucifer Yellow and
back-filled with 1 mol l–1 LiCl
(Stewart, 1978). Dye was
injected into a recorded cell by iontophoresis, with negative current pulses
applied to the electrode using an amplifier bridge circuit. The currents were
approximately 1–2 nA, and consisted of repetitive 500 ms pulses
delivered at 1 Hz. The dye-fills were viewed and photographed live, in the
recording dish, with a Nikon epifluorescence microscope and film camera. All
experiments were completed in natural seawater. Swimming was blocked with a
1:1 solution of isotonic MgCl2:seawater. This same solution was
used to anesthetize the animals prior to initial dissection. Electrical
stimuli were delivered by polyethylene suction electrodes (internal tip
diameters approximately 50 µm) and a Grass S9 stimulator (Astro-Med, Inc.,
West Warwick, RI, USA).
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For immunohistochemical labelling, tissue pieces were fixed in 4%
paraformaldehyde in phosphate buffer overnight, washed in phosphate buffer
with either 0.05% Triton X-100 or 1% Tween 20, and incubated in 5% goat serum
for 4 h. The tissue was then soaked in primary antibody (rabbit polyclonal
anti-FMRFamide; Chemicon International Millipore Corp., Billerica, MA, USA; or
mouse monoclonal 
-tubulin; Developmental Studies Hybridoma Bank,
University of Iowa, IA, USA) at 1:500 dilution in phosphate buffer for 24 to
48 h. In double labelling experiments, both primary antibodies were added
together. After washing with buffer, the tissue was incubated in secondary
antibody overnight (goat anti-mouse Alexa Fluor 488 for tubulin and goat
anti-rabbit Alexa Fluor 594 for FMRFamide; both from Molecular Probes,
Invitrogen, Carlsbad, CA, USA). In double labelling experiments, both
secondary antibodies were added together. The tissue was then washed in
phosphate buffer and cleared and mounted in a 1:9 mixture of Tris buffer and
glycerol. Specimens were mounted on glass slides using the same mounting
medium, and examined with a Nikon epifluorescence microscope and photographed
with a Spot Slider digital camera.
Staining in the subumbrellar neurons was not robust in either brightness and in resistance to fading, in particular, at the magnifications needed to view the neurons, fading was significant in the time for the camera exposure. In fact, if focusing the image took too long, most of the neurons in the visual field were too dim to show in the camera images. For this reason, confocal microscopy could not be used on these preparations. Also, in the higher magnification images (e.g. Fig. 7B,C), there is an under-representation of nerve net density.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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The subumbrella of a large medusa (approximately 8 cm bell diameter) is typically divided into over 50 interradial segments (Fig. 1). The segments are separated by 1 mm wide radial canals which are naked (no gonadal tissue) for their first 4 mm from the margin. The ridge of gonadal tissue extends from this point to the stomach as a 1.5 mm wide ridge that forms the roof of the radial canal. Each interradial muscular segment is thus bounded by two radial canals (with attached gonads), the marginal canal and the stomach. In an 8 cm diameter medusa, the interradial segments are nearly 30 mm tall, and up to 10 mm wide.
The ectoderm of the interradial segments includes two sheets of muscle, the outer one radial, and the inner one circular in orientation (Figs 2 and 3). The muscle sheets are totally interrupted in the gonadal regions of the radii, but are continuous with the musculature of the adjacent segment where the radial canals are naked. In the naked region, the radial canal is found beneath the muscular sheets. The subumbrellar side of the velum contains only circular epitheliomuscular cells, and is not divided by radii.
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Subumbrellar ectoderm
Two types of muscle cells make up the ectoderm of the interradial segments.
The outer layer is composed of surface epithelial cells with basal muscular
processes oriented in a radial direction. The myofibrillar organization within
the processes is of the smooth type (Fig.
2). Situated between the radial muscle cells and the mesoglea is a
layer of circularly oriented, striated muscle cells (swim muscle). The radial
muscle lies immediately over the striated muscle (Figs
2 and
3). Extensions of the circular
muscle cells, to the free surface of the epithelium, have not been found. The
ectoderm thus forms a pseudo-stratified epithelium.
Radial muscle cells
Radial muscle cells have a surface epithelial component and one or more
basal muscle processes (Fig.
2). Viewed from the surface, the cells are up to 50 µm in
diameter (normally around 25 µm). The cells are drawn out basally into one
or more muscle `feet' that taper to less than 1 µm and interdigitate with
similar processes of other radial muscle cells, and occasionally run between
circular muscle cells to the mesoglea. Radial muscle cells are very thin (mean
thickness, 1.39 µm; maximum 5 µm) with elongate nuclei and vacuolated
apical cytoplasm. The myofibrillar region of the radial cells includes thick
and thin filaments that are oriented in the long axis of the processes but
otherwise randomly interspersed. Since no evidence of cross or oblique
striations could be found, the radial muscle is considered to be smooth muscle
(Fig. 2). In cross section,
thick and thin filaments show no orderly or repetitive organization
(Fig. 3A). Thick filaments are
tubular in shape with a diameter of 20 nm, whereas the thin filaments appear
solid with a diameter of 5–7 nm. Elongate mitochondria are scattered
throughout the radial cells and are frequently found among the
myofilaments.
Radial muscle cells are interconnected by gap junctions, found primarily near the apical surface of the cells, but also occasionally deeper in the muscle layer (Fig. 4). In the junctional area, the intercellular space is 3–6 nm as compared with 20–25 nm in non-junctional areas. Similar junctions between radial muscle cells and the underlying circular muscle cells were not found.
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In cross section, thick and thin filaments are packed in an orderly array (Fig. 5). Thick filaments are hexagonally arranged with a centre-to-centre distance of 35–43 nm. The thick filaments are tubular with a diameter of 20 nm. Thin filaments are solid in appearance and are 5–7 nm in diameter (measurements derived from high magnification micrographs). The thick-to-thin filament ratio appears to be 1:8.
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Circular muscle cells are interconnected by gap junctions that appear structurally similar to those found between pairs of radial cells (Fig. 3A, Fig. 4A). Where two muscle cells are joined `end-to-end', a desmosome-like connection is usually found adjacent to one or more gap junctions. Circular muscle cells do not form a complete subumbrellar layer as radial cell processes occasionally extend to the mesoglea between the circular cells.
Subumbrellar neurites
Neurites and occasional neuron cell bodies are present throughout the
subumbrellar segments. Neurites are up to 1.5 µm in diameter and are
recognized by a relatively clear cytoplasm containing longitudinally oriented
microtubules and either large dense-core or clear vesicles. Neurite swellings,
including accumulations of dense-core vesicles (up to 140 nm in diameter) are
frequently encountered (Fig.
2). Despite a common occurrence of such swellings, definitive
neuromuscular junctions (including pre- and post-synaptic specializations)
have not been found.
Electrophysiology of circular muscle cells
Circular muscle cells were routinely penetrated with microelectrodes, and
were recognized by a one-to-one correspondence of depolarizing electrical
events and swim contractions. They were penetrated in all parts of the
subumbrellar segments and in the gonad-less regions overlying radial canals.
The mean resting potential for circular muscle cells was –62.3 mV
(N=54).
With each swim contraction, the muscle cells exhibited one (or both) of two
types of depolarizing potentials. During `full swims' in the recorded segment,
long duration action potentials (up to 125 mV above resting potential) were
recorded (Figs 6 and
7). As with epithelial cells of
the inner nerve ring (Satterlie,
1985b), muscle action potential duration was related to medusa
size, and was up to 700 ms in some animals. The action potentials were
variable in amplitude and duration, particularly when the area of recording
produced a weak contraction or was inhibited during a radial response
(Fig. 6). The second type of
recorded potential was an apparent junctional potential of relatively large
size (up to 56 mV amplitude) (Fig.
6). Junctional potential duration was 160–200 ms. Junctional
potentials were recorded alone, or immediately preceding (giving rise to)
action potentials in actively contracting segments
(Fig. 6). In partially
inhibited preparations, action potentials showed several types of variability.
In addition to variation in amplitude and duration, they sometimes exhibited
slow rise times with the reduced amplitudes, and either were not preceded by
junctional potentials or exhibited junctional potentials that were smaller
than those within the segments. Also, when junctional potentials were present
with muscle action potentials, slight `hitches' (delays) were sometimes
observed between the peak of the junctional potential and the rise of the
action potential (compare the action potential in
Fig. 6A,B). In some cases in
which swimming was suppressed in the recorded segment (via triggered
radial responses), the recorded muscle cell still exhibited a junctional
potential, but also showed a reduced-amplitude action potential followed by a
second action potential that lacked a junctional potential and had a slower
rise time (Fig. 6C). The second
action potential in this `saw-tooth' pattern is believed to result from
electrotonic wash from an adjacent, active segment, or if close to the margin,
from a velar action potential.
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). This `bursty' junctional activity decreased with distance
from the margin so within approximately 1 mm from the margin and beyond, only
a single junctional potential was recorded per swim contraction. Dual intracellular recordings from two circular muscle cells of the same segment showed identical activity in terms of the relative amplitudes of the action potentials (Fig. 7). Injection of hyperpolarizing currents into one of the recorded muscle cells showed time-locked hyperpolarizations in the other recorded cells, confirming electrical coupling between the cells (Fig. 7). Similar coupling was found between a circular muscle cell in a subumbrellar segment and another over the adjacent radial canal, as well as between cells of adjacent segments, although in the latter situation, very large currents had to be injected, and the associated hyperpolarization was extremely small. In dual recordings of a circular muscle cell and a radial muscle cell, no electrical coupling could be demonstrated (data not shown).
Injection of Lucifer Yellow into a single circular muscle cell produced widespread dye movement from the injected cell to other circular muscle cells (Fig. 8A). No dye spread was noted from a circular muscle cell to radial muscle cells.
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Pattern of spread of circular muscle activity
The recording of junctional potentials in circular muscle cells from all
parts of a subumbrellar segment following stimulation of the tissue within
that segment, and their presence at the beginning of muscle cell action
potentials, both electrically stimulated and spontaneous, suggest that a
subumbrellar nerve net participates in spread of electrical activity
throughout each segment. This is supported, in part, by the presence of
neurites in these regions (Fig.
2). Yet, such a nerve net raises questions about how swimming can
be `turned off' in a portion of the subumbrella during localized radial
responses. This problem required examination of nerve net conducting
properties within the subumbrella.
As shown in Fig. 9, a recording electrode was placed in a circular muscle cell in segment A, and an electrical stimulus was delivered at a point represented by the letter A. In trace A (stimulating and recording electrode in same segment), junctional potentials were recorded in a 1:1 relationship with electrical stimuli, and the junctional potentials were roughly the same size as those triggering the three spontaneous action potentials in the same trace.
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The delay between the stimulus artifact and junctional potential appearance was about 200 ms in trace B, which is several times greater than the expected delay of about 35 ms if the circular conduction velocity of swim contractions originating from the nerve ring is considered (43 cm s–1 conduction velocity). The delay in trace B was calculated as a circular conduction velocity between adjacent segments of about 7.5 cm s–1, a speed only two-thirds of the conduction speed in a radial direction in a single segment (12 cm s–1).
When the stimulating electrode was moved over one more segment (Fig. 9, segment C), so that it was two segments away from the recording site, electrical stimuli strong enough to trigger contractions in the stimulated segment did not produce detectable junctional potentials or contractions in the recorded segment (Fig. 9C). This entire experimental series was repeated in three preparations from three different animals with identical results, suggesting that the conducting system of an individual segment, responsible for production of circular muscle cell junctional potentials, was not through-conducting in a circular direction around the bell, but was restricted between segments. By contrast, there seemed to be no such restriction in a radial direction, over similar distances, within each segment.
Evidence for participation of a subumbrellar nerve net in the spread of swim contractions
When stimulating a subumbrellar segment (with a suction electrode),
junctional potentials in circular muscle cells appeared with a distinct
threshold, and when the stimulus was above that threshold, the size of the
junctional potential did not vary. Similarly sized junctional potentials gave
rise to full action potentials in the muscle cells when a spontaneous action
potential was triggered by normal inner nerve ring activity
(Fig. 6;
Fig. 9A). Furthermore,
identical junctional potentials could be stimulated in non-swimming
preparations when the stimulating electrode was placed well above (more apical
to) the recording electrode. When the preparation was bathed in high-magnesium
seawater, swimming was inhibited, but so was production of junctional
potentials from direct electrical stimulation of subumbrellar segments
(via a suction electrode). In these experiments, injection of current
via the intracellular recording electrode showed the muscle cell was
capable of electrogenesis. Finally, the presence of neurites detected in
electron microscope preparations was confirmed by immunohistochemical staining
using a commercial antibody to the neuroactive peptide, FMRFamide, which
identified a diffuse nerve net within the subumbrellar segments (Figs
10 and
11). The neurites of this
network of stained neurons were preferentially oriented in a radial direction.
Staining was noted over the radial canals between segments as well as within
the segments. It also extended to the inner nerve ring but could not be
followed within the nerve ring because of high background staining in the
region. Specifically, connections to immunoreactive neurons within the inner
nerve ring could not be resolved.
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). Mackie et al. noted that the stained neurons were a subset
of the total neuron number found in electron microscopical investigations of
these same leptomedusae, suggesting the presence of at least two independent
nerve nets in the subumbrella of these jellyfish. To test this, double-label
immunohistochemical staining was conducted using the polyclonal antibody to
FMRFamide (rabbit primary, Alexa Fluor 594 secondary) and a monoclonal
-tubulin antibody (mouse primary, Alexa Fluor 488 secondary). The
latter is a fairly non-specific neuronal marker in cnidarians (e.g.
Satterlie, 2002), presumably
staining neurotubules. In preparations using a combination of filters, both green (tubulin) and red (FMRFamide) cell bodies and neurites were visible in the same focal plane of the subumbrellar segments (Fig. 11A). When separate filters were used, both types of neurons were apparent with the FITC filter, whereas with the TRITC filter, only the FMRFamide-immunoreactive neurons were visible. For example, in Fig. 11B,C, one of the tubulin-immunoreactive neurons is clearly not stained with the FMRFamide antibody. Similar results were obtained in all parts of the subumbrellar segments in four different double-labelled preparations (four different animals), indicating the presence of two distinct nerve nets (at least based on immunohistochemical staining) in the subumbrellar of Aequorea.
An interesting aspect of junctional potential production is the
relationship between action potentials in the swim generating network of the
inner nerve ring, the putative nerve net that innervates the swim muscles, and
the action potentials in the circular muscle cells themselves. In the inner
nerve ring, each swim is triggered by a rapid burst of action potentials in
the swim-generating neural network
(Satterlie, 1985a). This is
translated into a burst of junctional potentials and a resulting single action
potential in the overlying epithelial cells
(Satterlie, 1985b). These
epithelial cells are in electrical contact with the circular muscle cells of
the subumbrella and velum via gap junctions. Yet, away from the
margin, only single junctional potentials were recorded in circular muscle
cells with each action potential (Fig.
6), suggesting the action potential bursts observed in the
motoneurons are converted to single action potentials in the putative
subumbrellar motor nerve net.
How does a local radial response turn off swimming in a restricted portion of the bell?
Input from the motor network of the inner nerve ring, to the swim muscle,
includes a direct synaptic activation of epithelial cells overlying the
neurons and electrotonic spread of activity from the epithelial cells to the
circular muscle cells. In addition, synaptic input from a putative
subumbrellar nerve net supplements the excitation to the muscle cells. During
a localized radial response, the burst production in the motor network was
partially or totally inhibited (Fig.
12), blocking the direct synaptic input to the epithelial cells,
and presumably the activation of the subumbrellar nerve net. However, if the
region of inhibition was not too broad, muscle cell action potentials (and
contractions) of the subumbrellar muscle cells could still be recorded,
although with lower amplitudes, an absence of junctional potentials and slower
rise times (Fig. 12). In
preparations in which the area of inhibition involved several segments on
either side of the recorded segment, muscle action potentials were not
recorded, and swimming in that segment was totally blocked. This suggests that
the electrotonic spread of action potentials through the subumbrellar gap
junctions is decreasing. If so, the putative motor nerve net may play a
crucial role in conduction of a full action potential (and contraction) within
each segment. This would allow a decrease, or total inhibition, of
contractility in the muscle sheet by a localized inhibition of activity in the
segmental regions of the subumbrellar nerve net.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Satterlie,
2002). The electrically coupled network of large neurons provides
a means for rapid circular distribution of motor activation around the bell,
and also a means of receiving symmetrical and asymmetric inputs from sensory
structures that are distributed around the margin of the animal
(Spencer and Arkett, 1984;
Arkett and Spencer, 1986a;
Arkett and Spencer, 1986b;
Arkett et al., 1988). This is
ultimately translated into locomotory activity that is dependent upon
coordinated contraction of circular musculature that lines the inside of the
bell – another common feature that produces a forceful ejection of water
from the bell cavity (Singla,
1978a; Spencer,
1978; Spencer,
1979; Spencer,
1981; Spencer and Satterlie,
1981; Satterlie,
1985b; Satterlie,
2002; Kerfoot et al.,
1985; Satterlie and Spencer,
1983; Mackie,
2004).
In the several medusae studied, the swim motor network of the inner nerve
ring is composed of a compressed network of oversized neurons that activate
overlying epithelial cells via chemical synapses
(Anderson and Mackie, 1977;
Anderson, 1979;
Spencer and Satterlie, 1980;
Satterlie and Spencer, 1983;
Spencer, 1981;
Spencer, 1982;
Satterlie, 1985a;
Satterlie, 1985b;
Satterlie, 2002;
Mackie and Meech, 2000;
Lin et al., 2001;
Mackie, 2004). Either local or
distributed nerve nets of smaller neurons may further conduct excitation from
the motor network to the muscles, as seen in the extreme in Aequorea
where a subumbrellar nerve net is found throughout the muscle sheets
(Fig. 13). Other
specializations are superimposed on the basic organization, as in
Aglantha, where motor giant neurons distribute impulses between the
motor network and the swim musculature
(Weber et al., 1982;
Kerfoot et al., 1985;
Mackie and Meech, 2000;
Mackie, 2004). Also, use of
the word `oversized' to refer to the motor network of Aglantha loses
some meaning since the neurons are dwarfed by the ring giant (in the outer
nerve ring) that is involved in escape swimming (see
Mackie, 2004). Again, however,
this illustrates how species-specific neuronal peculiarities are superimposed
on the basic system.
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). Other
medusae examined produce a single action potential per swim
(Anderson and Mackie, 1977;
Anderson, 1979;
Spencer and Satterlie, 1980;
Satterlie and Spencer, 1983;
Spencer, 1981). The action
potential burst in Aequorea induces a burst of junctional potentials
in the postsynaptic epithelial cells that overlie the inner nerve ring, and in
the immediately adjacent circular muscle cells, which are electrically coupled
to the epithelial cells (Fig.
13) (Satterlie,
1985b). Electrical coupling is found throughout the circular
muscle sheet; this represents another basic feature of hydromedusan locomotory
systems that aids in the spread of excitation between muscle cells. The degree
of `coupling' varies so aneural conduction is sufficient to transmit a muscle
action potential throughout the muscle sheet without decrement in
Polyorchis (Spencer and
Satterlie, 1981; Spencer,
1982), whereas the same conduction is apparently decremental in
Aglantha [in slow swimming
(Kerfoot et al., 1985;
Mackie, 2004)] and in
Aequorea (this study). This variation in muscle cell `coupling' may
be due to differences in gap junctional conductance, to threshold differences
for full action potential production, differences in the regenerative
properties of the action potentials, or a combination of these and other
properties. As an example, in Polyorchis, the muscle action potential
is conducted through the aneural circular muscle sheet without decrement when
it is generated via normal inner nerve ring activity. However, the
epithelial cells in the region of the inner nerve ring (the neuromuscular
synaptic region) form an incrementally conducting region that requires the
simultaneous input of junctional potentials from multiple sites to produce the
conducted action potentials in the muscle sheet. A compressed network of small
neurons is found in this area (Spencer,
1982) that presumably serves as a distributor network for
additional neuromuscular input. In this way, this restricted region may act
like the entire subumbrella of Aequorea, and the distributor network
may serve the same function as Aequorea's subumbrellar motor nerve
net. In this case, it is interesting to suggest that the motor system of
Aequorea represents a more diffuse organization of this
non-regenerative band of Polyorchis, or that the latter is a
compressed version of the former.
Regardless, the nature of subumbrellar conduction holds important consequences for the control of swimming, and in particular, the effect of other behaviours on swimming, as seen here in Aequorea. In other words, when the two basic properties of hydromedusan swimming systems are considered (electrically coupled motor network that synapses onto postsynaptic epithelial cells, and widespread electrical coupling throughout the circular muscle sheets), the organization and physiology of these two basic features is where we see significant variation that is related to the structure of the medusa as well as its lifestyle.
In terms of neuromuscular control, perhaps the simplest case is that of the
anthomedusa, Polyorchis. The electrically coupled motor network of
the inner nerve ring extends up the four radii and across the top of each
quadrant (Lin et al., 2001).
This provides synaptic activation of the four circular muscle sheets along the
entire periphery of each quadrant. As mentioned above, transmission in the
muscle sheet is strictly via gap junctions. Radial muscle is not
present in the quadrants, instead it is restricted to smooth muscle strips
overlying the radii, so that radial responses in these prolate medusae,
including crumpling, are triggered by radial contraction at the four radii
(Singla, 1978a;
Spencer, 1978;
Spencer, 1979).
In what is possibly the most complex elaboration, Aglantha (a
trachyline medusa) has the `basic' organization for slow swimming, however,
giant neurons are involved in this and in a faster escape response, with two
distinct kinds of action potentials in the motor giants serving as the muscle
input that determines the type of subumbrellar contraction [slow or escape
(Mackie and Meech, 1985;
Meech and Mackie, 1993;
Mackie, 2004)].
In many of the disc-shaped leptomedusae, including Aequorea,
radial muscle is not restricted to discrete bands, but rather forms sheets
that overlie the circular swim muscle
(Satterlie and Spencer, 1983).
Radial responses thus rely on more diffuse radial musculature
(Fig. 13), possibly because of
the challenge of the inward curling of the wide, oblate bell. A unique
behavioural peculiarity of Aequorea, with its large number of
subumbrellar segments, centres on its ability to invert a variable number of
segments, and to inhibit swimming in those segments only
(Satterlie, 1985a). Although
the behavioural significance of this is obvious, the mechanisms required to
turn off swimming in a variable portion of the subumbrella, with the basic
swim system organization mentioned above, is not so simple.
The excitability of the subumbrellar circular muscle sheet of Aequorea appears to be less than that of `normally' triggered contractions in Polyorchis. In the former species, muscle activity that spreads from one subumbrellar segment to another shows a decrease in amplitude and rise time characteristic of decremental transmission through gap junctions. For normal swimming, this electrotonic current spread could require `supplemental' synaptic input from a peripheral nerve net to bring muscle cell electrogenesis to that of full action potentials. A full contraction in a subumbrellar circular muscle cell would thus require a combination of electrotonic current spread and synaptic input.
Evidence for a subumbrellar motor nerve net (in addition to the electron microscopical data) includes both electrophysiological and immunohistochemical data. Although the motor network in the inner nerve ring produces a burst of action potentials per swim, and this is reflected in the junctional potentials seen in the postsynaptic epithelial cells, the synaptic inputs to circular muscle cells throughout each subumbrellar segment consist of a single junctional potential. This holds for normally initiated swims and for contractions initiated by direct electrical stimulation of the subumbrellar tissue. In the latter case, stimulation of any point of a subumbrellar segment produces a junctional potential and contraction in the muscle cells of that segment, indicating a non-directional transmission in the putative nerve net.
Immunohistochemical experiments with a commercial FMRFamide antibody show
the presence of a subumbrellar nerve net. Neuronal RFamides appear to be
ubiquitous within the Cnidaria
(Grimmelikhuijzen, 1983;
Grimmelikhuijzen et al.,
1996), and they are neuroactive
(Spencer, 1988). The presence
of an FMRFamide-immunoreactive nerve net in Aequorea has to be viewed
with caution, however, since data from a number of other hydromedusae suggest
that RFamide-immunoreactive subumbrellar networks are associated with radial,
smooth muscle rather than the striated swim musculature
(Grimmelikhuijzen and Spencer,
1984; Mackie et al.,
1985). Perhaps the best data, in this regard, come from the
anthomedusa, Podocoryne, where double labelling with an antibody
specific for cnidarian smooth muscle was coupled with a FMRFamide antibody to
directly show the structural correlation
(Weber, 1989). However, in
their comparative study of several hydromedusae, Mackie et al.
(Mackie et al., 1985) found
the density of subumbrellar neurites exceeded the density of
FMRFamide-immunoreactive neurons, suggesting the possibility of multiple,
separate nerve nets in the subumbrellar ectoderm. In Aequorea, this
possibility is backed up by data from double labelling experiments, which
clearly show the presence of two separate nerve nets in the subumbrellar
segments, only one of which stains with the FMRFamide antibody. This is
currently under further investigation in Aequorea using electron
microscopical examination of immunohistochemical preparations stained for
FMRFamide.
The extreme variability in muscle cell action potential size and shape in
Aequorea is not always completely `predictable' in either a normal
swimming preparation or in one that is undergoing a spontaneous or triggered
radial response. Current evidence suggests several neuromuscular properties of
the subumbrellar motor nerve net may contribute to this level of
electrophysiological (and contractile) variability. First, synchrony of inputs
to the subumbrellar muscle cells may be important in production of full muscle
electrogenesis. If an isolated muscle cell is depolarized, the surrounding,
coupled muscle cells may represent a current sink that could decrease the
amplitude and slow the rise time of the junctional potentials, thus decreasing
their efficacy. Simultaneous depolarization of all neighbouring muscle cells
would significantly decrease the current sink effect. Synchrony of activity
within the inner nerve ring motor network of Polyorchis was found to
influence action potential shape (Spencer,
1981), and the same could be true for the subumbrellar muscle
cells of Aequorea. Any inhibitory input that decreases the synchrony
of synaptic inputs to the muscle sheet could alter the amplitude and rise time
of muscle action potentials. This was found in the postsynaptic epithelial
cells of Aequorea during radial responses, where a delayed production
of motor network spike bursts produced smaller postsynaptic action potentials
with slow rise times compared to swims with unaltered synchrony
(Satterlie, 1985b). It may
also explain the slight delay between the peak of the junctional potentials
and the rise of the action potentials seen in
Fig. 6B (compared to the full
action potential in Fig. 6A),
and more clearly in the three full action potentials in
Fig. 12A.
Second, the subumbrellar nerve nets of Aequorea show a distinct
preferential orientation (radially; Fig.
10). This could also reflect a preferred physiological orientation
as well, particularly if the overall pathway in one direction is restricted,
as seen in the inter-segmental regions because of the radial canal gonadal
tissue. Restriction of conduction is suggested by the data in
Fig. 9. As an extreme case, a
nerve net could be through-conducting in the preferred direction (radial in
this case) and incrementally conducting in the perpendicular (circular)
direction. This could show up as a labile conduction of repetitive events in
the nerve net, including failures and re-appearances. An incrementally
conducting nerve net has been described in an anthozoan
(Anderson, 1976). Also
possible, however, are alterations in conduction patterns in a
through-conducting nerve net that merely alter the synchrony of activity
within the nerve net with consequences as previously mentioned.
Third, the action potentials of swim muscles of Aequorea are
variable in amplitude even during normal swimming bouts (see
Fig. 7). In a rested
preparation, a swimming bout is initiated by epithelial (and muscle) action
potentials that increase in amplitude with the first several swimming
contractions, reminiscent of neuromuscular facilitation
(Satterlie, 1985b). Despite
this, no significant correlation could be demonstrated between action
potential amplitude and the preceding inter-spike interval, suggesting that a
strong frequency-dependent facilitation is not in operation (unpublished
observations). Yet it appears that muscle cell electrogenesis is influenced by
the electrophysiological history of the cell, and this could contribute to
some of the observed variability.
Finally, as an unexplored possibility, the swim muscle cells may be
directly sensitive to the neuroactive substance (presumed an RFamide) released
from the FMRFamide-immunoreactive neurons during a radial response. In this
scenario, the nerve net associated with the radial muscle would have opposing
effects on the two muscle types. Thus two levels of chemical inhibition could
be acting on the swim system during a radial response: direct inhibition of
the inner nerve ring motor network
(Satterlie, 1985a;
Satterlie, 1985b) and a
peripheral modulatory action on the muscle cells. Thus far, permeability
difficulties posed by the tight epithelium of the subumbrella have prevented
direct testing of this possibility.
A combination of these nerve net and neuromuscular properties, in conjunction with the electrical coupling parameters of the swim muscle sheet, may be required to explain the full range of variability seen in the swim muscle cell recordings during normal swimming and during radial responses. In any event, the `turning off' of restricted subumbrellar segments appears to be based on both central (nerve ring) and peripheral (subumbrellar) mechanisms. Add to this the ability to totally inhibit activation of the subumbrellar nerve net (as shown in Fig. 12), sometimes with action potentials of apparently electronic origin, and it is apparent that a flexible range of muscle force suppression/inhibition can be achieved in this system.
As a final word on the swimming system of hydromedusae, the organization of
the motor network of the inner nerve ring is influenced by parallel sensory
networks of the inner and outer nerve rings
(Spencer and Arkett, 1984;
Arkett and Spencer, 1986a;
Arkett and Spencer, 1986b;
Arkett et al., 1988;
Mackie, 2004). Furthermore,
fourteen distinct conducting systems have been described in Aglantha
(reviewed by Mackie, 2004). In
Aequorea, multiple statocysts are found throughout the bell margin
(Singla, 1975), and righting
responses to tilt involve asymmetrical swimming contractions of the
subumbrella, possibly including the local inhibitory mechanisms discussed
here. These observations suggest that parallel and point-source pathways
provide excitatory or inhibitory input to the swim motor network in the inner
nerve ring of hydromedusae, pointing to an integrative organization that forms
what can be argued to be a true central nervous system in these radially
symmetrical organisms. In these animals, the `primitive' condition of diffuse,
non-directional nerve nets has been modified in some conducting systems into a
series of compressed, function-specific nerve networks that interact in the
complex neural structures of the inner and outer nerve rings. As an example of
the richness of function of the hydromedusan nervous system, one has to look
no further than the organization of Aglantha
(Mackie, 2004) to be impressed
with the neural complexity of this `simple' animal group.
|
Acknowledgments |
|---|
-tubulin antibody developed by J. Frankel and E.
M. Nelsen was obtained from the Developmental Studies Hybridoma Bank developed
under the auspices of the NICHD and maintained by The University of Iowa,
Department of Biological Sciences, Iowa City, IA 52242, USA. |
References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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