Biochemical and immunocytological localization of molluscan small cardioactive peptides in the nervous system of Aplysia californica

Abstract

High pressure liquid chromatography (HPLC) followed by bioassay on isolated snail hearts were used to locate two related peptides, termed small cardioactive peptides A and B (SCPA and SCPB) in each of the central ganglia of Aplysia. The peptides are most concentrated in the buccal ganglia, the ganglia involved in the control of feeding movements. Immunocytology with antisera raised to conjugated SCPB stained three groups of neurons in the buccal ganglia. One group consisted of relatively small neurons that were tightly clustered. The second group was comprised of larger neurons that were more scattered. The third group was made up of several neurons including the two largest in the ganglia, identified cells B1 and B2. B1 and B2 and other neurons in this group innervate the gut by way of the esophageal nerve. HPLC-bioassay of single, individually dissected B1 or B2 neurons demonstrated that the two peptides are present in a single cell. For B2, but not B1, choline injected into the cell body was converted to the conventional transmitter, acetylcholine. This indicates that, in addition to the two peptides, B2 also contains choline acetyltransferase, and raises the possibility that acetylcholine and the SCPs may act as co-transmitters in B2. Strong immunocytological staining of fibers and varicosities was observed in the neuropilar region of the cerebral, pleural, pedal, and abdominal ganglia. In addition to the buccal ganglia, immunoreactive neurons were observed in all of the other central ganglia. The high concentration of the SCPs and the relatively large number of immunoreactive neurons in the buccal ganglion suggest a particularly important role of these peptides specifically in feeding behavior. However, the widespread occurrence of the SCPs in fibers and neuronal cell bodies throughout the nervous system suggests that these peptides also may have additional behavioral functions in Aplysia.

Characterization of GdFFD, a d-Amino Acid-containing Neuropeptide That Functions as an Extrinsic Modulator of the Aplysia Feeding Circuit

“…Consistent with this complexity, previous work has shown that the feeding circuit is extensively modulated by neuromodulators, including neuropeptides that have been discovered in Aplysia (41,42,48,49,66,67,69). This study constitutes the first example of a DAACP acting as a neuromodulator in the Aplysia feeding circuit.…”

Frequency-dependent release of peptide cotransmitters from identified cholinergic motor neurons in Aplysia.

“…One class of peptides that have been commonly found to coexist with other transmitters in Aplysia neurons are the small cardioactive peptides (SCPs) (6)(7)(8). The SCPs are represented by two peptides, SCPA and SCPB, which are 11 and 9 amino acids in length, respectively.…”

“…These peptides have similar sequences, are derived from a common precursor polypeptide (9)(10)(11), and have been shown to satisfy many of the criteria required of neurotransmitters (12). They are present in, and synthesized by, individual neurons (6)(7)(8) and are contained in membrane-bound dense-core vesicles in central neurons and in varicosities in the central nervous system, muscle, and gut (13,14). Immunoreactive fibers and varicosities are present at both central and peripheral sites where the SCPs have been shown to potently modulate the efficacy of synaptic transmission (12,15,16).…”

Multiple neuropeptides in cholinergic motor neurons of Aplysia: evidence for modulation intrinsic to the motor circuit.

“…Buccal ganglia were incubated with 0.5 mCi of [35S]methionine (1 Ci = 37 GBq; Amersham) for 24 hr at 18°C in 1 ml of 50% artificial sea water (ASW)/50% sterile (0.2-,um filtered) hemolymph/100 ,l of antibiotics (penicillin and streptomycin each at 50 units per ml)/colchicine (2.5 ,ul of 1 M colchicine dissolved in Me2SO). Colchicine was added to inhibit axonal transport, which raised intrasomatic peptide levels and reduced, or eliminated, labeled peptides that might be present in fibers and terminals near the somata of dissected neurons (12). After 24 hr, ganglia were rinsed and incubated in 50% sterilized hemolymph containing antibiotics and colchicine as described: 50% L15 culture medium (Flow Laboratories) plus salts for 4-6 hr.…”