Morphology and physiology of multiglomerular olfactory projection neurons in the spiny lobster

Whole-cell patch-clamp recording was used to characterize olfactory projection neurons in an isolated brain preparation of the spiny lobster, Panulirus argus. Responses to electrical stimulation of the olfactory afferents were recorded from projection neuron somata using biocytin-filled electrodes. All projection neurons were multiglomerular, innervating up to 80% of all olfactory lobe glomeruli, but the innervation was heterogeneous. Most neurons densely innervated only 3–4 glomeruli; the remaining glomeruli in their dendritic arbor were sparsely innervated, thereby creating two distinct patterns of intraglomerular branching. Projection neurons responded to orthodromic stimulation with an initial depolarization and spiking followed by a 1–3 s hyperpolarization. The inhibitory phase of the response was lower in threshold and longer in latency than the excitatory phase, a response pattern also reported in olfactory projection neurons of insects and vertebrates. The somata of the projection neurons supported voltage-activated currents and TTX-sensitive action potentials, suggesting that the soma, although spatially separated from the axon and dendrites, may play a significant functional role in these cells. Dye coupling between some projection neurons correlated with the presence of multiple amplitude action potentials, suggesting that at least some projection neurons may be coupled via gap junctions.     

Cooperative Ca2+ removal from presynaptic terminals of the spiny lobster neuromuscular junction

Stimulation-induced changes in presynaptic free calcium concentration ([Ca2+]i) were examined by fluorescent imaging at the spiny lobster excitor motor nerve terminals. The Ca2+ removal process in the terminal was analyzed based on a single compartment model, under the assumption that the Ca2+ removal rate from the terminal cytoplasm is proportional to nth power of [Ca2+]i. During 100 nerve stimuli at 10-100 Hz, [Ca2+]i reached a plateau that increased in a less-than-linear way with stimulation frequency, and the power index, n, was about 2. In the decay time course after stimulation, n changed with the number of stimuli from about 1.4 after 10 stimuli to about 2 after 100 stimuli. With the change of n from 1.4 to 2, the rate became larger at high [Ca2+]i (>1.5 microM), but was smaller at low [Ca2+]i (<1 microM). These results suggest that a cooperative Ca2+ removal mechanism of n = 2, such as mitochondria, may play an important role in the terminal. This view is supported by the gradual increase in the [Ca2+]i plateau during long-term stimulation at 20-50 Hz for 60 s and by the existence of a very slow [Ca2+]i recovery process after this stimulation, both of which may be due to accumulation of Ca2+ in the organelle.     

Excitatory synapses of blue crab gastric mill muscles

Summary Physiological and ultrastructural studies were made of neuromuscular synapses in stomach muscles, especially two gastric mill muscles of the blue crab innervated by neurons of the stomatogastric ganglion. These muscles depolarized and contracted with application of glutamate, but not acetylcholine, whereas the dorsal dilator muscles of the pyloric region depolarized and contracted in acetylcholine, but not in glutamate. Large excitatory postsynaptic potentials (EPSP's) of 5–20 mV were recorded in the gastric mill muscles. At low frequencies of activation, individual synapses released on average about 2 quanta of transmitter for each nerve impulse. Facilitation of EPSP's after a single nerve impulse could be detected for at least 10 s. Synapses were found on enlarged terminals of the motor axon; their contact areas ranged from 0.2 m² up to 3 m². Both electron-lucent, round synaptic vesicles and dense-cored vesicles occurred near these synapses. A possible correlation between contact area of a synapse and output of transmitter, is discussed.Supported by grants from the National Research Council of Canada and the Muscular Dystrophy Association of Canada to H.L. Atwood and C.K. Govind. We thank Kazuko Hay, Eva Yap-Chung and Irene Kwan for technical assistance with electron microscopy and reconstruction of nerve terminals from micrographs     

Coordination and neuromuscular control of rhythmic behaviors in the blue crab,Callinectes sapidus

The stereotypical courtship display (CD) behavior of the male blue crab, Callinectes sapidus, includes an unusual component: the rhythmic waving of the swimming appendages above the carapace. This behavior occurs in a unique context but it resembles two other rhythmic behaviors performed using the swimming legs: sideways swimming and backward swimming. As a first step to understanding the mechanisms that allow the expression of apparently different rhythmic motor patterns, we have examined these behaviors using slow motion video analysis and electromyography of the basal muscles of the swimming legs in freely behaving crabs. The results show that these behaviors are distinguished by four parameters: the frequency of leg waving, the phase relationship between the legs, the presence of a stationary pause in basal muscle activity combined with rotation of the distal leg during CD, and an extended range of motion of these legs during CD and backward swimming, relative to sideways swimming. EMG analysis revealed that during sideways swimming, the sequence of muscular activity between the two legs was different. In contrast, during CD and backward swimming the sequence of activity for these legs is identical.Abbreviations CD courtship display - EMGs electromyograms - CD AMP courtship display in crabs with amputated fifth legs - CD1 crabs that voluntarily used one leg to perform courtship display waving - CD 1–3 courtship waving in cycles 1–3 - CD MID courtship waving after cycles 1–3 - M-C meral-carpal joint     

Anatomy and physiology of the swimming leg musculature in the blue crab,Callinectes sapidus

1) The anatomy and neurophysiology of muscles moving the fifth leg of Callinectes sapidus are described. Innervation of the muscles was studied using intracellular stimulating and recording techniques.

2) The movement of the leg is controlled by four sets of muscles composed of two, three, or four bundles each. The bundles which make up each muscle shared innervation with other muscle bundles within a functional muscle group. Muscle fibers characteristically displayed both “fast” and “slow” responses to stimulation.

3) Possible functional evolution of the neural changes which occurred with the morphological adaptations for swimming in C. sapidus is discussed.     

Cold-resistant changes in heartbeat of the Japanese spiny lobster

Heartbeat in Panulirus japonicus acclimated to 20°C is often augmented during cooling to 15^oC. Augmented contractions of the heart coincided with increasing amplitude of electrocardiogram. In cold saline, a pericardial hormone serotonin (10⁻⁷ M) increased both the amplitude and duration of the heartbeat while another hormone octopamine (10⁻⁶ M) slightly relieved the cold depression of heart rate despite a smaller increase in beat amplitude. In contrast, the application of the cold saline containing F1 (a FMRFamide-related peptide of pericardial hormones, 10⁻⁹ M) maintained the rate and amplitude of the heartbeat around the control level during cold exposure. This suggests that in the presence of F1, the lobster heart becomes cold resistant clearly. We previously reported that the pericardial organs of spiny lobsters are activated by a small fall in body temperature. The ligamental nerves, extensions of the pericardial organs, terminate in the heart beside the ostia and their ends remain in the isolated hearts. Therefore, the ligamental nerve ends might release their hormones into the ventricle with the fall in temperature even in the isolated hearts.     

An intrinsic distinction in neuromuscular junction assembly and maintenance in different skeletal muscles

We analyzed the formation of neuromuscular junctions (NMJs) in individual muscles of the mouse embryo. Skeletal muscles can be assigned to one of two distinct classes of muscles, termed "Fast Synapsing" (FaSyn) and "Delayed Synapsing" (DeSyn) muscles, which differ significantly with respect to the initial focal clustering of postsynaptic AChRs, the timing of presynaptic maturation, and the maintenance of NMJs in young adult mice. Differences between classes were intrinsic to the muscles and manifested in the absence of innervation or agrin. Paralysis or denervation of young adult muscles resulted in disassembly of AChR clusters on DeSyn muscles, whereas those on FaSyn muscles were preserved. Our results show that postsynaptic differentiation processes intrinsic to FaSyn and DeSyn muscles influence the formation of NMJs during development and their maintenance in the adult.     

Mouthpart and foregut ontogeny in larval, postlarval, and juvenile spiny lobster, Panulirus argus Latreille (Decapoda, Palinuridae)

The spiny lobster Panulirus argus has a life cycle consisting of a long-term (～9-12 months) planktonic larval period with 11 larval stages (the phyllosoma), a short (<1 month?) planktonic-to-benthic transitional postlarval stage (the puerulus), and benthic juvenile and adult phases. The mouthparts and foregut during these stages were examined and described by means of scanning electron microscopy (SEM) in an investigation of the species' developmental morphology, diet, and ecology. The phyllosoma mouthparts close to the esophagus are the labrum, mandibles, paragnaths, and first maxillae. The second maxillae and first and second maxillipeds are increasingly distant from the esophagus as the larva develops. The pair of asymmetrical mandibles bear many teeth and spines, and the molar processes form what appears to be an intricate toothed shear. The mandibles remain similar throughout the phyllosoma stages. During the molt into the puerulus, the mouthparts are greatly changed, and the second maxilla and the three maxillipeds join the other mouthparts near the esophagus. However, the transformation appears incomplete, and many of the mouthparts are not fully formed until the molt to juvenile completes their development. The phyllosoma foregut lacks a gastric mill and has but one chamber. In addition, the first two stages lack a gland filter. During the molt to puerulus, the foregut is greatly changed and subsequently is similar to typical decapod foreguts in having an anterior cardiac and posterior pyloric chamber. Only rudimentary internal armature is present. Following the molt to juvenile, the foregut is quite similar to that of the adult, which exhibits a substantial gastric mill. The 11 phyllosoma stages were separated into two groups (group A = stages 1-5, group B = stages 6-11) on the basis of changes in both mouthpart and foregut morphology. The puerulus has never been observed to feed. Nothing was observed in our investigations that would prevent feeding, though both mouthpart and foregut development appeared incomplete. The mouthpart and foregut structures of larval, postlarval and juvenile P. argus differ widely, possibly reflecting the extreme modifications for different habitats found among these life phases.     

Organization of the stomatogastric ganglion of the spiny lobster

Summary The Stomatogastric ganglion ofPanulirus interruptus contains about 30 neurons, and controls the movements of the lobster's stomach. When experimentally isolated, the ganglion continues to generate complex rhythmic patterns of activity in its motor neurons which are similar to those seen in intact animals.In this paper, we describe the synaptic organization of a group of six neurons which drive the stomach's lateral teeth (Figs. 2, 6). This group includes four motor neurons and two interneurons, all but one of which were recorded and stimulated with intracellular microelectrodes.One pair of synergistic motor neurons, LGN and MGN, are electrotonically coupled and reciprocally inhibitory (Figs. 9, 12). A second pair of synergistic motor neurons, the LPGNs, are antagonists of LGN and MGN. The LPGNs are electrotonically coupled (Fig. 14), and are both inhibited by LGN and MGN (Figs. 8, 11). The LPGNs inhibit MGN (Fig. 15) but not LGN. One of the two interneurons in the ganglion, Int 1, reciprocally inhibits both LGN and MGN (Figs. 10, 13). The other interneuron, Int 2, excites Int 1 and inhibits the LPGNs (Fig. 16). The synaptic connections observed in the ganglion are reflected in the spontaneous activity recorded from the isolated ganglion and from intact animals.From the known synaptic organization and observations on the physiological properties of each of the neurons, we have formulated some hypotheses about the pattern-generating mechanism. We found no evidence that any of the neurons are endogenous bursters.We thank D. Kennedy, Eve Marder, and D. Russell for criticizing early drafts of these papers, Nina Pollack and Betty Jorgensen for expert technical assistance, Diane Newsome, SanDee Newcomb, and Pattie Macpherson for typing the many drafts. The authors' research is supported by grant number NS-09322 from N.I.H. and by the Alfred P. Sloan Foundation. B. M. is an NINDS-NIH postdoctoral fellow.     

Metabolic characteristics of muscles in the spiny lobster, Jasus edwardsii, and responses to emersion during simulated live transport

The metabolic characteristics of five muscle groups in the spiny lobster Jasus edwardsii were examined in order to compare their anaerobic and oxidative capacities. Enzyme activities of phosphorylase, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase were highest in abdominal muscles supporting anaerobic burst activity. Hexokinase, citrate synthase, and HOAD activities in the leg and antennal muscles indicated higher aerobic potential. Arginine kinase activities were high in all muscle groups indicating that muscle phosphagens are an important energy reserve. Arginine phosphate concentrations in 4th periopod and abdominal flexor muscle from lobsters sampled in the field were higher than any values from captive animals, and approximately five times those for ATP. Muscle lactates were high in captive animals. Responses to emersion during simulated live transport appear to exploit the capacity for functional anaerobiosis and further differentiated the muscle groups. Abdominal muscles were especially sensitive and after 24 h showed significant increases in lactate, glucose, ADP, and AMP. ATP levels appeared to be maintained by muscle phosphagens and raised doubts about the efficacy of the adenylate energy charge in evaluating the emersion response. Haemolymph glucose, lactic acid, and ammonia peaked after 24 h emersion and were largely restored following re-immersion. We propose that arginine phosphate concentrations in the 4th periopod are an appropriate index of metabolic stress, and could lead to improved commercial handling protocols.     

Organization of the stomatogastric ganglion of the spiny lobster

Summary Three direct synaptic connections occur between neurons in the gastric and pyloric systems of the stomatogastric ganglion ofPanulirus interruptus. Two synapses are inhibitory, and one is electrical. This electrical synapse is both excitatory and inhibitory at different times. These synapses, and others within each system, let the two systems interact under some conditions. The synapses also form multisynaptic pathways which modulate the firing of many neurons in both systems. The consequences of these multisynaptic pathways are described and discussed.I thank Allen I. Selverston, Karen Sigvardt, Eve Marder, David Russell and Mary Chamberlin for criticizing a draft of this paper, Forrest Gompf and Doug Tissdale for technical support, and Nina Pollack and Betty Jorgensen for laboratory assistance. The research was supported by USPHS grant NS-12295 to BM and USPHS grant NS-09322 to Alien I. Selverston. BM was a USPHS NIH Postdoctoral Fellow in A.I. Selverston's laboratory during part of this research and is now a Research Fellow of the Alfred P. Sloan Foundation.     

Threshold determination for olfactory receptors of the spiny lobster

The shallow water caridean shrimps Heptacarpus pictus and H. paludicola are polymorphic in colour pattern. Populations of these species collected over colour‐variable substrates showed the greatest degree of coloration in terms of the proportion of individuals displaying a distinct colour pattern. The frequencies of H. Pictus colour morphs varied significantly between most sampling periods. Apostatic selection by fish predators is suggested as one hypothesis which could explain these changes in morph frequency.

Experiments with Heptacarpus pictus on rapid colour change showed that, although some pigment migration did occur in the chromatosomes studied, the macroscopic appearance of the colour patterns was not altered when shrimps were shifted from black to white backgrounds or vice versa. The results of background choice experiments with H. pictus suggest that these shrimp do not seek out colour backgrounds that would seem to be a matching background in a concealing coloration. This behaviour is consistent with the morphology of the colour patterns which appears to be a disruptive coloration rather than a concealing coloration that closely matches a particular substrate.     

Phenoloxidase activity in the hemolymph of the spiny lobster Panulirus argus

The prophenoloxidase activating system plays a major role in the defense mechanism of arthropods. In the present study, the phenoloxidase activity and its location in the hemolymph of the spiny lobster Panulirus argus is presented. Phenoloxidase activity was observed in the hemocyte lysate supernatant (HLS) and plasma after their incubation with trypsin. Higher amounts of trypsin were required to activate the HLS prophenoloxidase, due to the presence of a trypsin inhibitor in this fraction. Activation of prophenoloxidase was found when HLS was incubated with calcium, with an optimal pH between 7.5 and 8. This spontaneous activity is due to the prophenoloxidase activating enzyme, a serine proteinase that activates the prophenoloxidase once calcium ions were available. SDS was able to induce phenoloxidase activity in plasma and hemocyte fractions. Prophenoloxidase from HLS occurs as an aggregate of 300kDa. Electrophoretic studies combining SDS-PAGE and native PAGE indicate that different proteins produced the phenoloxidase activity found in HLS and plasma. Thus, as in most crustaceans, Panulirus argus contains a prophenoloxidase activating system in its hemocyte, comprising at least the prophenoloxidase activating enzyme and the prophenoloxidase. Finally, it is suggested that phenoloxidase activity found in plasma is produced by hemocyanin.     

Sensory physiology and behavior of blue crab (Callinectes sapidus) postlarvae during horizontaltransport

Adult blue crabs (Callinectes sapidus) live in estuaries and release larvae near the entrances to estuaries. Larvae are then transported offshore to continental shelf areas where they undergo development. Postlarvae, or megalopae, remain near the surface and undergo reverse diel vertical migration. The behaviors underlying this migration pattern are responses to light and a solar day rhythm in activity, in which megalopae are active during the day and inactive at night. Onshore transport probably occurs by wind‐generated surface currents. Once in the vicinity of an estuary, megalopae move up the estuary by selective tidal stream transport, in which they swim in the water column on rising tides at night and are on or near the bottom at all other times. Light inhibits swimming during the day. The ascent into the water column on nocturnal rising tides does not result from a biological rhythm in activity, but rather is cued by the rate of increase in salinity during rising tides. Megalopae have separatebehavioural responses in coastal/shelf areas and in estuaries, which are induced by chemical cues in offshore and estuarine waters.