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First published online March 12, 2009
Journal of Experimental Biology 212, 1021-1031 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.023507
Movement encoding by a stretch receptor in the soft-bodied caterpillar, Manduca sexta
Department of Biology, Tufts University, Medford, MA 02155, USA
* Author for correspondence (e-mail: Michael.Simon{at}tufts.edu)
Accepted 6 January 2009
 |
Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: locomotion, Manduca sexta, mechanosensors
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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)],
signal inspiration and lung volume [e.g. in birds
(Bouverot, 1978); in mammals
(Paintal, 1973)], coordinate
the timing and magnitude of muscle force generation during walking
(Stein et al., 2000) and
compensate for perturbations in gait due to ground irregularities and other
external factors (Dietz et al.,
2002). Stretch receptors also take a variety of forms in
invertebrates. The muscle receptor organ (MRO) of Cherax destructor,
for example, consists of pairs of stretch receptors that coordinate slow
abdominal movement and maintain tail position
(Fields and Kennedy, 1965) and
engage abdominal extension following the tail-flip response
(Cattaert and Le Ray, 2001).
Abdominal stretch receptors in the mosquito
(Gwadz, 1969) and Rhodnius
prolixus (Anwyl, 1972)
regulate feeding behavior to prevent abdominal rupture during blood meals.
And, motoneurons coordinating dorsal–ventral sinusoidal movement in the
nematode, Caenorhabditis elegans, appear to be stretch sensitive,
suggesting a role in coordinating forward and backward locomotion
(O'Hagan et al., 2005;
Syntichaki and Tavernarakis,
2004).
Invertebrate stretch receptors can be anatomically classified as either
muscle-associated or non-muscle-associated. One functional distinction between
the two types is that the response of muscle-associated stretch receptors can
be modulated in the periphery by efferent feedback. In the crustacean MRO,
pairs of innervated receptor muscles provide feedback on extensor muscle
movement, with separate `fast' and `slow' fibers responding quickly or slowly
to motoneuron activation and adapting quickly or slowly to changes in stretch
position, respectively (Alexandrowicz,
1967; Kuffler,
1954). Slow fibers respond tonically to degrees of stretch whereas
fast fibers respond phasically and briefly to rapid stretch. In both cases,
the associated muscle fibers are innervated by the same motoneurons that
innervate nearby non-sensory muscle fibers
(Fields, 1966;
Fields and Kennedy, 1965). The
presence of separate tonic and phasic sensors is not universal among
invertebrate stretch receptors, however. For example, the coxo-trochantal MRO
(CTMRO) of the locust is a muscle-associated receptor that is innervated by
only a single sensory neuron (Braunig and
Hustert, 1983). The associated muscle of the CTMRO is stimulated
by nearby non-muscle-associated proprioceptors and adjacent joints to maintain
sensory range (Braunig and Hustert,
1985). Such efferent feedback acts peripherally at the site of the
sensor in muscle-associated stretch receptors. By contrast, the
non-muscle-associated leg chordotonal organ in locust senses stretch in an
associated connective tissue that cannot be regulated by peripheral efferent
feedback.
Because such non-muscle-associated stretch receptors sense a connection
between hinged rigid points, the range of motion for these sensors is fixed.
In soft-bodied animals, although some rigid points exist [e.g. sclerotinized
cuticle (Snodgrass, 1935) and
quasi-jointed structures (Sumbre et al.,
2005)], such points are rare and movements generally have more
degrees of freedom. We expect that soft-bodied animals would require stretch
sensors with a greater dynamic range than that available through
non-muscle-associated stretch sensors. Such soft-bodied animals include the
caterpillar of the tobacco hawkmoth, Manduca sexta, and other
well-documented Lepidoptera such as Antheraea pernyii
(Osborne and Finlayson, 1965;
Weevers, 1965) and
Hyalophora cecropia (Beckel,
1958; Libby,
1961). The stretch receptor organ (SRO) of Antheria
pernyii responds tonically to slow stretch and phasically to rapid
stretch. Unlike the crustacean MRO, the stretch receptors described in
Antherea (Osborne and Finlayson,
1965) and in the locust
(Braunig and Hustert, 1983)
respond both tonically and phasically through the same sensory neuron and are
innervated by only one efferent motoneuron. This simplicity of structure and
control prompts questions about how the central nervous system (CNS)
integrates tonic and phasic information in the coordination of behavior.
In Lepidoptera, the most likely candidates for stretch feedback during
locomotion are the large longitudinal stretch receptors that span the length
of each abdominal segment and run parallel to the major intersegmental muscles
[illustrated in Hyalophora cecropia
(Beckel, 1958)]
(Fig. 1B). This SRO appears to
signal the length of tissues spanning its insertion points. Past studies have
shown that the SRO firing rate reflects both segment length and the velocity
of stretch (Lowenstein and Finlayson,
1960; Weevers,
1965). When the SRO is held in a fixed position or is stretched
slowly, the stretch receptor sensory neuron (SRSN) fires tonically, at a rate
proportional to the length of the SRO. When the SRO is stretched rapidly
(approximately >0.5 l s–1), the SRSN fires
phasically and at a rate proportional to the stretch velocity and far greater
than expected for a given position. The location and function of this SRO
suggest that it serves a proprioceptive function during abdominal movement.
SRO stimulation results in stretch reflexes [in Manduca
(Tamarkin and Levine, 1996);
in Antherea (Weevers,
1966c)] but it is unclear how such reflexes are incorporated into
spontaneous movements.
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; French,
1980); in cockroaches (French
and Wong, 1977; Gamble and
DiCaprio, 2003); in locust
(Kondoh et al., 1995); in cats
(Kroller, 1993); in flies
(Marmarelis and Mccann, 1973);
in chinchilla (Recio-Spinoso et al.,
2005)]. We used Wiener kernel analysis to test our results against
those observed in other organisms. Furthermore, to examine the role of the SRO
in behavior, we watched Manduca performing spontaneous and evoked
behavior following real or sham surgical SRO ablations. Finally, in the
present study, we discuss the implications of these findings in the context of
specific behaviors, including postural maintenance (stance), crawling and the
strike response to noxious stimuli. |
MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Male
and female fifth-instar larvae, in the first or second day following their
molt, were used for the experiments described in the present study.
Kinematics
In all cases, animals were anesthetized by chilling in ice for 30 min or
longer. When observing crawling kinematics, muscle insertion points on
proleg-bearing abdominal segments were marked with small (<200 µm)
fluorescent latex beads (e.g. see Movie 1 in supplementary material). Two
digital video cameras (Canon, Ota, Tokyo, Japan) then recorded crawling
movements. When observing strike reflex kinematics, intersegmental boundaries
were marked using fluorescent paint. One digital camera, directly over and
pointing down on the animal, recorded horizontal side-to-side flexing during
strike (see Movies 2A,B in supplementary material). Animals were gently
squeezed on their dorsal horn or their terminal ganglion with large forceps to
instigate the strike reflex. Digital video recordings of movements were
converted into 3-D (crawling) or 2-D (strike reflex) kinematic mappings by
linear transformation software (APAS, Ariel Dynamics, Trabuco Canyon, CA,
USA).
|
) and
the terminal and parathoracic ganglia were removed. Trachea and fat bodies
were then removed to clearly expose the underlying musculature. The SRO is connected to the CNS via the dorsal lateral nerve (DNL) (Fig. 1B). The ventral muscles were cut in order to expose the DNL, and the posterior and anterior DNL branches were cut close to the dorsal nerve. The ganglion for the segment under study, typically A4, was disconnected from the remainder of the nerve cord and body wall, leaving it still connected to the stretch receptor and dorsal musculature via the DNL. The entire dorsal hemisegment, composed of the appropriate segment and the two adjacent segments on either side, was entirely removed from the animal, with the DNL and ganglion still attached, and transferred to a separate elastomer-lined dish containing saline.
One side of the preparation was pinned cuticle-side up in the dish at one of the adjacent segments. Cuticle on the opposite end of the stretch receptor was pierced with a pin connected to the arm of a computer-controlled ergometer (Cambridge Technology, Watertown, MA, USA) and the tissue was stretched to approximately the resting length of the segment in the intact animal following chilling. The displacement of the hook and the force exerted by the ergometer were controlled by a customized LabVIEW program (National Instruments Corp., Austin, TX, USA), which was capable of generating arbitrary length and force signals.
DNL activity was monitored by an extracellular suction electrode placed on
the nerve and close to the SRO and amplified by a differential amplifier (A-M
Systems, Sequim, WA, USA). The DNL was cut proximal to the electrode so that
only afferent activity would be recorded. Afferent activity was sampled at
10 kHz and recorded by a computer-based data acquisition system (Dataq
DI-720 and Windaq Software, Dataq Instruments, Akron, OH, USA). Spikes were
identified by template or threshold detection using DataView software (W. J.
Heitler, University of St Andrews, UK). Except where otherwise noted,
instantaneous spike frequencies were determined from inter-spike intervals.
Phasic activity was typically quantified by mean maximum phasic frequency,
which was the mean of the maximum frequency observed during positive
stretching within at least 10 consecutive stretches.
Wiener kernel analysis
Preparations were mechanically stimulated with Gaussian white noise (GWN)
generated by a GWN function in LabVIEW (National Instruments Corp.) with three
different cutoff frequencies (fc) during the span of one
trial. By using multiple fc, we hoped to capture any
differences between response characteristics, particularly non-stationary or
adaptive responses to the higher frequency signals
(Kondoh et al., 1995;
Kroller, 1993). The GWN signal
was upsampled to the ergometer-length input rate (10 kHz) and passed through a
low-pass (10 kHz) finite impulse response (FIR) filter to avoid damage to the
preparation. Preparations were stimulated for approximately 90s at each
fc, twice the duration necessary for 95% complete tonic
adaptation at median adaptation time constant (see Discussion). Between GWN
stimuli, the preparations were held at rest length for approximately 90s.
Preparation lengths, as measured by an ergometer, and evoked spike response
were sampled as discussed earlier (Fig.
2A) and then downsampled to 2 kHz. SRO-evoked spikes were
converted to delta functions one sample long
(Marmarelis, 1978). First- and
second-order Wiener kernels were generated from 20 s-long stimulus/response
data series, beginning 30 s after the start of a stimulus, using traditional
cross-correlation techniques (French and
Marmarelis, 1999; Gamble and
DiCaprio, 2003; Marmarelis,
1978) and were computed by Lysis 7.1 software (BMSR-USC, Los
Angeles, CA, USA). Results were based on recordings from individual SROs from
12 different animals stimulated by GWN with fc=12.5, 25
and 50 Hz, in that order (Fig.
2B–D). These values were chosen as they encompassed the
range of velocities expected in behaviors of interest
(Fig. 3).
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Surgical SRO ablations
To confirm mobility and health, fifth-instar Manduca sexta in the
first day post-molt were observed for several hours crawling on various
substrates in different orientations. These animals were then chilled on ice
for at least 20 min. Holes were poked in the segments to be ablated using
insect pins, and superfine Vannas scissors (WPI Inc., Sarasota, FL, USA) were
inserted approximately 1 mm into the body to damage the SRO or DNL.
Following recovery of spontaneous activity, between 6 and 40 h
post-surgery, animals were tested for their performance in the following basic
behaviours; postural maintenance, righting reflex (return to upright posture
from side or back), horizontal crawling (upright), vertical crawling (up and
down) and strike reflex (instigated by poking the prolegs). When necessary,
crawling was evoked by anterograde dorsal stroking or gentle posterior
squeezing. After behavioral evaluation, animals were anesthetized by chilling
on ice, and SRO functionality was noted as described earlier (see
Electrophysiology). In cases where ablation or the presence of full SRO
function was unclear, the SRO morphology was assessed by staining with 0.02%
Janus Green in desheathing saline (Yack,
1993).
Based on the electrophysiological results, animals with non-functional SROs
were considered to be a successful ablation, animals with correctly functional
SROs were considered to be sham ablations and ambiguous cases were omitted
from analysis. Animals were categorized according to their behavioral scores,
and the difference between control and ablated animals in normal
versus atypical behavior was tested using a Chi-Squared
(
2) test.
Statistics
All statistical tests were carried out using Systat software (v. 10 or 11,
SPSS, Chicago, IL, USA). In all cases except for SRO ablation, data were first
tested for adherence to a normal distribution. Normally distributed data and
data transformable to a normal distribution in accordance with Tukey's ladder
(Tukey, 1977) were tested
using parametric tests. Most tests involving tonic or phasic frequency used
log-transformed frequency values. Log-transformed frequencies were normalized
in tests of tonic activity versus displacement and phasic activity
versus velocity. In the latter tests, velocities were also
log-transformed. Tests of phasic activity versus start-position used
normalized, but not log-transformed, phasic frequencies. Data that could not
be transformed to a normal distribution were tested using non-parametric
tests.
Correlation between endpoint of stretch and phasic activity was addressed differently. Because there were significant differences between the activity observed in different animals at given stretch velocities [Kruskal–Wallis test: by animal, U0.05(9,672)=508.386, P<0.0001; by stretch velocity, U0.05(5,672)=521.538, P<0.0001], we chose not to aggregate these data. Rather, we examined correlation between endpoint and phasic activity for each combination of animal and stretch velocity. Separate correlations between stretch endpoint and mean maximum phasic frequency were determined by animal and stretch velocity; these correlations were then averaged to observe the strength of correlation across multiple animals and stretch velocities.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Trimmer and Issberner, 2007);
for striking (Walters et al.,
2001)]. These motions were then classified according to amplitude
and speed of stretch of the abdominal body wall, as determined from kinematic
traces (Fig. 3Aiii,Biii).
The shortest range of motion was observed during the maintenance of
posture; aside from making small pulsing movements, the animal essentially
remained motionless. Net movements were close to zero and below levels
observed during crawling. During crawling, the abdominal segments maintained a
resting length (
5.0 mm) throughout stance phase but then shortened and
re-lengthened during the swing phase (Fig.
3A). During the rapid strike reflex, animals that were squeezed
gently on their terminal segment or on their anal claspers responded with a
rapid strike to the area of stimulus with their head
(Fig. 3B). Note that, in the
case of both behaviors, kinematic results reveal periods of constant segment
length and periods of rapid segment extension and contraction. Therefore,
experiments involving stretching stimuli utilized sustained stretch stimuli,
as well as stretch-and-hold stimuli, in order to cover the large range of
activity observed during behavior.
Tonic and phasic response in M. sexta
To compare the M. sexta SRO with that of A. pernyii, we
observed tonic output with the SRO stretched to different lengths
(Fig. 4). Tonic activity
increased as SRO length was increased (linear regression of log-transformed
normalized frequencies to displacement; t=23.8534, N=180,
P<0.0001). We also observed SRO output in response to
stretch-and-hold stimuli (Fig.
5). We confirmed that the mean maximum phasic frequency increased
with increasing stretch velocity (linear regression of log-transformed
frequencies to log-transformed velocities; t=14.942, N=38,
P<0.0001). We found that this log–log relationship was
particularly linear at stretch velocities between 1 and 64 mm
s–1. As with A. pernyii, the phasic response was
greater than what would be expected were the SRO to report displacement
tonically. During stretches slower than 1 mm s–1, phasic
activity was almost indistinguishable from tonic responses expected at a
similar length.
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).
To explore a full spectrum of behaviorally relevant stretch speeds, the
stimulus fc were varied and the responses to these stimuli
were examined separately.
All of the afferents responded similarly to mechanical GWN stimuli. At
fc=12.5 Hz, all 12 afferents had similar biphasic
first-order kernels (Fig. 6A).
At higher fc (25 Hz, 50 Hz), the SRO response was similar
to the response at the lowest fc (12.5 Hz), with the
exception that the lag prior to the first positive peak was greater at higher
frequencies. SRO second-order kernels were characterized by two peaks on the
diagonal and by symmetrical off-diagonal valleys, similar to the `four-eye'
configuration described by Kondoh and colleagues
(Kondoh et al., 1995) for
velocity-sensitive afferents. As with the first-order kernels, the
second-order kernels generated from all three fc were
similar, primarily differing in the degree of time lag prior to peaks and
valleys (Fig. 6B).
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; Weevers,
1965). Similarly, we report changes in tonic frequency observed at
a fixed interval following changes in SRO length
(Fig. 4). However, when the
tissue was stretched and held at a fixed length, the output frequency decayed
exponentially or rose exponentially following stretching or shortening,
respectively (Fig. 7A).
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In order to determine the factors influencing this tonic adaptation, we fitted each exponential decay to an exponential curve of the form f(t)=A–Bxe–Ct, where f(t) is the instantaneous tonic frequency over time (Fig. 7A). Quality of fit varied (Fig. 7B) but the median coefficient of determination (R2) was 0.80, with 69% of fits having R2>0.50. In real terms, the A parameter represented the steady-state level of tonic activity following the length change, the B parameter represented the degree of adaptation in tonic activity immediately following length change and the C parameter was the time constant indicating the duration of adaptation. Each (A,B,C) triplet was associated with a particular SRO length (Fig. 7C) and change in length (Fig. 7D).
We found a significant positive relationship between the tonic output level (A parameter) and segment length [linear regression of log(A) against length; N=58, t=2.717, P=0.0088] but not with a change in segment length [linear regression of log(A) against change in length; N=58, t=1.557, P=0.1252]. However, whereas there was no significant relationship between tonic adaptation and segment length [linear regression of log(B) against length; N=58, t=–1.199, P=0.2358], there was a significant negative relationship between tonic adaptation (B parameter) and change in segment length [linear regression of log(B) against change in length; N=58, t=–4.628, P<0.0001]. The time constant (C parameter) was not significantly affected by either segment length or change in length [linear regression of log(C) against length, N=58, t=–0.707, P=0.4827; change in length, N=58, t=–1.024, P=0.310].
Effects of displacement on phasic activity
Past studies have examined the phasic response to stretch velocity but not
the effect of displacement on phasic response during stretch. We tested two
ways by which displacement could be modified during stretch. First, we
adjusted the start-point of the stretch, changing the span of the displacement
but leaving the endpoint of the stretch fixed
(Fig. 8). All results were
normalized relative to the mean maximum phasic frequency observed in the case
of a 1 mm span of stretch. Varying the span of the stretch between 25% and
150% of the control span (1 mm) did not have any effect on mean maximum phasic
frequency [one-way analysis of variance (ANOVA);
F0.05(5,28)=0.3609, P=0.871].
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Tonic adaptation in the time domain
As predicted by tests of raw displacement
(Fig. 4), the steady-state
tonic response of the SRO to slow stretch was directly related to segment
length (Fig. 7C). However,
tonic SRO activity adapts slowly to changes in segment length, taking over 45
s to reach 95% of total adaptation at the observed median time constant of
0.065. Based on our observation that SRO displacement, and not length,
correlates significantly with the degree of adaptation
(Fig. 7D), we conclude that SRO
adaptation is largely a function of displacement, and not SRO length, for a
period of approximately 45 s following length changes. Thus, body-segment
length could be encoded by tonic activity in animals standing for long periods
of time; however, during any movement, patterned or otherwise, it seems
unlikely that the stretch receptor can accurately signal body segment length
in the short term.
Although neither SRO length nor change in length explain the time constant
of adaptation (Fig. 7C,D), we
observe that SRO adaptation is similar to the adaptation of passive
Manduca muscle to mechanical stimuli
(Woods et al., 2008). It is
possible that SRO adaptation arises from the internal mechanical properties of
the stretch receptor muscle fiber. However, we have not ruled out other
explanations such as neurochemical modulation as observed in acoustic sensory
systems (Herz et al., 2005).
Such a mechanism would permit the rate of adaptation to be modulated by
factors independent of movement such as by the stretch receptor motoneuron
(Weevers, 1966b).
Phasic response in displacement domain
We showed that the phasic response depends on the endpoint of the stretch
(Fig. 9) but it is not affected
by the start-point of the stretch (Fig.
8). These results suggest that as an abdominal segment stretches
rapidly, SRO activity will immediately increase with position, even at
constant velocity. Past tests of the response by the SRO to stretch velocity
typically involved stretching the SRO fixed distances between two points at
varying velocities. Finlayson and Lowenstein measured activity resulting from
sinusoidal stimulation (Lowenstein and
Finlayson, 1960); our present study (in
Fig. 5) and the study by
Weevers, measured activity resulting from fixed-velocity stretch-and-hold
patterns (Weevers, 1965).
However, previous stretch-and-hold experiments did not test the effect of
changes in the start- and endpoints of the stretch. Within the range of
stretches used in the present study, we find the unexpected result that span
does not affect phasic output.
First- and second-order Wiener kernels
The first-order kernel of an SRO afferent represents its response to an
impulse change in length of the SRO-bearing segment. We observed a
consistently biphasic first-order kernel
(Fig. 6A). The initial positive
peak represents the increase in activity in response to SRO stretch (length
increase), indicating that the SRO responds positively to stretch and not to
contraction or relaxation. The first-order kernel can also be interpreted as
the mean stimulus seen immediately before a spike
(Marmarelis, 1978). In that
way, we note that the negative peak seen after the initial positive peak
indicates that the receptor also responds to changes (negative-to-positive) in
stretch stimulus, i.e. it responds to stretch velocity. This positive-led
biphasic response matched the characteristic curve of other
position–velocity mechanosensors
(Gamble and DiCaprio, 2003;
Kondoh et al., 1995).
Second-order kernels represent the non-linear interactions between the
current response and two time points in the stimulus history,
1 and 2. SRO second-order kernels were
uniformally characterized by two peaks on the diagonal
1=2 and symmetrical off-diagonal troughs
(Fig. 6B). The on-diagonal
peaks and troughs represent amplitude-dependent non-linearities, which can be
compared directly with the linear system response of the first-order kernel
(Marmarelis, 1978). For
example, in the case of fc=12.5 Hz, the on-diagonal peak
observed at 1=2=25 ms in the second-order
kernel corresponds to a trough in the first-order kernel at the same time lag,
indicating a rectifying non-linearity. Off-diagonal peaks and troughs
represent interactions between historical stimuli and the current response.
For example, whereas a high amplitude stimulus immediately promotes a response
spike according to high amplitude in the first-order kernel at =0 s, the
interaction of the input at lag 1=0 s with the input at lag
2=25 ms results in a negative value, reducing the likelihood
of a spike; i.e. a high amplitude stimulus occurring 25 ms in the past reduces
the likelihood of a spike due to an immediate high amplitude stimulus. The
substantially reduced likelihood of a spike moments after a high amplitude
signal may be indicative of the refractory period seen after spiking
events.
Comparison with other proprioceptors
The Manduca SRO functions similarly to proprioceptors described in
other invertebrates. The most straightforward comparison is with the
Antherea SRO, which is similar anatomically to that of
Manduca (Lowenstein and
Finlayson, 1960; Weevers,
1966a). Antherea pernyii SROs produced tonic and phasic
outputs very similar to that of Manduca, although Weevers
(Weevers, 1965) also described
a phasic component due to stretch acceleration in the Antherea SRO
that we did not observe in Manduca SRO. In other animals,
similarities are seen in output but not structure. In crustaceans, for
example, the MRO response is tonic–phasic, like that of the
Manduca SRO; however, unlike in Manduca, the crustacean
tonic and phasic responces are divided between two wholly separate fibers, the
slow- and fast-adapting muscle fibers
(Eyzaguirre and Kuffler,
1955). Among the insects, a particularly interesting and
well-reported case comes in the form of the locust CTMRO, which reports
tonically proportional to angle but dynamically alters its response to nearby
non-muscle-associated proprioceptors
(Braunig and Hustert, 1985).
Although there are no nearby chordotonal or strand receptors in the case of
Manduca, it is an interesting and yet unexplored question as to
whether Manduca SROs directly influence each others' afferent
responses.
As described previously, GWN analysis has been used extensively for
characterizing mechanosensors. Our results describe a rectifying position- and
velocity-sensitive afferent; a characterization that agrees with our own
electrophysiology results as well as previous studies characterizing
lepidopteran stretch receptors (Lowenstein
and Finlayson, 1960; Weevers,
1966a). Furthermore, our results are very similar to those of two
non-muscle-associated sensors, the crab CB-chordotonal spiking neurons
(Gamble and DiCaprio, 2003)
and position-velocity sensing locust femoral chordotonal organs
(Kondoh et al., 1995).
Additionally, our findings on adaptation have precedent. In locust, both
the muscle-associated MROs and non-muscle-associated strand receptors adapt to
constant stimuli (Braunig and Hustert,
1985). As already noted, crustacean MROs are functionally
segregated as either slow- or fast-adapting fibers
(Cattaert and Le Ray, 2001).
However, unlike the fast-adapting fiber of crustacean MROs
(Eyzaguirre and Kuffler,
1955), the Manduca SRO did not have a threshold for
phasic spiking (Fig. 8).
SRO function in the context of postural maintenance
We have defined `postural maintenance' as the behavior of the animal during
a stance position even when faced with external forces. When a single
abdominal body segment stops moving longitudinally its constituent SROs should
cease to produce phasic activity (Fig.
5). Following tonic adaptation
(Fig. 7), the tonic activity
generated by these SROs could provide stable information about the length of
the body segment (Fig. 4). By
comparing spike rates from adjacent segments and across the longitudinal body
axis, a somatotopic map of relative body position could be formed in the CNS,
similar to that of the sensory hairs on M. sexta prolegs
(Levine et al., 1985;
Peterson and Weeks, 1988) and
on the abdominal body surface (Levine et
al., 1985).
One mechanism for postural maintenance could be through sensing strains
induced by adjacent segments. If a segment is not held in sufficient tension
by its intersegmental muscles, it will begin to lengthen under the force
exerted by adjacent segments. This local increase in stretch would cause an
increase in SRO tonic activity and could be used to restore segment length
through a stretch reflex (Tamarkin and
Levine, 1996). In this way, the SRO is a strain sensor that
indirectly measures tension on the body overall. If strain is related to
overall body tension, this could also provide a mechanism to regulate
turgor.
SRO function in the context of strike response
Our findings suggest a specific role for the SRO in controlling striking
behavior. Strikes to stimuli at the posterior segments are rapid and precise,
targeting the stimulus at specific points on the body
(Walters et al., 2001). During
the strike response, the sides of the abdominal segments stretch rapidly,
shortening at rates around 20 mm s–1 and lengthening at rates
double this (Fig. 3B). By
contrast, noxious stimuli applied to the thoracic and anterior abdominal
segments elicit a slower movement of the body away from the source of the
stimulus (Walters et al.,
2001).
In the range of stretch velocities observed during strikes (between 5 and
50 mm s–1), phasic activity increases very rapidly with
velocity (Fig. 5) and somewhat
less rapidly with increases in SRO length
(Fig. 9). Our results predict
that the combination of these two factors will cause a high frequency phasic
response as the SRO is stretched to the end of its dynamic range. This would
provide a powerful signal indicating the maximum strain of the body segment.
Furthermore, this signal would strongly excite ipsilateral intersegmental
muscles through the stretch reflex, bilaterally slowing the rate of stretch on
the segment (Tamarkin and Levine,
1996) and generating negative feedback control during this rapid
behavior.
SRO function in the context of crawling
One question in this study was whether or not the SRO plays a
proprioceptive role during crawling. Sensory feedback can be used to alter
locomotion in crawling animals (Weevers,
1965) and can coordinate condition-dependent reflexes during
crawling (Belanger et al.,
2000). However, because tonic activity is a poor indicator of body
segment length for nearly 45 s following a change in segment length, the SRO
cannot provide an accurate measure of body length during crawling. Phasic
activity may influence crawling by reporting the rapidity of stretch during a
crawl cycle; however, we have shown that the phasic response is positively
influenced by length, an effect that will skew the inferred rate of stretch.
Furthermore, we found that ablation of abdominal SROs does not have a
significant observable effect on crawling or any of the other major activities
we tested in Manduca (Table
1). Therefore, despite the position and size of the SRO, which are
suggestive of a role in crawling, our results do not support the hypothesis
that the SRO plays a critical role in the coordination of crawling.
Alternative roles of SROs in soft-bodied animals
It is possible that the SRO contributes to elements of behavior that are
not directly visible. For example, cat deafferentation results in changes that
can be detected by electromyography but not by the kinematics of undisturbed
and supported walking on a treadmill
(Hiebert and Pearson, 1999).
Fields, observing similar temporal lag in response to position in the
crustacean MRO, proposed that the stretch receptor acts as an error detector
rather than a length-measuring sensor during certain animal behaviours
(Fields, 1966). However, the
lack of any behavioral deficit in SRO-ablated Manduca shows that a
complete complement of SROs is not essential for most tasks; if information
from the SRO is used for motion control at all, it must be highly redundant or
easily compensated by other sensory systems.
Although not the focus of the present study, it is possible that a
developmental role is played by the longitudinal SROs in Manduca
sexta. Body size is a factor in gating final-instar molting in M.
sexta (Nijhout, 2003),
and abdominal stretch receptors are used for clearing size-regulated
checkpoints in Rhodnius prolixus
(Wigglesworth, 1934) and
Oncopeltus fasciatus (Nijhout,
1979). However, unlike in O. fasciatus, molting in
Manduca cannot be induced prematurely by abdominal inflation
(Nijhout, 1981), so size
monitoring by SROs in Manduca has yet to be demonstrated.
One possible behavioral role for SROs during crawling is that tonic and
phasic output are synthesized into a form of feature detection useful for
signaling the status of a crawl cycle. Such feature detection mechanisms have
been described in a variety of organisms, including crickets
(Marsat and Pollack, 2006),
electric eels (Oswald et al.,
2004) and humans (Lesica and
Stanley, 2004). Although we do not know if SROs play this role in
Manduca, SRO responses to repetitive and complex stretch cycles
(derived from the kinematics of crawling caterpillars) reveal consistent
patterns of activity (e.g. Fig.
1C). These patterns might convey features that are not essential
for immediate motor activity (and therefore cannot be resolved by SRO
ablation) but are nonetheless useful for adapting to environmental changes.
This possibility is the subject of an ongoing study.
LIST OF ABBREVIATIONS
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Footnotes |
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This research was supported by National Science Foundation grants IBN-0117135 and IOS-0718537 to B.A.T., and by a W. M. Keck Foundation Science and Engineering Program grant Biomimetic Technologies for Soft-bodied robots to B.A.T. Thanks to the Tufts Biology Writing Club for feedback during preparation of this manuscript. Many thanks, also, to the University of Southern California's Biomedical Simulations Resource (BMSR) center for providing their LYSIS 7.1 software free of charge and on the Internet (http://bmsrs.usc.edu/). The development of LYSIS is funded by Grant RRO1861 from the National Center for Research Resources of the National Institutes of Health. The BMSR holds the copyright of LYSIS.
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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