Postembryonic development of γ-aminobutyric acid-like Immunoreactivity in the brain of the sphinx moth Manduca sexta

We have investigated the distribution of immunocytochemical staining for the neurotransmitter γ-aminobutyric acid (GABA) in the brain of the sphinx moth Manduca sexta during larval, pupal, and adult development. In the larval brain, about 300 neurons are GABA-immunoreactive. All neuropil areas except the mushroom bodies and central complex show intense immunostaining. Only minor changes in the pattern of immunoreactivity occur during larval development. During metamorphosis, changes in immunostaining occur in two phases. Beginning in wandering fifth-instar larvae (stage W2), immunoreactivity appears in numerous neurons of the central body and optic lobe and becomes more intense during early pupal stages. At the same time, GABA-like immunoreactivity disappears in most neuropil areas of the brain and becomes faint in many immunoreactive somata. Neurons with arborizations in the ventrolateral protocerebrum, however, continue to exhibit intense immunostaining during this period, and strongly immunolabeled fibers connect these areas with the ventral nerve cord. The second phase of transformation begins around pupal stage P5/P6, when faint immunostaining appears in many previously nonimmunoreactive somata and most neuropil areas of the brain. In subsequent stages (P8–P10), this immunoreactivity disappears again in most somata, but in certain cell groups, it becomes more intense and gradually develops to the adult pattern. Most larval GABA-immunoreactive neurons appear to survive through metamorphosis into the adult. Neurons in the midbrain that acquire GABA-like immunoreactivity during metamorphosis usually lie adjacent to larval immunostained neurons, suggesting common lineages. The onsets of the two developmental phases of GABA-like immunoreactivity correlate with sharp rises in hemolymph titers of ecdysteroid hormones, suggesting a role for ecdysteroids in the regulation of GABA synthesis. We hypothesize that the disappearance of GABA in many areas of the brain starting 2 days prior to pupation dramatically alters its functional circuitry and thus may account for profound changes in the behavior of the animal. © 1994 Wiley-Liss, Inc.     

Serotonin immunoreactivity in the optic lobes of the sphinx moth Manduca sexta and colocalization with FMRFamide and SCPB immunoreactivity

In the optic lobes (OLs) of the sphinx moth Manduca sexta, 300-350 neurons per hemisphere are immunoreactive with an antiserotonin antiserum. Two groups of weakly serotonin-immunoreactive cells (OL1) appear to be amacrine cells of the medulla, whereas more intensely immunoreactive cells (OL2) are probably centrifugal neurons that innervate the lobula, medulla, and lamina, as well as the superior protocerebrum. At least one other OL2 cell is a local optic-lobe interneuron with arborizations in the dorsal medulla and lobula. The serotonin-immunoreactive cells are also immunoreactive with an antiserum against Drosophila melanogaster DOPA decarboxylase. All OL2 cells, but not the OL1 cells, are furthermore immunoreactive with an anti-FMRFamide antiserum and an anti-SCPB antiserum. This suggests that neuropeptides related or identical to FMRFamide and SCPB are localized and may serve as cotransmitters with serotonin in OL2 optic-lobe interneurons.     

Peptide-immunocytochemistry of neurosecretory cells in the brain and retrocerebral complex of the sphinx moth Manduca sexta.

Antisera against a variety of vertebrate and invertebrate neuropeptides were used to map cerebral neurosecretory cells in the sphinx moth Manduca sexta. Intense immunoreactive staining of distinct populations of neurosecretory cells was obtained with antisera against locust adipokinetic hormone, bovine pancreatic polypeptide, FMRFamide, molluscan small cardioactive peptide (SCPB), leucine-enkephalin, gastrin/cholecystokinin, and crustacean beta-pigment dispersing hormone (beta PDH). Other antisera revealed moderate to weak staining. Each type of neurosecretory cell is immunoreactive with at least one of the antisera tested, and most of these neurons can be identified anatomically. The staining patterns provide additional information on the organization of cerebral neurosecretory cells in M. sexta. Based upon anatomical and immunocytochemical characteristics, 11 types of neurosecretory cells have been recognized in the brain, one type in the suboesophageal ganglion, and one in the corpus cardiacum. Extensive colocalization experiments show that many neurosecretory cells are immunoreactive with several different antisera. This raises the possibility that these cells may release mixtures of neuropeptides into the hemolymph, as has been demonstrated in certain other systems. The immunocytochemical data should be helpful in efforts to identify additional peptide neurohormones released from the brain of this and other insects.     

Dopamine-like immunoreactivity in the brain and suboesophageal ganglion of the honeybee

The distribution of dopamine in the brain and suboesophageal ganglion of the honeybee Apis mellifera was investigated by means of immunocytochemistry with a well-characterized antiserum against dopamine. The binding of the antiserum in paraffin serial sections was studied with the peroxidase-antiperoxidase method. Dopamine-like immunoreactive neurons are present in most parts of the brain and in the suboesophageal ganglion. Only the optic lobes are devoid of label. There are ca. 330 dopamine immunoreactive somata in each brain hemisphere plus respective suboesophageal hemiganglion, which is less than 0.1% of the entire neuronal population. Most of the labelled somata are situated within three clusters: one below the lateral calyx and two in the anterior-ventral protocerebrum. Other labelled somata lie dispersed or in small groups around the protocerebral bridge, below the optic tubercles, proximal to the ventral rim of the lobula, and in the lateral and ventral somatal rind of the suboesophageal ganglion. Similar to neurons that react with an antiserum against serotonin, the fine processes of dopamine immunoreactive fibers have a varicose appearance which is typical for aminergic neurons. In addition to the neuronal staining, dopamine-like immunoreactivity is also present in the sheath surrounding the brain and in the retina, where it is not restricted to any particular cell type. A detailed account is given for those neurons and groups of neurons that could be traced and reconstructed in some detail. A common feature of all dopamine immunoreactive fibers is that each fiber invades large volumes of neuropil, suggesting that dopamine is more important in mediating distant rather than local neural interactions.     

Octopamine-immunoreactive neurons in the brain and subesophageal ganglion of the hawkmoth Manduca sexta

Octopamine is a neuroactive monoamine that functions as a neurohormone, a neuromodulator, and a neurotransmitter in many invertebrate nervous systems, but little is known about the distribution of octopamine in the brain. We therefore used a monoclonal antibody to study the distribution of octopamine-like immunoreactivity in the brain of the hawkmoth Manduca sexta. Immunoreactive processes were observed in many regions of the brain, with the distinct exception of the upper division of the central body. We focused our analysis on nine ventral unpaired median (VUM) neurons with cell bodies in the labial neuromere of the subesophageal ganglion. Seven of these neurons projected caudally through the ventral nerve cord. Two neurons projected rostrally into the brain (supraesophageal ganglion), and one of these was a bilateral neuron that sent projections to the gamma-lobe of the mushroom body and the lateral protocerebrum. Octopamine-immunoreactive processes from one or more cells originating in the subesophageal ganglion also form direct connections between the antennal lobes and the calyces of the mushroom bodies.     

Temporal tuning of odor responses in pheromone-responsive projection neurons in the brain of the sphinx moth Manduca sexta

By means of intracellular recording and staining, we studied the ability of several distinct classes of projection (output) neurons, which innervate the sexually dimorphic macroglomerular complex (MGC-PNs) in the antennal lobe of the male moth Manduca sexta, to encode naturally intermittent sex pheromonal stimuli. In many MGC-PNs, antennal stimulation with a blend of the two essential pheromone components evoked a characteristic triphasic response consisting of a brief, hyperpolarizing inhibitory potential (I₁) followed by depolarization with firing of action potentials and then a delayed period of hyperpolarization (I₂). MGC-PNs described in this study resolved pulsed pheromonal stimuli, up to about five pulses/second, with a distinct burst of action potentials for each pulse of odor. The larger the amplitude of I₁, the higher the pulse rate an MGC-PN could follow, illustrating the importance of inhibitory synaptic input in shaping the temporal firing properties of these glomerular output neurons. In some MGC-PNs, triphasic responses were evoked by antennal stimulation with only one of the two key pheromone components. Again, the maximal pulse rate that an MGC-PN could follow with that pheromone component as sole stimulus was high in MGC-PNs that responded with a strong I₁. These component-specific MGC-PNs innervated only one of the two principal glomeruli of the MGC, while MGC-PNs that were primarily excited by antennal stimulation with either key pheromone component had arborizations in both major MGC glomeruli. These observations therefore suggest that the population of antennal olfactory receptor cells responding to a single pheromone component is functionally heterogeneous: a subset of these sensory cells activates the excitatory drive to many uniglomerular MGC-PNs, while others feed onto inhibitory circuits that hyperpolarize the same PNs. This convergence of opposing inputs is a circuit property common to uniglomerular MGC-PNs branching in either of the major MGC glomeruli, and it enhances the ability of these glomerular output neurons to resolve intermittent olfactory input. Synaptic integration at the uniglomerular PN level thus contributes to the transmission of behaviorally important temporal information about each key pheromone component to higher centers in the brain. J. Comp. Neurol. 409:1–12, 1999. © 1999 Wiley-Liss, Inc.     

Metamorphosis of the cerebral neuroendocrine system in the moth Manduca sexta

We have examined the morphology and neuronal elements of the cerebral neuroendocrine system in the larval, pupal, and adult stages of the moth Manduca sexta with a variety of neuroanatomical techniques. The larval brain contains several discrete groups of neurosecretory and non-neurosecretory cells that project to the associated neurohemal organs (the corpora cardiaca-allata complex, or CC-CA) and to a variety of more peripheral structures. A previously undescribed set of cells in the subesophageal ganglion have also been found that project out the neurosecretory nerves. During metamorphosis, the cerebral neuroendocrine system undergoes a dramatic structural reorganization, including the reduction or loss of many larval nerves and a repositioning of the cell groups and their dendritic fields. Despite these changes, most of its central elements are retained. In addition, by the completion of adult development a new cluster of cells can be found on either side of the dorsal midline of the brain. We have also determined the relative contributions of the different cell groups to the moth neuroendocrine system by intracellular iontophoresis of dye into individual cells. Within the dorsal protocerebrum, five separate morphological types of cells can be recognized, each with a distinctive pattern of dendritic arborization in the brain and terminal neurohemal processes that project to the CC, the CA, the aorta, or to a combination of these regions. The large intrinsic cells of the CC have also been filled, revealing an unusual set of morphological features in these peripheral neurosecretory cells.     

Identification of distinct tyraminergic and octopaminergic neurons innervating the central complex of the desert locust,Schistocerca gregaria

The central complex is a group of modular neuropils in the insect brain with a key role in visual memory, spatial orientation, and motor control. In desert locusts the neurochemical organization of the central complex has been investigated in detail, including the distribution of dopamine‐, serotonin‐, and histamine‐immunoreactive neurons. In the present study we identified neurons immunoreactive with antisera against octopamine, tyramine, and the enzymes required for their synthesis, tyrosine decarboxylase (TDC) and tyramine β‐hydroxylase (TBH). Octopamine‐ and tyramine immunostaining in the central complex differed strikingly. In each brain hemisphere tyramine immunostaining was found in four neurons innervating the noduli, 12–15 tangential neurons of the protocerebral bridge, and about 17 neurons that supplied the anterior lip region and parts of the central body. In contrast, octopamine immunostaining was present in two bilateral pairs of ascending fibers innervating the upper division of the central body and a single pair of neurons with somata near the esophageal foramen that gave rise to arborizations in the protocerebral bridge. Immunostaining for TDC, the enzyme converting tyrosine to tyramine, combined the patterns seen with the tyramine‐ and octopamine antisera. Immunostaining for TBH, the enzyme converting tyramine to octopamine, in contrast, was strikingly similar to octopamine immunolabeling. We conclude that tyramine and octopamine act as neurotransmitters/modulators in distinct sets of neurons of the locust central complex with TBH likely being the rate‐limiting enzyme for octopamine synthesis in a small subpopulation of TDC‐containing neurons. J. Comp. Neurol. 521:2025–2041, 2013. © 2012 Wiley Periodicals, Inc.     

Segment-specific modifications of a neuropeptide phenotype in embryonic neurons of the moth, Manduca sexta

We have studied differences in the development of segmentally homologous neurons to identify factors that may regulate a neuropeptide phenotype. Bilaterally paired homologs of the peripheral neuron L1 were identified in the thoracic and abdominal segments in embryos of the moth Manduca: each bipolar neuron arises at a stereotyped location and, at 40% of embryogenesis, projects its major process within the transverse nerve of its own segment. Shortly after the initiation of axonogenesis (approximately 41%), L1 homologs in all but the prothoracic segment (T1) were labelled specifically by an antiserum to the molluscan neuropeptide Phe-Met-Arg-Phe-NH2 (authentic FMRFamide). Levels of peptide-immunoreactivity (IR) were comparable in all such segmental homologs up to the approximately 60% stage of embryogenesis, whereupon two distinct levels of peptide IR were displayed: homologs in the three most rostral segments (T2, T3, and A1; [abdominal segment 1]) showed high levels and were called Type I L1 neurons; homologs in the more caudal segments (A2-A8) typically showed low levels of IR and were called Type II L1 neurons. This segment-specific difference represented mature differentiated states and was retained in postembryonic stages. Intracellular dye fills of embryonic L1 neurons revealed that the morphogenesis of the Type I and II L1 neuron homologs was similar until approximately 48% of embryogenesis; thereafter it differed in two salient ways: (1) the cell bodies of Type II L1 neurons migrated approximately 150 microns laterally from their point of origin, and (2) the distal processes of the Type II L1 neurons contacted the heart, whereas those of Type I L1 neurons did not. Ultrastructural studies of both mature and developing L1 homologs showed that the FMRFamide-like antigen(s) localized specifically to secretory granules. Further, whereas the secretory granules in segmental homologs appeared similar initially (i.e., at approximately 50% of development), following the establishment of segment-specific differences, secretory granules found in mature Type I and II L1 neurons were cell type-specific.     

Sexual differentiation in the terminal ganglion of the moth Manduca sexta: role of sex-specific neuronal death

In the insect Manduca sexta the genitalia on the terminal abdominal segments are sexually dimorphic structures but they arise during metamorphosis from segments that are monomorphic in the larva. The motoneurons in the terminal ganglion that innervate these structures were examined by cobalt backfills of peripheral nerves. In the larval stage the population of motoneurons innervating the terminal segments was identical in both sexes. By contrast, the motoneuron populations in the terminal ganglia of adult males and females were strikingly different. No new motoneurons were produced during metamorphosis. Rather, this difference was the result of sex-specific cell death which occurred primarily during the early stages of adult differentiation. Possible mechanisms underlying this sex-specific degeneration of neurons are discussed.     

Localization of immunoreactive ubiquitin in the nervous system of the Manduca sexta moth

Selective neuronal death is a normal component of metamorphosis in the moth, Manduca sexta. In particular, the three unfused abdominal ganglia of the ventral nerve cord serve as a useful experimental preparation in which to study the regulation of the molecular mechanisms that mediate programmed cell death. Ubiquitin, a highly conserved 76-amino acid protein found in all eukaryotic cells, has previously been shown to be present in increased amounts in some tissue undergoing programmed cell death (e. g., larval intersegmental muscles inManduca sextamoths, dying cells in developing tunicates), but not in others (T-cells, Drosophila ommatidial cells, cultured sympathethic neurons deprived of nerve growth factor). It has been hypothesized that the need for ubiquitin-dependent proteolysis is increased in dying cells, and that the accumulation of ubiquitin might serve as an early marker for cells commited to die. Immunohistochemical localization of ubiquitin at the light microscopic level in the adbominal gaglia of Manduca sextasuggests that this protein plays a number of important roles in neuronal physiology and may be associated with the death of some neurons in this tissue. The most intense staining of neuronal cytoplasm, however, was found not in dying neurons, but instead in sets of persisting neurons that may serve a primarily neurosecretory or neuromodulatory function. The staining obtained in these cells with antibodies directed against ubiquitin was developmentally regulated. © 1994 Wiley-Lisx, Inc.     

Neurosecretory cells in the honeybee brain and suboesophageal ganglion show FMRFamide-like immunoreactivity.

Immunocytochemical analysis of the brain and suboesophageal ganglion of the honeybee Apis mellifera L. was combined with Lucifer Yellow backfilling from the corpora cardiaca and intracellular staining of single neurons. It is shown that more than one third of the cells that display FMRFamide-like immunoreactivity (F-LI) project to the corpora cardiaca, suggesting they are neurosecretory. Among the ca. 120 median neurosecretory cells (MNCs) in the pars intercerebralis about 32 show F-LI. The number of immunoreactive MNCs is highly variable and may depend on age and/or diet. Seven of at least 40 lateral neurosecretory cells display F-LI. They project through the brain via the medial branch of the bipartite nervus corporis cardiaci II. In the suboesophageal ganglion three types of immunoreactive neurosecretory cells were identified. Together with the median and the lateral neurosecretory cells in the brain these cells project through a single pair of nerves into the corpora cardiaca suggesting that the nervus corporis cardiaci (NCC) of the honeybee is a fusion of NCC I, II, and III described in other insects.     

Immunocytochemistry of dopamine in the brain of the locust Schistocerca gregaria.

Catecholamine-induced histofluorescence studies have suggested a rich innervation of the locust brain by dopamine-containing neurons. To provide a basis for future studies on dopamine action in this insect, the location and morphology of neurons reacting with antisera against dopamine were investigated in the supraoesophageal ganglion of the locust, Schistocerca gregaria. In each brain hemisphere, about 100 interneurons in the midbrain and approximately 3,000 cells in the optic lobe show dopamine-like immunoreactivity. All major areas of the brain except the calyces of the mushroom body, the antennal lobe, large parts of the lobula, and some areas in the inferior lateral protocerebrum contain immunoreactive neuronal processes. The arborization patterns of most dopamine-immunoreactive cell types could be identified through detailed reconstructions. The central body exhibits the most intense immunostaining. It is innervated by at least 40 pairs of dopamine-immunoreactive neurons belonging to three different cell types. Additional arborizations of these neurons are in the superior protocerebrum and in the lateral accessory lobes. A group of 4 immunoreactive neurons with ramifications in the antennal mechanosensory and motor center gives rise to a dense meshwork of varicose fibers in the pedunculus and parts of the alpha- and beta-lobes of the mushroom body. Other cell types innervate the ventrolateral protocerebrum, the inferior protocerebrum and the posterior optic tubercles. Three descending neurons originating in the tritocerebrum exhibit dopamine-like immunoreactivity. In the optic lobe, about 3,000 columnar intrinsic neurons of the medulla and a group of centrifugal tangential cells with arborizations in the medulla and lamina are dopamine-immunoreactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Copper/zinc superoxide dismutase-like immunoreactivity in the metamorphosing brain of the sphinx moth Manduca sexta

Cu/Zn superoxide dismutase (SOD) is part of the defense mechanism that protects cells from being damaged by reactive oxygen species. During metamorphosis of the nervous system, neurons undergo various fates, which are all coupled to high metabolic activities, such as proliferation, differentiation, pathfinding, and synaptogenesis. We describe the pattern of SOD immunoreactivity of identified neurons and neuron groups in the brain of Manduca sexta from the late larva through metamorphosis into adult. We focused on neurons of the developing antennal lobes, the optic lobes, and the central brain. Our results indicate the transient expression of SOD during phases in which the neurons develop their final adult identities. Our data also suggest that the SOD immunoreactivity may be used as an indicator for the period in which developing neurons form their synapses. We also observed SOD immunoreactivity within nitric oxide-sensitive cells as characterized by immunolabeling against 3'5'-cyclic guanosine monophosphate and soluble guanylyl cyclase, a novel finding in insects.     

Dopamine-synthesizing neurons include the putative H-cell homologue in the moth Manduca sexta

The catecholamine dopamine (DA) plays a fundamental role in the regulation of behavior and neurodevelopment across animal species. Uncovering the embryonic origins of neurons that express DA opens a path for a deeper understanding of how DA expression is regulated and, in turn, how DA regulates the activities of the nervous system. In a well-established insect model, Manduca sexta, we identified the putative homologue of the embryonic grasshopper "H-cell" using intracellular techniques, laser scanning confocal microscopy, and immunohistochemistry. In both species, this neuron possesses four axons and has central projections resembling the letter H. The H-cell in grasshoppers is known to be derived from the midline precursor 3 cell (MP3) and to pioneer the pathways of the longitudinal connectives; in Drosophila, the H-cell is also known to be derived from MP3. In the current study, we demonstrate that the Manduca H-cell is immunoreactive to antibodies raised against DA and its rate-limiting synthetic enzyme, tyrosine hydroxylase (TH). In larvae and adults, one DA/TH-immunoreactive (-ir) H-cell per ganglion is present. In embryos, individual ganglia contain a single midline TH-ir cell body positioned along side its putative sibling. Such observations are consistent with the known secondary transformation (in grasshoppers) of only one of the two MP3 progeny during early development. Although a hallmark feature of invertebrate neurons is the fairly stereotypical position of neuronal somata, we found that the H-cell somata can "flip-flop" by 180 degrees between an anterior and posterior position. This variability appears to be random and is not restricted to any particular ganglion. Curiously, what is segment-specific is the absence of the DA/TH-ir H-cell in the metathoracic (T3) ganglion as well as the unique structure of the H-cell in the subesophageal ganglion. Because this is the first immunohistochemical study of DA neurons in Manduca, we have provided the distribution pattern and morphologies of dopaminergic neurons, in addition to the H-cells, within the ventral nerve cord during development.     

Mandibular motor neurons of the caterpillar of the hawk moth Manduca sexta

As part of a planned study of the central neural basis of feeding behaviour in larval Manduca sexta, the morphology and physiology of the mandibular motor system is here described. The gross neuroanatomy of the postoral head segments has been investigated, especially the course and structure of the mandibular nerves. The electrophysiology of the mandibular opener and closer muscles has been investigated by extra- and intracellular recording during feeding behaviour and during electrical stimulation of the motor nerve. All the muscle fibres examined are of the "fast," twitch type. Contraction is associated exclusively with locally or completely propagated overshooting action potentials, never with local junctional potentials. Control of the muscles is by recruitment of more motor units and/or an increase of frequency of action potentials. No inhibitory synaptic potentials could be found. The motor neurons of the mandibular muscles have been identified by cobalt backfills of the mandibular nerve, and characterized by intracellular recording and dye injection. There are 12 closer and 8 opener motor neurons. All motor neurons recorded so far evoke 1:1 twitches in the muscle, and none appear to be inhibitory. No GABA-immunoreactive axons could be found in the mandibular nerve.     

Functionally distinct subdivisions of the macroglomerular complex in the antennal lobe of the male sphinx moth Manduca sexta.

Each antennal lobe in the brain of a male moth has a distinctive neuropil structure, the macroglomerular complex (MGC), which is specialized for primary processing of information about the conspecific female sex-pheromone blend. Olfactory interneurons with dendritic arborizations in the MGC were examined by means of tandem intracellular recording and staining with Lucifer Yellow. Neurons that responded selectively to stimulation of the antenna with the major pheromone component, (E,Z)-10,12-hexadecadienal, had arborizations that were restricted to a toroidal subdivision (the "toroid") of the MGC. Similarly, neurons that responded selectively to antennal stimulation with (E,Z)-11,13-pentadecadienal, a more stable mimic of a second essential but chemically unstable pheromone component, (E,E,Z)-10,12,14-hexadecatrienal, had arborizations confined to a globular subdivision (the "cumulus") of the MGC situated more proximally to the antennal nerve input. One neuron that responded to both of these stimuli clearly had arborizations in both subdivisions of the MGC. These anatomically distinct subdivisions of the MGC thus appear also to be functionally separate regions of pheromone-processing neuropil.     

Mapping of octopamine-immunoreactive neurons in the central nervous system of the lobster

It has been suggested that serotonin and octopamine serve important roles in behavioral regulation in lobsters. In this paper the locations of octopamine-immunoreactive neurons were mapped in wholemount preparations of the ventral nerve cord of 4th stage lobster (Homarus americanus) larvae. Approximately 86 neurons were found, distributed as follows: brain, 12; circumesophageal ganglia, 2; subesophageal ganglion, 38; thoracic ganglia, 6 each; and 4th and 5th abdominal ganglia, 2 each. All the octopamine-immunoreactive neurons are paired and located along the midline. Of the 86 neurons, 28 were identified as neurosecretory, and 26 as intersegmental ascending thoracic, ascending abdominal, or descending interneurons. The neurosecretory system is arranged segmentally and located entirely within the thoracic and subesophageal neuromeres with extensive terminal fields of endings along 2nd thoracic and subesophageal nerve roots. This set of neurons shares the features of central and peripheral endings with 2 pairs of large serotonin-containing neurosecretory neurons found in the fifth thoracic and first abdominal ganglia. The intersegmental neurons include: (1) two cells in the brain and 2 pairs of cells in the 3rd and 4th neuromeres of the subesophageal ganglion, which project to the 6th abdominal ganglion; (2) a segmentally organized group of ascending internurons found in the subesophageal and in all thoracic ganglia; and (3) pairs of ascending interneurons found in the 4th and 5th ganglia in the abdominal nerve cord. By means of a biochemical assay, the cell bodies of octopamine-immunoreactive neurosecretory cells in the thoracic segment of the nerve cord were found to contain 40–100 fmol of octopamine, while control neurons had none. © 1993 Wiley-Liss, Inc.     

Synaptic organization of the uniglomerular projection neurons of the antennal lobe of the moth Manduca sexta: A laser scanning confocal and electron microscopic study

The detailed branching pattern and synaptic organization of the uniglomerular projection neurons of the antennal lobe, the first processing center of the olfactory pathway, of the moth Manduca sexta were studied with laser scanning confocal microscopy and a technique combining laser scanning confocal microscopy and electron microscopy. Uniglomerular projection neurons, identified electrophysiologically or morphologically, were stained intracellularly with neurobiotin or biocytin. Brains containing the injected neurons were treated with streptavidin-immunogold to label the injected material for electron microscopy and with Cy3-streptavidin to lable the neurons with fluorescence for laser scanning confocal microscopy, and then embedded in Epon. Labeled neurons were imaged and reconstructed with laser scanning confocal microscopy (based on the retained fluorescence of the labeled neuron in the Epon block), and thin sections were cut at selected optical levels for correlation of light microscopic data and electron microscopic detail. Each neuron had a cell body in one of the three cell-body clusters of the antennal lobe, a primary neurite that extended across the coarse neuropil at the center of the antennal lobe and then formed a dense tuft of processes within a single glomerulus, and an axon that emanated from the primary neurite and projected from the antennal lobe via the antenno-cerebral tract to the lateral horn of the ipsilateral protocerebrum and, collaterally, to the calyces of the mushroom body. In the electron microscope, the fine dendritic branches in the apical zones of the glomeruli, where sensory axons terminate, were found to receive many input synapses. In deeper layers across the glomeruli, the processes participated in both input and output synapses, and at the bases of the glomeruli, the most proximal, thickest branches formed output synapses. In both of the protocerebral areas in which axonal branches terminated, those branches formed exclusively output synapses. Our findings indicate that, in addition to conveying olfactory information to the protocerebrum, uniglomerular projection neurons in the antennal lobes of M. sexta participate in local intraglomerular synaptic circuitry. J. Comp. Neurol. 379:2-20, 1997. © 1997 Wiley-Liss, Inc.