The stomatogastric nervous system (STNS) is a premiere model for studying modulation of motor pattern generation. Whereas the cellular and network responses to monoamines have been particularly well characterized electrophysiologically, the transduction mechanisms that link the different monoaminergic signals to specific intracellular responses are presently unknown in this system. To begin to elucidate monoaminergic signal transduction in pyloric neurons, we used a bioinformatics approach to predict the existence of 18 monoamine receptors in arthropods, 9 of which have been previously cloned in Drosophila and other insects. We then went on to use the two existing insect databases to clone and characterize the 10th putative arthropod receptor from the spiny lobster, Panulirus interruptus. This receptor is most homologous to the 5-HT 2 subtype and shows a dose-dependent response to 5-HT but not to any of the other monoamines present in the STNS. Through a series of pharmacological experiments, we demonstrate that this newly described receptor, 5-HT 2␤Pan , couples with the traditional G q pathway when expressed in HEK293 cells, but not to G s or G i/o . Moreover, it is constitutively active, because the highly conserved DRY motif in transmembrane region 3 has evolved into DRF. Site-directed mutagenesis that reverts the motif back to DRY abolishes this agonist-independent activity. We further demonstrate that this receptor most likely participates in the modulation of stomatogastric motor output, because it is found in neurites in the synaptic neuropil of the stomatogastric ganglion as well as in the axon terminals at identified pyloric neuromuscular junctions.

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.

The freshwater prawn Macrobrachium rosenbergii is a tropical crustacean with characteristics similar to those of lobsters and crayfish. Adult males develop through three morphological typessmall (SC), yellow (YC), and blue claws (BC)-with each representing a level in the dominance hierarchy of a group, BC males being the most dominant. We are interested in understanding the role played by neuropeptides in the mechanisms underlying aggressive behavior and the establishment of dominance hierarchies in this type of prawn. SIFamides are a family of arthropod peptides recently identified in the central nervous system of insects and crustaceans, where it has been linked to olfaction, sexual behavior, and gut endocrine functions. One of the six SIFamide isoforms, GYRKPPFNGSIFamide (Gly-SIFamide), is highly conserved among decapod crustaceans such as crabs and crayfish. We wanted to determine whether Gly-SIFamide plays a role in modulating aggression and dominant behavior in the prawn. To do this, we performed behavioral experiments in which interactions between BC/YC pairs were recorded and quantified before and after injecting Gly-SIFamide directly into the circulating hemolymph of the living animal. Behavioral data showed that aggression among interacting BC/YC prawns was enhanced by injection of Gly-SIFamide, suggesting that this neuropeptide does have a modulatory role for this type of behavior in the prawn.

Detection and toxicity assessment of waterborne contaminants are crucial for protecting human health and the environment. Development of easy-to-implement, rapid and cost-effective tools to measure anthropogenic effects on watersheds are critical for responsible management, particularly in times of increasing development and urbanization. Traditionally, environmental toxicology has focused on limited endpoints, such as lethality and fertility, which are directly affecting population levels. However, more sensitive readings are needed to assess sub-lethal effects. Monitoring of contaminant-induced behavior alterations was proposed before, but is difficult to implement in the wild and performing it in aquatic laboratory models seem more suited. For this purpose, we adapted a photo-dependent swimming response (PDR) that was previously described in zebrafish larva. We first asked if PDR was present in other aquatic animals. We measured PDR in larvae from two freshwater prawn species (Macrobrachium rosenbergii, MR, and Macrobrachium carcinus, MC) and from another fish the fathead minnow (FHM, Pimephales promelas). In all, we found a strong and reproducible species-specific PDR, which is arguing that this behavior is important, therefore an environmental relevant endpoint. Next, we measured PDR in fish larvae after acute exposure to copper, a common waterborne contaminant. FHM larvae were hyperactive at all tested concentrations in contrast to ZF larvae, which exhibited a concentration-dependent hyperactivity. In addition to this well-accepted anxiety-like behavior, we examined two more: photo-stimulated startle response (PSSR) and center avoidance (CA). Both were significantly increased. Therefore, PDR measures after acute exposure to this waterborne contaminant provided as sensitive readout for its detection and toxicity assessment. This approach represents an opportunity to diagnostically examine any substance, even when present in complex mixtures like ambient surface waters. Mechanistic studies of toxicity using the extensive molecular tool kit of ZF could be a direct extension of such approaches.

1. Endplate potentials (EPPs) and miniature endplate potentials (MEPPs) were recorded from frog neuromuscular junctions bathed in Ringer solutions containing normal (1P8 mM) or high (3-6 mM) Ca2". The peptide toxin /s-conotoxin GIIIA was added to the Ringer solution to prevent muscle action potentials and contraction. 2. The nerve was stimulated with conditioning trains of 200-4800 impulses applied at 20 impulses s' to characterize the effects of repetitive stimulation on changes in EPP amplitude and MEPP frequency under high quantal conditions. 3. MEPP frequency was dramatically increased during and immediately following repetitive stimulation under high quantal conditions, whereas EPP amplitude was greatly depressed. There was no effect of repetitive stimulation on MEPP amplitude. 4. Following the conditioning stimulation the increase in MEPP frequency decayed back to the control level with a time course that could be described by four exponentials. The time constants of these exponentials were very similar to those that describe the components of stimulation-induced increases in EPP amplitude and MEPP frequency observed under low quantal conditions when depression is absent. 5. The results of this study indicate that depression and the components of stimulationinduced increases in release (facilitation, augmentation and potentiation) can be present at the same time, suggesting that the mechanism of depression involves different underlying factors from the mechanism(s) responsible for increases in release. They also indicate either that depression selectively affects only those quanta destined to be released in direct response to the nerve action potential, which would suggest that EPPs and MEPPs arise from different pools of transmitter, or that depression in some way affects a step in the release process involved only in evoked release, and not asynchronous (spontaneous) release.