Quantification of Biogenic Amines from Individual GFP-Labeled Drosophila Cells by MALDI-TOF Mass Spectrometry.

Cell-cell communication plays a crucial role in orchestrating and modulating neural circuits. To understand such interactions, it is vital to determine and quantify the involved messenger molecules such as neuropeptides and biogenic amines on the level of single cells. In this study, we used single-cell mass spectrometry (SCMS) to qualify and quantify octopamine (OA) and tyramine (TA) from isolated single cells from intact brains of the fruit fly Drosophila melanogaster. Our workflow involved targeted GFP-guided single-cell microdissection, on-plate chemical derivatization with 4-hydroxy-3-methoxycinnamaldehyde (CA) or 2,5-dimethyl-1 H-pyrrole-3,4-dicarbaldehyde (DPD) for increasing ion stability and ion signal intensity, and isotopically marked internal standards for quantification by MALDI-TOF MS. We were able to determine a limit of detection for OA of 1 fmol/µL, for TA of 2.5 fmol/µL and a lower limit of quantification (LLOQ) of 10 fmol/µL for both substances. SCMS of GFP-labeled somata from ventral midline neurons of the labial neuromere (VMlb) of the gnathal ganglion revealed an OA titer of 17.38 fmol/µL and a TA titer (∼2.5 fmol/µL) lower than the LLOQ, independent of sex. However, using a genetically altered driver line devoid of OA, TßhnM18/Tdc2 > GFP, we confirmed TA in these cells. Furthermore, cold-anesthetization of flies caused a significant increase in OA content in VMlb somata. We compared OA titers of somata from two different OA/TA cell clusters to demonstrate the usefulness of targeted SCMS in advancing our understanding of OA/TA signaling in behavior and physiology. An influence on the detection of neuropeptides by our derivatized SCMS method could be excluded.

Predator exposure-induced immunosuppression: trade-off, immune redistribution or immune reconfiguration?

Although predator exposure increases the risk of wound infections, it typically induces immunosuppression. A number of non-mutually exclusive hypotheses have been put forward to explain this immunosuppression, including: trade-offs between the immune system and other systems required for anti-predator behaviour, redistribution of immune resources towards mechanisms needed to defend against wound infections, and reconfiguration of the immune system to optimize defence under the physiological state of fight-or-flight readiness. We tested the ability of each hypothesis to explain the effects of chronic predator stress on the immune system of the caterpillar Manduca sexta Predator exposure induced defensive behaviours, reduced mass gain, increased development time and increased the concentration of the stress neurohormone octopamine. It had no significant effect on haemocyte number, melanization rate, phenoloxidase activity, lysozyme-like activity or nodule production. Predator stress reduced haemolymph glutathione concentrations. It also increased constitutive expression of the antimicrobial peptide attacin-1 but reduced attacin-1 expression in response to an immune challenge. These results best fit the immune reconfiguration hypothesis, although the other hypotheses are also consistent with some results. Interpreting stress-related changes in immune function may require an examination at the level of the whole organism.

Learning, gustatory responsiveness and tyramine differences across nurse and forager honeybees.

Honeybees are well known for their complex division of labor. Each bee sequentially performs a series of social tasks during its life. The changes in social task performance are linked to gross differences in behavior and physiology. We tested whether honeybees performing different social tasks (nursing versus foraging) would differ in their gustatory responsiveness and associative learning behavior in addition to their daily tasks in the colony. Further, we investigated the role of the biogenic amine tyramine and its receptors in the behavior of nurse bees and foragers. Tyramine is an important insect neurotransmitter, which has long been neglected in behavioral studies as it was believed to only act as the metabolic precursor of the better-known amine octopamine. With the increasing number of characterized tyramine receptors in diverse insects, we need to understand the functions of tyramine on its own account. Our findings suggest an important role for tyramine and its two receptors in regulating honeybee gustatory responsiveness, social organization and learning behavior. Foragers, which were more responsive to gustatory stimuli than nurse bees and performed better in appetitive learning, also differed from nurse bees in their tyramine brain titers and in the mRNA expression of a tyramine receptor in the brain. Pharmacological activation of tyramine receptors increased gustatory responsiveness of nurse bees and foragers and improved appetitive learning in nurse bees. These data suggest that a large part of the behavioral differences between honeybees may be directly linked to tyramine signaling in the brain.

Octopamine and tyramine modulate the thermoregulatory fanning response in honey bees (Apis mellifera).

Biogenic amines regulate the proximate mechanisms underlying most behavior, including those that contribute to the overall success of complex societies. For honey bees, one crucial set of behaviors contributing to the welfare of a colony is involved with nest thermoregulation. Worker honeybees cool the colony by performing a fanning behavior, the expression of which is largely influenced by response thresholds modulated by the social environment. Here, we examined how changes in biogenic amines affect this group-performed thermoregulatory fanning behavior in honeybees. Concentrations of two biogenic amines, octopamine and tyramine, are significantly lower in active fanners than in non-fanners, but there is no difference in dopamine and serotonin concentrations. Direct feeding of octopamine and tyramine induced a decrease in fanning responses, but only when both amines were included in the treatment. This is the first evidence that fanning behavior is influenced by these two biogenic amines, and this result is consistent with the typical role of these neurotransmitters in regulating locomotor activity in other insects. Individual variation in amine expression also provides a mechanistic link that helps to explain how this group behavior might be coordinated within a colony.

Variability of Stimulant Levels in Nine Sports Supplements Over a 9-Month Period.

Many studies have found that some dietary supplement product labels do not accurately reflect the actual ingredients. However, studies have not been performed to determine if ingredients in the same dietary supplement product vary over time. The objective of this study was to assess the consistency of stimulant ingredients in popular sports supplements sold in the United States over a 9-month period. Three samples of nine popular sports supplements were purchased over the 9-month period. The 27 samples were analyzed for caffeine and several other stimulants (including adulterants). The identity and quantity of stimulants were compared with stimulants listed on the label and stimulants found at earlier time points to determine the variability in individual products over the 9-month period. The primary outcome measure was the variability of stimulant amounts in the products examined. Many supplements did not contain the same number and quantity of stimulants at all time points over the 9-month period. Caffeine content varied widely in five of the six caffeinated supplements compared with the initial measurement (-7% to +266%). In addition, the stimulants-synephrine, octopamine, cathine, ephedrine, pseudoephedrine, strychnine, and methylephedrine-occurred in variable amounts in eight of the nine products. The significance of these findings is uncertain: the sample size was insufficient to support statistical analysis. In our sample of nine popular sports supplements, the presence and quantity of stimulants varied over a 9-month period. However, future studies are warranted to determine if the variability found is significant and generalizable to other supplements.

Direct and mediated electrochemistry of peroxidase and its electrocatalysis on a variety of screen-printed carbon electrodes: amperometric hydrogen peroxide and phenols biosensor.

This study compares the behaviour of direct and mediated electrochemistry of horseradish peroxidase (HRP) immobilised on screen-printed carbon electrodes (SPCEs), screen-printed carbon electrodes modified with carboxyl-functionalised multi-wall carbon nanotubes (MWCNT-SPCEs) and screen-printed carbon electrodes modified with carboxyl-functionalised single-wall carbon nanotubes (SWCNT-SPCEs). The techniques of cyclic voltammetry and amperometry in the flow mode were used to characterise the properties of the HRP immobilised on screen-printed electrodes. From measurements of the mediated and mediatorless currents of hydrogen peroxide reduction at the HRP-modified electrodes, it was concluded that the fraction of enzyme molecules in direct electron transfer (DET) contact with the electrode varies substantially for the different electrodes. It was observed that the screen-printed carbon electrodes modified with carbon nanotubes (MWCNT-SPCEs and SWCNT-SPCEs) demonstrated a substantially higher percentage (≈100 %) of HRP molecules in DET contact than the screen-printed carbon electrodes (≈60 %). The HRP-modified electrodes were used for determination of hydrogen peroxide in mediatorless mode. The SWCNT-SPCE gave the lowest detection limit (0.40 ± 0.09 µM) followed by MWCNT-SPCE (0.48 ± 0.07 µM) and SPCE (0.98 ± 0.2 µM). These modified electrodes were additionally developed for amperometric determination of phenolic compounds. It was found that the SWCNT-SPCE gave a detection limit for catechol of 110.2 ± 3.6 nM, dopamine of 640.2 ± 9.2 nM, octopamine of 3341 ± 15 nM, pyrogallol of 50.10 ± 2.9 nM and 3,4-dihydroxy-L-phenylalanine of 980.7 ± 8.7 nM using 50 µM H2O2 in the flow carrier.

Nine prohibited stimulants found in sports and weight loss supplements: deterenol, phenpromethamine (Vonedrine), oxilofrine, octodrine, beta-methylphenylethylamine (BMPEA), 1,3-dimethylamylamine (1,3-DMAA), 1,4-dimethylamylamine (1,4-DMAA), 1,3-dimethylbutylamine (1,3-DMBA) and higenamine.

BACKGROUND: Weight loss and sports supplements containing deterenol have been associated with serious adverse events including cardiac arrest. OBJECTIVE: To determine the presence and quantity of experimental stimulants in dietary supplements labeled as containing deterenol sold in the United States. METHODS: Dietary supplements available for sale in the US and labeled as containing deterenol or one of its synonyms (e.g., isopropylnorsynephrine and isopropyloctopamine) were purchased online. For each brand, one container or subsample was analyzed by NSF International (Ann Arbor, MI) and one container or subsample by the Netherland's National Institute for Public Health and the Environment (RIVM, Bilthoven, The Netherlands). When differences existed between the two containers or subsamples of the same brand, both products were reanalyzed by Sciensano (Brussels, Belgium). NSF International carried out qualitative and quantitative analyses using ultra-high-performance liquid chromatography (UHPLC) quadrupole-Orbitrap mass spectrometry. RIVM performed qualitative and quantitative analysis using UHPLC quadrupole time-of-flight mass spectrometry. Sciensano carried out qualitative analysis using UHPLC quadrupole-Orbitrap mass spectrometry. RESULTS: Seventeen brands of supplements were analyzed. Many brands included more than one prohibited stimulant in the same product: 4 brands (24%, 4/17) included 2 stimulants, 2 (12%, 2/17) combined 3 stimulants, and 2 (12%, 2/17) combined 4 stimulants. The range of quantities per recommended serving size of the 9 stimulants detected were 2.7 mg to 17 mg of deterenol; 1.3 mg to 20 mg of phenpromethamine (Vonedrine); 5.7 mg to 92 mg of beta-methylphenylethylamine (BMPEA); 18 mg to 73 mg of octodrine; 18 mg to 55 mg of oxilofrine; 48 mg of higenamine; 17 mg of 1,3-dimethylamylamine (1,3-DMAA); 1.8 mg to 6.6 mg of 1,3-dimethylbutylamine (1,3-DMBA); and 5.3 mg of 1,4-dimethylamylamine (1,4-DMAA). CONCLUSION: Weight loss and sports supplements listing deterenol as an ingredient contained 9 prohibited stimulants and 8 different mixtures of stimulants, with as many as 4 experimental stimulants per product. These cocktails of stimulants have never been tested in humans and their safety is unknown.

Biogenic amines in cultured neuroblastoma and astrocytoma cells.

The presence of biogenic amines in cultured cells of mouse neuroblastoma C-1300 (clone NB-2a) was suggested by fluorescence-microscope histochemistry. Incubation in media containing L-[(14)C]tyrosine and L-[(14)C]tryptophan for 24 h, followed by high-voltage electrophoresis, radiochromatogram scanning, and scintillation counting, confirmed the presence of [(14)C]dopamine, [(14)C]norepinephrine, [(14)C]epinephrine, [(14)C]serotonin, [(14)C]tyramine, and [(14)C]octopamine. Dopamine, norepinephrine, epinephrine, and serotonin were demonstrated spectrophotofluorometrically in concentrations, expressed as micrograms amine per milligram protein, of 1.19, 0.027, 0.038, and 0.148, respectively, for cells in a stationary growth phase. Fluorescence-microscope histochemistry also suggested the presence of biogenic amines in cultured astrocytoma cells (cell line C6). Spectrophotofluorometric assay of cells in a stationary growth phase demonstrated intracellular dopamine, norepinephrine, epinephrine, and serotonin in concentrations significantly lower than those of neuroblastoma cells.

Octopamine: presence in single neurons of Aplysia suggests neurotransmitter function.

Octopamine has been identified and measured in individual neurons from Aplysia californica. Neither dopamine nor norepinephrine was detected in these cells. Thus, in Aplysia there may be separate populations of catecholaminergic and monophenolaminergic cells. Octopamine may have functions of its own in the central nervous system of mollusks.

Octopamine: normal occurrence in sympathetic nerves of rats.

Octopamine has been identified in several organs of normal rats by means of a sensitive enzymatic assay. It is localized within the sympathetic nerve endings.

Fast-scan cyclic voltammetry for the detection of tyramine and octopamine.

Tyramine and octopamine are biogenic amine neurotransmitters in invertebrates that have functions analogous to those of the adrenergic system in vertebrates. Trace amounts of these neurotransmitters have also been identified in mammals. The purpose of this study was to develop an electrochemical method using fast-scan cyclic voltammetry at carbon-fiber microelectrodes to detect fast changes in tyramine and octopamine. Because tyramine is known to polymerize and passivate electrode surfaces, waveform parameters were optimized to prevent passivation. No fouling was observed for octopamine when the electrode was scanned from 0.1 to 1.3 V and back at 600 V/s, while a small decrease of less than 10% of the signal was seen for 15 repeated exposures to tyramine. The technique has limits of detection of 18 nM for tyramine and 30 nM for octopamine, much lower than expected levels in insects and lower than basal levels in some brain regions of mammals. Current was linear with concentration up to 5 microM. This voltammetry technique should be useful for measuring tyramine and octopamine changes in insects, such as the fruit fly, Drosophila melanogaster.

Conformational study of octopamine in gas phase and effect of hydrochloride.

This work deals with the molecular modeling and vibrational spectra of all the twenty conformers of an important biomolecule octopamine which have been investigated using the DFT/B3LYP level of theory in combination with the 6-31++g(d,p) as a suitable basis set. The experimental FTIR and FTRaman spectra of octopamine neurotransmitter were recorded in the spectral region 400-4000 cm-1 and 50-4000 cm-1 respectively and correlated with the calculated spectra of the most stable conformer. The effect of hydrochloride on the important geometrical parameters of most stable conformer of octopamine was also studied. The normal coordinate analysis was performed to scale the theoretical frequencies and to calculate potential energy distributions for precise normal mode assignment. Most of the frequencies were in good agreement with experimental one. However, some have been modified. Natural bond orbital analysis was performed in order to confirm the stability of electronic structure of octopamine molecule. HOMO-LUMO analysis for all the twenty conformers was also performed to give the transition profile and to study the chemical reactivity of octopamine.

High sensitivity deep-UV LED-based z-cell photometric detector for capillary liquid chromatography.

A new high sensitivity deep-UV LED photometric detector with a z-type flow cell (45 nL or 180 nL) for miniaturised and portable capillary liquid chromatography (LC) was designed and fabricated to overcome sensitivity limitations due to short pathlength in on-capillary detectors. The new detector has a 10 mm geometric pathlength and uses high intensity light-emitting diodes (LED) as light sources in the deep-UV range (254 nm and 280 nm). No optical reference was necessary due to the low drift in the signal. Stray light was minimized by the use of an adjustable slit with a 0.5 mm pinhole. The direct relationship between absorbance and concentration was obtained using dichromate to evaluate the sensitivity and the linearity range of the detector. Performance of the miniaturised version was compared with that obtained from a commercial benchtop detector for capillary LC under the same conditions using the same optical z-cell. The miniaturised version exhibited a superior performance across all parameters, including 3 times higher effective pathlength, 4 times higher upper limit of detector linearity, and 2-5 times lower stray light levels. An application of the new detector was shown with the detection of l-dopa, l-tyrosine, norfenefrine, phenylephrine and tyramine, separated using capillary LC. The baseline noise level recorded was as low as 3.9 µAU. Further, the detector was applied in a miniaturised capillary LC for the indirect detection of common inorganic anions. In comparison to an on-capillary LED detector applied under similar chromatographic conditions, there was a 50 times higher signal to noise (S/N) ratio.

Determination of Bitter Orange alkaloids in dietary supplements standard reference materials by liquid chromatography with ultraviolet absorbance and fluorescence detection.

Four adrenergic amines [synephrine, octopamine, tyramine, and n-methyltyramine] were determined in a variety of Bitter Orange containing dietary supplements. Two extraction techniques were evaluated in detail: Soxhlet extraction and sonication extraction. A liquid chromatographic separation using a reversed-phase C(18) stationary phase and the ion-pairing reagent sodium dodecyl sulfate was developed to separate the Bitter Orange alkaloids. Ultraviolet absorbance detection at 220 nm and fluorescence detection with excitation at 273 nm and emission at 304 nm were used for the alkaloid detection. The method described was used for the assignment of the levels of the predominant alkaloids in three candidate standard reference materials containing Bitter Orange.

Determination of octopamine, synephrine and tyramine in Citrus herbs by ionic liquid improved 'green' chromatography.

Without adding any volatile organic solvents, aqueous solutions of room temperature ionic liquids (RTILs) were used as 'green' mobile phases to determine octopamine, synephrine and tyramine by liquid chromatography. The problems of the adrenergic amines separation, such as band tailing, low retention and low resolution were solved successfully by using RTIL. The effect of 1-ethyl-3-methylimidazolium tertafluoroborate ([EMIM][BF4]) was the best in the six investigated RTILs. The concentration of [EMIM][BF4], mobile phase pH and column temperature, which influenced the chromatographic behaviors of the analytes, were investigated in detail. The change of retention factors caused by pH shift was obviously suppressed by [EMIM][BF4]. The sensitivity, accuracy and repeatability of this method were found to be satisfactory. The contents of adrenergic amines in several Citrus herbs and extracts, such as Fructus aurantii immaturus, were simultaneously determined by this 'green' chromatographic method.

Immunocytochemical localization of octopamine in the central nervous system of Limulus polyphemus: a light and electron microscopic study.

We have determined the distribution and localization of the monoamine octopamine in the prosomal central nervous system of the horseshoe crab, Limulus polyphemus, by light and electron microscopic immunocytochemistry. Sixteen discrete clusters of octopamine-like immunoreactive neurons are situated bilaterally in the tritocerebrum and circumesophageal ring of fused thoracic ganglia. Two pairs of anterior clusters are located laterally in the cheliceral and first pedal ganglia; the remaining six pairs of clusters are located ventromedially in the second through fifth pedal ganglia, chilarial ganglia, and opercular ganglia. The immunoreactive somata range from about 40 to 100 microns in diameter and occur in clusters of 12-24 cells. There is extensive distribution of octopamine-immunoreactive nerve fibers in Limulus; dense fiber tracts course anteroposteriorly through the central nervous system, and most neuropil regions are innervated by immunoreactive processes and terminals. This wide distribution of octopamine-like immunoreactivity provides an anatomical basis for the several effects of octopamine in Limulus. We determined the subcellular localization of octopamine by postembedding immunoelectron microscopy. The immunogold-labelled terminals are morphologically unique; they contain large, distinctively shaped dense-core granules, typically cylindrical with a prominent indentation in one end. These large granules are 100-150 nm in diameter and range from 150-400 nm in length. The dense labelling of these unusual granules with immunogold particles indicates that octopamine is sequestered in or associated with the granules.

Octopamine-like immunoreactivity in the brain and subesophageal ganglion of the honeybee.

The organization of putative octopaminergic pathways in the brain and subesophageal ganglion of the honeybee was investigated with a well-defined polyclonal antiserum against octopamine. Five prominent groups of just over 100 immunoreactive (IR) somata were found in the cerebral ganglion: Neurosecretory cells in the pars intercerebralis innervating the corpora cardiaca via NCC I, one cluster mediodorsal to the antennal lobe, one scattered on both sides of the midline of the protocerebrum, one between the lateral protocerebral lobes and the dorsal lobes, and a single soma on either side of the central body. With the exception of the pedunculi and beta-lobes of the mushroom bodies, varicose immunoreactive fibers penetrate all parts of the cerebral ganglion. Strong labelling was found in the central complex and the protocerebral bridge. Fine networks of labelled processes invade the antennal lobes, the calyces and a small part of the alpha-lobes of the mushroom bodies, the protocerebrum, and all three optic ganglia. In the subesophageal ganglion, one labelled cell body was found in the lateral soma layer of the mandibular segment. Each of the three neuromeres contains a group of six to ten somata in the ventral median parts. Most of the ventral median cells send their neurites dorsally through the midline tracts, whereas the neurites of a few cells follow the ventral cell body neurite tracts. Octopamine-IR was demonstrated in all neuropils that contain pathways for proboscis extension learning in honeybees. Because octopaminergic mechanisms seem to be involved in the behavioral plasticity of the proboscis extension reflex, our study provides anatomical data on the neurochemical organization of an appetitive learning paradigm.

Developmental expression of the octopamine phenotype in lobsters, Homarus americanus.

We have used immunocytochemical methods to examine the sequence of appearance of octopamine-immunoreactive neurons during development, and to try to correlate that appearance with the emergence of behavioral or physiological capabilities. The first octopamine neurons express their transmitter phenotype at approximately 43% of embryonic development. The last cells show immunostaining at the 3rd larval stage. In the wild, therefore, immunoreactivity in cells appears over a 9-12 month period. In contrast, serotonin-immunoreactive neurons stain early in embryonic development and the last serotonin-immunoreactive cells appear at about the same time the first octopamine-immunoreactive neurons show staining. The pattern of appearance of octopamine-immunoreactive cells is cell type-specific. A pair of brain cells and the descending interneurons stain first. Additional brain cell staining is seen throughout embryonic development. The ascending interneurons appear next, and a general anterior-posterior gradient typifies their emergence over a relatively short portion of embryonic life (E 48-62%). The neurosecretory cell staining appears last, is segment-specific, begins at about 62% development, and continues to the 3rd larval stage. The emergence of immunostaining for amine neurotransmitters within groups of identified neurons at precise times in development may specify possible functional units. With at least one group of cells, this possibility seems plausible: the three pairs of claw octopamine neurosecretory cells show immunostaining as a unit.

Autoradiographic localization of newly synthesized octopamine to retinal efferents in the Limulus visual system.

The biogenic amine octopamine is synthesized from both tyrosine and tyramine in the lateral, median, and ventral eyes of Limulus. The autoradiographic studies presented here were designed to locate the sites of octopamine synthesis in the ventral and lateral eyes. We found that efferent fibers, which project to ventral and lateral eyes from the central nervous system, became intensely and selectively labeled during in vitro incubations with 3H-tyramine. In the ventral eye, more than 95% of the efferent fibers were labeled. Results of biochemical analyses suggested that most of the radioactive substance within these efferent fibers was newly synthesized octopamine. The selective labeling of efferent fibers during incubation with 3H-tyramine was used as an anatomical tool to study the number and distribution of efferent fibers within the ventral eye. Light microscopic (LM) reconstructions of the distribution of label in serial longitudinal sections through ventral optic nerves together with electron microscopic (EM) autoradiographic analyses revealed between 70 and 200 efferent axons. The results of these studies and of reconstructions of efferent innervation to photoreceptor somata suggest that each ventral photoreceptor cell or each small cluster of cells is innervated by a separate efferent fiber. Both LM reconstructions and EM analyses showed that efferent fibers ramify extensively and specifically in and near the internal rhabdom of ventral photoreceptor cells. In EM autoradiographs of lateral eyes incubated with 3H- tyramine, the silver grains that were located over ommatidia were concentrated exclusively over efferent fibers. All of these efferent fibers, which lay near rhabdoms and in partitions between retinular cells, were labeled. The results of our present studies support our hypothesis that octopamine is a neurotransmitter in Limulus retinal efferent fibers. This amine may modulate the biochemistry and physiology of ventral photoreceptor cells and may mediate many of the known effects of circadian efferent innervation to the lateral eye.

A new specific antibody reveals octopamine-like immunoreactivity in cockroach ventral nerve cord.

An antiserum was raised in rabbits immunized with octopamine conjugated to thyroglobulin. The specificity of this antiserum for octopamine is shown by dot blot immunoassay analysis. The antiserum does not crossreact with dopamine, noradrenaline, and serotonin, but slight crossreactivity with the amine tyramine at high concentrations was observed. The tyramine crossreactivity could be eliminated by preabsorption with a tyramine-glutaraldehyde-BSA conjugate. Using this antiserum, we describe the topographical distribution of octopamine-immunoreactive (ir) neuronal elements in wholemounts and paraffin sections of the ventral nerve cord of the American cockroach. The pattern of octopamine immunostaining is completely different from that obtained with an antidopamine serum, and can be blocked by preabsorbing the antioctopamine serum with BSA-conjugated octopamine. Cell bodies and dendritic processes of putatively octopaminergic dorsal (DUM) and ventral (VUM) unpaired median neurons were clearly octopamine-ir in all ganglia examined. The numbers of stained DUM somata in the mesothoracic, metathoracic, and terminal ganglion of females correspond to those of peripherally projecting DUM cells revealed previously by retrograde tracing (Gregory, Philos Trans R Soc Lond [Biol] 306:191, 1984; Tanaka and Washio, Comp Biochem Physiol 91A:37, 1988; Stoya et al., Zool Jb Physiol 93:75, 1989). In addition, various, previously unknown, paired cells with octopamine-like immunoreactivity were found in all ventral ganglia except abdominal ganglia 3-6. Some of these probably project intersegmentally.