Mushroom bodies of the cockroach: Activity and identities of neurons recorded in freely moving...

Mushroom Bodies of the Cockroach:Activity and Identities of Neurons

Recorded in Freely Moving Animals

MAKOTO MIZUNAMI,1 RYUICHI OKADA,2 YONGSHENG LI,1

AND NICHOLAS J. STRAUSFELD1*1ARL Division of Neurobiology, The University of Arizona, Tucson, AZ 857212Laboratory of Neurocybernetics, Research Institute for Electronic Science,

Hokkaido University, Sapporo 060, Japan

ABSTRACTThis article describes novel attributes of the mushroom bodies of cockroaches revealed by

recording from neurons in freely moving insects. The results suggest several hithertounrecognized functions of the mushroom bodies: extrinsic neurons that discriminate betweenimposed and self-generated sensory stimulation, extrinsic neurons that monitor motoractions, and a third class of extrinsic neurons that predict episodes of locomotion andmodulate their activity depending on the turning direction. Electrophysiological units havebeen correlated with neurons that were partially stained by uptake of copper ions and silverintensification. Neurons so revealed correspond to Golgi-impregnated or Lucifer yellow-filledneurons and demonstrate that their processes generally ascend to other areas of theprotocerebrum. The present results support the idea of multiple roles for the mushroombodies. These include sensory discrimination, the integration of sensory perception withmotor actions, and, as described in the companion article, a role in place memory. J. Comp.Neurol. 402:501–519, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: identified neurons; sensory integration; reafference; motor control; Periplaneta

americana

Traditionally, because they are so accessible for electro-physiological experiments, insect nervous systems havebeen considered ideal for understanding the neural basisof behaviors in terms of uniquely identifiable neurons.However, it is now recognized that even in such smallnervous systems the brain can contain more than 1.3million neurons, as in the honey bee (Witthoft, 1967). Mostof these neurons do not occur as unique pairs, but contrib-ute to ensembles of similarly shaped nerve cells as occursin vertebrates. Indeed, the cellular organization of sensoryneuropils in insects can resemble equivalent areas invertebrate brains, suggesting an unexpected degree ofevolutionary convergence. This finding is particularly no-ticeable in olfactory (Shepherd, 1992) and visual neuropils(Strausfeld, 1997).

In this context, the mushroom bodies are a brain regionof particular interest because their internal matrices arecomposed of many thousands, and in some taxa manyhundreds of thousands, of similarly shaped nerve cells.The mushroom bodies are present in most insect groupsand similar structures occur in many other arthropodssuch as chelicerates, chilopods, and diplopods (Strausfeldet al., 1998). Comparisons between the mushroom body

calyces and mammalian brain areas derive from therecognition that the position of the calyces in the olfactorypathway of neopterans is equivalent to the mammalianolfactory (piriform) cortex. The mushroom body lobes,which are supplied by the calyces, might then be comparedwith other olfactory-associated regions in mammals, suchas the hippocampus. Biochemical pathways thought to berelevant in learning and memory are common to bothhippocampus and mushroom bodies (Kandel and Abel,1995).

Grant sponsor: National Institutes of Health Fogarty InternationalFellowship; Grant number: F05 TW04390; Grant sponsor: National ScienceFoundation; Grant numbers: IBN-9316729, IBN-9726957; Grant sponsor:the Ministry of Education of Japan; Grant sponsor: Uehara MemorialFoundation; Grant sponsor: 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 402:501–519 (1998)

r 1998 WILEY-LISS, INC.

Traditionally the mushroom bodies have been describedas a center for intelligent actions (Dujardin, 1850; review,Strausfeld, 1998). Recent studies have stressed the crucialrole of the mushroom bodies in olfactory conditioning,learning, and memory (reviews, Erber et al., 1987; Davis,1993). There is some evidence that in certain insects themushroom bodies play a role in olfactory navigation(Vowles, 1964), place memory (Mizunami et al., 1993,1998), and the regulation of motor actions (Martin andHeisenberg, 1998). Other roles suggested for the mush-room bodies include their participation in mediating sex-specific behaviors (Ferveur et al., 1995) and the control ofother complex motor actions. For example, Huber (1959,1960) and Wadepuhl (1983) used electrical stimulation ofthe mushroom bodies and their surrounding neuropils toelicit extended episodes of courtship behavior in fieldcrickets. In honey bees, a change of behavior by the adultfrom nurse to active forager coincides with an increase ofthe juvenile hormone (JH) titer and is correlated withstructural changes in the mushroom bodies (Durst et al.,1994; Withers et al., 1995). Similarly, in crickets, a tran-sient increase in JH triggers neuroblast division in adultmushroom bodies and the birth of new intrinsic neurons(Cayre et al., 1994). These changes herald the onset ofbehaviors underlying courtship. If such a variety of func-tions is mediated by the mushroom bodies, as might besuggested, then mushroom body neurons should to somedegree reflect this by their electrophysiological activity.

We have examined neuronal activity in the mushroombodies of freely moving adult cockroaches, Periplanetaamericana. Here, we describe the identity and electricalactivity of 36 units recorded in the mushroom bodies fromamong 80 insects. Units responded to sensory inputs orwere activated in conjunction with motor actions. Record-ings were obtained from the brains of behaving cock-roaches in which 20-µm-diameter copper electrodes hadbeen chronically implanted. Neurons in the vicinity of theelectrodes were revealed after copper was released fromthe electrode. Units in the calyces responded exclusively tosensory stimulation, as would be expected from the re-ported structural organization of afferent supply to theseregions (Jawlowski, 1958; Mobbs, 1982, 1984; Gronenberg,1986; Homberg et al., 1989; Li and Strausfeld, 1997b;Strausfeld et al., 1998). Recordings from the pedunculusand the a- and b-lobes have detected types of nerve cellsthat have not been previously ascribed to the mushroombodies. Some of these distinguish self- from externalstimulation, whereas others report motor actions. Amongstthe latter are neurons whose activity predicts locomotionand whose spiking frequency is modified, depending on thedirection of turning during locomotion. Some of theseresults have been reported in a preliminary account (Mizu-nami et al., 1993).

MATERIALS AND METHODS

Experiments were performed on male American cock-roaches Periplaneta americana. Insects were obtainedfrom a mixed breeding colony generated from insectscaught in the wild as well as purchased from CarolinaBiological Supply Co. (Burlington, NC). Animals weremaintained in the laboratory at 20–24°C in moderatelycrowded conditions and a light-dark cycle of 11 and 13hours, respectively.

Electrode placement

The cockroach was restrained in a plastic tube, one endof which was closed the other open so as to expose theinsect’s head, which was temporarily fixed with sticky wax(Kerr, Romulus, MI). With its open end inclined upward,the tube was placed in a plastic box filled with ice. Thiscooled the insect’s body, thus slowing the heart rate andminimizing muscle contractions and blood loss while allow-ing access to the dorsal and posterior surface of the head.The insect remained cold-anesthetized during electrodeimplantation, which was achieved by first opening a smallarea of head cuticle to reveal the brain and the pairedmushroom bodies. The calyces of the mushroom bodies lielateral to the site of entry of the ocellar nerve and areflanked by the lateral protocerebrum and optic peduncu-lus. The medial (b) lobes of the mushroom bodies liesuperficially in the front of the brain, extending toward themidline some 350-µm above the entry of the antennalnerve into the brain. Appropriately angled light guidesallowed the paired calyces and the midline central body tobe distinguished during electrode placement. A smallincision was made on the brain surface, and an array of 2 32 or 2 3 3 Teflon-insulated 20-µm-diameter copper wireswere inserted into the brain. The wires were waxed to thehead capsule behind the opening, and a thicker (60-µm-diameter) copper wire, which served as the ground elec-trode, was implanted into the thorax. The cuticle wasreplaced over the opening and then waxed in place. Duringthe experiment, recording wires were connected to differen-tial AC amplifiers as described below.

Recording and stimulation

A total of 80 animals received electrode implantations.Thirty-six analyzable recordings and electrode markingsin the mushroom bodies were obtained from 67 insectsthat, together, provided recordings from the brain. After arecovery period of 15–30 minutes, the insect with itsimplanted electrodes was placed in an X-shaped dispos-able cardboard arena (see Fig. 1), each arm of which was8-cm wide and extended 10 cm from a central area of 8 3 8cm. The walls were smeared with Vaseline to preventescape. Each animal intended for recording had beenpreviously kept in a duplicate arena for 1 or 2 days beforethe operation to familiarize it with these surroundings andto reduce its tendency to escape.

Experiments commenced when an insect with im-planted electrodes showed normal mobility, usually 30–60minutes after reaching room temperature. Single- or multi-unit recordings were obtained differentially, between twoof the recording electrodes inserted into the mushroombodies, any of which could be remotely chosen for record-ing. The electrode showing the fewest units and the bestsignal-to-noise ratio was selected for the remainder of theexperiment. The recorded signals were stored on-line ontovideotape soundtrack, whereas the animal’s activitieswere video recorded. Analysis was made by using a Macin-tosh SE/Plus computer, with a WPI 121 window discrimi-nator and MacLab software for data analysis. Certain ofthe recordings are shown as unfiltered traces. Most othersare filtered recordings.

Sensory units are described with respect to only thesensory stimuli that elicited responses. In each experi-ment, the insect was subject to the same basic regimen,although the intervals between stimuli could vary, particu-

502 M. MIZUNAMI ET AL.

larly if a stimulus elicited locomotion. Stimuli were lighttouches to its abdomen, thorax, or antennae; a gentle airpuff to all these regions; and visual stimuli. An air puff or agentle touch to the body with a probe was usually effectivein inducing locomotion, although some units, describedtoward the end of this article, initiated their activity in theabsence of a given sensory stimulus. Stimuli includedhand-held visual patterns, olfactory cues, and tactile(mechanosensory) stimuli. Tactile and other mechanicalstimuli did not elicit escape responses. Behaviors weregenerally limited to simple body movements and locomo-tion: pronotal movements, antennation, cleaning behavior,forward and backward locomotion, and left and rightturns. Wind and tactile stimuli were applied to one of thefollowing body regions: antennae, head, thorax, abdomen,legs, and cerci. Visual stimuli were confined to ‘‘light on’’and ‘‘light off’’ or visual motion (by using a black and whitegrating). Olfactory stimulation consisted of a gentle aircurrent to the antennae from a syringe containing a filter

paper soaked with banana or orange extract. Controlstimulation was from a syringe with a blank filter paper.Elements of a behavioral sequence, recorded on videotape,were traced from stopped frames on the video monitor.

Histology

At the end of recording, copper ions were released intothe tissue by passing a positive 1–3 µAmp current betweenthe recording and the ground (negative) electrode for 3seconds. Between 5 and 20 minutes were allowed for iondiffusion, after which copper was precipitated to CuS byusing 5% ammonium sulfide carried in cockroach Ringer’ssolution (Yamasaki and Narahashi, 1959). The brain wasthen fixed for 1 hour in Gregory’s (1980) artificially agedBouin, removed from the head, washed in 70% alcohol, anddehydrated and permeabilized with propylene oxide. Brainswere next rehydrated for silver intensification by usingBacon and Altman’s (1977) procedure developed for orthop-

Fig. 1. Experimental design. The insect is attached to a bundle of4–6 recording electrodes and the ground electrode, but is free to movewithin an X-shaped arena. Electrophysiological recordings are ampli-fied (AMP), sent to an oscilloscope (CRO), and stored on a videotape

recorder (VTR) for quantitative evaluation or filtering with a MacLab(Mac) window discriminator. Behavior is videorecorded (CCD) on aseparate track of the recording tape.

MUSHROOM BODY ACTIVITY IN FREELY MOVING INSECTS 503

teran tissue. Afterward, the brains were dehydrated andembedded in Durcupan (Serva, Heidelberg, Germany) forserial sectioning at 30 µm. If intensified profiles werevisible when using transmitted light, reconstructions weremade with a camera lucida attached to a Leitz Diaplan,with an initial magnification of 2503. If neurons werevisible only by using darkfield illumination (Fig. 2), serialoptical sections were photographed onto Kodak Ekta-chrome 100 slide film (Eastman Kodak, Rochester, NY)and the profiles were reconstructed by using a slideprojector equipped with a front surface glazed mirror.Golgi-impregnated (Fig. 3) and Lucifer yellow–filled neu-rons (Fig. 4), used for comparison, were prepared accord-ing to previously published descriptions (see Strausfeldand Gilbert, 1992; Li and Strausfeld, 1997a). Figure 2 wasphotographed onto Kodak Ektachrome 100 ASA color slidefilm, which was digitized with a Polaroid SprintScan 45(Polaroid Corp., Cambridge, MA). Colors were transormedto a gray scale by using Adobe Photoshop (Adobe Systems,Inc., San Jose, CA), and the cotrast was adjusted toenhance silver-cobalt deposits. Reconstructed neurons (Fig.5) were montaged onto brain outlines drawn from Bodian(1936) reduced silver preparations with the drawing pro-gram ‘‘Smartsketch’’ (Futureware Software, Inc., San Diego,CA).

Terminology

Throughout, the terms ‘‘a’’ and ‘‘b’’ lobes are synonymouswith ‘‘vertical’’ and ‘‘medial’’ lobes. The latter are appli-cable to all species of insects, irrespective of whether theirlobes are simple and undivided, as in the cockroach, ordivided into several parallel entities none of which easilyfit the designations a and b (Strausfeld et al., 1998).The terms intrinsic neurons and Kenyon cells are usedinterchangeably.

RESULTS

Recovery from electrode implantation

After electrode implantation, about 80% of animalsappeared to behave normally and were used for subse-quent recordings. ‘‘Normal’’ is used to mean that individu-als showed behaviors seen before electrode implantation,the only observed difference being that operated animalsmoved less rapidly than unoperated ones because theycarried the weight of the electrodes. Animals exploredtheir arena while antennating the floors and walls. ‘‘Nor-mal’’ postoperative cockroaches ate and drank, groomedtheir antennae, cleaned their legs, and had periods ofinactivity during which they occupied the darkest part ofthe arena.

Extracellular recordingsand correlative histology

The use of an array of several electrodes allowed theexperimenter to choose from several possible recordingsites by switching from one electrode to another and thenselecting the electrode that provided the most stablerecording from the fewest units. Recordings culminatedwith the controlled release of copper. This method had thedouble advantage that it could be used on freely behavinginsects and that the recording electrode can be related to aspecific part of the mushroom bodies or even, in somecases, to neurons in the immediate vicinity of the electrode

(Fig. 2). Although it cannot be proven that the stainedneurons are the ones from which recordings were obtained,this is likely for the following reasons: (1) The stainedneurons are closest to the electrode tip, the position ofwhich can often be surmised from the electrode’s tract,whose cul de sac is surrounded by a darker halo ofsilver-copper deposits. (2) The morphologies of elementsstained by copper-silver correspond well to the shapes ofGolgi-impregnated (Fig. 3) or Lucifer yellow–filled neu-rons (Fig. 4) known to be associated with the mushroombodies (Li and Strausfeld, 1997a). (3) Although branchesfurthest from the electrode tip are rarely resolved, axonsleading away from the site of the electrode (Fig. 5) projectto brain regions known to receive the terminals of indi-

Fig. 2. Anatomical correlates to extracellular recordings. Darkfieldmicrograph of silver-intensified copper sulfide deposits in neuronalprocesses. The figure shows a frontal section showing a calyx (ca),pedunculus (ped), and medial lobe (b). A band of extrinsic neuronprocesses intersects the junction between the medial lobe and pedun-culus. Processes outside the mushroom body reside in one of themushroom body satellite neuropils (sat) that receives axon collateralsfrom b-lobe efferent neurons (Strausfeld, 1998). Ten laminae, eachindicated by a short arrow, are visible in this preparation. Eachrepresents a lamina of intrinsic cell axons (Li and Strausfeld, 1997a;Mizunami et al., 1997). Intrinsic cells synapse onto the dendrites ofefferent neurons (Strausfeld, 1998) and have here taken up copper.The cell bodies (cb) of copper-silver-stained Kenyon cells reside withinthe cup of one of the calyces (ca). A second area of intensification (i)represents recurrent processes into the a-lobe, which is almost out ofthe plane of section. Scale bar 5 100 µm.


vidual extrinsic neurons, as revealed by Lucifer yellowhistology. These axons are mainly in superior protocere-bral neuropils each side of the mushroom bodies (Li andStrausfeld, 1997a).

It was unusual to obtain an extracellular recording froma single neuron. This means that single units usually hadto be resolved by using a window discriminator. However,because the electrode array was small (60- to 80-µmdiameter) and because the raw unfiltered recordings sug-gested at most eight active units, we propose that therecorded activity was from within a small volume of themushroom body. When several units are recorded simulta-neously, their similar responses to the same sensory

stimuli suggest that the mushroom bodies comprise en-sembles of similar neurons responding in concert, ratherthan single uniquely identifiable neurons. This suggestionis supported by (1) observations of Bodian impregnationsthat show efferent neurons packed close together through-out the a- and b-lobes (Li and Strausfeld, 1997a; Straus-feld et al., 1998), and (2) Golgi preparations that identifysimilarly shaped dendritic trees within the same region ofthe pedunculus or lobes (Fig. 3) but show diverging axonprojections. Observations of copper-silver–intensifiedpreparations demonstrated that when copper was incorpo-rated into neurons, at least two, and often as many as eightneurons were stained in proximity (Fig. 5). This finding is

Fig. 3. Anatomical correlates to copper-silver staining. Partialimpregnation by the Golgi method reveals three similar and overlap-ping dendritic trees of b-lobe extrinsic neurons, two providing axon

collaterals to satellite neuropil (sat) but sending their axons todifferent protocerebral targets. s m pr, superior medial protocerebrum;ca, calyx; ant lob, antennal lobe.


again evidence that the electrode sampled neighboring oroverlapping extrinsic neurons whose axons diverge todifferent targets in the protocerebrum. However, it mustbe cautioned that Lucifer yellow fills have identifiednumerous afferent neurons terminating in the lobes (Liand Strausfeld, 1997a,b; Strausfeld et al., 1998). This

means that when referring here to an ‘‘extrinsic’’ neuron,there is a degree of uncertainty about whether the cell isan efferent or afferent.

Darkfield illumination was especially useful for reveal-ing faintly stained elements. This finding is illustrated inFigure 2, where copper has been taken up by four to six

Fig. 4. Anatomical correlates to copper-silver staining. Intracellu-lar Lucifer yellow fills of efferent neurons match many of the extracel-lular stainings, as shown by this reconstructed extrinsic neuron. Itsdendrites reflect the laminar organization of Kenyon cell axons in themushroom bodies, as demonstrated by silver staining and immunocy-tology (Li and Strausfeld, 1997; Strausfeld, 1998). The axon providesfive distinct arbors outside the mushroom bodies. At 1 and 3, processes

invade neuropils just medial to the lateral horn (l ho) and above theinferior lateral protocerebrum (i l pro). Spiny processes (at 2), sugges-tive of postsynaptic sites flank the a-lobe (2). Varicose processes,suggestive of presynaptic sites, lie in front of the a-lobe (4) and insatellite neuropil (5) in front of b. Other abbreviations: ca, calyx; ped,pedunculus; ant lob, antennal lobe; FB, fan-shaped body of the centralbody complex.


Fig. 5. Anatomical correlates to extracellular recordings. Six cam-era lucida drawings illustrate copper uptake and silver staining byneurons. A: Approximately seven extrinsic neurons are revealed, alloriginating from a cluster of cell bodies medial to the antennal lobes(ant lob). Three provide ascending axons to the superior medialprotocerebrum (s m pr). One axon extends into the pedunculus (ped)and corresponds to terminals, identified by Lucifer yellow injectionthat originate from the ventral bodies (Strausfeld, 1998). B: Threeextrinsic neurons with overlapping processes in the b-lobe providediverging axons. Two can be followed to the inferior lateral (i l pr) and

inferior medial protocerebrum (i m pr). C: Processes in the b-lobe,possibly corresponding to an afferent neuron revealed by Luciferyellow injection to originate in the superior medial protocerebrum (s mpr; Li and Strausfeld, unpublished data). D: Possible afferent neuronsin the a–b-lobe junction and part of an efferent within the a-lobe, theaxon of which extends to the lateral horn (l ho). FB, fan-shaped body.E: Partially stained efferent dendrites at the a–b-lobe junction withone axon projecting into the inferior lateral protocerebrum (i l pr).F: Mixed efferent and possibly afferent (in b-lobe) elements with axonsascending to the superior protocerebrum. ped, pedunculus.


neurons, all clustered together with dendrites invadingthe same restricted area of the b-lobe (see reconstruction offull depth of mushroom body, Fig. 5A). These cells areclearly identifiable as extrinsic neurons whose axons ex-tend into other protocerebral neuropils (see, Li and Straus-feld, 1997a). The neurons shown in Figure 2 arborizewithin the b-lobe, sending axons medially into the superiormedian protocerebrum and within a special satellite re-gion beneath and to the front of the medial lobe (Straus-feld, 1998). Neurons corresponding to this organization inthe b-lobe and satellite neuropil are revealed by Golgiimpregnations (Fig. 3) and by intracellular Lucifer yellowfills of single extrinsic cells, such as in Figure 4.

This neuron, which responds to visual and olfactory cues(Li and Strausfeld, 1997a), has dendrites at the tip of theb-lobe and what appears to be a second set of dendriteslaterally, in the superior lateral protocerebrum, flankingthe a-lobe. Axon collaterals also terminate in satelliteneuropil in front of the lobes. Its dendrites within theb-lobe show a characteristic pattern of banding, whichcorresponds to the laminar arrangements of intrinsicaxons in the lobe (Li and Strausfeld, 1997a; Mizunami etal., 1997). Electron microscopy shows that bands of effer-ent neuron dendrites are postsynaptic to profiles in alter-nating laminae (Strausfeld, 1998), a relationship that isdiscernible in Figure 2.

There, 10 silver-intensified laminae alternate with lami-nae that are silver-free. The stained laminae extend backfrom the stained dendritic branches of extrinsic neuronswithin the b-lobe into the pedunculus, toward the calyces.Numerous Kenyon cell bodies are intensified in one of thecalyces, demonstrating that it is these neurons that pro-vide lamination. Examples of silver-intensified copper-

labeled neurons are shown in Figure 5, with their relevantelectrophysiological and behavioral correlates describedbelow.

Functional activity in the mushroom bodies

Sensory units. Electrodes implanted into the calycesand pedunculi recorded units that were activated by thefollowing unimodal sensory stimuli: (1) visual stimulation,consisting of ‘‘light on’’ and ‘‘light off,’’ or moving gratings;(2) mechanosensory stimuli, consisting of tactile stimula-tion or the application of an air current to the legs,abdomen, thorax, or antennae; (3) olfactory stimulation,consisting of odor sources provided by fresh banana ororange, or their extracts in neutral oil. Recordings wereusually obtained from several units from which singleunits were isolated by using a window discriminator (seeMaterials and Methods). Responses to unimodal stimulicould be elicited both in walking and stationary individuals.

Responses to visual stimuli are shown in Figure 6.Visual units responded to ‘‘light off’’ (Fig. 6A), ‘‘light on,’’

or movement of gratings (Fig. 6B). Unimodal visual unitsdistinguished motion from steady state illumination (sus-tained firing versus a rapid decrement in firing rate) butdid not show directional selectivity. Subsequent copper-silver intensification revealed that the recording elec-trodes were located within the calyx neuropil and thatcopper could be traced from the calyces toward the lateralhorn. Lucifer yellow fills (Strausfeld et al., 1998) haveidentified afferents from the lateral horn to the calycesthat respond to visual stimuli.

Recordings from the calyces showed unimodal elementsthat responded to specific odors, discriminating these from

Fig. 6. Visual responses correlate to silver-copper staining of atract (stippled, as in other similar figures) between the calyces andlateral horn (right drawing). A: Peristimulus time histogram demon-

strates elevated activity in response to light off. B: Other unitsrecorded from a different animal responded to light-on (upper trace)and to motion, irrespective of direction.


Fig. 7. Units responding to odor and silver-copper staining in thecalyces and part of the antenno-calycal tract (right drawing).A: Peristimulus time histograms demonstrate olfactory responses ofcertain units to an air current carrying banana extract odor but not to

orange extract odor or the carrier stimulus. B: A second calycal unitresponds to both banana and orange odors, discriminating these fromthe carrier stimulus (air) alone.

Fig. 8. Unit responses of mechanosensory neurons in the calyx(right) responding to tactile stimulation of the antennae, the hindlegs,and abdomen. The same unit responded to a gentle air current onto the

cerci. This example demonstrates the surprisingly generalized repre-sentation of sensory information in the mushroom bodies from manyreceptive areas on the body.


the carrier stimulus, which was a constant air current tothe ipsilateral antenna. Other parameters, such as concen-tration or spatial location on the antenna, were not testedin these experiments. The results, summarized in Figure7A,B, show peristimulus histograms that compare theresponses of two recordings in two individuals with thetest extracts. The unit shown in Figure 7A was excited bybanana extract and showed no response to the odorlesscontrol wind stimulus or to the orange extract. The secondunit (Fig. 7B) responded to both banana and orangeextract but not to the control stimulus. Subsequent histo-chemistry revealed that, in both preparations, the record-ing electrode was located in the medial calyx. Silverintensification demonstrated copper uptake correspondingto the passages of tracts extending to the calyx from theipsilateral antennal lobe by means of the inner and outerantennocerebral tract (also known as the antennoglomeru-

lar tract), the organization of which has been describedfrom lepidopteran insects by Homberg et al. (1989), fromcockroaches by Malun et al. (1993), and from Diptera(Drosophila; Stocker, 1994). Copper-silver staining associ-ated with olfactory responses corresponds to the axonaltrajectories of intracellularly filled relay neurons thatproject from olfactory glomeruli to the lateral horn, withtributaries into the calyces (Malun et al., 1993; Li andStrausfeld, 1997a).

Recordings from the calyx and a-lobe (see below) haveidentified units responding to tactile stimuli applied todifferent body parts. Some units, recorded from the calyx,were activated by tactile stimuli applied to the antennae,cerci, abdomen, and hind leg, and to wind stimuli to theantennae and cerci (Fig. 8). Other units responded to bothtactile and wind stimulation. In some cases, their range ofsensitivity included responses to imposed stimulation

Fig. 9. Antenna-specific mechanosensory responses in the calyx.Among the unfiltered responses (a–c), at least two units can beidentified that respond to both tactile (a) and wind stimuli (b) to theipsilateral antenna. c: Both units are activated by self-stimulation

during active antennation of the substrate. Peristimulus time histo-grams demonstrate, in another cockroach, calycal units that areactivated by antennal movements, by antennation of the substrate,and by imposed tactile stimulation of the antennae.


(Fig. 9, recordings a and b) as well as responses to tactilestimuli caused by the insect’s own antennation (Fig. 9,recording c). Copper-silver staining of the brain demon-strated processes at the base of the pedunculus and originof the a-lobe (Fig. 5D). In contrast, other units, stained inthe b-lobe, discriminated between externally appliedstimuli, such as touch or wind onto the antenna, andself-stimulation. The units responded to tactile stimuli butwere suppressed when the insect groomed its antenna(Fig. 10B).

One class of responses that could not be interpretedsimply as mechanosensory units was by elements thatshowed activity only when an appendage or a body partsuch as the pronotum and head adopted a particularposition. Figure 11 shows three recordings (a–c), fromthree different individuals in which units were activatedonly when an antenna swept through a particular segmentof its sensory field. The recorded units are best interpretedas monitoring a motor action, possibly detected by proprio-ceptors at the base of the antenna, because the samedischarge could be elicited by an imposed mechanicaldisplacement of the antenna in the same direction. Silverintensification of the brains of these insects revealedcopper in tracts to the calyces leading from posteromedialneuropils in the deutocerebrum (the dorsal lobe), a locationcorresponding to known termination areas of primarymechanosensory neurons of the antennae (Sanchez, 1937)as well as termination areas of ascending interganglionicinterneurons from the thoracic-abdominal ganglia.

Motor reporting units. A variety of units, later identi-fied as originating from the b-lobe, were active duringlocomotory actions. These units either reported limb move-ments, or they began their activity before the onset oflocomotion and their subsequent activity was modulatedaccording to the direction of locomotion.

Because movement itself provides the organism withsensory stimulation, it is important to distinguish unitsthat report the sensory consequences of a motor output, asdescribed above, from units that seem to be directlyrelated to efferent control of the motor action itself. Thesedistinctions are not always clear-cut, as exemplified inFigure 12, where several units, all responding to mandibu-lar grooming of the foreleg (a in Fig, 12B), were alsoactivated during walking (b in Fig. 12B) and more vigor-ously so during running (c in Fig. 12B). That theseresponses reflect the tempo of leg movement is suggestedby differences in their firing rate. In the first example, theleg is drawn slowly across the mouthparts and unitsrespond with intermittent bursts of activity. In the walk-ing and running animal, as foreleg movements increasetheir tempo the spike rate increases accordingly. When theanimal stops, even briefly (C2 in Fig. 12B), the unitsreturn to their resting potential. Silver intensificationdemonstrated that the recording was made in the immedi-ate vicinity of a cluster of extrinsic neurons, the dendritesof which invade a small area of the b-lobe and transectparallel arrays of axons arriving there from the calyces(Figs. 2, 5E). The axons of these extrinsic neurons send

Fig. 10. Discrimination of an imposed stimulus versus a self-generated stimulus. Video frame drawings (A) show events duringrecording (B). Video frames (numbered in A) are indicated above eachtrace (1, 2, etc. in B). Wind to the right antenna (event 1 in A-a;response 1 in B-a), but not the left antenna, elicits phasic activity(unfiltered responses). Sustained wind from the same side, but to the

whole head (event 1 in A-b) elicits activity (1 in B-b) of a b-lobe unit.Activity was interrupted by a bout of cleaning (events 2, 3 in A-b)during which the unit was suppressed (2,3 in lower trace B-b). Windstimulus during cleaning (events 3 in A-b) elicited a response (in lowertrace B-b) only after cleaning had ceased (event 4 in A-b).


axons that ascend outside the lobes back toward the calyx,suggesting their identity as neurons linking the b-lobe tothe medial protocerebrum, just lateral to the calyces.

Some units, correlated with neurons in the a- or b-lobes,respond to a variety of sensory inputs and are trulymultimodal. The unit shown in Figure 13, which was anexceptionally clean recording, was histologically located tothe b-lobe. It responded to odor, tactile stimuli, andillumination. With respect to these modalities, the neuronis reminiscent of a multimodal afferent neuron recordedintracellularly that invades the lobe at the same location(Li and Strausfeld, 1997a,b).

Another unit that appeared to report motor actions isshown in Figure 14, later correlated to copper uptake byseveral cells partly reconstructed in Figure 5F. Activity inthe a- (or possibly b-) lobe was evoked during lowering ofhead by apparent flexion of the pronotum, and the unitdischarged as long as flexion was sustained. Whether headposition is reported to the brain by sensory feedback fromthe thorax or by motor corollary discharge is not known.If the latter, however, then the responses of these mush-room body units would represent an example of motorreafference.

Motor prediction units. Unambiguous responses ofthe mushroom body in conjunction with motor output,rather than sensory reafference, are shown by units thatsustain their spiking activity throughout motor actions.Some of these units are activated by sensory inputs, others

are not. Most typically they show elevated discharge ratessome 1–2 seconds before a specific motor action ensues,thereby, predicting motor output. Figures 15–17 illustratethe response properties of three such elements, in increas-ing complexity, and the behavior with which they areassociated.

In the first example (Fig. 15), the mushroom body unitresponded to tactile stimulation of the hindleg or thethorax. However, its discharge pattern differed, dependingon whether stimulation gave rise to a motor response ornot. Tactile stimulation, without a consequent behavior,resulted in a phasitonic response, lasting only about 1second, followed by occasional burstlike discharges (a inFig. 15B). However, when tactile stimulation elicited abehavioral response, such as a turning movement awayfrom the direction of stimulus (events 2–4 in b, Fig. 15B),unit activity persisted for the duration of the motor action,although the source of stimulation had been withdrawn.Interestingly, this unit also showed bursts of activity thatimmediately preceded an episode of behavior (walking),which lasted some seconds. During this behavior, the unitmaintained its activity except when the behavior itself wasinterrupted (c in Fig. 15B).

In the second and third examples (Figs. 16, 17), unitactivity preceded locomotion in the absence of any givensensory input stimulus. For the unit described in Figure16, three behavioral episodes are shown with the corre-sponding video frames, each indicated above one of the

Fig. 11. Units reporting antennal position. Recordings from threeindividuals. Units respond when the antenna moves through a specificarea within its tactile space (shaded zones in a–c): a, dorsally; b,

anteriorly; c, laterally. Record c shows a filtered phasitonic position-dependent response superimposed on a spontaneous backgrounddischarge.


accompanying electrophysiological traces. The initiation ofany movement (frame 1, a turning of the antennae) ispreceded by 300–800 msec of sustained spiking activity,which continues during forward motion and turning to theleft or right. Interestingly, the unit differed from that

shown in Figure 15 in that it sustained its firing rateduring a pause in locomotion, indicated by event 3 in traceb, Figure 16B.

A third class of motor-associated elements (Fig. 17) alsopredicted bouts of locomotory activity by exhibiting an

Fig. 12. A,B: Increasingly vigorous responses of mushroom bodyunits to self-stimulation, i.e., cleaning of a foreleg (a), walking (b), andthen running (c). Video frames (numbers) in these episodes (a–c in A)

are indicated above the relevant electrophysiological records (a–c inB). Each group of recordings shows unfiltered responses of about 6units above filtered data selecting two units.


abrupt two- to threefold increase in its firing rate, from aspontaneous level of about 18 spikes/second, startingabout 250 msec before the onset of locomotion, again in theabsence of any imposed stimulation. This high firing ratewas maintained during locomotion (episode a), but whenlocomotion ceased the firing rate diminished to its initialfrequency. After about 8 seconds of this low-level discharge

level, a second episode (episode b) of locomotion was againpreceded by 2 seconds of an elevated firing rate, which wasmaintained during forward motion and turns to the right.However, activity ceased completely during two left turns(events 5 and 7 in episode b). In a third episode (c, Fig.17B), unit activity again began before the onset of move-ment and again ceased during left turns.

Fig. 13. A multimodal unit in the b-lobe that responds to odor, to ipsilateral and contralateral tactilestimuli, and to visual stimuli to either compound eye. a: Unfiltered response. b–h: Filtered signals.Drawing to the right shows the position of copper-silver deposits.

Fig. 14. Peristimulus histogram of pedunculus–b-lobe units reporting head position (raised, 1;lowered, 2) probably due to movements of the pronotum rather than the head.


Copper-silver histology localized these motor reportingunits to clusters of perikarya flanking the neuropil nearthe junction between the pedunculus, a-, and b-lobes (Fig.5A,B). The dendrites of these neurons extend into thepedunculus and into the b-lobes but omit the a-lobes.Axons arising from these neurons project into the lateraland medial protocerebrum, on the same side as the re-corded pedunculus. As described from the cockroach Blatta(Sanchez, 1937), this region receives sensory inputs frommechanosensory afferents. Lucifer yellow fills into extrin-sic neurons from the b-lobes of Periplaneta demonstratethe same projection into this area (Li and Strausfeld,1997a).

DISCUSSION

Chronic extracellular recordings in freely moving andbehaving cockroaches have been used to record widelydiffering electrophysiological responses from different partsof the mushroom bodies. Silver-intensified copper deposi-tion and cell uptake correlate electrode locations to extra-cellularly recorded units. At their best, silver-copper depos-

its correspond to extrinsic neurons, recognized bycomparison with Lucifer yellow dye fills (Li and Straus-feld, 1997a). At their worst, copper-silver deposits fail toresolve cellular elements but indicate the general vicinityof the electrode within the mushroom bodies. This discus-sion considers evidence that the mushroom bodies areinvolved in multimodal sensory integration as well as inregulating motor actions.

Sensory units in mushroom bodies

Table 1 summarizes the results of recordings from unitsfor which copper-silver staining revealed the trajectories ofaxons to or from the mushroom bodies or the position of therecording electrode. In 14 preparations, copper-silver inten-sification revealed staining in the calyces and occasionallyinto one or more tracts extending to the calyces fromneuropils in the protocerebrum and deutocerebrum. Threecorollaries to these findings are (1) sensory elements canrepresent more than one modality, (2) responses to tactilestimuli are surprisingly generalized and do not representrestricted areas of the body, and (3) stained tracts to thecalyces correspond to previous descriptions of calycal

Fig. 15. A,B: Unfiltered signals from two to three sensory unitsresponding phasitonically (B-a) to tactile stimulation of either hind leg(A-a) and more vigorously (B-b) to stimulation of the thorax (A-b).Stimulation of the thorax initiated turning, locomotion, or both,

during which units continue their activity (1–4, B-b) until movementceased. The same units are activated (B-c) when the insect walksspontaneously (A-c).


Fig. 16. A,B: Filtered recordings from the pedunculus/b-lobe junc-tion of one individual showing a motor ‘‘predictive’’ unit. The unitbegan its activity 0.8–0.3 seconds before the onset of locomotion(indicated by 1 in each sequence/trace). The response persisted during

locomotion, increasing in frequency during a change in direction (3a,2b, 4b, 5b, 2c) and during accelerated forward motion (3c-4c) after aturn. Cessation of locomotion (4a, 4c) is accompanied by a return to theresting discharge frequency.

Fig. 17. A,B: Motor predictive unit recorded from the mushroombody of a single individual. The unit is spontaneously active duringrest (B-a). A vigorous tonic increase occurs 0.3–2.0 seconds before the

onset of motion (at 1) and persists during right turns and forwardmotion. Leftward turns inhibit this unit’s activity (episode b, events5–6, 7–8 and episode c, event 5).

supply from the olfactory (antennal) lobes of Periplaneta(Boeckh and Ernst, 1987; also, Gryllus bimaculatus, Schild-berger [1983]; Manduca sexta, Homberg et al. [1989]; Apismellifera, Mobbs [1982, 1984]; Drosophila, Stocker [1994])and from the dorsal lobes (Apis, Jawlowski [1958]). It hasbeen recently reported that the protocerebrum providesmany afferents to the calyces and lobes (Li and Strausfeld,1997b; Strausfeld et al., 1998), and some staining revealedhere, in association with sensory units, probably reflectsthis organization. Of interest in this context are units inthe medial lobes responding to bilateral stimulation, sug-gesting that the lobes receive heterolateral afferent inputs.Although dye-filled afferents so far identified are all unilat-eral (Li and Strausfeld, unpublished data), they may wellreceive bilateral inputs from sensory neuropils (Strausfeldet al., 1998).

It may seem counterintuitive that units interpreted assensory afferents can carry more than one modality. How-ever, intracellular recordings show many afferents to themushroom bodies to be multimodal (Li and Strausfeld,1997a,b; Strausfeld et al., 1998). This finding supports thepresent results because it suggests that mushroom bodies,at least in Periplaneta, are not integrators of pure unimo-dal sensory data but receive already integrated sensoryinformation.

Units in the mushroom bodiesassociated with motor actions

Intracellular recordings from mushroom body output(efferent) neurons in Orthoptera (Schildberger, 1984;Homberg, 1984), Hymenoptera (Gronenberg, 1987), andPeriplaneta (Li and Strausfeld, 1997a) demonstrate theirmultimodal nature, although none of the cited studiesdescribed neurons recorded during motor activity. Here, byusing extracellular recordings, units that were associatedwith motor events were recorded in 13 insects. Three ofthese were recorded from the calyx and are interpreted asresponding to antennal motion. These signals may beproprioceptive in origin. Units that relate to pronotalmovements that alter head position or to locomotion andthat were activated by tactile stimulation were identifiedin the pedunculus and b-lobe or in the a-lobe. Six units inthe pedunculus–b-lobe junction and two in the a-lobe wereassociated with motor actions and were recorded indepen-dently of a given sensory stimulus.

The mushroom bodies have long been suspected to playroles in controlling motor actions (see Huber, 1959, 1960).However, apart from a preliminary account (Mizunami etal., 1993), there has been little supporting evidence thatthey do so. The exception is a report on studies thatsuggests that Drosophila without mushroom bodies areunable to regulate motor actions, with the consequence

that periods of walking activity are far longer than inwild-type flies (Martin and Heisenberg, 1998).

One problem in relating the mushroom bodies to motoractions is that there is little evidence that efferent neuronsare organized so as to directly modify premotor pathways.To do so, mushroom body efferents would be expected tosynapse directly onto descending neurons. One reportdescribes two outputs from the mushroom bodies of Cal-liphora terminating amongst descending neurons (Straus-feld et al., 1984). And, in Periplaneta, only a single efferenthas been identified with a branch to a descending neuronaxon (Li and Strausfeld, 1997a). It is not known whetherany of these relationships involve synaptic connections.Most axons revealed by copper-silver staining ascend tovarious regions of the protocerebrum, as do efferents frommushroom bodies in Drosophila (Ito et al., 1998). Ingeneral, therefore, the evidence supports the view thatensembles of overlapping efferent neurons send divergentaxons to a variety of other ‘‘higher’’ neuropils of theprotocerebrum and that this activity represents the bulk ofthe mushroom body output.

Role of the mushroom bodiesin sensorimotor control

Paradoxically, many modern studies have assumed thatthe mushroom bodies have a relatively simple morphologyand that their main role is in supporting olfactory learningand memory (review, Davis, 1993; but see Erber et al.,1980). The structural basis for this notion is obvious: thereis a major supply to the calyces from the antennal lobes.Olfactory interneurons then contact many thousands ofKenyon cells, the axons of which project in parallel throughthe lobes where they are intersected by dendrites ofefferent neurons.

Although the mushroom bodies have been shown to playa role in learning and memory (review; Erber et al., 1980;Heisenberg, 1998), this is unlikely to be their only functionand certainly cannot be generalized across the Insecta.One reason is that our understanding of the internalorganization of mushroom bodies has recently been re-vised from genetic and immunocytologic studies. In Dro-sophila, enhancer trap lines reveal longitudinal subdivi-sions through the lobes that are correlated with subsets ofKenyon cells (Yang et al., 1995; Ito et al., 1997). Similarsubdivisions have been suggested for honey bees from theresults of affinities to FMRFamide (Schurmann and Erber,1990), and in cockroaches, as discussed above (Li andStrausfeld, 1997a; Mizunami et al., 1997; Strausfeld et al.,1998). If mushroom bodies are subdivided into discreteparallel modules, do these support different roles in sen-sory integration, one being that of integrating motoractions with sensory perception? The present accountdemonstrates a number of features new to mushroombodies that support this possibility: units that discrimi-nate autostimulation from external stimulation, as in thecase of antennal grooming versus imposed antennal stimu-lation, and units that either report, or predict and report,motor actions.

Possibly, such units reflect a role by the b-lobes inintegrating motor reafference with sensory inputs and inpatterning motor output. Already in 1896, Kenyon (1896)suggested that two sensory-motor pathways existed in thehoney bee brain, one direct and reminiscent of sensory-motor pathways in ganglia and the other indirect, bymeans of the mushroom bodies (review; Strausfeld, 1998).There have also been claims that the removal or lesioning

TABLE 1. Number and Location of Recordings from the Mushroom Bodiesof Active Animals

Type of unit(s)recorded

No. units recorded

CalyxPedunculus–

b-lobe a-lobe Total

Sensory 14 1 15Motor-associated

Antennal movement 3 1 4Head (pronotal) movement 2 1 3Locomotion 5 1 6

Motor-predictive 6 2 836


of mushroom body neuropils results in drastically alteredbehavior (van der Kloot and Williams, 1953; Vowles, 1964).Stimulation of neuropils at and around the mushroombodies evokes motor patterns (Huber, 1959; Howse, 1974).It is also significant that palaeopteran insects lackingantennal lobes and calyces possess mushroom bodies withprominent medial lobes, suggesting that phylogeneticallybasal mushroom bodies are not associated with olfactoryprocessing but with other functions (Strausfeld et al.,1998).

In conclusion, the present results suggest that mush-room bodies reveal a far greater variety of functional celltypes than would be expected if these structures supportedmainly olfactory perception and association. Further ex-periments are necessary to determine whether motoractions are represented in the mushroom bodies, so contrib-uting to mechanisms underlying sensory integration andmemory, or whether mushroom bodies plan motor actions.

ACKNOWLEDGMENTS

We have profited from discussions with Drs. Cole Gil-bert, and Wulfila Gronenberg and Kei Ito. We are gratefulto Ms. Carol Arakaki B.S. for assistance with the histologyand to Mr. Robert Gomez B.S. for photographic assistance.We thank Dr. Camilla Strausfeld for reading and correct-ing the manuscript.

LITERATURE CITED

Bacon, J.P. and J.S. Altman (1977) A silver intensification method forcobalt-filled neurones on wholemount preparations. Brain Res. 138:359–363.

Bodian, D. (1936) A new method for staining nerve fibres and nerve endingsin mounted paraffin sections. Anat. Rec. 65:89–97.

Boeckh, J. and K.-D. Ernst (1987) Contribution of single unit analysis ininsects to an understanding of olfactory function. J. Comp. Physiol. A161:549–565.

Cayre, M.C., Strambi, C., and A. Strambi (1994) Neurogenesis in an adultbrain and its hormonal control. Nature 368:57–59.

Davis, R. (1993) Mushroom bodies and Drosophila learning. Neuron111:1–14.

Dujardin, F. (1850) Memoire sur le systeme nerveux des insects. Ann. Sci.Nat. Zool. 14:195–206.

Durst, C., S. Eichmuller, and R. Menzel (1994) Development and experiencelead to increased volume of subcompartments of the honey bee mush-room body. Behav. Neural Biol. 62:259–263.

Erber, J., T. Masuhr, and R. Menzel (1980) Localization of short-termmemory in the brain of the bee, Apis mellifera. Physiol. Entomol.5:343–358.

Erber, J., U. Homberg, and W. Gronenberg (1987) Functional roles of themushroom bodies in insects. In A.P. Gupta (ed): Arthropod Brain. NewYork: Wiley-Interscience, pp. 485–511.

Ferveur, J.F., K.F. Stortkuhl, R.F. Stocker, and R.J. Greenspan (1995)Genetic femininization of brain structures and changed sexual orienta-tion in male Drosophila melanogaster. Science 267:902–905.

Gregory, G.E. (1980) The Bodian protargol technique. In N.J. Strausfeldand T.A. Miller (eds): Neuroanatomical Techniques: Insect NervousSystem. New York: Springer, pp. 75–95.

Gronenberg, W. (1986) Physiological and anatomical properties of opticalinput-fibers to the mushroom body of the bee brain. J. Insect Physiol.32:695–704.

Gronenberg, W. (1987) Anatomical and physiological properties of feedbackneurons of the mushroom bodies in the bee brain. Exp. Biol. 46:115–125.

Heisenberg, M. (1998) What do mushroom bodies do for the insect brain?Learning Memory 5:1–10.

Homberg, U. (1984) Processing of antennal information in extrinsic mush-room body neurons of the bee brain. J. Comp. Physiol. A 154:825–836.

Homberg, U., R.A. Montague, and J.G. Hildebrand (1989) Anatomy ofantenno-cerebral pathways in the brain of the sphinx moth Manducasexta. Cell Tissue Res. 254:255–281.

Howse, P.E. (1974) Design and function in the insect brain. In L.B. Browne(ed): Experimental Analysis of Insect Behavior. Heidelberg: Springer,pp. 180–195.

Huber, F. (1959) Auslosung von Bewegungsmustern durch elektrischeReizung des Oberschlundganglions bei Orthopteran (Saltatoria: Grylli-dae, Acridiidae). Z. Vgl. Physiol. 43:359–391.

Huber, F. (1960) Untersuchungen uber die Funktion des Zentralennerven-systems und insbesondere des Gehirns bei der Fortbewegung und derLauterzeugung der Grillen. Z. Vgl. Physiol. 44:60–132.

Ito, K., W. Awano, K. Suzuki, Y. Hiromi, and D. Yamamoto (1997) TheDrosophila mushroom body is a quadruple structure of clonal unitseach of which contains a virtually identical set of neurones and glialcells. Development 124:761–771.

Ito, K., K. Suzuki, P. Estes, M. Ramaswami, D. Yamamoto, and N.J.Strausfeld (1998) The organization of extrinsic neurons and theirimplications in the functional roles of the mushroom bodies of Dro-sophila melanogaster Meigen. Learning Memory. 5:52–77.

Jawlowski, H. (1958) Nerve tracts in the bee (Apis mellifica) running fromthe sight and antennal organs to the brain. Ann. Univ. Mariae Curie-Sklodowska Lublin. 12:265–268.

Kandel, E. and T. Abel (1995) Neuropeptides, adenylyl cyclase, and memorystorage. Science 268:825–826.

Kenyon, F.C. (1896) The meaning and structure of the so-called ‘‘mushroombodies’’ of the hexapod brain. Am. Nat. 30:643–650.

Li, Y.-S. and N.J. Strausfeld (1997a) Morphology and sensory modality ofmushroom body efferent neurons in the brain of the cockroach, Peripla-neta americana. J. Comp. Neurol. 387:631–650.

Li, Y.-S. and N.J. Strausfeld (1997b) Afferents supplying insect mushroombodies carry multimodal sensory information. Soc. Neurosci. Abstr.23:1571.

Malun, D., U. Waldow, D. Kraus, and J. Boeckh (1993) Connections betweenthe deutocerebrum and the protocerebrum, and neuroanatomy ofseveral classes of deutocerebral projection neurons in the brain of malePeriplaneta americana. J. Comp. Neurol. 329:143–162.

Martin, J.-P. and M. Heisenberg (1998) Effect of mushroom-body onlocomotor activity in Drosophila. In N. Elsner and R. Wehner (eds): NewNeuroethology on the Move (Proceedings of the 26th Gottingen Neurobi-ology Conference. Vol. II). Stuttgart: Thieme, p. 70.

Mizunami, M., J.M. Weibrecht, and N.J. Strausfeld (1993) A new role forthe insect mushroom bodies: Place memory and motor control. In R.D.Beer, R. Ritzmann, and T. McKenna (eds): Biological Neural Networksin Invertebrate Neuroethology and Robotics. Cambridge, MA: AcademicPress, pp. 199–225.

Mizunami, M., M. Iwasaki, M. Nishikawa, and R. Okada (1997) Modularstructure in the mushroom body of the cockroach. Neurosci. Lett229:153–156.

Mizunami, M., J.M. Weibrecht, and N.J. Strausfeld (1998) Mushroombodies of the cockroach: Their participation in place memory. J. Comp.Neurol. 402:520–537.

Mobbs, P.G. (1982) The brain of the honeybee Apis mellifera. I. theconnections and spatial organizations of the mushroom bodies. Philos.Trans. R. Soc. Lond. B 298:309–354.

Mobbs, P.G. (1984) Neural networks in the mushroom bodies of thehoneybee. J. Insect Physiol. 30:43–58.

Sanchez, S.D. (1937) Sur le centre antenno-moteur ou antennaire pos-terieur de l’abeille. Trav. Lab. Rech. Univ. Madrid 31:245–269.

Schildberger, K. (1983) Local interneurons associated with the mushroombodies and the central body in the brain of Acheta domesticus. CellTissue Res. 230:573–586.

Schildberger, K. (1984) Multimodal interneurons in the cricket brain:Properties of identified extrinsic mushroom body cells. J. Comp. Physiol.A 154:71–79.

Schurmann, F.W. and J. Erber (1990) FMRFamide-like immunoreactivityin the brain of the honeybee (Apis mellifera): A light and electronmicroscopical study. Neuroscience 38:797–807.

Shepherd, G.M. (1992) Modules for molecules. Nature 358:456–457.Stocker, R.F.M (1994) The organization of the chemosensory system in

Drosophila melanogaster. Cell Tissue Res. 275:3–26.Strausfeld, N.J. (1997) Oculomotor control in flies: From muscles to

elementary motion detectors. In P.S.G Stein and D. Stuart (eds):Neurons, Networks, and Motor Behavior. Oxford: Oxford UniversityPress, pp. 277–284.

Strausfeld, N.J. (1998) The insect mushroom body: A uniquely identifiableneuropil. In J. L. Leonard (ed): Identified Neurons in Model Systems.Cambridge, MA: Harvard University Press. (in press).


Strausfeld, N.J. and C. Gilbert (1992) Small-field neurons associated withoculomotor and optomotor control in muscoid flies: Cellular organiza-tion. J. Comp. Neurol. 302:954–972.

Strausfeld, N.J., U. Bassemir, R.N. Singh, and J.P. Bacon (1984) Organiza-tional principles of outputs from dipteran brains. J. Insect Physiol.30:73–93.

Strausfeld, N.J., L. Hansen, Y.-S. Li, R.S. Gomez, and K. Ito (1998)Evolution, discovery, and interpretations of arthropod mushroom bod-ies. Learning Memory 5:11–37.

van der Kloot, W.G. and C.M. Williams (1953) Cocoon construction by theCecropia silkworm. Behavior 6:233–255.

Vowles, D.M. (1964) Olfactory learning and brain lesion in the wood ant(Formica rufa). J. Comp. Physiol. Psychol. 58:105–111.

Wadepuhl, M. (1983) Control of grasshopper singing behavior by the brain:Responses to electrical stimulation. Z. Tierpsychol. 63:173–200.

Withers, G.S., S.E. Fahrbach, and G.E. Robinson (1995) Effects of experi-ence and juvenile hormone on the organization of the mushroom bodiesof honey bees. J. Neurobiol. 26:130–144.

Witthoft, W. (1967) Absolute Anzahl und Verteilung der Zellen im Hirn derHonigbiene. Z. Morphol. Tiere 61:160–184.

Yamasaki, T. and T. Narahashi (1959) The effects of potassium and sodiumions on the resting and action potentials of the cockroach giant axon. J.Insect Physiol. 3:146–158.

Yang, M.Y., J.D. Armstrong, I. Vilinsky, N.J. Strausfeld, and K. Kaiser(1995) Subdivision of the Drosophila mushroom bodies by enhancer-trap expression patterns. Neuron 15:45–54.


Mushroom bodies of the cockroach: Activity and identities of neurons recorded in freely moving...

Documents

Transcript of Mushroom bodies of the cockroach: Activity and identities of neurons recorded in freely moving...