Distribution of GABA-like immunoreactive neurons in insects suggests lineage homology

Distribution of GABA-LikeImmunoreactive Neurons in Insects

Suggests Lineage Homology

J.L. WITTEN1* AND J.W. TRUMAN2

1Department of Biological Sciences, University of Wisconsin-Milwaukee,Milwaukee, Wisconsin 53201

2Department of Zoology, University of Washington, Seattle, Washington 98105

ABSTRACTg-Aminobutyric acid (GABA) is an important inhibitory neurotransmitter in vertebrates

and invertebrates (Sattelle [1990] Adv. Insect Physiol. 22:1–113). The GABA phenotype islineally determined in postembryonic neurons in the tobacco hawkmoth, Manduca sexta(Witten and Truman, [1991] J. Neurosci. 11:1980–1989) and is restricted to six identifiablepostembryonic lineages in the moth’s thoracic hemiganglia. We used a comparative approachto determine whether this distinct clustering of GABAergic neurons is conserved in Insecta. Inthe nine orders of insects surveyed (Thysanura, Odonata, Orthoptera, Isoptera, Hemiptera,Coleoptera, Diptera, Lepidoptera, and Hymenoptera), GABA-like immunoreactive neuronswithin a thoracic hemiganglion were clustered into six distinct groups that occupied positionssimilar to the six postembryonic lineages in Manduca. On the basis of cell body position andaxon trajectories, we suggest that these are indeed homologous lineage groups and that thelineal origins of the GABAergic cells have been very conservative through insect evolution.The distinctive clustering of GABA-positive cells is shared with crustaceans (Mulloney andHall [1990] J. Comp. Neurol. 291:383–394; Homberg et al. [1993] Cell Tissue Res. 271:279–288) but is not found in the centipede Lithobius forficulatus. There is a two- to threefoldincrease in numbers of thoracic neurons between the flightless Thysanura and the mostadvanced orders of insects. Using the GABA clusters as indicators of specific lineages, we findthat only selected lineages have significantly contributed to this increase in neuronalnumbers. J. Comp. Neurol. 398:515–528, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: Manduca sexta; neurotransmitters; evolution; neuronal lineages;

immunocytochemistry

The mature nervous system of insects share manysimilar features, which likely reflect a common developmen-tal plan (Thomas et al., 1984; Tear et al., 1988). Stereotypicarrays of neuronal stem cells or neuroblasts are foundwithin each segmental ganglion, and this pattern is highlyconserved among evolutionarily distant insect orders, suchas grasshoppers and flies (Bate, 1976; Thomas et al., 1984;Doe and Goodman, 1985). This conservation of a commondevelopmental plan has led to studies on homologousneurons (reviewed in Kutsch and Breidbach, 1994; Kutschand Heckmann, 1995). Many such studies, however, havefocused on rare phenotypes, such as neuropeptides (re-viewed in Agricola and Braunig, 1995). We investigatedwhether a widely distributed neurotransmitter thathas important inhibitory functions in motor control i.e.,g-aminobutyric acid (GABA), is also conserved withinArthropoda.

GABA-like immunoreactivity (GLI) is found in the cen-tral nervous system (CNS) of many insects, includingcockroaches (Fuller et al., 1989), crickets (Honegger et al.,1990), grasshoppers (Watson, 1986), honeybees (Schaferand Bicker, 1986), moths (Hoskins et al., 1986; Homberg etal., 1987; Witten and Truman, 1991a), blowflies (Datum etal., 1986), and houseflies (Meyer et al., 1986). It functionsas an inhibitory transmitter in common inhibitory moto-neurons (Usherwood and Grundfest, 1965; Emson et al.,

Grant sponsor: National Institutes of Health; Grant numbers: NS07936and NS13079; Grant sponsor: University of Wisconsin–Milwaukee, Collegeof Letters and Science.

*Correspondence to: Jane L. Witten, Department of Biological Sciences,PO Box 413, University of Wisconsin–Milwaukee, Milwaukee, WI 53201.E-mail: [email protected]

Received 25 November 1997; Revised 28 April 1998; Accepted 7 May 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 398:515–528 (1998)

r 1998 WILEY-LISS, INC.

1974; Usherwood and Cull-Dandy 1975), specific subpopu-lations of spiking local interneurons (Burrows and Siegler,1982; Siegler and Burrows, 1984; Watson and Burrows,1987; Thompson and Siegler, 1991), and intersegmentalinterneurons (Laurent, 1987; Laurent and Burrows, 1988;Thompson and Siegler, 1991) in the grasshopper.

Comparative immunochemical studies in moth andgrasshopper thoracic ganglia show conservation in thedistribution of GLI. Neurons expressing GLI are clusteredinto five or six distinct groups (Watson, 1986; O’Dell andWatkins; 1988; Witten and Truman, 1991a). Cell lineagestudies for the postembryonic neurons in the moth Man-duca sexta demonstrate that the clustering results fromlineal restriction of the GABA phenotype (Witten andTruman, 1991b).

The postembryonic neurons are generated during larvallife and differentiate during metamorphosis (Booker andTruman, 1987; Witten and Truman, 1991a). Of the 24postembryonic lineages present in the second thoracicganglion (T2), GLI is restricted to only six lineages: threepaired ventral (K, M and N), two paired dorsal (E, T), andone unpaired dorsal (X) median lineage (Fig. 1; Witten andTruman, 1991a). All lineage groups remain tightly clus-tered in the adult except the K clonal group. Duringmetamorphosis, the K lineage neurons divide into medial(KM) and lateral (KL) subgroups based on their position inthe ganglion and insertion sites of their axons (Witten andTruman, 1991a). The majority of postembryonic neuronswithin each of the six lineage groups are GABA immu-nopositive. Adjacent to each of the six postembryoniclineage groups is a group of larger (25–35 µm) GABA-immunoreactive neurons. These large neurons are alsopresent in larval ganglia, but in contrast to the smaller (10µm) postembryonic neurons, they never incorporate DNAsynthesis markers during the larval stage (Witten andTruman, 1991b). We have assumed that these largerGABA-positive cells are born during embryonic neurogen-esis from the same neuroblasts that later produce thepostembryonic GABAergic neurons.

The positions of the six GABA-like immunoreactiveclusters in grasshopper thoracic ganglia are similar tothose in the moth (O’Dell and Watkins, 1988; Witten andTruman, 1991a). However, it is not known whether theclustering in the grasshopper results from the respectivegroups being the progeny of a single neuroblast or from themerging of two or more clonal groups (O’Dell and Watkins,1988). Additional anatomical observations suggest thatthese clusters may be homologous to those in Manducasexta. For example, the primary neurites of the cells withinthe midline ventral group in grasshoppers (MVG) and inmoths (KM) project into homologous tracts, ventral commis-sure II (VCII; Witten and Truman, 1991a). Also, theprojections of the lateral ventral group in grasshopper(LVG) neurons and in moth ventral lateral group (KL) cellsenter homologous tracts, the anterior perpendicular tract(aPT). Furthermore, the smallest progeny of the dorsalunpaired median (DUM) lineage of neurons in the grass-hopper and moth (X lineage) are GABA immunoreactive(Thompson and Siegler, 1991; Witten and Truman, 1991a).

We used a comparative approach to determine whethera lineal restriction of GABAergic phenotype is conserved ininsects. As a first step, GLI was mapped in the thoracicnervous systems of adults from nine orders of insects andone noninsect arthropod (the centipede Lithobius forficula-

tus). In all the insects we analyzed, GABA-immunoreac-tive neurons were clustered into six groups in positionssimilar to those of the lineage groups found in the moth.However, this distinctive clustering pattern was absent inthe centipede.

MATERIALS AND METHODS

Animals

Adult animals (males and females) as well as Manducasexta larvae were used in this study. Only winged adultswere analyzed for species in which both winged andnonwinged adults exist. Manduca sexta and Drosophilamelanogaster were obtained from our colonies. Manducasexta were reared under normal long-day photoperiods andon an artificial diet (Bell and Joachim, 1976). Drosophilamelanogaster (Canton-S strain) were raised at 22–25°C onstandard medium. All other animals were obtained fromour colleagues at the University of Washington and theUniversity of Wisconsin–Milwaukee.

GABA immunocytochemistry

Procedures for fixation, dissection, and anti-GABA im-munocytochemical staining have been described previ-ously (Witten and Truman, 1991a) and are detailed brieflyhere. Nervous systems were dissected from cold anesthe-tized animals and immediately fixed in a cold gluteralde-hyde–picric acid–acetic acid solution (GPA; Boer et al.,1979) for 3–4 hours. We used a polyclonal antiserum raisedagainst a GABA–keyhole limpet hemocyanin conjugategenerously supplied by Dr. Timothy Kingan (University ofCalifornia– Riverside).

Fig. 1. Distribution of g-aminobutyric acid (GABA)-like immunore-activity in second thoracic ganglion (T2) of the moth Manduca sexta.The positions and identities (left hemiganglion) of the six GABA-immunopositive lineage groups are shown in this schematized cameralucida drawing. There are three paired ventral lineages (open circles),K (subdivided in KM and KL), M, and N. The dorsal lineages (solidcircles) E and T are paired, and X is unpaired. Anterior is up. Scalebar 5 50 µm.

516 J.L. WITTEN AND J.W. TRUMAN

To aid in penetration of the ganglia and to reducenonspecific staining, nervous systems were exposed to 3%hydrogen peroxide in methanol for 15 minutes and thenincubated for 1 hour in 1 M ethanolamine (Sigma, St.Louis, MO), followed by a 1-hour incubation in collagenase-dispase (1 mg/ml; Boehringer-Mannheim, Indianapolis,IN) and a 2-hour incubation in 10% normal goat serum(NGS) in phosphate-buffered saline (PBS; 0.1 M sodiumphosphate buffer, pH 7.4; 0.15 M NaCl) containing 0.5%Triton X-100 (PBS-X). Nervous systems were incubated ina 1:5,000 dilution of the anti-GABA serum in PBS-X at 4°Cfor 3–4 days, then incubated for 36 hours in a 1:200dilution of the secondary antiserum (biotinylated goatanti-rabbit immunoglobulin G [IgG]; Vector Labs, Burlin-game, CA) in the cold.

After rinsing, the tissue was incubated for 2 hours in theavidin-biotin-peroxidase complex (Vector Labs). The glu-cose-oxidase method of Watson and Burrows (1981) with0.5 mg/ml diaminobenzidine (DAB; Sigma) as the sub-strate was used to visualize the antigen-antibody complex.Nervous systems from different animals always wereprocessed with control tissue, Manduca sexta, to ensurereproducibility of staining.

Three to six nervous systems from each insect andcentipede were processed for immunocytochemistry. Thetissues were mounted in Canada balsam between twocoverslips to permit microscopic analysis of both dorsaland ventral surfaces.

Specific GABA staining was blocked by preabsorbing thediluted antiserum with 8 µg/ml of GABA–bovine serumconjugates overnight before being processed for immunocy-tochemistry (Hoskins et al., 1986). Because we had limitednumbers of insects, only the Manduca sexta and Dro-sophila melanogaster nervous systems were used in thepreabsorption controls. All specific staining was blocked bythe preabsorption of the antiserum.

All preparations were analyzed from camera lucidadrawings of ventral and dorsal surfaces made using a LeitzOrtholux microscope. The preparations were photo-graphed on an Olympus BX50 microscope with Nomarskioptics. The camera lucida drawings shown are from indi-vidual preparations.

RESULTS

Localized distribution of GABA-likeimmunoreactivity in insects

The distribution of GLI was analyzed in adult thoracicganglia from nine orders of insects. We chose groups thatwould include one ametabolous order (firebrat, a thys-anuran), four orders of hemimetabolous insects, and thefour major holometabolous groups. The T2 was analyzed sowe could compare the pattern to the postembryonic lin-eages in the same ganglion of Manduca sexta.

We found six clusters of GABA-immunopositive neuronsin the thoracic ganglia of all insects examined (Figs. 2–5).These clusters were in positions similar to those of thepostembryonic GABAergic lineages in adult Manducasexta (Fig. 1), so we refer to them with the same letterdesignation, but in quotation marks. There were threedorsal GABA-immunoreactive clusters: The anterior ‘‘E’’and posterior ‘‘T’’ are paired and posterior-medial; ‘‘X’’ is

unpaired. The ‘‘X’’ cluster position varied from dorsal toventral in some insects (see below). There are threeventral paired clusters, ‘‘K’’ (including the lateral andmedial subgroups ‘‘KL’’ and ‘‘KM,’’ respectively), ‘‘M,’’ and ‘‘N.’’

Ametabolous and hemimetabolous insects. Ouranalysis included Thysanura (firebrat, Thermobia domes-tica) and four hemimetabolous orders: Odonata (dragonfly,Libellula quadrimaculata), Orthoptera (grasshopper,Schistocerca americana), Isoptera (termite, Zootermopisangusticollis) and Hemiptera (milkweed bug, Oncopeltusfasciatus). In these insects, the positions of the GABA-immunoreactive neuron clusters were very similar tothose of the postembryonic lineages in the moth (Figs.1–3). The distribution of the dorsal clusters (‘‘E,’’ ‘‘T,’’ and‘‘X’’) was unambiguous except that the midline ‘‘X’’ clusterin dragonflies (Fig. 2B) and in T2 only for grasshoppers(Fig. 2C) had a more ventral location. The ventral group-ings showed higher variability. Anteriorly, clusters corre-sponding to KM and KL were evident in all insects, but theK subgroups were essentially merged in the milkweed bug(Fig. 2E). The close proximity of the putative ‘‘M’’ and ‘‘N’’clusters made it difficult to resolve unambiguously theboundaries between these two groups. In grasshopper andmilkweed bug, the positions of the ‘‘M’’ and ‘‘N’’ clusterswere more medial-lateral relative to each other versustheir relative positions along the anterior-posterior axis infirebrats, dragonflies, and termites (Figs. 2, 3).

In addition to these positional similarities, axonal trajec-tories of the ‘‘KM’’ and ‘‘KL’’ cluster neurons were observedin whole-mount preparations of the firebrat (data notshown), dragonfly (Fig. 3B), and grasshopper (data notshown) and for ‘‘KL’’ only in the termite (data not shown).The axons of the ‘‘KM’’ clusters appear to be traveling in thesame tracts as those of the moth, VCII, and the ‘‘KL’’ clusterimmunoreactive axons travel in the aPT (Witten andTruman, 1991a). Our data are consistent with previousfindings of Watson (1986), which show similar trajectoriesfor the MVG (‘‘KM’’) and LVG (‘‘KL’’) immunopositive neu-rons in sectioned grasshopper thoracic ganglia.

Holometabolous insects. We analyzed the four majorholometabolous orders: Coleoptera (beetle, Tenebrio sp.),Lepidoptera (hawkmoth, Manduca sexta), Hymenoptera(bumblebee [Bombus sp.] and winged ant [Formica ob-suris]), and Diptera (fruitfly [Drosophila melanogaster]and blowfly [Calliphora vomitoria]). There was little distor-tion in the pattern of GLI in the beetle from the moth’spostembryonic lineages except for the ventral displace-ment of the ‘‘X’’ cluster (Figs. 4A, 5A). From whole-mountpreparations, it appeared that the beetle’s ‘‘KL’’ neuronssend axons into the aPT (Fig. 5A, open arrowhead), as dothe moth, firebrat, grasshopper, and termite. In Hymenop-tera and Diptera, the increase in total neuron number andcondensation of the ganglia resulted in less-definitiveboundaries between clusters. We observed some shifting ofcluster position within the anterior-posterior axis. In thewinged ant, the ventral clusters (‘‘K,’’ ‘‘M,’’ and ‘‘N’’)appeared to merge along the midline, and the ‘‘K’’ clusterneurons spread across the entire width of each hemigan-glion (Figs. 4C, 5C). Figure 4D shows the likely dorsalexpansion of the ‘‘N’’ cluster. In the bumblebee, the lateralexpansion of the ‘‘K’’ cluster was very prominent (Figs. 4E,5D). The positional displacement of the clusters was mostevident in flies (Figs. 4F, 5E). We analyzed the blowfly

DISTRIBUTION OF GABAERGIC NEURONS 517

Fig. 2. Distribution of g-aminobutyric acid (GABA)-like immuno-reactive clusters in hemimetabolous insects. Each camera lucidadrawing is a composite illustrating the positions of the immunoreac-tive neurons (right hemiganglion) and outlines and identities of theclusters (left hemiganglion) in T2. A: Firebrat (Thermobia domestica).B: Dragonfly (Libellula quadrimaculata). C: Grasshopper (Schisto-cerca americana). D: Termite (Zootermopis angusticollis). E: Milkweed

bug (Oncopeltus fasciatus). Six immunoreactive clusters can be identi-fied. The ‘‘K’’ cluster is divided into two subgroups (‘‘KM’’ and ‘‘KL’’) inall hemimetabolous insects except the milkweed bug (E). Dragonflies(B) are the only hemimetabolous insect with a distinctive ventralunpaired ‘‘X’’ cluster. Solid circles, dorsal surface; open circles, ventralsurface. Anterior is up. Scale bar 5 100 µm.


more extensively because it had larger ganglia than thefruitfly. The ‘‘X’’ cluster is displaced ventrally in theblowfly, as is the unpaired midline postembryonic neuro-

blast in Drosophila melanogaster (Truman and Bate, 1988),and the ‘‘M’’ cluster appears in a more anterior-posteriorrather than medial-lateral orientation (Figs. 4F, 5E).

Fig. 3. Distribution of g-aminobutyric acid (GABA)-like immunore-activity (GLI) in the T2 of hemimetabolous insects. Photographs ofventral surfaces of whole-mount preparations stained with anti-GABAserum. A: Firebrat (Thermobia domestica). B: Dragonfly (Libellulaquadrimaculata). C: Grasshopper (Schistocerca americana). D: Ter-mite (Zootermopis angusticollis). E: Milkweed bug (Oncopeltus fascia-tus). There are three paired clusters of GABA-immunopositive neu-rons on the ventral surface: ‘‘K,’’ ‘‘M,’’ and ‘‘N.’’ Arrows indicate the

positions of the medial and lateral subgroups of the ‘‘K’’ cluster (‘‘KM’’and ‘‘KL,’’ respectively). Asterisks in A–C indicate positions of putativecommon inhibitory motoneurons. Open arrowhead (B) points to poste-riorly projecting immunoreactive axons from the ‘‘KL’’ cluster. Theseaxons are likely to be traveling in the anterior posterior tract. Note thevery anterior position of ‘‘KL’’ in the termite (D). Small arrowheadindicates division between fused T2 and T3 segments in the milkweedbug (E). Anterior is up. Scale bar 5 50 µm.


Fig. 4. Camera lucida drawings of g-aminobutyric acid (GABA)-like immunoreactivity (GLI) in holometabolous insects. The positionsof the immunoreactive neurons are indicated on the right, and theidentities and outlines of the clusters on the left T2 hemiganglion.A: Beetle (Tenebrio sp.). B: Tobacco hawkmoth (Manduca sexta).C: Ant (Formica obscuris), ventral surface. D: Ant, dorsal surface.E: Bumblebee (Bombus sp.). F: Blowfly (Calliphora vomitoria). Thelarger numbers of neurons in holometabolous insects results in manyof the immunoreactive clusters shifting positions and merging to-

gether. For example, the M cluster in flies is oriented on the anterior-posterior axis (F), and the K subgroups appear continuous in allholometabolous insects. The ‘‘X’’ cluster is found dorsally in only twoholometabolous insect orders: Lepidoptera (moth; B) and Hymenop-tera (ant and bumblebee; D and E, respectively). The ventral anddorsal surfaces of the ant (C and D, respectively) are shown separatelyto facilitate cluster identification. Solid circles, dorsal surface; opencircles, ventral surface. Scale bar 5 100 µm.

Fig. 5. g-aminobutyric acid (GABA)-like immunoreactivity (GLI)in the T2 of holometabolous insects. Photomicrogaphs of the ventralsurface show the expansion, positional variability, and merging ofsome of the immunoreactive clusters. The ‘‘K’’ subgroups form acontinuous cluster across the mid-anterior portion of the ganglion(arrows indicate likely positions for the medial and lateral subdivi-sions). The asterisk (*) denotes the anterior-posterior change for the‘‘M’’ cluster in the blowfly (E). A: Beetle (Tenebrio sp.). B: Moth

(Manduca sexta). C: Ant (Formica obscuris). D: Bumblebee (Bombussp.). E: Blowfly (Calliphora vomitoria). Open arrowhead (A), posteriorprojecting immunoreactive axons of the beetle’s ‘‘KL’’ cluster. We alsosee similar axon trajectories from ‘‘KL’’ neurons in the hemimetabolousinsects. Small arrowheads, segmental boundaries between T2 and T3in bumblebee (D) and T1, T2, and T3 in blowfly (E) fused ganglia.Anterior is up. Scale bar 5 25 µm for A,B, 50 µm for C,D.


No distinctive clustering of GABAergicneurons in centipede

Because clustering of GLI is found in crustaceans (Mul-loney and Hall, 1990; Homberg et al., 1993), we wonderedwhether the clustering of GABAergic neurons was ageneral phenomenon of Arthropoda. To test this hypoth-esis, we examined the distribution of GLI in one noninsectarthropod from the class Chilopoda, the centipede Litho-bius forficulatus. GLI is found in centipedes, but theseimmunoreactive neurons are not found in six distinctiveclusters in any ganglia (Fig. 6A,C). Some clustering mayexist, especially on the dorsal surface, but the distributionbears no resemblance to that seen in insects.

Segmental specializations between the centipede andadult insects differ. The most obvious differences are thatcentipedes lack wings and that each body segment bearslegs. Therefore, we compared the pattern of GLI in centi-pedes to the thoracic ganglia of the nonwinged adult

firebrat (Figs. 2A, 3A) and the larval stage of Manducasexta (Fig. 6B). GABA-immunopositive neurons were foundin distinct clusters in thoracic ganglia of firebrats andManduca sexta caterpillars, suggesting the clusteringpattern is restricted to Insecta and Crustacea.

No common inhibitory motoneuronsin holometabolous insects

Common inhibitory motoneurons in grasshoppers arethe largest GABA immunoreactive cells on the ventralsurface, and their axons exit the ganglion in distinctivenerve roots (Watson, 1986; O’Dell and Watkins, 1988). Inour whole-mount preparations, the largest immunoposi-tive neurons on the ventral surface of the grasshopperganglion were between the ‘‘KM’’ and ‘‘M’’ cluster (Figs. 2C,3C). We analyzed the other insect ganglia to see whetherwe could identify putative homologues of common inhibi-tory motoneurons based on soma size (largest GABA-

Fig. 6. Clustering of g-aminobutyric acid(GABA)-like immunoreactivity (GLI) is notfound in a centipede. Photomicrographs ofventral surfaces of centipede (A) and T2 oflarval tobacco hornworm (B) stained withGABA antiserum. C: Camera lucida drawingof immunoreactive neurons in whole-mountpreparation of a centipede ganglion. The im-munoreactive neurons in the centipede gan-glia are not clustered into six distinctivegroups (A,C). Another difference between cen-tipede and insect ganglia is the presence oflarge numbers of GABA-immunoreactiveaxons traversing the ganglia and exiting innerve roots (A, arrows). Clustering is presentin thoracic ganglia of the fourth larval stageof Manduca sexta (B). Two clusters are indi-cated: ‘‘K’’ (solid arrowhead) and ‘‘M’’ (openarrowhead). Open circles, ventral surface;solid circles, dorsal surface (C). Small arrow-heads indicate the single pair of very largeGABA-immunoreactive neurons. Anterior isup. Scale bar 5 50 µm.


immunostaining neurons), cluster location, and presenceof immunoreactive axons in the nerve roots. In addition,we looked for putative GABAergic intersegmental neuronsthat would have axons in the connectives.

Hemimetabolous insects. As stated earlier, two orthree pairs of very large GABA-positive neurons (45–60µm) were found in the ‘‘KM’’ or ‘‘M’’ cluster of grasshoppers(Figs. 2C, asterisks in 3C). These immunopositive cellswere easily identifiable because they were at least twofoldlarger than any other GABA-immunoreactive neurons inthe ganglion. Furthermore, immunoreactive axons werefound in nerve roots with trajectories identical to thosedescribed for the grasshopper common inhibitory motoneu-rons (data not shown). Based on studies of Watson (1986)and Wolf and Lang (1994), it is likely that commoninhibitory motoneurons CI1 and CI2 are the most medialand CI3 the lateral immunopositive cells in Figure 2C.Dragonflies had relatively large (40 µm) pairs of neuronswithin the ‘‘M’’ cluster (Figs. 2B, asterisks in 3B). We foundlarge immunoreactive axons in the nerve roots of theseinsects (Fig. 7), suggestive of a peripheral function forsome of these GABA-staining neurons. Unfortunately, wewere not able to trace the immunoreactive axons in thenerve roots to specific cell bodies in the ganglion. Grasshop-per and dragonfly ganglia contain numerous immunoreac-tive processes in the connectives (data not shown). Theonly other hemimetabolous insect that showed any distinc-tively large GABA-positive neurons was the firebrat. Oneto four pairs of relatively large GABA-immunopositiveneurons (25 µm) were between the ‘‘KM’’ and ‘‘M’’ clusters(Figs. 2A, asterisks in 3A). One immunoreactive axon wasseen in the posterior nerve root of firebrats (data notshown), and as with the other insects, immunoreactiveprocesses in the connectives were numerous. Although wedid not find any unusually large neurons expressing GLIin termites or milkweed bugs, we did find immunoreactiveaxons in nerve roots (Fig. 7).

Holometabolous insects. Although GLI was detectedin neurons greater than 25 µm (Figs. 4, 5), no immunoreac-tivity was found in the nerve roots of holometabolousinsects. Immunoreactive axons were present in the connec-tives, which suggests that some of these larger immunoposi-tive neurons may be intersegmental interneurons (datanot shown).

Centipede. In the majority of preparations, one or twopairs of 25- to 30-µm GABA-immunoreactive neurons werefound in the anterior lateral margin of the centipedeganglia (Fig. 6A,C). These neurons were approximatelytwo to four times as large as the other GABA-stainingneurons. Centipede ganglia contained numerous immuno-reactive processes in the connectives (Fig. 6A). In contrastto the insects, large numbers of GABA-immunoreactiveprocesses were found in the nerve roots of centipedes. Allnerve roots contained at least one axon, but the majority ofaxons exited through the nerve root just posterior to themidpoint of the ganglion (Fig. 6A). It appeared as if some ofthese axons were traveling with the intersegmental axonsand then bifurcating and exiting the ganglion through thislarge midganglion nerve root.

Species-specific variations in cluster size

The number of GABA-immunoreactive neurons in thethree most easily identifiable groups (the anterior dorsal‘‘E,’’ midventral ‘‘K,’’ and unpaired medial ‘‘X’’) in T2 werecounted from camera lucida drawings. Because the ganglia

were mounted between two coverslips, separate drawingsof the ventral and dorsal surfaces could be made tofacilitate the analysis. These results are summarized inFigure 8.

The total number of neurons in adult T2 increasesamong the insects examined from about 1,500 in firebratsto about 4,000–5,000 in moths and flies (Booker andTruman, 1987; Truman and Bate, 1988; Truman, unpub-lished observations). In general, the holometabolous in-sects (beetles, ants, bees, moths, and flies) that had themost neurons tended to have the largest GABA-immunore-active clusters. However, there appeared to be a selectiveexpansion of specific cluster groups such as ‘‘K’’ and ‘‘X.’’

Segment-specific expression of GABAergicneuronal clusters

To test whether there was a segment-specific differencein the distribution of the GABAergic phenotype, we countedthe number of immunoreactive neurons in the most easilyidentifiable cluster (the unpaired medial or ‘‘X’’ cluster) inall three thoracic ganglia. This cluster also was chosenbecause thoracic-abdominal segmental differences in neu-ron number have been documented in this lineage in thegrasshopper (Thompson and Siegler, 1993). Because of thelimited supply of some insects, data points are missing fordragonflies (T1) and bumblebees (T1).

Fig. 7. g-aminobutyric acid (GABA)-like immunoreactivity (GLI)in nerve roots of hemimetabolous insects. Photographs from whole-mount preparations of milkweed bug (A) and dragonfly (B) gangliastained with antiserum raised against GABA. Based on the particulartrajectories of these axons, it is likely that some are the peripheralprojections of the common inhibitory motoneurons. Scale bar 5 25 µm.


There was little segmental variation in ‘‘X’’ cluster size(Fig. 9). Segment specificity in cluster sizes was more oftenseen in the holometabolous insects. We observed segmentspecificity in only one hemimetabolous group, the grasshop-pers. There was no consistent pattern for the size variationbetween thoracic segments. For example, the largest ‘‘X’’cluster in grasshoppers and moths was in T3, whereas inblowflies, T3 had fewer immunoreactive neurons than T1or T2.

DISCUSSION

Conservation in the organizationof GABAergic neurons in insects

In thoracic ganglia of insects, the neurons that exhibitGLI are organized into discrete clusters. In insects withunfused thoracic ganglia (Figs. 2A,B–D, 4A,C,D), thelocations of these clusters are remarkably similar, even ininsects of widely disparate orders. In addition to the cellbody location, the neurons of these clusters show character-istic axonal trajectories that are likewise conserved. Forexample, the primary neurites from the ‘‘KM’’ and ‘‘KL’’cluster subgroups of firebrats, dragonflies, grasshoppers,and beetles enter homologous tracts as those in the moth,the VCII, and aPT, respectively. On the basis of thesesimilarities in position and neurite trajectory for theneuronal clusters, we suggest that these six clusters ofGABA-immunopositive neurons are likely homologous andcan be compared across these diverse taxa.

Comparison of clusters of putative GABAergic neuronsbecomes more problematic in insects with fused thoracicganglia, such as bugs, bees, and higher flies (Figs. 2E,4E,F). Increased numbers of neurons and ganglion fusiondistort the positional relationships of the neuronal groups.Nevertheless, discrete dorsal and midline groups are al-ways present in these insects as are anterior-ventralclusters corresponding to ‘‘KM’’ and ‘‘KL.’’ The major uncer-tainty comes in the posterior-ventral region of the gan-glion, where the close packing of neurons makes it difficultto distinguish the boundaries of the two major clusters inthis region (‘‘M’’ and ‘‘N’’). Despite this ambiguity, theseinsects appear to have the same clusters of GABAergicneurons as other groups.

This conservation of GABA-like immunoreactive clus-ters likely reflects the importance of the function for thisneurotransmitter. GABA is a major inhibitory neurotrans-mitter in insects, and the presence of these neurons may becrucial for the functioning of appropriate neural circuits.For example, GABAergic neurons in two clusters in thegrasshopper probably are involved with leg movements.Some neurons within the MVG or ‘‘KM’’ cluster are spikinglocal inhibitory neurons, which integrate sensory informa-tion for the leg (Burrows and Siegler, 1982; Siegler andBurrows, 1984; Watson and Burrows, 1987), whereasthose in the MPG or ‘‘M’’ cluster are intersegmentalinterneurons involved with limb coordination (Laurent,1987; Laurent and Burrows, 1988). We think it likely thatthe functional roles of the GABA clusters are conservedacross Insecta, but this speculation requires direct physi-ological confirmation.

Relationship of clusters to neuronal lineages

Despite the great diversity of insect types, the earlydevelopment of the insect CNS is very conservative andthought to reflect a common developmental plan (Thomaset al., 1984; Tear et al., 1988). Within a segment, theneuronal stem cells, the neuroblasts, are invariant innumber and are found in stereotyped arrays (Bate, 1976)that are repeated in thoracic and abdominal segments(Doe and Goodman, 1985; Shepherd and Bate, 1990). Thenumber of neuroblasts per segment is remarkably con-served across the insects, showing little variation fromgrasshoppers to higher flies (Thomas et al., 1984). Thisconservation extends even into the primitive apterygote

Fig. 8. Comparison of cluster sizes in insect orders. The number ofg-aminobutyric acid (GABA)-immunoreactive neurons in three clus-ters (‘‘E,’’ ‘‘X,’’ and ‘‘K’’) of the T2 are graphed. We have ordered the dataalong the x-axis to reflect the trend of increasing size of the entireneuronal populations from hemimetabolous to holometabolous in-sects. The trend is that holometabolous insects have the largestGABA-like–immunoreactive clusters. However, there are cluster-specific differences. There is very little change in the cluster sizes ofthe ‘‘E’’ immunoreactive groups, yet the total neuronal populationsincrease almost threefold from the grasshopper to moth. The size ofthe ‘‘K’’ GABA-immunoreactive clusters in holometabolous insects(beetle, ant, bee, moth, blowfly) do seem to be larger than thehemimetabolous ones with the exception of the relatively largegrasshopper ‘‘K’’ cluster. The mean and standard error are plottedwhenever three or more preparations were analyzed. If only onepreparation was available for analysis, we plotted this value (indi-cated by the lack of error bars). Because we had a limited number ofmost insects, statistical analysis was inappropriate. Dgfly, dragonfly;Ghppr, grasshopper.


insects such as the silverfish Ctenolepisma longicauda-tum. Its thoracic neuroblast arrays are identical in bothnumber and position to those found in grasshopper em-bryos (Truman and Ball, 1998).

The conservation in the neuroblast arrays across ordershas been a cornerstone in establishing the homologiesbetween identified neurons in different species (reviewedin Kutsch and Briedbach, 1994; Kutsch and Heckman,1995). Besides rare transmitter phenotypes, the neuronstypically analyzed are large cells with peripherally project-ing axons; they usually are among the earliest born withina lineage. The cells born later are smaller neurons thatfunction as intersegmental, or local interneurons (Good-man et al., 1979; Thompson and Siegler, 1993). The sizes ofmost of the neurons in the GABAergic clusters are consis-tent with their inclusion in the latter group of cells.

Data from both grasshoppers and moths show that theclustering of small GABAergic neurons reflects their clonalorigins. In grasshoppers, for example, the GLI in thedorsal midline cluster is from the late-born progeny of themedian neuroblast (Thompson and Siegler, 1993). Also,neuroblast 5–5 produces two of the common inhibitorymotoneurons (CI1 and CI3) as well as about 60 smallerneurons that make the GABAergic cluster we have de-noted as ‘‘M’’ (Wolf and Lang, 1994). In some cases, though,a cluster seen in the adult grasshopper is thought to haveresulted from the fusion of two or more adjacent lineages(O’Dell and Watkins, 1988). In the moth Manduca sexta,the clustering of the smaller GABAergic cells is likewise aproduct of their clonal relationships (Witten and Truman,1991a). The seven clusters of GABAergic neurons that areborn during larval life in a thoracic hemisegment arisefrom six neuroblast lineages (the K lineage passively splitsin half during the growth of the ganglion).

The lineage relationships of the embryonically derivedneurons with GLI in Manduca are not as clear. Themajority of the cells are clustered around a growingpostembryonic lineage group and probably are the embry-onic progeny of the same neuroblast. Indeed, clonal studiesin Drosophila of the embryonic and postembryonic prog-eny from single neuroblasts typically show both sets ofneurons with similar sites of insertion of their primaryneurites into the neuropil (Prokop and Technau, 1991).More isolated larval GABAergic cells, however, may havebeen part of small lineages that only have an embryonicgeneration phase. In any event, the data from the thoracicganglia of the moth suggests that there are at least sixneuroblasts that produce primarily GABAergic neuronsand that these lineages are responsible for most, if not all,of the GABAergic cells.

For the insects other than grasshoppers and moths inthis study, the clusters of GABAergic cells presumablyidentify lineage groups that arise from the same neuro-blast(s). The similarity in the positions of the majorclusters across a diverse range of insects, coupled with theconservation in neuroblast arrays, then suggests thatsimilarly positioned clusters in different insects likelyresult from homologous neuroblasts. This relationship hasevolutionary implications that are discussed below. Al-though it has been long recognized that the large, uniquelyidentifiable neurons have been conserved through evolu-tion, the results of our study suggest that characteristics oflate-born cells in a lineage have been similarly ratherconserved through much of insect evolution. The stricthomologies of these lineages, though, need to be estab-lished directly by lineage tracing.

The conservation of the clustering of the GABAergicphenotype was detected in another arthropod class, Crus-

Fig. 9. Segmental specificity in ‘‘X’’ cluster neurons. The number ofg-aminobutyric acid (GABA)-immunopositive neurons in the ‘‘X’’ clus-ter from the three thoracic ganglia of each insect are plotted. Themajority of insects showed no distinctive variation in cluster size

between thoracic ganglia. Insects showing some thoracic segmentalspecificity were the grasshopper, bee, moth and perhaps ant. Mean 6S.E. are plotted when n $ 3. ND, No data. Open bars, T1 ganglia; solidbars, T2 ganglia; hatched bars, T3 ganglia.


tacea. Mulloney and Hall (1990) report the clustering ofGABA-immunopositive neurons in crayfish thoracic gan-glia into five distinct groups. Some of the crayfish clustersare in positions similar to those reported here for insects.For example, a pair of large clusters of neurons wasobserved on the ventral midline. The largest cluster of GLIin most of the insects we studied was found on the ventralmidline (the paired ‘‘KM’’ cluster). Another positional simi-larity is between the paired anterior-lateral cluster incrayfish, which extends dorsally, and our ‘‘E’’ cluster.Clustering of GLI and GAD-like immunoreactivity hasbeen found in the crab Eriphia spinofrons (Homberg et al.,1993). Homology has been suggested between thoraciccommon inhibitors in crayfish and grasshoppers (Wiensand Wolf, 1993). These similarities in neurochemical andanatomic data are consistent with molecular and develop-mental studies on homeotic genes that suggest that In-secta and Crustacea share common body and, probably,nervous system plans (Thomas et al., 1984; Akam et al,1994; Patel, 1994; Averof and Akam, 1995). Our datasuggest that this conservation of putative GABAergiclineages is not a general feature of all arthropods, becausethey are not evident in the centipede Lithobius forficula-tus. These observations support the sequence (Ballard etal., 1992) and neural developmental (Whitington et al.,1991) data that suggest insects may have closer affinitiesto crustaceans than to myriopods.

Fate of the common inhibitors

GABAergic common inhibitory motoneurons are presentin grasshoppers (Usherwood and Grundfest, 1965; Emsonet al., 1974; Watson, 1986) and crustaceans (Wiens andWolf, 1993) and are likely to play crucial roles in arthropodmotor control (Wiens, 1990). The three pairs of commoninhibitory motoneurons of grasshoppers are easily identi-fied because they are the largest GABA-positive neuronsin the thoracic ganglia and because they have characteris-tic peripheral trajectories. Clonal analysis of the grasshop-per common inhibitors show CI1 and CI3 are siblingsderived from the first division of neuroblast 5–5, whereasCI2 is produced from a neighboring neuroblast (Wolf andLang, 1994).

The largest GABA-immunoreactive neurons in the fire-brat and dragonfly are found in positions similar to thoseof the common inhibitory motoneurons of grasshoppers.These insects have immunoreactive axons in the nerveroots, but we were not able to trace the central source ofthe axons. We found peripherally projecting axons withGLI in termites and the milkweed bug. Thus, the presenceof common inhibitors was a feature of all the ametabolousand hemimetabolous insects that we examined. In con-trast, we found no GABA-like immunoreactive peripheralaxons in any of the holometabolous insects in our study.Our failure to find these cells suggests that these neuronsare either not produced (or do not survive) in the Holome-tabola or that they have acquired an interneuronal func-tion.

Implications for the diversification of theinsect thoracic CNS

An evolutionary trend found in insects is a strikingincrease in the number of neurons found in the thoracicganglia. The number ranges from about 1,500 neurons perganglion in the apterygote orders Thysanura and Archig-

natha (Truman, unpublished observations) to about 4,000–5,000 per ganglion in insects with well-coordinated flightsuch as Manduca (Booker and Truman, 1987) and Dro-sophila (Truman and Bate, 1988). As discussed earlier, thisincrease in neuronal numbers has been achieved withoutthe addition of new neuroblasts. Hence, it must have comeabout through increases in the number of neurons thatparticular neuroblasts produce.

Assuming that the various clusters of GLI cells doindeed represent homologous lineages, comparison of thenumerical size of a given cluster in different insectsprovides some insight into how CNS diversification hascome about. It should be stressed, though, that this kind ofcomparison may provide only an approximation of how agiven lineage has changed. Some lineages, such as 5–5 ingrasshoppers, appear to make only GABAergic cells (Wolfand Lang, 1994), whereas the midline lineage (‘‘X’’) in thisinsect makes an early group of octopaminergic cells beforeswitching to its GABA set (Thompson and Siegler, 1991,1993). In different insects, a pure GABA lineage may havechanged to one of mixed phenotypes, or a mixed lineagemay have changed its relative proportions of GABA-positive and GABA-negative progeny. At worst, the num-ber of GABAergic cells represents a minimum number ofcells produced by the neuroblast in a given species. An-other concern is that the final number of neurons is aproduct of both neurogenesis and cell death. Studies on themidline lineage in grasshoppers show that neuronal loss,even in the thorax, can be significant (Thompson andSiegler, 1993).

With these cautions in mind, it is nevertheless informa-tive to compare the sizes of various clusters in differentspecies. The data in Figure 9 show that the ‘‘E’’ lineage isrelatively stable in size through the various groups, de-spite overall neuronal numbers in the ganglia increasingby a factor of three moving from Thysanura to Manducaand higher flies. The ‘‘X’’ and ‘‘K’’ groups, however, haveexpanded three- to fourfold in some higher groups com-pared with firebrats. These differences between lineagessuggest that the increase in the neuron numbers seen inthe thorax of more advanced insects was not accomplishedby extended neurogenesis by all of the neuroblasts. In-stead, there has been a selective expansion of only some ofthe lineages. It seems reasonable to speculate that many ofthe lineages that underwent considerable expansion werethose that contributed to flight and its refinement.

Segmental differences in cluster size

Although there are substantial differences between sizesof homologous lineages in the thorax versus the abdomen(Shepherd and Bate, 1990; Thompson and Siegler, 1993),there is less variation between segments within a mainbody region. This is evident for many species when consid-ering the ‘‘X’’ cluster size in the three thoracic segments.There are notable exceptions, however, for example, the‘‘X’’ lineage in the grasshopper is much larger in T3 thanthe two anterior thoracic segments. Because cells in thiscluster respond to auditory stimuli (Thompson and Siegler,1991), the larger number of cells in the T3 cluster mayreflect proximity to the ear, which is located on the firstabdominal segment.

The similarity in cluster size among the thoracic gangliamay reflect similarity in their function. For example, ifclusters were involved with flight motor programs, one


might see more marked differences in specific clustersbetween insects with two pairs of wings than those withone (flies). It would be interesting to see whether changesin homeotic gene expression during evolution—as pro-posed by Carroll (1994) for the ultrathorax (Ubx) geneset—are reflected in changes in GABAergic cluster sizes inthese segments or in Ubx mutants. Comparisons of othercluster sizes in thoracic ganglia will provide more informa-tion concerning segmental differences of GABAergic neu-rons.

ACKNOWLEDGMENTS

The authors thank Drs. John Edwards and Mark Meyerat the University of Washington and Mr. Jody Barbeau atthe University of Wisconsin–Milwaukee (UWM) for provid-ing many of the insects used in this study. We alsoacknowledge the technical assistance of David Bailey andJosh Passman at UWM.

This study was supported in part by National Institutesof Health grants NS07936 (to JLW) and NS13079 (to JWT)and funds from the College of Letters and Science, Univer-sity of Wisconsin–Milwaukee (to JLW).

LITERATURE CITED

Agricola, H.-J. and P. Braunig (1995) Comparative aspects of peptidergicsignaling pathways in the nervous system of arthropods. In O. Breidbachand W. Kutsch (eds): The Nervous System of Invertebrates: An Evolu-tionary and Comparative Approach. Basel, Switzerland: BirkhauserVerlag, pp. 303–327.

Akam, M., M. Averof, J. Catelli-Gair, R. Dawes, F. Galciani, and D. Ferrier(1994) The evolving role of Hox genes in arthropods. Development[Suppl]:209–215.

Averof, M. and M. Akam (1995) Hox genes and the diversification of insectand crustacean body plans. Nature 376:420–423.

Ballard, J.W.O., G.J. Olsen, D.P. Faith, W.A. Odgers, D.M. Rowell, and P.W.Atkinson (1992) Evidence from 12S ribosomal RNA sequences thatonychophorans are modified arthropods. Science 258:1345–1348.

Bate, C.M. (1976) Embryogenesis of an insect nervous system. I. A map ofthe thoracic and abdominal neuroblasts in Locusta migratoria. J.Embryol. Exp. Morph. 35:107–123.

Bell, R.A. and F.G. Joachim (1976) Techniques for rearing laboratorycolonies of tobacco hornworms and pink bollworms. Ann. Ent. Soc. Am.7:413–442.

Boer, H.H, L.P.C. Schot, E.W. Roubos, A. Maat, J.C. Lodder, and D. Reichelt(1979) ACTH-like immunoreactivity in two electronically coupled giantneurons in the pond snail Lymnaea stagnalis. Cell Tissue Res. 202:231–240.

Booker, R. and J.W. Truman (1987) Postembryonic neurogenesis in the CNSof the tobacco hornworm, Manduca sexta. I. Neuroblast arrays and thefate of their progeny during metamorphosis. J. Comp. Neurol. 255:548–559.

Burrows, M. and M.V.S. Siegler (1982) Spiking local interneurons mediatelocal reflexes. Science 217:650–652.

Carroll, S.B. (1994) Developmental regulatory mechanisms in the evolutionof insect diversity. Development Suppl.:217–223.

Datum, K.H., R. Weiler, and F. Zettler (1986) Immunocytochemical demon-stration of g-aminobutyric acid and glutamic acid decarboxylase in R7photoreceptors and C2 centrifugal fibers in the blowfly visual system. J.Comp. Physiol. A 159:241–249.

Doe, C.Q. and C.S. Goodman (1985) Early events in insect neurogenesis. I.Developmental and segmental differences in the patterns of neuronalprecursor cells. Dev. Biol. 111:193–205.

Emson, P.C., M. Burrows, and F. Fonnum (1974) Levels of glutamatedecarboxylase, choline acetyltransferase and acetylcholinersterase inidentified motoneurons in the locust. J. Neurobiol. 5:33–42.

Fuller, H., M. Eckert, and K. Blechschmidt (1989) Distribution of GABA-like immunoreactive neurons in the optic lobes of Periplaneta ameri-cana. Cell Tissue Res. 255:225–233.

Goodman, C.S., M. O’Shea, R. McCaman, and N.C. Spitzer (1979) Embry-onic development of identified motoneurons: Temporal pattern ofmorphological and biochemical differentiation. Science 204:1219–1222.

Homberg, U., T.G. Kingan, and J.G. Hildebrand (1987) Immunocytochemis-try of GABA in the brain and suboesophageal ganglion of Manducasexta. Cell Tissue Res. 248:1–24.

Homberg, U., A. Bleick, and W. Rathmayer (1993) Immunocytochemistry ofGABA and glutamic acid decarboxylase in the thoracic ganglion of thecrab Eriphia spinifrons. Cell Tissue Res. 271:279–288.

Honegger, H.-W., B. Brunninger, P. Braunig, and K. Elekes (1990) GABA-like immunoreactivity in a common inhibitory neuron of the antennalmotor system of crickets. Cell Tissue Res. 260:349–354.

Hoskins, S.G., U. Homberg, T.G. Kingan, T.A. Christensen, and J.G.Hildebrand (1986) Immunocytochemistry of GABA in the antennallobes of the sphinx moth Manduca sexta. Cell Tissue Res. 244:243–252.

Kutsch, W. and O. Breidbach (1994) Homologous structures in the nervoussystems of Arthropoda. Adv. Insect Physiol. 24:1–113.

Kutsch, W. and R. Heckmann (1995) Homologous structures, exemplified bymotoneurons of Mandibulata. In O. Breidbach and W. Kutsch (eds): TheNervous System of Invertebrates: An Evolutionary Comparative Ap-proach. Basel, Switzerland: Birkhauser Verlag, pp. 221–248.

Laurent, G. (1987) The morphology of a population of thoracic intersegmen-tal interneurones in the locust. J. Comp. Neurol. 256:412–429.

Laurent, G. and M. Burrows (1988) A population of ascending intersegmen-tal interneurones in the locust with mechanosensory inputs from ahindleg. J. Comp. Neurol. 275:1–12.

Meyer, E.P., C. Matte, P. Street, and D.R. Nasal (1986) Insect optic lobeneurons identifiable with monoclonal antibodies to GABA. Histochemis-try 84:207–216.

Mulloney, B. and W.M. Hall (1990) GABAergic neurones in the crayfishnervous system: An immunocytochemical census of the segmentalganglia and stomatogastric system. J. Comp. Neurol. 291:383–394.

O’Dell, D.A. and B.L. Watkins (1988) The development of GABA-likeimmunoreactivity in the thoracic ganglia of the locust Schistocercagregaria. Cell Tissue Res. 254:635–646.

Patel, N. (1994) The evolution of arthropod segmentation: Insights fromcomparisons of gene expression patterns. Development Suppl.:201–207.

Prokop, A. and G. M. Technau (1991) The origin of postembryonic neuro-blasts in the ventral nerve cord of Drosophila melanogaster. Develop-ment 111:79–88.

Schafer, S. and G. Bicker (1986) Distribution of GABA-like immunoreactiv-ity in the brain of the honeybee. J. Comp. Neurol. 246:287–300.

Shepherd, D. and C.M. Bate (1990) Spatial and temporal patterns ofneurogenesis in the embryo of the locust (Schistocerca gregaria).Development 108:83–96.

Siegler, M.V.S. and M. Burrows (1984) The morphology of two groups ofspiking local interneurons in the metathoracic ganglion of the locust. J.Comp. Neurol. 224:463–482.

Tear, G., C.M. Bate, and A. Martinez-Arias (1988) A phylogenetic interpreta-tion of the patterns of gene expression in Drosophila embryos. Develop-ment 104:135–145.

Thomas, J.B., M.J. Bastiani, M. Bate, and C.S. Goodman (1984) Fromgrasshopper to Drosophila: A common plan for neuronal development.Nature 310:203–207.

Thompson, K.J. and M.V.S. Siegler (1991) The anatomy and physiology ofspiking local and intersegmental interneurons in the median neuro-blast lineage of the grasshopper. J. Comp. Neurol. 305:659–675.

Thompson, K.J. and M.V.S Siegler (1993) Development of segment specific-ity in identified lineages of the grasshopper CNS. J. Neurosci. 13:3309–3318.

Truman, J.W. and C.M. Bate (1988) Spatial and temporal patterns ofneurogenesis in the central nervous system of Drosophila melanogas-ter. Dev. Biol. 125:145–157.

Truman, J.W. and E.E. Ball (1998) Patterns of embryonic neurogenesis in aprimitive wingless insect, the silverfish, Ctenolepisma longicaudata:Comparison with those seen in flying insects. Dev. Genes Evol. (inpress).

Usherwood, P.N.R. and S.G. Cull-Candy (1975) Pharmacology of somaticnerve-muscle synapses. In: Usherwood, P.N.R (ed): Insect Muscle.Academic Press: London, pp. 207–280.

Usherwood, P.N.R. and H. Grundfest (1965) Peripheral inhibition inskeletal muscle of insects. J. Neurophysiol. 28:497–518.


Watson, A.H.D. (1986) The distribution of GABA-like immunoreactivity inthe thoracic nervous system of the locust Schistocerca gregaria. CellTissue Res. 246:331–341.

Watson, A.H.D. and M. Burrows (1981) Input and output synapses onidentified motoneurons of a locust revealed by the intracellular injec-tion of horseradish peroxidase. Cell Tissue Res. 215:325–332.

Watson, A.H.D. and M. Burrows (1987) Immunocytochemical and pharma-cological evidence for GABAergic spiking local interneurons in thelocust. J. Neurosci. 7:1741–1751.

Whitington, P.M., T. Meier, and P. King (1991) Segmentation, neurogen esisand formation of early axonal pathways in the centipede, Ethmostig-mus rubripes (Brandt). Roux’s Arch. Dev. Biol. 199:349–363.

Wiens, T.J. (1990) The inhibitory innervation of the walking leg of thelobster Homarus americanus. J. Comp. Physiol. 167:43–50.

Wiens, T.J. and H. Wolf (1993) The inhibitory motoneurons of crayfishthoracic limbs: Identification, structures, and homology with insectcommon inhibitors. J. Comp. Neurol. 336:261–278.

Witten, J.L. and J.W. Truman (1991a) The regulation of transmitterexpression in postembryonic lineages in the moth Manduca sexta. I.Transmitter identification and developmental acquisition of expression.J. Neurosci. 11:1980–1989.

Witten J.L. and J.W. Truman (1991b) The regulation of transmitterexpression in postembryonic lineages in the moth, Manduca sexta. II.Role of cell lineage and birth order. J. Neurosci. 11:1990–1997.

Wolf, H. and D.M. Lang (1994) Origin and clonal relationship of commoninhibitory motoneurons CI1 and CI3 in the locust CNS. J. Neurobiol.25:846–864.


Distribution of GABA-like immunoreactive neurons in insects suggests lineage homology

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