The heartbeat of the giant African snail Achatina fulica is modulated by extrinsic cardioregulatory neurons, and two RFamide peptides — FMRFamide and the atrial peptide ACEP-1 (Achatina cardio-excitatory peptide-1) — are prime candidates for this control. An immunohistochemical study by Fujiwara-Sakata and Kobayashi in 1994 mapped both immunoreactivities in the snail's ganglia and heart, and its findings and hedged functional conclusion leave a set of questions that remain open: which identified central neurons send the peptide-immunoreactive axons that reach the atrium and the aortic end of the ventricle; whether the two peptides coexist in the same neurons and in the same cardiac fibers; what molecules the immunoreactivities actually correspond to; how the peptide map relates to the electrophysiologically identified heart motoneurons, the giant dorsal motoneurons later named d-VLN and d-RPLN being reported to lack such immunoreactivity in work citing the 1994 study (Zhuravlev et al. 2002; Bugai et al. 2005); and whether these peptides are released in the heart to regulate the beat. Later work cloned the ACEP-1 gene and localized its transcript to candidate neurons and the atrium, identified additional heart motoneurons, and extended the atrial peptide map in Achatina and a congeneric species — but never connected specific central neurons to the peptidergic cardiac fibers, never performed FMRFamide/ACEP-1 double labeling in the heart, and never demonstrated release. The problem is a coherent, still-open cellular-neurobiology question in a small and largely dormant specialty.

Background

The giant African snail Achatina fulica is a pulmonate gastropod (an air-breathing land snail) whose central nervous system consists of cerebral ganglia above the gut and a ring of subesophageal ganglia (paired pedal, pleural and parietal ganglia plus the unpaired visceral ganglion). Its heart comprises a thin-walled atrium (auricle) that receives hemolymph and a muscular ventricle that pumps it onward; the heartbeat is myogenic (generated by the cardiac muscle itself) but is modulated by extrinsic cardioregulatory neurons whose axons reach the heart through peripheral nerves, the most prominent being the intestinal nerve arising from the visceral ganglion. In A. fulica, electrophysiological studies identified individual heart motoneurons by intracellular recording — tonically active neurons (TAN-1, TAN-2, TAN-3), a periodically oscillating neuron (PON) and neuron VG1 forming the excitatory unit (Furukawa and Kobayashi 1987; Zhuravlev et al. 1997), and the inhibitory neurons HI-1 and HI-2 (Bychkov et al. 1997) — but the chemical transmitters of these cells were not determined.

Two peptide candidates for cardiac control were known. FMRFamide (H-Phe-Met-Arg-Phe-NH2), the first neuropeptide isolated from molluscan neural tissue, is the prototype of a large family of FMRFamide-related peptides (FaRPs) that excite or relax molluscan hearts; FMRFamide-like immunoreactivity had been detected in the Achatina nervous system well before the study at issue (Takayanagi and Takeda 1988). ACEP-1 (Achatina cardio-excitatory peptide-1, H-Ser-Gly-Gln-Ser-Trp-Arg-Pro-Gln-Gly-Arg-Phe-NH2) is an eleven-residue amidated peptide isolated from Achatina atria (Fujimoto et al. 1990) that shares the Arg-Phe-NH2 (RFamide) C-terminus with FMRFamide and exerts strong positive inotropic effects on the ventricle.

Fujiwara-Sakata and Kobayashi (1994) mapped both immunoreactivities by immunohistochemistry, a method in which tissue is stained with antibodies raised against a peptide; 'immunoreactive' structures therefore contain substances recognized by the antiserum, not necessarily the exact peptide. They found more than 50 FMRFamide-immunoreactive neurons (30–50 micrometers) on the ventral surface of the right cerebral ganglion, many of which also reacted with ACEP-1 antiserum; FMRFamide-immunoreactive neurons in all subesophageal ganglia, including a reproducible set of large cells among which a roughly 200-micrometer neuron of the right parietal ganglion and two similar posterior visceral neurons are candidates for cardiac projection neurons; identifiable ACEP-1-immunoreactive neurons confined to the visceral and right parietal ganglia, two or three of which (ventral visceral cells) sent axons into the intestinal nerve and could be traced toward, but not continuously into, the atrium; and, in the heart, immunoreactive nerve fibers of both types abundant in the atrial wall and present only fragmentarily at the aortic end of the ventricle, with the ventricular midwall essentially free of fibers. Colocalization in individual neurons was inferred by comparing adjacent tissue sections rather than by double labeling; dot-blot tests showed no cross-reactivity of either antiserum with the other peptide even at high concentration despite the shared RFamide terminus; and the antisera were not tested against most other known Achatina neuropeptides, although the atrium itself is chemically known to contain further alpha-amidated peptides of the fulicin gene-related peptide family (Yasuda-Kamatani et al. 1997). These observations leave open the questions of which identified neurons actually supply the cardiac peptidergic fibers, whether both peptides coexist in the same neurons and the same cardiac axons, what the immunoreactive substances are molecularly, how the peptide map relates to the electrophysiologically identified heart motoneurons, and whether and how the peptides act on the heartbeat.

Problem Statement

Which neurons of the Achatina fulica central nervous system give rise to the FMRFamide-like and ACEP-1-like immunoreactive fibers of the heart, and what is the chemical coding and functional role of that peptidergic innervation? This source-derived problem concerns, for the atrium and the aortic end of the ventricle where the immunoreactive fibers terminate, the following constituent aspects of a single open characterization:

  1. Source-neuron identity: assign the peptide-containing cardiac fibers to specific identified central neurons, replacing the present circumstantial linkage through the intestinal nerve (the nerve running from the visceral ganglion toward the heart).
  2. Colocalization: determine whether FMRFamide-like and ACEP-1-like substances coexist in the same central neurons and in the same cardiac axons, given that cerebral neurons show apparent coexistence while large subesophageal neurons appear to contain only one of the two peptides.
  3. Molecular identity: identify the peptide molecule(s) responsible for each immunoreactivity in the heart and in the identified neurons, and establish antiserum specificity against the other known endogenous Achatina neuropeptides, such as achatin-I, fulicin and the fulicin gene-related peptides that occur in the atrium itself.
  4. Reconciliation with the motor network: establish the relation between the peptidergic map and the electrophysiologically identified heart motoneurons (TAN-1, TAN-2, TAN-3, PON, VG1, the inhibitory neurons HI-1 and HI-2, and the giant dorsal motoneurons d-VLN and d-RPLN of the visceral and right parietal ganglia, which were named after the source paper); the giant dorsal motoneurons are reported to lack immunoreactivity to these antisera in work citing the 1994 source (Zhuravlev et al. 2002; Bugai et al. 2005), whereas no immunoreactivity of TAN-1, TAN-2, TAN-3, PON, VG1, HI-1 and HI-2 to these antisera has yet been established.
  5. Function: demonstrate whether these peptides are released from the cardiac nerve fibers and modulate the heartbeat of the innervated heart in situ, as opposed to acting only as circulating or pharmacological agents.

These aspects share one scientific objective — establishing the cellular, molecular and functional organization of the peptidergic cardiac pathway of A. fulica. They arise from the 1994 study's findings and its hedged conclusion that the results "suggest that FMRFamide- and ACEP-1-like peptides are involved in regulating the heart beat." All five aspects are required for a resolution of the problem — none is optional and no single aspect suffices on its own.

References

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