Signal transduction in eclosion hormone-induced secretion of ecdysis-triggering hormone.

Inka cells of insect epitracheal glands (EGs) secrete preecdysis and ecdysis-triggering hormones (PETH and ETH) at the end of each developmental stage. Both peptides act in the central nervous system to evoke the ecdysis behavioral sequence, a stereotype behavior during which old cuticle is shed. Secretion of ETH is stimulated by a brain neuropeptide, eclosion hormone (EH). EH evokes accumulation of cGMP followed by release of ETH from Inka cells, and exogenous cGMP evokes secretion of ETH. The secretory responses to EH and cGMP are inhibited by the broad-spectrum kinase inhibitor staurosporine, and the response to EH is potentiated by the phosphatase inhibitor calyculin A. Staurosporine did not inhibit EH-evoked accumulation of cGMP. Changes in cytoplasmic Ca2+ in Inka cells during EH signaling were monitored via fluorescence ratioing with fura-2-loaded EGs. Cytoplasmic Ca2+ increases within 30-120 s after addition of EH to EGs, and it remains elevated for at least 10 min, corresponding with the time course of secretion. Secretion is increased in dose-dependent manner by the Ca2+-ATPase inhibitor thapsigargin, a treatment that does not elevate glandular cGMP above basal levels. The secretory response to EH is partially inhibited in glands loaded with EGTA, while cGMP levels are unaffected. These findings suggest that EH activates second messenger cascades leading to cGMP accumulation and Ca2+ mobilization and/or influx and that both pathways are required for a full secretory response. cGMP activates a staurosporine-inhibitable protein kinase. We propose that Ca2+ acts via a parallel cascade with a time course that is similar to that for cGMP activation of a cGMP-dependent protein kinase.

Ecdysteroids regulate secretory competence in Inka cells.

Ecdysis, or molting behavior, in insects requires the sequential action of high levels of ecdysteroids, which induce accumulation of ecdysis-triggering hormone (ETH) in Inka cells, followed by low levels of ecdysteroids, permissive for the onset of the behavior. Here, we show that high ecdysteroid levels suppress the onset of the behavioral sequence by inhibiting the development of competence to secrete ETH. In pharate pupae of Manduca sexta, Inka cells in the epitracheal glands normally develop competence to secrete ETH in response to eclosion hormone (EH) 8 h before pupation. Injection of 20-hydroxyecdysone (20E) into precompetent insects prevents this acquisition of competence, but does not affect EH-evoked accumulation of the second messenger cyclic GMP. Precompetent glands acquire competence in vitro after overnight culture, and this can be prevented by the inclusion of 20E at concentrations greater than 0.1 microg ml(-1)in the culture medium. Actinomycin D completely inhibits the acquisition of competence, demonstrating that it is dependent on transcriptional events. Cultured epitracheal glands become refractory to the inhibitory effects of 20E in the acquisition of competence at least 3 h earlier than for Actinomycin D, indicating that 20E acts on an early step in a sequence of nuclear events leading to transcription of a structural gene. Our findings suggest that declining ecdysteroid levels permit a late event in transcription, the product of which is downstream of EH receptor activation and cyclic GMP accumulation in the cascade leading to ETH secretion.

Detection of the mitochondrially encoded cytochrome c oxidase subunit I in the trypanosomatid protozoan Leishmania tarentolae. Evidence for translation of unedited mRNA in the kinetoplast.

With the aim of identification of kinetoplast-encoded proteins we investigated the subunit composition of cytochrome c oxidase (respiratory complex IV) from kinetoplast mitochondria of the trypanosomatid protozoan Leishmania tarentolae. Eleven stoichiometric subunits were visible in Coomassie-stained, two-dimensional Blue Native/Tricine-SDS electrophoretic gels. Their partial amino acid sequences indicated that these polypeptides are nuclear-encoded. The mitochondrial subunit I was detected with the polyclonal antibodies against an internal region of this polypeptide. In two-dimensional (9 versus 14%) polyacrylamide glycine-SDS gels this subunit is found as a series of spots located off the main diagonal, a property that can be explained by abnormal electrophoretic migration and aggregation. In gels loaded with high amounts of the purified, enzymatically active oxidase, the subunit I spots could be visualized by staining. The determined N-terminal amino acid sequence of the putative monomeric subunit I (MFXLCLVCLSVS) matched with the predicted sequence, thus indicating that the corresponding kinetoplast unedited mRNA is translated into a functional protein.

Identification of neuropeptides in the midgut of parasitized insects: FLRFamides as candidate paracrines.

Parasitism of Manduca sexta (Lepidoptera: Sphingidae) larvae by the braconid wasp Cotesia congregata (Hymenoptera: Braconidae) leads to accumulation of peptides in host neurons and neurosecretory cells of the central nervous system (CNS) and neurons and endocrine/paracrine cells of the midgut. This accumulation has now facilitated the characterization of two new members of the FLRFamide family from midguts of parasitized larvae. The peptides, given the names F24 and F39, are 24 and 39 amino acids in length with the sequences VRDYPQLLDSGMKRQDVVHSFLRFamide and YAEAAGEQVPEYQALVRDYPQLLDSGMKRQDVVHSFLRFamide. The sequence of F24 is identical to the C-terminal 24 amino acids of F39. The C-terminal 10-mer of each is identical to a previously characterized decapeptide neurohormone (F10). This sequence is preceded by a potential processing site. In nonparasitized insects F39 was present at several-fold the amount of F24. In parasitized insects F24 and F39 accumulate in the middle and posterior regions of the midgut, which are enriched in endocrine/paracrine cells reacting with FLRFamide antisera. In the combined brain and subesophageal ganglion F39 was not detected and the amount of F24 never exceeded 2 fmol per Br/SEG. Of the three peptides, only F10 was found in the hemolymph. Thus, F24 and F39 may be intermediates in the biosynthesis of F10 and may themselves be released locally from endocrine/paracrine cells in the midgut epithelium.

Regulation of ecdysis-triggering hormone release by eclosion hormone.

Ecdysis behavior in the tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae) is triggered through reciprocal peptide signaling between the central nervous system and the epitracheal endocrine system. Recent evidence indicates that eclosion hormone may initiate endocrine events leading to ecdysis through its action on epitracheal glands to cause the release of ecdysis-triggering hormone (ETH). Here, we report that direct exposure of epitracheal glands to eclosion hormone in vitro leads to secretion of ETH. The threshold concentration of eclosion hormone needed to evoke release of ETH is approximately 3 pmol l-1. Eclosion hormone also induces elevation of cyclic GMP, but not cAMP, concentration in epitracheal glands at concentrations similar to those causing release of ETH. Both cGMP and 8-Br-cGMP mimic the secretory action of eclosion hormone. The sensitivity of the secretory response to eclosion hormone occurs during a narrow window of development, beginning approximately 8 h prior to pupal ecdysis. However, eclosion hormone can cause elevation of cGMP levels in epitracheal glands long before they acquire competence to release ETH, showing that the initial portion of the signal transduction cascade is in place early in development, but that the absence of a downstream step in the cascade prevents secretion. Measurements of cGMP levels in epitracheal glands during the ecdysis sequence show a sudden elevation some 30 min after the onset of pre-ecdysis, well after ETH secretion has been initiated. ETH secretion can therefore be viewed as a two-step process, beginning at pre-ecdysis when cGMP levels are relatively low, followed by a massive release resulting from a logarithmic elevation of cGMP levels.

Identification of ecdysis-triggering hormone from an epitracheal endocrine system.

Developing insects repeatedly shed their cuticle by means of a stereotyped behavior called ecdysis, thought to be initiated by the brain peptide eclosion hormone. Here an ecdysis-triggering hormone, Mas-ETH, is described from the tobacco hornworm Manduca sexta. Mas-ETH contains 26 amino acids and is produced by a segmentally distributed endocrine system of epitracheal glands (EGs). The EGs undergo a marked reduction in volume, appearance, and immunohistochemical staining during ecdysis, at which time Mas-ETH is found in the hemolymph. Injection of EGs extract or synthetic Mas-ETH into pharate larvae, pupae, or adults initiates preecdysis within 2 to 10 minutes, followed by ecdysis. Sensitivity to injected Mas-ETH appears much earlier before ecdysis and occurs with shorter latency than that reported for eclosion hormone. The isolated central nervous system responds to Mas-ETH, but not to eclosion hormone, with patterned motor bursting corresponding to in vivo preecdysis and ecdysis. Mas-ETH may be an immediate blood-borne trigger for ecdysis through a direct action on the nervous system.

Parasitism-induced accumulation of FMRFamide-like peptides in the gut innervation and endocrine cells of Manduca sexta.

Manduca sexta larvae that are parasitized by the braconid wasp Cotesia congregata enter a state of developmental arrest following emergence of the wasp larvae from the host. These fifth instar hosts linger for 2 to 3 weeks without resuming feeding, molting, or metamorphosis once the wasps emerge. Immunohistochemical staining with antiserum against FMRFamide revealed dramatic accumulation of FMRFamide-like peptide(s) in the gut nervous and endocrine systems of the developmentally arrested larvae when compared to that observed in unparasitized feeding or starved larvae. Specifically, the number of immunopositive cells and the intensity of staining was enhanced in the neurons of the frontal ganglion, the axons and axon terminals on the midgut surface, and in the gastric endocrine cells. These results were confirmed using ELISA to show that the relative amounts of FMRFamide-like peptides in midgut extracts were highly elevated in the parasitized larvae relative to the fed or starved unparasitized larvae. These data suggest that FMRFamide-like peptides in developmentally arrested larvae are produced in a significantly larger number of gastric endocrine cells, and that the rate of release of the peptides may be suppressed, or the rate of their synthesis may be elevated. Localization of FMRFamide-like peptides in the gastric endocrine cells of C. congregata is also described.

Synthetic pheromone biosynthesis activating neuropeptide gene expressed in a baculovirus expression system.

A synthetic gene of the pheromone biosynthesis activating neuropeptide (PBAN) of corn earworm Helicoverpa zea, with and without a signal sequence of the cuticle protein of Drosophila melanogaster, was cloned behind the polyhedrin promoter of AcMNPV. Two recombinant baculoviruses were constructed and used to infect a number of insect cell lines including Sf9 and 5B1-4. High pheromonotropic activity was consistently obtained from 5B1-4 cell culture that was infected with the recombinant baculovirus vINV-4 containing the signal sequence. The PBAN gene-product was isolated by HPLC and analyzed by electrospray ionization mass spectrometry. Low levels of biological activity obtained from Sf9 cells infected with the recombinant virus vPBAN may be due to lack of proper amidation at the C-terminus of the expressed peptide or rapid proteolytic degradation of the product.

The loss of female sex pheromone after mating in the corn earworm moth Helicoverpa zea: identification of a male pheromonostatic peptide.

Female moths often become depleted of sex pheromone after mating as the various components of virgin behavior are switched off. In examining a potential male contribution to these events in the corn earworm moth Helicoverpa zea, we have characterized a basic polypeptide from the tissues producing (accessory glands) and storing (duplex) the seminal fluids. The peptide evokes the depletion of sex pheromone when injected into virgin females. This pheromonostatic peptide (PSP) is 57 amino acids long and contains a single disulfide bridge. It is blocked at the N terminus with pyroglutamate and at the C terminus by amidation. As little as 23 ng of peptide evokes the near-complete depletion of pheromone in decapitated (neck-ligated) females that had been injected with pheromone biosynthesis-activating neuropeptide. Activity is approximately 15-fold less in intact virgins, showing that the head limits the expression of activity in these injected females. Females mated to surgically impaired males, capable of producing a spermatophore but not transferring spermatozoa or seminal fluids, are depleted of pheromone by injected peptide. Females whose abdominal nerve cords have been severed are not depleted of pheromone after mating. Thus, neural signals either descending or ascending via the nerve cord are required for the depletion of pheromone after mating. PSP, from the seminal fluids, may participate in this process by direct or indirect action on the glandular tissue; if so, it represents an unusual mechanism in insects for the regulation by seminal fluids of postmating reproductive behavior.

Accumulation of neuropeptides in the cerebral neurosecretory system of Manduca sexta larvae parasitized by the braconid wasp Cotesia congregata.

Fifth instar larvae of Manduca sexta that were parasitized by the braconid wasp Cotesia congregata failed to develop after the parasitoid larvae emerged, and these host larvae lingered for 2-3 weeks in a quiescent, nonfeeding state without initiating a larval molt or metamorphosis. This study was focused on the neuroendocrine changes associated with the host's developmental arrest. Immunohistochemical studies suggested that the host brain neurosecretory cells as well as their axon terminals in the corpora cardiaca-corpora allata complex accumulated multiple neuropeptides. The extent of accumulation in cells and axons increased with time, so that hosts examined 7-14 days after the wasps emerged showed the most intense staining with antibodies against prothoracicotropic hormone, bombyxin, allatotropin, allatostatin, diuretic hormone, eclosion hormone, proctolin, and FMRFamide. Increased levels of prothoracicotropic hormone and FMRFamide-like peptides in the brains of parasitized larvae were confirmed using Western blots and enzyme-linked immunosorbent assay (ELISA), respectively. Starvation of the unparasitized larvae induced some accumulation of the neuropeptides; however, the intensity of staining and number of immunopositive cells and axons were in most cases clearly higher in the parasitized larvae. Our results suggest that accumulation of the neuropeptides is associated with developmental arrest of parasitized larvae. Because a similar developmental arrest occurs in a wide range of parasitized insects, our findings may have relevance for many other species. Moreover, these data illustrate the potential value of using parasitized M. sexta larvae as a model for studying the mechanisms governing the rates of neuropeptide expression, processing, packaging, and release, as well as providing a rich source of neuropeptides, thus facilitating their isolation and characterization.

Chemical signals from host plant and sexual behavior in a moth.

In the phytophagous corn earworm, Helicoverpa (Heliothis) zea, females delay their reproductive behaviors until they find a suitable host on which to deposit their eggs. Perception of volatile chemical signals from corn silk triggers the production of sex pheromone followed by its release, which leads to mating. Several natural corn silk volatiles, including the plant hormone ethylene, induced pheromone production in H. zea females. Because H. zea larvae feed on the fruiting parts of a wide variety of hosts, ethylene, which is associated with fruit ripening, could act as a common cue.

Isolation and identification of a new diuretic peptide from the tobacco hornworm, Manduca sexta.

A 30-amino acid diuretic peptide was isolated from the corpora cardiaca-corpora allata complexes and, separately, from medial neurosecretory cells of the Sphingid moth, Manduca sexta. The peptide was found to have the following sequence, determined by automated Edman degradation and mass spectrometry: SFSVNPAVDILQHRYMEKV AQNNRNFLNRV-NH2. We have named the peptide Mas-DP II. The peptide was synthesized and shown to possess diuretic activity in decapitated moths. Mas-DP II is related by sequence homology to a 41-amino acid diuretic peptide identified previously from M. sexta, and it belongs to the family of corticotropin releasing factor-like peptides.

A new peptide in the FMRFamide family isolated from the CNS of the hawkmoth, Manduca sexta.

We have purified a FMRFamide-like peptide from extracts of brain-subesophageal ganglion of the moth, Manduca sexta. The purification was monitored with a new, competitive ELISA, and accomplished with ion exchange and reverse-phase HPLC. The peptide structure was determined by a combination of tandem mass spectrometry and automated Edman degradation. The amino acid sequence of the peptide is less than Glu-Asp-Val-Val-His-Ser-Phe-Leu-Arg-Phe-amide (pEDVVHSFLRF-NH2). In a separate purification, an identical peptide was isolated from extracts of brain-associated neurohemal structures. We have named this peptide ManducaFLRFamide, to indicate its homology with other members of the "FMRFamide" family. In bioassays, chemically synthesized peptide increased the force of neurally evoked contractions in the major power-producing flight muscles, the dorsal longitudinal muscles. This observation suggests that hormonally released ManducaFLRFamide may play a role in sustaining or promoting the flight behavior necessary for mate-seeking (in males) or oviposition (in females) in sphingid moths.

Distribution of FMRFamide-like immunoreactivity in the brain and suboesophageal ganglion of the sphinx moth Manduca sexta and colocalization with SCPB-, BPP-, and GABA-like immunoreactivity.

Using an antiserum against the tetrapeptide FMRFamide, we have studied the distribution of FMRFamide-like substances in the brain and suboesophageal ganglion of the sphinx moth Manduca sexta. More than 2000 neurons per hemisphere exhibit FMRFamide-like immunoreactivity. Most of these cells reside within the optic lobe. Particular types of FMRFamide-immunoreactive neurons can be identified. Among these are neurosecretory cells, putatively centrifugal neurons of the optic lobe, local interneurons of the antennal lobe, mushroom-body Kenyon cells, and small-field neurons of the central complex. In the suboesophageal ganglion, groups of ventral midline neurons exhibit FMRFamide-like immunoreactivity. Some of these cells have axons in the maxillary nerves and apparently give rise to FMRFamide-immunoreactive terminals in the sheath of the suboesophageal ganglion and the maxillary nerves. In local interneurons of the antennal lobe and a particular group of protocerebral neurons, FMRFamide-like immunoreactivity is colocalized with GABA-like immunoreactivity. This suggests that FMRFamide-like peptides may be cotransmitters of these putatively GABAergic interneurons. All FMRFamide-immunoreactive neurons are, furthermore, immunoreactive with an antiserum against bovine pancreatic polypeptide, and the vast majority is also immunoreactive with an antibody against the molluscan small cardioactive peptide SCPB. Therefore, it is possible that more than one peptide is localized within many FMRFamide-immunoreactive neurons. The results suggest that FMRFamide-related peptides are widespread within the nervous system of M. sexta and might function as neurohormones and neurotransmitters in a variety of neuronal cell types.

A competitive enzyme-linked immunosorbent assay: applications in the assay of peptides, steroids, and cyclic nucleotides.

Indirect competitive enzyme-linked immunosorbent assays (ELISAs) that can be used to quantify several types of small, bioactive molecules, including peptides, steroids, and cyclic nucleotides, are described. The assays require no special expertise to perform, and the sensitivities are very high, equally or exceeding what is commonly achieved in radioimmunoassay (RIA). The molecule to be assayed or a synthetic derivative is coupled to a protein carrier (= conjugate). The conjugate is adsorbed to the wells of a microtiter plate where it is bound by antibody in inverse proportion to free hapten in a sample or standard. Bound antibody is then quantified with enzyme-labeled anti-immunoglobulin and appropriate substrate. The assay of peptides is illustrated for the sulfated cholecystokinin octapeptide, in which an ED50 of 20 fmol (2 x 10(-10) M in 100 microliters assay volume) is attained. The ED50's and slopes of the dose-response curves in the steroid and cyclic nucleotide ELISAs are compared with those parameters obtained earlier by RIA using the same antisera. This comparison indicates that a steroid, ecdysone, can be quantified with no apparent participation of the bridging group of the conjugate in the competitive assay. Furthermore, the ED50's in the ecdysone assays (ecdysone 2 beta, 3 beta, 14 alpha, 22R, 25-pentahydroxy-5 beta-cholest-7-en-6-one, 7.7 fmol; 20-hydroxyecdysone, 16 fmol) are 19- to 38-fold lower for ELISA than for RIA. In the cyclic nucleotide assay, the bridge of a cAMP conjugate (homologous with the bridge of the immunogen) decreases the slope of the dose-response curve. This effect is minimized by the use of short incubations with anti-cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Sexually dimorphic polypeptides in developing antennal sensory neurons of an insect.

Many insects have sexually dimorphic antennae with sensilla peculiar to one sex specialized to detect sex pheromones released by the other sex. In the moth Manduca sexta, the axons of receptor cells in the male-specific sensilla as well as those common to both sexes terminate in the antennal lobes of the brain. We have used 2-dimensional electrophoresis (2DE) to study some of the proteins that are produced by sensory receptor cells in developing antennae and transported through their axons to the brain. Extracts of antennal nerves from mature male and female M. sexta yield nearly identical 2DE patterns of proteins after staining or fluorography. Gels prepared from antennal nerves of developing animals, however, exhibit a sexual dimorphism in the quantitative pattern of at least 2 classes of proteins. One class consists of four 49 kDa proteins of similar charge, designated 49a, 49b, 49c, and 49d in order of decreasing mobility on nonequilibrium pH gradient electrophoresis gels. The total amount and apparent rate of synthesis of 49b and 49d are ca. 4- to 7-fold greater in antennal nerves of developing males than in those of females. Protein 49c is comparably enriched in female antennal nerves, while 49a is enriched ca. 1.2-fold in females. The second class consists of a single polypeptide of 24 kDa, which is nearly undetectable in silver-stained gels but was shown to be ca. 9-fold enriched in males by fluorography. At the end of adult development, male and female moths have similar patterns of the 49 kDa polypeptides, and synthesis of the 24 kDa polypeptide is reduced to nearly undetectable levels. The patterns of sexual dimorphism thus appear to be associated with the growth and maturation of antennal sensory axons into the antennal lobes. Biosynthesis of the 24 kDa polypeptide was nearly undetectable in antennal lobes or fragments of antennal nerves incubated in vitro with radiolabeled methionine. The 49 and 24 kDa polypeptides appear to belong to separate classes of rapidly transported proteins. The 24 kDa polypeptide is among the most rapidly transported proteins; it is found exclusively in a particulate fraction and is associated with plasma membrane but apparently not mitochondria. The 49 kDa polypeptides are found in both the particulate and soluble fractions; the more basic 49a and 49b are enriched in the particulate fraction, while the more acidic 49c and 49d are enriched in the soluble fraction.

Identification and distribution of a proctolin-like neuropeptide in the nervous system of the gypsy moth, Lymantria dispar, and in other Lepidoptera.

Although the neuropeptide proctolin has important functions in many arthropods, it is reported to be absent in Lepidoptera. Its possible occurrence in these insects was reinvestigated by bioassays of HPLC fractions and immunocytochemistry. A proctolin-like substance was recovered from the frontal and subesophageal ganglia of Lymantria dispar. This substance has the same chromatographic retention time as proctolin; enzymatic degradation indicates that it is a peptide; it is bound by proctolin antisera; and thus it is indistinguishable from authentic proctolin. A small subpopulation of proctolin-like immunoreactive (PLI) neurons was stained in the larval CNS of L. dispar, Manduca sexta, Trichoplusia ni, Galleria mellonella, and Vanessa cardui. Most prominent of these cells are median neurosecretory neurons in the brain, paired neurons in the frontal ganglion, two clusters of neurons in the subesophageal ganglion, paired lateral neurons in the thoracic ganglia, and dorsomedial neurons in the abdominal ganglia. Also, varicose PLI axons are found in the corpora cardiaca and perivisceral organs. In L. dispar, PLI cells also were found in the corpora cardiaca. The results of this study indicate that proctolin is of general occurrence in the Lepidoptera, that it has an important role in the stomatogastric nervous system, and that it may be released as a local neurohormone from various neurohemal organs.

Immunocytochemistry of GABA in the brain and suboesophageal ganglion of Manduca sexta.

We have used specific antisera against protein-conjugated gamma-aminobutyric acid (GABA) in immunocytochemical preparations to investigate the distribution of putatively GABAergic neurons in the brain and suboesophageal ganglion of the sphinx moth Manduca sexta. About 20,000 neurons per brain hemisphere exhibit GABA-immunoreactivity. Most of these are optic-lobe interneurons, especially morphologically centrifugal neurons of the lamina and tangential neurons that innervate the medulla or the lobula complex. Many GABA-immunoreactive neurons, among them giant fibers of the lobula plate, project into the median protocerebrum. Among prominent GABA-immunoreactive neurons of the median protocerebrum are about 150 putatively negative-feedback fibers of the mushroom body, innervating both the calyces and lobes, and a group of large, fan-shaped neurons of the lower division of the central body. Several commissures in the supra- and suboesophageal ganglion exhibit GABA-like immunoreactivity. In the suboesophageal ganglion, a group of contralaterally descending neurons shows GABA-like immunoreactivity. The frontal ganglion is innervated by immunoreactive processes from the tritocerebrum but does not contain GABA-immunoreactive somata. With few exceptions the brain nerves do not contain GABA-immunoreactive fibers.

Immunocytochemistry of GABA in the antennal lobes of the sphinx moth Manduca sexta.

We have prepared and characterized specific rabbit antisera against gamma-aminobutyric acid (GABA) coupled covalently to bovine serum albumin and keyhole-limpet hemocyanin. Using these antisera in immunocytochemical staining procedures, we have probed the antennal lobes and their afferent and efferent fiber tracts in the sphinx moth Manduca sexta for GABA-like immunoreactivity in order to map putatively GABAergic central neurons in the central antennal-sensory pathway. About 30% of the neuronal somata in the large lateral group of cell bodies in the antennal lobe are GABA-immunoreactive; cells in the medial and anterior groups of antennal-lobe cells did not exhibit GABA-like immunoreactivity. GABA-immunoreactive neurites had arborizations in all of the glomeruli in the antennal lobe. Double-labeling experiments involving tandem intracellular staining with Lucifer Yellow and immunocytochemical staining for GABA-like immunoreactivity demonstrated that at least some of the GABA-immunoreactive cells in the antennal lobe are amacrine local interneurons. Several fiber tracts that carry axons of antennal-lobe projection neurons exhibited GABA-immunoreactive fibers. Among the possibly GABA-containing projection neurons are several cells, with somata in the lateral group of the antennal lobe, that send their axons directly to the lateral protocerebrum.

Mushroom body feedback interneurones in the honeybee show GABA-like immunoreactivity.

The distribution of the transmitter substance GABA was investigated in the mushroom body neuropile of the honeybee by means of immunohistochemistry. The protocerebro-calycal tract (PCT) links a mushroom body output area with the calycal input sites. Interneurones contained within the PCT exhibit GABA-like immunoreactivity and may function as negative feedback loop.