Morphology and sensory modality of mushroom body extrinsic neurons in the brain of the cockroach,...

Morphology and Sensory Modalityof Mushroom Body Extrinsic Neurons

in the Brain of the Cockroach,Periplaneta americana

YONGSHENG LI AND NICHOLAS J. STRAUSFELD*Arizona Research Laboratories, Division of Neurobiology, The University of Arizona,

Tucson, Arizona 85721

ABSTRACTMushroom bodies are paired centers in insect brains that are thought to be crucial in

olfactory learning and memory. Early neuroanatomical descriptions suggested that themushroom bodies comprise rather simple arrangements of nerve cells. Intrinsic neuronswithin each mushroom body were believed to receive olfactory afferents and to supply long,branched axons to extrinsic neurons that lead from the mushroom body into the protocere-brum. More recent suggestions that the mushroom bodies integrate several sensory modali-ties find support from intracellular and extracellular recordings of extrinsic neurons in thebrains of crickets, honey bees, and cockroaches. Here, we describe two major classes ofextrinsic neurons, simple and complex cells, in the mushroom bodies of the cockroachPeriplaneta americana. Each class is defined by its pattern of branching in the brain. Simpleneurons correspond to extrinsic neurons described from other species that have one set ofdendrites only within the mushroom bodies. Complex extrinsic neurons possess dendrite-likebranches within and outside the mushroom bodies. This arrangement may account in part fortheir observed multimodality, as might newly identified afferent neurons that terminate inthe mushroom body lobes among the dendrites of extrinsic neurons and that respond tomultimodal stimuli. Organizational complexity within the mushroom bodies is suggested bythe grouping of intrinsic cell axons into discrete laminae. These are intersected by theblock-like arrangements of dendritic fields of extrinsic neurons in a manner reminiscent ofPurkinje cell dendrites intersecting parallel fibers in the cerebellum. The present results demon-strate that the cockroach mushroom body processes multimodal sensory information and that itsneural arrangements contribute to a precise architecture consisting of discrete longitudinal andtransverse subdivisions. J. Comp. Neurol. 387:631–650, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: insect brain; neuroanatomy; intracellular recordings; higher centers;

multimodality

The mushroom bodies of insects have attracted muchinterest since their discovery in 1850 by Felix Dujardin. Incomparative studies, by using clearing agents on whole-mounts, Dujardin discovered that mushroom bodies wereunusually large in social insects. He did not claim, as isoften suggested, that mushroom bodies are centers forlearning and memory. Instead, he proposed that mush-room bodies might be involved in ‘‘intelligent,’’ meaningadaptive, rather than instinctive behavior. Vowles (1964)was the first to devise lesion experiments that suggestedthat intact mushroom bodies were important for placelearning and memory. Bernstein and Bernstein (1969)suggested that efficiency of foraging and maze navigationby the wood ant Formica rufa is correlated, among other

factors, such as head size and receptor areas, with the sizeof the mushroom body’s calyces. Comparison of brains ofworker ants show that mushroom bodies are larger inworkers that undertake the complex task of brood carethan in those that do not (Gronenberg et al., 1996).

Grant sponsor: National Science Foundation; Grant number: IBN-931629; Grant sponsor: National Institutes of Health; Grant number:NS07309.

*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 7 March 1997; Revised 21 May 1997; Accepted 27 May 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 387:631–650 (1997)

r 1997 WILEY-LISS, INC.

Since Kenyon’s (1896a) original description, it has beensuggested that the afferent supply to the mushroom bodiescomprises olfactory inputs. Neuroanatomical descriptionsconcur that a discrete region of the mushroom body, calledthe calyces, receives axon collaterals from neurons originat-ing in the antennal lobes (Kenyon, 1896a,b; Holmgren,1916; Mobbs, 1982, 1984; Homberg et al., 1989). Conse-quently, most descriptions (see, e.g., Schurmann, 1987) of themushroom body refer to it as an olfactory neuropil, irrespec-tive of its possible functions as a memory center (Davis, 1993).

The honey bee Apis mellifera is well known for itslearning abilities (von Frisch, 1965), and this feature hascontributed significantly to the volume of research on therole of mushroom bodies in learning and memory (Menzelet al., 1996). A crucial experiment, performed by Erber etal. (1980), demonstrated that conditioning the proboscis-extension reflex to an odor could be blocked by focal coolingof the honey bee’s antennal lobes or its mushroom body’sneuropils.

A role for the mushroom bodies in olfactory learning hasbeen suggested from studies of Drosophila mutants, inwhich anatomically abnormal mushroom bodies show defi-ciencies in olfactory conditioning (Heisenberg, 1980, 1989;Han et al., 1992; Davis, 1993). The role of the mushroombodies in olfactory learning is supported by chemicalablation of neuroblasts at an early stage of development,leading to a depletion or absence of mushroom bodyKenyon cells in the adult brain and the abolition ofolfactory learning (deBelle and Heisenberg, 1994). Theassociation between the mushroom bodies and olfactorylearning and memory is supported by studies on thebehavioral mutant dunce (dnc). This mutant is deficient inolfactory conditioning (Dudai et al., 1976; Tully and Quinn,1985), habituation (Duerr and Quinn, 1982), and learning-dependent courtship (Hall, 1986). These defects are theresult of lesions in a gene encoding cAMP-specific phospho-diesterase (PDE; Davis and Kauvar, 1984; Han et al.,1992), and antibodies raised against dnc PDE reveal moreintense staining in wild-type Drosophila mushroom bodiesthan elsewhere in the central nervous system. In situhybridization demonstrates intense affinity of dnc RNA tothe cell bodies of intrinsic neurons (Nighorn et al., 1991;Davis, 1993). When it is preferentially expressed in mush-room bodies and adjacent areas in the protocerebrum, amutant form of protein, normally essential to the cAMP-PKApathway, preferentially perturbs Pavlovian-type olfac-tory conditioning (Connolly et al., 1996). Therefore, theconsensus is that, although a major role for the mushroombody is in odorant discrimination (Boeckh and Ernst, 1987;Laurent and Davidowitz, 1994), mushroom bodies are also‘‘higher’’ brain centers involved in olfactory learning andmemory (Erber et al., 1980; Schurmann, 1987; Hammerand Menzel, 1995; Davis, 1993, 1996).

It is likely, however, that mushroom bodies do more thanassociate and store information about odorants. For ex-ample, neuroanatomical observations demonstrate thatthere is a substantial input into the honey bee calyces fromthe optic lobes (Mobbs, 1982, 1984; Gronenberg, 1986). Incertain insects that lack olfactory antennae, such as theaquatic Hemiptera Notonecta and Gerris, large mushroombodies lacking calyces receive afferents from other cerebralneuropils that presumably serve visual and mechanosen-sory functions (Strausfeld et al., 1996).

Evidence that mushroom bodies play a role in planningmotor actions was first proposed by studies on crickets

(Acheta) and grasshoppers (Gomphocerripus) in whichfocal stimulation near the mushroom bodies elicited sing-ing and other courtship sequences (Huber, 1960; Wade-puhl, 1983). Evidence that mushroom bodies contribute tothe control of motor actions is suggested by studies ongynandromorph Drosophila (Hall, 1979, 1986), in whichneuropils associated with the mushroom body must bemale for expression of male behavior. In cockroaches,ablation experiments have shown that the integrity of themushroom bodies is essential for place memory (Mizunamiet al., 1993), and extracellular recordings from mushroombodies in intact foraging animals demonstrate increasedlevels of neural activity immediately before the onset oflocomotion and during locomotion. Although place memorycells (for review, see Muller, 1996) have not yet beenidentified, in neurons that are active during locomotion,discharge rates are modulated or suppressed by the direc-tion taken by the animal (Mizunami et al., 1993).

To what degree does the structural organization of themushroom body suggest that it can serve some, if not all, ofthe roles suggested for it? Does its role in place memory, asin Periplaneta, suggest that the cockroach mushroom bodyis fundamentally different from that of Drosophila? Do theanatomically distinct mushroom bodies of evolutionarilydivergent species, such as Apis and Gerris, serve differentfunctions? And, do different cellular organizations in themushroom bodies of different species find a common root inan evolutionarily basal pterygote, such as Periplaneta?

To begin to address these questions, we describe here theresults of intracellular recordings that determine whetherthe cockroach mushroom bodies process more than thesingle modality of olfaction. We identify two classes ofextrinsic cells and demonstrate their sensitivity to odors,to tactile and visual stimuli, and to sound. We demonstratethat some, and possibly many, extrinsic neurons, in addi-tion to their dendrites among the axons of Kenyon cells,have dendrite-like processes outside the mushroom bodiesin other areas of the protocerebrum. This organizationdeparts from previous descriptions (Kenyon, 1896a,b; Ry-bak and Menzel, 1993) in which extrinsic neuron dendritesappear to be contained exclusively within the mushroombodies. This present account also demonstrates that affer-ent supply to the mushroom bodies is not restricted tocalycal neuropil, as has been previously supposed (Mobbs,1982; Malun et al., 1993). We also show that the dendritesof extrinsic neurons interact with specific subsets of Kenyoncells, which, themselves, are subdivided into discretelaminae. Together, these results suggest unexpected struc-tural complexity. Some of the results described here haveappeared in abstract form (Vilinsky et al., 1994; Li andStrausfeld, 1995; Strausfeld et al., 1996).

MATERIALS AND METHODS

Experiments were performed on male American cock-roaches, Periplaneta americana, from a laboratory colonymaintained on IAMS cat food (The IAMS Company, Day-ton, OH) and water at 25°C on a 13:11 hour light:dark(L:D) cycle. Intracellular recordings were obtained duringtests with defined sensory stimuli. The recorded neuronswere filled iontophoretically with Lucifer yellow fluores-cent dye, fixed, and plastic embedded (Strausfeld et al.,1983) in order to examine their anatomical features with

632 Y. LI AND N.J. STRAUSFELD

conventional epifluorescence and laser confocal micro-scopy.

Preparation

Cockroaches were immobilized by cooling them on ice.The animal was put into a polyethylene tube and immobi-lized there with dental wax with its head, hindlegs, andcerci left exposed for stimulation. Mandibular muscleswere removed, and the esophagus was lifted to reducebrain movements. A small window was cut into the cuticleabove the brain, and fresh cockroach saline (O’Shea andAdams, 1981) was trickled onto the brain to keep it moist.Some muscles and a minimal amount of tracheae wereremoved to expose the brain’s frontal surface. Mechanicalstabilization of the brain was achieved by a AgCl groundelectrode that supported the brain from underneath.

Intracellular recordings

Recording electrodes (borosilicate glass: O.D., 1.0 mm;I.D., 0.78 mm) were pulled on a laser microelectrode puller(Sutter Instruments, Novato, CA). Electrode resistancevaried from 100 to 120 MOhm, as measured in tissue, forelectrode tips filled with 4% Lucifer yellow (L-453; Molecu-lar Probes, Eugene, OR) and backfilled with 0.1 M LiCl(Sigma, St. Louis, MO). Electrodes were mounted on aLeitz micromanipulator and were targeted to the positionof mushroom body lobes on one side of the brain. Theneural sheath at the entry point was scratched slightly tofacilitate electrode penetration.

Neuronal activity was amplified (Neuroprobe 1600; A-MSystems, Everett, WA) and viewed on a digital oscilloscope(Gould Electronics, Cleveland, OH). The responses weredigitized and recorded on a video recorder (Vetter 3000; ca.6 kHz effective sampling rate) throughout the experiment.Sensory stimuli were fed through three other channels.Voice commentaries describing tissue manipulation ornoting the stimulus type were recorded on a fifth channel.Electrophysiological data from successfully recorded andfilled neurons were analyzed off-line with a Macintosh-based computer data acquisition and analysis system (ADInstruments Inc., Milford, MA).

Stimuli and procedures

Olfactory. Odors of fresh apple and orange or offemale cockroaches were presented through an olfactorydelivery system. Odors were carried by a humidified airsource, cleared through a charcoal filter, and controlled bya solenoid valve (General Valve Corp., Fairfield, NJ). A0.5-ml glass tube (I.D., 8 mm) contained the test material.Delivery was through a pipette, the tip of which waspositioned at a distance of about 5 mm from the middle ofthe ipsilateral antenna. Stimulus duration was for 0.5seconds, usually followed by at least 30 seconds betweenthe next odor stimulus. A pure air puff was given prior toeach odor to achieve baseline activity (both as a blankcontrol and as a mechanosensory stimulus), and theresidual odors in the recording cage were continuouslysucked out of the room through a vacuum system. Thesignal from the solenoid was amplified and stored onvideotape along with the intracellular recording.

Visual. White light from a 150 W tungsten filamentbulb was manually shuttered and lead by a fiber optic lightguide through gratings mounted on a micromanipulatorand was projected onto the back of a matte translucentscreen at a distance of 2 cm from the compound eye on the

same side of the brain as the recording. Two black-and-white grating patterns could be moved in two orthogonaldirections: left-right and up-down. Stationary flashes andflickers of 10–15 Hz frequency were generated by a pulsegenerator (Wavetek, San Diego, CA) controlling an array of20 high-intensity green LEDs (E166; Gilway TechnicalLamp, Woburn, MA). The light from the LEDs was guidedthrough fiber optics to the compound eye. The signal froma photocell that monitored both flicker and stripe motionwas amplified and stored on videotape with the intracellu-lar recording.

Acoustic. A brief sound was delivered from a 3-cmbeeper with a fixed frequency of 2.8 kHz (Sonalert; NorthMallory Capacitor Co., Indianapolis, IN) mounted on anisolated stand and positioned 50 cm from the preparation(Shaw, 1994). The signal from a microphone mounted closeto the speaker was fed into one channel of the digitizer andwas recorded on the tape.

Tactile. A 15-cm-long, thin wood stick was used totouch the antenna, the tibia of the hindleg, and the cercuson the same side as the recording. Stimuli were presentedin the order listed above in most experiments.

Histology and reconstructions

After successfully recording a neuron, it was filled withLucifer yellow by injection with a hyperpolarizing current(21 to 22 nA) for 5–10 minutes. At the end of theexperiment, the brain was dissected under cockroachsaline and was fixed in 4% paraformaldehyde in Millonig’sbuffer (Strausfeld et al., 1983) for 6 hours at room tempera-ture (or at 4°C overnight). Fixed brains were rinsed inMillonig’s buffer (twice for 10 minutes each), dehydrated ina graded ethanol series, then placed in pure acetone beforeembedding in Spurr’s resin (1969). For the most part,polymerized blocks were oriented in order to seriallysection brains frontally into 14-µm-thick slices using asliding microtome. Sections were mounted on glass slidesfor viewing under a Leitz Diaplan epifluorescence micro-scope. Profiles of filled neurons were photographed onEktachrome 400 slide film at 2-µm optical steps througheach section. Ink drawings of neurons were obtained byprojecting slides via a surface-glazed mirror onto paper.Drawings were superimposed onto brain outlines obtainedby digitizing the perimeters of Bodian silver-stained neuro-pils by using the program ‘‘Smartsketch’’ (FuturewareSoftware Inc., San Diego, CA) to outline images capturedwith a Sony DC 5000 digital camera (Fig. 1A). Areas ofparticular interest were imaged and reconstructed fromserial plastic sections by using a laser confocal epifluores-cence microscope (MRC 600; Bio-Rad, Cambridge, UnitedKingdom) to capture stacks of 0.5–1.0 µm optical sections(Fig. 1B,C). Alignment of the stack obtained from oneplastic section with that obtained from the next wasaccomplished by false coloring one stack red and the nextyellow. Color contrast provides a visual ‘‘lock’’ when match-ing the bottom-most optical section of one stack with thetop-most section of the next (Fig. 1D). Merged stacks canthen be rendered yellow for alignment with a third stack,false colored red, and so on. The final reconstructed imagecan be redigitized as colors that typify Lucifer yellowfluorescence (Fig. 1E–J).

Bodian reduced-silver preparations (Fig. 2A,C) weremade by using Bodian’s (1936) original method for paraffinsections. Golgi preparations (Fig. 2B,D) were obtained bydissecting out the brain in 2.5% potassium dichromate

EXTRINSIC MUSHROOM BODY NEURONS IN THE COCKROACH BRAIN 633

Fig. 1. Extrinsic neuron morphology. A: Lucifer yellow-filled simpleextrinsic neuron leading from the a lobe (a) to the inferior lateralprotocerebrum (IL Pro). B,C: Confocal images showing an opticalsection (B) and details (C) of the spiny dendritic processes in the a lobe.The unusual density of these processes contrasts with the laminarstructure shown in E and F below and in Figure 2C,D. D: False colormatching of two confocal stacks. Detail from neuron shown in Figure7. E,F: Confocal ‘‘slices’’ showing layers of dendritic specializations

(denoted by arrows in F) corresponding to laminae of Kenyon cellaxons running at right angles to the dendritic tree (detail from neuronreconstructed for Fig. 9). G: Confocal detail of external dendrites of thecomplex neuron reconstructed in Figure 10. H,I: Details of b lobedendritic tree of neuron shown in Figure 10. J: Confocal imageshowing blebbed terminals of an afferent supplying the b lobe (see Fig.11). Scale bars 5 25 µm in B,E–H,J (J also applies to I), 10 µm in C,50 µm in D.

Fig. 2. Neural architectures in the cockroach mushroom bodies.A: Bodian (reduced silver) staining showing the block-like domains ofextrinsic neuron dendrites (one is bracketed) arranged across the blobes. B: Golgi-impregnated extrinsic neuron (bracketed) normal tothe passage of Kenyon cell axons. C,D: Laminar organization through-out the length of the mushroom body lobes is revealed in cross sectionsacross the a lobe, parallel to the plane of extrinsic neuron dendritesseen here entering the lobe from its anterior margin. Laminae(arrows) repeat across the lobes. Each comprises a pale band, a thin

coarse band, and a wide more intensely stained band of axons. In D,Golgi-impregnation shows rows (arrows) of dendritic specializationsthat belong to an extrinsic neuron and that correspond to Kenyon celllaminae. E,F: Local specializations of Kenyon cell axons. Confocalmicroscopy distinguishes between regions of varicosities (E) andregions of spine-like structures (F, arrows), possibly representing pre-and postsynaptic terminals, respectively. Scale bars 5 50 µm in A,B,25 µm in C,D, 10 µm in E,F.

with 1.3% w/v sucrose and then fixing the tissue in fivevolumes of this mixture and one volume of 25% glutaralde-hyde (Electron Microscopy Sciences, Ft. Washington, PA).After 6 days of incubation at 4°C, tissue was rinsed inseveral changes of 2.5% potassium dichromate during 1hour before immersion in 100 volumes 2.5% potassiumdichromate and 1 volume 1% osmium tetroxide. Afteranother 5 days at 4°C, tissue was swirled in distilled water(3–5 seconds) and then immersed in 0.75% silver nitratefor 3 days. Silver-chromated brains were dehydrated,embedded in Durcupan (Fluka, Heidelberg, Germany),and sectioned at 20 µm. Drawings were made by using acamera lucida attachment to a Leitz Laborlux microscope.

Figures

The standard format for Figures 5–12 depicts a recon-structed neuron with its modality-specific responses. Con-focal enlargements appear in Figure 1D–J. References tothese are given in the legends to Figures 5–12.

Terminology

Intrinsic neurons are referred to here as Kenyon cells,named after their discoverer. Neurons that extend axonsfrom the mushroom body to other brain regions arereferred to as extrinsic neurons, following Mobbs’s (1982)terminology. An extrinsic neuron is thus synonymous with‘‘output neuron’’ or a mushroom body ‘‘efferent.’’ Neuronssupplying the mushroom bodies are termed afferent neu-rons.

RESULTS

Mushroom body architecture and cell types

In the American cockroach P. americana, each of thepaired mushroom bodies (Fig. 3) consists of two cap-likeneuropils called the calyces, which reside posteriorly anddorsally in the protocerebrum, surmounting a system ofelongated, branched neuropils. Each calyx provides two

short stalks that fuse to form a columnar neuropil calledthe pedunculus, which projects forward in the brain andeventually divides into two lobes, one projecting dorsally(the a lobe) and one projecting medially toward the midline(the b lobe; Vowles, 1955).

According to its traditional description (Kenyon, 1896a;Pearson, 1971; Weiss, 1974; Mobbs, 1982; Schurmann,1987), the mushroom body typically has three classes ofneurons: afferent neurons, intrinsic neurons, and extrinsicneurons. 1) Afferent neurons, usually described as supply-ing the calyces, are derived mainly from the antennal lobes(but see below). In Periplaneta, each mushroom body issupplied by the terminals of approximately 400 antennallobe projection neurons (Malun et al., 1993). These syn-apse onto the dendrites of intrinsic neurons (Schurmann,1970a). 2) Intrinsic neurons (Flogel, 1876; Kenyon, 1896a,b)are also known as Kenyon cells (Strausfeld, 1976). Thereare about 400,000 Kenyon cells in the brain of an adultPeriplaneta (Neder, 1959). Kenyon cells originate fromchromatin-rich perikarya called ‘‘globuli cells’’ (Hanstrom,1928), which are situated above and also flank the calyces.Kenyon cells have dendritic trees in the calyces providingan axon that extends through the pedunculus to bifurcateinto each of the two lobes. 3) Extrinsic cells provideoutputs from the mushroom bodies to regions of the proto-and deutocerebrum (Kenyon, 1896a,b). Extrinsic cellshave been described as postsynaptic to Kenyon cell axons(Schurmann, 1987).

Neuronal organization withinthe a and b lobes

Intracellular Lucifer yellow injection demonstrates thatextrinsic neurons have densely packed dendrites that arearranged normal to the long axis of the lobes (Fig. 1A).Reduced-silver sections demonstrate that dendrites ofadjacent extrinsic neurons are arranged in discrete blocksacross the lobes (Fig. 2A) and that each block of dendritesintersects the passage of Kenyon cell axons (Fig. 2B).

Fig. 3. Schematic showing the main components and disposition ofthe cockroach mushroom bodies. The dorsally and medially extendinga and b lobes derive from a pedunculus (Ped) surmounted by twocalyces (Ca). Me, medulla; Lob, lobula; VGT, visual glomerular tract;

CB, central body; AGT, antennoglomerular tract leading from anten-nal lobes (Ant Lo) to the calyces; glob, globuli cells (perikarya ofKenyon cells); glom, glomeruli of antennal lobe; L Ho, lateral horn; VLDeu, ventrolateral deutocerebrum.


The axons of Kenyon cells are arranged into discretelaminae or ‘‘slabs’’ (see Mizunami et al., 1997). Theseextend through the pedunculus and the a and b lobes asrepeating subunits. Seen in cross section, each lamina iscomposed of an argyrophilic layer and an argyrophobiclayer in which may be embedded another thin argyrophiliclayer (Fig. 2C). Certain extrinsic neurons reflect theorganization of laminae, suggesting that their dendritesreceive inputs from specific subsets of Kenyon cells.

Kenyon cell axons provide patches of what appear to bepre- and postsynaptic specializations. In some places,axons give rise to swellings (blebs or varicosities; Fig. 2E),whereas, at others, they provide numerous spine-likestructures (Fig. 2F). Although these specializations havenot yet been examined by electron microscopy, otherstudies provide support that varicosities and spines indi-cate, respectively, pre- and postsynaptic sites (Strausfeldand Campos-Ortega, 1977), and Schurmann (1970b) firstconfirmed the presence of pre- and postsynaptic sitesamong Kenyon cell axons in the a lobes of the orthopteranAcheta.

Morphology of mushroom bodyextrinsic neurons

Most accounts (Kenyon, 1896a; Schurmann, 1973;Homberg, 1984; Schildberger, 1984) describe extrinsicneurons as having dendrites within the mushroom bodiesand axons that terminate in protocerebral neuropils or, insome cases, recurrently in the calyces (Gronenberg, 1987).

Golgi impregnations of Periplaneta suggest, however, thatextrinsic neurons can have elaborate dendritic morpholo-gies. An example is shown in Figure 4, where an extrinsicneuron in the b lobe gives rise to a second group ofdendrite-like branches immediately adjacent to the lobe.This finding suggests that some extrinsic neurons couldcombine information integrated in the mushroom bodywith information relayed by more direct routes to theirdendrites outside the mushroom body (see Discussion).

Lucifer yellow fills of single neurons confirm the pres-ence of two classes of mushroom body neurons, which weterm ‘‘simple’’ and ‘‘complex’’ neurons. Simple neuronshave a single dendritic tree that is situated in one of themushroom body lobes. These neurons provide an axonleading to one (Fig. 1A) or more (Fig. 6) terminal arbors.Complex neurons possess two or more dendritic trees, oneof which is inside the mushroom body, the other outside(Figs. 7, 9, 10). In both classes of extrinsic neurons, theirdendritic branches within the mushroom bodies are sofinely branched (Fig. 1C,D) that a single tree may contactmost if not all Kenyon cell axons.

Responses of extrinsic neurons

Recordings from a mushroom body neuron ranged from5 to 30 minutes, after which Lucifer yellow was iontopho-retically injected into the cell. All of the recorded neuronshad resting membrane potentials between 230 mV and250 mV. All neurons responded to at least two sensorymodalities.

Fig. 4. A–C: Serial reconstruction of a Golgi-impregnated complex extrinsic neuron in the b lobeshowing arrangement of internal dendrites (int de; B,C) across axons of Kenyon cells (K) and a system ofexternal dendrite-like processes (ext de; B,C) from the axon (ax) ramifying outside the b lobe.


Extrinsic neurons of the a lobes. We have recordedand filled three types of a-lobe neurons. Their dendritictrees have similar morphologies but occur at differentlocations along the length of the a lobe. Each had adifferent termination area, and each showed a differentmorphology with respect to its processes outside the a lobe,in the proto- or deutocerebrum (Figs. 1A, 5–7). For ex-ample, dendrites of the simple a-lobe neuron shown inFigure 1A intersect all of the laminae of Kenyon cell axons,and its axon provides a single terminal arbor in the brain’slateral deutocerebrum. Of the morphological types identi-fied from recordings, one (Fig. 5) was identified in twodifferent preparations, thus demonstrating that extrinsic neu-rons are uniquely identifiable and occur in all individuals.

Despite their similar dendritic morphologies, each cellhad a different and possibly characteristic physiologicalresponse to sensory stimulation. The simple neuron shownin Figure 5 has dendrites in the a lobe near its junctionwith the pedunculus and b lobe. Its axon projects to asingle terminal arbor in the protocerebrum’s lateral horn,an area that receives the terminals of the majority ofprojection neurons originating in the antennal lobes (Fig.5) and that is also supplied by axons originating in thevisual system’s lobula. Like most other extrinsic neurons,this cell has a variable background activity, with a meanfiring frequency around 17 Hz. Background activity wasdepressed in response to an odor pulse of orange extract(Fig. 5). However, when the cell’s background activity wasreduced by a 20.5 nA hyperpolarizing current, it re-sponded with a single spike associated with odor ‘‘on’’followed by inhibition when it was tested again with thesame odor. When it was tested for responses to visualstimulation, an upward vertical motion evoked a moderateand sustained increase (85%) in the spike frequency,whereas downward motion had little or no effect. Theneuron did not respond to 15-Hz flicker but responded tolight ‘‘off’’ with a delay of 30 msec and to light ‘‘on’’ with atransient increase in its firing rate (not shown). Acousticstimulation had no effect on the neuron’s firing rate (notshown). Multimodal neurons with similar morphology andprojection patterns have been identified in the mushroombodies of the cricket, Acheta (Schildberger, 1983, 1984). Incontrast, antennal lobe neurons that project to the calycesof the mushroom bodies respond exclusively to olfactorystimuli (Fig. 5).

Certain a-lobe neurons have unusual and sustainedbackground activity, manifested by ipsp-like negative po-tentials, as shown for the a-lobe neuron illustrated inFigure 6. This cell showed an increased frequency of smallhyperpolarizations in response to apple extract odor butwas unaffected by both light ‘‘on’’ and flicker (data notshown). In response to acoustic stimulation, spontaneousipsps were suppressed (Fig. 6), suggesting presynapticinhibition by other cells. Motion and tactile stimuli werenot tested due to the deterioration of the recording.Morphological observations demonstrate that this simpleneuron has elaborate spiny dendrites restricted to theupper middle section of the a lobe. Its axon projects to avariety of sites in the lateral protocerebrum, where it givesrise to varicose or swollen specializations that may beinterpreted as presynaptic terminals.

Many extrinsic neurons have elaborate shapes, as exem-plified by the element shown in Figure 7. This cell has twosets of spiny arborizations, interpreted here as dendrites,one within the a lobe and the other outside it. The cell has

extraordinarily fine dendritic processes in the upper middlepart of the a lobe (see also Fig. 1D), and all of the Kenyoncell axons in the lobe may be visited by its dendrites. Itsdendrite-like processes outside the mushroom body extendacross the inferior protocerebrum, behind the a lobe andjust above it, in neuropil corresponding to the mechanosen-sory dorsal lobes identified in Blatta (Sanchez and Sanchez,1937). The presence of two dendritic trees, one inside themushroom body and the other external to it, suggests thatthis neuron may integrate sensory inputs received outsideof the mushroom body with sensory information processedby the mushroom body. A second small group of branchesthat arises from the axon at a position lateral to the a lobeis equipped with varicose specializations, suggesting itspresynaptic nature.

This structurally complex neuron showed low-frequency(3 Hz) background spiking activity and was excited both byorange-extract odor and by female pheromone. The re-sponse to either odor was a brief increase in firing fre-quency, followed by a series of ipsps (Fig. 7). Light ‘‘on’’elicited brief (about 1.5 seconds) oscillatory potentials,followed by a transient increase in spike frequency. Light‘‘off’’ caused a transient increase in spike frequency. Tactilestimulation to either the antenna or the hindleg alsoelicited a brief increase in spike frequency. There was noresponse to visual motion (not shown).

Extrinsic neurons of the b lobes. All of the Luciferyellow-filled extrinsic neurons originating from the b lobehave densely branched dendritic trees within this neuro-pil. The b-lobe extrinsic neuron shown in Figure 8 has astratified arrangement of specializations among its den-drites, suggesting that it recruits inputs from specificlaminae of Kenyon cell axons, as shown in Figure 2C,D.This cell has three sets of varicose arborizations, inter-preted here as terminals. The smallest of these is situatedmedially, just beneath the b lobe, and comprises varicoseprocesses that arise from thick tributaries, which pen-etrate the b lobe and ramify into its dendritic tree. Asecond set of short axon collaterals is situated in theventrolateral deutocerebrum, from where a stout axonprojects out to the lateral superior protocerebrum andends there as an extensive terminal field. This region ofthe protocerebrum, as well as the ventral lateral deutocere-brum, is invaded by dendrites of premotor descendingneurons, as shown in the composite drawing of Figure 12.

On penetration, the neuron initially showed high-frequency background spiking activity (Fig. 8). The neuronresponded to orange odor with a slight change in firing rate(not shown). After stabilizing the cell with a 20.7 nAcurrent, it responded to a pulse of the same odor with abrief burst of spikes, followed by four to five subthresholdrebounding depolarizations. Sustained action potentialswere evoked after a long delay (1.5 seconds) in response tolight ‘‘on.’’ Flicker seemed to further increase spikingfrequency, returning it to the level seen after initialpenetration.Acoustic stimulation failed to elicit any changein this neuron’s activity (not shown).

b lobes also give rise to complex extrinsic neurons. Likethose from the a lobe, these are typified by their dendritesboth within and outside the mushroom body. An example isshown in Figure 9. Arborizations in the b lobe appear to beunusually finely tapered, and confocal microscopy demon-strates laminar stratification within its dendritic tree inthe b lobe (Fig. 1E,F). This tree gives rise to a stout processthat extends out to the posterior protocerebrum, where it


Fig. 5. Top: Anatomical reconstruction of a simple a-lobe extrinsicneuron (left) illustrated with an olfactory projection neuron (star,right), both of which respond to olfactory (orange) stimulation. Theextrinsic neuron has delicate dendritic processes at the a lobe andpedunculus junction from which arise short varicose processes (ar-row). The axon provides terminals in the inferior lateral protocere-brum (IL Pro). Bottom: The extrinsic neuron ceased firing in response

to orange odor (upper left graph). After hyperpolarization (upper rightgraph), the cell showed a biphasic response when tested with sameodor. Upward motion of a visual stimulus elicited a slight increase infiring frequency, but motion in opposite direction had no effect. Odorstimulation of the afferent neuron (lower right graph) had an inhibi-tory effect. For abbreviations, see Figure 3.

gives rise to a sparse arborization equipped with spinesthat we interpret as postsynaptic. Another process, alsooriginating from its dendritic tree in the b lobe, extendsanteroventrally, bifurcating in the inferior protocerebrum,where it provides a system of richly branched, varicoseterminals that we interpret as representing the neuron’soutput region. These branch in the same neuropil as theexternal dendrites of other b-lobe neurons, as exemplifiedin Figure 4.

Compared with other extrinsic neurons, backgroundactivity in this cell was low (Fig. 9), and the cell respondedto a variety of sensory stimuli. Orange-extract odor elicitedan initial burst of phasitonic excitation that was followedby intermittent DC shifts on which two or three spikeswere superimposed. The neuron responded to light ‘‘off’’with a brief burst of excitation. Acoustic stimulationelicited intermittent spikes accompanied by a slight posi-tive DC shift of the membrane potential. Tactile stimula-tion of the cercus elicited a complex burst of activity (Fig.9): two spikes were followed by an intense burst of firing,suggesting either two parallel inputs to the mushroombodies, one with a longer delay than the other, or recurrentactivity.

Another complex b-lobe neuron is illustrated in Figure10. Outside the mushroom body, this cell has two regions ofvaricose processes, one close to the b lobe and the otherflanking the pedunculus, which we interpret as presynap-tic. The neuron has densely arborizing and relatively largedendrites compared with other extrinsic neurons withinthe b lobe (Fig. 1H,I). In addition to dendrites inside the blobe, the axon leads to a tight cluster of spiny processes(Figs. 1G, 10) that extend out toward the lateral horn, aregion that is also visited by terminals of neurons originat-ing in the visual system’s lobula.

The morphology of this neuron (Fig. 10) suggests that itis likely to be supplied by a variety of sensory pathwaysoutside the mushroom bodies in addition to the input itreceives from Kenyon cell axons within the b lobes. Itsmorphology is partly reflected by its responses to sensorystimuli (Fig. 10). The neuron initially showed regularlow-frequency (5 Hz) spiking activity. In response to a puffof air onto the ipsilateral antenna, the cell underwent abrief hyperpolarization, followed by a positive DC shiftwith superimposed spikes. During the experiment, thespontaneous firing rate greatly diminished, revealing spo-radic subthreshold oscillations that were not associated

Fig. 6. Neuronal reconstruction and physiological responses of asimple a-lobe extrinsic neuron. The cell has dendritic processesramifying within the a lobe. Its diffuse terminals spread laterallyoutside the mushroom body. The neuron showed spontaneous inhibi-

tory postsynaptic potentials (ipsps) but no spikes. It responded toapple odor with increased ipsp frequency, showed no response to light,but responded to sound with ipsp suppression. For abbreviations, seeFigure 5.


Fig. 7. Anatomical reconstruction of a complex a-lobe extrinsicneuron demonstrates its densely ramifying dendrites within the alobe. Spiny external dendrite-like arbors (ext de) extend across theinferior protocerebrum and a small bush of varicose endings flanks thea lobe laterally. Confocal details of its dendritic processes are shown inFigure 1D. Although this neuron had complex endogenous activity, the

neuron briefly depolarized and then showed 3–5 ipsps in response toorange extract odor or female pheromone. Light ‘‘on’’ gave rise to a slowdepolarization, culminating in a transitory increase in firing rateabout 1.5 seconds after stimulus onset. Light ‘‘off’’ and tactile stimula-tion to antenna (first stimulus, lower right) and hindleg (secondstimulus) gave rise to a transitory increase in firing rate.

Fig. 8. Top: Simple b-lobe extrinsic neuron with stratified den-dritic processes that correspond to laminae of Kenyon cells. Shown aretwo sets of varicose terminals just outside the b lobe (arrows) and athird group of terminals in the inferior lateral protocerebrum (IL Pro).Bottom: Intracellular recordings demonstrate the neuron’s initial

high-frequency background activity. After stabilizing with a 20.7 nAcurrent, this cell responded to orange odor and to light ‘‘on.’’ Flickerappeared to increase the firing rate after the cell had returned to itsoriginal ‘‘background’’ frequency.


Fig. 9. Top: Complex b-lobe extrinsic neuron, reconstructed fromhorizontal sections and seen from below. External dendrites (ext de)reside under the calyces in the posterior protocerebrum. Its mainbranches to the b lobe provide varicose branches in the inferior medial

protocerebrum (IM Pro). Details of its dendrites in the b lobe areshown in Figure 1E,F. Bottom: The cell responded to orange extractodor, to light ‘‘off,’’ to sound, and to mechanical stimulation of thecercus.


Fig. 10. Top: Complex b-lobe extrinsic neuron with extensiveprocesses both within the b lobe (for details, see Fig. 1H,I) and in thelateral horn (L Ho; see Fig. 1G). The neuron has two regions ofputative presynaptic terminals (arrows; ter). Bottom: Recordings

show a high level of endogenous ‘‘background’’ activity. The neurondepolarized in response to air puffs to the ipsilateral antenna, toorange odor, and to tactile stimulation of the ipsilateral antenna.Visual flicker resulted in apparent inhibition of background activity.

with any particular stimulus. During this period, theneuron responded to orange-extract odor and tactile stimu-lation. Visual flicker suppressed background activity. Theneuron was unresponsive to acoustic stimuli or visualmotion (not shown).

Afferent supply to the b lobe

Although most previous studies assume that neuronslinking the mushroom body lobes with other brain areasare efferents, evidence from anosmic taxa (see Discussion)suggests that afferent neurons also terminate in the lobes.Afferent supply to Periplaneta’s b lobe is demonstrated bya Lucifer-filled neuron (Fig. 11) whose beaded and varicose

processes invade a domain that corresponds approxi-mately to the combined width of the dendritic trees of twoadjacent extrinsic neurons (Fig. 1J). The beaded appear-ance of these arborizations is clearly distinct from thefinely branched, spiny dendrites of extrinsic neurons (Fig.1H,I).

b-lobe terminals of this afferent are provided by an axonthat gives rise to spiny arborizations, which spread outwithin the superior medial protocerebrum, anterior to andbeneath the calyces. A second group of exaggeratedlyvaricose processes is situated dorsal to the b lobe (Fig. 11).The responses of this neuron were multimodal. Its back-ground spiking activity varied from 5 Hz up to 17 Hz.

Fig. 11. Afferent neuron terminating in the mushroom body b lobe.A dendrite branches within the superior medial protocerebrum (SMPro) in front of the a lobe. The axon gives rise to varicose processesbehind the b lobe and terminates as a blebbed a process within the b

lobe (see confocal details, Fig. 1J). This afferent neuron responded toseveral sensory modalities: female pheromone, orange extract odor,light ‘‘on,’’ and visual flicker. Tactile stimulation of the ipsilateralantenna also elicited a burst of spikes.


Female pheromone (Fig. 11) elicits robust bursting activitythat lasts more than 2 seconds after a 0.5-second phero-mone pulse. The neuron showed a shorter phasic responseto a pulse of orange odor. Response to light ‘‘on’’ was aslight increase in excitation. Sustained excitation waselicited during flicker, but there was no response to eithersound or visual motion (not shown). Tactile stimulation ofthe antenna elicited a brief but intense burst of depolariza-tions.

Mushroom body extrinsic neurons anddescending neurons

Several of the recorded extrinsic neurons project toneuropils in the lateral proto- and deutocerebrum. Theseareas are richly invaded by dendrites of descending neu-rons, the axons of which supply motor circuits in thethoracic and abdominal ganglia. Studies on Diptera(Gronenberg et al., 1995) and Orthoptera (Hensler, 1992)have demonstrated that, in the brain, descending neuronsreceive input from primary mechanosensory afferents andfrom optic lobe efferents. Evidence that descending neu-rons may also receive input from mushroom body extrinsicneurons is provided by intracellular dye fills. These demon-strate that certain extrinsic neurons provide terminalarborizations, which extend to and then fit the trajectoriesof descending neuron dendrites. Axon collaterals fromextrinsic neurons have also been observed encircling de-scending neuron axons or their main branches. This typeof arrangement is demonstrated by the reconstruction ofan extrinsic neuron, which is also illustrated alone inFigure 8, with a descending neuron recorded from apenetration in the opposite side of the same brain (Fig. 12).Here, a branch of the descending neuron extends contralat-erally with respect to its cell body and the descendingaxon. Dendrites from this branch overlap varicose collater-als from the b-lobe extrinsic neuron, suggesting a possibleregion of contact between the two. The extrinsic cell alsoprovides short collaterals that wrap around unstainedprofiles of other descending neurons. The descendingneuron showed low-frequency background activity. Likemushroom body extrinsic neurons, it responded to a vari-ety of sensory stimuli (Fig. 12), depolarizing to air ‘‘on’’ andto orange odor. It was also excited by both light ‘‘on’’ andlight ‘‘off’’ and to flicker, with prolonged excitation elicitedby the ‘‘off ’’ component of this stimulus. Acoustic stimula-tion as well as tactile stimulation of either the ipsi- orcontralateral antenna elicited brief bursts of depolariza-tion.

DISCUSSION

Multimodality and mushroom body responses

Mechanisms underlying the function of mushroom bod-ies have seldom been studied at the level of single neurons(Mauelshagen, 1993; Rybak and Menzel, 1993) and, then,usually with respect to its outputs (Homberg, 1984; Schild-berger, 1984). The studies by Homberg and Schildbergeragree that extrinsic neurons from the a and b lobesrespond to a variety of sensory inputs and are thusmultimodal. An elegant study on the honey bee (Mauelsha-gen, 1993) is unique, in that it demonstrates learning-dependent modifications of threshold and firing pattern inan identified b-lobe extrinsic neuron as a consequence ofsensory conditioning. Further studies on extrinsic neuronsin Periplaneta are needed to determine whether this is a

general feature or whether it relates to a specific subset ofextrinsic neurons. Cobalt injections, which resolve severalneurons (Rybak and Menzel, 1993), demonstrated that, inthe honey bee brain, extrinsic neurons target a widevariety of neuropils. Targets include their own calyces.Recordings (Gronenberg, 1987) from these recurrent extrin-sic neurons demonstrate that they respond to both olfac-tory and visual stimuli, but evidence that they are in-volved in adaptive functions is lacking.

The present results show that extrinsic neurons respondto odor mainly with excitation, except for one a-lobeneuron that responded with an increased frequency ofipsps (Fig. 6). Responses to tactile stimulation were briefand intense in most cases. A few neurons responded toacoustic stimuli. Responses to visual flicker usually oc-curred after much longer delays than to other stimuli.Some mushroom body neurons responded to stationarylight stimulus with a sustained increase of spiking fre-quency, also after a long delay of up to 1.5 seconds, possiblyindicating the intervention of more synaptic stations fromthe optic lobes than from the antennal and vertical (mecha-nosensory) lobes.

There are obvious differences between the ‘‘resting’’activity of sensory interneurons and mushroom body extrin-sic neurons. For example, antennal lobe and optic lobeprojection neurons (Hausen and Egelhaaf, 1989; Kanzakiet al., 1989; Strausfeld et al., 1995) show regular back-ground activity against which the responses to stimuli arepredictable and clear cut. The same generalization is truefor descending neurons (Fig. 12), such as those recorded inlocusts (Hensler and Rowell, 1990). In contrast, extrinsicneurons are characterized by extremely varied levels of‘‘background’’ activity. Some have high-frequency dis-charges, which mask the effects of sensory stimulationunless the cell is hyperpolarized (Fig. 5). Other neuronsshow episodic phasic activity, which makes it difficult todistinguish a bona fide response, because this may coin-cide with neural activity that has nothing to do with thestimulus. Also, even though some extrinsic neurons arerelatively quiet, a condition that should best reveal re-sponses to specific stimuli, even these can show unpredict-able bursts of spikes that are not very different from thoseelicited by an applied stimulus. In one example (top lefttrace in Fig. 9), it appears that the neuron responds justbefore the onset of a given stimulus, demonstrating thatthe stimulus fortuitously coincided with an episode of‘‘spontaneous’’ activity. Whether or not the response tosome stimuli might be enhanced by such endogenousevents needs to be investigated.

The high level and apparent complexity of endogenousactivity in extrinsic neurons suggest that cellular interac-tions between them and other elements is continuous andstructured. We suggest that such activity cannot simply bediscounted as ‘‘background’’ or ‘‘spontaneous,’’ because itmay reflect the unique structure and status of the mush-room bodies in associating not only stimuli provided by theexperimenter but all manner of ongoing sensory stimuliand, possibly, their recall. If the mushroom bodies areindeed involved in recording information about sensoryevents, then endogenous activity observed within a givenextrinsic neuron could be interpreted as reflecting a levelof ‘‘debate’’ occurring within, and outside, the mushroombody matrix.


Fig. 12. Top: Reconstruction of a descending neuron recorded fromone side of the brain with an extrinsic b lobe neuron recorded from theother side (see Fig. 8). A contralateral branch of the descending neuronreaches beneath the b lobe, where it converges with one of the varicoseterminals of the extrinsic neuron. Bottom: The descending neuron

typically showed low background activity. It was briefly excited by airto either antenna, orange odor, light ‘‘on,’’ and sound. Light ‘‘off’’ causedprolonged excitation. ax, descending axon; FB, fan-shaped body of thecentral body complex.

Polarity of extrinsic mushroom body neurons

Extrinsic neurons respond with different latencies todifferent stimuli. This observation would be surprising ifall sensory information was channeled to them over thesame route, via second-order interneurons to the Kenyoncells and then to extrinsic neuron dendrites. Long delays,such as those to certain visual inputs, between stimulusand extrinsic neuron response suggest that extrinsic neu-rons receive visual inputs via a route that does notnecessarily involve Kenyon cells.

A new finding is that certain extrinsic neurons havecomplex polarities that appear to involve a second den-dritic tree outside the mushroom body. Such ‘‘external’’dendrites have been observed in the lateral horn, an areathat is supplied by olfactory and visual interneurons.External dendrites have also been observed in the inferiorlateral protocerebrum and deutocerebrum, regions that, inPeriplaneta and in Diptera (Strausfeld and Gronenberg,1990), receive terminals from the optic lobes. These resultscaution against assuming that the multimodal characterof complex extrinsic neurons is due to their connectionsexclusively within the mushroom bodies.

If complex extrinsic neurons receive modalities otherthan olfaction outside the mushroom bodies, then how cansimple extrinsic neurons also be multimodal, because theirdendrites are restricted to within the mushroom bodies’neuropil? Possibly, the calyx of Periplaneta is supplied byvisual and mechanosensory afferents in addition to olfac-tory afferents, although this has not yet been demon-strated. Observations of honey bees (Mobbs, 1982; Gronen-berg, 1986) have identified terminals from the optic lobesin the calyces. A similar connection has been identified inthe moth Manduca sexta (Wicklein, unpublished observa-tion). Recently, we have recorded and filled several afferentneurons supplying the calyces from complex dendritictrees in the protocerebrum, including the anterior optictubercle (Li and Strausfeld, unpublished observation).Evidence that certain afferents to the mushroom bodiescan bypass the calyces comes from neurons similar to thatshown in Figure 11. So far, we have identified three ofthese afferent neurons, all of which originate in the lateralproto- and deutocerebra (Li and Strausfeld, unpublished),in neuropils where they could receive inputs from the opticlobes as well as from ascending acoustic interneuronsarising in the thoracic ganglia. Such neurons (Fig. 11)cannot be unique to Periplaneta. Observations of odonates(dragonflies) and aquatic Hemiptera (Aeschna sp., Noto-necta glauca, Gerris sp.), species in which the antennallobes are absent or greatly reduced, demonstrate that theirmushroom bodies either lack or have a greatly reducedcalyx. Yet these same mushroom bodies possess thousandsof globuli cells whose axons provide voluminous a and blobes that appear to be supplied by numerous afferentneurons (Strausfeld et al., 1996). This arrangement sug-gests that, although Kenyon cells in anosmic species lackobvious dendrites, they probably mediate local circuitswithin the a and b lobes for interactions between afferentsand extrinsic neurons.

Multiplex organization within themushroom bodies

An early view, stemming from Kenyon’s original studies(1896a,b), was that insect mushroom bodies compriseisomorphic arrays of functionally equivalent Kenyon cell

axons. This view, also espoused by Vowles (1955) andWeiss (1974), has undergone significant revision. Golgistudies on honey bees proposed that different types ofafferents (olfactory, visual) segregate to concentric, modal-ity-specific areas in the calyces. Mobbs (1982) termed thesethe lip, collar, and basal ring zones. Observations onGolgi-impregnated Kenyon cells (Mobbs, 1982, 1984) sug-gested that axons arising from each of these zones segre-gate out to discrete laminae that extend through the entirelength of the pedunculus and the a and b lobes.

Laminar organization in the pedunculus and lobes isrevealed by reduced silver stains (Mobbs, 1984) and byantibodies raised against neuropeptides or against certaintransmitter substances (for review, see Bicker, 1991).Immunocytochemistry reveals discrete lamina-like expres-sions of transmitters and peptides in the pedunculi andlobes of honey bees and crickets (Schurmann and Klemm,1984; Schafer and Bicker, 1986; Schafer et al., 1988).Three-dimensional reconstructions of laminae revealed byantibodies against gastrin-cholecystokinin demonstratedtheir passage from the calyx and throughout the peduncu-lus and lobes (Strausfeld et al., 1996). Thus, it is likely thatat least some of the immunoreactive bands described byother authors (see, e.g., Schafer and Rehder, 1989; Schur-mann and Erber, 1990) can be ascribed to subsets ofKenyon cell axons similar to those described from Golgistudies by Mobbs (1984). This suggestion finds supportfrom observations of extrinsic neurons whose dendritesmatch the widths and positions of certain immunoreactivelaminae (Strausfeld et al., 1996; see also Mobbs, 1984;Rybak and Menzel, 1993). Such an arrangement suggeststhat, in the honey bee, discrete subsets of Kenyon cellssynapse with certain extrinsic neurons but not with oth-ers.

In Periplaneta, a primitive expression of this type oforganization may be reflected in the laminar organizationof Kenyon cell axons in the pedunculus and lobes (Mizu-nami et al., 1997). In this species, laminae are arrangedapproximately equidistant from each other as repeatingsubunits (Fig. 2C) that are reflected by the anatomicalarrangements of the dendrites of certain extrinsic neurons(Fig. 2D). A much simplified version of this type of organi-zation is suggested by patterns of gene expression inDrosophila (Yang et al., 1995; Ito et al., 1997), in which atleast four discrete longitudinal subdivisions can be as-cribed to different subpopulations of Kenyon cells (Vilinskyet al., 1994). The recent discovery that nitrous oxidesynthase divides the locust a lobes into four parallelcomponents (Elphick et al., 1995) suggests that longitudi-nal subdivisions of insect mushroom bodies may be ubiqui-tous.

Longitudinal subdivisions are distinct from transversepartitioning of the a and b lobes by extrinsic neurondendrites (Fig. 2A), groups of which correspond to discretezones of catecholaminergic neuropil (Frontali and Man-cini, 1970; Mancini and Frontali, 1970; Klemm, 1983;Klemm et al., 1984). The recent observation of glial cellprocesses partitioning the mushroom body lobes (Hahn-lein and Bicker, 1996; Hahnlein et al., 1996) adds furthersupport that these centers are divided into discrete longitu-dinal and transverse regions.

Do these intriguing subdivisions of the mushroom body’sneuropil reflect a functional division of labor so thatsubsets of Kenyon cells and extrinsic neurons serve dis-tinct roles, such as planning motor actions (Mizunami et


al., 1993) or associating and memorizing odors? Evidencefor localized function has been suggested so far from onlytwo experiments. The first tested the role of the mushroombodies in place memory. In that study, Mizunami et al.(1993) demonstrated that ablations that target the pedun-culus-b lobe junction, but not other regions of the mush-room body, abolish the ability of Periplaneta to use distantolfactory or visual cues to locate a hidden goal. In thesecond experiment, expressing a sex-determination genein enhancer-trap lines specific to the Drosophila mush-room bodies (O’Dell et al., 1995) controlled the feminiza-tion of a longitudinal subdivision of the mushroom bodiesof male Drosophila. This resulted in male flies having asubset of sexually ‘‘incorrect’’Kenyon cells. The flies courtedboth females and males, suggesting their failure to discrimi-nate sex-specific odors. Both results suggest that futureexperiments must focus on identifying the range of func-tions for which different parts of the mushroom body are anecessary requirement.

ACKNOWLEDGMENTS

We thank Patricia Jansma, M.S., and Wendy Pott fortheir advice regarding confocal microscopy. Robert Gomez,B.S., provided excellent technical and photographic assis-tance. We thank Dr. John K. Douglass for helpful discus-sions concerning the manuscript. We are grateful to ananonymous referee for suggestions that much improvedthe paper.

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Morphology and sensory modality of mushroom body extrinsic neurons in the brain of the cockroach,...

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