3.1 Flies
The animals are kept on standard cornmeal/molasses medium
(recipe see Guo et al. 1996) at 25°C and 60% humidity with a 16hr light/8hr
dark regime. 24-48h old female flies are briefly immobilized by cold-anesthesia
and glued (Locktite UV glass glue) with head and thorax to a triangle-shaped
copper hook (diameter 0.05mm) the day before the experiment. The animals
are then kept individually overnight in small moist chambers containing
a few grains of sucrose.
3.2 Apparatus
The core device of the set-up is the torque meter. Originally
devised by Götz (1964) and repeatedly improved by Heisenberg and Wolf
(1984), it measures a fly's angular momentum around its vertical body axis.
The fly, glued to the hook as described above, is attached to the torque
meter via a clamp to accomplish stationary flight in the center of a cylindrical
panorama (arena, diameter 58mm), homogeneously illuminated from behind
(Fig. 2). Via the motor control unit K an electric motor can rotate the
arena according to the experimental procedures described below. The light
source is a 100W, 12V tungsten-iodine bulb. For green and blue illumination
of the arena, the light is passed through monochromatic broad band Kodak
Wratten gelatin filters (#47 and #99, respectively). Filters can be exchanged
by a fast magnet within 0.1 sec.
The angular position of an arbitrarily chosen point of
reference on the arena wall delineates a relative 'flight direction' of
0-360°. Flight direction (arena position) is recorded continuously
via a circular potentiometer (Novotechnik, A4102a306) and stored in the
computer memory together with yaw torque (sampling frequency 20Hz) for
later analysis. The reinforcer is a light beam (diameter 4mm at the position
of the fly), generated by a 6V, 15W Zeiss microscope lamp, filtered by
an infrared filter (Schott RG780, 3mm thick) and focused from above on
the fly. In all experiments the heat is life threatening for the fly: more
than 30s of continuous irradiation are lethal. Heat at the position of
the fly is switched on and off by a computer-controlled, magneto-electrical
shutter intercepting the beam (Fig. 2). The maximum temperature at the
point of the fly is measured separately after the experiments by a blackened
thermoelement of about 1mm3 after 10s of continuous irradiation.
3.3 Experimental procedures
Yaw torque learning. The fly’s spontaneous yaw
torque range is divided into a ‘left’ and ‘right‘ domain (approximately
corresponding to either left or right turns; for a justification of this
assumption see: Heisenberg and Wolf, 1993). Heat is switched on (input
voltage 6.0V) whenever the fly's yaw torque is in one domain and switched
off when the torque passes into the other (henceforth: yaw torque sign
inversion). There are no patterns on the arena wall, but the illumination
is spectrally restricted by a Schott daylight filter (BG18, glass, 3mm)
as it was used by Liu et al. (1999) to allow for context generalization.
Switch (sw)-mode: As in yaw torque learning, the
fly is punished whenever the fly’s yaw torque passes into the punished
range, but during yaw torque sign inversion not only temperature but also
a visual cue is exchanged. Visual cues can be either colors (blue/green)
or pattern orientations (up-right/inverted T in front). For color as visual
cue, the panorama consists either of 20 evenly spaced stripes (pattern
wavelength l=18°; transfer experiments)
or of no patterns at all (modified overshadowing) and the illumination
of the arena is changed from green to blue or vice versa. For pattern
orientation as visual cue, four black, T-shaped patterns of alternating
orientation (i.e. two upright and two inverted) are evenly spaced on the
arena wall (pattern width y=40°, height
J
=40°, width of bars=14°, as seen from the position of the fly).
One of the pattern orientations is presented stationarily in front of the
fly, the other at 90° and 270°. Whenever the range of the fly’s
yaw torque passes into the other half, the arena is turned by 90° to
bring the other pattern orientation in front. For technical reasons, a
hysteresis is programmed into the switching procedure: while pattern orientation
requires a ± 5.9·
10-10Nm hysteresis during yaw torque sign inversion, a ±
2.0· 10-10Nm hysteresis
is sufficient for color as visual cue if the striped drum is used. No hysteresis
is necessary if the patterns are omitted altogether.
Flight simulator (fs)-mode: Closing the feedback
loop to make the rotational speed of the arena proportional to the fly's
yaw torque (coupling factor K=-11°/s·
10-10Nm, Fig. 2) enables the fly to stabilize the rotational
movements of the panorama and to control its angular orientation (flight
direction). If pattern orientation is used as visual cue, the same black,
T-shaped patterns are used as in sw-mode (see above). For color as visual
cue (Wolf and Heisenberg, 1997) the arena either consists of 20 evenly
spaced stripes (l=18°; transfer experiments)
or of four identical vertical stripes (width y
=14°, height J =40°; compound experiments).
A computer program divides the 360° of the arena into 4 virtual 90°
quadrants. The color of the illumination of the whole arena is changed
whenever one of the virtual quadrant borders passes the frontal midline
(i.e. flight direction) of the fly. If a compound of colors and patterns
is used as visual cue, the vertical stripes are replaced by the four T-shaped
patterns and color is changed as described. Heat reinforcement (input voltage
6.0V) is made contiguous either with the appearance of one of the pattern
orientations in the frontal quadrant of the fly’s visual field or with
either green or blue illumination of the arena.
Transfer experiments. Visual discrimination learning
in fs-mode and sw-mode are carried out not only with patterns (upright
and inverted T) but, in a second series of experiments, also with colors
as visual cues. In each series six groups of flies were tested:
-
training and test in fs-mode;
-
training in fs-mode followed by test in sw-mode
-
training in fs-mode followed by familiarization training
and test in sw-mode
-
training and test in sw-mode
-
training in sw-mode followed by test in fs-mode
-
training in sw-mode followed by familiarization training
and test in fs-mode
Modified overshadowing (for an original overshadowing
experiment see below). Groups (4) to (6) of the transfer experiments constitute
one part of this experiment, yielding one group of flies that are trained
with a behavior (yaw torque) and a stimulus (colors) as the elements of
a compound (sw-mode). The 2x4 minutes of compound (composite) training
follow a 4 minute preference test. Then, either color learning is assessed
independently of motor learning in fs-mode (transfer experiment) or motor
learning is assessed independently of color learning by replacing the blue
and green color filters with the Schott daylight filter (BG18, glass, 3mm)
known to allow for context generalization or there is no change at all
(control). In an approach to directly measure the associability of the
behavioral vs. the sensory predictor, for one group the contingencies between
behavior and color are reversed in the final test (i.e. if ‘right’ turns
lead to blue illumination during training, they will lead to green illumination
during the final test phase and vice versa). To control for context generalization
effects, one group of flies is subjected to yaw torque learning in which
only the final test is carried out with the Schott BG18 filter, but in
the preceding training the fly receives behavior independent color changes
recorded from previously trained sw-mode flies (color replay). The control
for this group was a yaw torque learning experiment as described above.
Overshadowing. To test whether the flies are able
to separately process colors and patterns during compound (fs-mode) training,
the animals are trained in the following sequence. Four minutes of unreinforced
preference test are followed by 2x4 minutes of training, interrupted by
a 2 min test period (Table 1a). After these 14 minutes of compound presentation,
flies are either allowed to choose flight directions with the compound
as visual cue (control) or with colors or patterns alone (experimental
groups). A fourth group is presented a new compound in which the combination
between patterns and colors is exchanged (e.g. if during training flying
with an upright T in the frontal visual field led to green illumination
of the arena, it now, during the ‘exchanged’ test phase, would lead to
blue illumination).
Blocking. The two blocking experiments are designed
as between groups experiments, each with one blocking and one control group.
Both again consist of two half groups, one of which is presented with colors
alone in the first training phase (CS1+US) and the other with patterns
alone. The two experiments differ in the amount of compound training (CS1+CS2+US)
and the choice of control procedures. In the first experiment (Table 1bI),
flies receive equal amounts of first training and compound training. The
control groups are provided with the same amount of CS1 and US experience
as the blocking group. This is accomplished in two different ways: In the
control group stimulated by colors as CS1 during the first conditioning
phase flies are trained classically by recording the flight orientation
traces and heating regime of the corresponding blocking group and playing
them back to the naive flies (replay experiment; ; Wolf and Heisenberg,
1991). The other half of the control flies exposed to patterns as CS1 in
white light are operantly trained. It was observed that pattern memory
from training in white light is lost if colors are added to generate compound
stimuli (CS1+CS2). In the corresponding blocking group, a Schott BG18 3mm
thick broad-band blue-green filter allows for generalization upon compounding
the colors with the patterns . In this experiment as well as in the sensory
preconditioning and second-order conditioning experiments, the BG18 filter
is used throughout whenever patterns alone are presented, with the exception
of the control group mentioned above. In the second experiment (Table 1bII),
only half the amount of compound training is applied and the control groups
do not receive any reinforcement before the compound phase.
Second-order conditioning. Two second-order conditioning
experiments are conducted differing in the amount of second-order training
(CS1+CS2). The first (Table 1cI) is modeled closely after the first blocking
experiment (Table 1bI), except that the compound phase is shortened by
2 minutes. For the second experiment (Table 1cII) the second-order conditioning
phase was shortened even more to only 2x2 minutes (matching the second
blocking experiment most closely; see Table 1bII). Only colors are used
as conditioned reinforcer.
Sensory preconditioning. Two groups of flies are
allowed to fly without reinforcement using a compound of colors and patterns
as orientation cues (CS1+CS2) for 10 and 16 minutes, respectively (Table
1dI-II). The groups are then further subdivided into two half experiments
each, according to which stimulus (colors or patterns) is chosen as CS1
and is presented during the subsequent single stimulus phase. This phase
consists of 2x4 minutes of training (CS1+US), with an intermittent 2 minute
test (CS1 alone). The final 2 min test is conducted with the alternative
stimulus (CS2) alone (Table 1dI-II).
3.4 Analysis of Data
3.4.1 Arena position and yaw torque evaluation
The pattern, color or yaw torque range preference of
individual flies is calculated as the performance index: PI=(ta-tb)/(ta+tb).
During training, tb indicates the time the fly was exposed to
the reinforcer and ta the time without reinforcement. During
tests, ta and tb refer to the times when the fly
chose the situation designated as unpunished or punished, respectively.
3.4.2 Statistics
Tests for normal distribution of performance indices
yield varying results. Therefore, where possible, non-parametric tests
are used, i.e. a Kruskal-Wallis ANOVA to test the hypothesis that three
or more samples were drawn from the same population, a Mann-Whitney U-test
for comparing two independent samples and a Wilcoxon matched pairs test
to test single performance indices against zero. For more complicated two-way
designs, data are sufficiently close to being normally distributed to justify
a repeated measures ANOVA whenever within and between group comparisons
need to be carried out.
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