Habituated visual neurons in locusts remain sensitive to novel looming objects
Department of Biology, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E2
e-mail: jack.gray{at}usask.ca
Accepted 13 April 2005
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Summary |
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Key words: locust, Locusta migratoria, DCMD, visual cue, neuron, habituation
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Introduction |
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In a swarm, gregarious locusts fly 0.89.0 m apart at flight speeds
of about 3 m s-1, and those at the edge tend to turn inward toward
the centre of the swarm (Waloff,
1972). To avoid continual collisions with their nearest
neighbours, individuals must be able to react very quickly to generate
appropriate collision avoidance manoeuvres. While in flight, locusts also need
to detect potential predators, such as the fiscal shrike Lanius collaris
humeralis and the carmine bee-eater Merops nubicus, which can
capture locusts on the wing (see Rind and
Santer, 2004
). These predators have carpal-to-carpal pectoral
widths and wingspans of 5 cm and 28 cm, respectively, and often swoop in on
flying locusts with the wings held stationary in a gliding posture
(Fry et al., 1992
). Thus, they
present a larger image than a conspecific (wingspan of 7.510 cm) during
a looming approach. Recent studies examining locust collision avoidance and
looming responses of motion-sensitive neurons have used computer-generated
stimuli that incorporated aspects of objects thought to be biologically
relevant (Mohr and Gray, 2003
;
Rind and Santer, 2004
;
Santer et al., 2005
).
The visual system of locusts contains identified interneurons that respond
strongly to looming stimuli and provide a cue for impending collision
(Schlotterer, 1977;
Simmons and Rind, 1992
;
Rind, 1996
;
Judge and Rind, 1997
; Rind and
Simmons, 1997
,
1998
,
1999
; Gabbiani et al.,
1999
,
2001
,
2002
;
Gray et al., 2001
). The exact
mechanism by which one of these neurons, the Lobula Giant Movement Detector
(LGMD), encodes information about looming stimuli remains contentious. Two
current models predict how the output firing of the LGMD represents a looming
stimulus. One hypothesis states that the LGMD acts as an angular threshold
detector during approach of objects on a direct collision course from within
the horizontal plane up to 135°
(Gabbiani et al., 2001
).
According to this model, postsynaptic multiplication of excitatory and
inhibitory inputs that converge onto the LGMD produces a peak firing rate that
occurs with a fixed delay after the looming object reaches a fixed threshold
angular size (Gabbiani et al.,
1999
,
2002
). Accordingly, peak firing
occurs before collision (Hatsopoulos et
al., 1995
; Gabbiani et al.,
1999
,
2001
,
2002
;
Matheson et al., 2004a
).
Another model suggests that presynaptic inhibition shapes looming responses of
the LGMD (Rind, 1996
), which
produces a peak firing rate after object motion ceases (see
Rind and Simmons, 1999
;
Rind and Santer, 2004
).
In the locust brain, each right and left LGMD synapses onto a Descending
Contralateral Movement Detector (DCMD), which projects to the contralateral
ventral nerve chord. The DCMD axon branches bilaterally within the thoracic
ganglia, and synapses onto flight interneurons and motorneurons
(Burrows and Rowell, 1973;
Simmons, 1980
;
Robertson and Pearson, 1983
).
Each spike in the LGMD elicits a spike in the DCMD
(O'Shea and Williams, 1974
;
Rind, 1984
) and thus DCMD
activity reflects the spatiotemporal properties of a looming stimulus.
Many earlier studies describing the encoding properties of the DCMD
presented looming stimuli at 25 min intervals. More frequent
stimulation induces habituation of the response
(Horn and Rowell, 1968;
Rowell, 1971
;
Bacon et al., 1995
),
particularly if stimuli are presented 40 s apart
(Simmons and Rind, 1992
).
Recently, however, Matheson et al.
(2004a
) showed that DCMDs of
gregarious locusts maintain up to 85% of a nonhabituated response when
stimulated at 60 s intervals. Decreased sensitivity of the DCMD to repeated
small-field motion results from habituation of synapses between chiasmatic
visual afferents and the dendritic fan of the LGMD
(O'Shea and Rowell, 1976
;
reviewed by Rind, 2002
).
Moreover, Rowell et al. (1977
)
suggested that a phasic lateral inhibitory input impinging on the excitatory
afferents reduces input to the LGMD during large-field stimulation. The effect
of this network would be to reduce saturation of afferent neurons and fatigue
of excitatory inputs onto the LGMD during whole-field movements. Gregarious
locusts flying in a swarm would encounter many objects, including conspecifics
and flying predators, approaching frequently from many directions, which would
produce a combination of whole-field and small-field motion. A global
habituation mechanism would be maladaptive in these conditions since the
system would lose the ability to respond to small-field looming stimuli. In
this context, localized habituation of presynaptic inputs to the LGMD would
allow each locust within the swarm to remain sensitive to approaches of
individual objects within its field of view.
The purpose of the experiments described here was to examine the responses
of locust DCMDs to approaches of objects that emulate another locust or a
predatory bird at intervals of 34 s and 4 s. Approaches at 4 s intervals were
designed to further test whether habituated DCMDs were able to respond to the
same object approaching along a new trajectory or to an object with different
stimulus parameters approaching along the same trajectory. I show that the
DCMDs maintained responses to a simulated locust or bird approaching at 34 s
intervals and that habituated DCMDs were able to respond to the same object
approaching along a new trajectory and a larger object approaching along the
same trajectory. Moreover, internal object motion during an approach did not
affect habituation to repeated approaches. Some of the data presented here has
been published previously in abstract form
(Gray, 2004).
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Materials and methods |
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Visual stimuli
Computer-generated stimuli were created and rear projected onto a dome
screen using a Sony VPL-PX11 LCD data projector at 80 frames per second
(f.p.s.; Fig. 1A). Stimuli were
rendered at 900x768 pixels, which produced an actual pixel size of 0.70
mmx0.70 mm when projected onto the center of the dome screen
(radius=35.5 cm). Thus, each projected pixel subtended 0.36° of the
locust's field of view, which is below the spatial resolution of the
ommatidial array in the acute zone of the eye (1°;
Horridge, 1978). The stimuli
were designed to emulate the actual dimensions of either an approaching locust
(`locust') or bird (`bird'; Fig.
1C). Each projected object (luminance=170 cd
m-2=Imax) was set against a white background
(luminance=483 cd m-2=Imin), producing a
Michelson contrast ratio
(ImaxImin/Imax+Imin)
of 0.48. Luminance measurements were made using a Quantum Instruments PMLX
photometer (B & H Photo, New York, USA) placed at the position of the
experimental locust's head (see below). The luminance values were higher than
used in previous studies of DCMD responses to looming stimuli (see
Gabbiani et al., 1999
) and are
due to an overall brighter image from a LCD projector compared to a CRT or LCD
computer screen. Nevertheless, an object:background contrast ratio of 0.5 is
typical for experiments of DCMD responses to looming stimuli. Simulated
approaches were created using 3D Studio Max (version 5) animation software
(Autodesk Inc., Markham, ON, USA). Each object was scaled to real-world
coordinates and designed to approach from 9 m away at 3 m s-1. The
object remained stationary for 1 s after the end of approach, thus each
approach lasted 4 s. Approaches were rendered in AVI format at 80 f.p.s.,
slightly above the flicker fusion frequency of the locust eye
(Miall, 1978
), for a total of
320 frames. Power spectral density analysis of DCMD spike times showed no
discernible peaks at 80 Hz, suggesting that, even at the relatively high
luminance values produced by the LCD projector, the visual system did not
phase lock to individual frames during approach. Two pairs of wings on the
`locust' were designed to beat in antiphase to each other at 25 beats
s-1 to emulate flapping flight during approach. This produced a
temporal resolution of 3.2 frames wingbeat-1, which may have
produced artefacts during presentation. However, for approaches of 3 m
s-1 (i.e. locust flight speed), the rendering rate of the simulated
objects (80 f.p.s.) was the maximum that could be played back with high
fidelity using the existing hardware. Moreover, DCMD activity (see below) did
not phase lock to the motion of the `locust' wings, suggesting that this
relatively crude approximation of flapping flight did not produce confounding
artefacts during an approach. The dimensions of the `locust' (body diameter=1
cm, maximum wingspan during mid stroke=7 cm) are similar to those of real
locusts. The wings of the `bird' were fixed in place to emulate a gliding
approach. The dimensions of the `bird' (body diameter=5 cm, wingspan=28 cm)
are similar to those of small birds known to prey upon locusts (see
Fry et al., 1992
;
Rind and Santer, 2004
). The
experimental locust was placed such that its head was 11 cm from the apex of
the screen and therefore the final visual angle subtended by the general
regions of the simulated objects was: `locust' body=5.2°, `locust'
wings=35.3°, `bird' body=25.6°, `bird' wings=103.7°. Each
simulated object was rendered to compensate for distortions due to curvature
of the rear projection screen and placement of the experimental locust near
the apex.
|
The ratio of the half size of a symmetrical object (l) and the
absolute velocity |v| can be used to calculate a single
value that relates to the increase in angular subtense during an approach at
constant velocity (see Gabbiani et al.,
1999). Because the objects described here are composed of complex
shapes, I calculated the half size as half the length of the hypotenuse
(h) as defined by:
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The AVI files were played back with Windows Media Player (version
6.4.09.1128) set to full screen playback using a NVIDIA GeForce4 Ti4200 video
card with 128 MB of onboard memory. This configuration maintained the 80
f.p.s. of the original rendering and was confirmed by viewing the video
statistics of the player during playback as well as by recording projected
images with a high-speed video camera set at 250 f.p.s. The AVI files also
contained a 1 ms synchronization pulse that was aligned with the frame in
which the object approach stopped (i.e. at time = 3 s). The synchronization
pulse was played through the computer's sound card and connected to the TTL
channel of the multichannel recording system to allow for synchronization of
the stimulus and neurophysiological recordings. The angles subtended by the
`body' diameter and `wingspan' of each object during an approach (see
Fig. 1C) were aligned to the
time of collision and are shown in Fig.
1D. These stimuli reliably induce avoidance steering manoeuvres in
loosely tethered flying locusts (Mohr and
Gray, 2003).
A `locust' or `bird' approached along one of three trajectories: either from 0° azimuth in the horizontal plane (straight ahead) or from ±45° azimuth (to the right or left, respectively, of the experimental locust's longitudinal body axis). One sequence of presentations consisted of 30 consecutive approaches with a 34 s interval between the start of each 4 s approach. This sequence was repeated for each object and each trajectory.
Another sequence of approaches was designed to test whether a habituated DCMD was able to respond to the same object approaching along a different trajectory or to a new object approaching along the same trajectory. For each sequence a `locust' or `bird' approached 17 times with a 4 s interval between the start of each approach (i.e. no delay between the end of one approach and the start of the next approach). Approaches 115 were of the same object approaching along the same trajectory. To test for the effects of a new trajectory the 16th approach was of the same object approaching either from ±45° azimuth, if the first 15 approaches were from 0° azimuth, or of the same object approaching from 0° azimuth, if the first 15 approaches were from ±45° azimuth. The 17th approach was the same as that for the first 15. To test for the effects of a new object size the 16th approach was of a `bird' if the first 15 approaches were a `locust' and of a `locust' if the first 15 approaches were a `bird'. The effects of a new object size were tested for each trajectory.
A third sequence consisted of a `bird' approaching from 0° azimuth while it rotated ±45° about its longitudinal axis to emulate roll during approach. Rotations lasted 500 ms and continued until the time at which the approach stopped. This configuration was designed to test whether DCMD habituation is affected by low frequency, non-looming motion of an approaching object (i.e. internal object motion), which might occur during corrective steering manoeuvres of an approaching predator. The time interval between the start of consecutive approaches for this sequence was 4 s.
Including all experimental conditions, each locust (N=11) was presented with 433 approaches from 21 sequences for a total of 4763 presentations (see Table 1 for the order of sequence presentations to each experimental animal). The inter-sequence time interval was at least 5 min to reduce potential habituating effects of object size and trajectory between sequences of approaches. At least 5 min after the end of each experiment each locust was presented with a single approach of a `bird' from 0° azimuth to confirm that DCMD responses were not affected by the duration of the experiment or the total number of presentations (data not shown).
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Experimental setup and multichannel recording
Experimental locusts were mounted ventral side up onto a rigid tether using
low melting point beeswax. A small patch of ventral cuticle was removed to
expose the underlying mesothoracic ganglion. The exposed tissue was bathed in
a drop of locust saline (147 mmol l-1 NaCl, 10 mmol l-1
KCl, 4 mmol l-1 CaCl2, 3 mmol l-1 NaOH, 10
mmol l-1 Hepes, pH 7.2) and the preparation was transferred to a
flight simulator (Fig. 1B; see
Gray et al., 2002 for a
complete description). For multichannel recordings I used 2x2 tetrode
silicon probes provided by the University of Michigan Center for Neural
Communication Technology
(http://www.engin.umich.edu/facility/cnct/)
sponsored by NIH NCRR grant P41-RR09754
(Fig. 2Ai). The probes were
connected to a RA16AC 16 channel acute Medusa Bioamp System 3 workstation
(Tucker-Davis Technologies Inc., Alachua, FL, USA). Physiological signals were
sampled at 25 kHz/channel with Butterworth filter settings of 100 Hz(high
pass) and 5 kHz (low pass). An additional TTL channel was used to record
synchronization pulses from the visual stimuli (see above) to permit alignment
of the physiological recordings with the stimuli. Using a micromanipulator,
the probes were inserted ventrally into the mesothoracic ganglion until all
sets of tetrode recording sites were within the tissue
(Fig. 2Ai). After the initial
neuronal injury discharge from probe insertion had ceased I tested the
preparation for responses to visual stimuli by waving my hands in front of it.
The entire preparation was then rotated 180° to orient the locust
dorsal-side up. Following confirmation of responses to visual stimuli, the
rear projection dome screen was placed in front of the preparation such that
the head of the locust was 11 cm from the apex of the dome. In this
configuration, the dome occupied 250° of the locust's horizontal and
vertical field of view. The threshold settings for data capture were adjusted
to capture, selectively, spikes with the largest amplitude, which also showed
distinct responses to looming stimuli (see below). This configuration produced
stable recordings for the duration of each preparation, about 5 h. On
occasion, fixed preparations would generate flight-like rhythms and the
resulting neural activity in the ganglion would make it impossible to
distinguish DCMD activity. Therefore data were collected only if the
preparation did not generate these rhythms at least 2 min before and
throughout the stimulation sequence. Data were recorded onto disk and stored
for off-line analysis.
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Data analysis
To quantify DCMD responses, spike times
(Fig. 3, toprasters) were
transformed to instantaneous spike rates using a 50 ms Gaussian smoothing
filter (middle graph; see Gabbiani et al.,
1999). A Gaussian filter was used to reduce the artefacts caused
by temporal binning of the responses into peristimulus time histograms
(Richmond et al., 1990
). For
each approach I measured the time of the peak firing relative to collision
(asterisk in Fig. 3), the
amplitude of the peak and the number of spikes. Previous reports suggested
that if DCMD spikes are involved in initiating collision avoidance flight
manoeuvres then they must occur approximately
200 ms before collision
(Gray et al., 2001
;
Matheson et al., 2004a
). This
value takes into account known values of flight reaction times relative to
collision, the lag between visual input and the onset of a behavioural
response as well as conduction times of DCMD spikes from the brain to the
thorax. Moreover, although the DCMDs are likely not solely responsible for
initiating or triggering an escape jump, they could have a currently
undescribed role early in the underlying motor program
(Burrows, 1996
). Energy for a
jump is stored via co-contraction of tibial flexors and extensors
over a period of 200500 ms (Pearson
and O'Shea, 1984
). Therefore, I also measured the instantaneous
spike rate 200 ms before collision (arrow in
Fig. 3) as an approximation of
the latest time at which DCMD activity could influence escape behaviour.
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Results |
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Dishabituation of DCMDs between approach sequences
To determine that the DCMDs were fully dishabituated after a minimum 5 min
interval between approach sequences I plotted the number of spikes for the
first approach of a sequence against the order in which sequences were
presented (see Table 1,
Fig. 4A). A
KruskalWallis ANOVA on ranks showed that there was a significant
difference between treatments (H20=152.9,
P>0.001, N=11 approaches/treatment). A Dunn's
post-hoc multiple comparison showed that there were significantly
fewer spikes during approaches of a `locust' than during approaches of a
`bird' (P<0.05), but that there were no significant differences
within `locust' or `bird' approaches. To control for the type of object and
initial approach trajectory, the number of spikes were normalized to the
number for the first approach with a particular combination of parameters. For
example, the number of spikes for all sequences in which the initial approach
of a `locust' was from 0° azimuth was normalized to the first sequence in
which the first approach was a `locust' from the same trajectory
(Fig. 4B). A
KruskalWallis ANOVA on ranks (H20=46.3,
P<0.001) showed that there was a significant difference in the
median values of the groups (object type combined with trajectory). However, A
Dunn's post-hoc multiple pairwise comparison showed that there were
no significant differences between specific median values from approaches of
different objects or different trajectories. These data demonstrate that there
were no confounding effects of habituation due to the order of approach
sequences during each experiment.
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To quantify the extent of DCMD habituation within a sequence I normalized the values of the last approach to those of the first approach (Fig. 6) and used a two-way ANOVA to test the effects of object type and trajectory on the peak spike rate, the instantaneous spike rate 200 ms before collision and the number of spikes. Neither object type nor trajectory affected a decrease of the peak spike rate. While the object type or trajectory alone did not affect a decrease of the instantaneous spike rate 200 ms before collision there was a significant combined effect of these two parameters (F1,59=10.472, P=0.002). Holm-Sidak post-hoc multiple comparisons revealed that a `locust' approaching from 0° azimuth produced a greater decrease of the instantaneous spike rate 200 ms before collision than did a `bird' from the same trajectory or a `locust' approaching from ±45° azimuth. There was also a significant effect of the object type on a decrease of the number of spikes produced during an approach along either trajectory (F1,63=6.28, P=0.015). The same post-hoc multiple comparisons revealed that a `bird' approaching from ±45° azimuth produced a greater decrease of the number of spikes than did a `bird' from 0° azimuth or a `locust' from either trajectory. These data show that the DCMDs were able to maintain 6080% of the peak spike rate and 4060% of the number of spikes in response to repeated approaches of a `locust' or `bird' presented at 34 s intervals. However, the firing rate 200 ms before collision, when a behavioural response would be initiated, decreased to 60% or lower.
Responses to the same object approaching along a new trajectory
DCMD responses decreased dramatically during 15 approaches of a `locust' or
`bird' at 4 s intervals (Figs 7
and 9). In
Fig. 7A, approaches 115
and 17 are from 0° azimuth and approach 16 is from +45° azimuth. The
response to approach 15 of a `locust' or `bird' began later and was much
weaker than the response to approach 1. Qualitatively, the response decrement
was greater during the first 15 approaches than it was to approaches at 34 s
intervals (Fig. 5). The
response to approach 16 (new trajectory, arrows in
Fig. 7A) was much stronger than
to approach 15 and qualitatively similar to the response to approach 1 whereas
the response to approach 17 (original trajectory) was similar to the response
to approach 15.
Data from 11 animals showed that the peak spike rate, the instantaneous spike rate 200 ms before collision and the number of spikes during approaches 115 decreased with repeated approaches and were well-fit with an exponential decay (Fig. 7B) as described above. There was little qualitative difference in a decrease of the peak spike rate during repeated approaches of either object type from 0° azimuth whereas the DCMD habituated further during repeated approaches of a `bird' from ±45° azimuth (Fig. 7Bi). The decrease of the instantaneous spike rate 200 ms before collision was similar for a `locust' or `bird' approaching from 0° azimuth whereas during approaches from ±45° azimuth the spike rate was consistently low for each approach of a `locust' (Fig. 7Bii). This habituation profile is due to the fact that the DCMD firing rate does not increase until after a `locust' is within 200 ms of collision. The number of spikes also decreased during consecutive approaches of either object type (Fig. 7Biii). As with the 34 s interval data, an approaching `bird' evoked more spikes during the first approach, but these decreased to a level that was the same for either object type. For either object type or trajectory, the 16th approach evoked a higher peak spike rate, a higher spike rate 200 ms before collision and more spikes than approach 15, whereas the response to approach 17 was the same as to response 15.
Data for approaches 1517 were normalized to those for approach 1 (Fig. 8), as described above. To test the effects of the object type and approach number on the parameters measured I used a two-way ANOVA to compare data within each initial trajectory (0° or ±45° azimuth). For either trajectory there was a significant effect of the stimulus type or approach number on a decrease of the peak spike rate and number of spikes, whereas a decrease of the spike rate 200 ms before collision was affected by the approach number but not by the object type (see Table 2 for two-way ANOVA parameters). There was no combined effect of object type and approach number on habituation of the responses. I used a HolmSidak multiple comparison to test for significant differences between object types within a specific approach number or for differences between approach numbers within a given object type. Results showed that for all parameters measured, regardless of the object type, responses to the 15th and 17th approaches were lower than responses to the first approach (i.e. normalized values <1). Responses to the 16th approach were as high as or higher than responses to the first approach (i.e. normalized valuesx1) and were significantly greater than the normalized responses to approaches 15 and 17. The only significant differences between different objects were in responses of the peak spike rate (approaches 16 and 17 from 0° azimuth and approach 17 from ±45° azimuth) and the number of spikes (approach 16 from 0° azimuth). These differences should be interpreted with caution, however, as the P-values for each comparison ranged from 0.45 to 0.5 (with significance set at P=0.05), suggesting that they may not be biologically significant. Nevertheless, these data demonstrate that DCMD habituation was not maintained for targets approaching along a new trajectory which, in turn, did not dishabituate the response to the initial object parameters. Moreover, there were few differences between habituated responses to a `locust' or `bird'.
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Responses to a new object approaching along the same trajectory
Fig. 9A shows that
habituation of the responses during approaches 115 (the same object
traveling along the same trajectory) was consistent with that shown in
Fig. 7A. The 16th approach,
however, produced different responses based on the initial object type. For
responses that had habituated to a `locust' (black rasters), the 16th approach
(a `bird', red arrow) produced a more intense response, whereas for responses
that had habituated to a `bird' (red rasters), the 16th approach (a `locust',
black arrow) did not evoke a more intense response. For both conditions the
response to approach 17 was indistinguishable from the habituated response
(approach 15). In these examples all approaches were from +45° azimuth.
Data from 11 animals showed that a decrease of the peak spike rate, the
instantaneous spike rate 200 ms before collision and the number of spikes
during approaches 115 was qualitatively similar to that described in
Fig. 7B. However, when the new
object (approach 16) was a `bird', the responses were consistently larger than
the habituated responses compared to when the new object was a `locust'.
Data were normalized and plotted as in Fig. 8 (Fig. 10). A two-way ANOVA within each trajectory showed that there was a significant effect of the stimulus type or approach number as well as a combined effect of both factors on habituation of DCMD responses (Table 2). Multiple comparisons (HolmSidak) within the object type or approach number showed similar differences for responses as described in Fig. 8 (Fig. 10), except for one important point. For approach 16 the peak spike rate, the spike rate 200 ms before collision and number of spikes was significantly higher when the new object was a `bird'. When the new object was a `locust', neither parameter differed from the habituated state. These data show that habituated DCMDs are able to produce an increased response to a larger object approaching along the same trajectory and that a new object, of either type, did not dishabituate the response.
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For many initial and subsequent approaches of a `locust', DCMD activity did not increase until after 200 ms before collision. Therefore, these approaches could not be used to calculate the slopes of the transformed data. The resulting inequality of the number of observations eliminated the possibility of testing the effects of all three factors simultaneously. Therefore I used a two-way ANOVA within each trajectory to test for effects of the object type and approach interval on the rate of decrease of the instantaneous spike rate 200 ms before collision. The results suggest that, of the approach sequences presented here, DCMD habituation was more sensitive to different objects approaching from 0° azimuth and to the approach frequency along trajectories from ±45° azimuth. For approaches from 0° azimuth there was a significant combined effect of the object type and approach frequency (F1,89=8.50, P=0.004) and, individually, there was an effect of the object type (F1,89=8.76, P=0.004), but no effect of the approach frequency. For approaches from ±45° azimuth there was a significant effect of the approach frequency (F1,104=12.402, P<0.001), but no effect of object type or a combination of the two factors. Taken together, results from calculating habituation rates suggest that the DCMD is better able to respond upon repeated presentations of a `locust'.
Habituation of peak firing times
Matheson et al. (2004a)
showed that variability of the time of the peak DCMD spike rate was low for
gregarious locusts presented with objects approaching at 60 s intervals.
Therefore, I used a Pearson Product Moment Correlation to measure the
relationship between the approach number and the time of the peak DCMD spike
rate under the experimental conditions used here. Results revealed that during
repeated approaches at 34 s intervals, the mean peak firing time relative to
collision was invariant for a `locust' and `bird' from 0° azimuth, and a
`bird' from ±45° azimuth. The peak time was, however, significantly
negatively correlated to the approach number for a `locust' approaching from
±45° azimuth (r=0.669). For approaches at 4 s
intervals from 0° azimuth, the time of the peak was invariant for a
`locust' and positively correlated to a `bird' (r=0.733). For
approaches at 4 s intervals from ±45° azimuth, the time of the peak
was negatively correlated to a `locust' (r=0.771) and
positively correlated to a `bird' (r=0.595). Fitting a single
exponential decay function (Fig.
11A, see Materials and methods) showed that the peak time occurred
earlier during approaches of a `locust' from ±45° azimuth at 4 s
and 34 s approach intervals and, significant relationships described above
notwithstanding, was relatively insensitive to other combinations of object
type, approach trajectory and approach interval. To examine the variability of
peak firing time with respect to the object size, trajectory and the state of
habituation I plotted the standard deviation (S.D.) of the peak
time against the approach number (Fig.
11B). A Pearson Product Moment Correlation revealed that
variability of the time of peak firing was not affected by repeated
presentations at 34 s intervals of a `locust' approaching along either
trajectory or a `bird' approaching from 0° azimuth, whereas the
S.D. was positively correlated with repeated approaches of a `bird'
from ±45° azimuth, albeit weakly (r=0.429,
P=0.019). For 4 s interval data there was no effect of repeated
approaches of either object from 0° azimuth, whereas the variation
decreased for either object approaching from ±45° azimuth (`locust:
r=0.698, P=0.004, `bird': r=0.551,
P=0.03). These data suggest that the time of peak DCMD firing is
sensitive to repeated approaches of small objects from ±45° azimuth
and relatively insensitive to repeated approaches of a large object from
±45° azimuth, or to either object type from straight ahead.
Effects of internal object motion on habituation
To test for the effects of internal object movement on habituation I
normalized data from the 15th approach, as described above, of a `bird' that
rolled about its longitudinal axis (see Materials and methods) and compared
the mean to results from the pooled 4 s interval data of a `bird' approaching
from 0° azimuth (Fig. 12).
Internal motion during approach did not influence decreases of the peak spike
rate, the instantaneous spike rate 200 ms before collision or the number of
spikes (KruskallWallis ANOVA on ranks). The time of the peak for either
stimulus type was also invariant (data not shown).
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Discussion |
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DCMD responses to complex looming objects
For objects traveling along the same trajectory, lower
() values (i.e. a
simulated locust) generated shorter duration responses that began and ended
later during an approach (Figs
5A,
7A,
9A). These observations are
consistent with previous findings (Judge
and Rind, 1997
; Gabbiani et al.,
1999
,
2001
;
Gray et al., 2001
;
Matheson et al., 2004a
;
Rind and Santer, 2004
) and are
likely due to weak activation of the feedforward inhibition to the LGMD as the
expanding edges stimulate presynaptic inputs relatively late in the approach.
This may also explain why, for a constant object size, approaches from 0°
azimuth produced a later response compared to approaches from ±45°
(see Fig. 2B). However, the
frontal region of the eye is less sensitive to motion
(Rowell, 1971
;
Krapp and Gabbiani, 2004
;
Matheson et al., 2004b
) and
the relationship between l/|v| and LGMD firing
parameters is non-linear during frontal approaches
(Gabbiani et al., 2001
).
Therefore further experiments are needed to determine how presynaptic inputs
influence LGMD firing across different regions of the field of view.
Previous studies using either expanding circles or squares to stimulate the
LGMD/DCMD pathway have allowed investigators to describe precise relationships
between stimulus parameters and neuronal responses (see
Rind and Simmons, 1997;
Gabbiani et al., 2002
). While
the stimulus parameters of an expanding circle resemble the frontal profile of
a bird approaching with wings folded to the side, locusts in their natural
environment would encounter many complex objects in which the edges do not
expand uniformly across the retina. Although the angular threshold computation
of the LGMD is invariant for expanding circles and squares up to an azimuth of
135° within the horizontal plane
(Gabbiani et al., 2001
), it was
not known if DCMD responses are conserved for spatially complex objects or
objects that produce internal motion during an approach. To compare DCMD
responses to complex objects, presented here, with responses to simple
objects, reported previously, I calculated
thresh and
based on a linear measurement
(
) of the stimuli
used (see Materials and methods). The values for
thresh and
(see Results) are higher than reported by Gabbiani et al.
(1999
,
2001
,
2002
; their values:
thresh=1540° and
=1535
ms). In a separate study, Matheson et al.
(2004a
) reported that for two
populations of gregarious locusts presented with squares approaching from
90° azimuth,
thresh=1334° and
=1288 ms. The threshold angle is also larger here (see
Results), whereas the delay is within the range of the latter study. Two main
factors could contribute to the different values presented here and those
reported previously. First,
thresh is influenced by the
approach trajectory within the horizontal plane. Gabbiani et al.
(2001
) showed that the
two-dimensional linear model relating l/|v| and the
time of the peak spike rate does not explain approaches from 0° and
45° azimuth. Their data show that for a square approaching from 0° or
45° azimuth
thresh=180° or 90°, respectively
(compared to 122.7° and 38.6°, respectively, reported here). Secondly,
textured objects, containing multiple (>4) expanding edges, do not fit the
linear model (Gabbiani et al.,
2001
). Therefore, it is reasonable to expect that the relationship
between stimulation parameters of a looming object and DCMD responses would
vary depending on object trajectory and complexity. Although not within the
scope of this study, it would be interesting to explore how the LGMD responds
to multiple complex objects approaching from various trajectories at varying
time intervals within the entire field of view.
Rind and Simmons (1997)
have suggested that DCMD responses to looming stimuli decline dramatically
when a digitized object expands more than 3° in a given step (however, see
Gabbiani et al., 1999
). For the
objects described here, this jump occurs at distinct times before collision:
`locust' body = 37.5 ms, `locust' wings = 62.5 ms, `bird' body = 50 ms, `bird'
wings = 125 ms. The lag time from visual stimulation to the occurrence of a
DCMD spike in the cervical connective is approximately 40 ms (see
Matheson et al., 2004a
).
Assuming a DCMD conduction velocity of 3.3 m s-1 at 25°C (see
Money et al., 2005
), it would
take approximately 2 ms for a spike to travel from the midpoint of the
cervical connective to the mesothoracic ganglion (67 mm). Therefore,
with electrodes in the mesothoracic ganglion (see Materials and methods), a
rapid decrease of the DCMD spike rate would be detected approximately 42 ms
after an angular jump of 3°. Using the longest axis of the simulated
objects as an indication of when the angular subtense first jumps by 3°
(i.e. using the dimension of the `locust' or `bird' wings), then an effect on
DCMD firing, i.e. an artefactually induced peak, should occur, invariably, at
20.5 ms (`locust') and 85 ms (`bird'). To compare these times to
DCMD activity I pooled the time of peak firing during the first approach (to
avoid putative habituation) of each object from each trajectory. The peak
times for these stimulus parameters are: `locust' from 0° azimuth,
58.5±36 ms (range, 18414.4 ms), `locust' from
±45° azimuth, 21.3±111 ms (range, 233.0261.9
ms), `bird' from 0° azimuth, 77.9±60 ms (range,
298.523.7 ms), `bird' from ±45° azimuth,
80.5±47 ms (range, 22414.4 ms). While these ranges
bracket times at which the `locust' or `bird' wings jump by 3° there is a
high level of variability, suggesting that the peak times were not produced by
this single stimulus parameter (see also
Gabbiani et al., 1999
).
Discrepancies in the time of peak DCMD firing relative to collision have
received much attention in recent literature and are thought to be due to
differences in target size between different preparations. The data presented
here suggest that object complexity is also involved in defining the response.
In non-habituated locusts the mean time of peak firing occurred before the end
of the approach of the simulated bird, which was designed to emulate the size
and dimensions of small birds known to catch flying locusts on the wing (see
Materials and methods). Rind and Santer
(2004) used pectoral diameters
of avian locust predators to suggest the DCMDs are tuned to detect objects
with diameters in the range of 5090 mm, which would result in peak
firing occurring after the end of a loom. These birds, however, may also
approach flying locusts while gliding with outstretched wings
(Fry and Fry, 1992
), which, for
a given approach velocity, would produce a larger
l/|v| ratio and an earlier time of peak DCMD
firing, as reported here (see also Gabbiani
et al., 1999
). Further investigation is required to identify
specific effects of looming object complexity on DCMD firing responses.
Matheson et al. (2004a)
showed that in gregarious locusts the time of peak DCMD firing was invariant
during repeated approaches of a dark square
(l/|v|=20 ms) from the equivalent of 90°
azimuth. In agreement with their findings, I show that the mean and
S.D. of time of peak firing is stable during repeated approaches of
a `bird' (
ms), regardless of the approach trajectory or interval
(Fig. 11). However, the peak
time occurred earlier after repeated approaches of a `locust'
(
ms)
approaching from ±45° azimuth. It is difficult to attribute this
apparent discrepancy to a single factor since Matheson et al.
(2004a
) did not describe,
explicitly, stability of the time of the peak during habituation for each
value of l/|v| that they used. It would be
interesting to compare the stability of the time of peak firing during
habituation to their smallest sized objects
(l/|v|=10 ms, assuming their sample approach
speed of 2 m s-1) with the earlier peak times described above.
Although earlier peak times after repeated approaches of a small object has
been observed independently (F. C. Rind, personal communication), it is not
clear if it would be of any biological significance. This effect occurs in
conjunction with a decrease in the peak firing rate and the number of spikes
(Figs 5,
7,
9), which makes it difficult to
interpret the effects of habituation on this single parameter. Moreover, the
time of peak firing typically occurs after the initiation of escape responses
(see Materials and methods).
Mechanisms of habituation
Increased responses of habituated DCMDs to objects approaching along a new
trajectory (Fig. 8) or larger
objects approaching along the same trajectory
(Fig. 10) are consistent with
a presynaptic mechanism of habituation between chiasmatic visual afferents and
the dendritic fan of the LGMD (O'Shea and
Rowell, 1976; Rowell et al.,
1977
). Objects approaching from a new trajectory would stimulate a
different, nonhabituated, array of ommatidia and thus provide input to the
LGMD through a different series of visual afferents. Similarly, the edges of a
larger object approaching along the same trajectory would expand beyond the
subtense angle of the original, smaller, object and thus stimulate
nonhabituated local input elements to the LGMD. However, the edges of a new,
smaller object would expand within the visual field of a habituated array of
input elements, resulting in a continued `habituated' DCMD response (see
Fig. 10). In agreement with
previous findings (Rowell,
1971
; Bacon et al.,
1995
), a new trajectory or object did not dishabituate DCMD
responses. This is also consistent with a model of local synaptic habituation
because a new, larger object or the same object approaching from a new
trajectory would not influence a separate set of habituated synaptic inputs to
the LGMD. In this context, responses to internal object motion, such as the
beating wings of the simulated locust or the roll movements of the simulated
bird (Fig. 12), are not
surprising since the same regions of the retina would be stimulated often
enough to induce habituation to these specific motions.
Implications for avoidance responses
The ability of habituated DCMDs to respond to the same object on a new
trajectory could be advantageous for gregarious locusts. While there is no
information on specific patterns of visual stimulation experienced by
individual locusts in a swarm, there would be a complex combination of
translating, receding and looming stimuli produced by self motion (i.e.
whole-field optic flow, especially during flight) and object motion (e.g.
conspecifics and predators). On the ground or in the air, an individual within
a swarm would be surrounded by many conspecifics that would stimulate various
regions of the field of view. In the air, those that fly along non-colliding
trajectories would contribute to a background motion that is known to
influence LGMD responses to looming stimuli
(Gabbiani et al., 2002). The
lateral inhibition network among input elements to the LGMD prevents
saturation and fatigue of individual small-field elements
(Rowell et al., 1977
), and
optic flow sharpens the peak of LGMD firing during looming by activating
feed-forward inhibition pathways (Gabbiani
et al., 2002
). The limitations of the hardware used here for
stimulus presentation precluded the use of a visual flow field during
approach. Therefore, putative effects on DCMD habituation to the stimuli used
here are not known. However, the results are consistent with the mechanisms of
habituation described above.
Although the time of peak DCMD firing occurs after the initiation of escape
responses, earlier activity could be important for modulating avoidance
behaviours (Burrows, 1996;
Gray et al., 2001
;
Matheson et al., 2004a
).
Nonhabituated DCMD responses 200 ms before collision are lower during the
approach of a `locust' than during the approach of a `bird' (Fig.
5,
7,
9), which is consistent with
previous findings (Matheson et al.,
2004a
) and may be important in distinguishing a potential predator
from a conspecific. It should be noted, however, that in the presence of a
constant velocity visual flow field, the LGMD did not fire 200 ms before
collision of a looming object (Gabbiani et
al., 2002
).
The data presented here suggest that complex object shapes and differing
collision trajectories influence DCMD response parameters that define
responses to approaching objects (i.e. thresh and
).
This effect is likely due to non-linear integration of local inputs to LGMD as
images of looming objects expand differentially across the eye. The results
also demonstrate that the LGMD/DCMD pathway encodes looming approaches
irrespective of internal object motion, suggesting that fine movements may not
be involved in activating the collision detection circuitry. Responses of
habituated DCMDs to novel looming stimuli imply that the LGMD/DCMD pathway
should remain sensitive to multiple objects approaching along different
trajectories, which would occur in a swarm. Testing this assumption requires a
better understanding of a locust's visual input structure during swarming.
Flying locusts generate self-motion across the eye resulting from forward
motion and steering manoeuvres, making it necessary to incorporate complex
visual flow patterns into an experimental paradigm. Current studies are
underway to describe habituation of behavioural and kinematic responses of
loosely tethered locusts presented with the same stimuli used here, which will
allow us to emulate closed-loop visual stimuli while recording DCMD activity
during looming approaches. Stimulating the locust visual system with complex
objects and flow fields, in conjunction with multichannel recording
techniques, will make it possible to better understand how a motion sensitive
neuron, or a putative population of descending visual neurons, function in a
complex visual environment.
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Acknowledgments |
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