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Acetylcholinesterase activity in neurons of crayfish abdominal ganglia.

Acetylcholine is known to be a neurotransmitter in crustacean central nervous systems, but the numbers and distribution of cholinergic neurons in the segmental ganglia have not been described. To begin a census of cholinergic neurons in these ganglia, we used a histochemical assay for acetylcholinesterase to map neurons that contained this enzyme in the six abdominal ganglia of crayfish. In each abdominal ganglion, about 47 cell bodies were stained. The distributions of these stained cells in individual ganglia were similar, and the numbers were not significantly different. None of these stained cell bodies could be identified from their structures or locations as previously identified motor neurons or sensory neurons with central cell bodies. The process of one unpaired midline neuron that occurred only in the first three abdominal ganglia divided to send a pair of axons anteriorly into both halves of the connective. The central projections of afferent axons from many peripheral sensory neurons stained clearly as they entered each ganglion. Terminals of these axons were heavily stained in the horseshoe neuropil and the lateral neuropils. We labeled both gamma-aminobutyric acid (GABA) and acetylcholinesterase in individual ganglia. Only a few neurons in each ganglion were double-labeled. The unpaired midline neurons in the three anterior ganglia that stained for acetylcholinesterase did not show GABA-like immunoreactivity, but cells with similar shapes did label with the GABA antiserum. Acetylcholinesterase is not a definitive marker of cholinergic neurons, but its presence is often associated with the cholinergic phenotype. These stained cells should be considered as putative cholinergic neurons.

Abdomen↗

Morphology, physiology, and homology of the N-cell and muscle receptor organs in the thorax of the crayfish Cherax destructor.

The serial homology of arthropods, together with our ability to identify individual neurons from segment to segment, and from animal to animal, provides opportunities for studying the changes wrought by natural selection on specific neural elements when functional requirements change in different parts of the trunk. Using this concept as a guide, we studied the morphology and physiology of the thoracic N-cells and muscle receptor organs (MROs) of the crayfish Cherax destructor for evidence of serial homology and functional plasticity. Methylene blue staining, together with anterograde and retrograde filling with cobalt through cut axons, revealed the morphology of the receptors, disposition of their endings, and the pathways of their axons from receptor to ganglion. The seventh thoracic segment has tonic and phasic MROs with receptor muscles in parallel with different heads of the deep thoraco-abdominal extensor muscle. The sixth segment has a tonic MRO with a receptor muscle in parallel with one head of the abdominal abductor. These three receptors are typical MROs complete with accessory nerves. Thoracic segments 1-5 each give rise to one mechanosensory N-cell with a small cell body and long processes ramifying in a target muscle. N-cell 5 is associated with the abdominal-thoracic abductor muscle, and the other four are associated with parts of the epimeral attractor. The responses of N-cells 1-4 range from tonic to phasico-tonic and show a range of thresholds to passive muscle stretch and active contraction. Cobalt introduced into bundles of nerve fibers known to include N-cell axons reveals projections with branching patterns and morphology similar to abdominal MROs. The present findings, together with information on thoracic MROs and N-cells from other species, were tabulated according to neurotome. The clear pattern revealed leads us to propose that N-cells are derivatives of segmentally repeating MROs modified to monitor postural and locomotory movements in the less mobile thorax.

Animals↗

Inhibitory axoaxonal and neuromuscular synapses in the crayfish opener muscle: membrane definition and ultrastructure.

The specific inhibitory motoneuron to the crayfish (Procambarus clarkii) opener muscle provides neuromuscular synapses to the muscle fibers and axoaxonal synapses to the excitatory motor nerve terminals. Freeze fracture of the membrane in both types of synapses show that the presynaptic active zone consists of clusters of large particles (putative calcium channels), which are often encircled by large depressions representing fused synaptic vesicles on the internal leaflet or P face of the presynaptic membrane. Corresponding pits and protrusions mark the external leaflet or E face of the presynaptic membrane. The postsynaptic receptor-bearing surface, characterized for neuromuscular synapses only, consists of rows of particles on both leaflets of the muscle membrane. The organization differs from that seen at excitatory synapses where particles occur only on the E-face leaflet. Serial thin sections of nerve terminals reveal that neuromuscular synapses are significantly larger in proximal fibers than in their central counterparts and support a greater number of presynaptic dense bars (active zones). Axoaxonal synapses also show regional differences; almost three times as many occur in the proximal region compared with the central region. Most synapses possess a single dense bar. The majority of synapses formed by the inhibitory axon are neuromuscular; a minority are axoaxonal. The latter occur in various locations along the excitatory nerve terminals as well as on branches of the axon itself. This preterminal or "off-shore" location could act to cut off entire populations of excitatory synapses or reduce the amplitude of the preterminal action potential.

Animals↗

Crayfish brain interneurons that converge with serotonin giant cells in accessory lobe glomeruli.

Freshwater crayfish have well-developed olfactory systems with an array of receptors that project exclusively to areas in the brain that are functionally specialized for the processing of odors. The accessory lobes are large bilateral areas of neuropil that are anatomically associated with the olfactory lobes. The accessory lobes receive no primary afferents and do not contain the endings of motor efferents; thus, their role in olfaction is still obscure. Intracellular dye filling of interneurons in the deutocerebral commissure in the crayfish brain has shown that they end bilaterally in glomeruli in the accessory lobes, have cell somata in a dorsal cluster medial to the olfactory lobes, and have unilateral projections to the deutocerebral commissure neuropil. Each deutocerebral commissure interneuron has only 6 to 15 output glomeruli in each accessory lobe and does not share glomeruli with other deutocerebral commissure interneurons. The deutocerebral commissure interneurons converge with the dorsal giant serotonin neurons in the accessory lobe glomeruli. Deutocerebral commissure interneurons can be separated into classes according to their projections to the protocerebrum, central body, and deutocerebrum. Physiological responses of the deutocerebral commissure interneurons following photic stimulation of the eyes and electrical stimulation of the second antennae lead to the conclusion that the deutocerebral commissure represents an input to the accessory lobes from the protocerebral neuropils and that visual and tactile inputs are included in the processing performed in the accessory lobes.

Animals↗

"Satellite cells" and nerve terminals in the crayfish opener muscle visualized with fluorescent dyes.

Nerve terminals and associated cells on the muscle's surface were visualized in the crayfish opener muscle with several fluorescent dyes in conjunction with confocal microscopy and conventional fluorescence microscopy. The nerve terminals of the excitatory and inhibitory axons were best seen with 4-diethylaminostyryl-N-methylpyridinium iodide (4-Di-2-Asp). This dye is selectively accumulated in mitochondria, which are numerous both in the axons and in synapse-bearing terminal varicosities. Muscle nuclei were also clearly visualized, because they excluded 4-Di-2-Asp but were stained by acridine orange (AO). A positive attraction between muscle nuclei and nerve terminals was evident by visual inspection and was confirmed by spatial statistics. Additional flat cells on the muscle's surface appeared as bright rings with elongated processes that were often close to or overlapped nearby nerve terminals. The structure of these cells was established by electron microscopy after labeling them with fluorescent polystyrene beads, which could be found over structures on the muscle surface in sections of embedded specimens. The flat surface cells were distinct from peripheral glial cells closely associated with axons and nerve terminals. Nevertheless, spatial statistics showed that the surface cells were grouped near nerve terminals. They occupied a small fraction of the muscle cell's surface. Their functional role has not been determined in crustacean muscles.

Animals↗

Ultrastructure of the synaptic terminals of the dorsal giant serotonin-IR neuron and deutocerebral commissure interneurons in the accessory and olfactory lobes of the crayfish.

The olfactory and accessory lobes in the crayfish are large spherical neuropils found on each side of its brain. The olfactory lobes receive the afferent axons of chemoreceptors that are located along the outer branches of the biramous first antennae. The accessory lobes receive a large input from interneurons whose axons lie in the deutocerebral commissure. A pair of large serotonergic neurons (the dorsal giant neurons) branch unilaterally in the accessory and olfactory lobes of each side. From physiological recordings, it has been proposed that the deutocerebral commissure interneurons synapse with elements in the accessory lobes that in turn connect to the dorsal giant neuron. It has also been proposed that the dorsal giant neuron is activated by inputs in the accessory lobe and that its output is in the olfactory lobe. This ultrastructural study tests this hypotheses by examining the polarity of synaptic terminals on dorsal giant neurons and deutocerebral interneurons that have been filled with neurobiotin. In double-labelled preparations, we found the deutocerebral interneurons to be presynaptic to elements in the accessory lobes, but none of these postsynaptic elements was identifiable as the dorsal giant neuron. The dorsal giant neurons receive many more synaptic inputs in the accessory lobes than in the olfactory lobe. Very few giant serotonin neuron output synapses were found in either lobe.

Animals↗

Genetic analysis of the freshwater crayfish Cherax tenuimanus.

The marron (Cherax tenuimanus) is one of the few species of freshwater crayfish native to Australia that is suitable for aquaculture and occurs only in the southwest of Western Australia. This study describes polymorphic microsatellite markers which differentiate marron populations from several geographically distinct regions (including rivers and streams, dams, and commercial marron farms) throughout Western Australia. Twenty microsatellite loci, primarily of the (CA)n. (GT)n type, were isolated and sequenced from a marron cosmid library. Three of these loci were characterised further. Two loci exhibited extensive polymorphism and one was monomorphic. The polymorphic loci exhibited Mendelian codominant inheritance in the family group comprising two individual parents and approximately 100 offspring bred for this study. These loci permitted differentiation between the five geographically distinct populations studied and thus provide a basis for genetic characterisation of marron stock in Western Australia.

Animals↗

Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins.

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.

Animals↗

Mechanism of NMDA receptor contribution to axon-to-glia signaling in the crayfish medial giant nerve fiber.

Electrical stimulation of crayfish giant axons at high frequency activates group II metabotropic and NMDA glutamate receptors on adjacent glial cells via release of N-acetylaspartylglutamate and glutamate formed upon its hydrolysis. This produces a transient depolarization followed by a prolonged hyperpolarization of glial cells that involves nicotinic acetylcholine receptor activation. The hyperpolarization is nearly completely blocked by antagonists of metabotropic glutamate receptors but only slightly reduced by inhibition of NMDA receptors. We report that the NMDA-induced hyperpolarization of glial cells is reduced by decreased calcium in the solution bathing the giant nerve fiber, while removal of sodium ions or block of voltage-dependent calcium channels completely prevents the glial response to NMDA. Inhibition of nicotinic acetylcholine receptors or removal of extracellular Cl(-) converts the glial response from a hyperpolarization to a depolarization that is sensitive to NMDA receptor antagonist. We propose that NMDA receptor activation by glutamate, formed from extracellular N-acetylaspartylglutamate during nerve stimulation, contributes to glial hyperpolarization by increasing intracellular Ca(2+) via opening of voltage-sensitive Ca(2+) channels. Based on our previous work, we propose further that the added Ca(2+) supplements that produced by N-acetylaspartylglutamate and glutamate acting on group II metabotropic glutamate receptors to cause an increased release of acetylcholine and a larger hyperpolarization.

Acetylcholine↗

Involvement of adenylate cyclase and tyrosine kinase signaling pathways in response of crayfish stretch receptor neuron and satellite glia cell to photodynamic treatment.

Neuroglial interactions are most profound during development or damage of nerve tissue. We studied the responses of crayfish stretch receptor neurons (SRN) and satellite glial cells to photosensitization with sulfonated aluminum phthalocyanine Photosens. Although Photosens was localized mainly in the glial envelope, neurons were very sensitive to photodynamic treatment. Photosensitization gradually inhibited and then abolished neuron activity. Neuronal and glial nuclei shrank. Some neurons and glial cells lost the integrity of the plasma membrane and died through necrosis after the treatment. The nuclei of other glial cells but not neurons become fragmented, indicating apoptosis. The number of glial nuclei around neuron soma increased, probably indicating proliferation for enhanced neuron protection. Adenylate cyclase (AC) inhibition by MDL-12330A, or tyrosine kinase (TK) inhibition by genistein, shortened neuron lifetime, whereas AC activation by forskolin or protein tyrosine phosphatases (PTP) inhibition by sodium orthovanadate prolonged neuronal activity. Therefore, cAMP and phosphotyrosines produced by AC and TK, respectively, protected SRN against photoinactivation. AC inhibition reduced photodamage of the plasma membrane and subsequent necrosis in neuronal and glial cells. AC activation prevented apoptosis in photosensitized glial cells and stimulated glial proliferation. TK inhibition protected neurons but not glia against photoinduced membrane permeabilization and subsequent necrosis whereas PTP inhibition more strongly protected glial cells. Therefore, both signaling pathways involving cAMP and phosphotyrosines might contribute to the maintenance of neuronal activity and the integrity of the neuronal and glial plasma membranes. Adenylate cyclase but not phosphotyrosine signaling pathways modulated glial apoptosis and proliferation under photooxidative stress.

Adenylyl Cyclases↗

Activity-dependent change in morphology of the glial tubular lattice of the crayfish medial giant nerve fiber.

An evaluation of electron micrographs of stimulated nerve fibers used to investigate the effect of action potential generation on the structure-function relationship between axons and its associated glial cells revealed that what was at first thought to be stimulation-induced damage to the glia was, in fact, limited to volume expansion and disaggregation of the glial tubular lattice. All other structures appeared well preserved and otherwise normal. Using a 4-point subjective scale for evaluation by two investigators, 50-Hz stimulation for 2 min was observed to cause a volume expansion and disaggregation of the tubular lattice. Quantitatively, the internal diameter of the stimulated tubular lattice increased 65% above the unstimulated control (50.96 +/- 2.09 nm and 30.81 +/- 0.87 nm, respectively, P < or = 0.001). Stimulation had its greatest effect on tubular lattice volume and organization in the adaxonal glial layer and a decreasing effect as distance from the giant axon increased. These effects are reversible since the tubular lattice diameter and degree of disaggregation preserved 10 min after the cessation of stimulation were not found to be different from their unstimulated paired controls. Axons injected with TEA, a voltage-gated potassium channel blocker, prevented stimulation-induced volume expansion and disaggregation of tubular lattice structure. These results are consistent with an active uptake of K+ with obligated water or, alternatively, hyperosmotic K+ uptake and a fixation-induced increase in water permeation. Either mechanism of K+ uptake would result in tubular lattice volume expansion and disaggregation and suggests that the tubular lattice serves a larger role than a simple trans-glial diffusion pathway.

Action Potentials↗

Axon-glia transfer of a protein and a carbohydrate.

We have investigated the transfer of a fluorescent protein, the fluorescein isothiocyanate derivative of bovine serum albumin (FITC-BSA), and a fluorescent carbohydrate, FITC-dextran, from the crayfish medial giant axon (MGA) to the periaxonal glial cells. The dialyzed tracer was injected into one of the two MGAs, and, after a transfer period of 15-60 min, the tissue was fixed for histological examination of fluorescence distribution. With each tracer, the periaxonal sheath of the injected MGA was specifically labeled. Similar results were obtained with several different fixatives. During the transfer period, there was no appreciable change in the resting potential or conducted action potential of the MGA or in the resting potentials of the adaxonal glial cells. Polyacrylamide gel electrophoresis indicated that the axoplasmic and sheath fluorescence was produced by the intact tracers. These results suggest that "foreign" macromolecules can be exchanged from crayfish axons to glia under physiological conditions. Such transfers may indicate a substantial intercellular traffic of molecules or a means whereby neurons can eliminate waste materials.

Animals↗

Potassium channels in crustacean glial cells.

Unitary currents through single ion channels in the glial cells, which ensheath the abdominal stretch receptor neurons of the crayfish, were characterized with respect to their basic kinetic properties. In cell-attached and excised patches two types of Ca(++)-independent K+ channels were observed with slope conductances of 57 pS and 96 pS in symmetrical K+ solution. The 57 pS K+ channel was weakly voltage-dependent with a slope of the Po vs. membrane potential relationship of +95 mV for an e-fold change in Po. In addition to the main conductance level, the channel displayed conductance levels of 80 and 109 pS. In excised patches, channel activity of this "subconductance" K+ channel showed "rundown" that could be prevented with 2 mM ATP-Mg on the cytoplasmic side of the membrane. The 96 pS K+ channel was strongly voltage-dependent with a slope of +12 mV for an e-fold change in Po. Averaged single-channel currents elicited by voltage jumps proved the channel to be of the delayed rectifying type. Channel activity persisted in excised patches with minimal salt solution and in virtually Ca(++)-free saline. Because of its dependence on intracellular ATP-Mg, the subconductance K+ channel is discussed as a target of modulation by transmitters or peptides via phosphorylation of the channel.

Animals↗

Axon-glia interactions in the crayfish: glial cell oxygen consumption is tightly coupled to axon metabolism.

Oxygen consumption (QO2) of single isolated axons and their associated glial cell sheath was investigated under a variety of conditions to determine the contribution of each cell type to whole tissue QO2. It was found that the QO2 of the sheath, in the absence of a functional axon, represented approximately 30% of the total tissue QO2. When the axon was injected with carboxyatractyloside, an inhibitor of mitochondrial oxidative phosphorylation that is membrane impermeant, electrophysiological properties of the axon were not affected and glial sheath respiratory activity was stimulated by 1.7 to 2.7 times the untreated control level. These results suggest that glial cell metabolic activity is regulated by the metabolic activity of the axon. Depending on the experimental conditions the glial sheath accounts for 30% to nearly 100% of the QO2 of axon-glial cell tissue. On the basis of these and morphometric measurements we estimate that in a normally functioning axon-glial cell system the glial sheath accounts for 90% of the tissue QO2.

Animals↗

Glutamine cycle enzymes in the crayfish giant nerve fiber: implications for axon-to-glia signaling.

Two of the key enzymes involved in glutamate metabolism, glutaminase and glutamine synthetase, were quantitatively localized to axons and glia of the crayfish giant nerve fiber by immunocytochemistry and electron microscopy of antibody-linked gold microspheres. In Western blots, rabbit antisera for glutamine synthetase and glutaminase specifically recognized crayfish polypeptides corresponding approximately in size to subunits of purified mammalian brain enzymes. Glutamine synthetase immunoreactivity was found to be 11 times greater in the adaxonal glial cells than in the axon. Glutaminase immunoreactivity was found in somewhat greater concentration (2.5:1) in glia as compared to axoplasm. Glutamate immunoreactivity also was evaluated and found to be present in high concentration in both glia and axons, as might be expected for an important substrate of cellular metabolism. Using radiolabeled substrates it was demonstrated that glutamine and glutamate were interconverted by the native enzymes in the intact crayfish giant nerve fiber and that the formation of glutamine from glutamate occurred in the axoplasm-free nerve fiber, the cellular component of which is primarily periaxonal glia. The results of this investigation provide immunocytochemical and metabolic evidence consistent with an intercellular glutamine cycle that modulates the concentration of periaxonal glutamate and glutamine in a manner similar to that described for perisynaptic regions of the vertebrate central nervous system. These findings further corroborate previous electrophysiological evidence that glutamate serves as the axon-to-glial cell neurochemical signal that activates glial cell mechanisms responsible for periaxonal ion homeostasis.

Animals↗

Three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa: a two-domain protein with a calcium binding parallel beta roll motif.

The three-dimensional structure of the alkaline protease of Pseudomonas aeruginosa, a zinc metalloprotease, has been solved to a resolution of 1.64 A by multiple isomorphous replacement and non-crystallographic symmetry averaging between different crystal forms. The molecule is elongated with overall dimensions of 90 x 35 x 25 A; it has two distinct structural domains. The N-terminal domain is the proteolytic domain; it has an overall tertiary fold and active site zinc ligation similar to that of astacin, a metalloprotease isolated from a European freshwater crayfish. The C-terminal domain consists of a 21-strand beta sandwich. Within this domain is a novel 'parallel beta roll' structure in which successive beta strands are wound in a right-handed spiral, and in which Ca2+ ions are bound within the turns between strands by a repeated GGXGXD sequence motif, a motif that is found in a diverse group of proteins secreted by Gram-negative bacteria.

Amino Acid Sequence↗

Purification and catalytic properties of glutathione transferase from the hepatopancreas of crayfish macrobrachium vollenhovenii (herklots).

Glutathione transferase from the hepatopancreas of fresh water crayfish Macrobrachium vollenhovenii was purified to apparent homogeneity by ion-exchange chromatography on DEAE-cellulose and by gel filtration on Sephadex G-100. The enzyme appeared to be a homodimer with molecular weight (Mr) of 46.0 +/- 1.4 kDa and a subunit Mr of 24.1 +/- 0.35 kDa. Chromatofocusing of the apparently pure enzyme revealed microheterogeneity and resolved it into two isozymic peaks, which were eluted at pH 8.36 and 8.22 respectively. Inhibition studies showed that the I50 value for cibacron blue, S-hexylglutathione, hematin, and N-ethylmaleimide (NEM) were 0.01 microM, 340 microM, 5 microM and 33 mM respectively. Out of the several substrates tested, only 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole could be conjugated with glutathione. Chemical modification studies with DTNB revealed that two sulphydryl groups per dimer were essential to the activity of the enzymes. On the basis of structural and catalytic characteristics, M. vollenhovenii GST seems close, tentatively, to the omega and zeta classes of GST. Initial-velocity studies of the enzyme are consistent with a steady-state random kinetic mechanism. Denaturation and renaturation studies with guanidine HCl (Gdn-HCl) revealed that though low Gdn-HCl concentrations (less than 0.5 M) denatured the enzyme, the enzyme was able to renature completely (100%). At higher concentration of the denaturant (0.5-4 M), refolding studies indicated that complete renaturation was not achieved. The extent of renaturation was however a function of protein concentration. Our results are consistent with a three-state unfolding process.

Animals↗