Motor output characterizing thanatosis in the cricket Gryllus bimaculatus
Laboratory of Neurocybernetics, Research Institute for Electronic Science, Hokkaido University, Sapporo, 060-0812, Japan
e-mail: nishino{at}ncp8.es.hokudai.ac.jp
Accepted 3 August 2004
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Summary |
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Key words: cricket, Gryllus bimaculatus, common inhibitory motor neurone, accessory flexor, tonic immobility
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Introduction |
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Thanatosis in animals has been behaviourally characterized by (1)
suppression of the righting response
(Steiniger, 1936;
Godden, 1972
;
Nishino and Sakai, 1996
;
Faisal and Matheson, 2001
),
(2) maintenance of an unusual posture or of an unusual posture passively taken
(Steiniger, 1936
;
Godden, 1974
;
Bässler, 1982
;
Nishino and Sakai, 1996
). The
latter condition in (2) is termed `catalepsy', in which leg joint reflexes are
predominantly velocity-sensitive but not position-sensitive (e.g.
Bässler and Foth, 1982
;
Driesang and Büschges,
1993
; Wolf et al.,
2001
).
As thanatosis occurs in many postures due to its cataleptic nature, one
might ask what physiological characteristics distinguish the thanatotic state
from the normal quiescent resting states. Physiological differences have been
indicated by detailed behavioural studies. For example, during thanatosis in
orthopteran insects, all movements of the body (including ventilations in the
abdomen) and appendages are strongly suppressed and a rigid posture is
maintained (Nishino and Sakai,
1996). Most strikingly, the leg joints appear to be stiff, not
flaccid, and complete immobilization may continue for 1020 min
(Hoyle and Field, 1983a
;
Nishino and Sakai, 1996
). In
contrast, the voluntary resting state differs from the cataleptic state
because loss of muscle tonus (antennal and neck inclination) is prominent in
resting crickets (Nishino and Sakai,
1996
) and honeybees (Kaiser,
1988
; Sauer et al.,
2003
). This immobility is frequently interrupted by short bouts of
locomotor activity and by limb or antennal movements
(Kaiser, 1988
;
Nishino and Sakai, 1996
;
Sauer et al., 2003
). These
findings collectively indicate that during thanatosis the skeletal muscles
exhibit persistent rigidity but also plasticity when forcibly stretched.
However, no clear motor output characterizing the thanatotic state has yet
been defined.
Physiological studies focusing on peripheral motor control have revealed
two different mechanisms for maintaining persistent tonic immobility. A
primitive orthopteran, the weta Hemideina femorata, displays a
defensive posture with the metathoracic tibiae fully extended, maintaining
this posture for several minutes without any electrical activity in the
extensor tibiae muscle (Hoyle and Field,
1983a). This `catch-like tension' is triggered by a brief spike
burst from the excitatory motor neurones immediately after octopamine is
released from neuromodulatory, dorsal unpaired median (DUM) neurones
(Hoyle and Field, 1983b
).
Similar catch-like tension (i.e. prolonged maintenance of residual tension)
has been found in the claw opener muscle of the crayfish, Astacus sp.
(Hawkins and Bruner, 1979
) and
the metacoxal muscle of the cockroach, Periplaneta americana
(Chesler and Fourtner,
1981
).
In the stick insect Cuniculina impigra the thanatotic display is
maintained by the continuous activation of slow excitatory motor neurones. In
response to visual stimulation or to mechanical disturbance all six tibiae are
fully extended to form the stick posture. Although fast motor units are
reflexively activated by the tibial displacement, this does not cause arousal
(Godden, 1972). Thanatosis
occurs naturally during daylight and alternates to the camouflaging resting
posture, in which the tibiae are more flexed. The motor neuronal activity is
fundamentally similar in the two states except that the firing rate of the
slow extensor tibiae (SETi) is higher in the thanatotic state in order to
maintain the fuller tibial extension seen in the stick posture
(Godden, 1972
;
Bässler, 1982
).
In contrast, the cricket is an active walker in which the behavioural
switching from arousal to thanatosis, and vice versa, occurs rapidly.
This allows a reliable correlation between neural events and behaviour. A
preliminary study has shown that the thanatotic posture is maintained by
continuously active slow motor units, as in stick insects
(Nishino et al., 1999).
To determine the motor output typical of thanatosis, I have focussed on the
anatomy and physiology of the metathoracic flexor tibiae. This is one of the
principal posture-controlling muscles and is fundamental to the maintenance of
tibial flexion during thanatosis. Its motor innervation has been extensively
studied in locusts (Hoyle and Burrows,
1973; Phillips,
1980
; Sasaki and Burrows,
1998
), katydids (Theophilidis
and Dimitriadis, 1990
), tree wetas
(O'Brien and Field, 2001
) and
crickets (Nishino, 2003
).
Electrical recordings from the flexor motor neurones in minimally restrained
crickets revealed that stable tibial flexion depends on both mechanical and
physiological factors. The elaborated accessory flexor muscle stabilizes the
femorotibial (FT) joint, with the activity of the common
inhibitory motor neurones (CIs) being suppressed to maintain the muscle tonus
created by the activity of the slow excitatory motor neurones.
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Materials and methods |
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Behavioural experiments
Thanatosis can be induced in either the ventral-up position or the
dorsal-up body position. During thanatosis, the metathoracic FT joint
can be immobilized at any angle between 0° and 100°, corresponding to
the range in which the flexor tibiae muscle normally functions
(Nishino et al., 1999). To
standardise the experiments, only `flexed-leg thanatosis' (termed in
Nishino et al., 1999
) was
studied. This was induced by pressing both sides of the pronotum and forelegs
gently for 35 s with the thumb and forefinger, whereupon the cricket
enters thanatosis with all legs flexed
(Fig. 1A;
Nishino and Sakai, 1996
). The
cricket was gently released and placed on a flat wooden bar, 2 cm wide, with
its ventral side up to eliminate local reflexes caused by contact of the legs
with the substrate (Fig. 1A).
The switch from thanatosis to arousal was indicated by the righting response.
The bodily movements during thanatosis were monitored with a photo-coupler
placed beside the abdomen (P, Fig.
1A). The voluntary movements of animals were monitored verbally on
the voice channel of a data recorder or by a video camera (Handy-Cam, Sony,
Tokyo, Japan).
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Anatomy
The peripheral motor nerves were stained by forward-fills from the main leg
nerve (N5B2) while motor neuronal somata and dendrites in the metathoracic
ganglion were stained by back-fills from the peripheral motor nerves. The
dissection and staining procedures were adapted from Nishino
(2003). 6% solution of nickel
chloride hexahydrate (Merck) and 1% solution of fluorescent dyes such as
dextran, tetramethyl rhodamine (MW=3000, Molecular Probes, Eugene, OR, USA)
and dextran, fluorescein (MW=3000, Molecular Probes) were used as marker
substances. The nickel-filled specimens were reacted with rubeanic acid to
precipitate the nickel. The ganglia were fixed in Alcoholic Bouin and the
peripheral tissues in 5% neutral formaldehyde solution before being dehydrated
through an ethanol series, cleared in methyl salicylate and photographed under
a light microscope. The specimens were further processed by silver
intensification if necessary (Bacon and
Altman, 1977
). Fluorescent dye-filled specimens were fixed in 4%
neutral formaldehyde solution, dehydrated through an ethanol series, and
viewed under a confocal microscope (LSM510, Zeiss, Jena, Germany). Optical
sections (thickness: 5 µm each) were reconstructed two-dimensionally using
commercial software linked to the LSM.
Electrophysiology
To identify the physiological types of neurones sending axons into the
flexor nerve branches and to see their neural activity during thanatosis,
quiescence and walking behaviour, extracellular recordings were made from both
restrained and freely moving animals tethered by fine recording leads.
For recording under restrained conditions, the cricket was anaesthetized on
iced water for 10 min and then fixed ventral-side-up on a chamber filled with
beeswax. The dorsal halves of the body and pro- and mesothoracic legs and the
posterior halves of the metathoracic legs were embedded in PlasticeneTM
to prevent voluntary movements. The metathoracic FT joint to be
recorded was fixed at about 80°. The gut was removed via an
incision on the dorsal tip of the abdomen to eliminate body hemolymph, which
disturbs the observation of the recording sites due to its coagulation. To
expose the accessory flexor nerve the anterior cuticle covering the distal
femur and also the anterior muscle bundles of the accessory flexor were
removed. The cavity formed by removal of the muscle tissue was filled with
cricket saline (Nishino and Sakai,
1997). Care was taken not to cut the apodeme of the femoral
chordotonal organ (FCO) running very close to the nerve as cutting the apodeme
causes a severe reduction in the activity of slow excitatory motor neurones.
The accessory flexor nerve was cut proximally to the muscle and efferent
activity recorded from its proximal cut-end with a suction electrode. In
several recordings, the efferent activity was simultaneously recorded from the
proximal cut-end of N5B2 in the distal-end of the femur using a suction
electrode. Intracellular muscle recordings were made from the middle of the
accessory flexor muscle fibres using a borosilicate glass electrode filled
with 4 mol l1 potassium acetate to give a tip resistance of
1013 M
.
For recordings under tethered conditions, crickets anaesthetized with carbon dioxide were fixed onto the beeswax plate with stapler pins, leaving all body appendages intact. To record from one of the three main flexor motor nerves, a pair of copper electrodes (insulated except for the tip, 32 µm in diameter) was bound with fingernail lacquer and inserted through small holes in the cuticle (Fig. 1C). To record motor neuronal activity as directly as possible, the recording electrode was adjusted to obtain clear extracellular records from the targeted nerve. The reference electrode was placed on the surface of the muscle compartment innervated by that nerve. Each electrode was fixed in place with a wax-resin mixture. The cricket was placed in a plastic-walled observation arena (20 cmx20 cmx10 cm). The pair of electrodes was bound to an earth electrode inserted through the pronotum, and then all were fixed with wax resin onto the forewings and connected to the head stage of a differential amplifier (Fig. 1C). After the experiment, a conventional forward-fill from N5B2 was carried out, leaving the recording wire inserted, to check the distance between the nerve and the tip of the recording wire (Fig. 1D). Data derived from preparations in which the nerve and the tip of the recording wire were not attached were excluded.
To gain access to the accessory flexor nerve a small door-like incision was made on the posterior surface of the distal femur of the anaesthetized cricket and opened by inserting an insect pin between the cuticle flap and the muscle. The recording electrode (insulated copper wire, diameter: 22 µm) was scratched slightly around the tip to remove the insulation and then inserted through a small hole in the cuticle flap. It was tightly coiled twice around the posterior branch of the accessory flexor nerve and the body haemolymph around the recording site replaced with high vacuum grease (silicon lubricant, Toray Silicone, Tokyo, Japan), ensuring that the electrical activity was recorded only from the accessory flexor nerve. The reference electrode was inserted through another hole made on the cuticle flap and embedded in the silicon grease close to the nerve branch. After the cuticle flap was closed, each electrode was fixed in place with wax resin and then bound with the earth electrode as described above.
Electrical activity was recorded using an amplifier (Iso-DAM8A, WPI, Sarasota, USA) and displayed on an oscilloscope and a data acquisition system (Omniace, NEC, Tokyo, Japan). Data were stored on open reel tapes or DAT cassettes and analysed with the aid of custom-made Lab View v. 6I programs.
Definitions
The main body of the flexor tibiae muscle is arbitrarily named the `main
flexor muscle' to distinguish it from the `accessory flexor muscle'. As all
motor neurones innervating the flexor muscle in the cricket are
morphologically similar to those of locusts
(Nishino, 2003) the
nomenclature of the neurones follows that of the locust (for a review, see
Burrows, 1996
). Excitatory
motor neurones and inhibitory motor neurones are often abbreviated to
`exciters' and `inhibitors', respectively. Units of slow, intermediate and
fast exciters and inhibitors were discriminated by their spike amplitudes and
burst characteristics (Hoyle,
1980
; Hustert and Gnatzy,
1995
; Tauber and Camhi,
1995
; Berkowitz and Laurent,
1996
; Nishino et al.,
1999
). When the amplitudes of two units were similar (e.g.
inhibitor vs intermediate exciter), spike waveform shape was used as
an additional criterion.
During recordings from tethered animals, the deep resting state (termed
`sleep-like state'; Kaiser,
1988), characterized by antennal and head inclination, was not
observed because mechanical disturbances were given to the cricket to induce
thanatosis. Instead, brief quiescent episodes maintained from 3 to 30 s
appeared between voluntary movements. Accordingly, this immobile state was
termed simply the `quiescence' or `quiescent state'.
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Results |
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The main flexor muscle in the cricket is compartmentalized into three
regions, the proximal, middle and distal regions, each innervated by a
separate nerve branch diverging from the main leg nerve (N5B2): the proximal
flexor nerve, middle flexor nerve and distal flexor nerve, respectively
(Fig. 3). The proximal flexor
nerve bifurcates at its base to innervate the anterior and posterior muscle
bundles (Fig. 3B). This
morphologically simple innervation contrasts with the pattern in locusts
(Sasaki and Burrows, 1998) and
katydids (Theophilidis and Dimitriadis,
1990
), in which the homologous muscle is innervated by numerous
short branches diverging from N5B2.
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The accessory flexor muscle comprises two groups of 56 muscle bundles inserting onto the anterior and posterior edges of the cushion. These are innervated by bifurcated branches of the accessory flexor nerve diverging from N5B2 (Fig. 3B).
The four flexor nerve branches carry only efferent neurones, which are
excitatory, inhibitory and/or DUM neurones
(Nishino, 2003). As in locusts
(Hoyle and Burrows, 1973
;
Hoyle, 1980
), the excitatory
motor neurones are categorized into slow, intermediate and fast types
according to their physiological properties. The slow-type fires tonically to
maintain muscle tonus, while the fast-type fires very briefly but leads to a
strong twitch contraction. The intermediate-type fires in longer-lasting
bursts with a progressively declining frequency during visible movements
(Nishino et al., 1999
;
Nishino, 2003
). Recordings
from the motor nerve branches revealed that the proximal flexor nerve has 12
exciters (34 slow-, 78 intermediate- and 12 fast types),
the middle flexor nerve has three (two intermediate- and one fast-types), the
distal flexor nerve has four (one intermediate- and three fast-types) and the
accessory flexor nerve has four (three slow- and one intermediate-types). Thus
the main flexor muscle changes progressively from slow to fast innervation
distally, while the accessory flexor muscle is largely slow in nature. Two DUM
neurones were faintly stained only when the distal flexor nerve was
back-filled, suggesting that two thin axons from the DUM neurones supply the
distal region of the main flexor muscle
(Nishino, 2003
).
Differential nerve back-fills revealed that the three muscle compartments
of the main flexor muscle are innervated by different axons, except that one
intermediate exciter sends axons into both the proximal flexor nerve and the
middle flexor nerve. In total 18 exciters innervate the main flexor muscle,
the largest number so far reported from orthopteran insects
(Nishino, 2003). Overlapping
innervation is rather prominent in the muscle compartments that are distant to
each other. The proximal compartment of the main flexor muscle and the
accessory flexor muscle both receive overlapping-innervation from two exciters
and two inhibitors (Nishino,
2003
).
Effects of ablation of the motor nerves on maintenance of the thanatotic posture
In order to assess the roles of the different compartments of the flexor
muscle in maintaining tibial flexion, ablation experiments of nerves or muscle
bundles were performed in both metathoracic legs, and then the flexor muscle
tonus was evaluated by measuring the FT joint angles of the operated
legs when the cricket assumed both thanatosis during lying (see
Fig. 1A) and catalepsy during
hanging (see Fig. 1B). In
Table 1, all experimental data
are compared to the control FT joint angles of intact animals: 0°
(full flexion) during lying, and 33±11° during hanging. The data
are presented in order of increasingly greater disruption.
The legs with the middle flexor nerve-cut or the distal flexor nerve-cut showed no significant differences from intact legs (Table 1). However, the leg flexion response occurred more slowly than in intact legs. Cutting the posterior branch of the proximal flexor nerve resulted in a small but significant loss of muscle tonus: the operated FT joints were slightly opened compared to intact legs. Cutting the posterior branch of the accessory flexor nerve gave a similar effect to proximal flexor nerve-operated joints during lying. However, the FT joint was much more open during hanging. Removal of the muscle bundles innervated by the posterior branch of the accessory flexor nerve resulted in more severe deficiencies during both lying and hanging compared to legs with the posterior nerve branch cut. Cutting the anterior branch of the proximal flexor nerve or the accessory flexor nerve produced a similar effect to cutting the posterior branch of the respective nerves (data not shown). Finally, to confirm the role of the flexor muscle in mediating these effects, the flexor apodeme was cut. The FT joint remained half (lying) or fully open (hanging).
Activity of the motor neurones to the main flexor muscle during thanatosis
Cricket behaviour is disturbed by both chronic restraint and extensive
dissection. As thanatosis is seldom observed in these conditions, electrical
activity was recorded from free-moving crickets tethered by fine recording
leads. Extracellular recordings (neurograms) from the proximal flexor nerve
(N=8), middle flexor nerve (N=7) and distal flexor nerve
(N=6) revealed that probably all the intermediate and fast exciters
were recruited in the induction phase of thanatosis but ceased activity almost
completely during thanatosis (Fig.
4AC). When ventilatory movements occurred frequently during
thanatosis, intermediate exciters were likely to be activated more frequently
(Fig. 4B), but without apparent
coupling to the movements (arrows and time-stretched inset,
Fig. 4B). In other recordings
from the middle flexor nerve, whereas a barrage of spikes of an intermediate
exciter occurred during ventilation in the quiescent state
(Fig. 4C), activity of
intermediate exciters during ventilation was suppressed during thanatosis
(Fig. 4D), Typically, arousal
(righting response) was characterized by strong flexor muscle activation with
recruitment of fast exciters, as in locusts
(Faisal and Matheson, 2001).
However, behaviour just after arousal varied: crickets sometimes showed a long
quiescence before moving (Fig.
4A), running might begin immediately
(Fig. 4B), or slow walking
followed a brief quiescence (Fig.
4E). The latter two cases were most commonly observed. Neurogram
recordings from tiny axons of slow exciters and inhibitors running through the
proximal flexor nerve were extremely difficult, probably because the proximal
flexor nerve gives rise to fine terminal arborizations immediately after
diverging from N5B2 (see Fig.
3), making these axons too thin to be recorded above the noise
level.
Anatomy of the accessory flexor muscle
The above ablation experiments and physiological recordings indicate that
flexed-leg posture typical of thanatosis must result from slow muscle
activity, controlled by slow exciters and inhibitors. Because the accessory
flexor muscle is primarily a slow muscle, it was targeted for neural activity
recording. The accessory flexor nerve was isolated from other muscle or nerve
tissue before it reached the muscle bundles, where it gave rise to extensive
terminal arborizations decorated with rich varicosities
(Fig. 5A). A back-fill from the
accessory flexor nerve with dextran, tetramethyl rhodamine revealed that two
closely bound axons of CIs (these cell bodies were identified in the ganglion)
travelled distally through N5B2 to innervate the tarsal levator- and tarsal
depressor-muscles (Fig. 5B).
Though the posterior branch of the accessory flexor nerve was slightly thicker
than the anterior branch (Fig.
5B), back-fills from either branch labelled the identical set and
number of motor neurones. A differential back-fill using dextran, tetramethyl
rhodamine and dextran, fluorescein labelled neurones sending axons to the
accessory flexor nerve (red) and those to the distal flexor nerve (green;
Fig. 5C). The somata of all
excitatory motor neurones are grouped in the antero-lateral region of the
ganglion as in locusts (Burrows and Hoyle,
1972; Phillips,
1980
), while those of two moderate-sized CIs are located close to
the midline of the ganglion (Fig.
5C). The anterior and posterior CI somata were designated CI2 and
CI3, respectively, following the naming of morphological homologues in locusts
(Hale and Burrows, 1985
;
Watson et al., 1985
). The
somata of the four exciters sending axons to the accessory flexor nerve are
much smaller than those sending axons to the distal flexor nerve. No motor
neurones with axons in both nerves were detected
(Fig. 5C). Another specimen in
which the accessory flexor nerve was back-filled with NiCl2 and
silver-intensified, showed that there was no consistency in location of the
four exciters among individuals whereas the locations of the CIs were almost
invariable (compare Fig. 5D with
5C). One exciter (tentative intermediate-type) had a larger soma
(Fig. 5C,D) and a prominently
thicker axon (arrow in Fig. 5E)
compared with the others. The locations of the dendritic arborizations of the
exciters and CIs were largely segregated; those of the CIs were more
postero-dorsal (outlined by a thin broken line in
Fig. 5F) although some
overlapping appeared to exist in the dorso-lateral region of the posterior
half of the ganglion (Fig.
5F).
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Identification and discharge properties of accessory flexor motor neurones in restrained preparations
The number of efferent units recorded from the accessory flexor nerve in
restrained, dissected crickets was generally in good agreement with the actual
number of efferent axons (Fig.
6). Units recorded from quiescent crickets
(Fig. 6A, upper trace) were
categorized into three classes. (1) Small units fired tonically at relatively
high frequencies (520 Hz). Two to three units were usually
distinguishable. (2) Two moderate-sized units fired tonically at low
frequency, often synchronously. (3) One large unit was silent except for
occasional activation with abdominal ventilation. These physiological
characteristics indicate that the small units are slow exciters, the
moderate-sized units are CIs, and the single large unit is an intermediate
exciter.
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Several points indicate that the moderate-sized units are CIs. No more than two moderate-sized units were recorded simultaneously from N5B2 at the distal femur (Fig. 6A, lower trace) in which the CIs send axons (see Fig. 5B). Extracellular recordings from the accessory flexor nerve and simultaneous intracellular recordings from the accessory flexor muscle fibres (N=4) show action potentials of the two moderate-sized units corresponding exactly with inhibitory junctional potentials (IJPs) in the muscle (Fig. 6B). The amplitudes of the IJPs markedly decreased as more negative membrane potentials were imposed by current injection. Summation of IJPs was prominent in the muscle fibre when the two CIs fired synchronously (asterisks, Fig. 6B).
The slow exciters and the CIs show recruitment during disturbance stimuli. In Fig. 7A, each of three slow units (indicated by colour in the magnified inset) fired tonically at 612 Hz while the smaller unit of two CIs (indicated by 1) fired constantly at 25 Hz during quiescent state. When the cricket was slightly disturbed by tapping the substrate, the larger CI (indicated by 2) was recruited synchronously with the smaller unit (Fig. 7B). When the tarsus of the prothoracic leg ipsilateral to the recording site was touched with a wooden stick, both the CIs and the slow exciters were strongly activated and then the discharge gradually declined (Fig. 7C). When the same tarsus was pinched very strongly with forceps (for more than 5 s), the intermediate unit was initially activated but adapted quickly while the slow and the two CIs showed slowly adapting, tonic discharges lasting for more than 1 min (Fig. 7D). The larger CI (2) discharged more slowly than the smaller (1).
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As the ventilatory movements are known to influence motor neuronal activity
in quiescent locusts (Burns and Usherwood,
1979) and in crickets during thanatosis
(Nishino et al., 1999
), the
fluctuation of activity during ventilation in different types of motor
neurones was investigated in detail (Fig.
6C). On the expiration phase of the spontaneous ventilatory
movements, the CIs were the first to become active, and subsequently the slow
and intermediate exciters were activated in the expiration phase resulting in
hyperpolarization of the muscle fibre potential (EJPs caused by the
intermediate exciter were not detected in this muscle fibre). On the
inspiration phase, this activity quickly waned and the muscle potential
returned to the resting level.
Discharge properties of accessory flexor motor neurones in tethered crickets
In all recordings from tethered crickets, units of slow exciters,
intermediate exciters and CIs were readily discriminated by their spike
amplitudes, firing characteristics and spike shapes
(Fig. 8A). However, reliable
discrimination of any single units from the three slow exciters or from the
two CIs was difficult because their spike amplitudes and shapes were similar
and fluctuated.
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There was some variation in thanatotic posture from trial to trial in the
same cricket and from individual to individual
(Nishino and Sakai, 1996). In
many cases, a cricket that immediately entered thanatosis with all tibiae
flexed exhibited a low ventilation rate and a low responsiveness to mechanical
disturbance. However, stimulated crickets occasionally resisted the restraints
instead of assuming leg flexion and somehow entered immobility with all
FT joints opened during struggling. In this case, ventilation rates and
responsiveness to disturbance more closely resembled those of the quiescent
state than those of ordinary thanatosis
(Nishino and Sakai, 1996
).
Thus, thanatosis in which the FT joints were rigidly flexed was termed
`strong thanatosis' and thanatosis in which all FT joints were loosely
opened was termed `weak thanatosis' in this study
(Fig. 8B).
Activity of the slow excitatory motor neurones during thanatosis
In all the recordings derived from five individuals, slow exciters were
continuously active during both thanatosis and quiescence although their
discharge frequencies varied between individuals. In one recording, average
frequencies (Hz) of total discharges of slow exciters were measured from 5 s
samples when the cricket was in either thanatosis or the quiescent state at
various FT joint angles (Fig.
8A). Sample periods in which ventilatory movements occurred and
those just after arousal from thanatosis were excluded from the analysis
because slow exciter activity tended to fluctuate in both conditions (see
Fig. 6C). When the cricket was
quiescent, the slow exciters tended to discharge at progressively higher
frequencies as the FT joint was flexed (although there was fluctuation
even if the same FT joint angle was maintained.
Fig. 8B). However, when the
cricket was in strong thanatosis (FT joint angle was slightly opened to
15° due to the operation), the slow exciters fired at a somewhat lower
frequency compared to the quiescent cricket at the same FT joint angle
(Fig. 8B). This tendency was
more distinct when the cricket was in weak thanatosis with the FT joint
angle at 30°, where the slow exciters maintained a much lower frequency
than expected in the quiescent state (Fig.
8B).
Activity of the common inhibitory motor neurones during thanatosis
The CIs show distinctive pattern of activity during and immediately after
thanatosis. During strong thanatosis (Fig.
9A), the normal 0.52 Hz firing of the CIs during quiescent
state (Fig. 8A) was suppressed.
Only eight spikes from CIs were identified in 42 s of the maintenance phase of
thanatosis (asterisks in Fig.
9A). In contrast, strong CI firing (at about 15 Hz), possibly due
to disinhibition, occurred just after arousal, although the cricket was
motionless with the recorded FT joint maintained at 120°. The
intermediate exciter, which causes visible leg movements was not recruited. As
walking commenced, CI firing diminished rapidly while large motor neurones
were recruited (Fig. 9A). On
the other hand, during weak thanatosis (FT joint maintained at
95°), the CIs and the intermediate exciters fired at higher frequencies
(Fig. 9B), although these units
did not fire during ventilation (inset in
Fig. 9B). Again, strong CI
firing occurred just after arousal (Fig.
9B).
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The distinct pattern of CI discharge was not affected by joint position, duration of the CI discharge nor by position in which the cricket was placed. For example, when the FT joint angle was maintained at 120° (Fig. 9A), 90° (Fig. 9B) and 15° (Fig. 9C) immediately after arousal, CI discharge is similar in each case. A peri-stimulus time plot of CI activity in five trials of thanatosis (derived from three crickets) consistently showed that the CI activity was almost completely suppressed at the beginning of thanatosis but that the suppression declined gradually with the onset of ventilatory movements (Fig. 9D,F) and then strong recruitment of the CIs occurred on arousal (Fig. 9E,F). The CI discharge varied from 2070 s, but still the characteristic high onset frequency adapted to a plateau level in each case (Fig. 9F). The pattern was unaffected by placing the cricket ventral or dorsal side up (asterisk, Fig. 9F).
As an intriguing note, just after attempting to right on arousal, the cricket paused suddenly in the ventral-side-up position for 2.5 s on the wooden bar. During this pause only one CI spike was observed (asterisk, Fig. 9C).
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Discussion |
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General overview of the motor output defining thanatosis in the cricket
The main finding of this study is that motor output during thanatosis in
the cricket is characterized by the decreased tonic activity in slow
excitatory- and common inhibitory motor neurones to the flexor muscle.
Thanatosis does not depend on the absence of motor output, nor is there any
evidence for a catch-like mechanism in the muscle. Rather, muscle tonus
(created by the weak discharge of the slow exciters) is maintained by
suppression of CI activity that would otherwise relax the slow muscle fibres
(Usherwood and Grundfest,
1965). These characteristics are generally in good agreement with
those derived for other insects during thanatosis. In stick insects, only slow
units were active while most of the other units were inactive
(Godden, 1972
). In the locust,
there was little or no motor activity in any of the hindleg muscles
(Faisal and Matheson,
2001
).
The action of octopamine (released from DUM neurones) on the skeletal
muscle has been investigated in several orthopteran insects. In locusts,
octopamine reduces the amount of catch-like tension displayed by the extensor
tibiae muscle (Evans and Siegler,
1977). By contrast, in wetas, infusion of octopamine (released
from DUM neurones) into the slow extensor tibiae muscle and the subsequent
stimulation of the slow extensor motor neurone led to catch-like tension
(Hoyle and Field, 1983b
).
Similar effects were reported in the flexor tibiae muscle
(Field, 2001
). Although
further investigation is clearly necessary, effects of octopamine appeared to
be small for maintenance of the muscle contraction in crickets, as ablation of
the distal flexor nerve that contains the DUM axons did not affect the
flexed-leg posture during thanatosis (Table
1).
The most striking feature distinguishing thanatosis from other immobile
states is `active suppression (presumably central inhibition)' of the CIs,
which was inferred from their marked excitation just after arousal despite the
motionless of the leg (Fig. 9).
As the CIs act by reducing residual tension in slow muscle fibres and speeding
up ongoing leg movements (Usherwood and
Grundfest, 1965; Burns and
Usherwood, 1979
; Wolf,
1990
), tonic inhibition of the CIs during thanatosis promotes
muscle rigidity and the maintenance of a particular stable posture. The
post-arousal excitation (possibly post-inhibitory rebound) of the CIs reduces
the muscle rigidity in preparation for locomotion. It is interesting that this
period of the marked activation of CIs exactly coincides with that in which
explosive escape-running occurs when the cricket is disturbed
(Nishino and Sakai, 1996
).
The motor effects of catalepsy involve muscles of all movable joints and
segments (Nishino and Sakai,
1996), most of which are known to receive innervation from CIs
(all six legs, Burrows, 1973
;
Hale and Burrows, 1985
;
Watson et al., 1985
;
Schmäh and Wolf, 2003
;
the antennae, Honegger et al.,
1990
; body wall muscles, Yang
and Burrows, 1983
; Schmäh
and Wolf, 2003
). The inhibition of CIs is therefore very likely to
occur commonly in posture-controlling muscles during thanatosis. CIs that are
not associated with clusters of excitatory motor neurones
(Fig. 5C,D) are now regarded as
being clonally more closely related to inhibitory interneurones than to
excitatory motor neurones (Wolf and Lang,
1994
). This may underlie the anatomical observation that single
CIs innervate functionally different muscles and relax them synchronously. It
is known that even different CIs (e.g. CI1 innervates the extensor tibiae
muscle while CI2 and CI3 innervate the flexor tibiae muscle) show synchronous
activity because they receive many synaptic inputs in common, both in imposed
movements (Hale and Burrows,
1985
; Schmidt and Rathmayer,
1993
) and in active walking movements
(Wolf, 1990
). These features
have been difficult to understand in the control of active movements because
relaxation should occur out of phase between antagonistic muscles
(Hale and Burrows, 1985
).
However, the principle is potentially advantageous for the control of static
posture, because the rigidity of all movable joints could be controlled at the
same time by the activity of a relatively small number of CIs. Synchronizing
the inhibition of CIs is especially important for survival because even small
movements occurring in part of the body can elicit the attention of potential
predators such as praying mantises
(Yamawaki, 1998
).
During thanatosis, fast and intermediate exciters were almost completely
inactive while slow exciters were continuously active. This was also the case
for the quiescent state. However, detailed observations indicated that
exciters were held under weak inhibition during thanatosis. For example, fast
units recruited in the `leg flexion response (interganglionic response)'
caused by pressing the prothorax on induction of thanatosis were not recruited
when the same stimuli were applied during thanatosis
(Nishino et al., 1999). The
discharge of intermediate exciters that occurred during ventilatory phases in
the quiescent state (Figs 4C,
6C) was suppressed during
thanatosis (Fig. 4D, inset in
Fig. 9B). Asimilar inhibition
appeared to occur for the slow exciters because they discharged at a somewhat
lower frequency in thanatosis than in the quiescent state
(Fig. 8) and the enhanced
activity often occurred just after arousal
(Fig. 9E). It is known that
motor neurones to the same muscle usually have a high proportion of
postsynaptic potentials in common (Burrows
and Horridge, 1974
). This suppression of all exciter types may be
attributed to the inhibition of premotor elements that excite the entire motor
neuronal pool, as speculated by Godden
(1972
).
One might then ask why are only slow exciters continuously activated during
thanatosis? Activation of the slow exciters occurs largely through persistence
of the postural resistance reflex (Field
and Coles, 1994) mediated by the femoral chordotonal organ (FCO),
which contains position-sensitive sensory neurones as well as velocity- and
acceleration-sensitive neurones (Burns,
1974
; Matheson,
1990
,
1992
). This was revealed by an
ablation study in which removal of sensory cells in the ventral part of the
FCO scoloparia led to a severe loss of flexor muscle tonus in the respective
leg during thanatosis (Nishino et al.,
1999
). Similar effects from inactivation of the FCO have been
found in standing locusts (Usherwood et
al., 1968
).
I propose that the `muscle plasticity (catalepsy)' typical of thanatosis
depends on a weak discharge of the slow exciters producing a minimal force
sufficient only to maintain particular tibial positions. More intensive
recruitment of the slow exciters would produce a slow flexion movement instead
of catalepsy (Hoyle and Burrows,
1973; Hoyle,
1980
). In order to sustain a stable flexed-leg posture, despite
the weakness of the muscle contraction, the accessory flexor muscle acts to
mechanically stabilize the FT joint synergistically with the inhibition
of CI activity, as discussed below.
Functional roles of the main- and accessory flexor muscles in the cricket
Behaviourally, locusts do not walk quickly and escape by means of a
powerful jump, whereas crickets show a less powerful jump but can run quickly
(Tauber and Camhi, 1995).
Underlying these behavioural differences, there are certain morphological and
physiological differences in the flexor muscles of these two species. Most
prominently, the locust main flexor muscle has nine exciters, each of which
exhibits a unique and complex innervation to a restricted number of muscle
fibre bundles (Phillips, 1980
;
Sasaki and Burrows, 1998
); in
contrast, the cricket has double the number of flexor exciters (about 18) but
each innervates either the proximal-, middle or distal-muscle compartment with
a simple, almost non-overlapping pattern
(Fig. 3). Progressing distally,
these muscle compartments insert into more distal points on the flexor
apodeme/cushion complex at increasing angles and change from slow to
predominantly fast innervation (Nishino,
2003
). As a result, recruitment of the distal muscle compartment
accelerates tibial flexion dramatically by the synergistic action of its
increased effective leverage and rapid contraction. These properties must
underlie the agility of the cricket, enabling the tibia to move quickly to the
full flexion in running, jumping, kicking, and in the `leg flexion response'
on induction of thanatosis (Nishino et
al., 1999
). The cricket exhibits continuous acceleration of leg
movements when a pulse of air is given during slow walking
(Gras et al., 1994
). Jumping
and kicking readily occur, even during locomotion
(Tauber and Camhi, 1995
;
Hustert and Gnatzy, 1995
).
A unique morphology and neural innervation also occur in the accessory
flexor muscle. Compared with locusts, the cricket accessory flexor muscle is
well-developed (Fig. 2) and is
functionally separated from the distal muscle compartment of the main flexor
muscle due to the non-overlapping innervation of motor neurones
(Fig. 5C). The origin of the
accessory flexor muscle lies on the dorsal side of the FT joint, while
the origins of the main flexor muscle bundles lie on the same side (ventral)
as the apodeme (Figs 2B,
5A). Because the accessory
flexor muscle inserts onto the apodeme with such a large leverage angle, it
pulls the cushion dorsally and obliquely to the normal proximaldistal
path of action of the main flexor muscle. This off-axis, oblique pull of the
accessory flexor muscle has the effect of closing the joint with increasingly
greater leverage as the FT angle approaches zero
(Hustert and Gnatzy, 1995;
Nishino, 2003
). Hence, due to
this mechanical advantage (Heitler,
1974
; Hustert and Gnatzy,
1995
), the accessory flexor muscle effectively holds the FT
joint at 0° FT joint angle during thanatosis. This was clearly
demonstrated by the ablation study in which cutting the accessory flexor nerve
resulted in greater deficiency in maintenance of the tibial flexion than in
ablation of the proximal flexor nerve
(Table 1), although both nerves
have almost identical sets of slow exciters and although the proximal flexor
muscle was larger in volume than the accessory flexor muscle
(Nishino, 2003
).
Due to its oblique insertion onto the flexor apodeme, the accessory flexor
muscle has the joint-stabilizing role at any angle between 0° and
100°, corresponding to the range in which the flexor tibiae muscle
functions (Nishino et al.,
1999), by hindering the axial movement of the apodeme. Even with
no electrical activity, the contractile viscosity of the accessory flexor
muscle stabilized the joint, because cutting the accessory flexor nerve gave a
smaller deficiency than ablation of the muscle bundles, in the maintenance of
the tibial flexion during thanatosis (Table
1). This function seemed to be enhanced with suppression of CI
activity, which prevents relaxation of the muscle, resulting in stiffening the
joint. Indeed, the CI suppression exactly coincided with assuming
sudden-immobile posture such as `standing phase' (termed by
Gras and Hörner, 1992
)
that occurs in escape trials (Fig.
9C), or `thanatosis' that occurs immediately after forcible
restraint of leg movements (Nishino et
al., 1999
).
In other orthopteran insects, the development of the accessory muscle (or
comparable muscle bundles) seems to also relate to the joint stabilization
function. For example, the stick insect Cuniculina impigra has a
larger percentage of morphologically distinct bundles of slow fibres in the
distal portion of the extensor tibiae muscle compared with the locust
Locusta migratoria, leading to a stiffening of the leg joint to
assume effective catalepsy (Bässler et
al., 1996; Bässler and
Stein, 1996
). A particular tibial position of the metathoracic
leg, assumed through avoidance conditioning, is maintained stably without any
movements in the weta Hemideina femorata
(Hoyle and Field, 1983a
),
which has a well-developed accessory flexor muscle as in crickets, but is
`fidgeted' in the locust Scistocerca gregaria
(Hoyle, 1980
), which has a
small accessory flexor muscle (Matheson and Field, 2000).
CI activity may reflect arousal level
The present study enables the physiological characteristics of CIs to be
defined more exactly. CI2 and CI3 in the cricket have similar physiological
characteristics to those of the locust. For example, they had much lower tonic
firing frequencies than the slow units
(Burrows and Horridge, 1974).
The two CIs (CI2 and CI3) showed very similar activity patterns that were
often characterized by synchronous excitation (Figs
6,
7;
Hale and Burrows, 1985
;
Wolf, 1990
;
Schmidt and Rathmayer, 1993
).
One recording showed an apparent difference in threshold between the CIs, with
the smaller unit showing a higher background discharge and a higher tonic
discharge to mechanical stimuli than the larger unit
(Fig. 7).
However, in a strict comparison, background activity of CIs during
quiescent state was different between these two species; whereas activity of
the two CIs in minimally restrained locusts dropped 0 Hz
(Wolf, 1990), that of
minimally restrained crickets sustained tonically at ca. 0.52 Hz
(Fig. 9). This difference may
reflect the fact that the cricket tends to walk more actively than the locust,
so relaxation of the slow muscle fibres due to activation of CIs may be
important for starting locomotion smoothly. In the locust, the membrane
potentials of CIs are maintained at a high level, even in the stance phase
(static phase) of active walking (Wolf,
1990
). Similarly, the much higher background activity of CIs
(510 Hz, Figs 6,
7) detected in restrained
crickets, compared to that in minimally restrained crickets, may reflect a
high level of arousal driving an escape tendency.
These observations indicate that CI activity somehow increases with
progressive arousal as noted in locusts
(Wolf, 1990). The intensity
and duration of the increased excitation were proportional to the strength of
mechanical stimuli required for arousal
(Fig. 7). Similar CI activation
due to mechanical stimuli to sensory hairs on body appendages has been
reported in cockroaches (Fourtner and
Drewes, 1977
) and in locusts
(Runion and Usherwood, 1968
;
Hale and Burrows, 1985
;
Wolf, 1990
;
Schmidt and Rathmayer,
1993
).
In contrast, strong inhibition of the CIs occurred during thanatosis,
particularly in the beginning of the maintenance phase. The deeper in
thanatosis, the more inhibition occurred in CIs
(Fig. 9). Towards arousal,
inhibition gradually declined (Fig.
9F). The arousal was characterized by a long-lasting excitation of
the CIs (Fig. 9). Since this
state was similar to CI activation caused by very strong mechanical stimuli
such as pinching of the tarsus (Fig.
7D), one might speculate that self-stimulation involved with
righting causes a tonic increment in CI activity. However, this is less likely
for the following reasons. Firstly, the same excitation occurred in any other
cases even without a righting response
(Fig. 9F). Secondly, such a
long-lasting excitation never occurred when the cricket was rotated during
quiescent state. Hence, in thanatosis the cricket appears to be held at a very
low level of arousal. Interestingly, the cricket showed vigorous palpal and
antennal oscillation just after arousal
(Nishino and Sakai, 1996),
indicating `awakening' from a state of low arousal.
Investigation of premotor elements using as CI inhibition as a reliable index of the thanatotic state is a future step to probe into the central function that operates this unique arousal mechanism.
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