Uwe Homberg

Anatomy of antenno-cerebral pathways in the brain of the sphinx moth Manduca sexta

SummaryIn the moth Manduca sexta, the number and morphology of neuronal connections between the antennal lobes and the protocerebrum were examined. Cobalt injections revealed eight morphological types of neurons with somata adjacent to the AL neuropil that project in the inner, middle, and outer antenno-cerebral tracts to the protocerebrum. Neurons innervating the macroglomerular complex and many neurons with fibers in the inner antennocerebral tract have uniglomerular antennal-lobe arborizations. Most neurons in the middle and outer antenno-cerebral tracts, on the other hand, seem to innervate more than one glomerulus. Protocerebral areas receiving direct input from the antennal lobe include the calyces of the mushroom bodies, and circumscribed areas termed “olfactory foci” in the lateral horn of the protocerebrum and several other regions, especially areas in close proximity to the mushroom bodies. Fibers in the inner antenno-cerebral tract that innervate the male-specific macroglomerular complex have arborizations in the protocerebrum that are distinct from the projections of sexually non-specific neurons. Protocerebral neurons projecting into the antennal lobe are much less numerous than antennal-lobe output cells. Most of these protocerebral fibers enter the antennal lobe in small fiber tracts that are different from those described above. In the protocerebrum, these centrifugal cells arborize in olfactory foci and also in the inferior median protocerebrum and the lateral accessory lobes. The morphological diversity of connections between the antennal lobes and the protocerebrum, described here for the first time on a single-cell level, suggests a much greater physiological complexity of the olfactory system than has been assumed so far.

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

SummaryWe have prepared and characterized specific rabbit antisera against γ-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 protocerebmm.

Immunocytochemistry of GABA in the brain and suboesophageal ganglion ofManduca sexta

SummaryWe have used specific antisera against protein-conjugatedγ-aminobutyric acid (GABA) in immunocytochemical preparations to investigate the distribution of putatively GABAergic neurons in the brain and suboesophageal ganglion of the sphinx mothManduca sexta. About 20000 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-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.

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.

SummaryUsing an antiserum against the tetrapeptide FMRFamide, we have studied the distribution of FMRFamide-like substances in the brain and suboesophageal ganglion of the sphinx mothManduca 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 ofM. sexta and might function as neurohormones and neurotransmitters in a variety of neuronal cell types.

Serotonin-immunoreactive neurons in the median protocerebrum and suboesophageal ganglion of the sphinx moth Manduca sexta

SummarySerotonin-immunoreactive neurons in the median protocerebrum and suboesophageal ganglion of the sphinx moth Manduca sexta were individually reconstructed. Serotonin immunoreactivity was detected in 19–20 bilaterally symmetrical pairs of interneurons in the midbrain and 10 pairs in the suboesophageal ganglion. These neurons were also immunoreactive with antisera against DOPA decarboxylase. All major neuropil regions except the protocerebral bridge are innervated by these neurons. In addition, efferent cells are serotonin-immunoreactive in the frontal ganglion (5 neurons) and the suboesophageal ganglion (2 pairs of neurons). The latter cells probably give rise to an extensive network of immunoreactive terminals on the surface of the suboesophageal ganglion and suboesophageal nerves. Most of the serotonin-immunoreactive neurons show a gradient in the intensity of immunoreactive staining, suggesting low levels of serotonin in cell bodies and dendritic arbors and highest concentrations in axonal terminals. Serotonin-immunoreactive cells often occur in pairs with similar morphological features. With one exception, all serotonin-immunoreactive neurons have bilateral projections with at least some arborizations in identical neuropil areas in both hemispheres. The morphology of several neurons suggests that they are part of neuronal feedback circuits. The similarity in the arborization patterns of serotonin-immunoreactive neurons raises the possibility that their outgrowing neurites experienced similar forces during embryonic development. The morphological similarities further suggest that serotonin-immunoreactive interneurons in the midbrain and suboesophageal ganglion share physiological characteristics.

Neuroanatomy and immunocytochemistry of the median neuroendocrine cells of the subesophageal ganglion of the tobacco hawkmoth, Manduca sexta: Immunoreactivities to PBAN, and other neuropeptides

The median neuroendocrine cells of the subesophageal ganglion, important components of the neuroendocrine system of the tobacco hawkmoth, Manduca sexta, have not been well investigated. Therefore, we studied the anatomy of these cells by axonal backfills and characterized their peptide immunoreactivities. Both larvae and adults were examined, and developmental changes in these neuroendocrine cells were followed. Processes of the median neuroendocrine cells project to terminations in the corpora cardiaca via the third and the ventral nerves of this neurohemal organ, but the ventral nerve of the corpus cardiacum is the principal neurohemal surface for this system. Cobalt backfills of the third cardiacal nerves revealed lateral cells in the maxillary neuromere and a ventro‐median pair in the labial neuromere. Backfills of the ventral cardiacal nerves revealed two ventro‐median pairs of cells in the mandibular neuromere and two ventro‐median triplets in the maxillary neuromere. The efferent projections of these cells are contralateral. The anatomy of the system is basically the same in larvae and adults. The three sets of median neuroendocrine cells are PBAN‐ and FMRFamide‐immunoreactive, but only the mandibular and maxillary cells are proctolin‐immunoreactive. During metamorphosis, the mandibular and maxillary cells also acquire CCK‐like immunoreactivity and the labial cells become SCP‐ and sulfakinin‐immunoreactive. Characteristics of FMRFamide‐like immunostaining suggest that the median neuroendocrine cells may contain one or more of the FLRFamides that have been identified in M. sexta. The mandibular and maxillary neuroendocrine cells appear to produce the same set of hormones, and a somewhat different set of hormones is produced by the labial neuroendocrine cells. Two pairs of interneurons immunologically related to the neurosecretory cells are associated with the median maxillary neuroendocrine cells. These cells are PBAN‐, FMRFamide‐, SCP‐, and sulfakinin‐immunoreactive and project to arborizations in the brain and all ventral ganglia. These interneurons appear to have extensive modulatory functions in the CNS.

Olfaction in Manduca sexta: Cellular Mechanisms of Responses to Sex Pheromone

In insects, olfaction plays a major role in the control of many kinds of behavior. Orientation and movement toward, and interactions with, receptive mating partners, appropriate sites for oviposition, sources of food, and hosts for parasitism usually involve olfactory signals that initiate, sustain, and guide the behaviors. Because of their prominence in the zoosphere, their economic and medical importance, and their usefulness as models for both behavioral and neurobiological research, insects have been extensively studied by investigators interested in mechanisms of olfactory control of behavior. Insects respond to a variety of semiochemicals, including pheromones (chemical messengers within a species, such as sex attractants) and kairomones (chemical messengers between species and adaptively favorable to the recipient, such as attractants and stimulants for oviposition and feeding emitted by a host plant). Studies of insect responses to such biologically significant odors have shown that the quality and quantity of odorants in complex mixtures present in the environment are encoded in patterns of activity in multiple olfactory receptor cells (ORCs) in the antennae. These ‘messages’ are decoded and integrated in the olfactory centers of the central nervous system (CNS), and it is there that olfactorily induced changes in the behavior or physiology of the insect are initiated.

Neurotransmitters and Neuropeptides in the Olfactory Pathway of the Sphinx Moth Manduca Sexta

Like other animals, insects have many and diverse chemical “messengers” in their nervous systems. A growing list of synaptic neurotransmitters, neuromodulators, neuropeptides, and neurohormones -- collectively “transmitters” -- prompts efforts to seek physiological roles and mechanisms of action for these substances. An improved understanding of these chemical messengers in the insect nervous system promises to reveal key regulatory mechanisms, novel and accessible targets for pharmacological agents, and phyletic differences that can be exploited in new approaches to the manipulation of insect behavior and the selective destruction of harmful populations of insect pests and disease vectors. Toward such goals, we study the biochemistry, cellular distribution, and physiological actions of transmitter candidates in an experimentally favorable insect “model”, the sphinx moth Manduca sexta. In contrast with significant advances that have been made in many laboratories investigating peripheral neural and neuromuscular systems, relatively much less is known about chemical signalling in the insect central nervous system (CNS). With this in mind, and building upon substantial previous and ongoing studies of the anatomy, physiology, and development of the insect CNS in many laboratories including our own, we focus on the cellular neurochemistry of the CNS in Manduca. In particular we are exploring the olfactory pathway in the brain, for which we have accumulated much information about the types of neurons and their functional organization and development (e.g. see recent reviews: Hildebrand, 1985; Hildebrand and Montague, 1986; Christensen and Hildebrand, 1987).