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First published online October 7, 2004
Journal of Experimental Biology 207, 3899-3915 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01220

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Motor output characterizing thanatosis in the cricket Gryllus bimaculatus

Hiroshi Nishino

Laboratory of Neurocybernetics, Research Institute for Electronic Science, Hokkaido University, Sapporo, 060-0812, Japan

View larger version (96K): [in a new window]   Fig. 1. (A) Cricket in flexed-leg thanatosis placed on a flat wooden bar. See Materials and methods for induction procedure. The bodily movement during thanatosis was monitored with a photo-coupler (P) settled beside the abdomen. (B) Catalepsy during hanging. The cricket continued to be immobile with the femoro–tibial (F–T) joint opened (30–40°) by the body mass until arousal. (C) Set-up for extracellular recording in a minimally restrained cricket. A pair of insulated copper wires were inserted through small holes on the cuticle to the flexor tibiae muscle and are bound with an earth electrode extended from the pronotum and fixed on the forewings. Each insertion point of the wire was fixed by wax resin (grey). (D) The recording site, revealed by a forward-fill of the main leg nerve, nerve 5B2 (N5B2). The recording electrode remained inserted. The leg was cleared with methyl salicylate. This preparation, in which the tip of the recording wire contacts with the proximal flexor nerve, was used for Fig. 4A.

View larger version (27K): [in a new window]   Fig. 2. Camera lucida drawings of the flexor tibiae muscle in the metathoracic leg in the locust Locusta migratoria (A) and the cricket Gryllus bimaculatus (B). The relative size of the accessory flexor muscle (red) against the main flexor muscle (orange) in the cricket is larger than that in the locust. Whereas the locust has a thin flexor apodeme in the distal femur, the cricket has a cushion enlargement on which the accessory flexor muscle attaches.

View larger version (43K): [in a new window]   Fig. 3. Camera lucida drawings of motor nerve innervation of the flexor and extensor tibiae muscles in the cricket metathoracic leg, viewed posteriorly (A) and ventrally (B). The main flexor muscle (orange) is divided into the proximal-, middle- and distal regions, which are locally innervated by the proximal flexor nerve, the middle flexor nerve and the distal flexor nerve, respectively. A pair of muscle bundles of the accessory flexor muscle (red) are innervated by the anterior and posterior branches of the accessory flexor nerve diverging from the main leg nerve (N5B2).

View larger version (89K): [in a new window]   Fig. 4. Motor neuronal activity recorded from the three motor nerve branches (proximal-, middle- and distal-flexor nerves) supplying the main flexor muscle before, during and after thanatosis. The maintenance phase of thanatosis is shaded grey. During thanatosis, activity of intermediate- and fast-exciters was almost completely suppressed (A–E). The intermediate exciters tended to be activated more frequently when ventilatory movements (deflections, lower trace) occurred frequently during thanatosis (B). However, their activity was not necessarily coupled with ventilation phases but seen also in non-ventilatory phases (arrows and time-stretched inset in B). In other recordings, intermediate units vigorously activated with ventilatory movements during the quiescent state (C) were rather suppressed when ventilatory movements occurred during thanatosis (D). Fast exciter units are truncated in A, B and E. Note that voluntary leg movements immediately after arousal occurred in B but not in A and E.

View larger version (90K): [in a new window]   Fig. 5. Anatomy of the accessory flexor muscle and the motor neuronal innervation. (A) Camera lucida drawing of the motor innervation in the distal part of the metathoracic femur, viewed posteriorly. The accessory flexor muscle (red) is inserted diagonally (about 45°) onto the cushion, which is extended from the apodeme on which the main flexor muscle (orange) attaches. The accessory flexor nerve diverged from N5B2 gives rise to fine ramification with rich varicosities in the accessory flexor muscle. (B) Back-fill from the accessory flexor nerve with dextran, tetramethyl rhodamine, revealing that two inhibitor axons (whose somata are identified in the ganglion) pass through N5B2 distally to innervate the tibial muscles (indicated by broken boxes), thus are regarded as common inhibitors. The posterior branch of the accessory flexor nerve is slightly thicker than the anterior branch although both contain identical motor axons. (C) Motor neurones in the metathoracic ganglion, back-filled differentially from the distal flexor nerve and accessory flexor nerve with dextran, fluorescein (green) and dextran, tetramethyl rhodamine (red), respectively. There is no overlap of motor neurones supplying both motor nerves. Somata of four exciters sending axons to the distal flexor nerve (three are fast- and one is intermediate-type) are much larger than those sending axons to the accessory flexor nerve [one tentative intermediate (i) and three slow-type (s)]. Two somata of common inhibitors (CI2, CI3) are located closely to the midline (right edge of photo) and segregate from exciters that congregate in the antero-lateral part of the ganglion. Visible nerve roots are numbered. (D) Motor neurones supplying the accessory flexor nerve in the metathoracic ganglion, back-filled with NiCl2 and silver-intensified. Note that neither sensory afferents nor dorsal unpaired median (DUM) neurones were stained and there is no correlation of soma location of exciters between C and D. Visible nerve roots are numbered and the vertical broken line indicates midline of the ganglion. (E,F) Composite photomicrographs reconstructed from 19 µm transverse sections at the levels indicated in D. Three slow exciters arbitrarily numbered (s1–3), corresponded to those in D. The tentative intermediate exciter (i) is characterized by the larger soma and the thicker axon (arrow in E) compared with slow-type exciters. The primary dendritic area of CIs was circled by thin broken line. Visible nerve roots are numbered and the vertical broken line indicates midline of the ganglion.

View larger version (49K): [in a new window]   Fig. 6. Physiological characteristics of motor neurones innervating the accessory flexor muscle. (A) Efferent activity simultaneously recorded from accessory flexor nerve and N5B2 in the distal femur when the cricket was disturbed. When two small units, two moderate-sized units, and one large unit fire in the accessory flexor nerve (upper trace), only two moderate-sized units were recorded from N5B2 (lower trace), suggesting that the two moderate-sized units are common inhibitors. (B) Recording from the accessory flexor nerve efference (upper trace) combined with an intracellular recording from a fibre in the accessory flexor muscle (bottom trace) when the cricket is in the quiescent state, showing that spikes of two CIs evoke inhibitory junctional potentials (IJPs) in the muscle fibre. When the two CIs fired synchronously (asterisks), summation of IJPs occurred. (C) Activity pattern of the motor neurones in a ventilation phase. Initially two CIs and subsequently slow and intermediate exciters are activated in expiration, resulting in hyperpolarization of the slow muscle fibre. All units are declined in inspiration, resulting in resumed activity of the muscle potential. Excitatory junctional potentials corresponding to spikes of the intermediate exciter were not seen in this recording but were detected in recordings from other muscle fibres, as noted by Matheson and Field (1995).

View larger version (87K): [in a new window]   Fig. 7. Effects of external disturbances on activity of motor neurones innervating the accessory flexor muscle. (A) During the quiescent state, three slow units having different amplitudes (coloured in the magnified inset) were clearly discriminated. Only the small CI unit (1) fires tonically at about 3 Hz. (B) Tapping the recording substrate evoked brief activation of the large CI unit (2) additionally to the small CI unit (1) and slow excitatory units (especially in the smallest units). (C) Touching the prothoracic tarsus ipsilateral to the recording site evoked longer activation of two CI units and slow units. (D) Strong pinching of the same tarsus evoked a brief activation of intermediate units and subsequent long-lasting activation in two CIs and slow units. In this example, the tonic activation of CIs and slow units was sustained more than 70 s with slowly adapting discharges.

View larger version (29K): [in a new window]   Fig. 8. Activity of slow exciters during immobile postures of the cricket. (A) Raw data of activity of slow exciters when the cricket maintained the recorded F–T joint at various angles during thanatosis or quiescent state. During the quiescent state, at least one CI unit fired sporadically at low frequency (0.5–2 Hz). (B) Average spike frequencies of slow exciters sampled for 5 s when the cricket exhibited strong thanatosis, weak thanatosis (see drawings) or the quiescent state at various F–T joint angles were plotted quantitatively. To count spikes of slow motor neurones, the window (threshold) was set just above the noise level and below the CI spike amplitude. Activity during ventilatory phases and those just after arousal from thanatosis were excluded from sampling. Note that activity of slow exciters during thanatosis is rather suppressed compared with those during the quiescent state at the same F–T joint angle.

View larger version (58K): [in a new window]   Fig. 9. Activity of common inhibitory motor neurones (CIs) before, during and after thanatosis. The maintenance phase of thanatosis is shaded by grey. (A) Strong thanatosis in which the recorded F–T joint was maintained at 15°. Activity of CIs were strongly suppressed especially in the beginning of thanatosis, then increased gradually towards arousal. Only eight spikes of CIs were identified in 42 s of the maintenance phase of thanatosis. The strong recruitment of CIs occurred immediately after arousal despite lack of motion of the recorded F–T joint at 120° (note that large exciters are inactive). The increased activity suddenly waned once the cricket started walking with recruitment of intermediate exciters (Int.). Large truncated spikes are cross-talk from large motor neurones. (B) Weak thanatosis in which the recorded F–T joint was maintained at 95°. CI started firing soon after induction of thanatosis and occurred frequently during thanatosis. Nevertheless, strong recruitment of CIs occurred immediately after arousal when the F–T joint was maintained at 90°. Note that CIs fire in non-ventilatory phases but the smallest slow exciter is activated during ventilatory phases (inset). (C) Arousal from strong thanatosis. Intermediate exciter was recruited with some cross-talk from large motor neurones. Note that a brief pause (asterisk) occurred when the cricket was struggling to right itself. Large spikes were truncated. (D,E) Time-stretched activity of labelled bars indicated in C. Note that strong recruitment also occurred in the slow exciters (causing apparent increase in baseline thickness) although the F–T joint was maintained same angle (15°) as in thanatosis. (F) Peri-stimulus time plot of CI activity in the beginning, just before arousal, and just after arousal. Five trials of thanatosis derived from three individuals are plotted. One trial (asterisk) indicates thanatosis induced in normal dorsal-up posture.