Developmental and regional-specific expression of FLRFamide peptides in the tobacco hornworm, Manduca sexta, suggests functions at ecdysis.

The developmental profile of a family of three FLRFamide (Phe-Leu-Arg-Phe-NH2) peptides in the tobacco hornworm, Manduca sexta, revealed regional-specific expression patterns within the segmental ganglia. Levels of the three peptides-F7G (GNSFLRF-amide), F7D (DPSFLRFamide), and F10 (pEDVVHS-FLRFamide)-were always higher in the thoracic than abdominal ganglia. The predominant peptide also differed regionally, with F7G being highest in the thoracic ganglia and F7G and F100 being equivalent in the abdominal ganglia. Furthermore, we found regional-specific transient declines in ganglion peptide levels temporally correlated to ecdysis. Thoracic ganglion peptide levels declined at each molt, while abdominal ganglion levels declined over a period of 2 days after ecdysis. The decline in central levels was accompanied by an increase in levels in peripheral neurohemal sites, the transverse nerves (TNs). These observations suggest peptides were released from neurosecretory cells (NSCs) at ecdysis. Distinct sets of thoracic and abdominal NSCs and their processes in peripheral neurohemal sites were immunoreactive, supporting the biochemical data. These results also suggest the regional differences may arise from cellular-specific expression patterns for this family of peptides. In addition, fine immunoreactive processes were observed traveling between TNs and skeletal muscles, suggestive of myotropic actions. We propose that the release of different M. sexta FLRFamides from regionally distinct NSCs leads to a coordinated modulation of skeletal and visceral muscles that facilitate ecdysis.

Distribution of GABA-like immunoreactive neurons in insects suggests lineage homology.

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.

Developmental plasticity of neuropeptide expression in motoneurons of the moth, Manduca sexta: steroid hormone regulation.

Developmental changes in the expression of a FMRFamide-like (Phe-Met-Arg-Phe-NH2) peptide or peptides in motoneurons of the tobacco hornworm, Manduca sexta, were demonstrated using immunohistochemical techniques. The onset of FMRFamide-like immunoreactivity (FLI) was gradual during larval growth but by the final larval stage, immunoreactivity was present in the majority of motoneurons. FLI then declined during metamorphosis and was absent in all identified adult motoneurons. We used a novel in vivo culture system to demonstrate that the steroid hormone, 20-hydroxyecdysone, regulates the loss of FLI in motoneurons during metamorphosis. The small commitment peak of ecdysteroid appears to shut off the program of neuropeptide accumulation that is characteristic of the larval state of the motoneurons. The prepupal peak of steroid then causes the rapid loss of stored FLI. This steroid-induced change in the neuropeptide content of motoneurons may reflect major changes in neuromuscular functions between the larval and adult stages.

Fate of abdominal ventral unpaired median cells during metamorphosis of the hawkmoth, Manduca sexta.

Each of the unfused abdominal ganglia in the larval, pupal, and adult stages of the hawkmoth, Manduca sexta, has two large ventral median neurons with axons that bifurcate to innervate targets on both sides of the abdomen. Although the dendritic structures of the two neurons are similar, their axons branch to innervate distinct sets of target muscles. During metamorphosis both neurons undergo dendritic regression, followed by growth of new arborizations during adult development. The neurons must innervate different targets in the larva and adult, since many larval muscles degenerate and are replaced during metamorphosis. Both neurons were reactive with an antibody to the neuromodulatory compound, octopamine, in the larval and adult stages. Pairwise intracellular recordings in isolated nerve cords revealed spontaneous excitatory synaptic potentials that occurred in the ventral median neurons of each ganglion in an anterior-to-posterior sequence. The synaptic potentials were eliminated when the interganglionic connective was interrupted posterior to the subesophageal ganglion. The ventral median neurons were also excited by tactile stimulation of the body surface in larvae, pupae and adults.

The regulation of transmitter expression in postembryonic lineages in the moth Manduca sexta. I. Transmitter identification and developmental acquisition of expression.

The majority of the neurons in the adult nervous system of Manduca sexta are born postembryonically, during larval life. Stereotypic arrays of identifiable neuroblasts generate their clonal families or lineages commencing at the end of the second larval instar through pupal day 2, when the neuroblasts die (Booker and Truman, 1987a). We have used immunohistochemical techniques to follow the neurochemical differentiation of GABA and a peptide similar to molluscan small cardioactive peptide B (SCPB) in identified lineages. We report here the distribution and developmental acquisition of the expression of these putative transmitters. There are 24 postembryonic lineages in the second thoracic ganglion of the larvae (Booker and Truman, 1987a). Immunoreactivity against GABA and SCPB is seen only in a subset of these 24 clonal families. GABA immunoreactivity is confined to the progeny of the E, K, M, N, T, and X neuroblasts and is expressed by most or all of the neurons in these lineages. The SCPB-like immunoreactivity is found in a subset of the neurons in only two clonal groups, the K and M groups, and is colocalized with GABA. These results show that, though heterogeneity in transmitter type exists (GABA, GABA/SCPB), members of a given lineage share at least some features (GABA) in common. The onset of transmitter expression was followed in detail for the K- and M-lineage neurons. During the larval stages, the postembryonic lineage cells are developmentally arrested in a partially differentiated state (Booker and Truman, 1987a) and do not express transmitter immunoreactivity at this time. Their maturation resumes with the onset of metamorphosis.(ABSTRACT TRUNCATED AT 250 WORDS)

The regulation of transmitter expression in postembryonic lineages in the moth Manduca sexta. II. Role of cell lineage and birth order.

The expression of GABA is restricted to the progeny of only six of the 24 identified postembryonic lineages in the thoracic ganglia of the tobacco hornworm, Manduca sexta (Witten and Truman, 1991). It is colocalized with a peptide similar to molluscan small cardioactive peptide B (SCPB) in some of the neurons in two of the six lineages. By combining chemical ablation of the neuroblasts at specific larval stages with birth dating of the progeny, we tested whether the expression of GABA and the SCPB-like peptide was determined strictly by cell lineage or involved cellular interactions among the members of individual clonal groups. Chemical ablation of the six specific neuroblasts that produced the GABA-positive neurons (E, K, M, N, T, and X) or of the two that produced the GABA + SCPB-like-immunoreactive neurons (K, M) prior to the generation of their lineages resulted in the loss of these immunoreactivities. These results suggest that regulation between lineages did not occur. Ablation of the K and M neuroblasts after they had produced a small portion of their lineages had no effect on the expression of GABA, but did affect the pattern of the SCPB-like immunoreactivity. Combining birth-dating techniques with transmitter immunocytochemistry revealed that it was the position in the birth order and not interactions among the clonally related neurons that influenced the peptidergic phenotype. These results suggest that cell lineage is involved in establishing the GABAergic phenotype and that both cell lineage and birth order influence the determination of the peptidergic phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptidergic innervation of insect skeletal muscle: immunochemical observations.

Proctolin (Arg-Tyr-Leu-Pro-Thr) is a pentapeptide present in the hindgut or proctodeum of the cockroach Periplaneta americana where it may be a transmitter. Its widespread distribution among peripherally projecting neurons in the CNS (Bishop and O'Shea, '82) suggested that proctolin's motor function is not restricted to the hindgut, but has a variety of peripheral targets. This idea was further supported when proctolin was localized to an identified skeletal motoneuron, the slow coxal depressor, where it acts as a cotransmitter (O'Shea and Bishop, '82; Adams and O'Shea, '83). Our objective was to investigate the proctolinergic innervation of a variety of skeletal muscles of the cockroach Periplaneta americana. We used immunohistochemical and radioimmunochemical methods to map the distribution of proctolin immunoreactivity. This survey revealed that a subpopulation of skeletal muscles are innervated by proctolinergic motoneurons. The anatomical features of the peptidergic innervation and the levels of proctolinlike immunoreactivity of one muscle group, the coxal depressor system, are here described in detail. The source of the proctolin innervation to the metathoracic coxal depressor group is identified as the slow coxal depressor motoneuron. The results of a survey of fast and slow skeletal muscles revealed that proctolin is associated with slow motor function. The functional implications of the association of a peptide with motoneurons are discussed in relationship to the organization of the insect motor pool.

Structures of two cockroach neuropeptides assigned by fast atom bombardment mass spectrometry.

Amino acid sequences have been assigned to two cockroach neuropeptides (greater than Glu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2, M I, and greater than Glu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2, M II) by application of fast atom bombardment mass spectrometry, including high resolution and linked scan (metastable) studies. The peptides show considerable homology with two other invertebrate neuropeptides, adipokinetic hormone (AKH, from a locust) and red pigment concentrating hormone (RPCH, from a prawn), whose fast atom bombardment spectra were also studied. M I and M II are thus members of a family of structurally-related invertebrate neuropeptides.