Identification of orcokinins in single neurons in the stomatogastric nervous system of the crayfish, Cherax destructor

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Abstract
The orcokinins are a highly conserved family of crustacean peptides that enhance hindgut contractions in the crayfish Orconectes limosus (Stangier et al. [1992] Peptides 13:859–864). By combining immunocytochemical and mass spectrometrical analysis of the stomatogastric nervous system (STNS) in the crayfish Cherax destructor, we show that multiple orcokinins are synthesized in single neurons. Immunocytochemistry demonstrated orcokinin‐like immunoreactivity in all four ganglia of the STNS and in the pericardial organs, a major neurohaemal organ. Identified neurons in the STNS were stained, including a pair of modulatory interneurons (inferior ventricular nerve neuron, IVN), a neuron with its cell body in the stomatogastric ganglion that innervates cardiac muscle c6 via the anterior median nerves (AM‐c6), and a sensory neuron (anterior gastric receptor neuron). Five orcokinin‐related peptides were identified by matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS) post source decay fragmentation in samples of either the stomatogastric ganglion or the pericardial organs. Four of these peptides are identical to peptides derived from the cloned Procambarus clarkii precursor (Yasuda‐Kamatani and Yasuda [2000] Gen. Comp. Endocrinol. 118:161–172), including the original [Asn13]‐orcokinin (NFDEIDRSGFGFN, [M+H]+ = 1,517.7 Da), [Val13]‐orcokinin ([M+H]+ = 1,502.7 Da), [Thr8‐His13]‐orcokinin ([M+H]+ = 1,554.8 Da), and FDAFTTGFGHS ([M+H]+ = 1,186.5 Da). The fifth peptide is a hitherto unknown orcokinin variant: [Ala8‐Ala13]‐orcokinin ([M+H]+ = 1,458.7 Da). The masses of all five peptides were also detected in the inferior ventricular nerve of C. destructor, which contains the cell bodies and axons of the IVNs as well as the axons of two other orcokinin‐like immunoreactive neurons. In the oesophageal nerve, in which all the orcokinin‐like immunoreactivity derives from the IVNs, at least two of the orcokinins were detected, indicating that multiple orcokinins are synthesized in these neurons. Similarly, all four orcokinin masses were detected in the anterior median nerves, in which all the orcokinin‐like immunoreactivity derives from the AM‐c6 neuron. This study therefore lays the groundwork to investigate the function of the orcokinin peptide family using single identified neurons in a well‐studied system. J. Comp. Neurol. 444:245–259, 2002.