Download Sensory responses and movement-related activities in extrinsic

J Comp Physiol A (1999) 185: 115±129
Ó Springer-Verlag 1999
ORIGINAL PAPER
R. Okada á J. Ikeda á M. Mizunami
Sensory responses and movement-related activities
in extrinsic neurons of the cockroach mushroom bodies
Accepted: 5 May 1999
Abstract We have previously reported that most units
in the input regions of the cockroach mushroom bodies
have activities related to sensory inputs, while the majority of units in the output regions are related to
movements of the animal. In the present study, we were
able to attain a more satisfactory isolation of single units
by using thinner wires and further characterize the activities of units in the mushroom body output regions.
Forty-one units recorded here were classi®ed into three
types: sensory, movement-related, and sensori-motor
units. Di€erent units from each group exhibited a great
variety in activities. Some movement-related and sensori-motor units exhibited activity preceding the onset of
movements. We propose that the mushroom body participates in the integration of a variety of sensory and
motor signals, possibly for initiating and maintaining
motor action. While di€erent neurons displayed a great
diversity of responses, the activities of multiple neurons
recorded simultaneously exhibited similar, but not
identical, responses. These neurons appeared to locate
adjacent to each other and may represent a cluster of
extrinsic neurons that act synergistically to transmit a
speci®c set of mushroom body output signals.
Key words Mushroom body á Periplaneta
americana á Movement-related activity á
Multimodality á Motor control
Abbreviations MB mushroom body á KC Kenyon
cell á U1 unit 1 á U2 unit 2
R. Okada á J. Ikeda á M. Mizunami (&)
Laboratory of Neuro-Cybernetics,
Research Institute for Electronic Science,
Hokkaido University, Sapporo 060±0812, Japan
e-mail: [email protected]
Tel.: +81-11-706-2866; Fax: +81-11-706-4971
Introduction
Mushroom bodies (MBs), prominent neuropils in the
insect brain, are implicated in certain types of insect
behavior, including olfactory learning. In most insects,
the MB consists of four parts, the calyx (input region)
and the pedunculus and a and b lobes (output regions).
In honey bees, cooling of the a lobe of the MB shortly
after one-trial olfactory conditioning led to a decreased
conditioning response in later tests (Erber et al. 1980).
An extrinsic neuron in the pedunculus of the honey bee
MB has been found to change its odor responses transiently as a consequence of olfactory conditioning trials
(Mauelshagen 1993). Fruits-¯ies (Drosophila) in which
MBs were genetically or chemically ablated also
demonstrated de®cits in olfactory learning (Belle and
Heisenberg 1994; Connolly et al. 1996; Davis 1996).
Furthermore, cockroaches (Periplaneta americana)
whose MBs were surgically destroyed performed poorly
in place navigation trials (Mizunami et al. 1998d). In
honey bees, the volume of the MB begins to expand
around the time that the initiation of adult foraging
behavior occurs, and this expansion of MB volume is
thought to have both experience-expectant and experience-dependent components (Withers et al. 1993;
Fahrbach et al. 1997, 1998). Drosophila whose MBs
were genetically ablated were also found to be defective
in sex-speci®c courtship behavior (Hall 1994) and in the
control of locomotor activity (Martin et al. 1998).
The major sensory input into the MB arises from the
olfactory region (i.e., the antennal lobe), but the MB of
honey bees and cockroaches does also receive a variety
of sensory input other than olfaction and is considered
to be a multimodal center. In cockroaches, each MB
contains 200,000 intrinsic neurons (Fig. 1A; Neder
1959), called Kenyon cells (KCs) (Kenyon 1896;
Strausfeld 1976), the dendrites of which receive synaptic
connections from the axon terminals of extrinsic neurons in the calyx (Frontali and Mancini 1970; SchuÈrmann 1974). The majority of these extrinsic (input)
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neurons originate in the antennal lobe of the deutocerebrum (Weiss 1974; Malun et al. 1993), while others
originate in the lateral protocerebrum (Nishikawa et al.
1998; Nishino and Mizunami 1998) or the circumesophageal connective (Yamazaki et al. 1998). These extrinsic (input) neurons convey olfactory (Malun et al.
1993; Li and Strausfeld 1997), antennal mechanosensory
(Zeiner and Tichy 1998), and visual (Nishikawa et al.
1998) signals. KC axons run through the pedunculus
and lobes, where they are organized into a number of
subgroups (Li and Strausfeld 1997; Mizunami et al.
1997, 1998a, b). In the pedunculus and lobes, KC axon
terminals make synaptic connections with dendrites of
the extrinsic neurons (Fig. 1C; Frontali and Mancini
1970). The axons of these extrinsic (output) neurons
project into various areas of the protocerebrum (Li and
Strausfeld 1997). Signals from MBs are thought to be
transmitted, via protocerebral circuits, to descending
premotor neurons which supply the thoracic motor
centers (Strausfeld et al. 1998).
In order to further elucidate the functions of the MB,
we previously designed a technique which makes use of
thin copper wires to extracellularly record activities of
MB neurons in freely moving cockroaches (Mizunami
et al. 1993, 1998c). At the conclusion of recording,
copper ions were iontophoretically injected, often resulting in the partial staining of neurons in the vicinity of
the electrode tip. Successful recordings of unit activities
were obtained from 17 animals in the calyx and from 19
animals in the pedunculus or lobes. Most units recorded
in the calyx exhibited responses to a single modality of
sensory stimuli, and most units recorded in the pedunculus and lobes exhibited activities related to the
movement of the animal.
In this study, we further examined neural activity in
the pedunculus and lobes of the MB of cockroaches
using an improved recording technique. The major improvement to our system is use of thinner wire electrodes, which enable a more satisfactory isolation of
single unit activities and hence a more detailed analysis
of individual units.
Materials and methods
Preparation
All experiments were performed on adult male cockroaches
(P. americana), kept in colonies in the Laboratory of Neuro-Cybernetics, Hokkaido University at 27±29 °C and L:D=14:10 h. The
preparation for and the method of wire recording were slightly
modi®ed from those described previously (Mizunami et al. 1998c).
Each cockroach was anesthetized by cooling on ice. The head capsule was opened to expose the brain, and a small incision was made
on the brain surface to facilitate insertion of the electrodes. Polyurethane-coated copper wires (14, 17 or 20 lm in diameter), with a
coating of 1±2 lm, (Electrisola, Escholzmatt, Switzerland) were
used as electrodes. Four to six wires were formed into bundles by
applying a small amount of melted wax, and the bundles were then
cut to 30±35 cm in length. The diameter of the bundle was typically
40±70 lm. The tip of the bundle of wires was inserted into the MB of
the right hemisphere of the brain by using a micromanipulator. The
opened window was then covered with wax. About 30%, 40% and
30% of the units reported in this study were using wires of 14, 17 and
20 lm in diameter, respectively. For mechanical support, recording
electrodes were formed into bundles around a coated copper wire
with a diameter of 60 lm that was inserted into the thorax and ®xed
to the notum of the insect with wax. The thick copper wire was
grounded. The animal was placed in a cross-shaped arena, the area
of each arm and of the central section being 10 ´ 10 cm. The wall of
the arena was smeared with Vaseline to prevent escape. Each animal
had been kept in a duplicate arena for 1 or 2 days before the experiment for the purpose of familiarization.
Recording of neural activity and monitoring
of the animal's movement
After full recovery from anesthesia, the cockroaches exhibited long
sequences of grooming, and then began to explore the surroundings. Any animal that failed to show spontaneous locomotion,
grooming, and escape from tactile or wind stimuli applied to their
cerci was not used for unit recordings. To record unit activities, all
of the four to six wires were connected to a di€erential a.c. ampli®er (DAM 80, WPI, Sarasota, Fla.) in pairs of any combinations. Since the distance between the tips of the two electrodes was
very small (typically 3±40 lm) only the electrical signals generated
in close vicinity to the electrode tips could be detected. Indeed, all
units observed in this study were detected from only some speci®c
pairs of wires and, by comparing the amplitudes of units recorded
from the di€erent pairs, it was possible to determine which particular wire best contributed to the detection of the signal and,
therefore, was likely to be the closest to the source of the signal.
The wire thought to be the closest was used for copper impregnation (see below) after the conclusion of the recording. The electrical
signals were passed to a band-pass ®lter at 200±10,000 Hz and
stored in an audio channel of an 8-mm video tape. A window
discriminator (Nihon Kohden, Tokyo, Japan) was used to isolate
the activity of a single unit (i.e., to isolate the spike activity of a
single neuron). Isolation of a single unit was facilitated by observing the waveform of the spikes on a digital oscilloscope. The
behavior of the insect was monitored using a CCD camera, and the
images were stored on a video channel of 8-mm video tape at a rate
of 30 frames/s. Major features of the animal's behavior were recorded on an audio channel.
Sensory stimuli and observation of behavior
Visual, mechanosensory (tactile and air current), and olfactory
stimuli were routinely applied. The compound eyes were illuminated at 2000±2700 lx by a miniature bulb, with a background
luminance of 240 lx. For visual motion stimulation, the point
source, a black-and-white grating or a black spot, was manually
moved. For tactile stimulation, each antenna, leg or cercus was
gently touched with a stick. A current of air was applied to each
antenna or cercus for about 1 s from a delivery tube, the tip of
which was positioned about 1 cm from the antenna or the cercus.
For olfactory stimulation, a current of air was delivered to each
antenna for about 1 s from a syringe containing ®lter papers
soaked with an extract of orange or banana. As a control, a current
of air from a syringe containing no ®lter paper was also applied.
Each stimulus was applied three to ®ve times with an interval of at
least 20 s between applications. We noted large variations in the
responses to these stimuli, possibly due to the fact that the cockroaches moved their antennae spontaneously as well as in response
to stimulation and, thus, the parameters of the stimulus could not
be kept constant among trials. We judged a unit to have responded
to olfactory stimulation when a signi®cant di€erence in the spike
rate was detected between the responses to test and control stimuli.
Due to the small number of trials and large variation in the responses, many units whose olfactory response was only weak or
moderate may have been rejected.
The types of behavior observed included forward locomotion,
rotation, turning (forward locomotion accompanying a rotation),
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cleaning of each antenna or leg by licking, touching the ground
with antennae (antennation), swinging the antennae in the air, and
head or body ¯exion. Rightward and leftward rotations are referred to as ipsi- and contralateral rotations, respectively, since all
recordings were made from the right MB. Similarly, right and left
appendages are referred to as ipsi- and contralateral appendages,
respectively.
Marking of the recording site
When the examination of unit activity, which lasted for 15±60 min,
was concluded, copper ions were released from the recording
electrode by passing a positive current through the electrode.
Typically, a positive current of 20±40 nA was applied for 15±20 s.
The released copper ions were precipitated by ammonium sul®de.
The head of the cockroach was then isolated, and the brain was
pre®xed in 3% paraformaldehyde in cockroach saline for 30 min,
dissected out and then post-®xed in Bouin's solution for 30 min.
The tissue was dehydrated in a graded series of ethanol, treated
with propylenoxide, and intensi®ed with silver according to Bacon
and Altman (1977). Subsequently, the specimens were dehydrated
and were embedded in soft Araldite (Nisshin EM, Tokyo, Japan),
sectioned at 30±36 lm, and observed under a Nomarski interference or a conventional light microscope. The typical size of the
copper-silver deposits was only 50±70 lm in diameter (Fig. 2), thus
allowing for precise determination of the location of the
electrode tip.
Results
Extracellular recordings of MB units
in freely moving cockroaches
Among more than 250 cockroaches tested, over 15 min
of stable recordings of unit activities were obtained from
about 100 cockroaches. In 31 animals, the copper-silver
deposits were con®ned within the pedunculus or the
lobes of the MB (see Fig. 2), suggesting that the recorded electrical activities were from MB neurons. In
most of these 31 animals, several units were recorded
simultaneously, and single unit activities were separated
using a window discriminator. Here, we describe 41
units recorded from these 31 animals; from 1 of which 3
units that exhibited di€erent responses were isolated;
and from 8 of which, 2 units with di€erent responses
were isolated.
Identi®cation of recorded neurons
Several lines of evidence suggest that units recorded in
the pedunculus and lobes of the MB are, in large part,
from e€erent (output) neurons whose axons terminate in
various protocerebral regions (see Mizunami et al.
1998c). First, it must be stated that among the two types
of cells which make up the pedunculus and lobes, i.e.,
intrinsic neurons (KCs, Fig. 1A) and extrinsic neurons
(Frontali and Mancini 1970), the present recordings
were no doubt taken from the extrinsic neurons. It is
unlikely that the spike activities of KCs, the axons of
which are typically less than 0.5 lm in diameter (Iwasaki
et al. 1999), could be detected. In contrast, the axons of
extrinsic neurons are thick, often exceeding 7 lm
(Mizunami et al. 1997). Second, the majority of extrinsic
neurons in the pedunculus and lobes are output (e€erent) neurons (Fig. 1C), although some are thought to be
input (a€erent) neurons (Li and Strausfeld 1997). Third,
copper-impregnation often resulted in an uptake of
copper ions by neurons in the vicinity of the electrode
(Fig. 1B) and the pro®les of the copper-impregnated
neurons could be matched to those of output neurons
stained by the Golgi method (Mizunami et al. 1998a, b)
and by intracellular Lucifer yellow ®lls (Fig. 1C;
H. Nishino and M. Mizunami, unpublished data). The
relation between the neural activity and pro®les of the
copper-impregnated neurons has been examined in detail in our previous study (Mizunami et al. 1998c).
Classi®cation of recorded units
We classi®ed the 41 units isolated in this study into three
types according to sensory responses and activities
during the animal's movement. The ®rst type were (exteroceptive) sensory units that responded to olfactory,
mechanosensory and/or visual stimuli. The second type
were movement-related units, the activities of which
were related to the animal's movements and not to external sensory stimuli. The third type were sensori-motor
units that responded to external sensory stimuli and also
exhibited movement-related activity. When activity
during motion had a similar temporal pattern to a response to imposed sensory stimuli, we regarded the activity as response to external sensory stimuli that the
animal had received during motion (sensory rea€erence).
We were unable to ®nd any di€erences in the distribution between the three types of neurons (Fig. 3). About
50% of these units exhibited activities similar to those
recorded in the pedunculus and lobes in our previous
study (Mizunami et al. 1998c), and the remaining 50%
exhibited novel features. In this study, we will focus on
the latter units, although a few of the former units are
also described to demonstrate the reproducibility of
some of the major observations of our previous study.
Sensory units
Fifteen units responded to sensory stimuli and exhibited
no activities related to the animal's movement. Activities
during movement, if they occurred, could be explained
by sensory rea€erence. Four of these units exhibited
responses to at least two sensory modalities, while the
remaining units responded unimodally to the stimuli
tested. The majority of mechanosensory units were
found to be multisegmental; i.e., they responded to
stimuli applied to various segments of the body.
Figure 4A shows an example of a sensory unit that
exhibited multisegmental response to mechanosensory
stimulations. There were responses to tactile or air-current stimulus applied to the ipsilateral appendages (right
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antenna and legs) but not to contralateral (left) appendages. The unit showed a spontaneous spike discharge of 4±5 spikes s)1, and the spike rate increased
when tactile stimuli were applied to the ipsilateral antenna (Fig. 4A-a), hindleg (Fig. 4A-e) or midleg (not
shown), and when currents of air were applied to the
ipsilateral antenna (Fig. 4A-c). In contrast, there was no
response to tactile stimuli applied to the contralateral
antenna (Fig. 4A-b), foreleg (Fig. 4A-f), or cercus (not
shown), or to currents of air applied to the contralateral
119
b
Fig. 1 A Morphology of an intrinsic neuron (Kenyon cell) of the
mushroom body of the cockroach, reconstructed from serial frontal
sections of a Golgi-impregnated brain (modi®ed from Mizunami et al.
1997). B A photograph showing a group of copper-impregnated
extrinsic neurons at the tip of the recording electrode. The cell bodies
(arrows) of these neurons form a cluster ventrally to the pedunculuslobes junction. C Morphology of a Lucifer yellow-®lled extrinsic
neuron of the b lobe. The neuron has dendrite-like arborizations in the
lobe and its axon projects to the lateral horn (LH) and inferior lateral
protocerebrum. P pedunculus, LC lateral calyx, MC medial calyx, a a
lobe, b b lobe, AL antennal lobe, OL optic lobe. Scales: 100 lm in A
and C; 50 lm in B
antenna (Fig. 4A-d). This unit exhibited no response to
olfactory stimulation by banana or apple odor, nor to
illumination of the compound eyes.
Figure 4B shows a sensory unit that responded to
mechanosensory, olfactory and visual stimuli. This
multimodal unit exhibited a spontaneous spike discharge and the spike rate increased when currents of air
were applied to the ipsi- and the contralateral antenna
(Fig. 4B-a, b) and cerci (Fig. 4B-e, f), when orange
(Fig. 4B-c) or banana (Fig. 4B-d) odor was applied to
the ipsilateral antenna, and when light stimulus was
applied to the contralateral compound eye (Fig. 4B-g).
No response was observed to currents of air applied to
the contralateral cercus, to light stimulation of the
ipsilateral compound eye, or to tactile stimuli applied
to the ipsi- or contralateral antenna, legs, or cerci (not
shown). A notable feature of this unit was that the
latency of the response di€ered for di€erent sensory
stimuli, and to evaluate this latency, the time at which
the spike rate reached 70% of the peak rate from the
onset of stimulation was measured from a peri-stimulus
Fig. 3 A diagram showing the locations of 41 units recorded in the
present study. These units were recorded in the ventral half of the
output neuropil and were classi®ed into sensory units (rectangles,
indicated as SU), movement-related units (triangles, indicated as MU)
and sensori-motor units (circles, SMU). We were unable to ®nd any
di€erences in the distribution between the three types of neurons.
Preparatory activities, i.e., activities preceding the onset of movements, were found from one SMU in the a lobe, one MU in the
b lobe, and one SMU in the pedunculus-lobe junction. The dorsal side
is at the top. LC lateral calyx, MC medial calyx, P pedunculus, a a
lobe, b b lobe
Fig. 2 Copper-silver deposits (arrows) indicating the location of the
tip of the recording electrode, observed under a Nomarski interference
microscope. The deposits are seen in the pedunculus-lobes junction. lc
lateral calyx, mc medial calyx, p pedunculus, b b lobe. Scale: 100 lm
time histogram (bin width: 100 ms). The latency was
1000±1100 ms for the response to banana odor
(Fig. 4B-d). This unusually long latency can be explained if the response was formed by a combination of
a phasic inhibitory component and a tonic excitatory
component. The latency was 300±400 ms for the response to orange odor (Fig. 4B-c), 200±300 ms for the
response to currents of air applied to the ipsilateral
antenna (Fig. 4B-b), and 100±200 ms for the response
to currents of air applied to the ipsi- (Fig. 4B-e) and
contralateral (Fig. 4B-f) cerci and to the contralateral
antenna (Fig. 4B-a).
The unit shown in Fig. 4C exhibited a response when
the compound eyes were illuminated antero-dorsally.
This unit exhibited a high-frequency spontaneous discharge, which was suppressed at the onset of illumination of antero-dorsal parts of the compound eyes with a
latency of 0.2±0.3 s for 1±2 s. We were unable to determine whether the response was derived from the ipsior contralateral compound eye, or from both. This unit
did not respond to tactile stimulation of the ipsi- or
contralateral antenna, leg, or cercus, to currents of air
applied to the cerci, or to banana odor applied to either
of the antennae.
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Fig. 4A±C Three examples of sensory units. A Activities of a
multisegmental mechanosensory unit, recorded in the antero-medial
ventral region of the a lobe. The top trace is the original record, and
lower traces are unit activities isolated using a window discriminator.
The ®ring rate of the unit increased in response to air-current or tactile
stimulation of the ipsilateral antenna or legs (a, c and e), but
stimulation of contralateral appendages was ine€ective (b, d and f).
B Activities of a multimodal unit, recorded at the base of the a lobe.
The unit responded to air-current stimulation of the ipsi- (b) or
contralateral (a) antenna and to the ipsi- (e) or contralateral (f) cercus.
The unit also responded to olfactory stimulation by orange (c) or
banana (d) odor applied to the ipsilateral antenna and to light
stimulation of the contralateral compound eye (g). Note that the
latency of the response di€ered for di€erent sensory stimulations. C A
unit recorded at the lateral side of the pedunculus-lobes junction,
whose spontaneous spike discharge was inhibited when the compound
eyes were frontally illuminated. Scales: 5 s (A, C); 1 s (B, a±d); 2 s
(B, e±g)
Movement-related units
Twelve units exhibited activities associated with movement of the animal while exhibiting no response to the
sensory stimuli tested. The activities of 8 of these units
were thought to be due to signals from proprioceptors
which monitor the animal's posture or movement, but
the activities of the remaining units could not be explained in this way. These units were: a unit whose spike
activities changed depending on the direction of locomotion; a unit that showed long-lasting depression after
termination of locomotory movement; a unit that began
spike discharge prior to the onset of locomotion; and a
unit whose movement-related activities changed with
time during the recording (see Fig. 11).
Figure 5 shows examples of movement-related units
whose activities were best explained by signals from
proprioceptors monitoring the movement of forelegs.
The single-unit activities in this recording could not be
reliably isolated. The units exhibited a low frequency of
spontaneous spike discharge and generated a burst of
spikes when the animal rotated contralaterally (to the
left) (Fig. 5d, e). These units also ®red when the insect
moved the contralateral foreleg before licking the contralateral antenna (Fig. 5a, b, d, e) or before and after
licking the contralateral hindleg (Fig. 5c, f). These units
exhibited no spikes while the animal was licking an antenna or hindleg if its forelegs were not moving.
Figure 6 shows a movement-related unit whose activities could not be explained by proprioceptive signals.
121
Fig. 5a±f Units that were active during movement of the ipsi- or
contralateral foreleg, recorded at the pedunculus-lobes junction. In
this record, single-unit activities could not be reliably isolated.
Records d, e and f correspond to motor episodes a, b and c,
respectively. Numbers in each trace correspond to those in each
episode. The top trace in d is the original record, and other traces are
unit activities isolated using a window-discriminator. The units ®red
when the animal rotated contralaterally (1±2 in d; 1±2, 2±3 in e), and
the animal moved the contralateral foreleg before licking the
contralateral (left) antenna (2±3 in d; 3±4 in e) or before and after
licking the contralateral hindleg (f)
This unit exhibited little spontaneous discharge and
generated sporadic bursts of spikes during contralateral
(leftward) rotation and forward locomotion but not
during ipsilateral (rightward) rotation (Fig. 6c, d). Although locomotory actions accompanied movements of
the antennae, neck, and legs, no correlation between the
®ring and the position or movement of each body part
was found. Moreover, the movement of these body parts
did not elicit ®ring when they occurred in isolation. It is
therefore dicult to explain the activities of this unit by
signals of proprioceptors that monitor posture or
movement of the leg or the body. We concluded that the
activities of this unit were related to instructions concerning direction of movement; i.e., this unit may receive
an e€erence copy of motor instructions if not itself involved in the process of forming motor instructions (see
Discussion).
Figure 7 shows units that exhibited a long-lasting
depression in spike activity after the termination of locomotory movement. The single-unit activities in this
recording could not be reliably isolated. The units
Fig. 6 A unit that ®red during forward motion (1±2, 3±4 in c; 1±2, 4±
5 in d) and contralateral turn (2±3 in c; 5±6 in d) but not during
ipsilateral turn (4±5 in c; 3±4 in d) and ipsilateral rotation (5±6, 6±7 in
c; 2±3 in d). The unit was recorded at the pedunculus-lobes junction.
Records c and d correspond to episodes a and b, respectively
Fig. 7 Units that exhibited motor-after e€ects, recorded at the
pedunculus-lobes junction. Single-unit activities could not be reliably
isolated. The averaged spike frequencies before, during, and after ®ve
locomotory episodes (contralateral rotation, ipsilateral turn, ipsilateral
rotation, ipsilateral rotation followed by forward motion, contralateral turn followed by forward motion and contralateral rotation) are
shown as histograms. The averaged spike frequencies before and
during ®ve locomotory episodes were 4.1 and 3.5 spikes s)1,
respectively. The spike frequency remained low for several seconds
after termination of the movements
122
Fig. 8a±d A unit whose spike activities preceded the onset of
locomotion, recorded in the b lobe. Records c and d correspond to
episodes a and b, respectively. c The unit began to ®re about 500 ms
before the onset (1) of forward locomotion. The spike discharge was
maintained throughout the forward movement (1±2). d The unit
started to ®re 900 ms before the onset (1) of the contralateral turn.
The unit generated sporadic bursts of spikes during the contralateral
turn (1±2) and also during the subsequent ipsilateral turn (2±3) and
forward locomotion (3±4)
exhibited a discharge of about 4.1 spikes s)1 when the
animal was stationary, and the ®ring rate decreased to
3.5 Hz during spontaneously induced locomotory
movement. The ®ring rate remained below 3.5 Hz for
about 8 s after the termination of movement and returned to the resting level after 10±15 s.
Figure 8 shows a unit that exhibited spike activities
prior to the onset of locomotion. This unit rarely exhibited spikes when the insect was stationary but began
to generate spike discharge 500±1000 ms prior to the
onset of forward motion (1 in Fig. 8c) or a left turn (1±2
in Fig. 8d). The spike activity continued during locomotion (1±2 in Fig. 8c), although the discharge was often sporadic (1±4 in Fig. 8d). Fig. 8d shows that the
highest spike frequency occurred prior to the onset of
locomotion, not during locomotion.
Sensori-motor units
Fourteen units exhibited both sensory responses and
movement-related activities. The activities of these units
during movement could not be explained by sensory
rea€erence. The sensory responses of 4 of these units
were multimodal, while the remaining 10 units responded unimodally to the stimuli tested. Two of these
units exhibited activities prior to the onset of locomotion.
Figure 9 shows a sensori-motor unit that responded
to mechanosensory stimuli applied to the ipsi- and
contralateral antenna and also exhibited activities associated with the movement of the ipsilateral antenna. This
unit generated a spontaneous spike discharge and
Fig. 9a±g A unit that was active when the animal moved its
ipsilateral antenna and also in response to mechanosensory stimulation of each of the antennae, recorded at the pedunculus-lobes
junction. Record c corresponds to episode a; Records d and e
correspond to episode b. This unit ®red when the animal moved its
ipsilateral antenna to the posterior (1±2 in c) but not when it moved its
antenna to the anterior (2±3 in c). This unit also ®red in response to
tactile or air-current stimulation of the ipsi- (e, g) or contralateral (d, f)
antenna regardless of the direction of stimulation
exhibited an increase in ®ring rate when the animal
spontaneously moved the ipsilateral antenna to the
posterior, but not during spontaneous antennal motion
to the anterior (Fig. 9c). Tactile and air-current stimulation of the antennae produced an increase in the spike
rate, regardless of the direction of the stimulation
(Fig. 9d, e). The unit did not respond to tactile stimulation of the ipsilateral foreleg, midleg, hindleg, cercus, or
to air-current stimulation of either of the cerci. Stimulation with both banana and orange odor was also found
to be ine€ective. Further examples of sensori-motor unit
activity will be shown in the following section.
Activities of simultaneously recorded units
When two or more units were recorded simultaneously,
they usually exhibited similar responses; however, detailed observation of these units showed that their
activities were not the same.
Figure 10 shows a pair of sensori-motor units, which
we refer to as unit 1 (U1) and unit 2 (U2), recorded
simultaneously from the junction between the a and b
123
Fig. 10a±d A pair of sensori-motor units [unit 1 (U1) and unit 2 (U2)]
recorded simultaneously at the dorsal area of the junction between the
a and the b lobes. a±c Both units exhibited an increase in the spike rate
during forward locomotion (a and b) and contralateral rotation (c).
d Peri-stimulus time histograms showing responses of U1 (upper ones)
and U2 (lower ones) to illumination of the compound eyes.
Illumination of the contralateral compound eye produced a phasic
and prominent response in U2 (lower left) and a tonic and much less
prominent response in U1 (upper left). Both units exhibited phasic and
prominent responses to illumination of the ipsilateral compound eye
lobes. They exhibited irregular bursts of spontaneous
spike discharge, the ®ring rate of which increased during
forward locomotion (Fig. 10a, b) and contralateral rotation (Fig. 10c). When the contralateral compound eye
was illuminated, U2 exhibited a phasic and prominent
response and U1 exhibited a tonic and less prominent
response (Fig. 10d), while both units exhibited a phasic
response to illumination of the ipsilateral compound
eye. Neither of these units responded to tactile stimulation of the ipsi- or contralateral antenna, midlegs, hindlegs, the ipsilateral foreleg, the contralateral cercus, or to
air-current stimulation of the antennae or cerci. Stimulation with banana and orange odor was ine€ective.
A more prominent di€erence in the ®ring pattern was
observed between the pair of simultaneously recorded
units, U1 and U2, shown in Fig. 11. Both units were
active when the animal ¯exed its body, and U1 was
classi®ed as a sensori-motor unit and U2 as a movement-related unit. The ®ring rate of U1 increased when
the animal bent its body for cleaning (grooming) its
ipsilateral foreleg, midleg or hindleg, while U2 exhibited
spike activities only when the animal ¯exed its body for
cleaning the ipsilateral hindleg. Notably, the spike activities of U2 were observed on only two of the four
occasions that it cleaned its ipsilateral hindleg. This indicates that the activities of U2 do not simply re¯ect
signals from proprioceptors monitoring the ¯ex of the
thorax or the abdomen (Fig. 11e±h). Tactile stimuli
applied to the ipsi- and contralateral antenna produced
responses in U1 but not in U2 (Fig. 11i, j). Neither of
these units responded to tactile stimuli applied to midlegs, hindlegs, or cerci or to air currents applied to the
antennae or cerci. Odor and visual stimulations were
ine€ective. These units did not exhibit a change in ®ring
rate during forward movement or during ipsi- or contralateral rotation.
The ®nal example of a pair of simultaneously recorded units is shown in Fig. 12. Both units responded
to multimodal sensory stimuli and were also active
during locomotion, and activity preceding the onset of
movement was observed in U2 but not in U1. These
sensori-motor units exhibited spontaneous spike discharge, the frequency of which largely increased during
rotation toward the ipsilateral side (Fig. 12d, e) and
slightly increased during rotation toward the contralateral side (Fig. 12f). One critical di€erence between these
units is that U2 exhibited an increase in the ®ring rate
100±400 ms before the onset of ipsi- or contralateral
rotation, whereas U1 exhibited an increase in the spike
rate only after the onset of the rotation (Fig. 12d±f). The
®ring rate of these units increased in response to tactile
stimulation of the antennae, air-current stimulation of
the antennae and cerci (Fig. 12g±i), and illumination of
the compound eyes (data not shown).
Summary of the sensory properties of MB units
In this section, the observed sensory properties of neurons in the pedunculus or lobes (output regions of the
MB) are compared to those of the possible input neurons
of the calyx (input region) reported in our previous study
(Mizunami et al. 1998c). The percentages of 29 sensory
and sensori-motor units that exhibited responses to each
of the mechanosensory (tactile or air current), olfactory
or visual stimuli are shown in Fig. 13A. Twenty-®ve
(86%) of the units exhibited antennal mechanosensory
response, and the majority (14) of these 25 exhibited
124
Fig. 11a±k A pair of sensori-motor (U1) and movement-related (U2)
units recorded simultaneously from the b lobe. Traces e, f, g and h
correspond to episodes a, b, c and d respectively. Numbers in a, b, c
and d correspond to those in e, f, g and h, respectively. U1 exhibited
an increase in the spike rate when the animal ¯exed its body for
cleaning (grooming) the ipsilateral foreleg (a), midleg (b) or hindleg (f,
h, i). U2 exhibited an increase in the spike rate when the animal ¯exed
its body for cleaning the ipsilateral hindleg (e, f). Notably, an increase
in the spike rate of U2 was observed in only two out of four episodes
of ipsilateral hindleg cleaning (e±f). U1 responded to tactile
stimulation of the contra- (i) or ipsilateral (j) antenna, but U2 did
not. Scales: 2 s for d±h; 1 s for i and j
responses both to tactile and air-current stimulation.
Twelve units (41%) exhibited cercal mechanosensory
response, the majority (8) of which responded only to
air-current stimulation. Four units responded to tactile
stimulation of the legs, and 11 to light stimuli. Responses
to orange or banana odor were found in only 2 units. It is
puzzling that responses to olfactory signals are highly
underrepresented in our samples despite the fact that the
calyces receive massive olfactory inputs (Weiss 1974;
Malun et al. 1993). This is possibly because, ®rstly, our
experimental procedure was not e€ective in detecting
weak or moderate olfactory responses (see Materials and
methods), secondly, we tested only two kinds of odor,
and thirdly, our samples were small.
In Fig. 13B, the percentages of sensory units (n = 15)
and sensori-motor units (n=14) that responded to either
tactile, air-current, olfactory or visual stimulation are
compared with those of 14 possible input neurons of the
calyx recorded in our previous study (Mizunami et al.
1998c). This graph shows that the percentages of both
types of units that responded to each of the sensory
stimuli in this study is similar to those of the possible
input neurons of the calyx recorded previously.
Discussion
Advantages and disadvantages
of extracellular recording
In vertebrates, extracellular recording by implanting
coated wires has been widely used to study sensory and
motor correlates in the neural activity of various brain
areas of freely moving animals (e.g., toads: Ewerts 1980;
rats: Ranck 1973; Sharp and Green 1994). We have
shown that a similar recording technique is applicable to
much smaller animals, i.e., cockroaches (Mizunami
et al. 1993, 1998c). Although extracellular recording
does not usually enable identi®cation of the recorded
neurons, the cell type of the recorded neurons can often
be deduced when the recording is made in a highly
structured neuropil that consists of a small number of
cell types, such as the MB (see Results). Moreover,
partial staining of neurons by copper impregnation from
the electrode tip facilitates identi®cation of the recorded
neurons (see Mizunami et al. 1998c).
Recording of neural activity in freely moving animals
is a very e€ective method for clarifying the variety of
their sensory and motor correlates. However, recording
from freely moving animals is limited in its use in the
quantitative analysis of neural activities, because it is
125
Fig. 12a±i A pair of sensori-motor units (U1 and U2) recorded
simultaneously at the ventral part of the pedunculus-lobes junction.
Records d, e and f correspond to episodes a, b and c, respectively.
These units exhibited a prominent increase in the spike frequency
during ipsilateral rotation (d, e) and a less prominent increase during
contralateral rotation (f). U1 exhibited an increase in spike frequency
100±400 ms before the onset of rotation, but U2 exhibited no change
in spike activity prior to the onset of rotation. Both units responded to
tactile stimulation of the contralateral antenna (g) and to currents of
air applied to the ipsilateral antenna (h) and contralateral cercus (i)
dicult to repeatedly apply exactly the same sensory
stimuli and to repeatedly induce exactly the same
movements. A complementary study using a carefully
designed behavioral paradigm, in which a particular
movement of the animal can be repeatedly induced,
would aid in con®rming the observations of freely
moving animals made in this study.
Recordings from MB neurons
from freely moving cockroaches
In our earlier work (Mizunami et al. 1998c), most units
recorded in the pedunculus or lobes exhibited movement-related activities. Examples of these are: units
whose activities were explained by signals from proprioceptors in the legs; units whose activities changed
depending on the direction of locomotion; and units
whose activities preceded the initiation of locomotion.
In the present study, we further characterized the
activities of units in the MB output regions using thinner
wires that enabled better isolation of single units and,
hence, more detailed characterization of individual
neurons. We isolated 41 units from 31 animals. The
majority of these units are most probably from output
neurons whose axons exit the MB and project to
protocerebral neuropils (see Results). It is likely,
however, that some of the units recorded in this study
are from the presumed input neurons reported by Li and
Strausfeld (1997).
About half units recorded in the present study were
similar to those we reported previously, con®rming the
validity of our earlier observations. The other half
demonstrated novel features of (presumed) extrinsic
neurons. These features include: (1) discrimination between mechanosensory stimulations of left and right
appendages (Fig. 4A), (2) exhibition of motor after-effects (Fig. 7), (3) complex integration of external sensory
signals with signals concerned with the motion of the
animal (Fig. 9), and (4) changes in movement-related
activities with time during the recording (Fig. 11).
The possibility that some of the units recorded in this
study might have been from neurons outside the MB can
not easily be ruled out, even though the tips of the recording electrodes were located well within the MB and
the di€erential recording from these electrodes, the typical distance between the tips of which was only 3±
40 lm, assured that signals from remote sources were
not detectable. The neuropil surrounding the lobes is a
major termination area of the MB output neurons and
in where these neurons interact with local protocerebral
neurons (Strausfeld et al. 1998). It would be of great
interest to compare the activity of the neurons in this
higher protocerebral neuropil with that recorded from
the MB in the present study.
The main conclusion of the present study is that extrinsic neurons of the MB exhibit an unexpected degree
of diversity in their activities. This diversity can be interpretated in two ways. First, it may indicate that the
MB integrates diverse external sensory signals and internal motor-reporting signals, possibly to enable the
appropriate execution of motor action. In this respect,
the activities of MB neurons observed in this study may
be comparable to those in the higher motor areas of the
mammalian cortex which are known to exhibit a huge
variety of sensory and/or movement-related responses
126
e.g., sensory integration, memory, or motor control, is
the result of interaction of multiple brain areas, including
the MB. It is likely that these functions are so closely
interrelated with each other that they can not be allocated
to separate networks. The recent ®nding that both the
antennal lobe and the calyx of the MB may participate in
olfactory memory consolidation (Hammer and Menzel
1998) leads support to this hypothesis. In the following
sections, we will discuss the signi®cance of the present
observations of MB neurons, with an emphasis on their
possible roles in the control of locomotory movement.
Three classes of extrinsic MB neurons
Fig. 13A, B Summary of sensory properties of units recorded in the
present study. A The percentages of 29 sensory and sensori-motor
units that exhibited responses to each of the olfactory (olf), visual (vis),
and mechanosensory stimulation of the antenna (ant), cerci (cer), or
legs are shown. Units that responded to more than two categories of
sensory stimulations are counted repeatedly. Twenty-®ve (86%) of
these units responded to tactile or air current stimulation of the
antennae. ipsi, cont or bi indicates units that exhibited response to light
stimulation of the ipsi- or contralateral compound eye, or both. front
indicates units that exhibited response to frontal light stimulation; we
were unable to determine if the response was derived from the ipsi- or
contralateral compound eye, or from both. B The percentages of
sensory units (indicated as SU, n=15) and sensori-motor units
(indicated as SMU, n=14) that responded to each category of sensory
stimulation are compared to those of 14 units recorded in the calyx in
our previous study (Mizunami et al. 1998c)
(e.g., Alexander and Crutcher 1990a, b; Shen and
Alexander 1997). Second, the diversity of extrinsic neuron activities may support our previous suggestion that
the MB participates in multiple functions (Mizunami
et al. 1998c). This is to be expected if the insect brain is
not organized strictly hierarchically but with parallel and
distributed features so that a particular brain function,
The 41 units isolated in the present study were classi®ed
into three groups: sensory units (n=15, 37%), movement-related units (n=12, 29%) and sensori-motor units
(n=14, 34%). In a previous study (Mizunami et al.
1998c), we reported that the majority (13 of 19, 68%) of
units recorded in the MB output neuropil were movement-related, only 1 (5%) was sensory and 5 (26%)
sensori-motor. The exact reason why very few units that
responded to external sensory stimuli were encountered
in our previous study is not known. It may simply be due
to the small size of the sample.
The present results showing that a large majority
(71%) of units in the MB output neuropil exhibit sensory responses con®rms the suggestion that the MB
participates in sensory integration (Homberg 1984;
Schildberger 1984; Li and Strausfeld 1997), in addition
to other functions such as participation in associative
memory (Erber et al. 1980; Belle and Heisenberg 1994;
Davis 1996; Mizunami et al. 1998d) and the control of
behavior (Huber 1960; Hall 1994; Mizunami et al.
1998c; Martin et al. 1998). This sensory function may
include (1) the integration of multimodal sensory signals, as is indicated by the presence of multimodal units
(Fig. 4B), (2) the integration of signals received by the
left and right sensory organs (Figs. 4B, 9, 10), and (3)
the representation of somatosensory signals for the
whole body, as is exempli®ed by the presence of the
multisegmental mechanosensory unit which discriminates between stimuli applied to the left and right appendages (Fig. 4A).
One-third (34%) of the recorded units were sensorimotor units that exhibited sensory as well as movementrelated responses. Of course, the classi®cation of units in
the present study is based on their responses to a limited
range of sensory stimuli and simple movements; thus, it
is likely that some of the units described here as sensory
or motor-related units might also exhibit motor-related
activities or sensory responses if a much wider variety of
sensori stimuli were used and if we observed a much
wider variety of behavior. The roles of sensori-motor
neurons may be to discriminate between external sensory stimuli and stimuli evoked by movements of the
self (sensory rea€erence), as is exempli®ed by the unit
shown in Fig. 9, and to integrate motor actions with
127
sensory perception. The manner in which integrated
sensori-motor signals are utilized in the control of behavior would make for an interesting study.
Coding of exteroceptive and proprioceptive signals
in the MBs
The 17 units recorded in the calyx (input region) of
cockroach MBs in our previous study (Mizunami et al.
1998c) were categorized into ``exteroceptive'' sensory
units (n = 14) and ``proprioceptive'' units (n = 3). The
exteroceptive units responded to either visual, olfactory,
or mechanosensory stimuli, and the proprioceptive units
appeared to monitor the animal's movement by receiving signals from proprioceptors. The sensory and sensori-motor units recorded in the MB output regions in
the present study engaged in activities similar to the
above mentioned exteroceptive units (Fig. 13B). It is,
therefore, likely that signals are sent from the ``exteroceptive'' input neurons of the calyx, via KCs, to ``sensory'' and ``sensori-motor'' output neurons. Similarly, it
is likely that signals from ``proprioceptive'' input neurons of the calyx, via other KCs, to the ``movementrelated'' and ``sensori-motor'' output neurons. It has yet
to be determined whether there are di€erent populations
of KCs that speci®cally code for exteroceptive and
proprioceptive signals.
Possible roles of MB extrinsic neurons
in motor control
Some units among the movement-related units were
particularly notable because their activities changed
depending on the direction of locomotion (Fig. 5B).
Their directionally selective activities could not be explained as the reception of signals from proprioceptors.
In some insects, similar directionally selective activities
have been observed from descending neurons that
transmit motor instructions related to the ``turning''
movements to the thoracic motor circuits (moths:
Olberg 1983; Kanzaki et al. 1994; crickets: BoÈhm and
Schildberger 1992; HoÈrner 1992). Thus, it is likely that
the activities of these MB units re¯ect an e€erence copy
of motor instruction from descending premotor neurons, or from neurons of the motor center of the thoracic ganglia. The e€erence copy is thought to be
feedback, because output signals from the MBs are ®rst
transmitted to various protocerebral areas (Li and
Strausfeld 1997) and are then thought to be transmitted
to the premotor neuropil (e.g., posterior slope) from
which the descending neurons originate (Strausfeld et al.
1998). Alternatively, it might be that directionally selective extrinsic neurons are directly involved in the
process of forming motor instruction and that the instruction is sent to the premotor neuropil and triggers
the activities of the descending neurons. This possibility,
however, seems less likely as the protocerebral circuits
intervene between the output neurons of the MB and the
premotor descending neurons.
Other motor-associated units observed in the present
study that are worthy of special notice are those exhibiting activities for 100±1000 ms preceding the onset of
locomotion (Figs. 7, 12). A possible interpretation of
these activities is that they re¯ect e€erence copy from
descending neurons, since in some insects some descending neurons are known to exhibit activities preceding the onset of locomotion (see below). This
possibility, however, is unlikely since the onset of activities of these descending neurons precedes the onset of
locomotion for only 20±200 ms. In crickets, some descending premotor neurons initiate spike activity 20±
200 ms before the onset of walking (HoÈrner 1992; BoÈhm
and Schildberger 1992), while in grasshoppers, some
descending neurons begin spike discharge for up to
150 ms in advance of the onset of stridulatory movement (Hedwig 1994). Also, the descending neurons in
locusts begin activities 22±60 ms in advance of ¯ight
motor activity (Homberg 1994). Thus, we conclude that
the activities of extrinsic neurons of the MB far precede
the onset of the activities of descending neurons. In
mammals, extracellular recordings showed that neurons
in various motor-associated areas of the cerebral cortex
begin discharge in advance of the onset of voluntary
(self-induced) movement of the arm or hand (Tanji and
Evarts 1976, Alexander and Crutcher 1990a). These
neurons are considered to participate in the preparation
for voluntary movement (Tanji and Evarts 1976, Alexander and Crutcher 1990a, Shepherd 1994). We suggest
that motor-preceding activities in the cockroach MB are
similarly related to the preparatory processes for determination of locomotory behavior and that the preparatory signals of the MB are translated into actual motor
instructions as they are passed to the premotor neuropil,
via the protocerebral circuits. This suggestion accords
with earlier suggestions based on electric stimulation
studies in crickets and grasshoppers that it is the MBs
that determine the beginning and duration of stridulatory movement and that the decision made in the MB is
sent to the lower motor centers and then translated into
actual motor patterns (Huber 1960; Otto 1971; Wadepuhl 1983).
We have previously seen that cockroaches with bilateral MBs surgically ablated could still spontaneously
walk and ®nd shelter in a heated arena when the shelter
was visible (Mizunami et al. 1998d). This observation
indicates that the MB is not the only site for determining spontaneous locomotory behavior but, rather,
that it is one of multiple areas that participate in the
determination of spontaneous locomotion. It would be
of interest to clarify the roles of the protocerebral
circuits in deciding spontaneous locomotion, since they
are thought to intervene between the MB and the premotor neuropil (Strausfeld et al. 1998). It may be that
the protocerebral circuits themselves are responsible for
making the ®nal decision concerning spontaneous
locomotion.
128
In our previous report (Mizunami et al. 1998c), we
demonstrated that units recorded simultaneously from
the same pair of electrodes exhibit similar responses.
With the improvement in resolution of individual units in
the present study, due to the use of thinner wire electrodes, we were able to con®rm that simultaneouslyrecorded units did indeed exhibit similar responses, but
we were also able to demonstrate that these responses
were not identical. The ®nding that neurons from the
same vicinity exhibit similar activities is in sharp contrast
to the great di€erences in activities seen among neurons
recorded in di€erent locations. Copper-silver impregnation (Fig. 1B; Mizunami et al. 1998c), Golgi studies
(M. Mizunami et al., unpublished observations) and
Bodian staining (Li and Strausfeld 1997) all suggest that a
number of extrinsic neurons of the lobes of the cockroach
MB are organized into clusters, as are the extrinsic neurons of the a lobe of the honey bee (Rybak and Menzel
1993). We suggest that the extrinsic neurons forming each
cluster act synergistically to send a particular set of MB
output signals to their target areas. Further anatomical
and physiological characterization of individual clusters
of extrinsic neurons, using intracellular recording and
staining techniques, are necessary to clarify the functional
organization of the cockroach MB, and such study may
also allow researchers to match extracellularly recorded
units with a particular cluster of extrinsic cells.
In conclusion, we suggest that the MB of cockroaches
is a part of a protocerebral network that integrates
various external and internal signals and that possibly
plans motor action. In order to con®rm and further
extend this suggestion, sensory and motor correlates of
activities of neurons of the MB and of related neuropils
need to be quantitatively analyzed in an appropriate
behavioral paradigm.
Acknowledgements We thank M. Ohara for helpful comments and
Dr. H. Nishino for providing the previously unpublished data
shown in Fig. 1C. This study was supported by Sumitomo Science
Foundation, Nissan Science Foundation, The Japan Securities
Scholarship Foundation and the Ministry of Education, Science,
Culture and Sports of Japan.
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