A crustacean serotonin receptor: Cloning and distribution in the thoracic ganglia of crayfish and freshwater prawn

Abstract

Serotonin (5-HT) is involved in regulating important aspects of behavior and a variety of systemic physiological functions in both vertebrates and invertebrates. These functions are mediated through binding to 5-HT receptors, of which approximately 13 have been characterized in mammals. In crustaceans, important model systems for the study of the neural basis of behaviors, 5-HT is also linked with higher-order behaviors, associated with different 5-HT receptors that have been identified at the physiological and pharmacological levels. However, no crustacean 5-HT receptors have been identified at the molecular level. We have cloned a putative 5-HT(1) receptor (5-HT(1crust)) from crayfish, prawn, and spiny lobster and have raised antibodies that recognize this protein in all three organisms. 5-HT(1crust) immunoreactivity (5-HT(1crust)ir) was observed surrounding the somata of specific groups of neurons and as punctate staining within the neuropil in all thoracic ganglia of crayfish and prawn. In the crayfish, 5-HT(1crust)ir was also found in boutons surrounding the first and second nerves of each ganglion and on the 5-HT cells of T1-4. In the prawn, 5-HT(1crust)ir was also found in axons that project across the ganglia and along the connectives. We found examples of colocalization of 5-HT(1crust) with 5-HT, consistent with the short-term modulatory role of 5-HT, as well as cases of serotonergic staining in the absence of a 5-HT(1crust) signal, which might imply that other 5-HT receptors are found at these locations. We also observed receptors that did not possess counterpart 5-HT staining, suggesting that these may also mediate long-term neurohormonal functions of serotonin.

Circadian clocks in crustaceans: identified neuronal and cellular systems

“…was recently shown to display circadian rhythmicity, although a dependence upon 5-HT has not yet been analysed in this species (243). Three or more 5-HT receptors are present in the crustacean CNS (244,245), but only two, 5-HTR 1crust and 5-HTR 2_Pro (or 5-HTR 1alpha and 5-HTR 2beta ), have been analysed in more detail and localised in crayfish CNS structures (246,247).…”

Conservation of structure, signaling and pharmacology between two serotonin receptor subtypes from decapod crustaceans, Panulirus interruptus and Procambarus clarkii

“…Complete cloning and sequencing of 5-HT 2␤Pan and 5-HT 1␣Pan from Panulirus interruptus have been previously described (Clark et al, 2004;Sosa et al, 2004). We also previously cloned a large segment of 5-HT 1␣Pro spanning transmembrane domains III-VII from Procambarus clarkii .…”

“…Multiple cDNA preparations, each from a separate crayfish nervous tissue mRNA preparation, were used as templates for nested PCR experiments with these degenerate primers to amplify fragments of the crayfish ortholog, as described previously (Baro et al, 1994;Sosa et al, 2004). Primers specific to 5-HT 2␤Pro were designed to generate a large clone of 5-HT 2␤Pro .…”

“…Next, western blots were used to confirm tet-repressor-regulated 5-HT 1␣Pan expression as follows. Cells were plated in 60·mm dishes and induced with 1·mol·l -1 tetracycline 0, 6, 8, 12 and 24·h before collection and isolation of protein by previously described methods (Clark et al, 2004;Sosa et al, 2004). Protein preparations were run on a 10% SDS-PAGE gel, transferred to PVDF membranes and probed with a custom-made rabbit anti-5-HT 1␣Crust antibody .…”

“…This is not surprising as one would not expect selection for or against amino acids involved in binding synthetic ligands. We have recently identified and cloned two crustacean 5-HT receptors from the spiny lobster, Panulirus interruptus, infraorder Achelata: 5-HT 1␣Pan and 5-HT 2␤Pan (Clark et al, 2004;Sosa et al, 2004). We were interested to know whether the actions of pharmacological agents were conserved across crustacean species.…”