Surface Charge of Giant Axons of Squid and Lobster

A method is described for the determination of the electrophoretic mobility of single, isolated, intact, giant axons of squid and lobster. In normal physiological solutions, the surface of hydrodynamic shear of these axons is negatively charged. The lower limit of the estimated surface charge density is -1.9 × 10^-8 coul cm^-2 for squid axons, -4.2 × 10^-8 coul cm^-2 for lobster axons. The electrophoretic mobility of squid axons decreases greatly when the applied transaxial electric field is made sufficiently intense; action potential propagation is blocked irreversibly by transaxial electric fields of the same intensity. The squid axon recovers its mobility hours later and is then less affected by transaxial fields. Eventually, a state is reached in which the transaxial field irreversibly reverses the sign of the surface charge. In contrast, there is no obvious effect of electric field on the mobility of lobster axons. The mobility of lobster axons becomes undetectable in the presence of Th⁴⁺ at a concentration which blocks the action potential, and in the presence of La³⁺ at a concentration which does not affect propagation. Quinine does not alter lobster axon mobility at a concentration which blocks action potential conduction. Replacement of extracellular Na⁺ by K⁺ is without effect upon lobster axon mobility. The electrophysiological implications of the results are discussed.     

Effects of external ions on membrane potentials of a crayfish giant axon

Transmembrane potentials in the crayfish giant axon have been investigated as a function of the concentration of normally occurring external cations. Results have been compared with data already available for the lobster and squid giant axons. The magnitude of the action potential was shown to be a linear function of the log of the external sodium concentration, as would be predicted for an ideal sodium electrode. The resting potential is an inverse function of the external potassium concentration, but behaves as an ideal potassium electrode only at the higher external concentrations of potassium. Decrease in external calcium results in a decrease in both resting potential and action potential; an increase in external calcium above normal has no effect on magnitude of transmembrane potentials. Magnesium can partially substitute for calcium in the maintenance of normal action potential magnitude, but appears to have very little effect on resting potential. All ionic effects studied are completely reversible. The results are in generally good agreement with data presently available for the lobster giant axon and for the squid giant axon.     

Comparative study of the lipid composition of rabbit and lobster sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) membranes isolated from rabbit and lobster muscles have similar phospholipid classes, but they differ in plasmalogen content. The plasmalogenic species are mostly distributed among phosphatidylethanolamines (PE's) and make up about 62% of the total in rabbit SR and about 46% in lobster membranes. Lobster SR phospholipids contain large amounts of polyunsaturated fatty acids which are present in low amounts in rabbit membranes. The total unsaturated fatty acids of phosphatidylcholines (PC's) represent about 53% and 73% of the total fatty chains for rabbit and lobster SR, respectively. The values found for PE's were about 56% and 64%, respectively. Furthermore, lobster membranes contain significant amounts of PC and PE molecular species with unsaturated fatty acids in positions 1 and 2, whereas rabbit SR contain low amounts.     

A minimal biophysical model for an excitable and oscillatory neuron

Presented here is a minimal biophysical cell model, based on work by Hodgkin and Huxley and by Rinzel, that can exhibit both excitable and oscillatory behavior. Two versions of the model are studied, which conform to data for squid and lobster giant axons.     

Effects of calcium lack on action potential of motor axons of the lobster limb

The effects of external calcium deprivation on certain characteristics of the action potential of the lobster motor axon have been studied. Upon exposure to calcium-free solution the spike amplitude is rapidly decreased within a few minutes and is followed by a slow linear decline. The rates of spike rise and fall are proportionally reduced more than the spike but follow similar time courses during calcium lack. Associated with these phenomena are the loss in the normal slow spike repolarization process, the development of a large and lengthy undershoot, and the appearance of a high degree of refractoriness. The mean increase in the refractory period is 525 per cent upon 10 minutes' exposure to calcium-free solution. These effects are completely reversible upon returning the axons to normal solution. These results are compared to similar effects of calcium deprivation on frog myelinated axons and squid and lobster giant axons recently observed by other workers.     

The metabolism of gamma aminobutyric acid in the lobster nervous system. Enzymes in single excitatory and inhibitory axons

γ-aminobutyric acid (GABA) is the inhibitory transmitter compound at the lobster neuromuscular junction. This paper presents a comparison of the enzymes of GABA metabolism in single identified inhibitory and excitatory axons from lobster walking legs. Inhibitory axons contain more than 100 times as much glutamic decarboxylase activity as do excitatory axons. GABA-glutamic transaminase is found in both excitatory and inhibitory axons, but about 50% more enzyme is present in inhibitory axons. The kinetic and electrophoretic behavior of the transaminase activity in excitatory and inhibitory axons is similar. Succinic semialdehyde dehydrogenase is found in both axon types, as is an unknown enzyme which converts a contaminant in radioactive glutamic acid to GABA. In lobster inhibitory neurons, therefore, the ability to accumulate GABA ultimately rests on the ability of the neuron to accumulate the enzyme glutamic decarboxylase.     

Axon voltage-clamp simulations. II. Double sucrose-gap method.

This is the second in a series of four papers on the simulation of the voltage clamp of cylindrical excitable cells. In this paper we evaluate the double sucrose-gap voltage-clamp technique for the squid and lobster giant axons. Using the Crank-Nicolson method of solution of the cable equations and differential equations representing the voltage clamp circuit we studied the effect of length of the sucrose gap "node" on the voltage profile along an excitable cell during a simulated voltage clamp. The voltage gradients along the region of the cell within the node produce "notches" in the current recording as well as changes in the magnitude of the sodium and potassium current for a given voltage step. Our results show that good voltage clamp control requires node lengths less than one-half the axon diameter.     

Effects of narcotics on the giant axon of the squid

—Levorphanol (10^-3 M) reversibly blocked conduction in the giant axon of the squid and axons from the walking legs of spider crab and lobster. Similar concentrations of levallorphan and dextrorphan blocked conduction in the squid giant axon. Under the same experimental condition morphine caused an approximately 40 per cent decrease in spike height. Levorphanol did not affect the resting potential or resistance of the squid axon. Spermidine, spermine and dinitrophenol had little or no direct effect on the action potential nor did they alter the potency of levorphanol. Concentrations of levorphanol as low as 5 × 10^-5 M blocked repetitive or spontaneous activity in the squid axon induced by decreasing the divalent cations in the medium. After exposure to tritiated levorphanol, the axoplasm and envelope of the squid axon accumulated up to 500 per cent of the concentration of tritium found in the external medium, dependent on time of exposure, and other variables. At pH 6 the levels of penetration were 33-50% of those found at pH 8, which correlates with our observation that levorphanol is about 33 % as potent in blocking the action potential at pH 6. The penetrability of levorphanol was not affected by spermidine, dinitrophenol or cottonmouth moccasin venom. Levorphanol did not alter the penetration of [C¹⁴]acetylcholine nor did it render the squid axon sensitive to it. The block of axonal conduction by compounds of the morphine series is discussed both as to possible mechanisms and significance.     

Tetracycline fluorescence as calcium-probe for nerve membrane with some model studies using erythrocyte ghosts

Summary The tetracycline dyes, particularly chlorotetracycline, have been employed as probes of membrane-associated calcium during the excitation process of nerve. Both squid giant axons, stained internally, and lobster nerves, stained externally, show a small increase in fluorescent light during the action potential. Increasing the calcium concentration bathing a lobster nerve leads to a larger optical signal. Adding fluoride ion to the inside of a squid axon, which might be expected to influence the internal calcium-ion concentration, also leads to a larger optical signal. Squid axons have been studied under conditions of voltage clamp and the hyperpolarizing response. Model studies were done with erythrocyte ghosts to clarify the influence of membranes and calcium on the fluores-cence of the tetracyclines. Chlorotetracycline may be monitoring calcium concentration associated with the inner surface of the nerve membrane.     

The Ionic Mechanisms of Hyperpolarizing Responses in Lobster Muscle Fibers

Lobster muscle fibers develop hyperpolarizing responses when subjected to sufficiently strong hyperpolarizing currents. In contrast to axons of frog, toad, and squid, the muscle fibers produce their responses without the need for prior depolarization in high external K⁺. Responses begin at a threshold polarization (50 to 70 mv), the potential reaching 150 to 200 mv hyperpolarization while the current remains constant. The increased polarization develops at first slowly, then becomes rapid. It usually subsides from its peak spontaneously, falling temporarily to a potential less hyperpolarized than at threshold for the response. As long as current is applied there can be oscillatory behavior with sequential rise and subsidence of the polarization, repeating a number of times. Withdrawal of current leads to rapid return of the potential to the resting level and a small, brief depolarization. Associated with the latter, but of longer duration, is an increased conductance whose magnitude and duration increase with the antecedent current. Hyperpolarizing responses of lobster muscle fibers are due to increased membrane resistance caused by hyperpolarizing K inactivation. The oscillatory characteristic of the response is due to a delayed superimposed and prolonged increase in membrane permeability, probably for Na⁺ and for either K⁺ or Cl^-. The hyperpolarizing responses of other tissues also appear to result from hyperpolarizing K inactivation, on which is superimposed an increased conductance for some other ion or ions.     

The molecular organization of nerve membranes

Summary Plasma membranes were isolated from two types of squid nerves which have morphologically, different ratios of axolemma/Schwannlemma (A/S). These membranes were studied by means of differential and density gradient centrifugation.Thoroughly dissected giant axons were used as membrane source having low A/S ratio. Retinal fibers were used as membrane source with high A/S ratio. A similar procedure for the isolation of the plasma membranes was used for both types of squid axons.Differential centrifugation showed that at 1,500×g, the yield of membrane enzymes (Na, K-ATPase and NADH-ferricyanide oxidoreductase) from giant axon homogenates was 2 to 5 times greater than from retinal nerve homogenates, but at 105,000×g the opposite was the case, the yield from retinal axons being about two times greater. Thus, the major part of the membrane material from the retinal nerve seems to be less dense than the membrane material from giant axons.The behavior of the 105,000×g fraction from both types of fibers was studied by determining protein Na, K-ATPase, and NADH-oxidoreductase along a lineal sucrose gradient (10 to 40%; centrifuged at 40,600×g for 90 min). By any of the three measurements, retinal axons yielded a greater amount (2:1) of plasma membranes sedimenting at low sucrose concentration (16 to 25%) as compared to that observed at high sucrose concentration (35 to 38%). Giant axons, on the contrary, yielded a higher proportion of membranes (2.5:1) sedimenting at high sucrose concentrations (over 40%).The experimental data indicate that a different cellular origin can account for the behavior of nerve membranes along lineal gradient centrifugation. The membranes floating at low sucrose concentration (light membranes) can be tentatively ascribed to the axolemma; the membranes found at high sucrose concentration (heavy membranes) to the Schwannlemma and basement membranes.In accord with their high A/S morphological ratio, squid retinal axons yielded 5 times more light membranes (axolemma) than dissected giant axons.     

Regulatory proteins of lobster striated muscle.

The regulatory proteins of lobster muscles consist of tropomyosin and of troponin. Troponin contains a 17,000 chain weight component, two closely related components of about 30,000 and a 52,000 chain weight component. In addition to troponin, tropomyosin is required for the inhibition of the magnesium activated actomyosin ATPase activity in the absence of calcium and for the reversal of this inhibition by calcium. Lobster tropomyosin interacts with rabbit actin and lobster troponin interacts with rabbit tropomyosin. The 30,000 doublet component corresponds to the troponin-I of rabbit and inhibits the ATPase activity of actomyosin both in the presence and in the absence of calcium. The 17,000 component corresponds to the troponin-C of rabbit; it binds calcium and reverses the inhibition of the ATPase activity by troponin-I in the presence of calcium. No more than 1 mol of calcium is bound by a mole of troponin-C or by troponin. The 52,000 component interacts with tropomyosin and has been tentatively identified as troponin-T; however, it has not been demonstrated as yet that this component had a role in the regulation of lobster actomyosin.     

Current-Voltage Relations in the Lobster Giant Axon Membrane Under Voltage Clamp Conditions

The sucrose-gap method introduced by Stämpfli provides a means for the application of a voltage clamp to the lobster giant axon, which responds to a variety of different experimental procedures in ways quite similar to those reported for the squid axon and frog node. This is particularly true for the behavior of the peak initial current. However, the steady state current shows some differences. It has a variable slope conductance less than that of the peak initial current. The magnitude of the steady state slope conductance is related to the length of the repolarization phase of the action potential, which does not have an undershoot in the lobster. The steady state outward current is maintained for as long as 100 msec.; this is in contrast to a decline of about 50 per cent in the squid axon. Lowering the external calcium concentration produces shifts in the current-voltage relations qualitatively similar to those obtained from the squid axon. On the basis of the data available, there is no reason to doubt that the Hodgkin and Huxley analysis for the squid giant axon in sea water can be applied to the lobster giant axon.     

The polypeptide and the phospholipid components of axon plasma membranes.

The axon plasma membrane fraction isolated from garfish olfactory nerve was analyzed for its polypeptide composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There were present over 20 well-resolved polypeptide components in this membrane, and eleven of them, with an apparent molecular weight range of 22,000-130,000, accounted for most of the membrane proteins. None of the major polypeptide species present in the membrane appeared to be glycoprotein. Based on electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel, eight of the major polypeptides found in garfish nerve membrane appeared to be also present in the axon plasma membrane isolated from lobster walking leg nerve. Both garfish and lobster nerve membranes contained high concentration of lipids (66-76%) which were essentially cholesterol and phospholipids. The classes of phospholipids present were phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol and sphingomyelin. Lobster nerve membrane also contained about 3% phosphatidic acid. Assays for acetylcholinesterase in axon plasma membrane fractions isolated from different nerve sources showed a wide variation, ranging from a specific activity of 2.4 for garfish nerve to 312.5 for lobster nerve membrane.     

Intra- and interannual variation in the diet of the Magellanic penguin (Spheniscus magellanicus) at Martillo Island,Beagle Channel

Knowledge of seabirds’ diet at each breeding site and its temporal variation is key to understanding and evaluating how changes in marine resources affect each seabird population. In this study, we determined the diet of Magellanic penguins (MP, Spheniscus magellanicus) at Martillo Island, accounting for sex, breeding stage and year. We analyzed a total of 144 stomach contents during three consecutive breeding seasons (2006–2007, 2007–2008 and 2008–2009) and stages (incubation, early and late chick-rearing). MP fed mainly on fuegian sprat (Sprattus fuegensis), which represented 75 % of the biomass consumed by birds during the entire study. The next important prey was squat lobster (Munida gregaria), followed by Patagonian squid (Loligo gahi). Both sexes consumed similar prey items. We observed variation in diet relative composition among breeding years and stages. Fuegian sprat consumption decreased throughout the years whereas squat lobster increased. Penguins consumed a higher proportion of squat lobster and Patagonian squid during the incubation stage than in the chick-rearing stages, whereas fuegian sprat was almost the only prey item consumed during the late chick-rearing stage. MPs show certain flexibility in the use of resources probably as a response to changes in prey populations. Variability in the diet among different reproductive stages could be related to changes in the distribution and abundance of their main prey near the colony during the breeding season together with changes in the energy requirements of seabirds.     

Neurohormonal alteration of integrative properties of the cardiac ganglion of the lobster Homarus americanus.

The spontaneous burst discharges of isolated lobster (Homarus americanus) cardiac ganglia were recorded with a spaced array of electrodes. Small regions (less than 1 mm) of the ganglion were exposed to the cardioexcitor neurohormone in extracts of pericardial organs (XPO) or to 10(-5) M 5-hydroxytryptamine (5HT). All axons were excited (increased mean firing frequency, f) by both substances, but only by applications in the region between the soma (but excluding it) and proximal site of impulse initiation. Units not so exposed changed their f relatively little despite f increases of as much as threefold in exposed units and changes in burst rate and overall length. Regularity and grouping of all impulse activity into bursts was never disturbed. 5HT increases burst rate at any point of application. The increases are larger if small cells are affected than if only large cells are exposed. Burst length decreases except when the pacemaker is affected. In contrast, XPO affects neither burst rate or length unless small cells are affected. Length is increased if non-pacemaker small cells are affected; both rate and length increase if the pacemaker is affected. The pacemaker usually exhibits an f of intermediate value. Rate changes are not simply related to its f. A small cell can "burst" in the absence of impulses from any other cells. XPO may enhance endogenous "driver potentials," while 5HT may excite by depolarizing at limited sites.     

Overview,opportunities and outlook for Australian spiny lobster fisheries

Australia’s lobster fisheries are relatively small in volume (9500t) compared with global production (289,000t), but are the country’s most valuable in terms of both overall production and value of export (2014 Gross Value of Production of $610 million AUD). Further, they support commercial, recreational and indigenous fishers along most of the continent’s coastline. Here we review similarities and key differences between these lobster fisheries, based on biological characteristics, fishery data collection, assessment and management methods, and supply chain considerations. A diverse range of palinurid lobsters occur in Australia, but only three genera, distributed across eight different management jurisdictions, support significant fisheries. Catches of western rock lobster Panulirus cygnus dominate landings (61%), followed by southern rock lobster Jasus edwardsii, tropical lobster Panulirus ornatus and the eastern rock lobster Sagmariasus verreauxi. Large-scale environmental influences such as climate change are impacting on these fisheries in similar or different ways forcing new management and raising the need for greater resilience in current supply chains. Although these are separate fisheries, the integrated nature of the dominant Chinese export markets suggests potentially important economic and market-related interactions. Our overview highlights the critical role of continued monitoring of recruitment pulses, in combination with robust harvest strategies, to ensure that harvests respond adequately and fisheries achieve biological and economic sustainability. Approaches that also include socio-cultural considerations (triple bottom line) are important given many fisheries include indigenous Australians. Our integrated analysis of Australian lobster fisheries highlights differences and similarities with spiny lobster fisheries worldwide and lessons from opportunities, including adapting to new free trade agreements, enhancing the reputation of wild lobsters as a whole, sharing expertise, and better alignment of supply and demand.     

Permeability of Alkali Metal Cations in Lobster Muscle : A comparison of electrophysiological and osmometric analyses

Single muscle fibers from lobster walking legs are effectively impermeable to Na, but are permeable to K. They shrink in hyperosmotic NaCl; they swell in low NaCl media which are hyposmotic or which are made isosmotic with the addition of KCl. In conformity, the membrane potential is relatively insensitive to changes in external Na, while it responds according to the Nernst relation for changes in external K. When the medium is made isosmotic or hyperosmotic with RbCl the volume and membrane potential changes are of essentially the same magnitudes as those in media enriched with KCl. The time courses for attaining equilibrium are slower, indicating that Rb is less permeant than K. Substitution of CsCl for NaCl (isosmotic condition) produces no change in volume of the muscle fiber. Addition of CsCl (hyperosmotic condition) causes a shrinkage which attains a steady state, as is the case in hyperosmotic NaCl. Osmotically, therefore, Cs appears to be no more permeant than is Na. However, the membrane depolarizes slowly in Cs-enriched media and eventually comes to behave as an ideal Cs electrode. Thus, the electrode properties of the lobster muscle fiber membrane may not depend upon the diffusional relations of the membrane and ions, and the osmotic permeability of the membrane for a given cation may not correspond with the electrophysiologically deduced permeability. Comparative data on the effects of NH₄ and Li are also included and indicate several other degrees of complexity in the cell membrane.     

Sodium flux ratio in Na/K pump-channels opened by palytoxin

Palytoxin binds to Na(+)/K(+) pumps in the plasma membrane of animal cells and opens an electrodiffusive cation pathway through the pumps. We investigated properties of the palytoxin-opened channels by recording macroscopic and microscopic currents in cell bodies of neurons from the giant fiber lobe, and by simultaneously measuring net current and (22)Na(+) efflux in voltage-clamped, internally dialyzed giant axons of the squid Loligo pealei. The conductance of single palytoxin-bound "pump-channels" in outside-out patches was approximately 7 pS in symmetrical 500 mM [Na(+)], comparable to findings in other cells. In these high-[Na(+)], K(+)-free solutions, with 5 mM cytoplasmic [ATP], the K(0.5) for palytoxin action was approximately 70 pM. The pump-channels were approximately 40-50 times less permeable to N-methyl-d-glucamine (NMG(+)) than to Na(+). The reversal potential of palytoxin-elicited current under biionic conditions, with the same concentration of a different permeant cation on each side of the membrane, was independent of the concentration of those ions over the range 55-550 mM. In giant axons, the Ussing flux ratio exponent (n') for Na(+) movements through palytoxin-bound pump-channels, over a 100-400 mM range of external [Na(+)] and 0 to -40 mV range of membrane potentials, averaged 1.05 +/- 0.02 (n = 28). These findings are consistent with occupancy of palytoxin-bound Na(+)/K(+) pump-channels either by a single Na(+) ion or by two Na(+) ions as might be anticipated from other work; idiosyncratic constraints are needed if the two Na(+) ions occupy a single-file pore, but not if they occupy side-by-side binding sites, as observed in related structures, and if only one of the sites is readily accessible from both sides of the membrane.     

Modelling deep water habitats to develop a spatially explicit, fine scale understanding of the distribution of the western rock lobster, Panulirus cygnus

Background

The western rock lobster, Panulirus cygnus, is endemic to Western Australia and supports substantial commercial and recreational fisheries. Due to and its wide distribution and the commercial and recreational importance of the species a key component of managing western rock lobster is understanding the ecological processes and interactions that may influence lobster abundance and distribution. Using terrain analyses and distribution models of substrate and benthic biota, we assess the physical drivers that influence the distribution of lobsters at a key fishery site.

Methods and Findings

Using data collected from hydroacoustic and towed video surveys, 20 variables (including geophysical, substrate and biota variables) were developed to predict the distributions of substrate type (three classes of reef, rhodoliths and sand) and dominant biota (kelp, sessile invertebrates and macroalgae) within a 40 km² area about 30 km off the west Australian coast. Lobster presence/absence data were collected within this area using georeferenced pots. These datasets were used to develop a classification tree model for predicting the distribution of the western rock lobster. Interestingly, kelp and reef were not selected as predictors. Instead, the model selected geophysical and geomorphic scalar variables, which emphasise a mix of terrain within limited distances. The model of lobster presence had an adjusted D² of 64 and an 80% correct classification.

Conclusions

Species distribution models indicate that juxtaposition in fine scale terrain is most important to the western rock lobster. While key features like kelp and reef may be important to lobster distribution at a broad scale, it is the fine scale features in terrain that are likely to define its ecological niche. Determining the most appropriate landscape configuration and scale will be essential to refining niche habitats and will aid in selecting appropriate sites for protecting critical lobster habitats.