Mushroom bodies of the cockroach: Their participation in place memory

Mushroom Bodies of the Cockroach:Their Participation in Place Memory

MAKOTO MIZUNAMI,1 JOSETTE M. WEIBRECHT,1,2

AND NICHOLAS J. STRAUSFELD1*1ARL Division of Neurobiology, The University of Arizona, Tucson, Arizona 85721

2Arizona Health Science Center, The University of Arizona, Tucson, Arizona 85723

ABSTRACTInsects and other arthropods use visual landmarks to remember the location of their nest,

or its equivalent. However, so far, only olfactory learning and memory have been claimed to bemediated by any particular brain region, notably the mushroom bodies. Here we describe theresults of experiments that demonstrate that the mushroom bodies of the cockroach(Periplaneta americana), already shown to be involved in multimodal sensory processing, playa crucial role in place memory. Behavioral tests, based on paradigms similar to thoseoriginally used to demonstrate place memory in rats, demonstrate a rapid improvement in theability of individual cockroaches to locate a hidden target when its position is provided bydistant visual cues. Bilateral lesions of selected areas of the mushroom bodies abolish thisability but leave unimpaired the ability to locate a visible target. The present resultsdemonstrate that the integrity of the pedunculus and medial lobe of a single mushroom body isrequired for place memory. The results are comparable to the results obtained fromhippocampal lesions in rats and are relevant to recent studies on the effects of ablations ofDrosophila mushroom bodies on locomotion. J. Comp. Neurol. 402:520–537, 1998.r 1998 Wiley-Liss, Inc.

Indexing terms: microlesions; insect vision; learning; motor control; insect brain; b-lobes

The ability of many species of insect to locate the preciseposition of their home, foraging area, or prey has intriguednaturalists for well over a century. Dujardin alluded to thisin his classic paper on mushroom body structure (Dujar-din, 1850), and he again discussed this in his observationson the navigational skills of ants (Dujardin, 1853). Theidea that insects could remember and again locate theirnests was popularized by Fabre (1910) and von Frisch(1965) and given a quantitative foundation by Wehner(1992) and Dyer (1991).

The recognition of place from surrounding landmarks isalso a well-known attribute of mammals. Behavioral stud-ies and physiologic recordings have demonstrated thatplace memory and place navigation by using visual land-marks (or other sensory cues) can be ascribed to specificbrain areas (O’Keefe and Conway, 1978; Morris et al.,1982), of which the hippocampus appears to play a majorrole (Castro et al., 1980; Wilson and McNaughton, 1993;review, Muller, 1996). Place memory in mammals hasusually been considered in terms of cognitive maps, atheory that originally derived from psychological studieson rats, proposing that a detailed map of the rat’s worldwas centrally represented in the brain where it wascontinuously updated (Tolman, 1948). However, sugges-tions that a similar mechanism exists in insects (Gould,

1986) have been vigorously contested. Instead, homingbehaviors have been explained by the animal using othermechanisms (Dyer, 1991), such as polarized light orienta-tion, vector integration, and eidetic memory, instead ofcontinuously updating cues that are stored with referenceto a framework of coordinates somehow established withinthe central nervous system (review, Wehner and Menzel,1990).

In insects, the mushroom bodies (Fig. 1) have beencredited with crucial roles in olfactory learning and memory(Heisenberg, 1980, 1998; Erber et al., 1987; de Belle and

Grant sponsor: National Institutes of Health Fogarty InternationalFellowship; Grant number: F05 TW04390; Grant sponsor: National ScienceFoundation; Grant numbers: IBN-931629, IBN-9726957; Grant sponsor:Howard Hughes Undergraduate Biology Research Program; Grant spon-sor: the Ministry of Education of Japan; Grant sponsor: the UeharaMemorial Foundation; Grant sponsor: the Naitoh Foundation.

Dr. Mizunami’s current address: Laboratory of Neurocybernetics, Re-search Institute for Electronic Science, Hokkaido University, Sapporo 060,Japan.

*Correspondence to: Dr. Nicholas. J. Strausfeld, ARL Division of Neurobi-ology, 611 Gould Simpson Bldg., University of Arizona, Tucson, AZ 85721.E-mail: [email protected]

Received 30 June 1997; Revised 1 July 1998; Accepted 2 July 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 402:520–537 (1998)

r 1998 WILEY-LISS, INC.

Fig. 1. A: Mushroom bodies, situated on each side of the fan-shaped body (FB), are the dominant paired neuropils of the cockroachmidbrain. Each mushroom body consists of paired calyces (ca) sur-mounting a pedunculus (ped) and its vertical (V) and medial lobes (M).The right side of this figure shows three of the major afferent pathwaysto the mushroom body calyces. Afferents (asc) reach the calyces fromthe protocerebrum and dorsal lobes (d lob). Afferents from the anten-nal lobes (ant lob) reach the calyces by means of the inner antenno-glomerular (antennocerebral) tract (i act). Other afferents from proto-cerebral regions (e.g., optic tubercle) reach the calyces by means ofprotocerebral tracts (one shown: pr t), which also collect axons fromthe superior medial (s m pr) and superior lateral protocerebrum (s lpr), and lateral horn (l ho). op lob, optic lobe. B: Schematic relationship

of the mushroom bodies to other brain areas (adapted from Strausfeldet al., 1998). Olfactory, chemosensory, visual and tactile neuropilssupply interneurons directly to premotor centers (descending neu-rons). The same neuropils provide afferents to the mushroom bodieseither directly or indirectly (visual) by means of the protocerebrum.Inputs to the lobes (1–3) are multimodal and include visual signals (Liand Strausfeld, 1997; Strausfeld et al., 1998). Inputs to the calyces(4–6) mostly carry unimodal information except 7, which carries visualsignals. Afferents to the mushroom bodies interact with thousands ofparallel fibers of Kenyon cells. These synapse in the lobes onto efferentneurons (rectangles, E1–E3), the majority of which appear to targetthe protocerebrum. The protocerebrum, in turn, provides outputs topremotor centers.

Heisenberg, 1994). However, chemical stimuli are not theonly ones processed by the mushroom bodies. In cock-roaches and many other species such as crickets (Schild-berger, 1981, 1984; Homberg, 1984), mushroom bodiesintegrate many sensory modalities (Li and Strausfeld,1997), and extracellular recordings have identified neu-rons associated with motor actions (see companion paper:Mizunami et al., 1998). The suggestion (Mizunami et al.,1993) that mushroom bodies may play a role in placememory is intriguing for two reasons: first, in mammals,place memory has been attributed to a specific brain area,the hippocampus; second, the mammalian hippocampusand mushroom bodies (in Drosophila) seem to shareattributes of biochemical pathways that are thought tounderlie learning and memory (see, Kandel and Abel,1995).

The Drosophila mushroom body has been implicated inPavlovian-type olfactory conditioning (Heisenberg et al.,1985). A major difference between Drosophila and Peripla-neta is that in the former the predominant afferent supplyto the mushroom body calyces appears to be from theantennal lobes (Stocker, 1994; Ito et al., 1998). Thus,Drosophila might not be the most representative taxon inwhich to study mushroom body functions in learning andmemory, other than in olfactory conditioning (Quinn et al.,1974). Sensory information relayed to the mushroombodies of cockroaches, like that to the hippocampus ofmammals, includes most modalities. In Periplaneta, acous-tic, tactile, chemosensory, and visual interneurons fromthe protocerebrum supply the lobes and calyces (Li andStrausfeld, 1997; Strausfeld et al., 1998) as does informa-tion about motor actions (Mizunami et al., 1998).

Here, we present evidence from ablation studies of thecockroach mushroom bodies that these centers can play apivotal role in visual place memory under conditions thatimitate place memory tests for mammals (Morris et al.,1982; Wilson and McNaughton, 1993). Some of the presentresults have been presented as a preliminary study (Mizu-nami et al., 1993) and as an abstract (Weibrecht andStrausfeld, 1993).

The choice of Periplaneta for this study is not basedsimply on expedience. The subfamily Blattinae is bothphylogenetically ancient and, in terms of its geographicalradiation, the species Periplaneta is a success story by anydefinition. The order Blattodea is phylogenetically close tothe second largest group of eusocial insects, the termites(Kukalova-Peck, 1991), and amongst the Blattodea, cer-tain taxa, such the tropical species Cryptocercus, show alevel of social behavior (Cleveland et al., 1932). Periplanetaamericana is a domiciliary species that, in common withthe gray rat, lives in groups. Like rats, adult cockroachesare elusive and exploratory foragers.

MATERIALS AND METHODS

Adult male and female cockroaches, Periplaneta ameri-cana, were used for the study. The initial colony was basedon a moderately crowded population of mixed wild andpurchased individuals (Carolina Biological Supply Co.,Burlington, NC). Progeny from this population were raisedto adulthood in a 12-hour light/dark cycle and maintainedat 20–24°C.

Behavioral tests

Behavioral paradigms used to test spatial learning weremodeled after the Morris water maze (Morris et al., 1982),originally designed to test spatial memory in rodents. Inthe present study, the test arena comprised a 30-cm-diameter plate surrounded by a metal wall (Fig. 2A), bothmaintained at a constant temperature of 44–47°C (inearlier experiments, at 50–55°C: Mizunami et al., 1993)(Fig. 2B). A cold area (the target), 5 cm in diameter, wasmaintained at 17–20°C (in early experiments, 20–25°C) bymeans of an insulated thermos flask attached beneath thearena floor and containing water and crushed ice, asdiagrammed in Figure 2A. The arena was situated under awhite canopy, the top of which was fastened around thelens of a video camera. The arena floor was covered by athin, transparent, polyethylene sheet that could be rotatedor replaced after each trial, to control whether the animalwas using substrate cues, such as pheromones. The arenacould also be rotated independent of the canopy covering it,and visual cues placed on the arena wall (see below) couldalso be rotated independent of the arena. The pathwaytaken by an insect (Fig. 2B) was traced from stoppedframes on a video monitor.

Place memory was tested by using visual cues. Peripla-neta have large compound eyes, each composed of at least2,000 ommatidia (Mote, 1990), served by a large optic lobe,the organization of which suggests mechanisms for motionand form detection. Three types of test examined theability of the animal (1) to locate the invisible cold targetwithout any visible cue, (2) to recognize the target areawhen it provided visual contrast, and (3) to locate thetarget with reference to distant visual cues.

Control experiments (test 1) examined the behavior ofcockroaches when the target was invisible and the wallswere undecorated. In the second experiment (test 2), thetarget area and arena floor had different intensities (floorblack, target white) and the walls were undecorated. In thethird experiment (test 3), the surfaces of the arena wereuniformly black and the wall of the arena (14-cm high) wasdecorated with visual cues that had a specific geometricalrelationship to the arena during any one test series. Fourdifferent visual cues contrasted with the uniformly coloredarena wall: a white rectangle, a black rectangle, a rect-angle divided into equal vertical black and white stripes,and another, similarly divided horizontally. Preferencetests do not suggest that any one of these patterns is moreattractive than another, at least during the first trial ofany test (see Results). In trials 1 through 10, the coldtarget area was situated approximately between the verti-cal stripes and black rectangle or in front of the verticalstripes.

Before beginning a series of trials, each animal wasplaced on the cold target area for 5 minutes, covered by atransparent glass beaker (diameter, 10 cm) that was justlarge enough for the insect to antennate the heatedsurround. Omitting this pretraining provided similar learn-ing curves, but the animals required greatly extendedrunning times of up to 6 minutes in trial 1, and required upto 20 trials to reach times of between 30 and 60 seconds.Experiments without pretraining often resulted in heatshock, and many tests had to be abandoned. With pretrain-ing, trials could be limited to 10.

To initiate the first trial, the beaker was removed andtiming was started when the animal began to move from

522 M. MIZUNAMI ET AL.

the cold area to explore the heated arena. The animal wasscored as having located the cold area if it remainedimmobile on the area for at least 30 seconds. The cockroachwas allowed to remain on the target for up to a minute,after which it was then removed by covering it with anopaque beaker, and sliding a piece of black paper gentlybeneath it. After 2 minutes, this beaker was set down atsome arbitrary position in the arena (occasionally at ornear the target area). The paper was slid out from underthe insect, the beaker was removed and timing was begunfor that trial. Some animals were in an eleventh trial (see

below) in which the visual cues were rotated relative to theposition of the cold target.

Microsurgery

Cockroaches were immobilized by cooling. They werethen placed in a specially designed two part restrainingchamber, one part of which held the animal, the other partice. Cockroaches could thus be kept comatose with alowered heart rate during the operation, thereby minimiz-ing the loss of hemolymph. To expose the brain’s surface, asmall area of cuticle was removed from the top of the head

Fig. 2. Experimental design. A: The insect is placed in a heated30-cm-diameter arena, the walls of which are decorated with visualcues (as in test 3). An invisible target (indicated by double arrow in A,lower right) is maintained at low temperature by an insulated thermosflask, affixed beneath the arena and containing crushed ice. Thesurrounding arena is maintained by a hot bath. Surface temperature

distribution is shown in B, right. The arena is covered by a canopy.Behavior, such as running pathways (B, left), is recorded onto videotape. Transparencies of the arena and the location of the cold area areplaced over the monitor. Stopframe tracing of positions (asterisk,start; dot, end) and running tracks provides raw data for analysis (B,left).

MUSHROOM BODIES IN PLACE MEMORY 523

capsule between the antennae, avoiding the ocelli andother peripheral nerves. The piece of cuticle was also keptcool and was sealed back in place after the operation. Carewas taken not to disturb tracheation except when it wasunavoidably cut during lesioning.

Two small incisions were made in the brain’s perineuralsheath in preparation for lesioning. Lesions were made byimplanting slivers of aluminum foil into the protocere-brum. Slivers were made under the dissection microscopeand were approximately 100- to 150-µm wide, 20-µm thick,and 350-µm long, and had a pointed tip. After implanta-tion, the excised cuticle was put back in place and thewound sealed with low melting point dental wax.

After recovery, the animal was placed into the cage withother unoperated individuals. Behavioral tests were started1 week after surgery when the spontaneous behavior ofabout 40% of the operated animals was indistinguishablefrom unoperated ones. Animals that showed abnormalbehavior (were sluggish, or had locomotory impairment)were rejected. Histologic sections of two such abnormalanimals demonstrated lesions that had damaged the cen-tral body complex.

Histology

The precise locations of the lesions were ascertainedfrom postmortem histology. After its final behavioral test,the animal was cooled and its head capsule opened. A 3%paraformaldehyde solution in cockroach saline (O’Sheaand Adams, 1981) was used to fix the brain for 30 minutes.The brain was carefully removed, with the aluminumslivers still in place, and was postfixed for 30 minutes withartificially aged Bouin’s fluid (Gregory, 1980), then dehy-drated and embedded in Durcupan (Fluka, Heidelberg,Germany). Serial sections were cut at 30 µm. They werebriefly stained with toluidine blue, rinsed off with 70%alcohol, then absolute alcohol, and then with Xylol finallybeing cover-slipped under Permount (Fisher Scientific,Fair Lawn, NJ). The outlines of the mushroom bodies, asrecognized from Bodian (1936) reduced silver preparations(Fig. 3A), can be easily distinguished, and the location ofthe aluminum slivers can be clearly revealed by darkfieldmicroscopy (Fig. 3B).

Data evaluation

The time to reach the target in normal and in operatedanimals was used to evaluate performance. The results ofthe whole test group were plotted, as were those ofindividuals whose performances were tracked througheach test. The pathways were reconstructed from videorecordings. An acetate sheet, divided into quadrants,showing the outline of the arena, the position of the target,and the positions and types of visual cues, was placed overthe monitor. Tracks of individual animals were plotted onit from video recordings showing successively stoppedframes. Distances run and average speeds were calculatedfor one group of 18 animals. Groups of tested animals werealso evaluated by using x2 and Mann-Whitney U tests.

RESULTS

Normal behavior

Locomotory behavior. Irrespective of the type of test,individual cockroaches show the same basic type of locomo-tory behavior in the arena. Animals move rapidly, pausing

often to antennate the surface or walls of the arena. Aninitial tendency to walk around the perimeter of the arenadecreases from trial to trial. In tests using visual cues,individuals have been observed to stop, rock from side toside, or make tight turns (pirouettes), before continuing.

Test. 1: No cues. Control experiments in which thearena wall was undecorated, the target invisible, and aplastic floor cover rotated after each trial, demonstratethat, after 10 trials, there is no overall decrease in the timeto reach the target (Fig. 4). Nor was there any improve-ment observed by any individual animal. The same resultwas obtained when the arena floor was left undisturbed,suggesting that if individuals do put down chemical cues,these probably dissipate and provide no aid for targetlocation despite, as shown in video records, animals inter-mittently antennating the arena floor.

Test 2: Visible target. A test group of 10 animalsshowed a rapid improvement in performance if the targetitself was visible. However, as in the following experiment(test 3), best performance (that is the most rapid return tothe target from the position of release on the arena)occurred at trials 4–6, with trials 7 and 8 generally takingslightly longer. The results of trials with a visible target,compared with trials after mushroom ablation, is dis-cussed later.

Test 3: Distant visual cues and a hidden target. Whenthe wall is decorated with visual cues, animals reach thetarget faster after 10 trials than after the first 1–2 trials(Fig. 5A). As a group, the best performances seem to occurby trial 6, as in test 2. There is, however, considerablevariation of the performance of individual cockroacheswithin the test group of 21 animals. Analysis of individualperformances (asterisks, Fig. 5B) indicated that a few slowlearners caused such large standard deviations (Fig. 5B)that, without tracking individual performances, the differ-ence between the standard deviation of trial 1 and trial 10would not suggest a significant improvement. Trackingindividual performances demonstrated that 80–90% of thecockroaches improved their target location convincinglyfrom trial 1 to trial 10. Between 10–20% of the animals didnot, however. As shown in Figure 5B, after the tenth trial,all but 2 (asterisks in Fig. 5B) of the 21 test animals tookless than 45 seconds to reach the target compared with theinitial average run time of 120 seconds. The large standarddeviations derive from the behavior of two outlying indi-viduals, which took 201 and 297 seconds, respectively, toreach the target at the tenth trial and which performedconsistently poorly.

Cue preference. Typically, during a trial a cockroachwill intermittently pause, during which it may antennateor rock side-to-side, the latter reminiscent of locust peeringbehavior (Collett, 1978). Sometimes the insect will pirou-ette in a tight circle. Cockroaches paused more frequentlyin early trials than in later trials (Fig. 6A). After patternrotation (trial 11) an individual would again pause fre-quently at many positions across the arena (Fig. 6A). Totest whether individual cockroaches spontaneously pre-ferred one visual cue over another, insects were placed inthe center of an arena decorated with the cues describedabove. 20 animals were tested, noting in which segment ofthe arena, pertaining to one of the four patterns, anindividual first paused. By using the Rayleigh test fordirectional preference, this procedure showed no signifi-


Fig. 3. A: Frontal Bodian-stained section through the brain show-ing the vertical (a) and medial (b) lobes at a position immediatelyanterior to their junction with the pedunculus (out of section plane).Note the arrangement of extrinsic neuron dendrites at right angles tothe long axis of each lobe. B: Toluidine blue-stained semifrontalsection (tilted slightly upward to demonstrate the pedunculi (ped) and

b-lobes) viewed with darkfield illumination to show the location ofaluminum slivers (open arrows; equivalent position indicated by openarrows, right, in A) that had transected the b-lobe on one side, and thepedunculus on the other. This section is from a cockroach that wasunable to use distant visual cues for place memory.


cant preference for any one test pattern (P . 0.10).However, this result contrasts with observations of indi-vidual cockroaches during the place memory test 3 (Fig.6B). In trial one, there seemed to be no preference forwhere any individual paused, whereas, in later trials,pauses seemed to be clustered near certain cues (Fig. 6B).

Pattern rotation. A quantitative improvement in anindividual’s performance, such as shown in Figure 5B,suggests that cockroaches learn the position of the targetwith respect to the surrounding visual stimuli. As sug-gested by the results summarized in Figure 6A,B, it alsoappears that individual animals change their behavioralstrategy. However, as pointed out by Bitterman and Couvil-lon (1991), there is a difference between improving perfor-mance, such as fine tuning a motor pattern, in the absenceof real learning, and an improved performance that couldoccur only if learning occurs. If a cockroach indeed learnsthe position of the target during trials 1 through 10 then, ifthe patterns are rotated to a new position with respect tothe target (in an eleventh trial), when an individual isagain released into the arena, it would be expected to

initially run in the direction of the target’s old location, aswas previously indicated by the visual cues during trials1–10.

After cue rotation, a cockroach that had learned theposition of the target initially ran to its expected location(Fig. 7A). Certain individuals, as shown in Figure 7B, theninitiated a searching strategy in the vicinity of the ex-pected position of the target. This apparent resetting ofexploratory activity is also suggested by the number ofpauses made by an individual after cue rotation (Fig. 6A).Thus, the consequence of rotating the cues is essentiallysimilar to Tinbergen and Kruyt’s (1938) original experi-ment on Philanthus (see Discussion): the animal goes to, ortoward, the expected position of a learned target.

Distances and velocity. Does increased performance,as measured by decrease of time to reach the target, re-flect shorter pathways or faster running velocities, orboth? The distance traveled by an individual from onetrial to the next can show considerable variation, some-times great variation. The example provided by one indi-vidual (Fig. 7A) is extreme but not atypical. Dividing

Fig. 4. Results of test 1 (no cues). This and subsequent figures likeit demonstrate the time taken by individuals to reach the target in aparticular trial. Abscissa, time (seconds); ordinate, number of individu-

als to have reached the target. Although in trial 6 three individualsseem to reach the target sooner than in trial 1, trial 10 shows thisfinding is not maintained.


the length of the pathway by the time to reach the tar-get (discounting pauses) suggests that individual veloci-ties are more or less constant throughout each test sothat improved performance reflects primarily a decrease inthe distance run. For a single individual (solid bars, Fig. 8)the speed of locomotion increased only slightly midway

through the experiment, despite different distances cov-ered in each trial.

Mushroom body microlesions

Microlesions. Tests on unoperated animals, describedabove, demonstrate that individuals can use distant visual

Fig. 5. A: Histogram showing the average performance of a groupof 21 cockroaches in 11 trials of test 3. B: Tracking individualperformances demonstrates that averaging (A) can be misleading.

Here, trial 10 demonstrates that 19 of the 21 subjects reached thehidden target in less than 60 seconds, with two outliers (indicated byasterisks in trials) taking 201 seconds and 297 seconds, respectively.


cues to learn the location of a hidden target. To testwhether the mushroom bodies play a role in this ability,lesions were made within different parts of the protocere-brum, lateral to the mushroom bodies, and within themushroom bodies themselves. Lesions avoided the centralbody complex and the optic pedunculi, the former becauseof its possible role in coordinating locomotion (Strauss and

Heisenberg, 1993) and the latter because of their obviousrole in visual discrimination.

The locations of lesions are summarized in Figure 9.These were bilateral lesions to both mushroom bodies,

bilateral lesions avoiding the pedunculi and lobes of bothmushroom bodies, and bilateral lesions in which only onemushroom body suffered damage. Symmetrical mushroombody lesions were made at both a-lobes, or at both pedun-culi and their junction with the b-lobes. Lesions of themedial part of the b-lobes could not be made withoutdamaging the central body complex, which lies immedi-ately posterior and slightly dorsal to them. The location ofthe lesions was assessed after behavioral tests and afterhistologic examination, as described in the Materials andMethods section. By using dark-field microscopy, the lesionwas revealed as a rectangular area, filled with aluminum(Fig. 3B). Hemocytes surround this area, suggesting thatduring the period between lesioning and histology, therehas been some wound reaction by the tissue. However,there was no evidence for neuropil degeneration furtherdistant than the limit of hemocytes, which extend 20–50µm from the aluminum insert. Also, there was no evidenceof tissue distortion at the lesion site. Lesions appearedclean, and the tissue appeared cut cleanly rather thantorn.

Behavior. Animals were tested 7 days after surgery.Operated animals that appeared to walk normally, anten-nated, fed, sometimes copulated, and showed escape behav-ior were used for place memory tests. Operated animalswere subject to the two tests described in the Materialsand Methods section, i.e., location of a visible cold targetarea (test 2), and place navigation by using distant cues(test 3).

Animals were first tested for their ability to find a visiblecold target (test 2), and then 24 hours later the sameindividual was tested for place memory, by using test 3.Both tests were performed against the same ambientsensory background (elevated arena temperature, diffuselighting), and at the same time of day. Comparisons ofperformance were made over 10 trials. Normal animalswith sham operations (cuticle cut and then resealed) werecompared with control operated animals (bilateral lesionsthat omitted both mushroom bodies) and mushroom bodylesioned animals.

Test 2: Cockroaches with pedunculus–b-lobe ablations.Nine animals, later found to have sustained bilaterallesions to the pedunculi, or to the pedunculus–b-lobejunctions (Fig. 9A), were tested with a visible target. Allbut one of the individuals that completed all 10 trialsshowed a clear improvement in performance when testedwith a visible cold target in a hot arena (Fig. 10). Thisresult compared well with the performances in the sametest by sham-operated cockroaches (compare Fig. 12B andF), or cockroaches that had bilateral lesions outside themushroom body pedunculi and lobes (compare Fig. 12Dand F).

Test 3: Cockroaches with pedunculus–b-lobe lesions.Bilateral lesions to the pedunculi or to the b-lobes, neartheir junction with the pedunculus, abolished the ability ofthese insects to learn the location of a hidden cold targetwhen its position was provided by distant visual cues (Fig.11). Although there appears to be some slight improve-ment between trial 1 and trial 10, this is not significant,and no single individual of the 7 that completed all 10trials improved on its performance. Outliers included

Fig. 6. A: Pause positions of an individual cockroach, showing bodyorientations (lines, body axes; filled circles, head). Numbers representtrial numbers with rotation preceding trial 11. Positions of the cuesare indicted by the squares, the circle in the arena is the cold target.Pauses become less frequent as the trial progress. However, cuerotation (lower right in A) results in frequent pauses during aneleventh trial. B: Pause positions of six individuals combined, showingclustered pause in trials 4, 6, and, most obviously, in trial 8.


cockroaches that, in trial 10, did not reach the targetwithin 12 minutes or that, between trials 7–10, neverreached the target and were withdrawn from the experi-ment due to heat stress.

Comparisons of results of test 3 on bilaterally lesionedmushroom bodies and unoperated and control operatedanimals (Fig. 12) demonstrate that lesions affect only theability to remember the location of a target relative todistant visual cues. Figure 13 compares all operationalconditions for test 3, focusing on the results of trials 4 to10, trial 4 being the first to be statistically different fromtrial 1 in unoperated controls. Again, differences areapparent between animals with bilateral lesions of thepedunculi or pedunculus–b-lobe junctions (Fig. 13E), andanimals with bilateral a-lobe lesions (Fig. 13D), bilateralcontrol lesions (Fig. 13B), or unilateral b-lobe or peduncu-lus lesions (Fig. 13C,D). Cockroaches that had sufferedbilateral lesions of their pedunculi or b-lobes (Fig. 13E)were as ineffective at learning the position of a hidden goalas were unoperated insects denied any cues at all (Fig.13F).

The results are compared statistically in Table 1 (Mann-Whitney U test, P , 0.05). No significant differences werefound between: (1) unoperated and control-operated ani-mals; (2) unoperated and single pedunculus sectionedanimals; (3) unoperated animals and animals with bilat-eral a-lobe lesions; (4) control-operated animals and ani-mals with a single pedunculus lesion; or (5) animals withunilateral pedunculus lesions and bilateral a-lobe lesions.In contrast, the performance of unoperated animals in anarena without visual cues is indistinguishable from that ofanimals with bilateral pedunculus–b-lobe lesions tested inarenas with visual cues. Results 4 and 5 demonstrate thatanimals with unilateral lesions to one pedunculus or oneb-lobe (Fig. 13C) are able to locate the hidden target,suggesting that this task requires only a single mushroombody. Why this might be so is considered below.

DISCUSSION

Place memory and navigation by distant cues

Wehner and Wehner (1990) make the important distinc-tion between ego- and geocentric mechanisms for homing,the first relying on a continuous updating of vector informa-tion for relocating the home target by using dead reckon-ing, the second by using landmark features for periodicupdating and reorientation. Although navigation mayinvolve piloting along a physical or chemical gradientwithout involving memory (Papi, 1992), memory seems tobe implicit for other types of navigation, be they idiotheticor allothetic. For example, filter matching amongst asequence of local cues, or even the interpolation of avariety of sensory cues to provide vectorial integration,would seem to require memory.

Neural mechanisms underlying orientation are stillobscure. The one exception is that identified in the socialHymenoptera, which have specialized foraging tasks and

Fig. 7. A: Results of pattern rotation in trial 11 on path taken to thetarget. Conventions are the same as in Figure 6 except that, in A, thedistance and path run by the subject varies greatly from its startposition (asterisk) to its termination (spot on cold target) throughtrials 1–6, but assumes an almost direct trajectory in trial 10. In trial11, rotating the target to a new position (near the horizontal grating)results in an initial path to the original position of the target (finecircle), which was between the vertical grating and black rectangle. B:A second individual appeared to have learned the target location bytrial 6. Pattern rotation (trial 11) resulted in an extended bout ofsearching near the original position (fine circle) of the target.


are obligate diurnal navigators over long distances. In thisgroup, cues used for recording directions taken during theoutward journey, as well as course maintenance for thereturn, are provided by the pattern of celestial polarizedlight relative to the sun’s position (Rossel, 1989). Fieldobservations of Diptera, such as asilids, show the ability ofa non-social insect to return to its starting point after aforay. In Hymenoptera, a specialized dorsal array of photo-receptors at the rim of the compound eye is adapted for thistask and is served by specialized systems of optic-lobeneurons (Labhart, 1988). Likewise in Diptera there aresimilar sets of interneurons that relate specifically to thedorsal rim ommatidia (Strausfeld and Wunderer, 1985).However, it is likely that many other arthropod taxanavigate without the aid of polarized light cues. Individual

fiddler crabs, for example, forage hundreds of meters fromtheir nest but can rapidly return to their own burrowwithout confusing the visual signals provided by burrowsbelonging to other individuals. Although this mechanismhas been suggested to rely on the playback of a sequence ofmotor events (Hughes, 1966), similar geocentric homing byflying insects has been suggested to rely on eidetic memoryof visual cues.

Species that undertake active foraging are now recog-nized to use spatial recognition and, presumably, someform of landmark memory. Studies by Cartwright andCollett (1982, 1983), Cheng et al. (1989), and Zeil (1993a,b)have demonstrated that bees and wasps obtain visualinformation during interactive visual scanning (Lehrer etal., 1988; Srinivasan et al., 1989) and use eidetic memory

Fig. 8. Run times, distances (traveled from the starting point tothe target), and the mean velocities of 10 individuals in 10 trails of test3, showing mean and standard deviation (cross-hatched bars). Run-ning times improved from trial 1 to 10, velocities remained approxi-mately constant, and distance decreased. For a single individual (solid

bars), its running times intermittently increased (in trial 4 and 8), asdid the distance traveled in these trials. Its peak velocity was in trial 6,which showed the shortest run time and second shortest distancetraveled.


in navigation, apparently learning to recognize the spatialconfiguration of specific features of the visual surround-ings. Wehner (1987) has proposed that stored snapshotimages of these features are later compared with visuallandmarks so that a match between external cues and thestored snapshot allows the insect to recognize place, aphenomenon known as filter matching (Wehner, 1992).Evidence that insects use this type of visual memory isimplied by Tinbergen and Kruyt’s (1938) study of thesolitary hymenopteran Philanthus. When landmarksaround the nest of this species were displaced laterally, thewasp searched for its burrow where it should have beenwith respect to the displaced landmarks. In Tinbergen andKruyt’s experiments, home navigation relies upon a phe-nomenon that, in mammals, has been termed place memoryand which is characterized by the animal’s reference todistant cues (Morris et al., 1982). However, whereas placememory and place navigation in mammals are suggestedto be mediated, at least in part, by the hippocampus(O’Keefe and Conway, 1978), there has until recently(Mizunami et al., 1993) been no evidence in insects for abrain structure that could be crucial for a similar ability.

Place memory in cockroaches

The present results provide evidence that cockroachesare able to use visual cues to locate a hidden target andthat the mushroom bodies are essentially involved in thisability. Unambiguous and rapid learning of the hiddentarget location was demonstrated by about 80% of thecockroaches tested. Like individual rats, individual roacheswere able to learn the location of a target by reference tosurrounding landmarks. Bilateral lesions of a specific partof the mushroom bodies abolished this ability.

Analysis of the behavior of a few individuals suggeststhat, in later trials, cockroaches stop and scan the surround-

ings at preferred locations within the arena. Possibly, thisstrategy represents episodes of interactive vision, that is,active sampling of the visual surround at specific locationswithin the visual environment. This may be a generalphenomenon in arthropods with well-developed visualcenters, ranging from peering behaviors shown by locustsbefore jumping (Collett, 1978) to ‘‘stop and look’’ strategiesthat have been demonstrated in flying wasps and bees thatscan visual targets during foraging (Lehrer, 1993; Zeil,1993a,b).

Although it is not surprising that wide-ranging foragerssuch as the cockroach use place memory, previous experi-mental demonstrations of this capacity in insects havebeen rare (e.g., Tinbergen and Kruyt, 1938; Collett, 1992;Collett et al., 1993), and experiments on cockroach learn-ing and memory are few. For example, cockroaches havebeen shown to be able to associate a food source with anodor after only a single trial (Balderrama, 1980), andcockroaches have also been shown to use olfactory cues forlocating a hidden goal (Mizunami et al., 1993). The presentresults are superficially reminiscent of Vowles’s (1964)study, in which rather imprecise bilateral lesions in themushroom bodies had a more significant impact in anolfactory learning task than unilateral lesions.

Learning by individuals versus groupsof individuals

One technical difficulty in testing groups of animals is touse quantitative methods that are applicable to the popula-tion while allowing assessment of individual variation inbehavior. Such variation may be particularly apparent in aspecies such as Periplaneta where individuals are raised incrowded colonies. Within any group tested, some individu-als will have had more diverse sensory experiences thanothers. It is also to be expected that certain individuals are

Fig. 9. Schematic showing types and locations of lesions. A: Bilateral pedunculus–b-lobe lesions. B: Controllesions, lateral to mushroom bodies (these sometimes included parts of the lateral calyces). C: Bilateral a-lobelesions. D: Unilateral mushroom body lesion to one lobe with a control lesion on the other side of the brain.


less tolerant of heat than others. Thus, it is not surprisingthat, when assessing group behavior, the data show largestandard deviations. Only when assessing the perfor-mances of individual cockroaches is it apparent thatsome either seem not to learn at all, or learn very slowly.We conclude that in any group tested, there are out-lying poor performers. Outliers, such as shown inFigure 5, performed poorly in every trail and showedno improvement between the first and last of the tentrials.

Visual place memory and visual supplyto the mushroom bodies

An earlier report (Mizunami et al., 1993) showed thatolfactory cues as well as visual cues could provide informa-tion for place memory. However, considering that thecockroach calyx hardly receives visual information fromthe optic lobes, how is visual place memory possible?

In many Hymenoptera, the calyces are divided intothree zones, the lip, collar, and basal ring (Mobbs, 1982,1984), of which the collar receives a massive afferentsupply from the optic lobes (Gronenberg, 1986). In con-

trast, cobalt fills into one of the optic lobes of Periplanetareveal but a single axon to the calyces on the same side ofthe brain (Li and Strausfeld, unpublished observations).However, intracellular recordings have identified afferentinterneurons to the calyces and to the pedunculus thatcarry visual information. Such neurons originate in areasof the protocerebrum that receive collaterals and termi-nals of visual interneurons from the ipsilateral and contra-lateral optic lobes (Li and Strausfeld, 1997; Strausfeld etal., 1998). This organization would partly explain theability of the mushroom bodies to process high order visualinformation, such as pattern and orientation, as well asthe ability of the animal to perform visual place memoryeven when one of the two mushroom bodies has beenlesioned.

Are mushroom bodies crucialfor place memory?

Bilateral ablations of b-lobe–pedunculus junctions im-pair the cockroach’s ability to learn the position of a hiddentarget, suggesting that the medial lobes (b) and pedunculiare crucial for place memory in which distant cues have to

Fig. 10. Improvement in locating a visible target (test 2). Seven individuals (two outliers neverreached the target and were removed prior to trial 10) that had sustained bilateral lesions to thepedunculus–b-lobes acquire the target when it is itself visible.


be related to each other and an invisible target. However,the same lesions leave intact the ability of the cockroach tolocate the target when it is visible. This suggests thatlearning to associate a visual stimulus with a secondspatially coincident modality such as temperature does notrequire the participation of the mushroom bodies, whereasindirect associations do. It is useful to compare thesecontrasting results with the results of mushroom bodylesions in Drosophila (de Belle and Heisenberg, 1994). Inde Belle and Heisenberg’s experiments, chemical ablationswere used to delete the four embryonic neuroblasts eachside of the embryonic brain that would normally give riseto the adult mushroom bodies (Ito and Hotta, 1992). deBelle and Heisenberg (1994) showed that flies lackingmushroom bodies could not be conditioned to olfactorycues. Animals lacking mushroom bodies were, however,able to learn to avoid light in conditioning experimentswhere light coincided with a heat shock. In anotherexperiment, Martin and Heisenberg (1998) showed a mo-tor attribute of the Drosophila mushroom bodies: bilateraldeletions impair the ability of a walking fly to stopwalking. This suggests that even in the fruit fly, mushroombodies may play an important role in the regulation ofmotor actions.

Are mushroom bodies centersfor motor control?

The involvement of the mushroom bodies in controllingmotor repertoires was originally suggested by Huber’s(1960) demonstration that current injection into or nearthe mushroom bodies could elicit bouts of ritualized behav-ior. A role by the mushroom bodies in specific behaviors isalso suggested by structural changes that occur in themwhen a novel behavioral repertoire arises. For example, inadult crickets a transitory increase in the titer of juvenilehormone (JH) triggers the birth of new Kenyon cells. Thisis accompanied by the onset of courtship behaviors (Cayreet al., 1994, 1996). In worker honey bees, an increase inJH, concomitant with the behavioral change to forger fromnurse, is accompanied by a volumetric increase of themushroom body calyces (Withers et al., 1995). A role of themushroom bodies in multitasking (multiethism) is sug-gested by the difference in size of mushroom bodies in antsdenied a brood and in those allowed to care for one.Mushroom bodies are larger in ants with a brood (Gronen-berg et al., 1996).

These studies, along with the present results, suggestthat the mushroom bodies are crucial for a variety of

Fig. 11. This figure demonstrates that there is no improvement in locating a target by using distantcues (test 3) by individuals that have sustained bilateral lesions to the pedunculus–b-lobes.


functions, many of which are not so easily defined as, forexample, olfactory conditioning. It is therefore relevant toconsider the evolutionary history of the mushroom bodiesand to compare taxa that retain primitive features withthe main taxa used for learning and memory studies, suchas Drosophila, which may be highly derived (Strausfeld etal., 1998). Cockroaches represent evolutionarily basalneopterans (Kukalova-Peck, 1991). Their mushroom bod-ies receive multimodal inputs both to their calyces and totheir lobes, and activity in these regions can be triggeredby acoustic, tactile, visual and olfactory stimuli as well asby motor actions (Li and Strausfeld, 1997; Strausfeld et al.,1998; Mizunami et al., 1998).As mentioned above, interneu-rons that terminate in the cockroach mushroom bodylobes, coupled with the supply of antennal-lobe projectionneurons to the calyces, suggest convergence onto Kenyoncells of many different modalities. In contrast, the Dro-sophila mushroom body seems to be relatively simple in itsconstruction, receiving predominately antennal-lobe in-puts (Ito et al., 1998).

Despite taxonomic differences attributed to the mush-room bodies of various species, the variety of sensorymodalities that would be collectively represented in ageneric insect suggests that functions not obviously re-lated to each other may have common computationalrequirements based on the processing of distributed andoverlapping afferents by many thousands of parallel intrin-sic neurons. Possibly, different functions are parsed intoparallel subdivisions of the mushroom bodies, each repre-senting a discrete intrinsic cell population that represents,say, a specific zone of the calyces (Mobbs, 1982, 1984), agenetically and morphologically distinct set of Kenyoncells (Yang et al., 1995; Ito et al., 1997), or a population ofKenyon cells isolated from others by the neuromodulator itcontains (Bicker et al., 1987; Schurmann and Erber, 1990).

In considering the role of the mushroom bodies, adistinction should be made between their being crucial to awider neural system that supports learning and memoryand their being the center where memory is formed. Thelatter is sometimes assumed (Davis and Han, 1996) but a

Fig. 12. Comparison of performance by unoperated and lesionedanimals in test 3 (A,C,E) and test 2 (B,D,F). Unoperated animals (A)and animals with control surgery (lesions lateral to the mushroombodies, C) demonstrated improved run times over 10 trials in test 3;animals that had sustained bilateral lesions to the pedunculus–b-

lobes showed no improvement (E). The results contrast with thosefrom test 2, in which the target was visible and bilateral lesions to thepedunculus–b-lobe were not deleterious. Bi Ped, bilateral lesions inthe pedunculi.


broader perspective is suggested on the basis of recentstudies on Drosophila (Ito et al., 1998). These show thatrather than projecting to premotor descending neurons, sodirectly modifying commands from the brain, efferentneurons from the mushroom bodies distribute to otherareas of the protocerebrum about which virtually nothingis yet known. Studies on efferent neurons of Periplaneta(Li and Strausfeld, 1997; and unpublished data) and Apismellifera (Rybak and Menzel, 1993; Strausfeld, 1998)indicate a similar organization.

Although these findings might speak somewhat againstthe mushroom bodies as an exclusive neuropil for learningand memory, they do not detract from the observation thatin the cockroach mushroom bodies play a crucial role inplace memory, even if this role might be in providing

circuits for filter matching rather than for informationstorage. In conclusion, this and other recent studies (Mizu-nami et al., 1998; Li and Strausfeld, 1997; Strausfeld et al.,1998) demonstrate that the mushroom bodies are likely toplay cardinal roles in more types of higher functions thanhitherto accorded them.

ACKNOWLEDGMENTS

We thank Ms. Carol Arakaki, B.S., for assistance withthe histology and to Mr. Robert Gomez, B. S., for photo-graphic assistance. We also thank Dr. Camilla Strausfeldfor correcting the manuscript and to Dr. Yongshen Li forhelpful discussion of the results. J.M.W. was supported by

Fig. 13. The number of times (in trials 4 to 10) in which a cockroachreached the goal in less than 60 seconds was least for animals thathave suffered either bilateral lesions to the pedunculi or b-lobes (E), orunoperated animals that have no visual cues (F, test 1). There is little

difference between the results of test 3 on unoperated (A) or control-operated (B) cockroaches, or cockroaches that sustained either aunilateral lesion (C) or bilateral lesions to their a-lobes (D). Uni Ped,unilateral lesion to one pedunculus.

TABLE 1. Statistical Comparisons Between the Results of Trials 4–10 (Postlearning in Normal Subjects)1

Visual cues

No visual cuesVisual cues

UnoperatedBilateral pedunculus–

b-lobe lesionsBilateral a-lobe

lesions Unilateral pedunculusControlsurgery

Unoperated ,0.001 ,0.001 MW . 0.05 .0.05 .0.05M , 0.05

Control surgery ,0.001 ,0.001 .0.05 .0.05Pedunculus lesioned unilaterally ,0.001 ,0.001 .0.05

a-lobe lesioned bilaterallyMW , 0.001

M , 0.01 ,0.001Pedunculi lesioned bilaterally .0.05

1The results of trials on cockroaches in the absence of visual cues or cockroaches with bilateral pedunculus–b-lesions in the presence of visual cues differ significantly from trials onunoperated cockroaches or cockroaches with other types of lesions in the presence of visual cues. Unless otherwise stated (Median test, M), results derive from Mann-Whitney U tests(MW).Unoperated without visual cues: N (number of animals completing test) 5 7, n (data points) 5 34; bilateral pedunculus/b: N 5 9, n 5 60; bilateral a lobe: N 5 4, n 5 28; unilateralpedunculus: N 5 7, n 5 48; control surgery: N 5 9, n 5 63, unoperated with visual cues: N 5 10, n 5 69.


a Howard Hughes Undergraduate Biology Research Pro-gram fellowship.

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Mushroom bodies of the cockroach: Their participation in place memory

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