primary commissure pioneer neurons in the brain of the grasshopper schistocerca gregaria:...

Primary Commissure Pioneer Neurons inthe Brain of the Grasshopper

Schistocerca gregaria: Development,Ultrastructure, and Neuropeptide

Expression

PETER LUDWIG,1 LES WILLIAMS,1 DICK R. NASSEL,2 HEINRICH REICHERT,3

AND GEORGE BOYAN1*1Zoologisches Institut, Ludwig-Maximilians-Universitat, 80333 Munchen, Germany

2Department of Zoology, Stockholm University, S-10691 Stockholm, Sweden3Zoologisches Institut, CH-4051 Basel, Switzerland

ABSTRACTThe bilaterally paired primary commissure pioneer neurons in the median domain of the

grasshopper brain are large, descending interneurons that uniquely express the TERM-1antigen, even in the adult. After pioneering the primary interhemispheric brain commissure,these neurons extend TERM-1-immunoreactive collaterals into most parts of the brain exceptthe mushroom bodies. In this report, the authors show that the TERM-1 antigen is located inthe cell body cytoplasm of these neurons and not on the membranes. Screening with antiserato insect neuropeptides reveals that an antiserum recognizing peptides of the leucokininfamily labels the cell body cytoplasm of the primary commissure neurons. Leucokinin-relatedpeptides are known to modulate motility of visceral muscle, play a role in diuresis, and arelikely to be neuromodulators in the insect nervous system. The primary commissure neuronsdiffer ultrastructurally from median neurosecretory cells in that their cell body cytoplasm ismore extensive, contains high numbers of mitochondria and extensive endoplasmic reticu-lum, but does not contain neurosecretory granules. In the adult, the cell somata are envelopedby multiple glia membranes and associated trophospongia. According to these ultrastructuralcharacteristics, the primary commissure pioneers are not classical neurosecretory cells. J.Comp. Neurol. 430:118–130, 2001. © 2001 Wiley-Liss, Inc.

Indexing terms: axogenesis; primary commissure pioneer cells; leucokinin-1; TERM-1; insects

In recent years, the combination of molecular, histolog-ical, and electrophysiological techniques has greatly in-creased our understanding of the development and func-tion of insect nervous systems. Several such studiesinvolve identified neurons in the insect brain (Krautham-mer, 1985; Nassel et al., 1992; Bacon et al., 1995; Stern etal., 1995; Lundquist et al., 1998), and, in some cases, it hasproven possible to establish a clear function for the specificneurons involved (Agui, 1979, 1980; Copenhaver and Tru-man, 1986; Gammie and Truman, 1997; McNabb et al.,1999; Renn et al., 1999; Zitnan et al., 1999).

Despite these advances, only small numbers of neuronsin the insect brain can be shown to be identifiable based onthe fact that they are the only neurons that express agiven molecule (Truman and Copenhaver, 1989; Thomp-son et al., 1991; Meier et al., 1993). Neurons of the grass-

hopper brain, such as the TERM-1-expressing primarycommissure pioneer (PCP) cells (Meier et al., 1993; Boyanet al., 1995a; Ludwig et al., 1999) or the vasopressin-like-immunoreactive (VPLI) cells of the subesophageal gan-glion (Tyrer et al., 1993), belong to this category. Theirspecific antigen expression allows them to be easily iden-tified at all developmental stages, and, because they are

Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: BO1434/1-3; Grant sponsor: Swedish Natural Science Research Council.

*Correspondence to: Prof. Dr. George Boyan, Zoologisches Institut derLMU, Luisenstrasse 14, 80333 Munchen, Germany.E-mail: [email protected]

Received 23 August 2000, Revised 18 October 2000; Accepted 18 October2000

THE JOURNAL OF COMPARATIVE NEUROLOGY 430:118–130 (2001)

© 2001 WILEY-LISS, INC.

large enough to be characterizable electrophysiologically,such neurons probably offer the most promising prospectsfor linking development and function in the insect brain.

The PCP neurons are located in the dorsal median do-main (dMD) in the midline of the embryonic grasshopperbrain and differentiate directly from the neuroectoderm(Ludwig et al., 1999). The first known function of the PCPneurons is to establish the primary interhemisphericbrain commissure at 31% of embryogenesis (Boyan et al.,1995a). Meier et al. (1993) previously described cells in thebrain midline that uniquely express the TERM-1 antigen.They demonstrated that TERM-1 is a glycosylated, 48-kDprotein accumulated in the growth cones of the axons andcollaterals during early embryogenesis. Subsequently, wewere able to show that the TERM-1-expressing cells andthe PCP neurons are one and the same (Ludwig et al.,1999).

In this study, we combined immunocytochemical, histo-logical, and electron microscopic techniques to analyze thedevelopment of the TERM-1-expressing PCP neurons(Boyan et al., 1995a,b) in the brain of the grasshopperSchistocerca gregaria from late embryogenesis, throughthe larval instars, to adulthood. We show that the PCPcells form extensive collaterals that innervate almost allmajor regions of the grasshopper brain, with the exceptionof the mushroom bodies. TERM-1 expression continues toadulthood, and semithin sections reveal that the antigenis located in the cytoplasm of cell somata, not on theirmembrane surface. Our ultrastructural investigationsshow that the PCP neurons lack electron-dense granules,but they possess densely packed mitochondria and ER andhave extensive glial wrapping around the cell somata.Thus, they can be distinguished easily from their neigh-boring median neurosecretory cells, which have a differentontogeny (Ludwig et al., 1999). Finally, we were able tolabel the TERM-1-immunoreactive PCP cells with an an-tiserum against the cockroach neuropeptide leucokinin-1(LK-1). This antiserum recognizes known members of afamily of leucokinin-like peptides (Nassel et al., 1992;Nassel, 1993), including the locust peptide locustakinin(Schoofs et al., 1991). Members of this peptide family areknown to induce contractions in visceral muscle, stimulatesecretion in Malphigian tubules, and are likely to be mod-ulators in the central nervous system (for reviews, seeHolman et al., 1986; Torfs et al., 2000). Their presence in

PCP cells suggests a central neuromodulatory or neuro-trophic role of the peptide in this particular neuronalsystem.

MATERIALS AND METHODS

Animals

S. gregaria were raised in crowded cages at 26°C andwith a 12 hour light/12 hour dark cycle. Eggs were incu-bated at 30°C in moist, aerated containers. Embryos werestaged at time intervals equal to the percentage of embry-ogenesis according to Bentley et al. (1979). Each experi-ment described below was repeated several times. Allexperiments were conducted according to the guidelinesfor animal care in force with the Deutsche Forschungsge-meinschaft.

Immunocytochemistry

Embryos were dissected out of the egg in phosphate-buffered saline (PBS), pH 7.4, and fixed in a solutioncontaining 100 mM Pipes, 2 mM EGTA, 1 mM MgSO4, and3.7% formaldehyde, pH 7.0 for 30 minutes. Embryos wereused as wholemount preparations in subsequent proce-dures. After anesthesia with CO2, larval and adult brainswere dissected out of the head, the brain sheath wasremoved, and the tissue was fixed for 1 hour as describedabove. Preparations were then washed in PBS, pH 7.4, for1 hour. A 4% agarose solution (A 0169; type I-A; Sigma, St.Louis, MO) in PBS was made up by heating in a micro-wave oven. Embedding of the specimens was performed at50°C, and the agarose blocks were then cooled to allowfurther jelling. After trimming and mounting, 100-mm-thick sections were cut on a Vibratome (series 1000; Tech-nical Products International, St. Louis, MO). The sectionswere held in PBS for subsequent immunocytochemistry.For semithin sectioning, selected specimens were post-fixed in 2% osmium tetroxide for 2 hours and dehydratedin a graded acetone series. After embedding in hard Epon,semithin sections (1.5 mm) were cut using glass knifes andcounterstained with Toluidine blue.

TERM-1 immunocytochemistry. Embryos were pre-incubated for 1 hour in a solution comprised of 0.5% PBS,0.5% Triton X-100, and 0.5% bovine serum albumin (BSA;PBT) with 5% normal goat serum (NGS). Preparations

Abbreviations

1°com primary commissureA anteriorAL antennal lobea-lobe a lobe of the mushroom bodyCB central bodyDC deutocerebrumdMD dorsal median domainER endoplasmic reticulumgl glial cellLa laminaLc lateral cellLK-1 leucokinin-1LPI lateral lobe of the pars intercerebralisLo lobulaM midlineMB mushroom bodyMe medullamlt midline tractmnc median neurosecretory cells

mocn median ocellar nervemocnr median ocellar nerve rootn nucleusNB2–NB3 neuroblasts 2 and 3 of the pars intercerebralisncc 1 corpora cardiaca nerve 1OL optic lobePB protocerebral bridgePC protocerebrumPCP primary commissure pioneerped pedunculus of the mushroom bodyPI pars intercerebralispn perineuriumSEG subesophageal ganglionstom stomodeumTC tritocerebrumTyp1–Typ3 median neurosecretory cell types 1–3V ventralX second optic chiasma

119PCP NEURONS IN GRASSHOPPER BRAIN

were exposed to the primary antibody (Mab 4G5; 1:10dilution) for 24 hours at 4°C in the same incubating solu-tion. In adult brain slices and wholemount preparationsfrom embryos older than 55% of embryogenesis, the incu-bation was for 2 days at 4°C. After washing in PBT,embryos were incubated either for 3 hours at room tem-perature or at 4°C overnight with either Dako EnVision™secondary antibody (goat anti-mouse; 1:1 dilution; Dako,Carpinteria, CA) or an fluorescence isothiocyanate(FITC)-coupled secondary antibody (goat anti-mouse;1:400 dilution; Dinova, Hamburg, Germany). After furtherwashing, preparations were either stained with diamino-benzidine (DAB) using Sigma Fast DAB tablets or directlymounted in glycerol, as appropriate.

Leucokinin immunocytochemistry. LK-1 immunocy-tochemistry followed the same protocol as described forTERM-1 above. The primary antibody (code 9228-7) wasused at a concentration of 1:2,000 (for details, see Nasselet al., 1992).

Other antisera. Allatostatin and perisulfakinin anti-sera (see also Agricola and Braunig, 1995) were tested ongrasshoppers of all ages at a concentration of 1:1,500using the protocol described for TERM-1 above.

Histology

Wigglesworth’s osmium tetroxide-ethyl gallate methodwas performed as described in Boyan et al. (1993).

Microscopy

Sections and wholemount embryos were viewed usingdifferential interference contrast optics and a video cam-era. For fluorescent preparations, an FITC filter was em-ployed as appropriate. The video images were digitizedwith a Scion VG-5 frame-grabber card and processed withpublic domain imaging software (NIH Image, Bethesda,MD). Confocal microscopy was performed with a LeicaTCS SP2 photon laser-scanning microscope (Leica, Wet-zlar, Germany). Confocal reconstructions of TERM-1-expressing PCP cells were made from sequential 2-mmscans through 100-mm-thick Vibratome sections. Thestaining present in each focal plane was drawn into sep-arate windows of a graphics program (SXM Image) andthen focused into a single stack.

For electron microscopy, embryonic, larval, and adultbrains were dissected and fixed in a solution comprised of2% glutaraldehyde and 1% osmium tetroxide in 0.1 Mcacodylate buffer, pH 7.1, at 4°C for 1 hour followed bypostfixation in 1% osmium tetroxide in buffer for 2 hours.Specimens were thoroughly washed in buffer, dehydratedin a graded acetone series, and embedded in hard Epon.Ultrathin sections (100–120 nm) were cut with glassknifes, counterstained with uranyl acetate and lead ci-trate, and inspected on a Philips CM 10 electron micro-scope at 80 kV.

Neuraxes

The axes applied in this study are neuraxes, not bodyaxes (see Boyan et al., 1993). Accordingly, the top of thebrain (in the head) is neurally anterior, the front of thebrain (sic) is neurally ventral, the back of the brain (sic) isneurally dorsal, and the base of the brain (sic) is neurallyposterior.

RESULTS

Identification of the primary commissurepioneers in the median domain

A functional analysis of brain morphology is consider-ably facilitated if individual cells can be identified andtheir ontogeny established. For this reason, the presentstudy focuses on the embryonic and postembryonic devel-opment of the PCP neurons that have been identifiedpreviously in the median domain of the grasshopper brain(Boyan et al., 1995a) and whose lineage is known (Ludwiget al., 1999).

The PCP neurons are located as bilaterally arrangedpairs on both sides of the exact midline of the brain in thedMD, between the two lobes of the pars intercerebralis(PI; Fig. 1A). These neurons differentiate without identi-fied precursor cells from the epithelium of the mediandomain (Ludwig et al., 1999). Subsequent to their differ-entiation at about 28% of embryogenesis, the PCP neuronsdirect axonal growth cones contralaterally across thebrain midline and, in so doing, pioneer the primary inter-hemispheric brain commissure (Fig. 1B; Boyan et al.,1995a; Ludwig et al., 1999). The axons then join the dorsalfascicle of the circumesophageal connective and descend tothoracic neuromeres of the ventral nerve cord.

At around 40% of embryogenesis, the PCP neurons be-gin to express the TERM-1 antigen (Fig. 1B) and are theonly cells in the brain to do so (Meier et al., 1993; Ludwiget al., 1999). This TERM-1 expression, as shown below,continues throughout subsequent embryonic and postem-bryonic development and is present in the adult. Theirunique and completely consistent staining pattern (over250 preparations) allows the PCP neurons to be unequiv-ocally identified at all stages of development and providesa perfect tool with which to study the contribution ofidentified cells to the functional organization of the devel-oping brain.

Embryonic projection patterns of thePCP neurons

Having previously described their neurogenesis (Lud-wig et al., 1999), we now investigated axogenesis by thePCP neurons during later embryonic and larval develop-ment. The PCP cells are present as a bilateral pair on eachside of the brain midline (Fig. 1). We observe below thatthe axonal projections established by each sibling cellthroughout the brain are so similar that they are insepa-rable at this level of resolution; thus, we assume that bothsiblings have a comparable morphology and the sameinnervation field. In addition, the bilateral pairs of PCPcells produce mirror-image projection patterns in thebrain hemispheres and the ventral nerve cord (Fig. 3). Wewill therefore refer to the morphology of a single PCP cellas representative for all.

Progressive axogenesis of the PCP cells, for ease ofanalysis, may be divided into a discrete series of steps(Fig. 2A). After crossing the midline at 31% of embryogen-esis and thereby establishing the primary brain commis-sure, a given PCP axon grows posteriorly in the dorsalfascicle of the circumesophageal connective first to thetritocerebrum (TC), which it reaches at 42% of embryo-genesis, and then the subesophageal ganglion, which itreaches at 45% of embryogenesis. The axon continues togrow posteriorly in the ventral nerve cord and reaches thelast abdominal ganglion at about 60% of embryogenesis

120 P. LUDWIG ET AL.

(not shown). The axon, as it grows, gives off collateralsthat then, beginning at 55% of embryogenesis, innervatethe brain hemispheres. The first collaterals are formed atthe level of the stomodeum (Figs. 2, 3A) and project to thePI, protocerebrum (PC) and optic lobes (OL). Subse-quently, at 60% and 65%, of embryogenesis (see Figs. 2and 3B, respectively), further collaterals appear that in-nervate the deutocerebrum (DC) and the TC. Collateralsare progressively formed in the subesophageal ganglion(at 60% of embryogenesis), the thoracic ganglia (65% ofembryogenesis), and the abdominal ganglia (between 65–75% of embryogenesis). The bilateral pairs of PCP cellsproduce mirror-image projection patterns in the brainhemispheres and the ventral nerve cord (Fig. 3A).

By 70% of embryogenesis, a given PCP axon hasinnervated extensive regions of the brain, such as the

PI, PC, DC, TC, and OL (Fig. 2B). The major collateralsthen arborize further to form a fine meshwork of TERM-1-expressing branches throughout the brain (Fig. 3C,D).The densest TERM-1 expression is found at dis-crete varicosities located at intervals along thesebranches. We found no evidence for any further concen-tration of TERM-1 expression in growth cones as op-posed to the axons after these had innervated theirtarget regions.

During late embryogenesis (.75% of embryogenesis),the axonal projection pattern assumes the form seen inthe larva and the adult (Figs. 3C,D, 4A,B, 6). Apart frominnervating the diffuse neuropil associated with the PC,DC, TC, and OL, the PCP neurons innervate more highlystructured regions, such as the central body (CB) and the

Fig. 1. Cellular organization of the dorsal median domain (dMD)in the grasshopper brain. A: Schematic showing the dorsal mediandomain (dMD) in the midline (M) of the brain between the two lobesof the pars intercebralis (PI). The locations of the identified primarycommissure pioneer neurons (PCP; stars) and lateral cells (LC;dashed circles) are shown at 40% of embryonic development. Theneurites of the four PCP cells (solid arrows) are the first neurons tocross the midline at 30% of embryonic development and, thus, pioneerthe primary interhemispheric brain commissure (1°com). The growthcones (solid arrows) pass by those of their contralateral homologs andsubsequently turn posteriorly to project into the circumesophgealconnectives. Further identified cells in this brain region are the lat-eral cells (Lc; see Graf et al., 2000) and neuroblasts 2 and 3 (NB2 andNB3) of the PI. The neurites of the Lc join the 1°com ipsilaterally anddescend to the subesophageal ganglion (dashed arrows). The PI isdelimited from the dMD by a glial border (shown schematically as ablack curve). The arrow marked A indicates anterior for A and B.B: TERM-1 immunocytochemistry at 40% of embryonic developmentreveals the bilaterally paired PCP neurons (stars) in the dMD withtheir initial neurites in the 1°com. No other cells in the brain express theTERM-1 antigen. For other abbreviations, see list. Scale bar 5 30 mm.

Fig. 2. Axogenesis of the PCP neurons in the brain. A: Schematicsummarizing the progressive axogenesis of the PCP neurons (comparewith B). Numbers represent the percent of embryonic development atwhich growth cones reach each brain region indicated (1, 37%; 2, 40%;3, 42%; 4, 43%; 5, 45%; 6, 55%; 7, 60%; 8, 65%; and 9, 70%). Note thatthe main axonal scaffold is established first and that the first collat-erals (6) do not appear until 50% of embryonic development. Majorarborizations are represented by thicker arrows. B: Drawing of anembryo at 70% of development after TERM-1 immunocytochemistryshows the major arborizations of the PCP neurons in the brain at lateembryogenesis. The initial neurites cross into the contralateral side ofthe brain, from which the axons (arrows) descend within the dorsalfascicle of the circumesophageal connectives to the subesophagealganglion (SEG) and the thoracic ganglia (not shown). The axons of thepaired PCPs on each side run closely bundled, as do their axonalcollaterals in the brain. Two major branches (arrowheads and openarrows) on each side innervate the pars intercerebralis (PI), the pro-tocerebrum (PC), and the optic lobe (OL). The deutocerebrum (DC)and the tritocerebrum (TC) are innervated by minor collaterals. Thearrow marked A indicates anterior. For other abbreviations, see list.Scale bar 5 250 mm.


protocerebral bridge (PB). No collaterals, by contrast, in-nervate the mushroom bodies (Figs. 3D, 4).

Postembryonic development of thePCP neurons

Throughout larval development, the TERM-1-expressingPCP cell bodies are clearly seen at the ventral surface of thebrain midline (Fig. 4A,B). Although their gross axon projec-tion remains largely unchanged, finer collaterals continue to

extend into more remote brain regions, such as the glomeruliof the antennal lobes (see Fig. 6D) and the medulla of theoptic lobes (see Fig. 6F).

Morphogenetic changes in brain structure during lateembryogenesis and early larval development (see Boyan etal., 1995b) now force the PCP cell bodies to assume aposition between the median neurosecretory cell clusters(Figs. 4, 5). These latter cell groups derive in part from thedMD and in part from the median parts of the PI (Ludwig

Fig. 3. Confocal images of PCP neurons at different stages ofembryonic development after TERM-1 immunocytochemistry revealthe progressive innervation of brain regions by fine arborizations.Each image is an extended focus picture representing about 100 mmdepth through the brain. Note that the intensity of TERM-1 expres-sion remains high throughout embryogenesis. A: At 55% of embryonicdevelopment, the PCP cell bodies (stars) are located in the mediandomain between the two lobes of the pars intercerebralis (PI). Theinitial neurites (small arrows) run posterior before they form a chi-asma as they cross to the contralateral side in posterior fascicle XVII(large arrow in A–D).The axons (open arrowheads) descend in thedorsal fascicle of the circumesophgeal connectives to the thoracicganglia (not shown). At this age, two pairs of axonal collaterals (solidarrowheads and open arrow) project from the main axons. These will

innervate the PI, PC, and optic lobes, respectively (see Fig. 2). Arrowsmarked M and A indicate the midline (M) and anterior (A) through-out. B: Image taken at 65% of embryonic development showing thePCP cell bodies (stars), neurites (small arrows), and collaterals (solidarrowheads) at higher magnification. A mesh of fine collaterals (solidarrowheads) now extends across the midline. The open arrowheadpoints to the main axon. C: At 75% of embryonic development, finearborizations are visible in the PI, PC, DC, and central body (CB).Dense collaterals obscure the major axons (open arrowheads). D: At95% of embryonic development, the immunostaining reveals more finecollateral arborizations in the PI, PC, and DC. Note the lack ofTERM-1 immunoreactivity in the anterior part of the brain, where themushroom bodies (MB) have started to form. For other abbreviations,see list. Scale bar 5 150 mm in A; 100 mm in B; 160 mm in C,D.


et al., in preparation). The dMD ceases to remain a sepa-rate entity and fuses with neighboring PI regions as mor-phogenetic forces push the two lobes of the PI toward oneanother. Despite these profound changes, the PCP cellsretain their proximity to the median ocellar nerve root, andthis serves us as a useful landmark for locating them inhistological sections of the larval and adult brain (Figs. 4, 5).

The PCP cell bodies expand in size during development.To document this, we measured cell body diameters in ten

preparations each at 30% of embryogenesis, at 60% ofembryogenesis, and in the adult brain. The soma diameterchanges from approximately 16 mm (S.D., 0.88 mm) indiameter at birth (30% of embryogenesis), to 26 mm (S.D.,3.27 mm) at 60% of embryogenesis, and 55 mm (S.D., 8.74mm) in the adult, by which time they are among the largercell bodies in the brain. The neurites leave the PCP cellbodies dorsally, parallel to the fibers in the median ocellarnerve root, in the so-called midline tract. This tract loopsdorsally around the central body and then turns posteri-orly beneath the PB to enter the central neuropilar regionof the brain. The only TERM-1 immunoreactivity in thebrain (Fig. 5D) maps exactly onto this anatomical projec-tion (Fig. 5A–C).

Adult brain

The fact that the PCP cells continue to express theTERM-1 antigen strongly in the adult and are the onlycells in the brain to do so makes their identification sim-ple. We took advantage of this to reconstruct the entireprojection of the PCP neurons in the adult brain from aseries of optical sections through a brain slice at the levelof the central body (Fig. 6A). The extent and density of thearborization in the adult brain is immediately obvious.TERM-1 immunoreactivity in the PCP neurons is so densethat it delimits major neuropilar regions in the brain fromone another. Whereas neuropil in the PC (Fig. 6A,B), thecentral body (Fig. 6C), and PB (Fig. 6E) is extensivelyinnervated, the a-lobes and pedunculi of the mushroombodies appear as negative images that are completelydevoid of TERM-1 expression. This clear restriction ofTERM-1 expression is true of the antennal lobes (Fig. 6D),where the glomeruli are innervated but not the medulla.

Localization of the TERM-1 antigen

An exact cellular location of the TERM-1 antigen canprovide insights into possible functions of this molecule forthe cell. We used the DAB precipitation product to visu-alize TERM-1 immunoreactivity in the cell somata of thePCP neurons in an embryo at 60% of embryogenesis andin a second instar larva (Fig. 7). Semithin sagittal sectionsof intensely labeled PCP neuron cell somata examinedwith the light microscope reveal that the TERM-1 antigenis located in their cytoplasm. The DAB reaction product ispresent in the form of discrete spots distributed homoge-neously throughout the cytoplasm. There is no immuno-reactivity evident in the nucleus. However, we are not yetable to establish whether the TERM-1 antigen is associ-ated with any specific cell organelle. More importantlyperhaps, there is no cell surface immunoreactivity associ-ated with the cell bodies (Fig. 7B). The intensity of DABstaining (and, by implication, TERM-1 expression), as ageneral feature, appeared to be greater in postembryonicpreparations.

Identification of a putative secretedmolecule

Testing the PCP neurons with antibodies against sev-eral candidate neuropeptides (allatostatin, perisulfakinin,LK-1) resulted in only one positive result, namely, LK-1(Fig. 8). LK-1 is an insect myotropic octapeptide that wasisolated first from the cockroach (Holman et al., 1986) andbelongs to the family of cephalotropins. An LK-1-like im-munoreactivity is found together with TERM-1 in the PCP

Fig. 4. Postembryonic development of the PCP neurons as re-vealed by TERM-1 immunocytochemistry. A: Second instar. Videomontage from different focal depths of the same horizontal section(100 mm) of the brain at the level of the protocerebral bridge (PB). Dueto brain morphogenesis, the PCP cell bodies (stars) come to lie be-tween the median neurosecretory cells (mnc) on each side along withtheir axons in the corpora cardiaca nerves 1 (ncc1). The medianneurosecretory cells (mnc) partly arise from the median part of thepars intercerebralis and the dorsal median domain. The PCP neurites(arrows) run in the midline tract dorsally around the central body(CB) and turn posteriorly to enter the central neuropil of the brainbetween the CB and the PB. TERM-1-immunoreactive arborizationsare present in the PC (arrowheads), CB, and PB but not in the a-lobesor pedunculi of the mushroom bodies. This and the medulla of theantennal lobe (see Fig. 6) are the only brain regions in which noTERM-1 immunoreactivity is observed. The arrow marked V indi-cates ventral for A and B. B: Confocal image that was taken afterfluorescent TERM-1 immunocytochemistry in a plane of section com-parable to that shown in A but in a fifth instar larva. The PCP cellbodies and neurites (white arrows) are located as in the second instarimage shown above. Extensive arborizations (arrowheads) are nowfound in the neuropil regions surrounding the a-lobes and the pedun-culi of the mushroom bodies. Scale bar 5 100 mm in A; 120 mm in B.


neurons at specific stages of development, but its inten-sity, extent, and temporal expression differ from that ofTERM-1 (Fig. 8).

Compared with to TERM-1 (Fig. 8A), no LK-1-like im-munoreactivity is found in the PCP neurons during em-bryogenesis (Fig. 8B). Other, unidentified cells do showLK-1-like expression in the PI at this time (Fig. 8B).Postembryonically, however, LK-1-like expression isclearly evident in the PCP cells at the second instar stage(Fig. 8D). LK-1-like expression is limited to the cell bodies,whereas TERM-1 is present throughout the cell (Figs. 3,8C). The intensity of LK-1-like expression in the secondinstar is less than that seen in other unidentified, smaller

cells in the brain, and this remains true throughout sub-sequent development. In later larval instars, coexpressionof TERM-1 (Fig. 8E) and LK-1 (Fig. 8F) continues; how-ever, LK-1-like expression is consistently restricted to thecell bodies of the PCP cells and is never seen in theirneurites or axons. This situation continues into the adult(Fig. 8G,H) and is at variance with the LK-1-like expres-sion in the cell bodies, neurites, and axons of other neu-rons of the brain, such as the median neurosecretory cells,at the same stages of development (Fig. 8H).

Attempts at double labeling for TERM-1 and LK-1 inthe PCP neurons failed in our hands. This may have beendue to the overall low level of LK-1-like expression, or,

Fig. 5. Sagittal sections through the midline of the grasshopperbrain at different developmental stages reveal the location of the PCPcell bodies and their initial axonal projection. A–C are from Wiggles-worth histology, and D was taken after TERM-1 immunocytochemis-try. A: An embryo at 80% of development showing three large, pear-shaped PCP cell bodies (stars) that are located posterior to the medianneurosecretory cells (mnc) and ventral to the corpora cardiaca nerve 1(ncc1). The PCP neurons can be easily identified according to theirsize, shape, and axonal projection. This axonal projection is includedschematically (curved black arrow) here and in B and C. The axonsloop around the central body (CB) running in the midline tract(straight white arrow), first dorsally, then project posteriorly between

the CB and the protocerebral bridge (PB), and finally cross to thecontralateral side at the bottom of the CB in the posterior fascicleXVII. B,C: In the second instar (B) and adult (C), the brain hasincreased in size, and differentiated tracts and commissures are nowclearly visible in the CB (open arrows). The cell body position andneurite projection established early are maintained. D: TERM-1 im-munostaining of the PCP cells (stars) in a sagittal section of an adultbrain confirms the morphology of the PCP neurons described above forA–C. The projection of the axons in the midline tract is marked witharrows. Note extensive TERM-1-immunoreactive arborizations in theCB and the PB (arrowheads). For other abbreviations, see list. Scalebar 5 40 mm in A; 50 mm in B; 60 mm in C; 75 mm in D.


because both TERM-1 and LK-1 are cytoplasmic mole-cules, due to competition between the antibodies for over-lapping antigenic sites.

Ultrastructure of the PCP neurons

The anterior midbrain of the insect has long been rec-ognized as a site where neurosecretory cells are especiallypredominant (Geldiay and Edwards, 1973; Raabe, 1989).We have demonstrated here that the PCP cells are clearlydistinguishable from their neighbors on the basis of cellbody size (Figs. 7, 8H) and TERM-1 expression (Fig. 8E).The next logical step was to determine whether thesevarious cells also differ at the ultrastructural level. Using

standard electron microscopic techniques, we were able toidentify several ultrastructural features that were pecu-liar to the PCP neurons in the embryonic and adult brainand that clearly distinguish them from neighboring cells.

At 60% of embryogenesis, a low-power electron photomi-crograph of the dMD (Fig. 9A) shows the PCP cell bodiesalong with a number of unidentified median neurosecre-tory cells. Compared with their neighboring cells, the PCPcells have an extensive cytoplasm, and their nuclei show ahomogeneous matrix with no condensed chromatin (Fig.9A). This is also the case in the adult brain (Fig. 10A). Themedian neurosecretory cells, glia cells of the central body,and other cells of the dMD all are consistently smaller in

Fig. 6. Expression of the TERM-1 antigen by the PCP neurons invarious regions of the adult grasshopper brain. A: Reconstruction ofTERM-1 immunoreactivity present in 50 focal planes of a horizontalsection (100 mm) through the adult brain at the level of the CB. AllTERM-1 expression shown is from the two pairs of PCP cells. Thedashed outline marks the PCP neurites running in the midline tract(mlt). The four PCP cell bodies are marked with stars. Note theabsence of arborizations in the a-lobes and the pedunculi of themushroom bodies. The arrow marked V indicates ventral in A–C, E,and F. B–F are video images of different parts of the adult grasshop-per brain that were taken after TERM-1 immunocytochemistry. The

arrowheads point to fine collateral arborizations of the PCP neurons.B: Protocerebrum (PC) with the a-lobe and the pedunculus (ped) ofthe mushroom body. C: Central body (CB; dashed outline). D: Anten-nal lobe showing that immunoreactive arborizations are present onlyin the glomeruli (arrowheads) but not in the medulla (triangle) of theAL. The arrow marked A indicates anterior. E: TERM-1-expressingarborizations (arrowhead) within the protocerebral bridge (PB;dashed outline) and the lateral ocellar nerve roots (arrows). F: TERM-1-expressing arborizations within the medulla (Me; dashed outline).The X marks the second optic chiasma. Scale bar 5 60 mm in A,B; 50mm in C; 125 mm in D,F; 80 mm in E.


size than the PCP neurons. Compared with PCP cells,embryonic neurosecretory cells have only a thin cytoplas-mic region surrounding the nucleus and have condensedchromatin in the nuclei, suggesting that they are stillundergoing mitosis (Fig. 9A). To test whether the differ-ences in cytoplasmic and nuclear dimensions we observedin electron microscopic sections have a quantifiable basis,we calculated the nuclear and cell body volumes for thesevarious cell types in the dMD of the 60% embryo andadult, as previously undertaken for the DUMETi neuron(Goodman et al., 1979). We found that, in the embryonicPCP neurons, the nucleus occupies almost two-thirds ofthe cell body, whereas, in the adult PCP cells, the ratioapproaches 1:1, indicating that the cytoplasmic volumehas increased compared with that of the nucleus. A simi-lar trend was seen in the median neurosecretory cells.

These data are consistent with the findings of Goodman etal. (1979).

The embryonic PCP cells are characterized cytoplasmi-cally by extensive ER and a high density of mitochondriaof varying lengths and sizes (Fig. 9B,C). However, notypical granular vesicles are found that could serve asstorage sites for the TERM-1 antigen (Fig. 10A, inset). Thehigh density of mitochondria is maintained in the adultPCP cells (Fig. 9E), but the extensive ER is now missing.The PCP cell bodies are enveloped by multiple layers ofglial membranes (Fig. 9D), and trophospongia, generallyconsidered to have a trophic function, are located on theinner surface of the PCP cell membranes (Fig. 10A, inset).

Comparative ultrastructure of the PCPneurons and median neurosecretory cells

Neurosecretory cells can often be recognized by the ul-trastructural organization of their cell bodies: for exam-ple, the presence of granular vesicles in the cytoplasm(Geldiay and Edwards, 1973; Schooneveld, 1974). A com-parison between the PCP cells and the median neurose-cretory cells (Figs. 9A, 10) shows that the PCP cells do notpossess typical granular vesicles, are enveloped by multi-ple glial membranes with trophospongia, and have a cy-toplasm with dense mitochondria and ER (Figs. 9D,E,10A). The median neurosecretory cells of the adult braincan be divided into three subtypes. In contrast to the PCPcells, all three types possess abundant granular vesiclesbut appear different at the ultrastructural level (Fig. 10B–D). Type 1 cells contain a large number of small, granularvesicles of varying sizes (Fig. 10C). Type 2 cells containtwo different types of granular vesicles: small granularvesicles, similar to type 1 cells but fewer in number, and asmall number of large, granular vesicles (Fig. 10D). Type3 cells possess a high density of electron-translucent spots(Fig. 10B) that may be granular vesicles containing neu-rosecretory material. Compared with the PCP cells, noneof these three median neurosecretory cell types is sur-rounded by glial membranes.

Fig. 7. The TERM-1 antigen is located in the cytoplasm of the PCPcell bodies. Sagittal, semithin sections through the midline of thebrain in a 60% embryo (A) and a second instar larva (B) show indi-vidual PCP cell bodies (stars). Note the increase in cell body diameterfrom the embryo to the second instar. TERM-1 antigen expression isseen as a black DAB-reaction product (white arrows) in the cytoplasmof the PCP cell bodies (white dashed outline in A) at both ages. Theintensity of expression in the second instar is significantly greaterthan that in the embryo. No antigen expression is present on the cellbody surface (black arrows in B) or in the nuclei (n; black dashedoutline in A). Black arrowheads point to condensed chromatin, notTERM-1 expression. Condensed chromatin is also present in the me-dian neurosecretory (mnc) and the glia cells (gl) of the CB of the 60%embryo. The arrow marked A indicates anterior in A and B. For otherabbreviations, see list. Scale bar 5 10 mm.

Fig. 8. TERM-1-immunoreactive PCP neurons also express aleucokinin-1-like (LK-1) antigen. A and B are video images and C–Hare video montages of different focal planes through the brain. A: At70% of embryogenesis, the PCP neurons clearly express the TERM-1antigen in their cell bodies (stars), neurites (arrow), axons (whitearrowheads), and collaterals (black arrowheads). B: No LK-1-likeexpression is detectable at this age in the region where the PCPneurons are found (dashed rectangle), but other LK-1-immunoreactive neurons (open arrow) already are present in the PI.The arrow marked A indicates anterior for A and B. C: Second instar.TERM-1 expression continues in the PCP cell bodies (stars), neurites(arrows), and collaterals (arrowheads). Fine TERM-1-immunoreactivearborizations are present in the protocerebrum (PC; arrowheads). Thearrow marked V indicates ventral in C–H. D: LK-1 immunocytochem-istry in the second instar reveals that antigen expression is restrictedto the PCP cell bodies (stars). No LK-1-like immunoreactivity is foundon the neurites of the PCP neurons or on the collaterals. The LK-1-like expression in the PC is not related to the PCP neurons. E,F: Fifthinstar larva shows that the PCP neurons continue to be TERM-1- andLK-10-immunoreactive throughout larval development, as describedabove. Whereas the TERM-1 antigen (E) is expressed by cell bodies(stars), neurites (arrows), and collateral arborizations (arrowheads),LK-1-like expression (F) is restricted to the cell bodies (stars). G,H: Inthe adult, the expression pattern described for earlier stages is main-tained. Some of the median neurosecretory cells (mnc) have started toexpress LK-1 (H). Scale bar 5 100 mm in A,B; 45 mm in C–F; 40 mmin G,H.


Figure 8

DISCUSSION

Our study provides some insights into the immunocyto-chemistry, ultrastructure, and development of a set ofunique interneurons in the dMD of the brain of the grass-hopper Schistocerca gregaria. The PCP neurons areunique, in that they are the only cells in the entire central

Fig. 9. Ultrastructure of the PCP neurons in the brain of a 60%embryo and adult grasshopper brain. Data are from ten sections andthree preparations for each age. A: Overview of the dorsal median do-main (dMD) with the PCP neurons (stars), the neighboring CB, medianneurosecretory cells (mnc), and glia (gl). Note that the PCP neurons haveconsiderably larger cell bodies and nuclei than the other cells. Theregions outlined with the dashed rectangles are shown in high-magnification photomicrographs in B and C for the embryo and in D andE for the adult. The arrow marked A indicates anterior. B: Extensive ER(white arrows) is a typical feature of the embryonic PCP cell bodies andmay be the production site of the TERM-1 antigen. The solid black arrowmarks the cell membrane, and the open black arrow indicates the doublemembrane of the nucleus (n). C: Characteristic of the PCP neurons is thehigh number of mitochondria (arrowheads) of varying lengths and forms(see also E). The white arrow again marks ER. D: In the adult, glia cellsenvelope the PCP cell bodies with multiple layers of membranes (ar-rows). Trophospongia in the cytoplasm of the PCP cells are indicated byopen white arrows. Extensive ER, as seen in the embryo, is not obviousin the adult PCP neurons. E: The adult PCP neurons still contain thehigh numbers of mitochondria (arrowheads) seen in the embryo. Openarrow indicates the nuclear membrane. n, Nucleus. For other abbrevia-tions, see list. Scale bar 5 12 mm in A; 1.25 m in B,D; 1 mm in C,E.

Fig. 10. Fine structure of PCP neurons compared with that of themedian neurosecretory cells (mnc). A: The two PCP cell bodies (PCP1and PCP2) have axons in the midline tract (mlt) running dorsally overthe CB. Some of the median neurosecretory cells (mnc) also areshown. Inset shows a higher power view of the region enclosed by thedashed rectangle in A. The nucleus (n) of PCP1 and the nuclearmembrane (solid arrow) are surrounded by cytoplasm that is delim-ited from PCP2 by a multilayered glial envelope (triangle). In thecytoplasm, trophospongia (open arrows) are arranged along the glialborder with the neighboring PCP2 neuron. Note the absence ofelectron-dense granular vescicles in the cytoplasm of the PCP neu-rons. B: Three different types of median neurosecretory cells can bedistinguished. Types 1 and 2 contain granular vescicles of differentsize; type 3 cells do not contain any granular vescicles but contain ahigh density of electron-translucent spots (arrows). C: Type 1 cells arecharacterized by a high number of small granular vescicles (arrow-heads). Such granular vescicles are typical for neurosecretory cellsand are considered to contain neuropeptides. Black arrow points tothe double membrane of the nucleus. D: Type 2 cells contain two typesof granular vescicles: small vesicles like those shown in C but fewer innumber (arrowhead) and a small number of large granular vescicles(white arrow). The black arrow indicates the cell membrane. Scalebar 5 20 mm in A; 2 mm in inset; 8 mm in B; 1.25 mm in C,D.


nervous system of this grasshopper that express theTERM-1 antigen (Meier et al., 1993; Ludwig et al., 1999).Exclusive expression of a single molecule in a few individ-ual cells is rare in the insect nervous system; other exam-ples include the two VPLI neurons in the subesophagealganglion of the grasshopper (Thompson et al., 1991; Tyreret al., 1993), the four eclosion hormone-expressing neu-rons in the larva (Truman and Copenhaver, 1989), and thefour prothoracicotropic hormone-producing cells in thepupa (Agui et al., 1979) of Manduca sexta.

The PCP cells arise directly from the neuroectodermand establish the first interhemispheric commissure of theembryonic brain (Boyan et al. 1995a; Ludwig et al., 1999).Expression of the TERM-1 antigen appears at about 40%of embryogenesis and continues into adulthood (Figs. 1, 6),making it relatively simple to follow axogenesis in thesecells over a broad time span (Figs. 3, 4). Indeed, using acombination of intracellular staining, immunocytochemis-try, and classical histology, we can now follow the devel-opment of the PCP neurons from their birth in the mediandomain (Ludwig et al., 1999) through to their adult mor-phology (Fig. 6).

The exact cellular site of TERM-1 expression and itsdynamics have remained unclear up to now. TERM-1 is aglycosylated, 48-kD protein that has not been furthercharacterized biochemically. Meier et al. (1993) observed ahigh TERM-1 expression in the growth cones of the axonsand collaterals during early embryogenesis followed by aredistribution of expression in the axons later in develop-ment. On this basis, they suggested a role in pathfindingor target recognition for the TERM-1 antigen. Althoughthis may be consistent with a role in pathfinding, the onlydocumented case of pioneering by these neurons is that ofthe primary brain commissure (Boyan et al., 1995a; Lud-wig et al., 1999), in which our data suggest that TERM-1is unlikely to play a role. Early in embryogenesis, TERM-1immunoreactivity does not appear in the PCP cells untilwell after they have pioneered the primary commissure.Where we have been able to localize the expression ofTERM-1 (in the cell somata), it was found exclusively inthe cytoplasm and not on the cell surface (Fig. 7). Pioneer-ing mediated by molecules expressed cytoplasmically, atleast in the cell body, is most unlikely. On the other hand,a role for TERM-1 in intercellular signaling (for example,as a neurotrophin) cannot be precluded. Support for thiscomes from the fact that we observed no change in thestrength of the TERM-1 immunoreactivity of these axonsduring subsequent development, as may be expected ifTERM-1 is implicated in pioneering this commissure.Rather, intense immunolabeling in the cell bodies, axons,and the extensive meshwork of collaterals is presentthroughout all of development and in the adult (Figs. 3, 4,6), suggesting a further, as yet unidentified role forTERM-1 in development in addition to that proposed byMeier et al. (1993).

Subsequent to their pioneering of the primary commis-sure, the PCP cells innervate extensive neuropilar re-gions, such as the central body, PB, antennal lobe, and OL(Figs. 5, 6), as well as projecting into thoracic and abdom-inal neuromeres (Meier et al., 1993; Ludwig et al., 1999).Such an extensive network of TERM-1-immunoreactivearborizations in the central nervous system makes it dif-ficult to associate these neurons with a single function or,indeed, to recognize obvious sites for synaptic input in thebrain. The only neurons described so far with comparable

extensive arborizations in the brain are the two VPLIneurons in the subesophageal ganglion (Thompson et al.,1991; Tyrer et al., 1993). It is noteworthy that Thompsonet al. (1991) showed that immunostaining does not revealthe entire VPLI neuron morphology, because there aresmall midline arborizations close to the cell bodies in thesubesophageal ganglion that do not express vasopressin.Thompson et al. (1991) interpreted these arborizations asregions of synaptic input. Only intracellular dye injectionof PCP neurons in the adult brain will reveal whethersimilar non-TERM-1-immunoreactive arborizations existfor the PCP neurons.

We have shown that the PCP cells coexpress TERM-1and a LK-1-like neuropeptide in their cell body cytoplasm(Fig. 8). The cells are not allatostatin or perisulfakininimmunoreactive. LK-1 is an insect myotropic octapeptidethat was isolated first from the cockroach Leucophaeamaderae (Holman et al., 1986) and acts there as a circu-lating hormone modulating visceral muscle contractionsand diuresis (Cook et al., 1989; Hayes et al., 1989; Torfs etal., 2000). Their ultrastructure (see below) suggests thatthe PCP neurons are unlikely to function as neurosecre-tory cells. However, the leucokinin-like peptides are alsothought to be neuromodulators in the CNS of insects (Nas-sel et al., 1992; Nassel, 1993; Torfs et al., 2000), so that thePCP cells may be using this peptide as a neurotransmitteror a neuromodulator. It is interesting to note that LK-1-like expression in the PCP cells appears only after thesecond larval instar and continues into adulthood (Fig. 8).Its appearance during early larval stages rather thanembryonic stages may be associated with metabolicchanges accompanying the switch in feeding behaviorfrom egg yolk to plant material over this period.

Electron microscopy reveals that the PCP neurons areunique in the midline region of the brain, in that theypossess extensive ER and a high density of mitochondria,both probably indicative of a high metabolic rate. Furtherdistinguishing features are the absence of neurosecretorygranules typical of neurosecretory cells (Figs. 9, 10; seeGeldiay and Edwards, 1973), their large cell body size (dueto a large cytoplasmic volume), and the presence of mul-tiple layers of glia membranes forming trophospongia(Figs.9, 10). The PCP neurons and the median neurose-cretory cells also have different axonal projections. Theaxons of the median neurosecretory cells project throughthe corpora cardiaca nerves I to the corpora cardiaca,where their neurosecretory products are released (Agui etal., 1979; Copenhaver and Truman, 1986). By contrast,the PCP axons descend to the ventral nerve cord. Coupledwith our immunocytochemical findings above, these datasuggest that the PCP cells, anatomically, are wide-field,descending neurons that use a neuropeptide as a neuro-modulator in addition to another, as yet unidentified, pri-mary transmitter.

A survey of the literature suggests that cells comparable tothe PCP neurons have been observed previously in otherinsects (Calliphora erythrocephala, giant neurons: Thomsen,1965; Achaeta domesticus, type 4: Geldiay and Edwards,1973; Periplaneta americana, type 2: Krauthammer, 1985).All of these cells have some characteristics in common: a cellbody in the ventral midline region of the brain close to themedian neurosecretory cells, large cell body diameters, theidentical number of neurons (4; except for Calliphora, with10 neurons), multiple glial layers with trophospongia aroundthe cell soma, and extensive ER. The cells in A. domesticus


contain neurosecretory granular vesicles and were consid-ered to belong to the median neurosecretory cells. Those in P.americana do not contain granular vescicles, and Krautham-mer considered them to be nonneurosecretory. The authorscited above all assumed that the axons of these large neu-rons join the corpora cardiaca nerve 1. However, a compar-ative study of PCP-like neurons in different insects (Ludwiget al., in preparation) shows that the cells described byThomsen (1965), Geldiay and Edwards (1973), andKrauthammer (1985) have a projection into the ventralnerve cord just like the PCP neurons of the grasshopper. Ourfindings, together with those of Thomsen (1965), Geldiay andEdwards (1973), and Krauthammer (1985), lead us to spec-ulate that the PCP neurons have putative homologs in otherinsects and that they may have a common neuromodulatoryfunction in the central nervous system. Future studies in-volving electrophysiological recordings and screening for co-expression of TERM-1 with other neuropeptides and trans-mitters aim to elucidate this function further.

ACKNOWLEDGMENTS

The authors thank Dr. H. Agricola for kindly providingallatostatin and perisulfakinin antibodies. DAKO Diag-nostika GmbH (Hamburg, Germany) kindly provided freesamples of their EnVision™ goat anti-mouse HRP-conjugated polymer. The authors also thank Dr. R. Melzerfor kindly providing assistance with electron microscopy.

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