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J Neurophysiol (March 1, 2003). 10.1152/jn.0846.2002
Submitted on Submitted 24 September 2002; accepted in final form 25 October 2002
Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Poulet, J.F.A. and
B. Hedwig.
A Corollary Discharge Mechanism Modulates Central Auditory
Processing in Singing Crickets.
J. Neurophysiol. 89: 1528-1540, 2003.
Crickets communicate using loud (100 dB
SPL) sound signals that could adversely affect their own auditory
system. To examine how they cope with this self-generated acoustic
stimulation, intracellular recordings were made from auditory afferent
neurons and an identified auditory interneuron
the Omega 1 neuron
(ON1)during pharmacologically elicited singing (stridulation). During
sonorous stridulation, the auditory afferents and ON1 responded with
bursts of spikes to the crickets' own song. When the crickets were
stridulating silently, after one wing had been removed, only a few
spikes were recorded in the afferents and ON1. Primary afferent
depolarizations (PADs) occurred in the terminals of the auditory
afferents, and inhibitory postsynaptic potentials (IPSPs) were apparent
in ON1. The PADs and IPSPs were composed of many summed,
small-amplitude potentials that occurred at a rate of about 230 Hz. The
PADs and the IPSPs started during the closing wing movement and peaked in amplitude during the subsequent opening wing movement. As a consequence, during silent stridulation, ON1's response to acoustic stimuli was maximally inhibited during wing opening. Inhibition coincides with the time when ON1 would otherwise be most strongly excited by self-generated sounds in a sonorously stridulating cricket.
The PADs and the IPSPs persisted in fictively stridulating crickets
whose ventral nerve cord had been isolated from muscles and sense
organs. This strongly suggests that the inhibition of the auditory
pathway is the result of a corollary discharge from the stridulation
motor network. The central inhibition was mimicked by hyperpolarizing
current injection into ON1 while it was responding to a 100 dB SPL
sound pulse. This suppressed its spiking response to the acoustic
stimulus and maintained its response to subsequent, quieter stimuli.
The corollary discharge therefore prevents auditory desensitization in
stridulating crickets and allows the animals to respond to external
acoustic signals during the production of calling song.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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A key question in
sensory neuroscience is how animals deal with self-generated, or
reafferent, sensory information. It has been suggested that a neural
signal, an "efference copy" (Holst and Mittelstaedt
1950
) or "corollary discharge" (Sperry
1950), from the motor network is used to anticipate and cancel
out reafferent responses. In this way animals could prevent reafferent
information from desensitizing their own sensory pathway and/or from
being confused for external stimuli.
A modulation in the sensitivity to reafferent stimulation by centrally
generated neural signals has been identified in a variety of sensory
systems, e.g., visual (Zaretsky and Rowell 1979),
electroreceptive (Bell 1981, 1982; Bodznick et
al. 1999), proprioceptive (Gossard et al. 1991;
Sillar and Skorupski 1986; Wolf and Burrows
1995), and mechanoreceptive (Blakemore et al.
1998; El Manira et al. 1996; Murphey and
Palka 1974; Sillar and Roberts 1988). A
reduction in the responsiveness of auditory neurons in the brain has
been recorded in humans (Creutzfeldt et al. 1989,
Numminen et al. 1999) and other vertebrates
(Kirzinger and Jürgens 1991; McCasland and
Konishi 1981; Metzner 1989, 1993;
Müller-Preuss and Ploog 1981; Schuller
1979; Suga and Schlegel 1972; Suga and
Shimozawa 1974) during vocalization, but the nature and source
of the inhibition has never been characterized. Here we take advantage
of the simple auditory circuit of a cricket to analyze how this
acoustically communicating insect deals with self-generated auditory stimulation.
Male crickets (Gryllus bimaculatus) sing for hours at over
100 dB SPL (Nocke 1972) to attract females. Songs are
generated by rhythmically rubbing the forewings together: a form of
sound production called stridulation. As crickets' ears are located on
the forelegs, they are fully exposed to the self-generated sounds. Many
animals reduce the responsiveness of their peripheral auditory system
during sound production (Borg and Counter 1989; Hennig et al. 1994; Narins 1992;
Suga and Jen 1975), but crickets do not (Poulet
and Hedwig 2001). Despite this, behavioral experiments have
shown that singing crickets can respond to external sounds (Heiligenberg 1969; Jones and Dambach
1973). To examine how crickets' central auditory system deals
with reafferent auditory information, we made intracellular recordings
of auditory afferents (Eibl 1978; Michel
1974) and an identified auditory interneuronthe Omega 1 neuron (ON1) (Casaday and Hoy 1977; Popov et al.
1978; Wohlers and Huber 1978)during
stridulation. A summary of part of this work has been published
(Poulet and Hedwig 2002).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
All experiments were performed on adult male G. bimaculatus selected from a cricket colony maintained on a 12 h light:12 h dark cycle. Prior to dissection they were chilled at 4°C
for 
30 min. They were then fixed in a standing position on a holder
that allowed free rotation of the animal (Fig.
1A). To allow for silent stridulation, the left wing of the crickets was removed. When recording
fictive stridulation, the cricket was placed upside down in a
Plasticene well and its ventral cuticle was removed to expose the
abdominal and thoracic ganglia. The thoracic or thoracic and abdominal
nerves were cut, except for prothoracic nerve 5, which contains the
auditory afferents. Care was taken not to damage the main ventral
trachea. To deafen crickets, the forelegs were removed just distal to
the coxa. Experiments were done at room temperature (18-22°C).
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Pharmacological stimulation
The antennae and a piece of head cuticle were removed to reveal
the brain for pharmacological stimulation. Glass capillaries, filled
with the acetylcholine esterase inhibitor Eserine Salicylate (10
2 mol l1 in insect
saline), were connected to a WPI PV 820 pneumatic Pico Pump and
positioned in the anterior protocerebrum of the brain. Having located
the auditory neuron for intracellular recording, a 0.3-s pulse of
Eserine (about 1 nL) was injected into the anterior protocerebrum to
elicit singing (Otto 1978; for details of
pharmacological injection techniques, see Wenzel and Hedwig
1999). Injection of acetylcholine and cholinergic agonists into
the cricket's anterior protocerebrum activates the stridulatory
command neurons, which spur the thoracic central pattern generator into
action (Hedwig 2000a).
Acoustic stimulation
Auditory stimuli were generated by the software Cool Edit 2000 running on a Toshiba Laptop (Satellite 4010) and delivered from two piezo-electric speakers through brass tubes with frontal openings of 14 mm which were positioned 20 mm from the posterior tympanic membrane. Sound tubes were used to avoid echoes associated with free field stimulation. All acoustic stimuli had a carrier frequency of 4.5 kHz and rising and falling ramps of 2 ms. Stimuli either had a duration of 8 ms, a period of 15 ms, and an intensity of 75 dB SPL re. 20 µPa root-mean-square (RMS) or, when mimicking cricket calling song, they had a duration of 21 ms, a period of 42 ms, and an intensity of 100 dB SPL re. 20 µPa (RMS). Sound amplitude was calibrated using a Bruel and Kjaer amplifier (Type 2610) and a 1/2 in. microphone (Type 4191) positioned at the ears of the cricket.
Neuron recordings
For intracellular recording, the holder with the cricket
attached was rotated so that the crickets were upside down, and the prothoracic ganglion was exposed (Fig. 1A). A pair of
forceps, which acted as the indifferent electrode, was modified so that one tine could be placed under the ganglion to act as a platform, while
the other was a stabilizing ring that could be lowered onto the
ganglion. Exposed nervous tissue was bathed in insect saline (ionic
composition in mmol l1: 140 NaCl; 10 KCl; 4 CaCl2; 4 NaHCO3; 6 NaHPO4). Recordings and stainings of auditory
neurons were made in the auditory neuropil of the prothoracic
ganglion using thick-walled glass micropipettes filled with 5% Lucifer
yellow and LiCl at a resistance of 100-150 M
. Auditory afferents
were recorded close to their terminal branches, whereas ON1 was
recorded in its dendritic region. Neurons were held slightly
hyperpolarized during an experiment to stabilize the recording. After
the experiments, ganglia were dehydrated in an alcohol series and
cleared in methyl salicylate, and stained neurons were identified under
a UV fluorescence microscope.
Behavioral recordings
Sonorous singing was monitored with a microphone
(Audio-Technica, AT853A) positioned 5 cm from the wings. Wing movements
were measured with an optoelectronic camera focused onto a reflective disk (2 mm diam, 3M Scotchlite 7610) glued to the right wing
(Hedwig 2000b). Motor activity during fictive
stridulation was recorded with a suction electrode placed on
mesothoracic nerve 3A, which contains motor axons that innervate wing
closer and opener muscles (Kutsch and Huber 1970).
Data sampling and analysis
All data were sampled on-line at a rate of 10 kHz per channel
transferred directly onto a computer, via a high-speed Data Translation
AD board (DT 2821 F8DI). Recordings were analyzed off-line using
Neurolab software (Knepper and Hedwig 1997) and Microsoft Excel 2000. Wing movement, mesothoracic nerve 3A activity, or
the acoustic stimuli were used as a temporal reference when averaging
data (time = 0 ms in the figures). Microphone recordings were
full-wave-rectified before averaging. Data values are given as
mean ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Pharmacologically elicited stridulation
Natural calling song consists of a series of chirps, 250 ms long,
separated by a 300-ms chirp interval (Doherty 1985).
Each chirp is composed of four 21-ms syllables that are produced during the closing wing movement and increase in intensity throughout the
chirp (Fig. 1B). Pharmacological stimulation normally
elicited calling song with very similar temporal and acoustic
properties to the natural song (Otto 1978; Wenzel
and Hedwig 1999). One difference was the appearance of very
quiet sounds during wing opening (arrow 1, Fig. 1B). These
were probably a result of the animals singing upside-down. Stridulation
started roughly 10 min after injection of Eserine Salicylate into the
brain. Sonorous stridulation lasted for 10-15 min, whereas fictive
stridulation lasted for 30-45 min. Most intact animals had several
pauses in their singing, which lasted for several minutes. Periods of
fictive stridulation, however, continued uninterrupted for a few
minutes longer.
Activity of auditory neurons during sonorous singing
About 60 auditory afferents project from each ear along the dorsal
part of prothoracic nerve 5 and terminate in the auditory neuropile of
the prothoracic ganglion (Eibl 1978; Michel
1974). Two mutually inhibitory (Selverston et al.
1985), local, auditory interneuronsthe Omega 1 neurons
(ON1)are located within the prothoracic ganglion and have dendritic
branches that overlap with the output branches of the auditory
afferents (Casaday and Hoy 1977; Popov et al.
1978; Wohlers and Huber 1978). These identified interneurons receive auditory information from the ear ipsilateral to
their soma and are most sensitive to a sound frequency of 4.5 kHz, the
carrier frequency of male calling song (Casaday and Hoy 1977; Popov et al. 1978; Wohlers and
Huber 1978).
During sonorous singing, auditory afferents were activated by the sound (Fig. 2A). The afferents responded with bursts of spikes both to the loud syllables produced during wing closing (asterisks in Fig. 2Ai) and to the quieter sound during wing opening (arrow 1 in Fig. 2Ai). This is clearly demonstrated by the quantitative evaluation, which shows the peristimulus time (PST) histogram overlaid with the average spike rate (top), the average wing movement (middle), and the sound pattern (bottom) during the sonorous chirps (Fig. 2Aii). Loud sound pulses occurred in-phase with the closing movements and caused the large maxima in the PST histograms and spike frequency plots (asterisks in Fig. 2Aii) that occurred at the transition from wing closing to opening (dashed line Fig. 2Aii). The softer syllables, in-phase with the opening movement, caused only weak afferent activity (arrow 1 in Fig. 2Aii). On average, the afferents reached a maximum spike frequency of 246 ± 24 Hz (n = 6 afferents) during sonorous chirps. During some chirps, with a silent initial wing opening movement, a depolarization of the afferent membrane potential was evident prior to the spikes (arrow 2 in Fig. 2Ai). Since these recordings were obtained close to the terminals of the afferents, such a depolarization could indicate the presence of a primary afferent depolarization (PAD).
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ON1 was also activated during sonorous singing with rhythmic membrane potential oscillations in-phase with the syllables (Fig. 2B). At the beginning of a chirp, the first one to two spikes were elicited by the quiet sound caused by the initial opening wing movement (arrow 3 in Fig. 2Bi). Further spike activity then occurred only in response to the loud syllables produced during the closing wing movements (asterisk in Fig. 2Bi). ON1's 15- to 20-ms response delay time meant that these maxima occurred during the subsequent opening wing movements (dashed line in Fig. 2Bii). The response latency of ON1 to the first quiet sound at the start of a chirp is about 20 ms. The response to the following louder syllables has a shorter latency because the cell is already depolarized by the preceding quieter sound that moves the membrane potential nearer to spiking threshold. The quantitative evaluation of ON1's response demonstrated that the average ON1 maximum spike frequency to self-generated syllables was 176 ± 28 Hz (n = 10 crickets). Immediately after ON1's response during the chirp, it was slightly hyperpolarized and during the chirp interval the membrane potential gradually repolarized. These experiments suggest that the afferents and ON1 respond to reafferent sound. To test this hypothesis, recordings were made in crickets with one wing removed so that they stridulated silently.
Responses of auditory neurons during silent stridulation
During silent stridulation, the afferents only occasionally generated action potentials, indicating that their high-frequency spiking during sonorous stridulation was indeed a response to the cricket's own song. With the major source of excitation removed, PADs were now evident in all of the 13 auditory afferents recorded (Fig. 3, A, B, and C). During silent stridulation, PADs were recorded both in afferents which responded best to 4.5 kHz (n = 12 crickets) (Fig. 3, A and C) and in an afferent that responded best to 10-30 kHz (Fig. 3B). In four of these crickets, the average duration of the opening to closing wing movement cycle underlying the production of syllables was 36.0 ± 1.8 ms. The PADs started 4.2 ± 0.4 ms after the start of wing closing and reached a maximum at 19.3 ± 1.4 ms, just after the transition between wing closing and opening at 17. 8 ± 1.0 ms (n = 4 crickets) (dashed lines in Fig. 3, Aii, Bii, Cii). This corresponds to the phase of maximum response to reafferent sound. On average, in six crickets, the PADs increased in size from 2.6 ± 0.5 mV (measured from chirp interval baseline membrane potential to peak amplitude), at the start of the chirp (arrow 1 in Fig. 3B), to 3.3 ± 0.7 mV at the end. Although wing movement often decreased in amplitude during singing, the timing and amplitude of the PADs were not affected, indicating that they did not depend on the intensity of sensory feedback associated with wing movements (Fig. 3, B and C).
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ON1 only occasionally spiked during silent stridulation. Inhibitory
postsynaptic potentials (IPSPs) occurred in all 21 ON1s recorded that
started and built up in-phase with the closing wing movements of each
syllable (Fig. 4Ai). The
average duration of a wing movement cycle underlying a syllable was
38.1 ± 1.5 ms (n = 5 crickets). IPSPs started
5.0 ± 0.2 ms after the start of wing closing and reached a
maximum at 23.3 ± 1.4 ms, just after the transition between wing
closing and opening at 19.0 ± 0.7 ms (dashed lines in Fig. 4,
Aii and Bii). The average mean amplitude of the
IPSPs increased from 
2.3 ± 0.3 mV at the first syllable to
5.3 ± 0.5 mV at the last (average from 8 crickets, measured from chirp-interval baseline membrane potential to peak amplitude). Following the last IPSP, the membrane potential gradually repolarized to the resting level. Similar to the PADs, the amplitude and timing of
the IPSPs was independent of the amplitude of wing movements (Fig.
4B).
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The PADs and IPSPs were composed of many small-amplitude potentials (Fig. 4C). The PADs were built up by the summation of small depolarizations (arrow 2 in Fig. 3Ai, arrow 1 in Fig. 4Ai), while the IPSPs were composed of summated hyperpolarizing potentials (arrow 1 Fig. 4, Ai and C). The average maximum frequency with which these small potentials occurred were very similar in both the PADs (228 ± 29 Hz, n = 5 crickets) and the IPSPs (232 ± 22 Hz, n = 6 crickets), implying that they could originate from the same source.
Thus the responses of ON1 in sonorously singing crickets must be mixture of excitation from reafferent sound and additional inhibition. The inhibitory synaptic inputs may have two sources: reafferent feedback from nonauditory sense organs or the network of neurons generating the stridulation motor pattern. To examine this further, we recorded the responses of the neurons in fictively stridulating crickets, devoid of sensory feedback and efferent muscle control.
Activity of auditory neurons during fictive stridulation
During fictive stridulation, the afferents and ON1 rarely produced spikes (Fig. 5). PADs were present in low-frequency afferents (n = 17) and in a high-frequency afferent. The data collected from six low-frequency afferents were analyzed further. Although wing closer and opener motor activity is not as clear an indicator for the timing of stridulation as wing movement, the PADs seemed to have a similar timing and amplitude to those seen in intact animals (Fig. 5A). Just as in intact animals, the PADs were composed of smaller amplitude depolarizing potentials that occurred in-phase with the syllables. The PADs thus increased in amplitude from 1.9 ± 0.3 mV at the start of the fictive chirp to 3.5 ± 0.1 mV at the end (n = 6 crickets).
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IPSPs were present in ON1 that were also composed of many smaller
amplitude hyperpolarizing potentials. They occurred in-phase with the
syllables of the fictive chirps, which were indicated by the motor
activity. The maximum amplitude of the IPSPs increased from 2.4 ± 0.6 mV in-phase with the first syllable of the fictive chirp to
5.1 ± 0.7 mV at the last (n = 5 crickets) (Fig.
5Bii). As during silent singing, following the last IPSP of
a chirp, the membrane potential gradually repolarized back to a resting level. To prove that the IPSPs and PADs do not originate in the auditory pathway, in some of the fictively singing crickets the front
legs, which contain the ears and other sensory systems, were removed.
During fictive stridulation, with the ears removed, both PADs
(n = 2 crickets) and IPSPs (n = 4 crickets) persisted, with the same overall temporal structure and
amplitude as in the intact animal (Fig.
6, A and B). We
conclude that the PADs in the afferents and IPSPs in ON1 were not the
result of any reafferent feedback but were generated within the CNS of
the crickets and therefore represent an efferent input from a corollary
discharge generated by the singing motor network.
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Responses of auditory neurons to acoustic stimulation during stridulation
To examine the effect of the efferent synaptic input on auditory processing in the afferents and ON1, trains of 4.5 kHz, 75 dB SPL acoustic stimuli with a short period (15 ms) and duration (7 ms) were played to silently singing crickets (Fig. 7). This stimulus paradigm revealed the time course of the auditory response during stridulation, as sound stimuli were present at all phases of stridulation (Fig. 7, Aiii and Bii). In the afferents spikes elicited by the sound were superimposed on the PADs caused by the corollary discharge (asterisk in Fig. 7Ai). The PADs did not have an effect on the frequency of spiking, which was the same in chirps and the chirp interval (Fig. 7Aiii) (n = 5 crickets). In two recordings, however, spikes riding on top of the PADs were reduced in amplitude by approximately 3 mV (Fig. 7, Ai and Aii).
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ON1 spiked consistently to the continuous sound stimuli while the cricket was at rest. During silent stridulation, ON1 was activated by the acoustic stimuli in the chirp intervals, but its response was strongly inhibited during the silent chirps (Fig. 7Bi) (n = 21 crickets). Inhibition started at the first closing wing movement of a chirp and occurred in the rhythm of the syllable pattern, as seen before (Fig. 4). Excitatory postsynaptic potentials (EPSPs) were elicited by the sound pulses during the chirp (asterisk in Fig. 7Bi), and occasionally spikes were triggered by the larger amplitude EPSPs at the transition from wing opening to closing (arrow 1 in Fig. 7Bii) when the inhibition slightly decreased (Fig. 4). The inhibition followed the structure of the IPSPs in ON1: it had a rapid onset at the start of the chirp (arrow 2, Fig. 7Bii) and at the end of the chirp its effect gradually waned (arrow 3, Fig. 7Bii, compare with Figs. 5Bii and 6Bii).
Afferent and ON1 recordings also showed these inhibitory effects on auditory processing in fictively stridulating crickets: afferents received PADs during fictive chirps but constantly responded to sound stimuli throughout fictive stridulation (Fig. 8A) (n = 10 crickets), whereas ON1 was rhythmically inhibited during fictive chirps (Fig. 8B) (n = 6 crickets). In one fictively stridulating cricket, the spikes on top of the PADs were reduced in amplitude by approximately 3 mV (Fig. 8Ai). These experiments clearly demonstrate that the corollary discharge has a major influence on auditory processing during stridulation at two levels. It presynaptically inhibits the auditory afferents, thereby reducing the amount of excitation forwarded to the central auditory pathway, and additionally it inhibits auditory interneurons in-phase with wing closing movement.
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The impact of the inhibition on sound patterns similar to cricket song
To examine the efficacy of the corollary discharge, we presented five silently stridulating crickets with acoustic stimuli that mimicked the species-specific calling song (4.5 kHz, 100 dB SPL, 21-ms duration, 42-ms interval) (Fig. 9). Sound pulses presented at rest and during the chirp intervals evoked a depolarization and a burst of six spikes in ON1 with an average maximum spike frequency of 376 ± 50 Hz. During the chirps, the compound EPSPs elicited by the sound stimuli were superimposed with small deflections, indicating the efferent IPSPs (arrows, Fig. 9A). Alongside the presynaptic inhibition, these IPSPs reduced the amplitude and duration of the sound-evoked EPSPs. During the chirps, these depolarizations triggered only one to two action potentials/stimulus with a spike rate of only 123 ± 29 Hz (n = 5 crickets) (responses to gray stimuli in Fig. 9A). The response during the chirps, as reflected by the discharge rate and the number of spikes per stimulus, was only 33% of the response during the chirp intervals, which was significantly lower (2-tailed paired t-test: P < 0.002, t = 7.71, df = 4) (Fig. 9B).
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Responses to the 100 dB SPL stimuli varied in amplitude depending on the phase of occurrence within the silent chirp, e.g., compare response to the first stimulus in the chirp to the response to the second (Fig. 9A). To examine the exact time course of the response modulation throughout the silent chirp, we plotted the maximum spike frequency of each response (represented as a dot) against the averaged wing movement and then calculated the average maximum spike frequency during the chirp (solid line, Fig. 10A). ON1 responded with regular high-frequency bursts of spikes (in this example, at about 280 Hz) during the chirp interval. The response of ON1 to the acoustic stimuli was minimal (in this example, about 10 Hz) during the opening wing movements (gray bars, Fig. 10; compare with Fig. 4A). A weak spiking response to the acoustic stimuli could occur during the closing movement when the inhibition decreased (compare with Figs. 4A and 7B). Thus the effect of the efferent inhibition is maximum in-phase with wing opening.
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We then compared the timing of the inhibition (Fig. 10A) with ON1's response to reafferent sound (Fig. 10B). Sound is generated during the closing movement, but due to ON1's latency of 15-20 ms its response occurred in-phase with the following wing opening movement (Fig. 10B). If the wing movements from a sonorously and a silently singing cricket were aligned, the phase of maximum inhibition coincides with the phase in which ON1 is maximally driven by reafferent sound stimulation (gray bars in Fig. 10). Thus the inhibition, mediated by the corollary discharge, is precisely timed to reduce reafferent auditory stimulation.
The functional role of the corollary discharge
In female crickets, intense acoustic stimulation causes ON1 to
spike but it also causes a gradual activity-dependent hyperpolarization that reduces the neuron's sensitivity to sound (Pollack
1988). This inhibition is thought to be due to activation of
Ca2+-dependent K+ channels
by the spikes (Sobel and Tank 1994). The duration and amplitude of the hyperpolarization is dependent on the duration and
amplitude of acoustic stimulation (Pollack 1988). If a
similar mechanism is present in males, self-generated acoustic
stimulation during singing should gradually hyperpolarize ON1 and
desensitize it to external sounds. We tested whether such a
desensitization is present and whether it is prevented by the corollary
discharge. Reducing ON1's spiking response to loud chirps should
increase its response to subsequent, quieter sounds.
We examined the responses of ON1 to sound pulses that mimicked the cricket's own song and to quieter test stimuli. A control series of test sound pulses (80 dB SPL) each elicited a burst of spikes with an average maximum frequency of 203 ± 24 Hz (n = 8 crickets) (Fig. 11, A and D). The sound of normal singing was then mimicked with 100 dB SPL chirps, which were presented immediately before a series of test stimuli. The response to these stimuli was significantly weaker than the response to the control series with an average maximum spike frequency of 30 ± 11 Hz (2-tailed paired t-test: P < 0.001, t = 8.70, df = 7) (Fig. 11, A, B, and D). The reduction persisted for the whole of the artificial chirp interval but was greatest just after the 100 dB SPL chirp (Fig. 11B).
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We then mimicked the effects of the inhibition from the corollary
discharge by injecting hyperpolarizing current into ON1 to prevent it
from spiking during the 100 dB SPL chirp. The average maximum response
to the subsequent 80 dB SPL test stimuli was now a burst of spikes at
143 ± 32 Hz (n = 8 crickets) that was significantly higher than the response to the test pulses after the 100 dB SPL chirps without hyperpolarizing current injection (2-tailed
paired t-test: P < 0.004, t = 4.21, df = 7) (Fig. 11, A, C, and
D). ON1's response following current injection did not reach the same amplitude as to the test stimuli because afferent adaptation occurs at high sound intensities (Givois and Pollack 2000; Nocke 1972). The effect was still present
after deafening the ear contralateral to the soma of ON1 and was
therefore independent from inhibition from the contralateral ON1. In
three one-eared crickets, the average spike frequency to 80 dB SPL
stimuli was 263 Hz. When the stimuli were preceded by a 100 dB SPL
chirp, the response to the same stimuli was reduced to 23 Hz. If the neuron was hyperpolarized during the chirp, the response increased to
168 Hz. If a burst of spikes was generated by a depolarizing current
injection in a completely deafened cricket, the graded hyperpolarization following spiking persisted. Therefore a reduction in
the spiking response of ON1 to intense sounds helps to maintain the
neuron's sensitivity to quieter, subsequent sounds. In this way the
corollary discharge will prevent auditory desensitization during stridulation.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Crickets sing so loudly that reafferent sound could be confused
with external sound and/or desensitize the cricket's own auditory system. One solution to this problem could be to modulate the biophysical sensitivity of the ear during sound production. However, the tympanic membrane of the cricket remains fully responsive during
stridulation (Poulet and Hedwig 2001). Therefore we
examined how their central auditory system copes with the intense
reafferent stimulation.
Inputs to auditory afferents and ON1 during singing
The auditory afferents and ON1 spiked in response to sound production during sonorous chirps (Fig. 2). Spiking was greatly reduced in both types of neuron in silently stridulating crickets (Figs. 3 and 4); therefore, during sonorous stridulation, the response of the neurons was a reaction to reafferent auditory stimulation. Excitation from reafferent sound stimulation, however, was not the only type of input to these neurons. During silent chirps, PADs became evident in the afferents and IPSPs in ON1 (Figs. 3 and 4).
These inhibitory inputs could have had two sources: reafferent
nonauditory feedback generated during stridulation or a centrally generated pattern of neuronal activity. As both the PADs and the IPSPs
were presented during fictive chirps with all forms of reafferent feedback removed, the major source of the inhibition must be a central
one (Figs. 5 and 6). Vibrating the foreleg of the cricket inhibits ON1
(Wiese 1981; unpublished observations). In the
intact animal, therefore, vibrations conducted from the wing via the exoskeleton to the foreleg may result in reafferent inhibition superimposed on the central inhibition.
Both the PADs and the IPSPs were present during the chirps and occurred
at the same phase of the syllable cycle. Both were composed of many,
summed, smaller potentials that were generated at regular intervals
(every 4-5 ms, 230 Hz). This suggests that they may have a common
source. Furthermore, the unitary nature of the small potentials
suggests that they are the result of input from a single neuron. The
neuron(s) causing these inputs is unlikely to be the descending command
neuron(s) for stridulation as it spikes during the chirp at about 35 Hz
with little modulation of its firing rates (Hedwig
2000a). Interneurons participating in stridulatory motor
pattern generation in crickets and grasshoppers reach phasic spike
rates of 
200 Hz (e.g., Hedwig 1992a,b; Hennig 1990). The most likely candidate neuron(s) mediating the
inhibitory corollary discharge is therefore those contained within the
so-far uncharacterized stridulatory motor network in the thoracic
ganglia. The amplitude of the PADs and IPSPs remained the same during
low-amplitude wing movement (Figs. 3 and 4). This implies that the
neuron(s) mediating the corollary discharge is involved in central
rhythm generation rather than in the finer control of the stridulatory wing movement (for a distinction between these types, see Hedwig 1992a,b).
Neural signals generated within motor networks that affect the
processing of reafferent information were termed efference copies by
Holst and Mittelstaedt (1950) or corollary discharges by
Sperry (1950). According to Bell (1984),
all neural discharges from the networks of neurons forwarding motor
activity to a sensory pathway should be termed corollary discharges,
since an efference copy depends on the properties of reafferent
information. One well-studied example of a corollary discharge occurs
in the knollenorgan electroreceptive pathway, which is involved
with electrocommunication between mormyrid electric fish (Bell
1989; Bell and Grant 1989). To prevent
reafferent stimulation from interfering with the detection of external
signals, this pathway is inhibited by a corollary discharge during the
electric organ discharge, which is independent from the intensity of
reafferent stimulation. The inhibition in the cricket auditory pathway
is very similar to that seen in the electric fish, as it is also the
result of a corollary discharge that is independent from the production
of reafferent feedback (Figs. 3, 4, 5, and 6). Furthermore, it is
stable, in that it continued for as long as we could record from a
singing cricket (up to 1 h). Inhibition by an invariant corollary
discharge is a sensible strategy for crickets to adopt as they may sing
continuously for many hours with a very stereotyped, regular sound
pattern, so the afferent response to self-generated sound is also
unlikely to change.
Effects of central inputs in the cricket and other systems
The responses to acoustic stimulation during silent and fictive
singing indicate that both the PADs in the afferents and the hyperpolarizations in ON1 are inhibitory. PADs in many other systems are inhibitory synaptic inputs with a reversal potential more positive
than the resting membrane potential (Clarac and Cattaert 1996). If the auditory nerve was intact (Figs. 3 and 5), spikes were occasionally recorded in the afferents even when the cricket was
silent. These spikes originated in the periphery and were not generated
by the PADs, as spikes were never recorded in afferents once the ears
had been removed (Fig. 6A). In many systems, PADs are
thought to be mediated by GABA-ergic inputs to the afferent terminals
(Burrows and Laurent 1993; Cattaert et al.
1992; Eccles et al. 1963). Consistent with this
observation, GABA-immunoreactive boutons have been identified on the
terminals of cricket auditory afferents (Hardt and Watson
1999). PADs can cause a reduction in the spike amplitude
invading the afferent terminal (Burrows and Matheson
1994; Cattaert et al. 1992), as seen in this
study (Figs. 7 and 8). This is in agreement with the shunting effect of
PADs on action potentials due to an increased conductivity of the
afferent terminal's membrane (Clarac and Cattaert
1996). By reducing the afferent spike height, the PADs could
reduce the amount of excitatory neurotransmitter released from the
afferent synapses during spiking, thus reducing the amplitude of the
EPSP evoked in postsynaptic cells (Burrows and Matheson
1994; Cattaert et al. 1992).
During silent and fictive chirps, ON1's response to acoustic stimuli is inhibited in-phase with the IPSPs generated by the corollary discharge (Fig. 7). Hyperpolarizing ON1 inhibits its response to acoustic stimulation (Fig. 11). During sonorous chirps, ON1 receives a simultaneous drive of excitation from reafferent sound stimulation and inhibition from the centrally generated IPSPs.
The strength of the centrally generated inhibition was tested by playing sounds, which mimicked the calling song, to silently stridulating crickets. ON1's response during silent chirps was reduced to 33% of the response at rest and during chirp intervals (Fig. 9). The response at rest was also much higher than the response to self-generated sounds, confirming the response reduction during stridulation. The response during the chirps, however, was slightly lower than the response to self-generated sounds. This may be due to differences in the stimulus design compared with natural sounds.
The PADs and IPSPs occur throughout the chirp in-phase with the syllables: they start during each closing wing movement and reach a peak during the consecutive opening wing movement (Figs. 3 and 4). We therefore examined the responses to sound pulses played during silent chirps. The largest response reduction coincides with the opening wing movement, which is the time when ON1 would respond to self-generated sounds (Fig. 11). As the inhibition is generated within the nervous system, it can be precisely timed to certain phases of motor activity.
Studies made in stridulating grasshoppers demonstrated a reduction of
the spiking response to acoustic stimuli during sound production
(Hedwig 1986; Wolf and Helversen 1986).
This reduction is, however, due to a desynchronization of receptor
activity in response to the self-generated sound and not the result of
neural inhibition (Hedwig 1990; Hedwig and Meyer
1994). A reduction in auditory neural responses during sound
production has also been recorded in vertebrates (Creutzfeldt et
al. 1989; Kirzinger and Jürgens 1991;
McCasland and Konishi 1981; Metzner 1989,
1993; Müller-Preuss and Ploog 1981;
Numminen et al. 1999; Schuller 1979;
Suga and Schlegel 1972; Suga and Shimozawa
1974). In these studies, however, the CNS has always been
intact or semi-intact; therefore, inhibitory influences from
nonauditory reafferent information during sound production cannot be
completely ruled out. As these experiments used extracellular
recordings, a reduction in spiking during vocalization could be the
result of a reduction in excitatory input, rather than neural
inhibition; therefore, a complete characterization of any inhibitory
mechanisms involved is lacking.
Centrally generated inhibitory mechanisms that affect proprioceptive
and mechanoreceptive pathways have been characterized during other
motor behaviors, such as walking (Gossard et al. 1991;
Murphey and Palka 1974; Sillar and Skorupski
1986; Wolf and Burrows 1995) and swimming
(El Manira et al. 1996; Sillar and Roberts
1988). These types of sensory information have a direct impact
on the ongoing motor activity and contribute to the maintenance of an
adaptive motor output (Pearson 1993). We show that the
cricket's stridulatory motor activity modulates the responsiveness of
a sensory pathway that is not involved in regulating the ongoing motor
activity. Inhibition of proprioceptive and mechanoreceptive pathways
acts presynaptically at the afferent terminals (El Manira et al.
1996; Gossard et al. 1991; Murphey and
Palka 1974; Sillar and Skorupski 1986;
Wolf and Burrows 1995), but has also been observed at
the level of a sensory interneuron (Sillar and Roberts 1988). Our study highlights the finding that a centrally
generated neural mechanism, which controls sensory responsiveness, can
act in parallel both at the afferent and at the interneuron level.
Biological function of the corollary discharge
Spiking in ON1 causes a graded hyperpolarization and a reduction
of its response to acoustic stimuli. This mechanism is thought to allow
female crickets to selectively attend to the louder of two singing
males (Pollack 1988; Römer and Krusch
2000). Just as in females, we show that presentation of loud
stimuli to male crickets desensitizes ON1 and prevents it from
responding to subsequent, quieter stimuli (Fig. 11B). This
mechanism may allow males to listen selectively to the louder, and
therefore nearer, of two conspecific males, which may pose a threat to
their mating success with nearby females. This hyperpolarization is
present if ON1 is made to spike with depolarizing current injection,
which adds further weight to the proposal that this mechanism is an
inherent property of ON1 (Sobel and Tank 1994).
Despite the desensitizing effect of loud sounds on the responsiveness
of ON1, behavioral studies have shown that singing male crickets
respond to acoustic stimuli that occur in the chirp intervals (Heiligenberg 1969; Jones and Dambach
1973). We show that the corollary discharge inhibition during
the chirps will prevent desensitization in ON1 and allow the cricket to
hear quieter, subsequent sounds in the chirp intervals (Fig.
11C). In a group of stridulating male G. bimaculatus, where individuals are spaced apart by 2 m on
average, the ability to hear during singing will be an advantage. It
would allow males to defend their territory from rival singing males,
to maintain a fixed distance from each other, and to hear noisy
predators (Simmons 1988).
Auditory information ascends from the prothoracic ganglion, where these
recordings were performed, to the brain via at least two identified
interneurons. Experiments are underway to examine whether auditory
sensitivity is also modulated in these neurons during stridulation. A
reduction in auditory responses has been recorded in walking
(Schildberger et al. 1988; Staudacher and Schildberger 1998) and flying (Tomioka and Yamaguchi
1984) crickets; these reductions were suspected to be the
result of central and peripheral effects. We aim to identify the
neuron(s) causing the inhibition during singing and examine whether
they are responsible for similar effects in other behaviors.
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ACKNOWLEDGMENTS |
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We thank T. Matheson and D. Parker for constructive comments on the manuscript, J. Rodford for help with Fig. 1A, and S. Ellis and G. Harris for technical assistance.
This work was supported by a Biotechnology and Biological Sciences Research Council studentship and grants from the Wellcome Trust and the Royal Society.
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FOOTNOTES |
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Address for reprint requests: J. Poulet, Department of Zoology, University of Cambridge, Cambridge, CB2 3EJ, UK (E-mail: jfap2{at}cam.ac.uk).
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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