Central pattern generator for swimming in Melibe

The nudibranch mollusc Melibe leonina swims by bending from side to side. We have identified a network of neurons that appears to constitute the central pattern generator (CPG) for this locomotor behavior, one of only a few such networks to be described in cellular detail. The network consists of two pairs of interneurons, termed `swim interneuron 1\u27 (sint1) and `swim interneuron 2\u27 (sint2), arranged around a plane of bilateral symmetry. Interneurons on one side of the brain, which includes the paired cerebral, pleural and pedal ganglia, coordinate bending movements toward the same side and communicate via non-rectifying electrical synapses. Interneurons on opposite sides of the brain coordinate antagonistic movements and communicate over mutually inhibitory synaptic pathways. Several criteria were used to identify members of the swim CPG, the most important being the ability to shift the phase of swimming behavior in a quantitative fashion by briefly altering the firing pattern of an individual neuron. Strong depolarization of any of the interneurons produces an ipsilateral swimming movement during which the several components of the motor act occur in sequence. Strong hyperpolarization causes swimming to stop and leaves the animal contracted to the opposite side for the duration of the hyperpolarization. The four swim interneurons make appropriate synaptic connections with motoneurons, exciting synergists and inhibiting antagonists. Finally, these are the only neurons that were found to have this set of properties in spite of concerted efforts to sample widely in the Melibe CNS. This led us to conclude that these four cells constitute the CPG for swimming. While sint1 and sint2 work together during swimming, they play different roles in the generation of other behaviors. Sint1 is normally silent when the animal is crawling on a surface but it depolarizes and begins to fire in strong bursts once the foot is dislodged and the animal begins to swim. Sint2 also fires in bursts during swimming, but it is not silent in non-swimming animals. Instead activity in sint2 is correlated with turning movements as the animal crawls on a surface. This suggests that the Melibe motor system is organized in a hierarchy and that the alternating movements characteristic of swimming emerge when activity in sint1 and sint2 is bound together

The Ability of Horseshoe Crabs (Limulus polyphemus) To Detect Changes in Temperature

Previous studies have suggested that horseshoe crabs prefer warm water, suggesting that they may be able to detect changes in water temperature. The overall goal of this study was to test this hypothesis. Our specific objectives were to: 1) find out if horseshoe crabs can detect temperature changes; 2) determine the magnitude of temperature change they can detect, and; 3) determine whether their temperature receptors are located internally or externally. Animals were placed in a light-tight chamber that received a constant flow of cooled seawater. Their heart rates were continuously recorded and a change in heart rate following the addition of warmer water was used as an indicator that they sensed the change in temperature. The results showed that 50% of horseshoe crabs responded to a temperature change of 1°C, while 100% responded to a temperature change of 2.6°C. Over half of the horseshoe crabs also responded to a rate of temperature change of less than 1.5°C. Both of these results indicate that horseshoe crabs can, indeed, sense temperature changes. Also, the horseshoe crabs typically showed a response before their internal temperature changed, indicating that their temperature receptors are most likely located externally

Thermosensitivity of the lobster, Homarus americanus, as determined by cardiac assay

It is generally accepted that crustaceans detect, and respond to, changes in water temperature, yet few studies have directly addressed their thermosensitivity. In this investigation a cardiac assay was used as an indicator that lobsters (Homarus americanus) sensed a change in temperature. The typical cardiac response of lobsters to a 1-min application of a thermal stimulus, either warmer (n = 19) or colder (n = 17) than the holding temperature of 15 degrees C, consisted of a short bradycardia (39.5 +/- 8.0 s) followed by a prolonged tachycardia (188.2 +/- 10.7 s). Lobsters exposed to a range of rates of temperature change (0.7, 1.4, 2.6, 5.0 degrees C/min) responded in a dose-dependent manner, with fewer lobsters responding at slower rates of temperature change. The location of temperature receptors could not be determined, but lesioning of the cardioregulatory nerves eliminated the cardiac response. Although the absolute detection threshold is not known, it is conservatively estimated that lobsters can detect temperature changes of greater than 1 degree C, and probably as small as 0.15 degrees C. A comparison of winter and summer lobsters, both held at 15 degrees C for more than 4 weeks, revealed that although their responses to temperature changes were similar, winter lobsters (n = 18) had a significantly lower baseline heart rate (34.8 +/- 4.4 bpm) and a shorter duration cardiac response (174 s) than summer lobsters (n = 18; 49.9 +/- 5.0 bpm, and 320 s respectively). This suggests that some temperature-independent seasonal modulation of cardiac activity may be occurring

Modulation of swimming in the gastropod Melibe leonina by nitric oxide

Nitric oxide (NO) is a gaseous intercellular messenger produced by the enzyme nitric oxide synthase. It has been implicated as a neuromodulator in several groups of animals, including gastropods, crustaceans and mammals. In this study, we investigated the effects of NO on the swim motor program produced by isolated brains and by semi-intact preparations of the nudibranch Melibe leonina. The NO donors sodium nitroprusside (SNP, 1 mmol l–1) and S-nitroso-N-acetylpenicillamine (SNAP, 1 mmol l–1) both had a marked effect on the swim motor program expressed in isolated brains, causing an increase in the period of the swim cycle and a more erratic swim rhythm. In semi-intact preparations, the effect of NO donors was manifested as a significant decrease in the rate of actual swimming. An NO scavenger, reduced oxyhemoglobin, eliminated the effects of NO donors on isolated brains, supporting the assumption that the changes in swimming induced by donors were actually due to NO. The cGMP analogue 8-bromoguanosine 3′,5′-cyclic monophosphate (1 mmol l–1) produced effects that mimicked those of NO donors, suggesting that NO is working via a cGMP-dependent mechanism. These results, in combination with previous histological studies indicating the endogenous presence of nitric oxide synthase, suggest that NO is used in the central nervous system of Melibe leonina to modulate swimming

Skewed sex ratio in an estuarine lobster (Homarus americanus) population

A total of 19,485 lobsters were caught sites in the estuarine and coastal waters of New Hampshire from 1989 to 1992, and their size and sex were determined. The sex ratio of lobsters caught farthest from the coast, in Great Bay, was heavily skewed in favor in males. Sex ratios in other estuarine and river sites were also skewed toward males, and there was a tendency for the number of males per female to decline as one moved down the estuary toward the coast, where the sex ratio was nearly 1:1. The single offshore site was dominated by females, with about 0.6 males for each female. There were also seasonal trends in the sex ratios in the upper estuarine sties, where the number of males per female tended to decline from summer through autumn. In general, differences in the sex ratios between sites were those of primarily adult lobsters larger than 80 mm carapace length (CL). At all sites, the sex ratio of lobsters smaller than this size was close to 1:1, whereas in the upper estuary the mean sex ratio of lobsters greater than 80 mm CL was more than 14:1. These data, in conjunction with seasonal variations of sex ratios, suggest that differential movements of adult male and female lobsters is the primary cause of skewed sex ratios in the Great Bay Estuary

Gills as Possible Accessory Circulatory Pumps in Limulus polyphemus

Heart electrical activity (ECGs), gill closer muscle potentials (EMGs), and blood pressures in the heart and the branchiocardiac canals, were measured in adult horseshoe crabs (Limulus polyphemus) during various activities. During ventilation, hyperventilation, and swimming, large transient increases in pressures (10-35 cm H2O) occur in the branchiocardiac canals, which carry blood from the gills to the heart. These pulses of positive pressure are related to, and apparently caused by, gill plate closing. During quiescent periods, with no ventilatory activity, there are no pressure pulses in the canals, but the pressure is still greater than zero. We found covariation of heart and ventilation rates during intermittent ventilation, hyperventilation, gill cleaning, and swimming, as well as evidence of transient periods of phasic coordination. The heart appears to be weakly entrained to the gill rhythm by phasic cardioregulatory nerve input. The preferred phase of heartbeats, with respect to gill rhythm, was 0.5, or 180 degrees out of phase. In some animals, intra-cardiac pressures were enhanced when the heart and gill rhythms were entrained. We suggest that rhythmic movements of the gill plates enhance the flow of low pressure blood returning from the body to the heart. Thus, ventilatory appendage movements may constitute an accessory blood pumping mechanism in Limulus

Second messenger systems underlying amine and peptide actions on cardiac muscle in the horseshoe crab, Limulus polyphemus

The biochemical mechanisms by which octopamine, catecholamines and the peptide proctolin exert their actions on Limulus cardiac muscle were investigated. Amines produced long-lasting increases in the amplitude of contractions evoked by electrical stimulation. At 10(−5) mol l-1, the apparent order of potency for amine-induced increases in evoked contraction amplitude was dopamine approximately equal to octopamine greater than norepinephrine approximately equal to epinephrine. At this dose, amines produced long-lasting increases in the levels of cyclic AMP (octopamine greater than dopamine approximately equal to norepinephrine approximately equal to epinephrine), but not of cyclic GMP, in Limulus cardiac muscle. Like the amines, the adenylate cyclase activator forskolin enhanced cardiac muscle contractility and increased levels of cyclic AMP, but not of cyclic GMP. The phosphodiesterase inhibitor IBMX produced a transient increase in cardiac muscle contractility, but typically produced long-lasting negative inotropy. This agent increased levels of both cyclic AMP and cyclic GMP in Limulus cardiac muscle. Proctolin and the protein kinase C activator phorbol dB increased the contraction amplitude of the intact heart and the electrically stimulated myocardium. These compounds, as well as dopamine, elicited sustained contractures and rhythmic contractions when applied to deganglionated Limulus cardiac muscle rings. Unlike the amines, proctolin and phorbol dB did not increase cardiac muscle cyclic AMP levels. These results suggest that several second-messenger systems may be utilized by amines and peptides to produce excitatory actions on cardiac muscle fibers of the Limulus heart. Cyclic AMP appears to be an important second messenger underlying the effects of amines to enhance cardiac muscle contractility. Pharmacological data suggest that proctolin may alter cardiac muscle contractility and excitability by a mechanism which involves the phosphatidylinositol pathway. Dopamine, unlike the other amines, produces a number of proctolin-like effects and may activate both the cyclic AMP and the phosphatidylinositol systems in Limulus cardiac muscle

Influence of Natural Inshore and Offshore Thermal Regimes on Egg Development and Time of Hatch in American lobsters, Homarus americanus

Some egg-bearing (ovigerous) American lobsters (Homarus americanus) make seasonal inshore-to-offshore movements, subjecting their eggs to different thermal regimes than those of eggs carried by lobsters that do not make these movements. Our goal was to determine if differences in thermal regimes influence the rate of egg development and the subsequent time of hatch. We subjected ovigerous lobsters to typical inshore or offshore water temperatures from September to August in the laboratory (n = 8 inshore and 8 offshore, each year) and in the field (n = 8 each, inshore and offshore), over 2 successive years. Although the rate of egg development did not differ significantly between treatments in the fall (P ∼ 0.570), eggs exposed to inshore thermal regimes developed faster in the spring (P \u3c 0.001). “Inshore” eggs hatched about 30 days earlier (mean = 26 June) than “offshore” eggs (mean = 27 July), and their time of development from the onset of eyespot to hatch was significantly shorter (inshore = 287 ± 11 days vs. offshore: 311.5 ± 7.5 days, P = 0.034). Associated growing degree-days (GDD) did not differ significantly between inshore and offshore thermal treatments (P = 0.061). However, eggs retained by lobsters exposed to offshore thermal regimes accumulated more GDD in the winter than did eggs carried by inshore lobsters, while eggs exposed to inshore temperatures acquired them more rapidly in the spring. Results suggest that seasonal movements of ovigerous lobsters influence the time and location of hatching, and thus the transport and recruitment of larvae to coastal and offshore locations

Swimming Behavior of the Nudibranch Melibe leonina

Swimming in the nudibranch Melibe leonina consists of five types of movements that occur in the following sequence: (1) withdrawal, (2) lateral flattening, (3) a series of lateral flexions, (4) unrolling and swinging, and (5) termination. Melibe swims spontaneously, as well as in response to different types of aversive stimuli. In this study, swimming was elicited by contact with the tube feet of the predatory sea star Pycnopodia helianthoides, pinching with forceps, or application of a 1 M KCl solution. During an episode of swimming, the duration of swim cycles (2.7 ± 0.2 s [mean ± SEM], n = 29) and the amplitude of lateral flexions remained relatively constant. However, the latency between the application of a stimulus and initiation of swimming was more variable, as was the duration of an episode of swimming. For example, when touched with a single tube foot from a sea star (n = 32), the latency to swim was 7.0 ± 2.4 s, and swimming continued for 53.7 ± 9.4 s, whereas application of KCl resulted in a longer latency to swim (22.3 ± 4.5 s) and more prolonged swimming episodes (174.9 ± 32.1 s). Swimming individuals tended to move in a direction perpendicular to the long axis of the foot, which propelled them laterally when they were oriented with the oral hood toward the surface of the water. The results of this study indicate that swimming in Melibe, like that in several other molluscs, is a stereotyped fixed action pattern that can be reliably elicited in the laboratory. These characteristics, along with the large identifiable neurons typical of many molluscs, make swimming in this nudibranch amenable to neuroethological analyses

Involvement of cyclic AMP in multiple, excitatory actions of biogenic amines on the cardiac ganglion of the horseshoe crab Limulus polyphemus

Cyclic AMP appears to be involved in several excitatory actions of amines on neurones of the Limulus cardiac ganglion. Amines selectively increase levels of cardiac ganglion cyclic AMP with a magnitude and time course similar to that observed for amine-induced excitation of cardiac ganglion burst rate. With respect to either the physiological or biochemical effect, the apparent order of potency is octoparnine\u3eepinephrine==dopamine\u3enorepinephrine. Elevation of cardiac ganglion cyclic AMP levels by octopamine or dopamine is dose-dependent and is potentiated by the phosphodiesterase inhibitor 3-isobutyl 1-methylxanthine (IBMX). Several pharmacological agents which influence cyclic nucleotide metabolism, including forskolin, IBMX and 8-substituted cyclic AMP analogues, have amine-like effects on the Limulus cardiac ganglion. These effects include increased burst rate of the isolated cardiac ganglion and decreased burst duration, interburst interval and number of spikes per burst in follower neurones. Forskolin and IBMX increase levels of cardiac ganglion cyclic AMP, and IBMX also increases cyclic GMP levels in this tissue. Amines, forskolin and IBMX have direct effects on follower neurones pharmacologically isolated from pacemaker cell input. Octopamine, forskolin and IBMX depolarize follower neurones, while dopamine hyperpolarizes these cells. Amines, forskolin and IBMX elicit burst-like potentials in follower neurones, and increase the size of evoked, unitary junction potentials recorded in cardiac muscle fibres. These pharmacological and biochemical data suggest that multiple, excitatory effects of biogenic amines on the Limulus cardiac ganglion are mediated by simultaneous increases in cyclic AMP at several loci within this neural network