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Characterization of a G-protein from the mandibular organ of the lobster Homarus americanus (Nephropidae, Decapoda).

1. GTP-binding activity was found in both calf brain and male lobster mandibular organ (MO). There was approximately two to three times as much binding in the calf brain. 2. The GTP-binding activity could be extracted from the calf brain with sodium cholate, but not from the MOs. 3. Using ADP-ribosylation catalyzed by pertussis toxin, GTP-binding was shown to be the result of the presence of G-protein. In the lobster MO the G-protein alpha subunit has a molecular weight of about 42 kDa and may be of the Go or Gi varieties.

Adenosine Diphosphate Ribose

Growth of lobster giant axons: correlation between conduction velocity and axon diameter.

The conduction velocity and axon diameter of medial and lateral giant axons in lobsters was followed during growth from early juvenile (13 mm) to adult (250 mm) forms. In both axons, conduction velocity increased more than 4-fold in the growth period examined. Correlated with the increase in conduction velocity is an increase in axon diameter and the relationship between these two parameters is expressed by the equation CV = krX, where X equals 0.5 for the lateral and 0.7 for the medial giant axons. The sheath surrounding the giant axons also increases in size during growth with that of the medial giant axon being considerably thicker than that of the lateral giant axon. The significance of the increase in axon diameters is to increase conduction velocity and thereby reduce the latency of the escape response mediated by these giant axons.

Animals

Comparative strategies in the investigation of neural networks.

Comparative studies on nervous systems, though infrequently undertaken for the purpose of comparison, have yielded some important generalities about the formats of nervous networks, and about the cell biology of certain neural types. In the first category, it is clear that convergent evolutionary processes arrived at very similar networks to accomplish reciprocal and lateral inhibition, and load-compensation in "resistance reflexes." A newer general network format is described, command-derived inhibition, in which the central nervous elements controlling a rapid movement deliver presynaptic inhibition to the terminals of sensory neurons that carry reafferent excitation from the movement. It is argued that such circuits occur in several groups of animals, and that they include as a special class the efferent inhibitory neurons innervating acoustico-lateralis receptors in vertebrates. The properties of circuit elements that now seem to constitute useful generalizations include size principle (the inverse relationship between size and excitability in a variety of neurons), and the late differentiation of sensory neurons, failure to decussate, and their inability to mediate inhibition. Many other generalities have emerged, only to fall; one conclusion from such searches is that many supposedly "basic" properties of cell types or neural circuits are in fact not phylogenetically conservative, however much the physiologist may expect them to be.

Action Potentials

Photoreceptor processes: some problems and perspectives.

Visual photoreceptors from both vertebrates and invertebrates are characterized by extensive elaboration of membrane which contains visual pigment (rhodopsin). Visual pigments in all phyla examined are chemically similar: the chromophore is 11-cis retinaldehyde attached by an aldimine linkage (Schiff base) to a membrane protein, opsin. The effect of light is to isomerize the chromophore to the all-trans configuration. Beyond these fundamental similarities, several specific areas are discussed in which variations and differences appear. (1) Light causes vertebrate visual pigments to bleach, liberating the chromophore. Most invertebrate visual pigments do not bleach in the light, but instead form a thermally stable metarhodopsin, with the chromophore in the all-trans configuration still attached to the opsin. (2) In the disk membranes of vertebrate rod and cone outer segments, the rhodopsin molecules are oriented with their chromophores nearly coplanar with the disks. Within this plane, however, both rotational and translational diffusion are possible. In the microvillar membranes of arthropod and cephalopod rhabdoms, on the other hand, the situation is less clear. There is evidence for some preferential orientation of chromophores that implies restrictions on Brownian rotation. (3) In the outer segments of vertebrate receptors, absorption of light by rhodopsin causes the plasma membrane to hyperpolarize due to a decrease in sodium conductance, possibly mediated by calcium ions. In most invertebrate photoreceptors, light causes a depolarization due to an increase in conductance, principally to sodium ions. A subsequent entry of calcium causes a partial repolarization of the membrane, due to a decrease in sodium conductance. (4) For vertebrate receptors, log threshold is directly proportional to the fraction of rhodopsin bleached (Dowling-Rushton relationship). The proportionality constant varies in different preparations from less than four to more than 30, and the physical basis for the relationship is unknown. For invertebrates, by contrast, the dependence of sensitivity on rhodopsin concentration is much less dramatic and may well depend simply on the probability of quantum catch. (5) In most species, vertebrate and invertebrate, the accumulation of photoproduct probably has no effect on membrane conductance, but several possible exceptions exist. (6) Photoregeneration of rhodopsin from metarhodopsin is likely an important mechanism of recovery in certain arthropods such as diurnal insects, but dark mechanisms of recovery also exist in all phyla. In no single case are they adequately understood.

Adaptation, Ocular

Adaptation of muscle pyruvate kinases to environmental temperatures and pressures.

Kinetic and structural properties of muscle pyruvate kinases from species adapted to different temperatures and hydrostatic pressures were examined. Most muscle pyruvate kinases can exist in two temperature-dependent conformational states. For the low temperature conformers, activation free energies and enthalpies were proportional to the species' adaptation temperatures. The interspecific differences in activation parameters may be due to different amounts of weak bond formation/rupture during the rate-limiting event(s) in catalysis. When comparisons of substrate binding ability at physiological temperatures are made, a marked conservation in apparent Km values is observed among species, Structural rigidity, as estimated by heat inactivation temperature, is positively correlated with adaptation temperature. The apparent size of the conformational change which occurs during catalysis is also positively correlated with cell temperature, except for enzymes for deep-sea species.

Adaptation, Physiological

Aspects of the embryology and neural development of the American lobster.

It is feasible to study the anatomical, physiological, and biochemical properties of identifiable neurons in lobster embryos. To exploit fully the advantages of this preparation and to lay the foundation for single-cell studies, our recent goals have been to 1) establish a quantitative staging system for embryos, 2) document in detail the lobster's embryonic development, 3) determine when uniquely identifiable neurons first acquire their transmitter phenotypes, and 4) identify particular neurons that may serve developmental functions. Behavioral, anatomical, morphometric, and immunocytochemical studies have led to a detailed characterization of the growth and maturation of lobster embryos and to the adoption of a percent-staging system based upon the eye index of Perkins (Fish. Bull., 70:95-99, 1972). It is clear from these studies that the lobster nauplius molts at approximately 12% embryonic development (E12%) into a metanauplius, which subsequently undergoes a complete molt cycle within the egg. This molt cycle climaxes with the emergence of the first-stage larva shortly after hatching. Serotonin and proctolin, neurohormones widely distributed in the lobster nervous system, appear at different times in development. Serotonin immunoreactive neurons begin to appear at approximately E10%, with the adult complement being established by E50%. In contrast, proctolin immunoreactive neurons appear later and attain their full complement over a protracted period including larval and juvenile stages. The development of serotonergic deutocerebral neurons and their targets, the olfactory and accessory lobes in the brain, are also examined. The olfactory lobes are forming by E10% and have acquired their glomerular organization by E50%, whereas the formation of the accessory lobes is delayed; the early rudiments of the accessory lobes are seen by E50%, and glomeruli do not form until the second larval stage.

Animals

Histochemical heterogeneity of fibers in the abdominal superficial flexor muscles of the Norway lobster, Nephrops norvegicus (L.).

The superficial flexor muscle in the abdomen of the Norway lobster Nephrops norvegicus (L.), comprises medial and lateral bundles with distinct fiber type composition. Fibers of the medial bundle have long sarcomeres (> 9 microns) and a thick fringe of subsarcolemmal mitochondria. In histochemical tests they have a low total myofibrillar ATPase activity, a pH-stable isoform of myosin ATPase, and a high level of oxidative enzyme activity. A few fibers of the lateral bundle also display these morphological and histochemical properties. However, the majority of lateral fibers have shorter sarcomeres (< 8 microns), no subsarcolemmal mitochondria, but a well-developed tubular system. They also have a higher total myofibrillar ATPase activity, a pH-labile isoform of myosin ATPase, and a low level of oxidative enzyme activity. The heterogeneous pattern of different fiber types in the lateral bundle of this muscle is similar but not identical in the different abdominal segments and in different individuals.

Abdominal Muscles

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster.

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage-current relationship of the membrane. By including gamma-aminobutyrate (GABA) in the bathing medium, the voltage-current relationship was made linear in the depolarizing direction over a range of 15-20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and aspartate functioning synergistically.

Animals

Content of amino acids in axons from the CNS of the lobster.

The contents of alanine, proline, glycine, GABA, glutamate, and aspartate were measured in four bundles of axons (designated areas A through D) from the circumesophageal connective of the lobster (Homarus americanus). The contents of these amino acids were also determined in individual axons within specific bundles and in the external sheath covering the circumesophageal connective. Within the nerve bundles the levels of aspartate were highest of the amino acids measured, ranging from 1.95 +/- 0.12 mumol/mg protein in area C to 7.55 +/- 0.54 mumol/mg protein in area B. On the other hand, GABA had the lowest value in the four bundles; its highest level was found in area C (0.083 +/- 0.006 mu mol/mg protein) and the lowest in area B (none detected). The content of glycine ranged from 1.63 +/- 0.14 (area C) to 2.52 +/- 0.32 mumol/mg protein in area A; that for glutamate ranged from 0.390 +/- 0.019 (area C) to 1.01 +/- 1.03 (area B). The contents of alanine and proline changed relatively little from bundle-to-bundle. The content of aspartate was the highest of any of the amino acids assayed in individual axons (with diameters in the range of 40 to 65 mu) dissected from areas B and C. Glycine had the next highest content followed in order by glutamate, proline, and alanine. GABA was not detected in these axons. With the exception of GABA (which could not be detected), aspartate had the lowest level (0.066 +/- 0.017) and glycine had the highest level (2.00 +/- 0.498 mumol/mg protein) in the external sheath covering the the circumesophageal connective.

Amino Acids

Effects of thiamine antagonists on nerve conduction. I. Actions of antimetabolites and fern extract on propagated action potentials.

To assess the hypothesis that thiamine is directly involved in the permeability changes at the sodium channel during nerve conduction, the effects of thiamine antagonists on lobster giant axon resting and action potentials were determined. Thiamine antimetabolites, in millimolar concentrations, reversibly decreased the maximum rate of rise and amplitude of the action potential while increasing its duration. In particular, thiamine tert-butyl disulfide (TTBD) elicited the formation of pronounced shoulders during repolarization, lengthening the action potential by 2-50 times, depending on dose. Antimetabolites also depolarized the resting membrane, but this change was poorly reversible and may indicate a dual mechanism for antimetabolite action. An extract of the fern, Pteris aquilina, reversibly decreased the maximum rate of rise of the action potential and depolarized the resting potential. It also elevated and prolonged the action potential after-depolarization, sometimes causing repetitive activity. The strength of these actions was correlated with the antithiamine potency of the extract, and was diminished by addition of thiamine to the extract.

Action Potentials

Agonistic and antagonistic activity of glutamate analogs on neuromuscular excitation in the walking limbs of lobsters.

Forty-six analogs of L-glutamate were tested for activity on muscle fibers in the walking limbs of lobsters. Effects on the membrane potential, input resistance, and amplitude of neurally evoked EPSPs and IPSPs were studied as well as effects on applied L-glutamate. Seventeen of the compounds studied depolarized the muscle fibers in a manner indicative of an agonistic action on receptors in the neuromuscular excitatory membrane. Six analogs selectively reduced the amplitude of evoked EPSPs, and at least three of these (kainic acid, D-glutamate, and D-aspartate) antagonized the excitatory action of applied L-glutamate. Kainic acid was the most potent of the blockers of neuromuscular excitation, but even it was relatively weak since a concentration of 1 mM was required for an apparent effect. Generally those analogs in the L-configuration which possessed activity, had agonistic actions, whereas those in the D-configuration were usually antagonistic. These observations provide pharmacological evidence for the concept that L-glutamate is the transmitter agent which mediates neuromuscular excitation in the walking limbs of lobsters. In addition, our results are consistent with recent studies which indicate that L-aspartate may also function in this neuromuscular excitatory process.

Animals

Lobster claw motorneurons respond to contralateral sensory stimuli.

Unilateral sensory stimuli in an isolated lobster claw--ganglion preparation elicits soma and axon spikes in homologous motorneurons in the ipsilateral as well as the contralateral hemiganglion. This cross excitation of the motorneurons provides a basis for bilateral reflexes in the claw and is likely mediated through interneuron(s).

Animals

Mg++-sensitivity of neuromuscular transmission in two crustaceans: correlation with blood Mg++ levels.

Neuromuscular transmission was measured in muscles of spider crabs (Hyas areneus) and lobsters (Homarus americanus). Solutions containing 40 and 10 mM/1 Mg++, which were approximately the same as those measured in the blood of Hyas and Homarus, respectively, were used to soak the preparations prior to testing. In Homarus, neuromuscular transmission was severely depressed by 40 mM Mg++. In spider crabs, neuromuscular transmission was not severely depressed. Although the amount of transmitter released by nerve impulses was reduced, total membrane depolarization during trains of impulses was not reduced because a compensating increase in muscle fiber membrane resistance occurred in Hyas preparations exposed to M Mg++. Hyas, but not Homarus, is physiologically adapted to function at relatively high blood Mg++ concentrations.

Animals

Claw asymmetry in lobsters: case study in developmental neuroethology.

An enduring debate in the study of development is the relative contribution of genetic and epigenetic factors in the genesis of an organism, that is, the nature vs. nurture debate. The behavior of the paired claws in the lobster offers promising material for pursuing this debate because of the way they develop. The paired claws and their closer muscles are initially symmetrical; both are slender in appearance and have a mixture of fast and slow fibers in their closer muscles. During a critical period of development, they become determined into a major (crusher) and minor (cutter) claw and during subsequent development acquire their final form and behavior: The crusher becomes a stout, molar-toothed claw capable of closing only slowly because its closer muscle has 100% slow fibers while the cutter becomes a slender, incisor-toothed claw capable of closing rapidly because its closer muscle has 90% fast fibers. Our initial hypothesis was that the more active claw became the crusher and its less active counterpart the cutter. Presumably, nerve activity would influence muscle transformation, which in turn would influence the exoskeleton to which they attach and hence claw morphology. Curtailing nerve activity to the claw prevented crusher development, while reflex activation of a claw promoted its development; both results support the notion that nerve activity directly regulates claw form and function. This is not, however, the case, for when both claws were reflexly exercised neither formed a crusher, signifying rather that bilateral differences in predominantly mechanoreceptive input to the paired claws somehow lateralized the claw ganglion [central nervous system (CNS)] into a crusher and cutter side. The side experiencing the greater activity becomes the crusher side while the contralateral side becomes the cutter and is also inhibited from ever becoming a crusher. This initial lateralization in the CNS is expressed, via as yet unknown pathways, at the periphery in claw morphology, muscle composition, and behavior. The critical period defines a time when the CNS is susceptible to being lateralized into a crusher and cutter side. Such lateralization is dependent upon experience of the environment in the form of mechanoreceptive input. In the absence of such experience, the CNS is not lateralized and paired cutter claws develop. Thus, while the critical period for crusher determination is genetically determined the actual trigger is influenced by experience.

Animals

Larval and adult eye of the western rock lobster (Panulirus longipes).

A number of differences exists between the compound eyes of larval and adult rock lobsters, Panulirus longipes. The larval eye more closely resembles the apposition type of compound eye, in which retinula cells and rhabdom lie immediately below the cone cells. The adult eye, on the other hand, is a typical clear-zone photoreceptor in which cones and retinula cell layers are separated by a wide transparent region. The rhabdom of the larval eye, if cut longitudinally, exhibits a "banded" structure over its entire length; in the adult the banded part is confined to the distal end, and the rhabdom is tiered. Both eyes have in common an eighth, distally-located retinula cell, which possesses orthogonally-oriented microvilli, and a peculiar lens-shaped "crystal", which appears to focus light onto the narrow column of the distal rhabdom. Migration of screening pigment on dark-light adaptation is accompanied by changes in sensitivity and resolution of the eye. Retinula cells belonging to one ommatidium do not arrange into one single bundle of axons, but interweave with axons of four neighbouring facets in an extraordinarily regular fashion.

Animals