The first Aplysia californica insulin gene is characterized and its proteolytic processing from prohormone to final peptides elucidated using a combination of biochemical and mass spectrometric methods. Aplysia insulin (AI) is one of the largest insulins found, with a molecular weight of 9146 Da, and an extended A chain compared with other invertebrate and vertebrate insulins. The AI prohormone produces a series of C peptides and also a unique N-terminally acetylated D peptide. AI-producing cells are restricted to the central region of the cerebral ganglia mostly within the F and C clusters, and AI is transported to neurohemal release sites located on the upper labial and anterior tentacular nerves. The expression of AI mRNA decreases when the animal is deprived of food, and injections of AI reduce hemolymph glucose levels, suggesting that the function of insulin-regulating metabolism has been conserved.

Neuropeptides are a ubiquitous class of signaling molecules. In our attempt to understand the generation of feeding behavior in Aplysia, we have sought to identify and fully characterize the neuropeptides operating in this system. Preliminary evidence indicated that Mytilus inhibitory peptide (MIP)-like peptides are present and operating in the circuitry that generates feeding in Aplysia. MIPs were originally isolated from the bivalve mollusc Mytilus edulis, and related peptides have been identified in other invertebrate species, but no precursor has been identified. In this study, we describe the isolation and characterization of novel Aplysia MIP-related peptides (AMRPs) and their precursor. Several AMRPs appear to have some structural and functional features similar to vertebrate opioid peptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to confirm that all 14 AMRPs predicted by the precursor are processed in isolated neurons. Northern analysis, whole-mount in situ hybridization, and immunohistochemistry are used to map the abundant expression of these peptides in the CNS and peripheral tissues such as the digestive tract, vasculature, and the reproductive organs. Physiological studies demonstrate that the rank order of the inhibitory actions of these peptides is different for three target muscles. These results underscore the importance of using a multidisciplinary approach to identifying and characterizing the actions of neuropeptides in an effort to gain understanding of their role in systems of interest. The widespread distribution of the AMRPs indicates that they may be operating in many different systems of Aplysia.

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Intracellular concentrations of L-citrulline (Cit) and its metabolites are related to nitric oxide synthase (NOS) activity, an enzyme producing the intercellular messenger NO in animal tissues including the nervous system. A capillary electrophoresis system using laser-induced fluorescence detection is described, and methods are developed to monitor the levels of L-arginine (Arg), Cit, and related molecules in identified neurons of the marine slugs, Pleurobranchaea californica and Aplysia californica. The limits of detection for Arg, Cit, L-arginino-succinate, L-ornithine, and L-arginine phosphate range from 50 amol to 17 fmol (5 nM to 17 microM in the neurons under study); these detection limits are significantly lower than actual intracellular levels of the metabolites, allowing the direct assay of single cells. The levels of NOS metabolites in individual neurons varied form 6 (Arg) and 4 mM (Cit) in putative NOS-containing neurons down to < 1 microM (undetectable) levels in many putative NOS-negative cells. The Arg/Cit ratio is independent of cell volume, correlates with NADPH-diaphorase staining, and appears to be a characteristic parameter for the presence of NOS activity in identified neurons.

Matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry is used to examine the formation of N-pyroglutamate (pGlu) in single, identified neurons from Aplysia. Six pGlu peptides are identified in the R3-14 and the R15 neurons that result from in vivo processing of peptides containing either Glu or Gln at their respective N-termini. Moreover, we show that Glu-derived pGlu is not a sample collection or measurement artifact. The pGlu peptides are detected in isolated cell bodies, regenerated neurites in culture, interganglionic connective nerves, cell homogenates, and collected releasates. We also demonstrate that R3-14 cells readily convert a synthetic N-Glu peptide to its pGlu analogue, indicating the presence of novel enzymatic activity.

An HPLC NMR system is presented that integrates a commercial microbore HPLC system using a 0.5-mm column with a 500-MHz proton NMR spectrometer using a custom NMR probe with an observe volume of 1.1 microL and a coil fill factor of 68%. Careful attention to capillary connections and NMR flow cell design allows on-line NMR detection with no significant loss in separation efficiency when compared with a UV chromatogram. HPLC NMR is performed on mixtures of amino acids and small peptides with analyte injection amounts as small as 750 ng; the separations are accomplished in less than 10 min and individual NMR spectra are acquired with 12 s time resolution. Stopped-flow NMR is achieved by diversion of the chromatographic flow after observation of the beginning of the analyte band within the NMR flow cell. Isolation of the compound of interest within the NMR detection cell allows multidimensional experiments to be performed. A stopped-flow COSY spectrum of the peptide Phe-Ala is acquired in 3.5 h with an injected amount of 5 micrograms.

The isolation, characterization, and bioactivity in the feeding circuitry of a novel neuropeptide in the Aplysia californica central nervous system are reported. The 17-residue amidated peptide, NGGTADALYNLPDLEKIamide, has been termed cerebrin due to its primary location in the cerebral ganglion. Liquid chromatographic puri®cation guided by matrix-assisted laser desorption/ionization time-of-¯ight mass spectrometry allowed the isolation of the peptide with purity adequate for Edman sequencing. The cerebrin cDNA has been characterized and encodes an 86 amino acid prohormone that predicts cerebrin and one additional peptide. Mapping using in situ hybridization and immunocytochemistry showed that cerebrin containing neuronal somata are localized almost exclusively in the cerebral ganglion, mostly in the F-and C-clusters. Both immunostaining and mass spectrometry demonstrated the presence of cerebrin in the neurohemal region of the upper labial nerve. In addition, immunoreactive processes were detected in the neuropil of all of the ganglia, including the buccal ganglia, and in some interganglionic connectives, including the cerebral-buccal connective. This suggests that cerebrin may also function as a local signaling molecule. Cerebrin has a profound effect on the feeding motor pattern elicited by the command-like neuron CBI-2, dramatically shortening the duration of the radula protraction in a concentration-dependent manner, mimicking the motor-pattern alterations observed in food induced arousal states. These ®ndings suggest that cerebrin may contribute to food-induced arousal in the animal. Cerebrin-like immunoreactivity is also present in Lymnaea stagnalis suggesting that cerebrin-like peptides may be widespread throughout gastropoda. Keywords: Aplysia californica, cDNA cloning, in situ hybridization, immunocytochemistry, Lymnaea stagnalis, MALDI MS. Invertebrate model systems are used extensively to gain insight into neural mechanisms that are responsible for the expression and plasticity of behavior. In these systems, characterization of behavior-mediating circuits is greatly facilitated by the fact that neural circuits contain a limited number of neurons. Consequently, in many invertebrates, the neural circuits that mediate a variety of behaviors have been extensively characterized. However, in order to understand the neural basis of behavior it is also necessary to have fairly complete knowledge of signaling molecules that affect the circuits from within and without. Neuropeptides represent the largest and most diverse group of signaling molecules in the nervous system. Advantageous features of invertebrate nervous systems have greatly facilitated identi®cation of neuropeptides that are involved in generating and modifying a number of behaviors. Several important insights into the role of various neuropeptides have been obtained and the functional consequence of the large peptide diversity in several well-de®ned neuronal networks is actively being investigated (Marder et al. 1995;Brezina and Weiss 19...

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