Shaun D. Cain

Hormone complement of the Cancer productus sinus gland and pericardial organ: An anatomical and mass spectrometric investigation

In crustaceans, circulating hormones influence many physiological processes. Two neuroendocrine organs, the sinus gland (SG) and the pericardial organ (PO), are the sources of many of these compounds. As a first step in determining the roles played by hemolymph‐borne agents in the crab Cancer productus, we characterized the hormone complement of its SG and PO. We show via transmission electron microscopy that the nerve terminals making up each site possess dense‐core and/or electron‐lucent vesicles, suggesting diverse complements of bioactive molecules for both structures. By using immunohistochemistry, we show that small molecule transmitters, amines and peptides, are among the hormones present in these tissues, with many differentially distributed between the two sites (e.g., serotonin in the PO but not the SG). With several mass spectrometric (MS) methods, we identified many of the peptides responsible for the immunolabeling and surveyed the SG and PO for peptides for which no antibodies exist. By using MS, we characterized 39 known peptides [e.g., β‐pigment‐dispersing hormone (β‐PDH), crustacean cardioactive peptide, and red pigment‐concentrating hormone] and de novo sequenced 23 novel ones (e.g., a new β‐PDH isoform and the first B‐type allatostatins identified from a non‐insect species). Collectively, our results show that diverse and unique complements of hormones, including many previously unknown peptides, are present in the SG and PO of C. productus. Moreover, our study sets the stage for future biochemical and physiological studies of these molecules and ultimately the elucidation of the role(s) they play in hormonal control in C. productus. J. Comp. Neurol. 493:607–626, 2005.

Identification and characterization of a tachykinin-containing neuroendocrine organ in the commissural ganglion of the crab Cancer productus

SUMMARY A club-shaped, tachykinin-immunopositive structure first described nearly two decades ago in the commissural ganglion (CoG) of three species of decapod crustaceans has remained enigmatic, as its function is unknown. Here, we use a combination of anatomical, mass spectrometric and electrophysiological techniques to address this issue in the crab Cancer productus. Immunohistochemistry using an antibody to the vertebrate tachykinin substance P shows that a homologous site exists in each CoG of this crab. Confocal microscopy reveals that its structure and organization are similar to those of known neuroendocrine organs. Based on its location in the anterior medial quadrant of the CoG, we have named this structure the anterior commissural organ (ACO). Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry shows that the ACO contains the peptide APSGFLGMRamide, commonly known as Cancer borealis tachykinin-related peptide Ia (CabTRP Ia). Using the same technique, we show that CabTRP Ia is also released into the hemolymph. As no tachykinin-like labeling is seen in any of the other known neuroendocrine sites of this species (i.e. the sinus gland, the pericardial organ and the anterior cardiac plexus), the ACO is a prime candidate to be the source of CabTRP Ia present in the circulatory system. Our electrophysiological studies indicate that one target of hemolymph-borne CabTRP Ia is the foregut musculature. Here, no direct CabTRP Ia innervation is present, yet several gastric mill and pyloric muscles are nonetheless modulated by hormonally relevant concentrations of the peptide. Collectively, our findings show that the C. productus ACO is a neuroendocrine organ providing hormonal CabTRP Ia modulation to the foregut musculature. Homologous structures in other decapods are hypothesized to function similarly.

Magnetic Orientation and Navigation in Marine Turtles, Lobsters, and Molluscs: Concepts and Conundrums

Abstract The Earths magnetic field provides a pervasive source of directional information used by phylogenetically diverse marine animals. Behavioral experiments with sea turtles, spiny lobsters, and sea slugs have revealed that all have a magnetic compass sense, despite vast differences in the environment each inhabits and the spatial scale over which each moves. For two of these animals, the Earths field also serves as a source of positional information. Hatchling loggerhead sea turtles from Florida responded to the magnetic fields found in three widely separated regions of the Atlantic Ocean by swimming in directions that would, in each case, facilitate movement along the migratory route. Thus, for young loggerheads, regional magnetic fields function as navigational markers and elicit changes in swimming direction at crucial geographic boundaries. Older turtles, as well as spiny lobsters, apparently acquire a “magnetic map” that enables them to use magnetic topography to determine their position relative to specific goals. Relatively little is known about the neural mechanisms that underlie magnetic orientation and navigation. A promising model system is the marine mollusc Tritonia diomedea, which possesses both a magnetic compass and a relatively simple nervous system. Six neurons in the brain of T. diomedea have been identified that respond to changes in magnetic fields. At least some of these appear to be ciliary motor neurons that generate or modulate the final behavioral output of the orientation circuitry. These findings represent an encouraging step toward a holistic understanding of the cells and circuitry that underlie magnetic orientation behavior in one model organism.

Identification of magnetically responsive neurons in the marine mollusc Tritonia diomedea

SUMMARY Behavioral experiments have demonstrated that the marine mollusc Tritonia diomedea can use the Earths magnetic field as an orientation cue. Little is known, however, about the neural mechanisms that underlie magnetic orientation behavior in this or any other animal. In previous studies, two neurons in the brain of Tritonia, known as LPd5 and RPd5, were shown to respond with enhanced electrical activity to changes in earth-strength magnetic fields. We report evidence that two additional neurons, known as LPd6 and RPd6, also respond with increases in electrical activity when the magnetic field around the animal is altered. Anatomical analyses revealed that prominent neurites from the Pd6 cells are located within two ipsilateral nerves, pedal nerves 1 and 2. These nerves extend to the periphery of the animal and innervate tissues of the anterior ipsilateral foot and body wall. Electrophysiological recordings demonstrated that action potentials generated by the Pd6 cells propagate from the central ganglia toward the periphery. These results imply that the Pd6 cells play an efferent role in the magnetic orientation circuitry. Given that these cells contain cilio-excitatory peptides and that Tritonia crawls using ciliary locomotion, the Pd6 neurons may control or modulate cilia used in crawling, turning, or both.

Identifiable neurons inhibited by Earth-strength magnetic stimuli in the mollusc Tritonia diomedea.

SUMMARY Diverse animals use the Earths magnetic field as an orientation cue, but little is known about the sensory, processing and motor elements of the neural circuitry underlying magnetic orientation behavior. The marine mollusc Tritonia diomedea has both a magnetic compass sense and a simple nervous system accessible to electrophysiological analysis. Previous studies have revealed that four identifiable neurons, known as LPd5, RPd5, LPd6 and RPd6, respond with enhanced electrical activity to changes in Earth-strength magnetic fields. Here we report that two additional neurons, LPd7 and RPd7, are inhibited by magnetic stimuli. Cobalt fills of the Pd7 neurons indicated that two prominent neurites emerge from the soma and project to the periphery through the ipsilateral cerebral nerves CeN6 and CeN3; in some cases, a third neurite was visible in CeN2. The nerves extend to the anterior region of the animal where they innervate the lateral body walls, oral veil and mouth region. Action potentials in the Pd7 neurons propagate from the central ganglia toward the periphery. Thus, the Pd7 cells have characteristics of efferent neurons. The precise function of these cells during magnetic orientation behavior, however, remains to be determined.

Immunochemical and electrophysiological analyses of magnetically responsive neurons in the mollusc Tritonia diomedea

Tritonia diomedea uses the Earth’s magnetic field as an orientation cue, but little is known about the neural mechanisms that underlie magnetic orientation behavior in this or other animals. Six large, individually identifiable neurons in the brain of Tritonia (left and right Pd5, Pd6, Pd7) are known to respond with altered electrical activity to changes in earth-strength magnetic fields. In this study we used immunochemical, electrophysiological, and neuroanatomical techniques to investigate the function of the Pd5 neurons, the largest magnetically responsive cells. Immunocytochemical studies localized TPeps, neuropeptides isolated from Pd5, to dense-cored vesicles within the Pd5 somata and within neurites adjacent to ciliated foot epithelial cells. Anatomical analyses revealed that neurites from Pd5 are located within nerves innervating the ipsilateral foot and body wall. These results imply that Pd5 project to the foot and regulate ciliary beating through paracrine release. Electrophysiological recordings indicated that, although both LPd5 and RPd5 responded to the same magnetic stimuli, the pattern of spiking in the two cells differed. Given that TPeps increase ciliary beating and Tritonia locomotes using pedal cilia, our results are consistent with the hypothesis that Pd5 neurons control or modulate the ciliary activity involved in crawling during orientation behavior.

The anterior cardiac plexus: an intrinsic neurosecretory site within the stomatogastric nervous system of the crab Cancer productus

SUMMARY The stomatogastric nervous system (STNS) of decapod crustaceans is modulated by both locally released and circulating substances. In some species, including chelate lobsters and freshwater crayfish, the release zones for hormones are located both intrinsically to and at some distance from the STNS. In other crustaceans, including Brachyuran crabs, the existence of extrinsic sites is well documented. Little, however, is known about the presence of intrinsic neuroendocrine structures in these animals. Putative intrinsic sites have been identified within the STNS of several crab species, though ultrastructural confirmation that these structures are in fact neuroendocrine in nature remains lacking. Using a combination of anatomical techniques, we demonstrate the existence of a pair of neurosecretory sites within the STNS of the crab Cancer productus. These structures, which we have named the anterior cardiac plexi (ACPs), are located on the anterior cardiac nerves (acns), which overlie the cardiac sac region of the foregut. Each ACP starts several hundred μm from the origin of the acn and extends distally for up to several mm. Transmission electron microscopy done on these structures shows that nerve terminals are present in the peripheral portion of each acn, just below a well defined epineurium. These terminals contain dense-core and, occasionally, electron-lucent vesicles. In many terminals, morphological correlates of hormone secretion are evident. Immunocytochemistry shows that the ACPs are immunopositive for FLRFamide-related peptide. All FLRFamide labeling in the ACPs originates from four axons, which descend to these sites through the superior oesophageal and stomatogastric nerves. Moreover, these FLRFamide-immunopositive axons are the sole source of innervation to the ACPs. Collectively, our results suggest that the STNS of C. productus is not only a potential target site for circulating hormones, but also serves as a neuroendocrine release center itself.

Advances in the neural bases of orientation and navigation

The ability to locomote in one direction (oriented movement), and the ability to navigate toward a distant goal are related behaviors that are phylogenetically widespread. Orientation behaviors include finding the source of an odor or acoustic signal, using a sun-compass for guidance, and moving relative to fluid-dynamic cues. Such abilities might require little more than directionally selective sensors coupled to a turning mechanism. This type of behavior, therefore, can be implemented by relatively simple circuits. In contrast, navigation involves both the ability to detect direction, as well as a map-sense that provides position. Navigation is less common and arguably requires greater brain computation than does simple orientation, but may be present in arthropods as well as in vertebrates. Great progress has been made in exploring the biophysical and sensory bases for these behaviors, and in recent years the locations and the identity of the cellular transducers of the sensory stimuli (for example, geomagnetic fields) have been narrowed in some taxa. Similarly, neurons within the central nervous that most likely function in higher order computational processes have been identified. For example, direction-selective and position-responsive brain cells have been located in the brains of mammals and birds, and these cells might contribute to a cognitive map that can enable navigation. One model organism in which orientation and navigation has been extensively studied is the sea slug Tritonia diomedea. This animal orients its crawling to chemical, hydrodynamic, and geomagnetic cues. The brain of Tritonia has ∼7000 relatively large neurons that facilitate circuit analysis. Recent work has characterized both peripheral and central neural correlates of orientation signals, as well as the control of thrust and turning, and studies of their field behavior have suggested how these disparate orientation systems may be integrated. These findings provide the basis for future studies to determine how the nervous system combines multiple sensory cues into a complex hierarchy of signals that can direct motor output and therefore guide navigational tasks.

Do chitons have a compass? Evidence for magnetic sensitivity in Polyplacophora

Several animals and microbes have been shown to be sensitive to magnetic fields, though the exact mechanisms of this ability remain unclear in many animals. Chitons are marine molluscs which have high levels of biomineralised magnetite coating their radulae. This discovery led to persistent anecdotal suggestions that they too may be able to navigationally respond to magnetic fields. Several researchers have attempted to test this, but to date there have been no large-scale controlled empirical trials. In the current study, four chiton species (Katharina tunicata, Mopalia kennerleyi, Mopalia muscosa and Leptochiton rugatus, n = 24 in each) were subjected to natural and artificially rotated magnetic fields while their movement through an arena was recorded over four hours. Field orientation did not influence the position of the chitons at the end of trials, possibly as a result of the primacy of other sensory cues (i.e. thigmotaxis). Under non-rotated magnetic field conditions, the orientation of subjects when they first reached the edge of an arena was clustered around 309–345° (north–north-west) in all four species. However, orientations were random under the rotated magnetic field, which may indicate a disruptive effect of field rotation. This pattern suggests that chitons can detect and respond to magnetism.

OPENING THE BLACK BOX

By Gunther K. H. Zupanc Oxford University Press (2004) 360pp. ISBN 0-19-870056-3 £23.99 (pbk) ![Figure][1] In Behavioral Neurobiology: An Integrative Approach , Gunther Zupanc provides an excellent introduction to the field of neuroethology, the study of the