No abstract

Although the medicinal leech is a well-studied system in which many neurons and circuits have been identified with precision, descriptions of the distributions of some of the major biogenic amines, such as dopamine (DA) and octopamine (OA), have yet to be completed. In the European medicinal leech Hirudo medicinalis and the American medicinal leech Macrobdella decora,we have presented the first immunohistochemical study of DA neurons in the entire central nervous system, and of OA-immunoreactive (ir) neurons in the head and tail brains. Dopaminergic neurons were identified using the glyoxylic acid method and antisera to DA and its rate-limiting synthetic enzyme tyrosine hydroxylase (TH). Octopaminergic neurons were recognized using a highly specific antiserum raised against OA. An antibody raised against DA-beta-hydroxylase (DbetaH), the mammalian enzyme that converts DA to norepinephrine (NE), was found to immunostain OA-ir neurons. This antibody appears to cross-react with the closely related invertebrate enzyme tyramine-beta-hydroxylase, which converts tyramine to OA, suggesting that the OA-ir cells are indeed octopaminergic, capable of synthesizing OA. Because the DbetaH antiserum selectively immunostained the OA-ir neurons, but not the DA-synthesizing cells, our results also indicate that the DA-ir neurons synthesize DA and not NE as their end product. The expression of TH immunoreactivity was found to emerge relatively early in development, on embryonic day 9 (47-48% of development). In contrast, OA expression remained absent as late as embryonic day 20. Higher order processes of some of the dopaminergic and octopaminergic neurons in the adult brain were observed to project to a region previously described as a neurohemal complex. Several TH-ir processes were also seen in the stomatogastric nerve ring, suggesting that DA may play a role in the regulation of biting behavior. By mapping the distributions and developmental expression pattern of DA and OA neurons in the leech, we aim to gain a better understanding of the functional roles of aminergic neurons and how they influence behavior.

Octopamine, a biogenic amine analogous to norepinephrine, plays an important role in the orchestration and modulation of invertebrate behavior. In the leech, the behavioral actions of octopamine have been demonstrated; however, identification of octopaminergic neurons had not been determined by using immunohistochemical techniques. Thus, we used an antibody highly specific to octopamine to examine the distribution of octopamine-immunoreactive neurons in the segmental ganglia of American and European medicinal leeches (Macrobdella decora and Hirudo medicinalis). One pair of octopamine-immunoreactive neurons was located in the dorsolateral ganglionic region of anterior ganglia 1-6 and posterior ganglia 15-21. No corresponding octopamine-immunoreactive neurons were found in midbody ganglia 7-14. Using Neutral Red staining in combination with intracellular Neurobiotin injections and octopamine immunostaining, we determined the identity of the dorsolateral octopamine-immunoreactive cells. The dorsolateral octopamine-immunoreactive neuron (the DLO) was not cell 21, the only previously reported Neutral Red staining neuron in the dorsolateral position. We also determined that the Leydig neuron was not octopamine immunoreactive in either of the two medicinal leech species. Octopamine immunostaining in the sex ganglia revealed hundreds of immunoreactive neurons in sexually mature leeches. Such neurons were not observed in juvenile leeches. The developmental time course of octopamine immunoreactivity in the dorsolateral octopamine-immunoreactive neurons was also investigated by staining embryonic Hirudo medicinalis. Octopamine expression occurred relatively late as compared with the detectable onset of serotonin expression. Octopamine expression in the dorsolateral octopamine-immunoreactive cells was not detectable at early to mid-embryonic stages, and must commence during late embryonic to early juvenile stages. The identification of octopamine-immunoreactive cells now sets the stage for further investigations into the functional role of octopamine in leech behavior and the development of behavior.

The major membrane protein of the bovine lens fiber cell is a 26-kilodalton (kD) protein (MP26), which appears to be a component of the extensive junctional specializations found in these cells. To examine the arrangement of MP26 within the junctional membranes, various proteases were incubated with fiber cell membranes that had been isolated with or without urea and/or detergents. These membranes were analyzed with electron microscopy and SDS-PAGE to determine whether the junctional specializations or the proteins were altered by proteolysis. Microscopy revealed no obvious structural changes. Electrophoresis showed that chymotrypsin, papain, and trypsin degraded MP26 to 21-22 kD species. A variety of protease treatments, including overnight digestions, failed to generate additional proteolysis. Regions on MP26 which were sensitive to these three proteases overlapped. Smaller peptides were cleaved from MP26 with V8 protease and carboxypeptidases A and B. Protein domains cleaved by these proteases also overlapped with regions sensitive to chymotrypsin, papain, and trypsin. Specific inhibition of the carboxypeptidases suggested that cleavage obtained with these preparations was not likely due to contaminating endoproteases. Since antibodies are not thought to readily penetrate the 2-3 nm extracellular gap in the fiber cell junctions, antibodies to MP26 were used to analyze the location of the protease-sensitive domains. Antisera were applied to control (26 kD) and proteolyzed (22 kD) membranes, with binding being evaluated by means of ELISA reactions on intact membranes. Antibody labeling was also done following SDS-PAGE and transfer to derivatized paper. Both assays showed a significant decrease in binding following proteolysis, with the 22 kD product showing no reaction with the anti-MP26 sera. These investigations suggest that MP26 is arranged with approximately four-fifths of the primary sequence "protected" by the lipid bilayer and the narrow extracellular gap. One-fifth of the molecule, including the C-terminus, appears to be exposed on the cytoplasmic side of the membrane.

Bursicon is the final neurohormone released at the end of the molting cycle. It triggers the sclerotization (tanning) of the insect cuticle. Until now, its existence has been verified only by bioassays. In an attempt to identify this important neurohormone, bursicon was purified from homogenates of 2,850 nerve cords of the cockroach Periplaneta americana by using high performance liquid chromatography technology and two-dimensional gel electrophoresis. Bursicon bioactivity was found in four distinct protein spots at approximately 30 kDa between pH 5.3 and 5.9. The protein of one of these spots at pH 5.7 was subsequently microsequenced, and five partial amino acid sequences were retrieved. Evidence is presented that two of these sequences are derived from bursicon. Antibodies raised against the two sequences labeled bursicon-containing neurons in the central nervous systems of P. americana. One of these antisera labeled bursicon-containing neurons in the crickets Teleogryllus commodus and Gryllus bimaculatus, and the moth Manduca sexta. A cluster of four bilaterally paired neurons in the brain of Drososphila melanogaster was also labeled. In addition, this antiserum detected three spots corresponding to bursicon in Western blots of two-dimensional gels. The 12-amino acid sequence detected by this antiserum, thus, seems to be conserved even among species that are distantly related.

Immunoglobulin-related molecules have been shown to play important roles in cell-cell recognition events during the development of both vertebrate and invertebrate nervous systems. In the moth, Manduca sexta, we report the presence of novel, mouse, immunoglobulin G (mIgG)-like immunoreactivity in a discrete population of identified neurosecretory neurons (the NS-Ls also known as the cell 27s) and interneurons (the IN-704s). A number of polyclonal anti-mIgG antibodies were used to immunostain these cells in wholemount. The mIgG-like-immunoreactive (IR) neurons were present during embryogenesis through the developing adult stages, but disappeared in the postemerged adult. Biochemical analysis of M. sexta ventral nerve cords revealed that the mIgG-like antigen is a membrane-associated 27-kDa protein which is likely responsible for the mIgG-like immunostaining observed. Unambiguous identification of the mIgG-like-IR neurons was based on neuronal morphology and our ability to demonstrate conclusively that these neurons expressed immunoreactivity to an antiserum against crustacean cardioactive peptide (CCAP). The NS-Ls and IN-704s were both shown to colocalize the CCAP and mIgG-like immunoreactivities. The mIgG-like and CCAP-IR neurons were identical to a subset of CCAP-IR neurons recently described by Davis et al. [(1993) J. Comp. Neurol., 338:612-627] in pupae. We found these CCAP-IR neurons, however, also to be present in larvae. The mIgG-like- and CCAP-IR neurons included the NS-L pair of the subesophageal maxillary neuromere, which projected anteriorly to the corpora cardiaca, and the NS-L of the labial neuromere whose axons projected out the dorsal nerve of the next posterior ganglion. The mIgG-like and CCAP-IR NS-Ls were also observed throughout the three thoracic ganglia, and all shared strikingly similar structural features. These cells exited out the dorsal nerve of the next posterior ganglion and eventually projected to the neurohemal release sites of the perivisceral organs. These neurons appear to be the homologues of the abdominal CCAP-IR NS-Ls, neurons that in the adult switch their neurotransmitter and release the neuropeptide bursicon. Our description of the distribution and developmental expression of this novel mIgG-like immunoreactivity may provide new insights into the regulation of neurotransmitter plasticity and/or recognition-signaling events involved in the embryonic and postembryonic assembly of the nervous system.

No abstract

Octopamine (OA), a biogenic amine similar to norepinephrine, has profound and well-documented actions on the nervous systems of invertebrates. In the insect, Manduca sexta, we examined the developmental plasticity of OA synthesis, studied its endocrine regulation, and observed previously undescribed OA-immunoreactive (ir) neurons. We found that levels of tyramine beta-hydroxylase (TbetaH), an essential enzyme for the biosynthesis of OA, increase during metamorphosis. Based on the established and influential roles of the steroid hormone 20-hydroxyecdysone (20-HE) during development, we tested the hypothesis that increases in TbetaH levels and OA immunoreactivity are regulated by the rise in 20-HE occurring during pupal-adult development. We determined that the levels of TbetaH in the terminal abdominal ganglion (neuromeres 6-9) remain at a constant level during pupal development and the early stages of adult development. Beginning at ca. pupal stage 8, however, the levels of TbetaH begin to rise, reaching a maximum level by pupal stage 12. By removing the source of ecdysteroid hormone through ligation, and by subsequent replacement of 20-HE via infusion, we found evidence indicating that the preadult rise of 20-HE is both necessary and sufficient for the increased levels of TbetaH. During the course of our study, we also identified previously unreported OA-ir neurons. In particular, adult-specific OA-ir lateral cells were found, as were relatively small OA-ir dorsal median pairs that doubled in size during adult development. Abdominal ganglia not exposed to the preadult rise in 20-HE possessed neither the OA-ir lateral neurons nor the somatic growth of the smaller OA-ir median neurons. These newly described OA-ir neurons probably contribute to the steroid-induced elevations of TbetaH observed at the end of metamorphosis.