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First published online June 13, 2008
Journal of Experimental Biology 211, 2123-2133 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.019125
Front leg movements and tibial motoneurons underlying auditory steering in the cricket (Gryllus bimaculatus deGeer)
Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
* Author for correspondence (e-mail: bh202{at}cam.ac.uk)
Accepted 14 April 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: cricket, phonotaxis, motoneuron, auditory processing
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Ball et al., 1989;
Schildberger et al., 1989;
Pollack, 2001). The auditory
afferents transmit the auditory information from the ears located in the front
legs to a small number of auditory interneurons in the prothoracic auditory
neuropiles. The auditory information is then passed on by few ascending
neurons to local and descending brain neurons which may form a pattern
recognition network (Schildberger,
1984).
Little is known about the motor performance during phonotaxis, especially
upon changes in sound direction. Trackball recordings show that
phonotactically walking females turn towards attractive sounds with a delay of
55–60 ms (Hedwig and Poulet,
2004; Hedwig and Poulet,
2005). Pollack and Hoy
(Pollack and Hoy, 1980)
reported a clear response of a flight muscle to acoustic stimulation during
phonotaxis in flying crickets (Teleogryllus oceanicus). During both
phonotactic flying (Pollack and Hoy,
1980; Nolen and Hoy,
1986; Brodfuehrer and Hoy,
1989) and walking (Poulet and
Hedwig, 2005) steering responses may be achieved by a pattern
recognition system regulating the gain of a more direct auditory-to-motor loop
to the steering motor network. In phonotactically active animals it should
therefore be possible to observe specific motor outputs as a direct result of
auditory stimulation.
An effective method for steering during walking
(Dürr and Ebeling, 2005;
Rosano and Webb, 2007) and
jumping (Santer et al., 2005)
in insects is to change the positioning of the front legs. To identify
movement components and consequently motoneurons mediating auditory steering
responses we therefore first analysed the movement of a front leg tibia during
phonotaxis, and related this to the direction of the sound patterns presented.
This identified a critical involvement of front leg movements, and in
particular of tibial extension and flexion movements, in auditory steering.
Using electromyogram recordings we then analysed the activity of the tibial
extensor and flexor motoneurons during phonotaxis. Based on behavioural data
and electromyogram recordings from tibial muscles, we identified these
intracellular motoneurons and investigated if any direct or indirect auditory
input exists and if it can be gated by descending interneurons or local
pharmacological stimulation.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Trackball system
For phonotaxis experiments crickets were placed on top of a trackball
system and held by a small metal pin waxed onto their back. For details see
Hedwig and Poulet (Hedwig and Poulet,
2005).
Optical measurements of leg movements
A custom-build optoelectronic system was used to measure front leg
movements (Hedwig, 2000;
Hedwig and Becher, 1998). A
modified SLR camera with a 2D photodiode (United Detector Technology,
Hawthorne, CA, USA; PIN DLS-20) in the plane of the film was used to record
the movements of a small piece of reflective material (Scotchlite 7610; 3M
Laboratories, Neuss, Germany) fastened around the distal part of the tibia
using a small drop of beeswax. We recorded the frontal projection of left
tibial movements during walking; i.e. its left–right and up–down
movements. This required animals to walk towards the light source of the
optical recording system, which reduced the phonotactic performance, even when
long wavelength (LED at 630 nm) illumination was used (N=28).
For relating electromyogram (EMG) recordings to the step cycle the forward–backward motion of the femur was recorded from above the animal and used as an indication of the swing and stance phase. Here a one-dimensional version of the optoelectronic system (Laser Components, Olching, Germany; Type 1L30) was used with infra-red illumination (LED at 850 nm; N=4).
Acoustic stimulation
An artificial calling song at a carrier frequency of 4.8 kHz, syllable
duration of 21 ms, syllable period of 42 ms, chirp duration of 250 ms and
chirp period of 500 ms was used (Thorson
et al., 1982). Crickets were presented with alternating six-chirp
sequences from the left and the right at 75 dB sound pressure level (SPL)
relative to 2x10–5 Pa. Sound stimuli were digitally
generated at 22.05 kHz sampling rate (CoolEdit 2000, now: Adobe Audition;
Syntrillium, Phoenix, USA) and were presented by PC audio boards via
two active speakers (SRS A57; Sony, Tokyo, Japan) positioned 60 cm frontal to
the cricket each at an angle of 45° to the animal's length axis. Sound
intensities were calibrated with an accuracy of 1 dB at the position of the
cricket using a Bruel and Kjaer (Naerum, Denmark) free field microphone (Type
4191) and measuring amplifier (Type 2610).
Electromyogram recordings
Electromyograms (EMG) of tibial extensor and flexor muscles were obtained
using two varnish-coated steel wires (30 µm diameter) inserted distally
into the extensor tibia muscle or proximally into the flexor tibiae muscles
(Fig. 4A). Large amplitude
extensor muscle potentials were recorded while at the same time activity in
the flexor muscles was reliably picked up at lower amplitude. This occurred
vice versa in flexor recordings
(Fig. 4B). In all further
recordings we consequently used this cross talk to identify flexor activity in
extensor recordings, avoiding the need for separate flexor recordings. A
gliding length filter (Hedwig and Knepper,
1992; Römer et al.,
2002) was applied to EMG recordings and EMG peaks were sorted by
amplitude of voltage change over time into four classes
(Fig. 4C). Signals were picked
up using an amplifier (Differential AC Amplifier Model 1700; A-M Systems,
Sequim, WA, USA).
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Thick-walled borosilicate micropipettes with resistances of 60–120
M
, filled with 5% Lucifer Yellow (Molecular Probes, Eugene, OR, USA) in
water (tip) and 1 mol l–1 LiCl (shaft) were used to record
from the main neurites of motoneurons. Recordings lasted for up to 1 h. For
intracellular staining with Lucifer Yellow a 1–9 nA hyperpolarising
current was injected for 5–20 mins. Signals were recorded using an
SEC-10L amplifier (NPI, Tamm, Germany) and digitised at 10 kHz. Motoneurons
were characterised and identified according to morphology, the impact of
spiking on tibial movement and the size of evoked EMG potentials. A total of
250 crickets were used, of which 93 yielded the presented data.
Sensory stimulation during intracellular recordings
Auditory
Sound stimuli were presented using a small speaker (ø=2 cm) attached
to the wide end of a 15 cm conical copper tube, the narrow end of which was
placed 2 cm from the opening of the ipsilateral auditory spiracle. Intensities
of stimuli were calibrated to an accuracy of 0.5 dB SPL at the position of the
spiracle. The carrier frequency of sound stimuli was 4.8 kHz, and the
amplitude used throughout was 90 dB SPL. Background noise in the room was
<45 dB SPL.
Air currents
Air currents stimuli were generated using a Picopump (PV 820 Pneumatic
PicoPump; WPI, Sarasota, FL, USA) connected to a rubber tube (i.d. 0.5 mm),
the other end of which was positioned 5 cm in front of the animal.
Tactile
Tactile stimuli were applied manually using a small paintbrush. In all
recordings the tibia and tarsus were gently touched at several positions, and
the largest response recorded.
Visual
Visual stimuli were generated using a white LED (Nichia 1100 mcd, 50°
divergent angle; Tokushima, Japan) positioned at a distance of 3 cm from the
head of the animal pointing at the eye ipsilateral to the front leg
investigated.
Activation of descending pathways
A small bipolar hook electrode was placed underneath the connective
ipsilateral to the recorded motoneuron between the prothoracic and
suboesophageal ganglia and insulated with a mixture of 90% VaselineTM and
10% paraffin. Stimuli were generated using a stimulus isolation unit (WPI A360
SIU), triggered by a custom-built pulse generator. Current pulses were of 2 ms
duration and between 1 and 50 µA amplitude, applied at 1–100 Hz
(N=19).
Pharmacological stimulation
To disinhibit or activate thoracic motor networks the ganglion was bathed
in the GABA blocker picrotoxin (10–4 mol l–1
in saline) or the muscarinic receptor agonist pilocarpine
(10–3 mol l–1 in saline), respectively
(Ryckebusch and Laurent,
1993). This elicited increased motor activity after 20–30 s,
which persisted until the entire thoracic cavity was washed with saline. To
ensure all activity recorded was generated within the prothoracic ganglion,
the connectives towards the suboesophageal ganglion were cut in
N=3/23 experiments.
Processing of neurons stained with Lucifer Yellow
After intracellular staining of a motoneuron it was left for 5–20
mins to allow the dye to diffuse throughout the cell. The ganglion was then
dissected and placed in 4% formaldehyde for 1 h. The specimens were then
dehydrated and cleared in methyl salicylate (Sigma-Aldrich, St Louis, MO,
USA). The ganglion was photographed using a digital SLR camera (Canon EOS
350D) attached to a Zeiss (Axiophot) fluorescence microscope with a
ultraviolet light source (Zeiss VHW 50f-2b). For graphical projections of
neural arborisations photo-stacks were traced manually using Adobe Photoshop
(CS 8.00).
Data sampling and analysis
An A/D board (MIO 16E4, National Instruments, Austin, TX, USA) linked to
custom-built software running under LabView 5.01 (National Instruments) was
used in all experiments. Behavioural and electrophysiological data was
analysed in Neurolab (Hedwig and Knepper,
1992). Further data analysis was performed using MatLab 6.5
(Mathworks, Natick, MA, USA).
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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It may be advantageous for phonotactic steering to synchronise the stepping
cycle with the rhythm of incoming chirps
(Hedwig and Poulet, 2004).
Step cycle durations ranged between 250 ms and 600 ms (21–23°C).
Fig. 3A shows the distribution
of step cycle durations for one representative animal. We tested animals with
chirp rates between 1 and 5 Hz. There was no coupling between the step rhythm
and the chirp rhythm at any repetition rate tested when the sound direction
was constant (Fig. 3B). We
furthermore tested if there is a phase-dependent effect of incoming sounds on
the leg movements using a double pulse paradigm, where the first and last two
syllables of each chirp were presented from contralateral speaker, and the
middle two syllables from ipsilateral speaker
(Fig. 3C inset). The leg
movement traces were sorted into 20 bins depending on their phase relative to
the onset of the ipsilateral sound, and averaged within each bin, centred at
the peak of swing phase (t=0 ms). Leg movement traces from four
representative phase relations between ipsilateral sounds presentation and the
step cycle are shown (Fig. 3C).
Ipsilateral acoustic stimulation during swing phase, but not during stance,
reduced the amplitude of the left–right leg movement during the
following step (red, asterisk).
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Movements in at least three joints can contribute to the measured movements of the front leg. However, owing to the nature of our recording method we cannot directly identify the contribution of coxal rotations, tibial extension and flexion movements or overall bending movements of the body to the observed front leg movement patterns (see Discussion).
Tibial musculature
To investigate the control of tibial movements we identified the tibial
musculature and its innervation. Nomenclature was based on the description of
the hind leg musculature in locust
(Snodgrass, 1929). A single
tibial extensor muscle (dorsal: 135) and four tibial flexor muscle bundles
(one antero-ventral, two ventral, one postero-ventral: 136a–d) were
identified (Fig. 4A). The
proximal ends of flexors 136a and 136d attached to multiple points along the
anterior and posterior cuticle, respectively. A single retractor unguis (139)
was positioned antero-dorsal to the acoustic trachea.
EMG recordings during phonotaxis
Extracellular recordings from tibial muscles were used to monitor tibial
motoneuron activity. A single pair of EMG electrodes inserted into the
extensor muscle made it possible to monitor both extensor and flexor muscle
potentials (see Materials and methods). We recorded the muscle activity during
walking and simultaneously measured forward–backward movements of the
femur to monitor the step cycle. Amplitude sorting of gliding length filtered
(see Materials and methods) EMG potentials allowed us to separate at least
four different motor units contributing to tibial movements during walking
(Fig. 4C). These were
characterised by their typical activity during walking. The description of EMG
activity presented in this paragraph relates to motor units. An intracellular
identification of the associated motoneurons is presented below.
Fig. 4D shows the average spike
occurrence of each motor unit during the step cycle. Fast extensor tibiae
(FETi) activity was present in less than 1 out of 20 steps and occurred just
prior, or during, early swing phase. This motor unit only showed increased
activity (1–2 spikes per step) during escape running, elicited by wind
stimulation of the cerci. Slow extensor tibiae (SETi) activity also occurred
just prior and during early swing phase. Fast flexor tibiae (FFTi) potentials
occurred during late swing and early stance phase. Finally, slow flexor tibiae
(SFTi) activity was high throughout the step cycle, but reduced just prior and
during early swing phase.
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).
Increases in SETi activity always preceded changes in the trackball movements,
and reliably increased with a delay of 35–40 ms after ipsilateral sound
presentation.
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Identification of tibial motoneurons
To identify the motoneurons underlying tibial EMG activity during
phonotactic walking, we intracellularly recorded and identified the front leg
tibial motoneurons and revealed their morphology and synaptic inputs.
Identification criteria included the effect of depolarisation on tibial
movement, the amplitude of the elicited EMG signal and their morphology.
Two extensor tibiae motoneurons, the FETi and the SETi (Fig. 7A), were individually identified. Each spike of the FETi elicited a >10 mV EMG potential and gave rise to a rapid tibial extension. By contrast, SETi spikes gave rise to 3–5 mV EMG potentials and resulted in slower, graded extension movements, dependent on spike rate. One fast flexor motoneuron (FFTi) was also identified (Fig. 7B left). In addition a group of at least four FFTi motoneurons was morphologically distinct from the latter FFTi (Fig. 7B right) and a group of at least three SFTi motoneurons (Fig. 7C) were distinguished. The minimal number given for these groups of FFTi and SFTi are derived from sequential stainings of the respective neuron type in the same specimen. The single identifiable FFTi was labelled FFTi1 and the morphologically distinct group of four FFTi was labelled FFTi2–5 (Fig. 7B). Spike activity in either type of FFTi gave rise to 2–3 mV EMG potentials and resulted in graded flexion movements of the tibia. SFTi spikes elicited the smallest (1 mV) EMG potentials, and alone were insufficient to move the tibia.
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Morphology of tibial motoneurons
Somata of all motoneurons were located antero-ventrally with the somata of
SETi, FETi and the group of FFTi2–5 typically adjoining the
anterior-most border of the ganglion, whereas somata of the group of
SFTi1–3 and that of FFTi1 were located more posteriorly. The most
prominent neurite of all motoneurons runs 150–200 µm beneath the
dorsal surface of the ganglion between the midline and the point where the
axon left the ganglion through the respective side nerve. A second large
neurite runs posteriorly in all motoneurons except for FFTi1, where it runs
antero-medially. All motoneurons exit the ganglion via nerve 5, with
exception of the FETi which exits via nerve 3
(Fig. 7A left).
The dendrites of both FETi and SETi extend throughout the entire
ipsilateral dorsal surface of the ganglion, with extensive medial branching
(Fig. 7A). The main processes
and the posterior dendrite of SETi were thicker than those of FETi. The main
processes of FFTi1 were very large (ø=20–30 µm), with the
main, thickest neurite almost reaching the midline. The main branches gave off
very short secondary neurites (Fig.
7B left). By contrast, the main neurites of FFTi2–5
(Fig. 7B right) were much
thinner (ø=5 µm) than of any other tibial motoneuron, with secondary
and tertiary branching patterns similar to the extensor motoneurons, but very
sparse. The morphology of the main neurites of SFTi1–3 varied
substantially and only one example is given
(Fig. 7C). The extent of the
branching patterns of their secondary and tertiary neurites was similar to
SETi. None of the motoneurons exhibited any overlap with the ventrally located
auditory neuropile (Schildberger et al.,
1989; Imaizumi and Pollack,
2005).
Sensory and central inputs to tibial motoneurons
Sensory inputs to tibial motoneurons were investigated during rest and
activity. SETi, FETi and FFTi2–5 did not spike at rest. By contrast,
SFTi1–3 and FFTi1 were active with a spike rate between 0.5–2 Hz
and generated frequent excitatory and inhibitory postsynaptic potential (EPSPs
and IPSPs; Fig. 8A) in resting
animals. We did not detect any auditory or visual inputs in this state.
However, all neurons received both wind and tactile inputs
(Fig. 8B), also demonstrating
that auditory evoked responses were unlikely to have been missed because of a
lack of sensitivity of the recordings. Only tactile inputs to SFTi1–3
could elicit spikes.
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).
Previous studies suggested that the brain may be involved in auditory pattern
recognition (Schildberger,
1984): successful recognition of species-specific song may lead to
phonotactic steering by a descending pathway acting on the thoracic motor
system (Poulet and Hedwig,
2005; Pollack and Hoy,
1980). To reveal any descending control over tibial motoneuron
activity we extracellularly stimulated the ipsilateral descending connective
between the prothoracic and the suboesophageal ganglia. This allowed us to
elicit EPSPs and spikes in all tibial motoneurons
(Fig. 8C). EPSPs occurred with
a delay of <3 ms and were dependent on the amplitude of the stimulation
current, indicative of a direct, parallel polyneural input from descending
pathways.
During walking, leg motoneurons are under the control of local central
pattern generating networks (Burrows,
1996, Büschges et al.,
2008). We therefore tested for local prothoracic inputs to tibial
motoneurons. Prothoracic motor networks were pharmacologically activated by
the muscarinic receptor agonist pilocarpine (10–3 mol
l–1) or the GABA antagonist picrotoxin (10–4
mol l–1) (Ryckebusch and
Laurent, 1993; Büschges et
al., 1995). In all motoneurons both picrotoxin and pilocarpine
elicited powerful motor bursts that exceeded spike threshold. However, bursts
were irregular and occurred at lower frequency (0.1–1 Hz) than during
walking (2–3 Hz; Fig.
8D). This experiment demonstrated inputs to all tibial motoneurons
from prothoracic motor networks.
Can auditory inputs be gated?
Our data from EMG recordings during phonotaxis demonstrate an auditory
input to SETi and to at least one of the two groups of FFTi motoneurons.
However, we did not detect any auditory inputs to these motoneurons at rest.
Poulet and Hedwig (Poulet and Hedwig,
2005) suggested a descending modulatory pathway, which may gate
auditory inputs towards the motor system in phonotactically active animals. It
may therefore be possible to unmask auditory inputs to SETi or either class of
FFTi by stimulating descending pathways. A strong hyperpolarising current (5
nA) was injected into SETi to both prevent spiking and reveal potentially weak
auditory inputs during and after stimulation of the connectives. However,
neither single trial nor continuous activation of descending pathways
upregulated any putative auditory inputs to any of the tibial motoneurons
(Fig. 9A). Furthermore no
auditory responses were apparent during pharmacologically elicited motor
activity (Fig. 9B). The trace
presented is a section between ongoing motor activity and was chosen as it
lacks motor burst activity that could obscure any auditory inputs.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Front leg movements during phonotaxis
We studied front leg movements during phonotaxis in order to identify the
motor pathway involved in phonotactic steering. During forward walking
left–right front leg movements were remarkably small, but during
steering leg movements showed a clear dependency on sound direction (Figs
1,
2). Throughout stimulation from
either left or right, steering movements were unchanged
(Fig. 1A,
Fig. 2A), however, in response
to a change in sound direction animals switched the steering pattern within a
single step cycle, and frequently after less than 60 ms
(Fig. 1B,
Fig. 2A). This remarkably fast
response is in agreement with rapid steering movements of 55–60 ms
measured with a trackball system (Hedwig
and Poulet, 2004; Hedwig and
Poulet, 2005) and indicates the importance of the front legs in
steering.
There are three possible, non-exclusive modes of steering using the front
legs: (1) changing the femoral/tibial angle
(Dürr and Ebeling, 2005),
(2) changing the positioning of the femur
(Laurent and Richard, 1986a;
Laurent and Richard, 1986b;
Dürr and Ebeling, 2005),
and (3) overall bending movements of the prothoracic segment passively moving
the front legs into a steering position (unlike in many species of
grasshoppers or phasmids, the prothoracic segment of cricket is not rigidly
connected to the mesothorax and could be used in steering). Although effects
due to positioning of the femur and bending of the prothoracic segment are
superimposed on the tibial movements due to the nature of our recording
technique, we judge these effects as less significant: during visual
inspection of front leg steering movements, tibial extension and flexion
movements could clearly be identified. Detailed analysis of leg positioning
using high-speed video analysis did clearly demonstrate the contribution of
tibial extension and flexion movements in steering (A. Witney and B.H.,
unpublished data).
Movement in the femoral-tibial joint provides a powerful way to affect the movement direction of the animal: increased tibial extensions shift the anterior end point (AEP) towards a more extreme position along the axis of the femur and therefore allow consequent tibial flexion to pull the animal towards that point. For tibial movements to allow for steering, the overall step cycle must be taken into account: whereas an extension during swing phase will shift the AEP forwards, an activation of extensor motoneurons during stance will push the animal away from the AEP, resulting in a sideways or even backwards movement of the cricket. Similarly flexion during swing phase would decrease the step size and therefore limit the steering, whereas flexion during stance would pull the animal forwards, towards the AEP. High speed video recordings will be necessary to clarify the details of sound-induced leg steering movements.
During acoustic stimulation, animals did not couple their overall step
rhythm to the chirp rhythm (Fig.
3B). This is in contrast to locust flight pattern generators,
which are under pivotal control of rhythmic wind inputs to synchronise wing
beats between animals (e.g. Camhi et el.,
1995). The absence of coupling between sounds and the step cycle
in crickets indicated that here steering commands are probably integrated with
the walking central pattern generator (CPG). They do not modify the overall
stepping pattern, but instead modulate the amplitude of steering responses.
The phase dependency of motor effects caused by sounds during swing phase
supports this.
Anatomy and morphology
The muscles of the front leg tibia and their innervation patterns showed
several parallels to that of the locust front leg
(Hoyle, 1955a;
Hoyle, 1955b), stick insect
middle leg (Bässler, 1993)
and the cricket middle leg (Nishino,
2003). The single large extensor muscle was driven by two
excitatory extensor motoneurons, a FETi and a SETi. These corresponded to
tibial extensor motoneurons in cricket and locust hind legs and stick insect
middle legs. The flexor system was more complex, but acted as a single
functional unit because of a common distal attachment point. At least eight
excitatory flexor motoneurons exit. The cricket hind leg tibia is innervated
by at least 19, the locust hind leg by nine and the stick insect by 14
excitatory flexor motoneurons. All motoneurons identified, except for the
FETi, which projected its axon through nerve III, projected their axons
through nerve V. This corresponds to the arrangement of the front tibial
motoneurons in locusts (Burrows,
1996).
EMG recordings
We investigated the role of the tibial extensor motoneurons and flexor
motor units in walking and phonotactic steering. During walking, tibial
extensions were carried almost entirely by SETi. By contrast, SFTi was
tonically active throughout stance phase but reduced activity during swing
(Fig. 4D). Flexion was strongly
driven by FFTi activity, which peaked at the beginning of stance phase. We did
not attempt to identify any common inhibitor motoneurons or dorsal unpaired
median cells innervating tibial muscles. In particular the low activity of the
tibial extensors is in clear contrast to similar studies performed on locust
legs (Burns and Usherwood,
1979) and cockroach hind legs
(Krauthamer and Fourtner,
1978) where at high stepping rates SETi is tonically active and
bursts of FETi activity support the step rhythm. Because of its very low spike
rate during phonotactic walking FETi is unlikely to contribute to steering
under normal circumstances. It remains open whether FETi supports steering at
very high stepping rates. However, the low SETi activity during walking in
G. bimaculatus leaves room for its recruitment during steering: SETi
responded to ipsilateral acoustic stimulation during phonotaxis. EMG
recordings also demonstrate an auditory input to at least one type of FFTi
motoneurons (Fig. 5). It is
unclear which, if not both types of FFTi motoneurons underlie these auditory
responses during phonotaxis. Previously Pollack and Hoy
(Pollack and Hoy, 1980)
demonstrated activity in dorsal longitudinal muscles in response to individual
sound pulses in flying crickets (Teleogryllus oceanicus) (see also
Nolen and Hoy, 1986). These
inputs result in bending of the abdomen towards the direction of the sound
during flight. It is, however, unclear to what extent abdominal movements
contribute to auditory steering during walking. While these findings emphasise
the likely involvement of multiple motor systems of the body other than tibial
extension and flexion movements in supporting cricket phonotaxis behaviour,
our behavioural experiments highlight both the SETi and either FFTi1 or
FFTi2–5 as key output neurons for phonotactic steering during
walking.
Auditory control over tibial motoneurons
As demonstrated in EMG recordings tibial motoneurons integrate auditory
inputs with activity from walking-pattern-generating networks during
phonotactic steering. However, intracellular recordings revealed no auditory
inputs to any motoneuron identified at rest or during pharmacologically
elicited motor activity. Furthermore, motoneurons were located dorsally and
did not extend any neurites towards the ventrally located auditory neuropiles
(Schildberger et al., 1989).
The auditory input to motoneurons therefore has to be indirect, leaving two
options: (1) it may be entirely local, via interneurons in the
prothoracic ganglion or (2) it may reach motoneurons via the brain.
In this respect the timing is crucial: increased spike rate in SETi in
response to acoustic stimulation during EMG recordings occurred after
35–40 ms. Subtracting 4 ms to allow for spikes to be generated and
propagated towards the muscles (Fig.
7) this leaves 31–36 ms for the synaptic input to be evoked
in the motoneurons following acoustic stimulation. First order prothoracic
auditory interneurons such as the omega 1 neuron (ON1) or ascending neuron 1
(AN1) respond to acoustic stimulation with a latency of 15–17 ms
(Wohlers and Huber, 1978).
This leaves 14–21 ms for the information to reach motoneurons
via either a thoracic or a cephalic pathway. This delay is rather
long for an entirely prothoracic pathway, but it also leaves only little time
for a loop via the brain: AN1 activity in the brain occurs with a
latency of 20–22 ms (Schildberger,
1984) (M. Zorovi
, personal communication), implying a
propagation time of auditory signals between the prothoracic ganglion and the
brain of 5 ms. Two way propagation to and from the brain therefore costs
a total 10 ms leaving 4–11 ms for local processing in the brain. In
support of a cephalic pathway, extracellular stimulation of descending
pathways clearly indicated a direct, parallel polyneural descending input to
tibial motoneurons. The physiological relevance of the extracellularly evoked
synchronous spike activity in several descending axons remains unclear. In
addition several multimodal descending brain neurons respond to auditory
stimuli and are known to terminate dorsally in all thoracic ganglia
(Staudacher, 2001). These
respond with latencies between 25–47 ms at the level of the connectives
between the suboesophageal and prothoracic ganglia, and may therefore be
candidate neurons for a descending auditory control of front tibial
motoneurons. Similarly, Brodfuehrer and Hoy
(Brodfuehrer and Hoy, 1989;
Brodfuehrer and Hoy, 1990)
identified several ultrasound-sensitive descending brain neurons of T.
oceanicus that may mediate negative phonotactic steering responses. An
entirely prothoracic auditory loop towards the motoneurons would require
descending gating control (Pollack and
Hoy, 1980; Poulet and Hedwig,
2005) to enable the pathway only during steering. However,
stimulation of descending interneurons did not unmask any auditory inputs to
the motoneurons. Furthermore, despite decades of research, no prothoracic
auditory interneurons have been identified that project from the ventral
auditory neuropiles towards the dorsal motoneurons. Instead the gating
mechanism may exist in the brain, with a descending pathway mediating the
steering responses. Most of AN1's presynaptic terminals project
antero-ventrally in the brain, laterally of the 
-lobes
(Schildberger, 1984), however,
most descending brain neurons extend their dendritic fields in the ventral
posterior deutocerebrum (Staudacher,
1998). We therefore anticipate a cephalic auditory loop to require
at least two synapses within the brain, involving local brain neurons
forwarding the auditory information from AN1 towards descending pathways.
Future experiments will aim at a more comprehensive understanding of auditory processing in the brain. The identification of the descending pathways to the SETi and FFTi motoneurons as well as the postsynaptic targets of AN1 will be crucial. Furthermore, the identification of the mechanism underlying the gating of the auditory-to-motor pathway during phonotaxis is a central question.
LIST OF ABBREVIATIONS
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Acknowledgments |
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and Olivier Faivre for
critical reading of the manuscript. This study was supported by the BBSRC,
Royal Society and Cambridge University European Trust. |
References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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