Immunocytochemical demonstration of locustatachykinin-related peptides in the central complex of the...

Immunocytochemical Demonstration ofLocustatachykinin-Related Peptides in

the Central Complex of the Locust Brain

HARM VITZTHUM AND UWE HOMBERG*Institut fur Zoologie, Universitat Regensburg, 93040 Regensburg, Germany

ABSTRACTThe central complex, a highly ordered neuropil area in the insect brain, plays a role in

motor control and spatial orientation. To further elucidate the neurochemical architecture ofthis brain area, we have investigated the distribution and morphology of neurons containinglocustatachykinin I/II-related substances in the central complex of the locust Schistocercagregaria. The central complex is innervated by at least 66 locustatachykinin I/II-immunoreactive neurons, which belong to two sets of tangential neurons and four sets ofcolumnar neurons. These neurons give rise to immunostaining in the protocerebral bridge, inseveral layers of the upper division of the central body, and in all layers except layer 5 of thelower division of the central body. Double-label experiments show colocalization of immunore-activity for both locustatachykinin I/II and octopamine in tangential neurons of theprotocerebral bridge. A pair of tangential neurons of the lower division of the central bodyexhibits both locustatachykinin I/II and g-aminobutyric acid (GABA) immunoreactivity. A setof 16 columnar neurons of the lower division of the central body shows colocalizedimmunoreactivity for locustatachykinin II, leucokinin, and substance P. This study revealsnovel features of the anatomical organization of the locust central complex and suggests aprominent role for locustatachykinin-related peptides as neuromediators and cotransmitterswithin this brain area. J. Comp. Neurol. 390:455–469, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: g-aminobutyric acid; immunocytochemistry; insect brain; neuropeptide;

octopamine

In contrast to the ganglia of the ventral nerve cord, theinsect brain contains a number of neuropil structures withhighly regular arrangements of neuronal elements. Themost prominent of these neuropils are the antennal andoptic lobes, sensory brain centers that are involved inolfactory and visual signal processing, and two brain areaswithout direct input from sensory afferents, the mushroombodies and the central complex. The mushroom bodiesreceive olfactory input and play a prominent role inolfactory learning and memory formation (Davis, 1993;Menzel et al., 1994). In contrast, the anatomical organiza-tion and functional role of the central complex is less wellunderstood. The central complex occupies the center of theinsect brain and is composed of four major subunits: theprotocerebral bridge, the upper and lower divisions of thecentral body, and the paired noduli (Williams, 1975;Homberg, 1987, 1994a). Electrical stimulations, intracellu-lar recordings, activity labelling techniques, and studieson structural brain mutants have suggested a variety offunctions for the central complex, including visual integra-tion (Homberg, 1985; Milde, 1988; Bausenwein et al. 1994;Homberg and Muller, 1995) and the control of motor

activities, such as walking (Strauss et al., 1992; Straussand Heisenberg, 1993; Heisenberg, 1994) and flight(Homberg, 1994b; Ilius et al., 1994). Recent evidence fromthe locust specifically proposes a role of the central com-plex in polarized light detection and compass navigation(Muller and Homberg, 1994; Homberg and Muller, 1995).

A striking feature of the central complex in all insectspecies studied so far is its cristalline-like, three-dimen-sional organization into layers and columns. In the locust,the protocerebral bridge and the central body are con-nected through sets of topographically organized columnarneurons that subdivide both neuropils into linear rows of16 columns (Williams, 1975; Homberg, 1991; Vitzthum etal., 1996; Muller et al., 1997). The second major category of

Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: Ho950/4.

*Correspondence to: Dr. Uwe Homberg, Institut fur Zoologie, UniversitatRegensburg, D-93040 Regensburg, Germany.E-mail: [email protected]

Received 18 March 1997; Revised 14 August 1997; Accepted 15 August1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 390:455–469 (1998)

r 1998 WILEY-LISS, INC.

neuronal cell types, tangential neurons, has arborizationsthroughout single layers of the central complex (Homberg,1991; Vitzthum et al., 1996; Muller et al., 1997). Bothcolumnar and tangential neurons connect the centralcomplex to adjacent areas in the insect brain, most promi-nently the lateral accessory lobes of the protocerebrum.

Immunocytochemistry suggests a rich diversity of neuro-active substances in the central complex and has beenused as a valuable tool for analysis of its anatomicalorganization. Among the many substances detected immu-nocytochemically in the central complex of the locust andother insects are the classical neurotransmitter,g-aminobutyric acid (GABA); biogenic amines, such asoctopamine, serotonin, and dopamine; and neuropeptidesrelated to FMRFamide, Dip-allatostatins, myomodulin,leucokinins, tachykinins, and pigment-dispersing hor-mones (for review, see Homberg, 1994a). Neuropeptides ofthe tachykinin family appear to be particularly abundantand have been demonstrated immunocytochemically inthe central complex of all insect species studied so far(Lundquist et al., 1993, 1994b; Muren et al., 1995; Nassel,1993, 1994; Nassel et al., 1995a; Wegerhoff et al., 1996).

To date, insect members of the tachykinin peptide familyhave been sequenced from brains of the locust Locustamigratoria, termed locustatachykinins I–V (LomTK I–V);from the mosquito Aedes aegypti, termed sialokinins I andII; from the mosquito Culex salinarius, termed culetachy-kinins I and II; and from the blowfly Calliphora vomitoria,termed callitachykinins I and II (Schoofs et al., 1990a,b,1993; Clottens et al., 1993; Champagne and Ribeiro, 1994;Lundquist et al., 1994a). All insect tachykinins havemyotropic activity on smooth muscles (Schoofs et al., 1993;Nassel, 1994). LomTK I is involved in the release ofadipokinetic hormone from locust corpora cardiaca (Nasselet al., 1995a) and has pheromonotropic activity in the silkmoth Bombyx mori (Fonagy et al., 1992). The wide distribu-tion of tachykinins detected immunocytochemically inbrains of a locust, a cockroach, a beetle, and several speciesof flies suggests that these peptides also function asneuroactive compounds in the central nervous system ofinsects.

To gain further insight into the anatomical and neuro-chemical organization of the locust central complex, wehave investigated the number and distribution of LomTKI- and II-like-immunoreactive neurons in this brain areain detail. Double-label experiments revealed colocalizationof LomTK II-like immunostaining with GABA-, octopa-mine-, leucokinin-, and substance P-related substances,particularly in central-complex neurons. Parts of thisstudy have been published in abstract form (Vitzthum andHomberg, 1996).

MATERIALS AND METHODS

Animals

Immunocytochemical staining was performed on brainsof adult Schistocerca gregaria. Animals were obtainedfrom crowded laboratory cultures at the University ofRegensburg. No differences in immunostaining were ob-served between males and females. All protocols used inour study were approved by the animal care and usecommittee of the University of Regensburg and conformedto the German law on the Protection of Animals.

Antisera

The anti-LomTK II antiserum (K1-50820091) was a giftfrom H. Agricola (University of Jena, Germany). Its speci-ficity has been tested by enzyme-linked immunosorbentassay (ELISA) with synthetic LomTK I and II and calli-tachykinins I and II as antigens (Nassel et al., 1995b). Theantiserum cross reacts with all four tachykinins, with aslightly higher affinity to the LomTKs. The anti-LomTK I(9207-7) and antileucokinin (9028-7) antisera, gifts fromD. Nassel (University of Stockholm, Sweden), have beencharacterized previously on brains of the locust L. migrato-ria (Nassel, 1993). The anti-LomTK I antiserum showssimilar specificities for synthetic locusta- and callitachyki-nins I and II but requires higher antiserum concentrationsthan the anti-LomTK II antiserum (Nassel et al., 1995b).The antisubstance P antiserum (TG2-4), a gift from M.R.Brown (University of California, San Diego, CA), has beencharacterized by Mancillas and Brown (1984). Its specific-ity in locust brain tissue has been shown by Wurden andHomberg (1995). The anti-GABA antiserum (KLH-GABA9/24) was a gift from T.G. Kingan (University of Arizona,Tucson, AZ). Its specificity was determined by Hoskins etal. (1986) on brains of Manduca sexta. The antiserumagainst octopamine, a gift from H.G.B. Vullings (Univer-sity of Utrecht, The Netherlands), has been characterizedby Sporhase-Eichmann et al. (1992).

Specificity of immunostaining in thelocust brain

The specificities and cross reactivities of the antisera inimmunostaining of S. gregaria brain sections were testedby liquid-phase preadsorption of the diluted antisera withvarious concentrations of synthetic LomTK II (Peninsula,Heidelberg, Germany), GABA-glutaraldehyde conjugate,synthetic leucokinin (Peninsula), synthetic substance P(Sigma, Deisenhofen, Germany), and octopamine-bovineserum albumin (BSA) conjugate. Preadsorption of thediluted antisera with 10 µM of their specific antigen (100µM in case of octopamine-BSA conjugate) abolished allimmunostaining in brain sections of S. gregaria. No reduc-tion of LomTK II-like immunostaining was detected bypreadsorption with 100 µM GABA-glutaraldehyde conju-gate, 100 µM leucokinin, or 100 µM substance P. Likewise,immunostaining with antisera against GABA, leucokinin,substance P, and octopamine was not affected after pread-sorption of the diluted antisera with 100 µM syntheticLomTK II. Preadsorption of the anti-LomTK I antiserumwith 100 µM synthetic LomTK II abolished all immuno-staining in brain sections of S. gregaria.

Immunocytochemistry

After animals were anesthetized by cooling, Zamboni’sfixative (4% paraformaldehyde, 7.5% picric acid in 0.1 Mphosphate buffer, pH 7.4) was injected into the headcapsule. Brains were dissected out of the head capsule andimmersed for an additional 3 hours at 4°C in fixative. Afterfixation, brains were embedded in gelatin/albumin andsectioned at 30 µm with a Vibratome (Technical Products,St. Louis, MO) in the frontal, horizontal, or sagittal plane.Immunostaining was performed on free-floating sectionsby using the indirect peroxidase-antiperoxidase (PAP)technique (Sternberger, 1979). The LomTK II antiserumwas diluted at 1:16,000 and 1:18,000, and the antiserumagainst LomTK I was used at 1:6,000. Primary antisera

456 H. VITZTHUM AND U. HOMBERG

were diluted in 0.1 M Tris HCl/0.3 M NaCl (SST), pH 7.4,containing 0.5% Triton X-100 (Sigma) and 1% normal goatserum. They were applied to the sections for at least 18hours at room temperature. The PAP staining procedurewas carried out as described previously (Vitzthum et al.,1996).

Double immunofluorescent staining was performed, asdescribed by Wurden and Homberg (1993), by using biotin-ylated rabbit LomTK II antibodies. Immunoglobulin frac-tions from the anti-LomTK II antiserum were biotinyl-ated, as described by Bayer and Wilchek (1980), by usingbiotinamidocaproate N-hydroxy succinimide ester (Sigma;provided by Dr. S. Wurden, University of Konstanz, Ger-many). Preparation, fixation, embedding, and sectioningprocedures were the same as those used in the PAPtechnique. Sections were incubated first with polyclonalrabbit antisera against leucokinin (1:1,000), substance P(1:600), or LomTK I (1:6,000) for 18 hours at room tempera-ture followed by dichlorotriazinyl amino fluorescein(DTAF)-conjugated goat-anti rabbit immunoglobulin (IgG;Jackson ImmunoResearch, Avondale, PA) at 1:80 for 1hour. Nonspecific binding sites were blocked by incubationwith rabbit IgG (Jackson ImmunoResearch) at 1:25 for 2hours. Subsequently, the sections were treated with biotin-ylated rabbit anti-LomTK II at 1:650 for 18 hours, followedby incubation with streptavidin-Texas red (AmershamBuchler, Braunschweig, Germany) at 1:100 for 2 hours.Finally, the sections were mounted in Elvanol (Rodriguezand Deinhardt, 1960) under glass coverslips.

Immunostaining for GABA and octopamine requiredmodifications in the fixation and staining procedure. ForGABA immunofluorescence, we used Boer’s GPA fixative(Boer et al., 1979) instead of Zamboni’s fixative. Theanti-GABA antiserum was diluted at 1:1,000. Backgroundfluorescence that resulted from glutaraldehyde fixationwas eliminated by treating the sections with sodiumborohydride (0.45 M in SST with 0.5% Triton X-100 for 30minutes) after the incubation with the anti-GABA antise-rum.

For double-labeling experiments involving the antioc-topamine antiserum, the fixative consisted of 2.5% glutar-aldehyde in 0.1 M phosphate buffer containing 1% sodiummetabisulfite, pH 4.0. Vibratome sections were treatedfirst with 0.45 M sodium borohydride in SST containing0.5% Triton X-100 for 15 minutes to saturate double bonds.After rinsing, the sections were incubated with 2% normalgoat serum in 0.1 M phosphate buffer/0.45 M NaCl with0.5% Triton X-100 and 1% sodium metabisulfite. Theantioctopamine antiserum was diluted at 1:1,000 in 0.1 Mphosphate buffer/0.45 M NaCl containing 1% normal goatserum with 0.5% Triton X-100 and 1% sodium metabisul-fite.

Data analysis

LomTK II-like-immunoreactive neurons with arboriza-tions in the central complex were reconstructed from serialfrontal sections with a Zeiss microscope (Oberkochen,Germany) equipped with camera lucida attachment. Theterminology for brain structures largely follows Strausfeld(1976) and, for central-complex subdivisions, followsHomberg (1991, 1994a) and Muller et al. (1997). Positionalinformation is given with respect to the body axis of theanimal. Immunofluorescent preparations were analyzedby using a confocal laser microscope (LSM 310; Zeiss)equipped with an argon (488 nm) and a helium/neon (543

nm) laser. DTAF immunofluorescence was detected with a515–565 nm filter, and Texas red fluorescence was detectedwith a 575–640 nm filter. Images were processed on apersonal computer by using Corel Draw 4 and wereprinted with a digital color printer (UP-D8800; Sony,Tokyo, Japan).

RESULTS

General staining pattern with antiseraagainst LomTK I and II

The antisera against LomTK I and II revealed virtuallyidentical patterns of immunostaining in the brain of S.gregaria. Double-label experiments showed that all so-mata and neuronal arborizations that were immuno-stained by the anti-LomTK II antiserum were also stainedby the anti-LomTK I antiserum. The staining quality,however, particularly the signal-to-background ratio, wassuperior with the anti-LomTK II antiserum. Therefore,the anti-LomTK II antiserum was used throughout thisstudy for analysis and reconstructions of LomTK-like-immunoreactive neurons in the central complex.

LomTK I/II immunostaining in the brain of S. gregariaresembled the staining pattern for LomTK I in L. migrato-ria, as described by Nassel (1993). We counted about 950immunostained somata throughout the brain (200 in eachoptic lobe and 550 in the brain proper), with processesinnervating most neuropils (Fig. 1). The higher number ofsomata in the brain of S. gregaria compared with 400 cellbodies in L. migratoria (Nassel, 1993) may result fromincluding faintly stained somata in our counts.

Immunostaining in the central complex

The central complex is densely innervated by at least 66LomTK I/II-like-immunoreactive neurons with processesin most subdivisions of the central complex. We coulddistinguish six distinct types of LomTK I/II-like-immuno-reactive neurons with ramifications in the central com-plex. Four of these are columnar neurons, and two typesare groups of tangential neurons. The protocerebral bridgeis innervated by probably four types of columnar and byone type of tangential neuron (Figs. 2B, 3C,D). The upperdivision of the central body exhibits immunostaining inthe anterior lip and in layers I and II originating from twosets of columnar neurons (Figs. 2, 3E, 5), whereas denseimmunostaining in the lower division of the central bodyresults from one type of columnar neuron and one type oftangential neuron (Figs. 2, 3B,E, 4A, 6A). Faint immuno-staining was also found in the lower subunits of the noduli(Figs. 2B, 3E), but the neuronal origin of this stainingcould not be determined.

Columnar neurons

Four systems of columnar neurons of the central com-plex exhibit LomTK I/II-like immunostaining. All of theseneurons have somata in the pars intercerebralis. Ramifica-tions of columnar neurons inside the central complex aremore weakly immunostained than terminal arborizationsof these neurons in the lateral accessory lobes.

Sixteen neurons with large somata (27 µm diameter) inthe posterior pars intercerebralis, termed LTC I, sendneurites into the protocerebral bridge (Figs. 2B, 4A). Themain fibers of these neurons leave the bridge ventrally andjoin four pairs of fiber bundles termed w-, x-, y-, andz-bundles (Williams, 1975). In each bundle, we detected

LOCUSTATACHYKININS IN THE CENTRAL COMPLEX 457

two large immunoreactive fibers. All except the mostlateral fibers of the w-bundle continue through the verticalfiber bundles of the central body (Williams, 1972) andenter the lower division of the central body dorsally. LTC Ineurons appear to innervate all layers of the lower divisionof the central body except layer 5 (Fig. 3E; for nomencla-ture, see Muller et al., 1997). The dorsalmost layer 1 seemsto be innervated more densely than the other layers. In theventral groove fiber complex (for nomenclature, see Wil-liams, 1972), LTC I fibers leave the central body and run tothe lateral triangle of the lateral accessory lobes, wherethey form terminal arborizations (Figs. 2A,C, 4A).

Sixteen small somata (15 µm diameter) in the parsintercerebralis give rise to a second system of columnarneurons of the central complex (LTC II; Fig. 4B). The mainfibers of LTC II neurons run in close proximity to LTC Ifibers between the protocerebral bridge and the lowerdivision of the central body, but they have a much smaller

diameter (Fig. 4B). Because of the superposition of immu-nostaining, we could not determine whether LTC II fibersactually enter the lower division of the central body. In theventral groove fiber complex, up to eight small fibers perhemisphere, probably from LTC II neurons, again join theLTC I fibers toward the lateral triangles of the lateralaccessory lobes (Fig. 4B).

Twenty-four additional somata in the pars intercerebra-lis, eight large (27 µm in diameter) and sixteen small (15µm in diameter) perikarya, apparently correspond to twocolumnar neuron systems of the upper division of thecentral body (LTC III and LTC IV, respectively; Fig. 5).Because of the intense immunostaining in the centralbody, however, LTC III and LTC IV neurons could not betraced completely.

A set of eight small somata in the pars intercerebralisappears to give rise to fine fibers of LTC III neurons, whichproject to the central body via the posterior chiasma (Figs.

Fig. 1. Frontal diagram of the brain of Schistocerca gregariashowing the distribution of Locusta migratoria tachykinin I/II (LomTKI/II)-like-immunoreactive perikarya and major neuropils. For clarity,somata in the posterior protocerebrum are shown separately (top).Intensely immunostained perikarya are black, moderately labeledcells are stippled, and faintly immunoreactive somata are drawn in

outline. Several cell groups could be identified: four types of columnarneurons of the central complex (LTC I–IV) and two types of tangentialneurons of the central complex (LTT I and LTT II). aL, a-lobe; AL,antennal lobe; bL, b-lobe; Ca, calyx; CB, central body; Lo, lobula; Me,medulla; P, pedunculus; Tc, tritocerebrum. Scale bar 5 200 µm.


2B, 5A). Although no LTC III fiber was detected in thew-bundle, the x- and y-bundles each contain one fiber, andthe z-bundle contains two fibers. In the upper division ofthe central body, the fine neurites enlarge and form densearborizations mainly in layers I and II. Main fibers runthrough the posterior vertical bundles of the central bodyand, thus, use the same fiber paths as the LTC I and LTC IIneurons (Fig. 3E). Terminal arborizations of these neuronscould not be identified due to dense immunostaininglaterally from and in front of the central body.

Eight large fibers of LTC IV neurons have denselyimmunostained arborizations in layers I and IIa of theupper division of the central body and in the anterior lip(Figs. 2D, 3E, 5B). Main fibers run through the ventralgroove in front of the lower division of the central body toterminal varicose arborizations concentrated in the ante-rior dorsal shell of the lateral accessory lobes. Primary

neurites of LTC IV neurons to their presumed cell bodies inthe pars intercerebralis were not immunostained.

Tangential neurons

In addition to the four sets of columnar neuron, twotypes of tangential neuron of the central complex showLomTK I/II-like immunostaining. The first type of neuron(LTT I) consists of two bilateral pairs of tangential cellsthat innervate the lower division of the central body (Figs.3A,B, 6A). The neurons have somata (16.5 µm diameter) inthe inferior median protocerebrum. Their primary neu-rites send weakly stained side branches into the lateraltriangles of the lateral accessory lobes (Figs. 3A, 6A).Axonal fibers continue through the isthmus tract to thelower division of the central body. The neurons havestrongly immunostained, fan-like terminals in ventral

Fig. 2. LomTK II-like immunoreactivity in the central complex ofthe locust S. gregaria. A: Frontal section showing intense LomTKII-like immunostaining in the lower division of the central body (CBL)and in layers Ia and IIa of the upper division of the central body (CBU).The vertical fiber bundles through the CBU consist of immunoreactiveprocesses of LTC I, II, and III neurons (single arrowheads). One pair ofimmunostained somata in each hemisphere of the ventromedianprotocerebrum gives rise to fibers that connect both lateral accessorylobes (LAL). The other two somata belong to LTT I neurons (arrows).Note intense immunostaining in the lateral triangle (double arrow-head) of the LAL originating from LTC I, II, and LTT I neurons. AL,antennal lobe; aL, a-lobe. B: Section through the posterior central

complex showing immunostaining in the protocerebral bridge (PB)and in fibers of columnar neurons connecting the bridge with thecentral body. Arrows indicate faint immunostaining of the noduli. C:Frontal section through the anterior central body and the LALshowing dense immunostaining in the CBU and the CBL. Note thestrongly immunostained fibers of LTC I and II neurons in the isthmustract of the LAL (arrows). Arrowheads indicate arborizations of LTCIV neurons in the anterior dorsal shell of the LAL. D: Frontohorizontalsection through the central body. Arrows indicate processes of LTC IVneurons in layer II of the CBU. In the CBL, all layers except layer 5(asterisk) exhibit dense immunostaining. Scale bars 5 100 µm.


Fig. 3. LomTK II-like immunoreactivity in the central complex ofthe locust S. gregaria. A: Frontal section through the right lateralaccessory lobe (LAL) showing somata, primary dendrites, and arbori-zations of LTT I neurons in the lateral triangle (LT). B: Frontohorizon-tal section showing fibers of LTT I neurons (arrows) entering the lowerdivision of the central body (CBL). C,D: Frontal (C) and horizontal (D)sections through the pars intercerebralis showing primary neurites(arrows in C), apparently dendritic arborizations (arrowheads in C),and axonal processes of LTT II neurons (arrows in D) in the PB. E:Sagittal section through the central complex slightly off the midline.

LTC I, II, and III neurons have fibers in the vertical fiber bundles(arrow) of the central body. Fibers of LTC III neurons arborize in layersI and II of the upper division of the central body (CBU). LTC IVneurons have large, intensely immunostained processes in the ante-rior lip (ant L) as well as in layers I and IIa. Dense immunostaining inthe CBL originates from processes of LTC I and LTT I neurons. Notethe absence of immunostaining in layer 5 of the lower division of thecentral body (asterisk). The lower subdivision of the noduli (NoL)shows weak immunostaining. NoU, upper subdivision of the noduli,PB, protocerebral bridge. Scale bars 5 100 µm in A–D, 50 µm in E.


Fig. 4. Frontal reconstructions of LTC I (A) and LTC II (B)neurons. Somata of both sets of neurons are in the pars intercerebra-lis. Primary neurites enter the protocerebral bridge (PB) dorsally. A:From the PB, immunoreactive fibers run via the w-, x-, y-, andz-bundles to the lower division of the central body (CBL) and give riseto dense immunoreactive processes in the CBL. Terminal varicosearborizations are in the lateral triangle (LT) of the lateral accessory

lobes (LAL). B: LTC II neurons arborize in the PB and send fine fibersvia the w-, x-, y-, and z-bundles to the CBL. Arborizations of theseneurons inside the CBL could not be identified because of superposi-tion of immunoreactivities. Axonal terminals of LTC II neurons areconfined to the LT of the lateral accessory lobes (LAL). Scale bars 5100 µm.


layers (probably layers 3 and 4) of the lower division of thecentral body (Figs. 3B, 6A).

The second type of immunostained tangential neuron(LTT II) consists of three bilateral pairs of neurons thatinnervate the protocerebral bridge (Figs. 3C,D, 6B). Their

somata (diameter 16.5 µm) lie in the posterior pars interce-rebralis near the calyces of the mushroom bodies. Theneurons have wide arborizations in the posterior medianprotocerebrum (Figs. 3C, 6B). Axonal fibers enter theprotocereberal bridge laterally and, as they run along the

Fig. 5. Camera lucida drawings of LTC III (A) and LTC IV (B)neurons. A: Eight large and 16 small somata dorsal to the protocere-bral bridge (PB) send primary neurites into the PB. They probablygive rise to two systems of columnar neurons of the upper division ofthe central body (CBU). Eight fine fibers in the x-, y-, and z-bundles ofthe posterior chiasma between the PB and the CBU enlarge in theCBU and form arborizations in layers I and II. Main fibers of LTT IIIneurons could be traced in the vertical fiber bundles of the central body

toward the lower division of the central body (CBL). Terminal arboriza-tions could not be identified because of superposition of immunostain-ing in the CBL and lateral accessory lobes. B: Eight large columnarfibers in the ventral groove in front of the CBL form dense arboriza-tions in layers I and IIa of the CBU. Arborizations in the lateralaccessory lobe (LAL) are confined to its anterior dorsal shell. Scalebar 5 100 µm.


Fig. 6. Frontal reconstructions of LTT I (A) and LTT II (B) neurons.A: Fibers of LTT I neurons pass through the lateral accessory lobes(LAL), send off side branches into the lateral triangle (LT), and ramifyin ventral layers of the lower division of the central body (CBL). B:Three pairs of tangential LTT II neurons of the protocerebral bridge

(PB) give rise to axonal fibers that could be traced through thecomplete longitudinal axis of the bridge. Fine and, thus, probablydendritic arborizations are in the posterior median protocerebrum(PMP). Scale bars 5 100 µm.


posterior edge of the bridge, give off strongly immunoreac-tive, wide ramifications into both hemispheres of thebridge (Figs. 3D, 6B).

Double-staining experiments

Double-label experiments show that certain subpopula-tions of LomTK I/II-like-immunoreactive neurons of thecentral complex also exhibit immunoreactivity for GABA,octopamine, leucokinin, and substance P (Figs. 7, 8).Homberg (1994a) showed that tangential neurons of thelower division of the central body exhibit dense GABAimmunostaining. Double-label experiments for GABA- andLomTK II-like immunostaining showed that the two pairsof LomTK-like-immunoreactive tangential LTT I neuronsindeed also exhibit GABA immunoreactivity (Fig. 7A–F).

Tangential neurons of the protocerebral bridge resem-bling the LTT II neurons have been reported to exhibitoctopamine immunostaining (Homberg et al., 1992; Hom-berg, 1994a). Double-label experiments revealed that allLTT II neurons share octopamine- and LomTK II-likeimmunoreactivity. Interestingly, octopamine immunostain-ing does not seem to be evenly distributed throughout thecytoplasm (Fig. 7G–L). Three additional octopamine-immunoreactive neurons of the same type do not showLomTK II-like immunostaining.

Nassel (1993) reported that leucokinin- and LomTKI-like immunostaining patterns are similar but are notcolocalized in the central complex of the locust L. migrato-ria. Double-label experiments in S. gregaria, in contrast,revealed that all 16 LTC I neurons exhibit immunoreactiv-ity for leucokinin and LomTK II, although leucokinin-likeimmunostaining, especially in the protocerebral bridge, ismuch fainter than LomTK II-like immunostaining (Fig.8A–C).

Finally, double-label experiments showed that LomTKI/II-like-immunoreactive LTC I neurons also exhibit sub-stance P-like immunostaining (Fig. 8D–F). No substanceP-like immunostaining was found in the other types ofLomTK I/II-like-immunoreactive central complex neu-rons.

DISCUSSION

The present study shows that LomTK-related sub-stances are distributed widely throughout the brain of thelocust S. gregaria. We found no obvious differences be-tween the patterns of immunostaining in S. gregaria andL. migratoria, as investigated by Nassel (1993). In con-trast to analysis of the general distribution of LomTK-likeimmunostaining in the brain of L. migratoria (Nassel,1993), we focussed our investigation on the mapping ofimmunoreactive neurons in the central complex of S.gregaria and revealed new details that are important for afunctional analysis of this brain structure.

Neuroarchitecture of the central complex

The central complex of the locust S. gregaria is denselyinnervated by LomTK I/II-like-immunoreactive processesin the protocerebral bridge, in the lower division of thecentral body, and, less intensely, in the upper division ofthe central body. At least six different cell types of thecentral complex are immunostained. They can be classifiedas four types of columnar neurons and two types oftangential neurons. The tangential neurons have weaklyimmunostained processes in the lateral accessory lobes

(LTT I) or in the posterior median protocerebrum (LTT II)and more intensely stained varicose terminals within thecentral complex. In contrast, arborizations of columnarneurons in the central complex are weakly immuno-stained, and terminals in the lateral triangles or in thedorsal shell of the lateral accessory lobes are varicose andstrongly immunoreactive. Varicose terminals are usuallyinterpreted as axonal presynaptic endings (Schurmann,1974; Strausfeld, 1976), whereas weakly immunostainedarborizations are likely to be dendritic (Thompson et al.,1991). Therefore, LomTK I/II-like-immunoreactive tangen-tial neurons probably mediate signal input into the centralcomplex, whereas the columnar neurons might be outputsfrom the central complex into the lateral accessory lobes.This corresponds with observations on other tangentialand columnar neurons of the central complex in locusts(Homberg, 1991, 1994a,b; Vitzthum et al., 1996) and inflies (Strausfeld, 1976; Hanesch et al., 1989).

Because of the dense supply of the central complex byLomTK I/II-like-immunoreactive neurons, only ensemblereconstructions of the branching patterns of the differentcell types were feasible. In addition, the projections ofsome of the LomTK I/II-like immunoreactive cell typesmight be more extensive than shown in the drawings,because fine terminal arborizations often could not betraced completely. Nevertheless, Golgi impregnations andsingle-cell dye fills (Muller et al., 1997; personal observa-tions) strongly support the validity of the reconstructions,particularly those of the LTC I, LTC II, LTT I, and LTT IIneurons.

Each fiber bundle of the posterior chiasma contained twolarge immunoreactive fibers and two (w-bundle), three (x-and y-bundles), or four (z-bundle) small fibers. This corre-sponds to 16 large LTC I, 16 small LTC II, and 8 small LTCIII somata in the pars intercerebralis. Eight additionalsmall somata detected in the pars intercerebralis mightconstitute an additional incompletely stained set of LTCneurons. Fibers of LTC IV neurons between the protocere-bral bridge and the central body were not stained by theanti-LomTK II antiserum, but the columnar organizationof processes in the upper division of the central bodystrongly suggests that these neurons correspond to eightadditional large somata of the pars intercerebralis. A set of64 central-complex neurons, termed CCI or CL1 neurons,

Fig. 7. Confocal photomicrographs of 1 µm optical sections show-ing double immunofluorescent staining for LomTK II-like (red in A, D,G, and J) and g-aminobutyric acid (GABA; green in B and E) oroctopamine (green in H and K) immunoreactivity. Yellow areas in C, F,I, and L show colocalization or superposition of immunoreactivities forLomTK II and GABA (C and F) and for LomTK II and octopamine (Iand L). A–C: Sagittal section through the central complex of S.gregaria showing superposition of GABA- and LomTK II-like immuno-reactivities in the lower division of the central body (CBL). In layer 2 ofthe CBL, GABA immunostaining is not as dense as LomTK II-likeimmunoreactivity (arrowhead in C). Note the complete lack of LomTKII-like immunoreactivity in layer 5 of the CBL (arrow in A). D–F:Colocalization of GABA- and LomTK II-like immunoreactivities in onesoma corresponding to an LTT I neuron (arrows in E and F). Thenon-GABA-immunoreactive neuron does not innervate the centralbody. G–L: Main fibers (arrow in I) and somata (arrowheads in L) ofLTT II neurons show colocalization of LomTK II-like (red in A) andoctopamine immunoreactivities (green in B). Octopamine immunostain-ing seems to be concentrated in special compartments of the somata.Scale bars 5 100 µm in C (also applies to A,B), 50 µm in F (also appliesto D,E), I (also applies to G,H), and L (also applies to J,K).


Figure 7


closely resembles the LTC I neurons (Williams, 1972, 1975;Muller et al., 1997). Like LTC I neurons, CCI/CL1 colum-nar neurons ramify in the protocerebral bridge, in thelower division of the central body, and in the lateraltriangle of the lateral accessory lobes (Williams, 1972,1975; Muller et al., 1997). The 16 LTC I neurons, therefore,seem to be a subsystem of the CCI/CL1 neurons. The LTCII neurons might be an additional subset of the 64 CCI/CL1 neurons, but, because their fiber diameters are muchsmaller than those of the LTC I neurons, they are morelikely to be the small CCI accessory fibers (Williams, 1972)that bypass the lower division of the central body.

Two previously described sets of columnar neurons ofthe upper division of the central body of S. gregaria (ASC 1and ASC 2) are immunoreactive to the insect peptideDip-allatostatin I, and one of those sets of neurons alsoexpresses serotonin immunoreactivity (Vitzthum et al.,1996). Those neurons are clearly different from the LTC IIIand LTC IV neurons described here. The ASC 1 neurons(Vitzthum et al., 1996) reveal an organization of the upperdivision of the central body into 16 columns. In contrast,LTC III and LTC IV neurons as well as ASC 2 neurons(Vitzthum et al., 1996) only give rise to an eightfoldcolumnar organization of the upper division of the centralbody. Because ASC 1 neurons as well as LTC III and LTC

IV neurons arborize in layer I of the upper division of thecentral body, this difference in columnar organization doesnot correspond to different layers being innervated bythese neurons.

Tangential neurons of the protocerebral bridge of thelocust have previously been shown to exhibit immunostain-ing for Dip-allatostatin I and serotonin (Vitzthum et al.,1996). In contrast to the LTT II neurons, however, thoseneurons connect the protocerebral bridge with the poste-rior optic tubercles; therefore, they belong to a differenttype of tangential neuron of the protocerebral bridge. Inthe lower division of the central body, five types of tangen-tial neuron have recently been described by Muller et al.(1997). The LTT I neurons of our study show similarities toa particular subtype of TL 2 neuron (Muller et al., 1997)innervating layers 3 and 4 of the lower division of thecentral body.

Comparison with other insect species

Although the central complex shows prominent tachyki-nin immunostaining in all insects studied so far, there areconspicuous species-specific differences in the patterns ofimmunostaining and the types of immunoreactive neu-rons. In contrast to LomTK immunostaining that is

Fig. 8. Confocal photomicrographs of 10 µm optical sections throughthe central complex showing LomTK II-like (red in A and D) andleucokinin-like (green in B) or substance P-like (green in E) immunore-activity. Yellow areas in C and F show colocalization or superpositionof immunoreactivities. A–C: Colocalization of LomTK II-like andleucokinin-like immunoreactivities was found in all LTC I neurons

(arrowheads in C). Superposition of both immunoreactivities in theCBU results from distinct groups of leucokinin-like and LomTK II-likeimmunoreactive neurons. D–F: All LTC I neurons are also immunore-active to an antiserum against substance P (arrowheads in F). Scalebars 5 100 µm in C (also applies to A,B) and F (also applies to D,E).


stronger in the lower than in the upper division of thecentral body in S. gregaria, the upper division of thecentral body is more densely supplied by tachykinin-like-immunoreactive neurons than its lower division in acockroach, a beetle, and two species of flies (Lundquist etal., 1994b; Nassel, 1994; Muren et al., 1995; Nassel et al.,1995b; Wegerhoff et al., 1996). In the meal beetle Tenebriomolitor, 64 LomTK II-like-immunoreactive central com-plex neurons (RCL neurons) connect the protocerebralbridge to the upper division of the central body. Theseneurons send axonal projections to small areas in thelateral accessory lobes that appear to be equivalent to thelateral triangles of the locust (Wegerhoff et al., 1996). TheRCL neurons, therefore, closely resemble the LTC I andLTC II neurons of the locust, except that they ramify in theupper and not in the lower division of the central body.Sixty-four tachykinin-immunoreactive columnar neurons(P5 neurons) likewise connect the protocerebral bridge tothe upper division of the central body in the cockroachLeucophaea maderae, but it was not determined whetherthose neurons send fibers to the lateral accessory lobes(Muren et al., 1995). In contrast, tachykinin-immunoreac-tive columnar neurons appear to be completely absent inthe central complex of flies (Lundquist et al., 1994b;Nassel, 1994; Nassel et al., 1995b). Instead, two types ofLomTK I-like-immunoreactive tangential neuron of theupper division of the central body were reported in C.vomitoria and Drosophila melanogaster (Lundquist et al.,1994b; Nassel, 1994), but those neurons show no apparentsimilarities to any of the LomTK I/II-like-immunoreactiveneurons of the central complex in S. gregaria. In conclu-sion, the comparison of tachykinin-like-immunoreactivefiber systems in the central complex shows considerablevariations among different insect species. Future physi-ological studies might show whether these differencesreflect specializations in the functional organization of thisbrain area.

Colocalization experiments

Our double-label experiments show that LTC I neuronsin the locust S. gregaria are also immunoreactive tosubstances related to leucokinin and substance P. Thisresult contrasts with data from Nassel (1993), who foundno colocalization between LomTK I- and leucokinin-likeimmunostaining in the central complex of the locust L.migratoria. Preadsorption of the anti-LomTK II antiserumwith leucokinin or substance P as well as preadsorption ofthe antisubstance P or antileucokinin antisera with LomTKII had no effect on immunostaining, although the threepeptides share a phenylalanine residue in position fivefrom the amidated C-terminus and may therefore bedistantly related. Nassel (1993) also reported that theLomTK I antiserum does not cross react with leucokininand substance P. In contrast, antisera against LomTK Iand II show full cross reactivity when they are tested inbrain sections of S. gregaria. Therefore, we suggest thatdistinct substances or epitopes related to leucokinin, sub-stance P, and LomTKs are colocalized in LTC I neurons,whereas the antisera against LomTK I and II recognizethe same populations of antigens.

Colocalization of LomTK I/II-like and GABA immunore-activities shows that the two pairs of LTT I neurons arealso GABA-immunoreactive and appear to be a smallsubsystem of GABA-immunoreactive tangential neuronsof the lower division of the central body (Homberg, 1994a).

Similarly, the three pairs of LTT II neurons are also asubset of at least 20 octopamine-immunoreactive tangen-tial neurons of the protocerebral bridge (Homberg et al.,1992; Homberg, 1994a).

The present study, together with previous immunocyto-chemical investigations, shows a rich diversity of neuroac-tive compounds in the central complex of the locust S.gregaria. In addition to GABA and the amines serotonin,octopamine, and dopamine, neuropeptides related to adipo-kinetic hormone, crustacean cardioactive peptide (CCAP),Dip-allatostatin I, FMRFamide, gastrin/cholecystokinin,leucokinin, locustamyoinhibiting peptide, LomTK I/II, myo-modulin, and pigment-dispersing hormone (PDH) havebeen mapped in the locust central complex (Myers andEvans, 1987; Homberg and Binkle, 1989; Homberg, 1991;Homberg et al., 1991; Wendt and Homberg, 1992; Nassel,1993; Wurden and Homberg, 1995; Dircksen and Homberg,1995; Swales and Evans, 1995; Schoofs et al., 1996;Vitzthum et al., 1996). This large variety of putativeneuromediators reflects the large number of neuronal celltypes in the central complex (see, e.g., Muller et al., 1997)and, in addition, appears to result from the abundance andcomplex combinations of colocalized substances found inthis brain area.

Recent intracellular recordings showed that neurons ofthe lower division of the locust central body resemblingLTC I, LTC II, and LTT I neurons are sensitive to polarizedlight (Muller and Homberg, 1994; Homberg and Muller,1995). These neurons might be involved in spatial orienta-tion and navigation of the locust with respect to the skypolarization pattern. The immunocytochemical evidencefor the presence of LomTKs, leucokinins, substance P, andGABA in these neurons is an important step towardfurther analysis of the role of the central body in polarized-light detection and should facilitate future ultrastructuralstudies on the synaptic organization of this brain area.

ACKNOWLEDGMENTS

We are grateful for the generous gifts of antisera re-ceived from Drs. H. Agricola, M.R. Brown, T.G. Kingan,D.R. Nassel, and H.G.B. Vullings. We thank Dr. M. Stenglfor valuable comments on the paper, Dr. S. Wurden for thebiotinylation of the LomTK II antibodies, and S. Buch-hauser and H. Halmer for photographic assistance.

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