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First published online March 2, 2007
Journal of Experimental Biology 210, 1025-1035 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02717
Temperature dependent modulation of lobster neuromuscular properties by serotonin
Department of Neuroscience, University of Virginia Health Science Center, Charlottesville, VA 22908-0230, USA
* Author for correspondence (e-mail: mkw3k{at}virginia.edu)
Accepted 21 December 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: neuromuscular, serotonin, lobster, crustacean, muscle tension, temperature
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). In the lobster
Homarus americanus serotonin acts both as a neurotransmitter and a
circulating hormone to modulate skeletal and cardiac neuromuscular systems
(Glusman and Kravitz, 1982;
Goy and Kravitz, 1989;
Goy et al., 1984;
Heinrich et al., 1999;
Huber et al., 1997;
Kravitz, 1988;
Kravitz et al., 1980;
Livingstone et al., 1980;
Worden et al., 2006;
Worden et al., 1995). However,
the environmental temperature surrounding the lobster is another major factor
that influences lobster physiology and behavior. Like other invertebrates
lobsters are cold blooded, and their internal body temperature closely follows
that of the surrounding water [see fig. 2 of Worden et al.
(Worden et al., 2006)].
Estuary and coastal water temperatures within the habitat of H.
americanus vary over a 25°C range according to the winds, the tides
and the seasons (Cowan et al.,
2007; Lawton and Lavalli,
1995; Manning,
2005). Temperature influences multiple parameters of lobster
biology, including growth and reproduction
(Ennis, 1995;
Waddy et al., 1995), cardiac
and respiratory performance and its thermal limits
(Camacho et al., 2006;
Worden et al., 2006), and
locomotion into lobster traps (Drinkwater
et al., 2006). Lobsters can detect thermal change with exquisite
resolution, exhibiting alterations in heart rate in response to thermal
changes as small as 0.15°C (Jury and
Watson, 2000).
The goal of this study was to examine how changes in environmental
temperature might alter the effectiveness of neurohormonal modulation of
neuromuscular transmission and muscle physiology. A number of previous studies
have characterized the temperature dependence of synaptic output and muscle
movements in crustacean neuromuscular systems
(Fischer and Florey, 1981;
Harri and Florey, 1977;
Harri and Florey, 1979;
Stephens, 1990;
Stephens and Atwood, 1982).
However, surprisingly little is known about how temperature might alter the
sensitivity of neuromuscular systems to endogenous neuromodulatory hormones.
In the crayfish skeletal phasic abdominal neuromuscular system the
neurohormone DF2 potentiates synaptic transmission much more
strongly at cold temperatures compared to warm
(Friedrich et al., 1994),
suggesting that hormonal modulation might help compensate for low synaptic
output in part of the temperature range inhabited by the animal in
vivo.
Compensating for temperature effects on synaptic transmission may be
particularly important in species that spend considerable periods of time
living at temperature extremes. H. americanus overwintering in the
Gulf of Maine waters, for example, spend 6 months of each year at frigid
temperatures <5°C (Cowan et al.,
2007). H. americanus are currently in high abundance in
the Gulf of Maine [Atlantic States Marine Fisheries Commission
(ASMFC, 2005)], suggesting that
colder temperatures are favorable for this species in the wild. In contrast,
the same trawl and landings data show that fishing mortality is high and
lobster abundance is low in warmer waters south of New England, particularly
in Long Island Sound, where bottom water temperatures can average 20°C in
August and September (Howell et al.,
2005) and the commercial fishery has collapsed. Understanding the
thermal limits of lobster physiological function will be important for
predicting the environmental consequences of global warming for the health and
survival of this commercially valuable species.
In this study we examine how the environmental temperature and thermal change affects neurotransmission and contraction in the lobster dactyl opener skeletal muscle. We demonstrate that neuromuscular function is strongly temperature dependent and that 5-HT modulates the system to extend the effective temperature range for neuromuscular function.
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). The
first or second walking legs were dissected in chilled saline (462 mmol
l–1 NaCl, 16 mmol l–1 KCl, 26 mmol
l–1 CaCl2, 8 mmol l–1
MgCl2, 11 mmol l–1 glucose and 5 mmol
l–1 Hepes buffer adjusted to pH 7.4). Part of the exoskeleton
and the entire dactyl closer muscle were removed from the propopodite segment
of the leg to expose the dorsal surface of the dactyl opener muscle. The
specific opener inhibitory (OI) axon and the opener excitatory (OE) axon
innervating the dactyl opener were exposed in the meropodite. The axon of the
common inhibitory motoneuron (CI) also provides innervation to all the fibers
of the lobster dactyl opener (Worden and
Camacho, 2006), but we did not examine its physiological effects
in this study.
After dissection, the preparation was pinned to SylgardTM in the
bottom of a 1.5 ml saline-filled chamber made of resin, through which flowed
refrigerated coolant. Saline was superfused over the preparation at a rate of
1–2 ml min–1; the tubing through which this saline
flowed was also chilled by refrigerated coolant. Using this system, the
temperature of the bath could be regulated over the range 1–23°C. In
experiments in which temperature was varied, the rate of change of bath
temperature was approximately 0.67°C min–1. All
experiments began at 2°C and the temperature was warmed over the course of
the experiment. In experiments examining the temperature dependence of 5-HT
effects, the following protocol was used. The bath was warmed from 2°C to
20°C while physiological measurements were made in control saline. The
bath was then chilled to 2°C. Serotonin (5-HT) (Sigma Chemical, St Louis,
MO, USA) was added to the saline superfusing the preparation at 2°C and
the effects monitored for at least 15 min to ensure that intracellular
responses and neurally evoked contractions had stabilized. Finally, the bath
was warmed again from 2°C to 20°C while physiological measurements
were repeated. The concentrations of 5-HT used in these experiments were in
the range 50–100 nmol l–1, approximately one order of
magnitude higher than the concentration found in lobster hemolymph under
resting conditions (Livingstone et al.,
1980).
Electrophysiological and tension recording
Using suction electrodes, excitatory junction potentials (EJPs) and
inhibitory junction potentials (IJPs) were elicited in the muscle by selective
stimulation of the axons of the dactyl opener excitatory motoneuron (OE) and
specific opener inhibitory motoneuron (OI), which run separate courses in the
meropodite. Synaptic responses were recorded intracellularly in areas of the
muscle in which IJPs appeared largest; these included fibers in the most
proximal region of the muscle as well as the most proximal fibers of the
central region. Intracellular recordings were performed using 3–5
M
pipettes filled with 3 mol l–1 potassium acetate.
Changes in muscle membrane potential were recorded differentially between the
microelectrode and the bath, grounded through a silver/silver chloride
electrode, and amplified by an Axopatch-1D amplifier (Axon Instruments, Union
City, CA, USA). Input resistance was measured by determining the change in
muscle membrane potential elicited by a hyperpolarizing current injection.
Tension was measured almost isometrically by attaching a surgical thread
from the apodeme of the dactyl opener muscle to a tension transducer (model
FT03, Grass Instruments, West Warwick, RI, USA) connected to an amplifier
(Cyberamp 320, Axon Instruments). At the start of each experiment muscles were
stretched to a degree that resulted in 2 g tension at 2°C. Outputs from
the microelectrode and the tension transducer were digitized using a Digidata
1200 interface (Axon Instruments) at sampling rates of 2.9–13.3 kHz,
recorded on VCR tape via a digital data recorder (Model 10B,
Instrutech Corp., Port Washington, NY, USA), and analyzed on- and off-line
using pClamp software (versions 6.0-7.0, Axon Instruments). Tension recordings
were filtered at 300 Hz and calibrated in grams (g). In this study we examined
muscle contractions stimulated by the excitatory motoneuron (OE) as well as
muscle relaxations stimulated by the specific inhibitory motoneuron (OI). Both
inhibitory motoneurons that innervate the dactyl opener (OI and CI) can
inhibit muscle contractions stimulated by the excitatory motoneuron OE
(Worden and Camacho, 2006),
but we did not explore these physiological effects in this study.
Data analysis
In some experiments, stimulation of the excitatory and/or the specific
inhibitory motoneuron innervating the dactyl opener muscle was applied at a
constant low frequency (0.5–1 Hz), and the amplitude of junction
potentials was determined by averaging recordings from ten consecutive trials.
The effect of 5-HT on junction potential amplitude was determined by comparing
the average of ten trials in the presence of 5-HT to control responses at the
same temperature. EJP amplitudes were corrected for non-linear summation
according to the equation
Ejp'=Ejp/(1–Ejp/Er)
(Martin, 1955) where the
corrected value for the size of the EJP (Ejp') is determined
from the measured value of the EJP (Ejp) and the reversal potential
(Er) of the synaptic current for the opener excitor motoneuron,
assumed to be +11.5 mV as demonstrated in crayfish
(Onodera and Takeuchi, 1975).
In experiments in which IJPs were elicited by stimulus trains, the amplitude
of the last IJP in the inhibitory postsynaptic response was measured with
respect to baseline. In some experiments, muscle movements were elicited by
applying brief (200–600 ms) stimulus trains (10–30 Hz) to the
axons of the motoneurons. In each experiment, identical stimulus protocols
were used under control conditions and in the presence of 5-HT. Neurally
evoked muscle contractions and relaxations were measured by averaging ten
consecutive tension responses to stimulus trains and determining the peak of
the contraction, or relaxation, with respect to the baseline tension. Resting
muscle tension was determined as the level of muscle tension during periods
when motoneurons were not being stimulated and any muscle movements due to
prior motoneuron stimulation had decayed. Graphing and statistical analysis
were performed using Origin Software (Microcal Software, Inc., Northampton,
MA, USA). All quantitative data are reported as means ± s.e.m., if not
indicated otherwise.
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Results |
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Serotonin enhances EJPs and twitch contractions
Serotonin (5-HT) potentiates both EJP amplitude and neurally evoked
contractions of lobster dactyl opener muscle
(Fig. 3A). Its potentiating
effects on EJPs have a threshold of approximately 1 nmol l–1,
and saturate at approximately 1 µmol l–1
(Fig. 3B). Serotonin does not
affect the muscle resting potential significantly; 5 nmol l–1
and 50 nmol l–1 serotonin change muscle resting potential by
2.0±2.1 mV (mean ± s.d.; N=6) and –0.8±3.0
mV (N=27), respectively. The potency of serotonin's effect on EJP
amplitude strongly depends on the size of the EJP before serotonin is added;
small (<1 mV) EJPs are potentiated more strongly than larger (>2.5 mV)
EJPs (Fig. 3C). Analysis of
data from 15 experiments in which 5-HT potentiated EJP amplitude over a
temperature range of 2–20°C showed no evidence for a
temperature-dependent effect of 5-HT on EJP (data not shown), in agreement
with data in Fig. 3C showing
that serotonin's effect on excitatory neurotransmission is similar at 2°C
and 16°C (Fig. 3C).
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Serotonin increases both muscle relaxation and the temperature range over which IJPs are effective in relaxing muscle
To examine the temperature dependence of inhibitory neuromuscular function,
trains of IJPs were elicited and the corresponding motor movements measured as
a function of temperature. 5-HT increases IJP amplitude as well as the
magnitude of the IJP-evoked muscle relaxations
(Fig. 4A,B). In addition, 5-HT
shifts the temperature at which IJPs reverse polarity to warmer temperatures
(Fig. 4B), enabling relaxation
in a temperature range where IJPs would otherwise be depolarizing and
ineffective in triggering relaxation. For example, in the preparation
illustrated in Fig. 4B, muscle
relaxations could not be evoked in the absence of 5-HT at temperatures
>4°C where IJPs were depolarizing. However, in the presence of 5-HT,
the IJP reversed polarity at 9°C and relaxations could be observed up to
temperatures <8°C. The traces in
Fig. 4C demonstrate that IJPs
are depolarizing under control conditions but hyperpolarizing in the presence
of 5-HT when recorded in the same muscle fiber at 6°C. Data averaged from
four experiments examining muscle relaxation are shown in
Fig. 4D. 5-HT most effectively
potentiates muscle relaxations at the coldest temperature tested (by an
average of 17.6-fold at 2°C) and enables neurally evoked relaxations of
muscle at temperatures between 4°C and 10°C.
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To test whether 5-HT might change the reversal potential for GABA-gated chloride flux through the postsynaptic muscle membrane the temperature and membrane potential at which IJPs reversed polarity were measured in the presence and absence of 5-HT. Compared to that measured under control conditions, 5-HT (100 nmol l–1) shifts the temperature at which IJPs reverse from 6.6±2.6°C (mean ± s.d.) to 13.2±6.1°C (N=8; P<0.05 in a paired t-test) without changing the membrane potential at which the IJP reverse (–64.8±8.7 mV compared to –66.5±7.6 mV; P>0.05). To control for the possibility that the temperature of IJP reversal might shift as a result of warming the preparation twice (see Materials and methods), another set of experiments was performed in the absence of 5-HT. In the control experiments, warming the preparation twice did not significantly change the temperature at which IJPs reversed (7.0±3.5°C versus 5.3±2.7°C (N=6; P>0.05) (see below).
The increase in IJP amplitude elicited by 5-HT has been reported to be due
to an increase in GABA release (Vyshedskiy
et al., 1998); however, it is also possible that 5-HT increases
the driving force for chloride flux through the GABA-gated channel. To test
this possibility, the effects of 5-HT on resting membrane potential were
measured. At 2°C, 100 nmol l–1 5-HT depolarized muscle
fibers by a mean (± s.d.) value of 4.8±4.0 mV (N=8)
(Fig. 5).
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The potentiation of resting muscle tension and neurally evoked contractions by serotonin depends differentially on temperature
The temperature dependence of 5-HT modulation of neurally evoked
contractions was examined by stimulating trains of EJPs and monitoring the
corresponding contractions of the muscle. Results from a typical experiment
are illustrated in Fig. 6A, and
data pooled from multiple experiments are shown in
Fig. 6B,C. Under control
conditions, neurally evoked contractions evoked with brief stimulus trains are
maximal at 2°C and are virtually abolished as the temperature approaches
18°C (lower traces of Fig.
6A, open symbols in Fig.
6B). In the presence of 5-HT, neurally evoked contractions are
potentiated over the entire temperature range (upper traces of
Fig. 6A, solid symbols in
Fig. 6B). Quantitatively, the
effect of 5-HT in potentiating neurally evoked contractions was significantly
stronger at 18°C than it was at other temperatures
(Fig. 6C). This result is
consistent with the raw data traces shown in
Fig. 6A, which show that at
18°C 5-HT enabled large neurally evoked contractions that were nearly
undetectable under control conditions.
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To examine whether 5-HT might directly modulate muscle independently of its modulatory effects on neurotransmission, resting muscle tension was monitored in the absence of neural input both in the presence and absence of 5-HT. Results from a typical experiment are shown in Fig. 7. During the first 30 min in control conditions, resting muscle tension decreases by half from its original value of 2 g (Fig. 7B) as temperature increases from 2°C to 22°C (Fig. 7A). This effect reverses as the temperature cools back to 2°C (over 30–100 min). 5-HT (hatched bar above Fig. 7A) added to the bath at a constant temperature of 2°C increases muscle tension significantly to 2.8 g (over 100–117 min). As temperature warms to 22°C in the presence of 5-HT, muscle tension decreases to reach a value at 22°C comparable to that observed at 22°C under control conditions. Finally, as the temperature chills back to 2°C (160–210 min), muscle tension recovers nearly to its potentiated value of 2.8 g. Thus, in this experiment, 5-HT maximally increased resting muscle tension at the coldest temperature of 2°C and had almost no effect at 22°C.
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The temperature dependent modulation of resting muscle tension by 5-HT was highly reproducible. Fig. 7C shows results from seven preparations in which the experimental protocol shown in Fig. 7A was repeated. Under control conditions muscle tension decreased significantly (by an average of 26.9%) as temperature warmed from 2°C to 22°C (Fig. 7B) and recovered when the muscles were chilled to 2°C. Adding 50 nmol l–1 5-HT (hatched bar in Fig. 7C) at 2°C significantly increased muscle tension (by an average of 46.9%). As the temperature warmed from 2°C to 22°C in the presence of 5-HT muscle tension relaxed, becoming significantly lower (by an average of 28.2%) than it had been at 2°C before 5-HT was added and approximately the same as it was at 22°C in the absence of 5-HT. Thus, 5-HT strongly potentiates resting muscle tension at the coldest temperatures, and has no effect on muscle tension at the warmest temperature.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). Here we describe the temperature-dependent
physiology of the lobster dactyl opener muscle and its modulation by the
circulating neurohormone serotonin (5-HT). The lobster dactyl opener is a
popular model system for studies of synaptic plasticity
(Bykhovskaia et al., 1999;
Bykhovskaia et al., 2001;
Bykhovskaia et al., 2004;
Glusman and Kravitz, 1982;
Goy and Kravitz, 1989;
Hamilton et al., 2006;
Kravitz et al., 1980;
Vorob'eva et al., 1999;
Worden et al., 1997), as is
the homologous crayfish dactyl opener muscle
(Beaumont and Zucker, 2000;
Delaney et al., 1991;
Dixon and Atwood, 1989a;
Dixon and Atwood, 1989b;
Qian and Delaney, 1997;
Vyshedskiy et al., 1998;
Vyshedskiy and Lin, 1997;
Wang and Zucker, 1998). We
demonstrate that (1) thermal change within a biologically relevant range of
temperatures profoundly affects the neurophysiological properties of the
lobster dactyl opener neuromuscular system, and (2) the modulatory effects of
serotonin on neuromuscular function have distinctly different temperature
dependencies and extend the thermal range over which motor movements are
effective. Fig. 8 summarizes
the temperature dependence of lobster neuromuscular function and behavior over
the biologically relevant temperature range for H. americanus in the
wild.
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) and
the crayfish claw opener and stretcher muscles
(Jacobs and Atwood, 1981a;
Jacobs and Atwood, 1981b).
Further, the temperature dependence of both the resting membrane potential and
the muscle input resistance in the dactyl opener
(Fig. 1D,E) is similar to that
reported in previous studies of neuromuscular systems in crayfish
(Harri and Florey, 1977;
Harri and Florey, 1979;
Fischer and Florey, 1981),
lobster (Colton and Freeman,
1975b) and crab (Stephens,
1990; Stephens and Atwood,
1982).
Our results differ from those reported in previous studies, however, in
that the pattern of temperature dependence of EJP amplitude is biphasic with
two temperature optima: EJP amplitudes are maximal both in the temperature
range from 14–18°C and at very cold temperatures (around 2°C)
(Fig. 1A,B). Other studies of
crayfish muscles report different patterns of temperature dependence of EJPs.
EJP amplitudes are maximal at 2°C but progressively smaller as the
temperature warms in dactyl opener muscle
(Fischer and Florey, 1981;
White, 1983), whereas they
increase in size over the temperature range 10–20°C in deep
abdominal extensor muscle (Dunn and
Mercier, 2003).
The cellular mechanism underlying the temperature dependence of EJP
amplitude in lobster dactyl opener muscle cannot be explained by the
temperature dependence of the muscle input resistance (compare
Fig. 1B,E). However, there are
several other possibilities. For example, the synaptic conductance underlying
the EJP may change as a function of temperature, as suggested by the
observation that the ionic response of lobster muscle to bath-applied
glutamate is relatively more selective for sodium at 3°C compared with
18°C (Colton and Freeman,
1975a) and by reports that sodium influx is critical for
hormone-induced membrane potential oscillations at 15°C but not at
21°C in a lobster stomatogastric neuron
(Johnson et al., 1992).
Another factor is that the time course for neurally evoked synaptic vesicle
release depends on temperature. Quantal neurotransmission at dactyl opener
muscle synapses is asynchronous at cold (<5°C) temperatures
(Bykhovskaia et al., 1999;
Worden et al., 1997) and
becomes more synchronous at warmer temperatures. Finally, it is also possible
that temperature regulates the relative amount of neurotransmitter released
from synaptic terminals, as demonstrated in crayfish
(Dunn and Mercier, 2003).
Since IJPs in the lobster dactyl opener result from the release of GABA
from the inhibitory motoneurons (Otsuka et
al., 1966), the temperature dependence of the IJP amplitude and
polarity can be understood in terms of the driving force for chloride flux
through the GABA-gated chloride channel. At the coldest temperatures where the
resting potentials are relatively depolarized (see
Fig. 1D), we suspect that IJP
amplitudes are large and hyperpolarizing because the driving force for
chloride influx is large. Cold (2°C) temperatures have been used
experimentally in studies of inhibitory neurotransmission in the dactyl opener
because these temperatures enhance the signal-to-noise ratio for inhibitory
synaptic signals and favor large inhibitor-evoked muscle relaxations
(Worden and Camacho, 2006). As
temperature warms to approximately 6°C and the muscle fibers
hyperpolarize, the resting membrane potential approaches the reversal
potential for chloride flux when the IJP becomes indistinguishable from the
baseline (see data at 6°C in Fig.
1A). At warmer temperatures, IJPs are depolarizing, presumably
because the muscle resting potential become more negative than the reversal
potential for chloride flux. Notably, depolarizing IJPs do not generate motor
movements, contradicting the idea that the classical inhibitory signals
generated by OI in the dactyl opener will act as `excitatory inputs' under
experimental conditions where IJPs are depolarizing
(Segev and Parnas, 1983). The
decline in amplitude of IJPs and EJPs at temperatures warmer than 15°C may
reflect several factors, including a temperature-dependent reduction of
transmitter output, a decrease in muscle fiber input resistance and the
metabolic stress of warm temperatures for this species
(Chang et al., 1998).
Finally, it is important to note that many aspects of physiology are
temperature dependent in crustacea. For example, thermal change alters the
firing rate of specific lobster neurons
(Konishi and Kravitz, 1978),
the release of hormones from lobster neurosecretory organs
(Kuramoto and Tani, 1994), the
strength of long-term facilitation at crayfish neuromuscular synapses
(Jacobs and Atwood, 1981a;
Jacobs and Atwood, 1981b), the
physiological properties of crayfish gap junctional synapses
(Heitler and Edwards, 1998),
and the conduction velocity of action potentials in crabs
(Young et al., 2006).
Temperature dependent modulation of neuromuscular movements by serotonin
In agreement with previous studies of the lobster dactyl opener muscle
(Kravitz et al., 1980;
Glusman and Kravitz, 1982;
Goy and Kravitz, 1989;
Worden et al., 1995) 5-HT has
multiple modulatory effects on the dactyl opener neuromuscular preparation.
Interestingly, although 5-HT modulated neurally evoked contractions and
relaxations as well as resting muscle tension in temperature-dependent ways,
it potentiated excitatory neurotransmission in a temperature-independent
manner. In contrast, the neuromodulatory effect of peptide DF2 on
crayfish phasic abdominal extensor muscles is three times as strong at
7–9°C as it is at 15–17°C
(Friedrich et al., 1994), an
effect that has been attributed to the greater ability of the peptide to
increase calcium influx into presynaptic terminals of the motoneurons at low
temperatures (Dunn and Mercier,
2003).
However, it is important to note that the electrophysiological results
reported in this study were collected mainly in the most proximal fibers of
the dactyl opener muscle. Different regions of the dactyl opener have fibers
with different properties (Mykles et al.,
2002) and we cannot rule out the possibility that the central and
distal fibers of the lobster dactyl opener may exhibit different synaptic
responses to serotonin and/or temperature. On the other hand, our tension
recordings reflect the physiological output of all the fibers of the entire
muscle. Therefore, the thermal and hormonal modulatory effects we observe on
contractility are characteristic of the output of the dactyl opener as a
whole.
In the presence of 5-HT, neurally evoked contractions are particularly
large not only at 2°C but in the temperature range 12–16°C,
which includes the thermal preferendum of lobsters allowed to freely move in
thermal gradients (16°C) (Crossin et
al., 1998). Quantitatively, the maximal potentiation of
contractions by 5-HT occurs at 18°C, a temperature that nearly abolished
neurally evoked contractions triggered by the brief stimulation protocols
employed in this study. Thus 5-HT extends the upper thermal limit for neurally
evoked contractions of the dactyl opener muscle. In contrast, the potentiating
effect of serotonin on the strength and frequency of the lobster heartbeat
does not depend on temperature (Worden et
al., 2006).
In dactyl opener skeletal muscle 5-HT also acts by shifting the temperature
at which IJPs elicited by the specific inhibitory motoneuron reverse polarity
to warmer temperatures, thereby extending the upper limit of the temperature
range over which the specific inhibitor acts to actively relax resting muscle
tension. In agreement with an earlier report
(Kravitz et al., 1980), we
find that 5-HT does not modulate the reversal potential for chloride flux
through the GABA channel. Transient neurally evoked muscle relaxations [which
can be triggered by both of the inhibitory motoneurons innervating the dactyl
opener (Worden and Camacho,
2006)] have also been observed in crayfish muscle [for example,
see fig. 12 of Harri and Florey (Harri and
Florey, 1977)], and may serve in vivo to facilitate
contractions of the antagonist dactyl closer muscle and movement of the
dactyl. These relaxations may be particularly important under the frigid
(<5°C) environmental conditions endured by some lobster populations for
over 6 months each year in the wild (Cowan
et al., 2007), because in this thermal range resting muscle
tension is high and the effects of 5-HT in potentiating resting muscle tension
are strong (see Fig. 7). It is
interesting to speculate that a relatively high level of resting muscle
tension at cold temperatures may be physiologically adaptive for tonic muscles
in that it permits sustained contractions under thermal conditions where
metabolism is significantly slowed and the energetic costs of neural
electrical activity are therefore high.
Modulation of muscle tone by temperature and by serotonin
Contraction of crustacean striated muscle is proportional to the level of
muscle fiber depolarization (Harri and
Florey, 1977; Orkand,
1962), but the mechanism by which resting muscle tension is
generated is poorly understood. An early suggestion that the relatively
depolarized muscle membrane potentials recorded at cold temperatures might be
sufficient to exceed the threshold for excitation–contraction
(e–c) coupling (Harri and Florey,
1977) was contradicted by a subsequent report that the threshold
for e–c coupling in crayfish dactyl opener muscle is both temperature
independent and at least 15 mV more depolarized than the resting membrane
potential over the entire temperature range [see fig. 3 of Fischer and Florey
(Fischer and Florey, 1981)].
It is unlikely that resting muscle tension in the lobster dactyl opener is due
to calcium influx through voltage-dependent ion channels because the threshold
for the voltage-dependent calcium current is –35 to –45 mV, which
is approximately 20 mV more depolarized than the resting potentials of the
muscle fibers (Kravitz et al.,
1980) (and see Fig.
1D).
Our observation that 5-HT strongly modulates resting muscle tension at cold
temperatures (see Fig. 7B,C) is
also difficult to understand in terms of an effect on ion channels. Although
5-HT-mediated increases in the resting muscle tension of dactyl opener muscle
depend on extracellular calcium levels
(Kravitz et al., 1980), 5-HT
changes neither the threshold for e–c coupling in crayfish dactyl opener
(Fischer and Florey, 1983) nor
the threshold for the voltage-dependent calcium current in lobster dactyl
opener (Kravitz et al., 1980).
Resting muscle tension in crayfish superficial extensor muscle also increases
at colder temperatures (Quigley and
Mercier, 1997), although it has not been reported whether
neuromodulators are more effective at modulating tension at colder
temperatures. It is likely that one or more muscle membrane conductances in
the dactyl opener is temperature dependent, given that muscle input resistance
is highest at the coldest temperatures. Thermosensing ion channels have been
described previously both in invertebrate and in vertebrate species
(Viswanath et al., 2003).
Given the muscle fiber heterogeneity in the dactyl opener muscle
(Mykles et al., 2002), one
confounding issue is whether muscle tone and changes in tone might arise from
the action of a small number of fibers of a special type. In order to
elucidate the physiological pathway linking membrane excitation and tension it
will be important to measure electrophysiological and contractile properties
at the level of single muscle fibers and to determine the mechanism by which
temperature alters the muscle resting membrane potential. Previous studies of
crustacean muscles have suggested that temperature might alter the activity of
sodium pump or the relative permeability of the muscle to potassium and sodium
ions (Florey and Hoyle, 1976;
White, 1983). In addition,
temperature may alter the activity of the Na+/Ca2+
exchanger, which has previously been implicated as a mediator of tonic muscle
contractions (Goblet and Mounier,
1982; Mounier and Goblet,
1987). Elucidating the cellular mechanism(s) mediating resting
muscle tension will be an important first step in understanding how muscle
contractility can be modulated by temperature and by 5-HT.
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Acknowledgments |
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