Mesothoracic ventral unpaired median (mesVUM) neurons in the blowflyCalliphora erythrocephala

Mesothoracic Ventral Unpaired Median(mesVUM) Neurons in the Blowfly

Calliphora erythrocephala

MICHAEL SCHLURMANN* AND KLAUS HAUSEN

Zoologisches Institut, Universitat Koln, 50923 Koln, Germany

ABSTRACTThe study describes five ventral unpaired median neurons in the mesothoracic neuro-

mere of the fused thoracic ganglion of Calliphora identified by biocytin staining (mesVUMneurons). The group comprises four efferent neurons and one interneuron which are charac-terized by a common soma cluster in the ventral midline of the neuromere, bifurcatingprimary neurites and bilaterally symmetrical arborizations. Respective soma clusters ofnot-yet-identified VUM neurons were also found in the prothoracic, metathoracic, and ab-dominal neuromeres. The efferent mesVUM neurons are associated with the flight system.Their main arborizations are located in the mesothoracic wing neuropil and their bilateralaxons terminate at the flight control muscles, the flight starter muscles, the flight powermuscles, or at myocuticular junctions of the latter. In contrast, an association of the inter-neuron with a particular functional system is not apparent. The arborizations of the neuronare intersegmental and invade all thoracic neuromeres. A further difference between the twotypes of neurons regards their somatic action potentials, which are overshooting in theefferent neurons and strongly attenuated in the interneuron. Immunocytochemical stainingsrevealed four clusters of octopamine-immunoreactive (OA-IR) somata in the thoracic gan-glion, which reside in the same positions as the VUM somata. We regard this as strongevidence that all groups of VUM neurons contain OA-IR cells and that, in particular, theidentified efferent mesVUM neurons are OA-IR. Our results demonstrate that the mesVUMneurons of Calliphora have similar morphological, electrophysiological, and presumably alsoimmunocytochemical characteristics as the unpaired median neurons of other insects. J.Comp. Neurol. 467:435–453, 2003. © 2003 Wiley-Liss, Inc.

Indexing terms: octopamine; neuroanatomy; neuromuscular junction; insect;

immunocytochemistry; flight muscle

In recent years considerable attention has been paid tothe unpaired median neurons of insects, which were firstdescribed in locusts (Plotnikova, 1969) and have beenfound since then in various hemimetabolous and holo-metabolous species. The somata of these bilaterally sym-metrical neurons are located in the midline of the CNSand reside either in the dorsal or the ventral cortex. Ac-cordingly, they are termed dorsal unpaired median (DUM)neurons (Hoyle et al., 1974) or ventral unpaired median(VUM) neurons (Lange and Orchard, 1986). Both DUMand VUM neurons are either efferent neurons with axons,leaving the ganglia in at least one nerve on each side, orinterneurons.

By means of biochemical methods an efferent DUMneuron was first shown to contain (Evans and O’Shea,1978) and to release (Morton and Evans, 1984) octopam-ine (OA) at its peripheral target sites. Later, the numbers

and arrangements of putative octopaminergic somata inthe brains and segmental ganglia of different insect spe-cies were revealed by immunocytochemical studies (e.g.,locust: Stevenson et al., 1992; cricket: Sporhase-Eichmannet al., 1992; cockroach: Konings et al., 1988; bee: Kreissl etal., 1994; moth: Pfluger et al., 1993; fly: Monastirioti et al.,1995; review: Stevenson and Sporhase-Eichmann, 1995).

*Correspondence to: Michael Schlurmann, Zoologisches Institut der Uni-versitat Koln, Weyertal 119, 50923 Koln, Germany.E-mail: [email protected]

Received 26 September 2002; Revised 9 June 2003; Accepted 6 August2003

DOI 10.1002/cne.10930Published online the week of October 27, 2003 in Wiley InterScience

(www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 467:435–453 (2003)

© 2003 WILEY-LISS, INC.

It turned out that only efferent unpaired neurons showOA-immunoreactivity, whereas unpaired interneuronsare GABAergic (Stevenson and Pfluger, 1992; Thompsonand Siegler, 1993; Sinakevitch et al., 1996). In locusts,most thoracic DUM neurons were individually identifiedby combining immunocytochemistry and neuroanatomicaltracing methods (Stevenson and Sporhase-Eichmann,1995; Braunig, 1997; Duch et al., 1999).

In insects, OA has a broad spectrum of—usuallyenhancing—functions. For instance, as a transmitter itaffects endocrine glands, as a neurohormone it has meta-bolic effects, and as a neuromodulator it has effects onmuscles, sensory structures, and central circuits (reviews:Evans, 1985; Bicker and Menzel, 1989; Erber et al., 1993;Orchard et al., 1993; Braunig and Pfluger, 2001). Thisvariety of effects and the finding that DUM neurons aresensitive to any arousing stimuli (Hoyle and Dagan, 1978)led initially to the assumption that unpaired median neu-rons are activated collectively at the onset of motor activ-ity, and that the peripheral release of OA plays a role ingeneral arousal mechanisms (review: Orchard et al.,1993). Recent investigations in locusts revealed, however,that the activity of identified efferent DUM neurons, andthus the release of OA, is coupled to specific motor pat-terns (Burrows and Pfluger, 1995; Duch and Pfluger,1999) and is controlled by specific inputs (Duch et al.,1999; Morris et al., 1999).

In flies, the existence of unpaired median neurons wasindicated by staining of median somata in the CNS orlabeling them immunocytochemically (Coggshall, 1978;Bothe and Rathmeyer, 1994; Monastirioti et al., 1995), butthe structures of the neurons remained unknown. Fur-thermore, it was doubtful whether these neurons werehomologs of the unpaired median neurons in other spe-cies, since it was claimed that their development wasdifferent (Klambt et al., 1991; Bossing and Technau,1994). In the present study we identified ventral unpairedmedian neurons in the mesothoracic part of the fusedthoracic ganglion of the blowfly Calliphora (mesVUM neu-rons) by means of intracellular biocytin injections anddemonstrate that most of them innervate flight muscles.In addition, we performed immunocytochemical labelingexperiments in order to investigate whether the mesVUMneurons show octopamine-immunoreactivity.

MATERIALS AND METHODS

Experimental animals

The study was performed on wild-type female blowflies(Calliphora erythrocephala, Meig.) obtained from labora-tory stocks (22°C; 12/12 hour light/dark cycle). Cobaltlabelings and immunocytochemical stainings of the mes-VUM neurons were performed in animals age 7–10 days.Young animals age 2–4 days were taken for intracellularrecordings and biocytin fillings of the VUM neurons, astheir ganglion sheath is easier to penetrate with micro-electrodes.

Staining protocols

Cobalt. Retrograde or anterograde mass impregna-tions of all motoneurons and VUM neurons terminatingon particular flight muscles were performed using thecobalt method (Pitman et al., 1972; Tyrer and Bell, 1974).The neurons were filled by cutting the respective motor

nerves and placing the ends into microchambers insertedinto the thoracic cavities and containing 5% (w/v) cobaltchloride. The chambers were then sealed with Vaselineand the animals were stored in a moist chamber (3–5hours, 20°C). After infusion, the incorporated cobalt wasprecipitated by ammonium-sulfide and the whole animalswere fixed for 2 hours with AAF (10 ml 37% formaldehyde,5 ml pure acetic acid, 85 ml 100% ethanol; Lillie, 1965).The thoracic ganglia or the respective muscles were sub-sequently removed, hydrated in a descending series ofethanol, and silver-intensified (Bacon and Altmann, 1977;Strausfeld and Hausen, 1977). Finally, the ganglia ormuscles were dehydrated in an ascending series of etha-nol, embedded via propylene oxide in soft araldite (Dur-cupan, Fluka, Buchs, Switzerland), and cut in horizontalor longitudinal serial sections of 16 �m.

Biocytin. The animals were anesthetized with CO2and glued with beeswax ventral-side-up on a glass slide.The legs of the animals were excised and the wings andthe heads were fixed with wax. The thoraces were ven-trally opened to expose the thoracic ganglia and wereflooded with Ringer’s solution (Hausen, 1982). In order toprevent movements of the ganglia, the foreguts and themidguts, as well as the furcas, were removed and theganglia were stabilized with a small glass hook.

Somata of mesVUM neurons were impaled with micro-electrodes containing 3% biocytin (Sigma, St. Louis, MO)in 0.1% KCl and their spontaneous activity was recordedusing standard electrophysiological techniques. The neu-rons were then iontophoretically filled with biocytin, usingconstant depolarizing currents of 2–5 nA for 5–10 min-utes, and the animals were stored for 2–4 hours in a wetchamber (20°C). The thoraces were isolated, fixed for 4hours in a solution of 4% paraformaldehyde and 0.8%saccharose in 0.07 M phosphate buffer, pH 7.0, and theganglia and all mesothoracic flight muscles were dissectedout. The latter were stored in phosphate buffer (PB) at 4°Cfor later examination, while the ganglia were rinsed in PBfor 2 hours and transferred for 16–20 hours into a solutionof 1.5% streptavidin-HRP (Amersham, Arlington Heights,IL) and 0.5% TritonX-100 in PB. After being rinsed againin PB, the ganglia were preincubated for 3–4 hours in0.05% diaminobenzidine (DAB) in PB, and then incubatedfor 3–5 hours in a solution of 3 ml 0.05% DAB in PB with15 �l 35% H2O2 (Merck, Darmstadt, Germany), both at4°C in the dark. After being rinsed overnight in PB theganglia were contrasted with 1% osmium tetroxide in PBfor 1 hour in the dark, rinsed again in PB, dehydrated ina graded series of ethanol, embedded via propylene oxidein araldite (Durcupan, Fluka), and cut in horizontal serialsections of 16 �m. In the case of successful staining of aVUM neuron, the stored flight muscles of the animal weretreated as the ganglia and cut in transversal or longitu-dinal serial sections of 20 �m in order to reveal the axonterminals of the neuron.

Immunocytochemistry

Flies were obtained from the laboratory stock just be-fore the beginning of the light cycle. Each was immedi-ately anesthetized with CO2 in order to prevent stress-induced OA-release (Orchard et al., 1993) and the thoraxwas isolated. The thoraces were mounted in Petri disheswith dental cement (Protemp, Espe) and were opened andflooded with freshly prepared cold fixative (30 ml satu-

436 M. SCHLURMANN AND K. HAUSEN

rated aqueous solution of picric acid, 10 ml 25% glutaral-dehyde, 0.2 ml glacial acetic acid and 1% Na-metabisulfite, adjusted to pH 3–4 with 3 N NaOH) for 12hours at 4°C. Then the ganglia were dissected out andwashed for 6 days at 4°C in daily changed 0.07 M PB of pH7.6, with 1% TritonX-100 (Aldrich, Milwaukee, WI) added.Thereafter, either the whole ganglia or vibratome slices ofthe ganglia (thickness: 40–60 �m; agarose-embedding)were treated as follows: 1) 10 minutes in 0.1 M Na-borohydride (Sigma) in PB; 2) 1 hour in 10% normal goatserum diluted in PB with 0.25% TritonX 100; 3) incuba-tion for 12 hours with anti-OA serum (monoclonal anti-bodies from mice, BIOMAR Diagnostic Systems, Marburg,FRG) diluted 1:500 in PB with 0.25% TritonX-100 and0.25% bovine serum albumin; 4) incubation for 12 hourswith goat antimouse IgG (Sigma) diluted 1:50 in PB; 5)incubation for 12 hours with peroxidase antiperoxidasecomplex (Sigma) diluted 1:100 in PB. The antibody incu-bations were performed at 4°C. The slices or the wholeganglia were washed for 2 hours in PB after each step andwere finally treated with DAB. The whole ganglia wereadditionally incubated in osmium tetroxide to intensifythe peroxidase staining, as described in the section onbiocytin staining, and cut in horizontal serial sections of16 �m. Apart from some modifications, the immunocyto-chemical method corresponds to that described bySporhase-Eichmann et al. (1992). As controls, further gan-glia, which were not incubated with anti-OA-serum, wereroutinely processed together with the specimens. In noneof the experiments did this treatment lead to cell labeling.

Antibody specificity

The antibody used against OA reportedly shows nocrossreactivity with dopa, dopamine, tyrosine, and seroto-nin, but some crossreactivity with adrenaline, noradrena-line, and tyramine (data sheet, BIOMAR). As discussed byEckert et al. (1992), tyramine is a precursor of octopaminein the biosynthetic pathway, but apparently it does notoccur in the synthesis of other biogenic amines.

Reconstruction of neurons andmicrophotography

Labeled neurons were graphically reconstructed fromthe horizontal serial sections of the ganglia using a pro-jection microscope that allowed imaging of the sectionsonto a drawing table. All reconstructions were fitted into astandard average outline of the thoracic ganglion obtainedfrom perfectly oriented horizontal serial sections. Sectionswere photographed using a compound photomicroscope.The photographs were processed with standard image pro-cessing software (Adobe PhotoShop, Mountain View, CA).

Nomenclature of thoracic neuropils, nerves,and flight muscles (Fig. 1)

The flight muscles of Calliphora consist of two distinctsets of power muscles and control muscles (reviews: Heide,1983; Dickinson and Tu, 1997). The large indirect andasynchronous power muscles insert at the scutum andcomprise one pair of dorsolongitudinal muscles (DLM) andthree pairs of dorsoventral muscles (DVM 1–3), whereasthe 17 pairs of synchronous control muscles (CM) insert atthe lateral sclerites of the thorax. In addition, there is onepair of large indirect and synchronous tergotrochantermuscles (TTM), which serve as starter muscles at theonset of flight.

The thoracic ganglion of Calliphora comprises the fusedpro-, meso-, and metathoracic neuromeres (T1, T2, T3) aswell as the abdominal neuromeres (ABD). The dorsal me-sothoracic neuropil contains the dendrites of flight mo-toneurons and is designated the wing neuropil (Merrittand Murphey, 1992). All flight muscles are innervated bythe dorsal anterior and posterior mesothoracic nerves(AMN and PMN) and the dorsal mesothoracic accessorynerve (MAN). The power muscle DVM 1 is supplied by theAMN, whereas DVM 2 and DVM 3 are supplied by theMAN and the PMN, respectively (Schlurmann, in prep.).The PMN also innervates the DLM and the TTM. Thecontrol muscles are innervated by the AMN and MAN.The AMN supplies the basalar muscles (b 1–3), the tergo-pleural muscles (tp 1–2), the pteral muscles (I 1–2 and III2), and the muscles of the posterior notal wing process (hg1–2). The MAN innervates the pteral muscles (III 1 and

Fig. 1. Innervation pattern of the flight muscles of Calliphora. Thefigure shows the fused thoracic ganglion of Calliphora in dorsal viewand the branching pattern of the three mesothoracic nerves supplyingall flight muscles. The arrangement of the prothoracic, mesothoracic,and metathoracic neuromeres T1, T2, T3 within the ganglion is indi-cated by their outlines at the bases of the leg neuropils. See text forfurther explanations. ABD, abdominal neuromeres; CEC, cervicalconnective; FLN, frontleg nerve; HLN, hindleg nerve; MLN, midlegnerve. Scale bar � 200 �m.

TABLE 1. Nomenclature, Axonal Pathways, and Target Muscles ofIdentified Mesothoracic Ventral Unpaired Median Neurons of Calliphora

NeuronMain axonal

pathwaysTarget muscles on both sides of

the flight motor

mesVUM-CM AMN, MAN Control musclesmesVUM-TT AMN, PMN, MLN Tergo-trochanter muscle (starter

muscle) and targets within themidlegs

mesVUM-MJ PMN Myocuticular junctions of powermuscles DLM and DVM 3.

mesVUM-PM AMN, PMN Power muscles DLM, DVM 1–3.mesVUM-IN — Interneuron, no peripheral targets

437VUM NEURONS IN C. erythrocephala

Figure 2


III 3–4), the muscles of the posterior notal wing process(hg 3–4), and the pleurosternal muscles (ps 1–2). TheAMN and PMN contain also sensory axons from the wingsensilla (W) and the mesothoracic bristles (MB).

RESULTS

The mesVUM neurons of Calliphora were identified byiontophoretic biocytin injections and were reconstructedfrom horizontal serial sections of the thoracic ganglia.Since axonal profiles of the neurons were found to projectvia the dorsal mesothoracic nerves to the flight muscles(see Fig. 1), the latter were also sectioned in order toidentify the targets of each neuron. In addition, the mes-VUM neurons and motoneurons of some flight muscleswere simultaneously labeled by retrograde or anterogradecobalt infusion into the cut ends of the mesothoracicnerves in order to reveal the spatial arrangement of theirarborizations in the thoracic ganglion or their terminals inthe muscles.

The analysis of 71 successful biocytin labelings and 21cobalt labelings demonstrated that the mesVUM neuronsof Calliphora comprise four efferent neurons and one in-terneuron, which could be clearly distinguished by theirdistinct morphologies. The neurons were named accordingto their peripheral targets and are listed in Table 1.

General morphologyof the mesVUM neurons

The mesVUM neurons show individual central ar-borization patterns but also have some common structuralcharacteristics, which are illustrated by the retrogradecobalt staining and the intracellular biocytin stainingscompiled in Figure 2. The somata of the neurons (so, Fig.2A) are rather large, reaching diameters of 20–35 �m, andform a cluster in the ventral cortex of the mesothoracicneuromere, situated in the midline between the two mid-leg neuropils. The single primary neurites (pn) emerging

from the somata ascend in a bundle to the mesothoracicwing neuropil, which contains the dendrites (de) of themotoneurons of the flight muscles. In this area, each pri-mary neurite bifurcates (bi) into two bilaterally symmet-rical lateral neurites (ln in Fig. 2B), which project to thelateral margins of the thoracic ganglion and give rise tothe prominent arborizations. In the efferent mesVUMneurons, the lateral neurites split into axons projecting onboth sides through the mesothoracic nerves into the pe-riphery.

In general, two distinct groups of thin profiles, termedlongitudinal and ventral branches, arise from the primaryneurites between the somata and the main bifurcations.The anterior and posterior longitudinal branches (al andpl in Fig. 2A,C) are situated at the basis of the wingneuropil and run horizontally along the midline of theganglion towards or even into the prothoracic and met-athoracic neuromeres, respectively. The ventral branches(vb in Fig. 2A) emerge from the posterior sides of theneurites just above the somata, project dorsally, and ter-minate between the posterior longitudinal branches. Sincethe ventral branches are extremely fine and could hardlybe traced in the horizontal serial sections of the ganglia,they were in most cases not included in the reconstruc-tions of the neurons.

As judged from their light microscopical appearance,the bulk of the finer central arborizations of the neuronsappear to be dendritic.

mesVUM-CM neuron

Central arborizations (Fig. 3A). The mesVUM-CMis an efferent neuron which shows distinctly fewer rami-fications in the thoracic ganglion than the other efferentmesVUM neurons described below. Typical features of theneuron are numerous very short side branches arisingfrom the primary neurite over the whole length from somato main bifurcation, rather short ventral branches (vb),and, in particular, only very short longitudinal branches,which may even be lacking, as in the neuron shown inFigure 3. The lateral neurites (ln) in the wing neuropilgive rise to a number of secondary processes and to onerelatively small arborization on each side (a) which reachthe posterior border of the prothoracic neuromere. At thelateral edges of the wing neuropil, the neurites split intotwo pairs of axons (ax) which traverse the AMN and MANon either side. On their way into the periphery the axonsof the mesVUM-CM give rise to thin varicose collateralswhich meander through the nerves and terminate withinthem (not shown in Fig. 3).

Peripheral targets (Fig. 3B,C). The axons of themesVUM-CM innervate the control muscles of the flightmotor, some of which are sketched in Figure 3B. The axonrunning through the AMN terminates at the basalar mus-cles with exception of b 1, the tergopleural muscles tp 1–2,the pteral muscles I 1–2 and III 2, as well as the musclesof the posterior notal wing process hg 1–2. The axon tra-versing the MAN terminates at the pteral muscles III 1,III 3–4, and the muscles of the notal wing process hg 3–4.The innervation of the pleurosternal muscles ps 1–2 couldnot be investigated since the muscles were destroyedwhen removing the furca during dissection, but it is verylikely that they are also innervated by the mesVUM-CMneuron. The neuron would, hence, innervate all flightmuscles except for b 1.

Fig. 2. Central arborizations of the mesVUM-neurons. A: Generalarrangement of the mesVUM neurons in the thoracic ganglion. Mid-sagittal section of the thoracic ganglion containing mesVUM neurons,motoneurons of power muscles, and sensory neurons of thoracic bris-tles labeled by a cobalt backfill of the PMN. The somata (so) of themesVUM neurons are situated in the midline of the ventral mesotho-racic neuromere, just posterior to a ventral commissure (vc). A furthercommissure, situated more anteriorly, is marked by an asterisk. Theprimary neurites (pn) project in dorsofrontal direction into the wingneuropil, where they bifurcate (bi). The main arborizations of theneurons invest the same area as the dendrites (de) of the motoneuronsof power muscles in the wing neuropil. The primary neurites give riseto two types of side branches, the ventral branches (vb) and theanterior and posterior longitudinal branches (al, pl). Arrowheads in-dicate sensory projections of mesothoracic bristles. ABD, abdominalneuropils; CEC, cervical connective; T1, T2, T3, prothoracic, mesotho-racic, and metathoracic neuropils; a, anterior; d, dorsal. B: Bilaterallysymmetric lateral neurites (ln) and higher-order branches of abiocytin-labeled mesVUM-TT in the wing neuropil. The neuron ishorizontally sectioned at a level just above the main bifurcation (bi) ofthe primary neurite. Note that tracts of other neurons are clearlyvisible due to osmium treatment of the tissue. CEC, cervical connec-tive; a, anterior; l, lateral. C: Horizontal section of biocytin-labeledanterior and posterior longitudinal branches (al, pl) of the mesVUM-PM, emerging from the primary neurite (pn) at the ventral border ofthe wing neuropil. CEC, cervical connective; ht, haltere tract; a,anterior; l, lateral. Scale bars � 100 �m.


The axons of the mesVUM-CM use the same pathwaysas the motoneurons of the individual control muscles,which were recently identified (Schlurmann, in prep.; Dro-sophila: Trimarchi and Schneiderman, 1994). Even afterentering the muscles the main branches of themesVUM-CM neuron follow the axonal ramifications ofthe respective motoneurons, which were clearly visible inthe serial sections due to the osmium treatment of themuscles. Figure 3C shows some of the axonal branches ofthe mesVUM-CM and branches of the motoneuron (arrow-heads) within the second basalar muscle (b 2) as an ex-ample. Although the very thin profiles of themesVUM-CM appear to be smooth in the section shown,they exhibit numerous varicosities, which are only visibleunder higher magnification. The density of terminalbranches of the mesVUM-CM in the target muscle is sig-nificantly higher than that found in the other efferentmesVUM neurons.

Interestingly, one of the five successfully labeledmesVUM-CM was found to innervate not only the controlmuscles listed above, but also the ventral half of the mus-cle b 1. Since none of the labeled mesVUM-CM showedobvious indications of incomplete staining, this could ei-ther be due to a particular structural variability of theneuron, which was not found in the other mesVUM neu-rons, or could indicate that there are two subtypes of themesVUM-CM neuron having virtually the same centralarborization patterns and axon pathways but differingwith regard to the number of their target muscles.

mesVUM-TT neuron

Central arborizations (Fig. 4A). The mesVUM-TTshows prominent longitudinal side branches of the pri-mary neurite reaching into the prothoracic and the met-athoracic neuromeres (al, pl), and dense arborizationsemerging from the lateral neurites (ln), which invest pref-erentially the anterior part of the wing neuropil. Charac-teristic side branches of the lateral neurites protrude intoprothoracic and metathoracic areas (a1, a2). The axons ofthe mesVUM-TT project on either side into the PMN andthe midleg-nerve MLN, where thin varicose collateralsspread off. Further varicose profiles (co) enter the AMNand seem to terminate within the nerve.

Peripheral targets (Fig. 4C,D). The axon travelingthrough the PMN innervates the tergo-trochanter muscleTTM. On its way into the periphery it courses through abranch of the nerve, which is described as containing thelargest of three motoneurons supplying the TTM (Baconand Strausfeld, 1986; Schouest et al., 1986). The axonshows large varicosities and meanders through the nerve,sometimes reaching the surface. In addition, it gives off anumber of varicose collaterals, which enter tiny sidebranches of the nerve. The terminal arborization withinthe TTM consists of about 100 equally distributed profilesthat closely follow branches of the motoneurons of theTTM. However, since the large TTM motoneuron alonehas about 4,000 terminal branches (Bacon and Strausfeld,1986), only a few of them are accompanied by profiles ofthe mesVUM-TT.

Fig. 3. Structure and target muscles of the mesVUM-CM. A: Ar-borizations of the mesVUM-CM in the thoracic ganglion. The primaryneurite of the neuron shows short ventral branches (vb). Prominentsecond-order branches (a) split off from the lateral neurites (ln) in thewing neuropil. The axonal projections of the neuron (ax) leave theganglion via the dorsal mesothoracic nerves AMN and MAN. Recon-struction after biocytin labeling from horizontal serial sections.B: Target muscles of the mesVUM-CM. The axons of the recon-

structed neuron are assumed to terminate on all control musclesexcept the basalar muscle 1 (b 1). For simplicity, only eight of theinnervated control muscles in the right side of the thorax are shown.Pathways and branching patterns of the nerves AMN and MAN areslightly schematized. C: biocytin labeled axonal branches (arrow) ofthe mesVUM-CM in the muscle b 2. The branches follow the axonalprofiles of the single motoneuron of the muscle (arrowheads). Scalebars � 200 �m in A; 100 �m in C.


The axons in the MLN on either side could only befollowed to the bases of the midlegs, which were routinelyexcised in the course of preparation. It seems reasonableto assume that they terminate on the muscles of the mid-legs.

mesVUM-MJ neuron

Central arborizations (Fig. 5A). The soma of themesVUM-MJ has a diameter of about 35 �m and is one ofthe largest cell bodies in the mesVUM cluster. The pri-mary neurite of the neuron shows pronounced anteriorand posterior longitudinal side branches (al, pl), whichreach the posterior margin of the prothoracic neuromereand extend into the metathoracic neuromere, respectively.The main arborizations cover the whole wing neuropil.The branches originate from the lateral neurites (ln) andproject with no obvious preference in anterior and poste-rior directions, whereby the anterior branches are locatedmore dorsally than the posterior branches. Some mediananterior branches invade the prothoracic neuromere (a).The curved lateral neurites project into the two PMN. Atthe root of each PMN thin varicose collaterals arise, whichpass frontally through the cortex layer into the AMN,where they seem to terminate. Further collaterals arefound within the PMN.

Peripheral targets (Fig. 5B,C). The axons of themesVUM-MJ project to the DLM and the DVM 3 on bothsides. While running over the surfaces of their targetmuscles, they divide into thinner branches and finallyreach the attachment sites of the muscles at the cuticula.In contrast to the terminations of the other mesVUMneurons, the axon terminals of the mesVUM-MJ are found

exclusively in the myocuticular junctions of the two powermuscles, which are constituted of specialized epidermalcells (epi) connecting the individual muscles fibers to thecuticula (Auber, 1963). The terminals of the mesVUM-MJform a loose meshwork of varicose profiles between thesecells. Interestingly, the myocuticular junctions of thepower muscles DVM 1 and 2, which are light-microscopically identical to those of DLM and DVM 3,were not found to be innervated by the neuron.

mesVUM-PM neuron

Central arborizations (Fig. 6A). The mesVUM-PMresembles the mesVUM-MJ with regard to the particu-larly large soma (diameter �35 �m), the pronounced lon-gitudinal branches emerging from the primary neurite (al,pl), and the curved lateral neurites (ln) in the wing neu-ropil. The main arborizations emerging from themesVUM-PM in the wing neuropil are, however, less ex-tended than those of the mesVUM-MJ. Characteristic ofthe mesVUM-PM are one or two major anterior sidebranches (a) of the lateral neurites, which bend to themidline and project far into the prothoracic neuromere,and long posterior side branches (b), which project into thelateral wing neuropil. The axons of the mesVUM-PM runthrough the AMN and PMN, and give rise to some shortvaricose collaterals in the latter.

Peripheral targets (Fig. 6B,C). The mesVUM-PMinnervates the DLM and the DVM 1–3 on either side of thethorax. The axon in the PMN projects to the DLM and theDVM 3, whereas that in the AMN projects to the DVM 1.Both follow the pathways of the motoneurons of the re-spective muscles. The axonal pathway to the DVM 2 has

Fig. 4. Structure and target muscles of the mesVUM-TT. A: Ar-borizations of the mesVUM-TT in the thoracic ganglion. Characteris-tic of the mesVUM-TT are pronounced longitudinal side branches ofthe primary neurite (al, pl), and dense arborizations emerging fromthe lateral neurites (ln), which give rise to long lateral branches (a1,a2). Axonal profiles (ax) leave the ganglion via the PMN and MLN andbeaded axonal collaterals (co) leave the ganglion via the AMN. Recon-

struction after biocytin staining from horizontal serial sections.B: Target muscles of the mesVUM-TT. The axon running through thePMN terminates on the tergotrochanter muscle TTM, as illustrated.The targets of the axon passing through the MLN were not identified,but are presumably muscles of the midleg. C: Biocytin-labeled termi-nals of the mesVUM-TT (arrows) in close vicinity to a motoneuron(arrowheads) within the TTM. Scale bars � 200 �m in A; 100 �m in C.


not yet been identified, but it definitely differs from that ofthe motoneurons of the DVM 2, which run through theMAN (see Fig. 1). In most of the labeled mesVUM-PM, theaxon traversing the PMN was found to have a diameter of5 �m, which is about twice the diameter of the axon in theAMN. At their target muscles, the axons ramify and enterthe individual muscle fibers (Fig. 6C).

Axon terminals (Fig. 7). In order to study the inner-vation of the power muscles in more detail, the PMNanterogradely labeled with cobalt and the power muscleswere sectioned in longitudinal or transverse direction andexamined light-microscopically. The terminal patterns ofthe labeled mesVUM-PM and the motoneurons at theindividual muscle fibers are discussed using a dorsal fiberof the dorsolongitudinal muscle, the DLM e, as an exam-ple.

Figure 7A shows the gross morphology of the DLM e incross section. In general, the muscle fibers of the powermuscles are composed of polygonal columns of myofibrils.In the case of the DLM e, there are about 50 columns (co),arranged in two rows and separated by intercolumnarclefts of about 4 �m width (arrows). Depending on theirdifferent diameters, the individual columns contain about200–450 myofibrils, which are clearly visible under thelight microscope when using Nomarski optics.

Branches of the PMN invade the space below and abovethe DLM e and project longitudinally over its dorsal andventral surface. Side branches are given off from the lon-gitudinal branches at more or less regular intervals, pro-jecting into the intercolumnar clefts and also invading thecolumns of myofibrils (Fig. 7B), most probably via the

membrane invaginations of the T-system described previ-ously (Smith and Sacktor, 1970). At higher magnificationtwo types of terminal branches can be discriminated in theclefts (Fig. 7C). The first type exhibits varicosities withdiameters of about 0.5–1.5 �m. The second type is thinner,has smaller varicosities (�0.5 �m) and always accompa-nies branches of the first type, although by no means all ofthem. In both types of branches the varicosities are spacedat about 5–15 �m. The ratio of thick branches and thinbranches is about 20:1.

A comparison with muscle sections containing exclu-sively biocytin-labeled mesVUM-PM terminals (Fig. 7D)demonstrates that the thick and thin profiles belong to themotoneuron of the DLM e and the mesVUM-PM, respec-tively. In the motoneuron the varicosities were exclusivelyfound in the profiles invading the muscle fiber and not inthose running on its surface. Hence, apparently all outputsynapses of the neuron are located in the vicinity of themyofibrils. In contrast, both the surface profiles and theinvading profiles of the mesVUM-PM were found to bevaricose (see Fig. 6C).

As estimated from counts in selected sections, the totalnumber of motoneuron varicosities in the DLM e fiberamounts to �40,000. Respective counts of themesVUM-PM varicosities could not be performed. How-ever, because of the lower density of its terminal profiles,it can be assumed that the number of its varicosities is atleast one magnitude lower than that in the motoneuron.Qualitatively, the same arrangement of terminal profilesis found in all muscle fibers innervated by the mesVUM-PM.

Fig. 5. Structure and target muscles of the mesVUM-MJ. A: Ar-borizations of the mesVUM-MJ in the thoracic ganglion. The neuronis characterized by a large soma, distinct longitudinal side branches(al, pl) of the primary neurite, and curved bilateral neurites (ln).Frontal branches (a) in the middle part of the main arborizations inthe wing neuropil bend towards the midline of the ganglion. Theaxons of the neuron (ax) project through the PMN, varicose axoncollaterals are found in the PMN as well as in the AMN. Reconstruc-tion of a biocytin stained cell from horizontal serial sections. B: Target

sites of the mesVUM-MJ. The axon of the mesVUM-MJ projectsthrough the PMN-branches over the surface of the DLM and the DVM3 and terminates exclusively in the myocuticular junctions of themuscles (shaded areas). C: Terminal branch (arrows) of a biocytin-labeled mesVUM-MJ between the epidermal cells (epi), constitutingthe myocuticular junction of a DLM-fiber with the cuticle (blackstrand on the left side of the epidermis). Scale bars � 200 �m in A; 50�m in C.


mesVUM-IN neuron

The mesVUM-IN (Fig. 8) has the smallest soma in thegroup of the mesVUM neurons (diameter 20 �m). Thestructure of the neuron conforms to that of the othermesVUM neurons with regard to the cell body location, aswell as the overall bilateral symmetry of its arborizations.It lacks, however, the characteristic ventral and longitu-dinal branches emerging from the primary neurite andany axonal projections into the periphery. Furthermore,the main bifurcation (bi) of the primary neurite occursabout 200 �m ventral of the bifurcations of the efferentmesVUM neurons. Immediately after the bifurcation eachof the resulting two neurites splits off a branch, whichfurcates and projects horizontally into the prothoracic andmetathoracic neuromeres (b1, b2). The two neurites as-cend in dorsal direction, thereby flanking the primaryneurites of the efferent mesVUM neurons. Then, bothneurites project laterally and bifurcate several times intomain branches, which descend into the three leg neuropils(a1–a3), and a dorsal branch running through the lateralwing neuropil into the dorsal prothoracic neuropil (a4).Light-microscopically, all higher-order ramifications ofthe neuron appear to be smooth, without any indicationsof varicosities.

The reconstruction of the mesVUM-IN shows only themain branches of the neuron, since its extended higher-order profiles are very thin and dense, and could not betraced. These arborizations seem to invest all layers of thethoracic neuropils.

Soma cluster of the mesVUM neurons

The cluster of mesVUM somata (Fig. 9) represents aunique group of cell bodies in the ventral mesothoraciccortex, which are distinguished by their size and spatialarrangement, and which can be unequivocally identifiedeven when they are not stained with neuroanatomicaltracers.

As stated above, the diameters of the mesVUM somatareach from about 20 �m (mesVUM-IN) to 35 �m(mesVUM-PM), whereas the diameters of the neighboringsomata in this cortex area do not exceed 15 �m. In gen-eral, the somata of the mesVUM neurons are pear-shapedand are even larger in vertical direction. The entire somacluster has a characteristic elongated form with a lengthof �100–150 �m, a width of �50 �m, and a depth of �60�m. The individual somata of the cluster do not seem to bearranged in a constant pattern, although that of themesVUM-PM was usually found in the dorsalmost posi-tion.

The cluster resides anteriorly in the midline of the cor-tex area between the midleg neuropils and lies directlyposterior to a neuropil bridge between the latter (called“ventral commissure” in the following). The lower edge ofthe ventral commissure is situated 70 �m above the ven-tral surface of the ganglion and represents the lowermostconnection between the midleg neuropils, which fuse inmore dorsal layers and are completely separated belowthis point, as shown in Fig. 9A (see also Fig. 2A). Hence,the ventral commissure is a clearly detectable structural

Fig. 6. Structure and target muscles of the mesVUM-PM. A: Ar-borizations of the mesVUM-PM in the thoracic ganglion. ThemesVUM-PM has a large soma and typically curved lateral neurites(ln). Characteristic of the mesVUM-PM are pronounced side branchesof the lateral neurites (a, b), and long anterior and posterior longitu-dinal side branches (al, pl) of the primary neurite. The axons of theneuron project into the AMN and PMN. Reconstruction after biocytinstaining from horizontal serial sections. B: Target muscles of the

mesVUM-PM. The neuron innervates the entire set of power muscleson each side of the thorax. The figure shows the branches of the PMNon the median surface of the six fibers of the DLM, all of which containaxonal branches of the mesVUM-PM. C: Biocytin-labeled varicoseterminal branches of the axon of the mesVUM-PM on the outersurface of the DLM a (arrows). tr trachea. Scale bars � 200 �m in A;50 �m in C.


landmark defining the position of the soma cluster pre-cisely. Reconstructions of the soma clusters of differentanimals containing single biocytin-labeled efferent mes-VUM neurons illustrate their constant spatial relation-

ship to the ventral commissure (Fig. 9B–E). Examinationof the cortex area between the ventral commissure and thetwo midleg neuropils revealed no further group of largesomata in this region. Counts in the preparations showed

Fig. 7. Innervation of thepower muscles. A: Cross-section ofthe muscle fiber DLM e, recon-structed from a serial section afterparaformaldehyde fixation. Themuscle fiber consists of polygonalcolumns of myofibrils (co), sepa-rated by intercolumnar clefts (ar-rows). The structure is representa-tive of all six muscle fibers of theDLM. l, lateral; m, median. B: Lon-gitudinal section of a DLM e withcobalt-labeled branches of thenerve PMN. The PMN branchesrun longitudinally over the dorsaland ventral surface of the musclefiber and are accompanied by tra-chea (tr). Side branches of thenerve (arrows) enter the muscle fi-ber. C: Higher magnification of thesame preparation, demonstratingthat the labeled branches shown inB consist of two types of profiles.Thick profiles are terminalbranches of the motoneuron (ar-rows), thin profiles are terminalbranches of the mesVUM-PM (ar-rowheads). D: Longitudinal sec-tion of the DLM e with a varicoseterminal branch of the mesVUM-PM, stained by intracellular biocy-tin injection. The terminal branchoriginates from a main branch pro-jecting over the dorsal muscle sur-face (arrowheads) and runsthrough several columns of myofi-brils separated by intercolumnarclefts containing trachea (tr). Themyofibrils and clefts are visibledue to the paraformaldehyde fixa-tion used in biocytin histology.Scale bars � 100 �m in A,B; 20 �min C,D.


consistently that the mesVUM cluster comprises 6–7large somata (diameter �20 �m), which exceeds the num-ber of the neurons described above. Hence, there are ap-parently one or two further mesVUM neurons, which arenot yet identified.

Interestingly, similar clusters of large cell bodies werealso found at constant positions in the ventral midline ofthe prothoracic neuromere (2–3 somata), the metathoracicneuromere (2 somata), and the abdominal neuromeres(8–12 somata), which are assumed to also be VUM neu-

rons, although their structural symmetry remains to bedemonstrated. Sections and a reconstruction of the tho-racic clusters are compiled in Figure 11A.

Immunocytochemistry

To investigate whether the efferent mesVUM neuronsshow octopamine-like immunoreactivity as reported forefferent unpaired median neurons in other insects, tho-racic ganglia of Calliphora (n � 28) were labeled with acommercial monoclonal antibody against octopamine. Theexperiments revealed that a cluster of OA-IR somata islocated in the ventral midline of each thoracic neuromereand the fused abdominal neuromeres. The arrangement ofthe four clusters is illustrated in Figure 10.

In a first series of experiments the immunocytochemicalprocedure was performed on vibratome slices of the gan-glia. As demonstrated by the photomicrograph in Figure10, the somata revealed in this way were only weaklystained but could be clearly discriminated from the trans-parent background. However, their positions within theneuromeres could not be exactly determined since prop-erly oriented slices could not be routinely achieved andanatomical landmarks for orientation were barely detect-able in the tissue. A second series of labeling experimentswas therefore performed with whole ganglia, which wereadditionally contrasted with osmium after the immuno-staining and were cut in horizontal serial sections of 20�m. The osmium treatment enhanced the contrast of thelabeled somata considerably and also revealed the struc-ture of the surrounding tissue. Examples of sections ob-tained from two different ganglia are compiled in Figure11B,C.

The four clusters of OA-IR somata were consistentlylabeled in all preparations, irrespective of the stainingmethods employed. The prothoracic cluster is located pos-terior of the frontleg neuropils, whereas the elongatedmesothoracic cluster and the metathoracic cluster resideanteriorly between the respective leg neuropils. The longabdominal cluster (not shown in Fig. 11, but seen in Fig.10) extends over the full length of the cortex layer belowthe row of fused abdominal neuropils. No further clustersof labeled somata were detected.

In general, the immunoreactive somata had diametersof 20–30 �m, whereas the unstained somata surroundingthe four clusters were significantly smaller (diameters�15 �m). However, single immunoreactive somata resid-ing within or somewhat apart from the thoracic clusterswere found to have diameters of only 10–15 �m. Exami-nation of the clusters revealed further that usually one orseveral large somata of the clusters remained unlabeled.The compositions of the clusters were evaluated in theosmium-treated preparations and are compiled in Table 2.

The table shows that the total number and the numberof labeled somata in the four clusters differed somewhatbetween preparations, which is at least partly due to tech-nical effects such as variabilities in the immunocytochem-ical staining process and uncertainties in the counts ofunstained somata. The possibility cannot be excluded,however, that the actual numbers of somata also variedamong preparations. The results demonstrate that theclusters consist of 2–3 large somata in the pro-and met-athoracic neuromeres, of only one soma in each of thefused 11 abdominal neuromeres, but of 6–7 large somatain the mesothoracic neuromere, which supplies the wing-bearing segment. The data show, in addition, that the

Fig. 8. Gross morphology of the mesVUM-IN. The soma of theneuron resides in the common soma cluster of the mesVUM neuronsand gives rise to a single ascending primary neurite, which splits offtwo pairs of main branches (b1, b2) in a median layer of the ganglion,and bifurcates (bi) into two neurites ascending further to the wingneuropil. The neurites divide into main branches, which project intoall leg neuropils (a1, a2, a3), or run through the lateral wing neuropilinto the dorsal prothoracic neuropil (a4). Higher-order profiles of theneuron are neglected. Reconstruction after biocytin labeling fromhorizontal serial sections. Scale bar � 200 �m.


total number of large somata exceeds the number of la-beled large somata in all but the prothoracic cluster, sug-gesting that the clusters also contain neurons which arenot detected by the OA-antibody. While the arrangementof the large somata along the ventral midline of the gan-glion is compatible with the view that they are unpairedneurons, the small OA-IR somata residing distinctly apartfrom the clusters in lateral positions may well be pairedOA-IR neurons.

A comparison of the immunolabeled sections shown inFigure 11B,C with respective sections of the biocytin prep-aration shown in Figure 11A indicates that the threethoracic clusters containing the large OA-IR cell bodiesare identical with the three clusters of VUM somata de-scribed above. The obvious difference regarding the diam-eters of the large somata of respective clusters in the twotypes of preparations are due to different fixation. Theidentity of the mesothoracic cluster with OA-IR somataand the mesVUM cluster is particularly evident, sinceboth are situated directly behind the ventral commissure(see arrows in the left panel) in the midline between the

midleg neuropils and consist of 6–7 large somata. Al-though the positions of the prothoracic, metathoracic, andabdominal clusters of OA-IR somata and the respectivegroups of VUM somata in the biocytin preparations arenot as precisely defined by a prominent landmark as themesothoracic cluster, it seems reasonable to assume thatthese clusters are identical as well, since their arrange-ment relative to the respective leg neuropils and the fusedabdominal neuropil agree closely, and since they comprisesimilar numbers of their large somata.

We therefore regard the results of the present immuno-cytochemical labeling experiments as strong evidence thatall four clusters of VUM neurons in the thoracic ganglionof Calliphora comprise OA-IR cells and that, in particular,at least the majority of the identified mesVUM neuronsshows OA-immunoreactivity.

Signals of the mesVUM neurons

In the course of the biocytin-staining experiments, thesignals of the impaled somata were routinely recordedprior to the injections. In about half of the preparations,

Fig. 9. Spatial arrangement of mesVUM somata in the ventralmesothoracic cortex. Ai–Aiii: Successive horizontal sections of thecortex area between the midleg neuropils (ml) containing parts of abiocytin-labeled soma of an mesVUM-MJ (2, black) and accompanyingunstained large somata of the mesVUM cluster (3–6). The sectionsdemonstrate that the soma cluster is situated in the midline of thecortex, directly posterior to the lowermost commissure between themidleg neuropils (ventral commissure, arrows in Aii and Aiii). Afurther commissure between the midleg neuropils is situated slightlymore frontally and dorsally (asterisk in Aiii). Section planes: 62 �m

(Ai), 78 �m (Aii), 94 �m (Aiii) above the ventral surface of the gan-glion. B: Reconstruction of the soma cluster shown in A. ThemesVUM-MJ (2) occupies the most frontal position in this cluster andresides below the ventral commissure. Somata 1 and 6 are locatedbelow and above the sections shown in A. The cluster comprises sixsomata. C–E: Reconstructions of three further mesVUM clusters con-sisting of six (C,E) and seven (D) somata. Biocytin-labeled somata areblack (C: mesVUM-CM, D: mesVUM-PM, E: mesVUM-TT). All clus-ters are arranged along the midline and are situated posterior to theventral commissure. Scale bars � 50 �m.


the mesVUM neurons had constant resting potentials of–55 mV to –60 mV, and did not show any spike activity. Inthe other preparations, the somata were depolarized dueto synaptic inputs and exhibited low-frequency spike ac-tivity (1–5 Hz). In the efferent mesVUM neurons, theobserved action potentials were always overshooting, in-dicating that the primary neurites or the somata of theneurons have excitable membranes (Fig. 12A–D). At halfamplitude the spikes had a duration of 1.8 � 0.2 ms (meanand SD of 85 spikes of 10 neurons each, see Fig. 10F, forexample) and were followed by a pronounced afterhyper-polarization. In contrast, the action potentials recordedfrom somata of the mesVUM-IN were strongly attenuatedand always nonovershooting, indicating that they werepassively conducted into the somata. In addition, thespikes of the mesVUM-IN were observed several times tooccur in duplets (Fig. 12E).

DISCUSSION

Morphology, number and arrangement ofVUM neurons in Calliphora

The mesVUM neurons identified in the present studycomprise one interneuron and four efferent neurons. Ofthe efferent neurons, one innervates all indirect dorsolon-gitudinal and dorsoventral flight muscles (mesVUM-IF),one the flight control muscles (mesVUM-CM), one thetergotrochanter muscles and unknown targets in the mid-leg (mesVUM-TT), and one supplies the myocuticularjunctions of all the indirect dorsolongitudinal flight mus-cles and of the third dorsoventral flight muscles(mesVUM-MJ).

Common morphological characteristics of the efferentneurons are large somata clustered in the ventral midlineof the mesothoracic neuromere, single primary neuritesascending in dorsofrontal direction to the mesothoracicwing neuropil, bilaterally symmetrical central arboriza-tions, and bilateral axonal projections through the dorsalmesothoracic nerves. Apart from these general features,the four neurons exhibit distinctly different central ar-borizations. In contrast to the efferent neurons, the pro-cesses of the interneuron are confined to the thoracic gan-glion. Its main branches establish intersegmentalconnections between the wing neuropil and all leg neuro-pils as well as further dorsal and median areas in theprothoracic and mesothoracic neuromeres. These struc-tural specificities allowed the unequivocal identification ofthe individual neurons. Each was stained at least fivetimes to confirm the identification.

Although only five mesVUM neurons were identified,their soma cluster was consistently found to comprise 6–7large cell bodies in all preparations. This discrepancycould either indicate that particular mesVUM neuronswere never impaled in the staining experiments, or thatthere are subtypes of the neurons, which show virtuallyidentical central arborization patterns and axon projec-tions, and which were therefore not recognized. Theformer seems rather unlikely when considering the largenumber of stainings performed (n � 71). However, the factthat one of the identified mesVUM-CM was found to in-nervate the muscle b 1, whereas the other four neuronsdid not, could indicate that subtypes of the identified neu-rons having slightly different terminal organizations may

Fig. 10. OA-IR somata in thethoracic ganglion of Calliphora.The photomicrograph shows partof a 60-�m thick vibratome slice ofthe ganglion containing two pro-thoracic and five mesothoracicOA-IR somata (indicated by ar-rows). Dark structures in the sliceare trachea, some of which aremarked by arrowheads. The recon-struction of all somata labeled inthe ganglion shows that clusters ofimmunolabeled cells reside in theventral midline of all thoracic neu-romeres (T1–T3) and the abdomi-nal neuromeres (ABD). The gan-glion is shown in ventral aspect.AMN, anterior mesothoracicnerve; CEC, cervical connective;FLN, frontleg nerve; HLN, hindlegnerve; MLN, midleg nerve. Scalebars � 200 �m, left; 50 �m, right.


exist in fact and may account for the additional 1–2 so-mata in the mesVUM cluster.

The analysis of the biocytin preparations has furtherrevealed that groups of cell bodies, of the same size as themesVUM somata, are also located in the ventral midline

of the prothoracic, metathoracic, and abdominal neuro-meres. The structures of these neurons have not yet beeninvestigated but the size and positions of their somatasuggest strongly that they are also VUM neurons. Thisnotion is supported by the results of the present immuno-

Fig. 11. A: Arrangement of thesoma cluster of the mesVUM neu-rons and the prothoracic and met-athoracic clusters of large somatain the ventral midline of the tho-racic ganglion. The two successivehorizontal sections were obtainedfrom an osmium-treated biocytinpreparation at the levels indicated(d: distance to ventral border ofganglion) and show somata of thethree clusters (arrowheads) as wellas the frontleg, midleg, and part ofthe hindleg neuropils (fl, ml, hl).The reconstruction at right showsall large somata of the clusters re-vealed in the preparation. ThemesVUM cluster is closely associ-ated with the ventral commissureof the midleg neuropils (arrow)and a second, more frontal com-missure (asterisk) and contains apartially visible biocytin-injectedmesVUM-TT (black in the sec-tions, dark gray in the reconstruc-tion). The prothoracic cluster re-sides posterior of the frontlegneuropils, the metathoracic clusteris situated anteriorly between thehindleg neuropils. B,C: Arrange-ment of OA-IR somata in the ven-tral midlines of two ganglia. Thesections were taken at about thesame levels as in A and showgroups of immunolabeled somata(arrowheads) in the three neuro-meres. The entire clusters areshown in the reconstructions (darkgray: labeled somata; light gray:accompanying unlabeled large so-mata). The mesothoracic clustersof OA-IR somata reside in exactlythe same position relative to theventral commissure (arrows) asthe mesVUM cluster in the abovebiocytin preparation. The locationsof the other two clusters of OA-IRsomata agree also with those in A.Differences of the soma diametersin the two types of preparationsare due to different fixations. Scalebar � 50 �m.


labelings and respective findings in Drosophila, which willbe discussed next, and by a previous study which revealedthe existence of two prothoracic VUM cells in the callipho-rid fly Sarcophaga (Bothe and Rathmeyer, 1994). Thesomata of these neurons are located posterior to the pro-thoracic leg neuropils, which conforms to the position ofthe prothoracic cluster of ventral midline somata found inthe present study.

Immunoreactivity of the VUMneurons of Calliphora

The immunolabelings of the thoracic ganglion of Calli-phora were performed with a monoclonal antibody againstOA, which was stated to have high specificity (no crossre-activity with dopa, dopamine, tyrosine, and serotonin),and which was successfully employed previously (Schnei-der et al., 1996). In the latter account the selectivity of theantibody was stated to be superior to that of a polyclonalantibody against OA (Eckert et al., 1992), which has beenused in Drosophila (Monastirioti et al., 1995) and a num-ber of other insect species (e.g., Stevenson and Sporhase-Eichmann, 1995).

The present immunostainings consistently revealedfour clusters of large OA-IR somata and associated unla-beled large somata situated in the ventral midline of thethree thoracic neuromeres and the fused abdominal neu-romeres. Further immunopositive soma clusters were notencountered. Comparison of the mesothoracic cluster con-taining OA-IR somata with the cluster containing themesVUM somata identified by the biocytin injections dem-onstrated clearly that both clusters are identical. We con-clude, therefore, that most of the mesVUM neurons showOA-immunoreactivity, and it can be assumed that theseare the efferent mesVUM neurons as found in other in-sects (see below). In addition, it seems most likely that themajority of the neurons constituting the VUM clusters inthe other thoracic and abdominal neuromeres are alsoOA-IR cells. The immunostainings demonstrated furtherthat single, small OA-IR somata are associated with thethree thoracic clusters. These somata were found to besituated either in line with or between the large OA-IRsomata, but also distinctly apart from the cluster in morelateral positions, suggesting that the latter are pairedOA-IR cells.

The present findings agree partially with the results ofa previous immunocytochemical study in Drosophila,which also revealed four clusters of OA-IR VUM somata inthe thoracic ganglion (Monastirioti et al., 1995). Judgingfrom a micrograph of the immunostained somata in thatarticle (fig. 10), the locations of the clusters in the neuro-

meres coincide rather well with those in Calliphora. How-ever, there are also several differences between the twospecies: 1) the numbers of large OA-IR somata in the pro-,meso-, metathoracic and abdominal clusters amount to4/5-6/3/5-6 in Drosophila and 2-3/4-6/1-2/4-8 in Callipho-ra; 2) the prothoracic cluster of Drosophila contains ananterior median soma, which lies separately in front of theother three somata, and which was not found in Calli-phora; and 3) the small OA-IR somata associated with thethoracic clusters in Calliphora were not detected in Dro-sophila. While the numerical differences between respec-tive clusters, and different spatial arrangements of theprothoracic clusters, may be species-dependent, the dis-agreement regarding the small OA-IR somata could havetechnical reasons. These somata were clearly detectable inCalliphora since the immunolabeling did not lead to un-specific background staining. However, in Drosophilasuch background staining was reported to be significant(Monastirioti et al., 1995) and it cannot be excluded that itmasked the small somata.

Comparison of the unpaired median neuronsin flies and other insects

The morphologies of median unpaired neurons havebeen investigated in locusts (Watson, 1984; Campbell et

Fig. 12. Spontaneous activity recorded in the somata of mesVUM-neurons. The action potentials of the efferent neurons are overshoot-ing (A–D,F), whereas those of the interneuron are strongly attenu-ated. Resting potentials as indicated. A: mesVUM-MJ. B: mesVUM-PM. C: mesVUM-MJ. D: mesVUM-TT. E: mesVUM-IN. F: Average of85 spikes of the mesVUM-TT shown in D. Calibration � 250 ms, 20mV in A–E; 2.5 ms, 20 mV in F.

TABLE 2. Composition of OA-IR Soma clusters in the Ventral Midline ofthe Thoracic Ganglion of Calliphora1

Total numberof large somata

(�20 �m)

Large OA-IRsomata

(�20 �m)

Small OA-IRsomata

(�15 �m)

Prothoraciccluster

3 2–3 0–1

Mesothoraciccluster

6–7 4–6 0–1

Metathoraciccluster

2–3 1–2 0–1

Abdominalcluster

10–12 4–8 —

1Values based on counts in 15 preparations.


al., 1995; Duch et al., 1999), crickets (Davis and Alanis,1979; Grass et al., 1990; Sporhase-Eichmann et al., 1992),cockroaches (Crossman et al., 1971; Sinakevitch et al.,1996), moths (Brookes and Weevers, 1988; Pfluger et al.,1993; Consoulas et al., 1999), and other insect species(Davis, 1977; Christensen and Carlson, 1981). Combinedwith immunocytochemical stainings of unpaired medianneurons (review: Sporhase-Eichmann and Stevenson,1995), these studies led to the general concept that groupsof unpaired median neurons are located in all segmentalganglia of insects, except the supraesophageal ganglia,and comprise efferent neurons as well as local and inter-segmental interneurons, as shown in locusts (Thompsonand Siegler, 1991, 1993). According to their soma positionsthe unpaired median neurons may be either DUM or VUMneurons, which is attributed to displacement of the latterduring development (Kondoh and Obara, 1982). The so-mata of individual unpaired median neurons of moths andlocusts were even shown to occupy either dorsal or ventralpositions (Kondoh and Obara, 1982; Watson, 1984; Pflugerand Watson, 1988; Stoya et al., 1989; Pfluger et al., 1993;Siegler et al., 2001). A peculiarity of the efferent DUM/VUM neurons is that their somata have excitable mem-branes which generate overshooting action potentials(Hoyle and Dagan, 1978; Goodman and Heitler, 1979;Lange and Orchard, 1986), whereas unpaired interneu-rons show small passively conducted soma spikes (Thomp-son and Siegler, 1991).

All efferent DUM/VUM neurons investigated in thisrespect show OA-immunoreactivity and are structurallycharacterized by a single primary neurite bifurcating intosymmetrical lateral neurites in dorsal neuropil areas thatgive rise to arborizations restricted to their own ganglionor neuromere, and one or several axons exiting the CNS.Targets of the thoracic DUM/VUM neurons are skeletal orvisceral muscles.

Hence, the efferent mesVUM neurons identified in Cal-liphora agree closely with the efferent unpaired medianneurons described in other species concerning their struc-ture and their overshooting somatic action potentials, andmost likely also with regard to their OA-immunoreactivity. However, there are also some notewor-thy differences: 1) the numbers of the mesVUM neuronsand the numbers of somata in the pro-and metathoracicVUM clusters of Calliphora are significantly smaller thanthose in locusts (Campbell et al., 1995; Duch et al., 1999),crickets (Sporhase-Eichmann et al., 1992), and cock-roaches (Eckert et al., 1992), although in all these speciesthe pterothoracic ganglia or neuromeres contain highernumbers of unpaired median cells than the other seg-ments. 2) The efferent VUM neurons of Calliphora, apartfrom their dorsal main arborizations in the wing neuropil,have additional longitudinal and ventral branches indeeper layers of the ganglion, which have so far not beendescribed in other species. 3) The arborizations of mostefferent mesVUM neurons extend into adjacent neuro-meres. 4) The soma spikes of efferent VUM neurons inflies are faster than those in locusts and also in moths(mean duration of 1.8 ms in mesVUM neurons of Calli-phora vs. mean duration of 4.4 ms in DUMETi of locusts,Goodman and Heitler, 1979, and 20–25 ms in moths VUMcells, Brookes and Weevers, 1988; Pfluger et al., 1993). Itshould be mentioned, finally, that at least in the metatho-racic ganglion of locusts there are multiples of certainmorphological types of efferent neurons (e.g., individuals

of DUM 3,4,5, Watson, 1984), which have so far not beendistinguished according to their central arborizations,their axon pathways, and their peripheral targets. If thereare subtypes of the mesVUM-CM or other efferent mes-VUM neurons of Calliphora as discussed above, thesesubtypes can also be assumed to have undistinguishablecentral arborizations and axonal projections.

Whereas the efferent mesVUM neurons of Calliphorashare fundamental structural characteristics with DUM/VUM cells in other insects, the structure of the identifiedintersegmental mesVUM-IN differs significantly from de-scribed intersegmental DUM interneurons in locusts(Thompson and Siegler, 1991). The latter have only asingle primary neurite, which bifurcates in a similar wayto those of efferent DUM neurons, and their lateral neu-rites form branches, which project into adjacent neuro-meres. In contrast, the short primary neurite of themesVUM-IN bifurcates into two parallel, dorsally project-ing neurites, and the intersegmental main branches arisenot only from the lateral neurites but also from the pri-mary neurite. Additionally, projections of intersegmentalDUM interneurons in locusts are restricted to dorsal areasof the neuropil, whereas the mesVUM-IN sends main neu-rites into dorsal neuropils and all ventral leg neuropils,and fine arborizations are found in nearly all areas of thethoracic neuromeres. Hence, the morphology of themesVUM-IN is not compatible with that of the interseg-mental DUM interneurons of locusts. Rather, it seems tobe reminiscent of a mesothoracic catecholamine contain-ing unpaired ventral cells revealed in Drosophila (Budnikand White, 1988), which is presumably dopaminergic,since dopamine is thought to be the only catecholamine inthe CNS of insects (Orchard, 1990; Monastirioti, 1999).Similar to the mesVUM-IN, the neuron sends two neuritesin the dorsal direction, where both project laterally to theopposite borders of the neuropil. In addition, the mainbranches of the neuron have a characteristic curved ap-pearance, as found in the mesVUM-IN, and the position ofthe cell body matches that of the mesVUM-IN. In allinvestigated insects, only one of these neurons was foundper segment (Goodman et al., 1981; Budnik and White,1988; Orchard, 1990; Mesce et al., 2001). The neurons areintersegmentally projecting unpaired cells with a mor-phology resembling the letter H and were consequentlynamed H-cells (Goodman et al., 1981). Cells of this typewere reported to exist also in the fly embryo (Schmid et al.,1999). The mesVUM-IN seems to be somewhat more com-plex than the H-cells described in Schistocerca and Rhod-nius (Goodman et al., 1981; Orchard, 1990) and does notshow the typical H-form, possibly because of the conden-sation of the ganglia. However, in its complexity it resem-bles the H-cells identified recently in Manduca (Mesce etal., 2001), especially with respect to the fine and extremelydense arborizations, which extend into all thoracic neuro-meres. Dopamine immunocytochemistry should finallyshow whether the mesVUM-IN is a dopaminergic neuronor not.

Terminals and functional role of themesVUM neurons

All efferent mesVUM neurons of Calliphora terminateat flight muscles, suggesting strongly that they are func-tionally involved in flight control. The mesVUM-PM,mesVUM-CM, and the mesVUM-TT terminate with vari-cose profiles at the surfaces and within their target mus-


cles, thereby following closely the terminal branches of therespective motoneurons. Closer examination of the termi-nals within the power muscle DLM demonstrated that theterminals of the motoneurons show varicosities that aredistinctly larger than those of the mesVUM-PM. A similarsituation is found in the larval body wall muscles of Dro-sophila (Monastirioti et al., 1995) and Manduca (Consou-las et al., 1999) in which motoneurons and median un-paired neurons constitute large and small varicositiestermed type I and type II boutons, respectively.

The close association of the mesVUM terminals and themotoneuron terminals in all flight muscles suggests thatthe mesVUM neurons could affect their target musclesdirectly as well as indirectly by modulating the transmit-ter release of their motoneurons. However, since in thepower muscles and the tergotrochanter muscle, and mostlikely also in the control muscles, the density of the mo-toneuron terminals was found to exceed the density of theVUM terminals, only a fraction of the former could bedirectly affected by the VUM neurons.

The functional significance of mesVUM neurons inflight control and, in particular, the effects of OA on themuscle activity, have not yet been investigated. However,in locusts and moths application of OA into the hemo-lymph is well known to have both pre-and postsynapticeffects on dorsolongitudinal flight muscles. In particular,it has been demonstrated that OA enhances the neuro-muscular junction potentials, increases the resting poten-tials of the muscles (O’Shea and Evans, 1979; Klaassen etal., 1986), and increases the amplitude of twitch tension,the rate of twitch contraction, and the rate of twitch re-laxation (Whim and Evans, 1988; Malamud et al., 1988).In locusts, all thoracic DUM neurons, including thoseassociated with the flight muscles, were originally thoughtto be active during flight and to affect by overall OA-release the central and sensory circuits of the flight sys-tem, as well the contraction kinetics and the energy me-tabolism of the flight muscles (Orchard et al., 1993).Recent investigations demonstrated, however, that theDUM neurons innervating the flight muscles are inhibitedin flight (Duch and Pfluger, 1999). It has been demon-strated that OA-release of the latter at rest facilitateseffective carbohydrate metabolism of the flight musclesrequired, especially during the energy-demanding start offlight, but not during prolonged flight, in which the mus-cles are known to switch to lipid oxidation as the primaryenergy source (Mentel et al., 2003). Whether OA-releaseby the mesVUM neurons of flies has similar effects on thecontraction kinetics and the energy metabolism of theflight muscles remains to be investigated. It should bementioned, however, that the flight muscles of flies areexclusively fueled by carbohydrates (Beenakkers et al.,1985), and that preliminary recordings of the mesVUM-PM, mesVUM-CM, and mesVUM-MJ indicate that theyactivate during take-off and seem to remain active duringthe whole flight.

In contrast to the other efferent mesVUM neurons, themesVUM-MJ terminates exclusively between the special-ized epidermis cells linking the myofibrils of power mus-cles to the cuticle. So far, this kind of innervation has notbeen described in any other species. Interestingly, only themyocuticular junctions of the DLM and the DVM 3, andnot those of the DVM 1 and DVM 2, are innervated. Thismight be attributed to the fact that the DVM 3 is a dorsaloblique muscle, and belongs, hence, to the dorsolongitudi-

nal muscles, although it functions as a dorsoventral mus-cle (see Wisser and Nachtigall, 1984). Electron-microscopystudies of the myocuticular junctions of the DLM in Cal-liphora (Auber, 1963) and Drosophila (Reedy and Beall,1993) have demonstrated that the myofibrils of the DLMterminate with specialized z-disks at the epidermis, andthat each epidermal cell links 7–20 myofibrils to the cuti-cle. Within the epidermal cells numerous microtubulispan between cuticle and myofibrils. As structural ele-ments, microtubuli are involved in defining and maintain-ing cell shape and are found, in particular, in specializedmechanically stressed structures. In the myocuticularjunctions of dipteran power muscles, they apparentlytransmit the power of muscle contractions to the cuticularthoracic box, the oscillations of which drive the wingmovements in flight. It is tempting to assume that OArelease by the mesVUM-MJ could modulate the mechan-ical properties of the microtubules and, thus, could influ-ence the power transmission of asynchronous flight mus-cles to the thorax, which in turn should alter wingkinematics.

Apart from their varicose terminals at the flight mus-cles, all efferent mesVUM neurons show further varicoseprofiles, which branch off from the axons either in thethoracic ganglion or within the peripheral nerves. Thesecollaterals meander through the peripheral nerves andcould, hence, affect the axons of neighboring motor orsensory neurons as well as glia cells in some way not yetknown. Since the axons of the mesVUM neurons usuallyproject through the same nerves as the motoneurons oftheir target muscles, the latter may be affected in partic-ular. Some axons of efferent DUM neurons in locusts wererecently reported to form dense meshworks of varicoseramifications at the surface of peripheral nerves servingas neurohemal areas (Braunig et al., 1994; Braunig,1997). Such meshworks are not observed in Calliphora,although the possibility cannot be excluded that the su-perficial parts of the axon collaterals of the mesVUMneurons may also have neurohemal functions. Alterna-tively, the fine-beaded neurites may have no functionalsignificance at all and represent only developmental relicsof outgrowing axons, as discussed by Watson (1984).

Are unpaired neurons in Calliphorahomologous to unpaired neurons

in other insects?

Developmental studies have demonstrated that un-paired median neurons of Orthoptera are progenies of asingle unpaired median neuroblast (MNB) in each seg-ment (e.g., Goodman and Spitzer, 1979; Thompson andSiegler, 1991, 1993). In Drosophila, lineage analysis of thetiny neuroblasts proved to be difficult until Bossing andTechnau (1994) developed an elegant method for in vivolineage tracing of neuroectodermal cells. They reportedthat VUM neurons are not progenies of the MNB but ofseparate VUM precursors, as proposed by an earlier study(Klambt et al., 1991). Unpaired median neurons of Dipteraand Orthoptera would be, thus, not homologous but cells ofdifferent developmental origin. However, Bossing andTechnau found also progenies of the MNB in late Drosoph-ila embryos, which had similar morphological propertiesas unpaired median neurons.

In a more recent study, Schmid et al. (1999) reinvesti-gated the fate of embryonic neuroblasts in Drosophila.


The results of the MNB lineage analysis largely confirmedthe findings of Bossing and Technau, but the VUM neu-rons were concluded to be early-born progeny of the MNBrather than originating from a different cell lineage, sincedistinct VUM neuron precursors were not found. Follow-ing this line of evidence, the VUM neurons of flies arehomologs of the unpaired median neurons of Orthopteraand other insects, as suggested also by the close agree-ment of their morphological, immunocytochemical, andelectrophysiological characteristics revealed in thepresent study.

ACKNOWLEDGMENTS

We thank H. Doring for expert technical assistance andfor preparing the figures, and Prof. H.-J. Pfluger, for read-ing an earlier version of the article.

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Mesothoracic ventral unpaired median (mesVUM) neurons in the blowflyCalliphora erythrocephala