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Antennal mechanosensors play a key role in control and stability of insect flight. In addition to the well-established role of antennae as airflow detectors, recent studies have indicated that the sensing of antennal vibrations by Johnston's organs also provides a mechanosensory feedback relevant for flight stabilization. However, few studies have addressed how the individual units, or scolopidia, of the Johnston's organs encode these antennal vibrations and communicate it to the brain. Here, we characterize the encoding properties of individual scolopidia from the Johnston's organs in the hawk moth, Manduca sexta, through intracellular neurophysiological recordings from axons of the scolopidial neurons. We stimulated the flagellum-pedicel joint using a custom setup that delivered mechanical stimuli of various (step, sinusoidal, frequency and amplitude sweeps) waveforms. Single units of the Johnston's organs typically displayed phaso-tonic responses to step stimuli with short (3-5 ms) latencies. Their phase-locked response to sinusoidal stimuli in the 0.1-100 Hz frequency range showed high fidelity (vector strengths>0.9). The neurons were able to encode different phases of the stimulus motion and were also extremely sensitive to small amplitude (<0.05 deg) deflections with some indication of directional tuning. In many cases, the firing frequency of the neurons varied linearly as a function of the stimulus frequency at wingbeat and double wingbeat frequencies, which may be relevant to their role in flight stabilization. Iontophoretic fills of these neurons with fluorescent dyes showed that they all projected in the antennal mechanosensory and motor center (AMMC) area of the brain. Taken together, these results showcase the speed and high sensitivity of scolopidia of the Johnston's organs, and hence their ability to encode fine antennal vibrations., (© 2014. Published by The Company of Biologists Ltd.)

By means of single-cell matrix assisted laser desorption/ionization time-of-flight mass spectrometry, we analysed neuropeptide expression in all FXPRLamide/pheromone biosynthesis activating neuropeptide synthesizing neurons of the adult tobacco hawk moth, Manduca sexta. Mass spectra clearly suggest a completely identical processing of the pheromone biosynthesis activating neuropeptide-precursor in the mandibular, maxillary and labial neuromeres of the subesophageal ganglion. Only in the pban-neurons of the labial neuromere, products of two neuropeptide genes, namely the pban-gene and the capa-gene, were detected. Both of these genes expressed, amongst others, sequence-related neuropeptides (extended WFGPRLamides). We speculate that the expression of the two neuropeptide genes is a plesiomorph character typical of moths. A detailed examination of the neuroanatomy and the peptidome of the (two) pban-neurons in the labial neuromere of moths with homologous neurons of different insects indicates a strong conservation of the function of this neuroendocrine system. In other insects, however, the labial neurons either express products of the fxprl-gene or products of the capa-gene. The processing of the respective genes is reduced to extended WFGPRLamides in each case and yields a unique peptidome in the labial cells. Thus, sequence-related messenger molecules are always produced in these cells and it seems that the respective neurons recruited different neuropeptide genes for this motif.

Three large median cell bodies with a diameter between 40 and 70 microm that exhibit octopamine immunoreactivity were identified in the posterior part of the suboesophageal ganglion of the tobacco hawkmoth larva, Manduca sexta. These neurons possess bilaterally symmetrical axons in the posterior neck connectives, and at least one of them extends through the whole ventral nerve cord to the terminal abdominal ganglion. Therefore, these neurons belong to the class of descending ventral unpaired median neurons. From each cell body, a primary neurite ascends anteriorly, which after bending dorsally turns posteriorly and then bifurcates to give rise to two descending axons. From the primary neurite two main dendritic branches ascend anteriorly, and four characteristic branches can be distinguished originating from them: two descending dendritic branches and two ascending dendritic branches. Dense arborizations from all these branches exist in all neuromeres of the suboesophageal ganglion. Intracellular recordings from these neurons show that in contrast to the ventral unpaired median neurons of thoracic and abdominal ganglia, they do not produce overshooting action potentials but exhibit passive soma spikes only. During pharmacologically evoked fictive motor patterns these neurons show coupling to various motor patterns such as crawling, feeding and molting.

Eph receptor tyrosine kinases and their ephrin ligands participate in the control of neuronal growth and migration in a variety of contexts, but the mechanisms by which they guide neuronal motility are still incompletely understood. By using the enteric nervous system (ENS) of the tobacco hornworm Manduca sexta as a model system, we have explored whether Manduca ephrin (MsEphrin; a GPI-linked ligand) and its Eph receptor (MsEph) might regulate the migration and outgrowth of enteric neurons. During formation of the Manduca ENS, an identified set of approximately 300 neurons (EP cells) populates the enteric plexus of the midgut by migrating along a specific set of muscle bands forming on the gut, but the neurons strictly avoid adjacent interband regions. By determining the mRNA and protein expression patterns for MsEphrin and the MsEph receptor and by examining their endogenous binding patterns within the ENS, we have demonstrated that the ligand and its receptor are distributed in a complementary manner: MsEphrin is expressed exclusively by the migratory EP cells, whereas the MsEph receptor is expressed by midline interband cells that are normally inhibitory to migration. Notably, MsEphrin could be detected on the filopodial processes of the EP cells that extended up to but not across the midline cells expressing the MsEph receptor. These results suggest a model whereby MsEphrin-dependent signaling regulates the response of migrating neurons to a midline inhibitory boundary, defined by the expression of MsEph receptors in the developing ENS.

Neuronal differentiation is characterized by stereotypical sequences of membrane channel and receptor acquisition. This is regulated by the coordinated interactions of a variety of developmental mechanisms, one of which is the control by steroid hormones. We have used the metamorphosis of the holometabolous insect, Manduca sexta, as a model to study effects of 20-hydroxyecdysone on the maturation of thoracic neuron membrane channel expression. To test for direct hormone action, neurons were dissociated into primary cell culture on the first day of pupal life. In situ hybridization demonstrated that the amount of expression of the acetylcholine receptor alpha subunit, MARA1, was not affected by 20-hydroxyecdysone. Immunocytochemistry with an antibody directed against the SP19 segment of voltage-gated sodium channels revealed no effect of 20-hydroxyecdysone treatment during the first 6 days in culture. SP19 sodium channel protein was evenly distributed along all neurites. In contrast, after 8 days in culture, 20-hydroxyecdysone increased the amount of SP19 protein expression and strongly affected its distribution in differentiating neurons. In the presence of 20-hydroxyecdysone, patches of high densities of SP19 sodium channel protein were found in growth cones close to the base of filopodia. This is a further step toward unraveling the blend of membrane proteins under the control of steroids during the development of the central nervous system of postembryonic Manduca. Our results, taken together with previous studies, indicate that 20-hydroxyecdysone does not affect the expression of potassium membrane current or of the nicotinic acetylcholine receptor but instead regulates the amplitude of the calcium membrane current and the amount and distribution of SP19 sodium channel protein.

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., ((c) 2005 Wiley-Liss, Inc.)

The antennal lobe (AL) of the sphinx moth Manduca sexta is a well-established model system for studying mechanisms of neuronal development. To understand whether neuropeptides are suited to playing a role during AL development, we have studied the cellular localization and temporal expression pattern of neuropeptides of the A-type allatostatin family. Based on morphology and developmental appearance, we distinguished four types of AST-A-immunoreactive cell types. The majority of the cells were local interneurons of the AL (type Ia) which acquired AST-A immunostaining in a complex pattern consisting of three rising (RI-RIII) and two declining phases (DI, DII). Type Ib neurons consisted of two local neurons with large cell bodies not appearing before 7/8 days after pupal ecdysis (P7/P8). Types II and III neurons accounted for single centrifugal neurons, with type II neurons present in the larva and disappearing in the early pupa. The type III neuron did not appear before P7/P8. RI and RII coincided with the rises of the ecdysteroid hemolymph titer. Artificially shifting the pupal 20-hydroxyecdysone (20E) peak to an earlier developmental time point resulted in the precocious appearance of AST-A immunostaining in types Ia, Ib, and III neurons. This result supports the hypothesis that the pupal rise in 20E plays a role in AST-A expression during AL development. Because of their early appearance in newly forming glomeruli, AST-A-immunoreactive fibers could be involved in glomerulus formation. Diffuse AST-A labeling during early AL development is discussed as a possible signal providing information for ingrowing olfactory receptor neurons.

The metamorphosing antennal lobe (AL) of the sphinx moth Manduca sexta serves as an established model system for studying neuronal development. To improve our understanding of mechanisms involved in neuronal plasticity, we have analyzed the size, shape, and localization of ten identified glomeruli at three different time points during development and in the adult, viz., (1) 13 days after pupal eclosion (P13), which reflects a time when the basic glomerular map has formed, (2) immediately after adult eclosion (A0), which represents a time when the newly formed glomeruli are uninfluenced by external odors, and (3) 4 days after adult eclosion (A4), which reflects a time when the animals have been exposed to surrounding odors. Our data from normally developing ALs of male M. sexta from P13 to A0 revealed an increase in size of all examined glomeruli of between 40% and 130%, with the strongest increases occurring in two of the three sex-specific glomeruli (cumulus, toroid). From A0 to A4, the cumulus and toroid increased significantly when correlated to AL volume, whereas the other glomeruli reached the sizes gained after A0. This study was based on antibody staining against the ubiquitous synaptic vesicle protein synaptotagmin, confocal laser scan microscopy, and the three-dimensional (3D) analysis tool AMIRA. Tissue permeability and therefore reliability of the staining quality was enhanced by using formalin/methanol fixation. The standard 3D glomeruli introduced in this study can now be used as basic tools for further examination of neuronal plasticity at the level of the identified neuropil structures, viz., the glomeruli of the AL of M. sexta.

Recently, both nitric oxide synthase (NOS), and nitric oxide (NO)-sensitive guanylyl cyclase were cloned in Manduca sexta and implicated in several cellular, developmental, and behavioral processes (Nighorn et al. [1998] J Neurosci 18:7244-7255). However, NO is a highly diffusive gas, and little is known about the range and specificity of its actions on neurons. To begin examining the role of NO as a neurotransmitter in the central nervous system (CNS) of larval Manduca, we have mapped potential NO-producing neurons using fixation-resistant NADPH-diaphorase staining and antisera that recognize a NOS-specific epitope. In addition, to detect NO-responsive neurons, we treated the CNS with NO donors and used antibodies that recognize elevated levels of cyclic 3;,5;-guanosine monophosphate (cGMP). Many potential NO-producing neurons were mapped, including the ventral unpaired median cells and three pairs of lateral cells in each abdominal ganglion. Additional neurons in the dorsal midline of ganglia A5-7 (PM2) appear to express NOS in a segment-specific manner. At the larval-to-pupal transition, this staining pattern changes; the PM2 neurons stain weakly or are undetectable and there is novel expression of NOS in cell 27. In response to NO donors, a small number of neurons produce detectable cGMP accumulation in a segment-specific pattern. These include a pair of posteriodorsally positioned interneurons (IN505) in all the abdominal ganglia, PM2 neurons in A5, and PM1 and PM2 neurons in A7. Hence, PM2 neurons in A5 and A7 are potentially capable of producing and responding to NO. These identified NO-producing and responding neurons provide a tractable model system for studying the dynamics and specificity of NO signaling in the CNS., (Copyright 2000 Wiley-Liss, Inc.)

Each hemisegment of the Manduca sexta larva is supplied with a subepidermal plexus of approximately 350 multidendritic neurons. An initial set of neurons, the primary plexus neurons, arise at 35-45% of embryogenesis. These neurons comprise 12-16 uniquely identifiable neurons per hemisegment that have homologues in other insect larvae. Each spreads processes across a characteristic portion of the body wall and has an axon that projects into the central nervous system. Secondary plexus neurons are born in two waves: the first between 70% and 80% of embryogenesis and the second during the molt to the second larval stage. The secondary plexus neurons are multidendritic, spread uniformly across the body wall, and appear to make contacts with the primary plexus neurons. Each secondary plexus cell arises as part of a five-cell cluster; the other cells produce a sensory bristle and socket along with the bristle sensory neuron and a glial cell. Application of nitric oxide (NO) donors induces plexus neurons to produce cyclic 3',5' guanosine monophosphate (cGMP), suggesting the presence of soluble guanylate cyclase. With few exceptions, plexus neurons become sensitive to NO stimulation approximately 10 hours after their birth and remain so throughout larval life. Cyclic GMP is detected primarily in the cytoplasm of plexus neurons and extends into the finest peripheral dendrites. Our results suggest that cGMP participates in the development and/or physiology of this peripheral neural plexus.

Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in vertebrates and invertebrates (Sattelle [1990] Adv. Insect Physiol. 22:1-113). The GABA phenotype is lineally determined in postembryonic neurons in the tobacco hawkmoth, Manduca sexta (Witten and Truman, [1991] J. Neurosci. 11:1980-1989) and is restricted to six identifiable postembryonic lineages in the moth's thoracic hemiganglia. We used a comparative approach to determine whether this distinct clustering of GABAergic neurons is conserved in Insecta. In the nine orders of insects surveyed (Thysanura, Odonata, Orthoptera, Isoptera, Hemiptera, Coleoptera, Diptera, Lepidoptera, and Hymenoptera), GABA-like immunoreactive neurons within a thoracic hemiganglion were clustered into six distinct groups that occupied positions similar to the six postembryonic lineages in Manduca. On the basis of cell body position and axon trajectories, we suggest that these are indeed homologous lineage groups and that the lineal origins of the GABAergic cells have been very conservative through insect evolution. The distinctive clustering of GABA-positive cells is shared with crustaceans (Mulloney and Hall [1990] J. Comp. Neurol. 291:383-394; Homberg et al. [1993] Cell Tissue Res. 271:279-288) but is not found in the centipede Lithobius forficulatus. There is a two- to threefold increase in numbers of thoracic neurons between the flightless Thysanura and the most advanced orders of insects. Using the GABA clusters as indicators of specific lineages, we find that only selected lineages have significantly contributed to this increase in neuronal numbers.