TheAplysia MytilusInhibitory Peptide-Related Peptides: Identification, Cloning, Processing, Distribution, and Action

Abstract

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.

Identification and Characterization of the Feeding Circuit-Activating Peptides, a Novel Neuropeptide Family ofAplysia

“…Standard molecular techniques (Sambrook et al, 1989) were used, except where noted. Semi-nested rapid amplification of cDNA ends (R ACE) was performed as described previously (Fujisawa et al, 1999;Furukawa et al, 2001). Briefly, two bluescript primers (BS1 ϭ ACCAT-GATTACGCCAAG and then BS2 ϭ AATTAACCC TCAC TAAAG) were used sequentially with a single degenerate oligo (GAY WSY I TI GGI GGI TTY GAR GTI CA), designed to a partial sequence of the 1387 peptide, in PCR on a random-primed Apl ysia C NS cDNA library.…”

“…Northern analysis were performed as described previously (Fujisawa et al, 1999;Furukawa et al, 2001). Briefly, blots were hybridized overnight at 50°C with [ 32 P]dC TP-labeled FCAP cDNA, washed, and exposed to film.…”

“…The rabbit anti-small cardioactive peptide (SC P) antibody was a gift from Dr. Richard Scheller (GeneTech, San Francisco, CA). Antibodies to FCAP were generated in rats as described previously (Fujisawa et al, 1999;Furukawa et al, 2001), but using FCAPb (ALDSLGGFQV HGW; Protein Core Facility, University of I llinois, Urbana, IL) coupled to BSA (Sigma A0281) using 1-ethyl-3-(dimethylaminopropyl)carbodiimide (Sigma E7750). Testing of the rat FCAP antibodies showed that immunostaining was abolished by preincubation with 10 Ϫ4 M ALDSLGGFQV HGW (data not shown).…”

“…Immunoc ytochemistr y. Immunocytochemistry (IMM) was performed on whole mounts as described previously (Vilim et al, 1996;Fujisawa et al, 1999;Furukawa et al, 2001). Briefly, fixed tissues were exposed to primary antibodies (4 -7 d), washed for 2 d, and exposed to secondary antibodies for 2-3 d. The longer incubations and washes used here have been found to improve the signal-to-noise ratio in our whole mounts.…”

“…Mass spectrometr y. M ALDI-TOF MS of neurons was performed as described previously (Garden et al, 1996;Fujisawa et al, 1999;Furukawa et al, 2001). Analysis of the CBC s was done on short sections (Ͻ1 mm) of the desheathed cut ends of CBC s after varying periods of incubation in Leibovitz's L -15 media (Life Technologies).…”