Axotomy-Induced Changes of the Protein Profile in the Crayfish Ventral Cord Ganglia.

We suggest novel experimental model of nerve injury-bilaterally axotomized ganglia of the crayfish ventral nerve cord (VNC). Using proteomic antibody microarrays, we showed upregulation of apoptosis execution proteins (Bcl-10, caspases 3, 6, and 7, SMAC/DIABLO, AIF), proapoptotic signaling proteins and transcription factors (c-Myc, p38, E2F1, p53, GADD153), and multifunctional proteins capable of initiating apoptosis in specific situations (p75, NMDAR2a) in the axotomized VNC ganglia. Simultaneously, anti-apoptotic proteins (p21WAF-1, MDM2, Bcl-x, Mcl-1, MKP1, MAKAPK2, ERK5, APP, calmodulin, estrogen receptor) were overexpressed. Some proteins associated with actin cytoskeleton (α-catenin, catenin p120CTN, cofilin, p35, myosin Vα) were upregulated, whereas other actin-associated proteins (ezrin, distrophin, tropomyosin, spectrin (α + ß), phosphorylated Pyk2) were downregulated. Various cytokeratins and ßIV-tubulin, components of intermediate filament and microtubule cytoskeletons, were also downregulated that could be the result of tissue destruction. Downregulation of proteins involved in clathrin vesicle formation (AP2α and AP2γ, adaptin (ß1 + ß2), and syntaxin) indicated impairment of vesicular transport and synaptic processes. The levels of L-DOPA decarboxylase, tyrosine, and tryptophan hydroxylases that mediate synthesis of serotonin, dopamine, norepinephrine, and epinephrine decreased. Overexpression of histone deacetylases HDAC1, HDAC2, and HDAC4 contributed to suppression of transcription and protein synthesis. So, the balance of multidirectional processes aimed either at cell death, or to repair and recovery, determines the cell fate. Present data provide integral, albeit incomplete, view on the nervous tissue response to axotomy. Some of these proteins can be probably potential markers of nerve injury and targets for neuroprotective therapy.

Septin 4, the drosophila ortholog of human CDCrel-1, accumulates in parkin mutant brains and is functionally related to the Nedd4 E3 ubiquitin ligase.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Although most PD cases are sporadic, several loci have been involved in the disease. parkin (PARK) is causative of autosomal recessive juvenile Parkinsonism (ARJP) and encodes an E3 ubiquitin ligase associated with proteasomal degradation. It was proposed that loss of PARK function may lead to the toxic accumulation of its substrates in the brain, thus causing dopaminergic (DA) neuron death. Indeed, the first identified PARK substrate was CDCrel-1, a protein of the Septin family that accumulates in ARPJ brains. Drosophila has been used as a successful model organism to study PD broadly contributing to the understanding of the disease. Consistently, park mutant flies recapitulate some key features of ARJP patients. In this scenario, we previously reported that overexpression of Septin 4 (Sep4), the Drosophila ortholog of CDCrel-1, is toxic for DA neurons and interacts physically with Park, thus suggesting that Sep4 could be a Park substrate in Drosophila. Confirming this hypothesis, we show that Sep4 accumulates in park mutant brains as its human counterpart. Furthermore, we demonstrate that Nedd4, another E3 ubiquitin ligase that may have a role in PD, is functionally related to Sep4 and could be involved in regulating Sep4 subcellular localization/trafficking.

Leucopenia associated with abalone viral ganglioneuritis.

OBJECTIVE: To compare microscopic lesion severity with circulating total haemocyte counts (THC) in abalone affected by abalone viral ganglioneuritis (AVG). RESULTS: A herpes-like virus led to severe mortality in a number of Australian abalone farms in 2006. The infection was associated with severe necrotising ganglioneuritis. The microscopic lesions were well demarcated, affecting the neural tissue almost exclusively and were characterised by necrosis and increased cellularity in affected ganglia and nerves. On two farms, the presence or absence of typical AVG pathology was compared with THC. Those abalone with microscopic lesions of AVG had significantly lower haemocyte counts. The mean THC in abalone with no evidence of AVG from both farms was 4.6 × 10(6)/mL (±0.3 SE). The THC in AVG-affected abalone in farm 1 was 2.8 × 10(6)/mL (±0.5 SE) and farm 2 was 0.98 × 10(6)/mL (±0.4 SE). CONCLUSIONS: Severe AVG is associated with leucopenia in affected abalone.

Sublethal effect of copper toxicity against histopathological changes in the spiny lobster, Panulirus homarus (Linnaeus, 1758).

The tissue damage induced by various organic pollutants in aquatic animals is well documented, but there is a dearth of information relating to the histological alterations induced by copper in the spiny lobster. In the present study, intermoult juveniles of the spiny lobster Panulirus homarus (average weight 150-200 g) were exposed to two sublethal concentrations of the copper (9.55 and 19.1 µg/l) for a period of 28 days. The muscle, hepatopancreas, midgut, gills, thoracic ganglion and heart of the lobsters were then dissected out and processed for light microscopic studies. Exposure to copper was found to result in several alterations in the histoarchitecture of the muscle, hepatopancreas, midgut, gills, thoracic ganglion and heart of P. homarus. The alterations included disruption and congestion of muscle bundle in muscle tissue; blackened haemocytes; distended lumen and F cell; necrosis of the tubules of the hepatopancreas; disarrangement of circular muscle of the midgut; accumulation of haemocytes in the haemocoelic space; swelling and fusion of lamellae; abnormal gill tips; hyperplastic, necrotic, and blackened secondary gill lamellae of the gills; damaged neurosecretory cell and sensory and motor fibre; necrotic of the thoracic ganglion; dispersedly arranged muscle bands; clumped satellite cells and nucleus of the heart. The results obtained suggest that the muscle, hepatopancreas, midgut, gills, thoracic ganglion and heart of lobsters exposed to copper were structurally altered. Such alterations could affect vital physiological functions, such as absorption, storage and secretion of the hepatopancreas, digestion of gut and respiration, osmotic and ionic regulations of the gills, which in turn could ultimately affect the survival and growth of P. homarus. Thus, all possible remedial measures should be adopted to prevent the occurrence of copper contamination in the aquatic environment.

Multiple changes in peptide and lipid expression associated with regeneration in the nervous system of the medicinal leech.

BACKGROUND: The adult medicinal leech central nervous system (CNS) is capable of regenerating specific synaptic circuitry after a mechanical lesion, displaying evidence of anatomical repair within a few days and functional recovery within a few weeks. In the present work, spatiotemporal changes in molecular distributions during this phenomenon are explored. Moreover, the hypothesis that neural regeneration involves some molecular factors initially employed during embryonic neural development is tested. RESULTS: Imaging mass spectrometry coupled to peptidomic and lipidomic methodologies allowed the selection of molecules whose spatiotemporal pattern of expression was of potential interest. The identification of peptides was aided by comparing MS/MS spectra obtained for the peptidome extracted from embryonic and adult tissues to leech transcriptome and genome databases. Through the parallel use of a classical lipidomic approach and secondary ion mass spectrometry, specific lipids, including cannabinoids, gangliosides and several other types, were detected in adult ganglia following mechanical damage to connected nerves. These observations motivated a search for possible effects of cannabinoids on neurite outgrowth. Exposing nervous tissues to Transient Receptor Potential Vanilloid (TRPV) receptor agonists resulted in enhanced neurite outgrowth from a cut nerve, while exposure to antagonists blocked such outgrowth. CONCLUSION: The experiments on the regenerating adult leech CNS reported here provide direct evidence of increased titers of proteins that are thought to play important roles in early stages of neural development. Our data further suggest that endocannabinoids also play key roles in CNS regeneration, mediated through the activation of leech TRPVs, as a thorough search of leech genome databases failed to reveal any leech orthologs of the mammalian cannabinoid receptors but revealed putative TRPVs. In sum, our observations identify a number of lipids and proteins that may contribute to different aspects of the complex phenomenon of leech nerve regeneration, establishing an important base for future functional assays.

Retention and tissue damage of PSP and NSP toxins in shrimp: Is cultured shrimp a potential vector of toxins to human population?

Toxic microalgae outbreaks have caused significant economic losses in the Mexican aquaculture industry. Blooms that involve PSP and NSP phycotoxins are two of the most dangerous, causing harmful effects to the environment, economy and public health. The exact metabolic mechanism of these toxins in shrimp still remains unknown. Because shrimp consume microalgae their edible tissues are clearly possible vectors for human toxic syndrome. This study examined and verified the toxicological effects for white leg shrimp (Litopenaeus vannamei) exposed to different cell densities of Gymnodinium catenatum and Karenia brevis. Acute assays demonstrated good survival rates of shrimp at low densities of dinoflagellates (10(3) cell/L), while mortality and abnormal behavior were observed with higher densities (>10(4) cell/L). Chronic assays showed significant differences in survival rates, percentage of feed and weight gain of organisms exposed to the dinoflagellates with respect to controls. Furthermore, PSP and NSP toxins were detected in all the edible tissues. Gastric glands and muscle retained toxins for a longer period of time compared to other tissues, even after a depuration period. Histology damages were observed in the heart, gastric gland and brain. This study strongly supports that shrimp represent a potential risk for humans as unconventional vectors of phycotoxins.

Nitric oxide regulates axonal regeneration in an insect embryonic CNS.

In higher vertebrates, the central nervous system (CNS) is unable to regenerate after injury, at least partially because of growth-inhibiting factors. Invertebrates lack many of these negative regulators, allowing us to study the positive factors in isolation. One possible molecular player in neuronal regeneration is the nitric oxide (NO)-cyclic guanosine-monophosphate (cGMP) transduction pathway which is known to regulate axonal growth and neural migration. Here, we present an experimental model in which we study the effect of NO on CNS regeneration in flat-fillet locust embryo preparations in culture after crushing the connectives between abdominal ganglia. Using whole-mount immunofluorescence, we examine the morphology of identified serotonergic neurons, which send a total of four axons through these connectives. After injury, these axons grow out again and reach the neighboring ganglion within 4 days in culture. We quantify the number of regenerating axons within this period and test the effect of drugs that interfere with NO action. Application of exogenous NO or cGMP promotes axonal regeneration, whereas scavenging NO or inhibition of soluble guanylyl cyclase delays regeneration, an effect that can be rescued by application of external cGMP. NO-induced cGMP immunostaining confirms the serotonergic neurons as direct targets for NO. Putative sources of NO are resolved using the NADPH-diaphorase technique. We conclude that NO/cGMP promotes outgrowth of regenerating axons in an insect embryo, and that such embryo-culture systems are useful tools for studying CNS regeneration.

Ganglioneuritis causing high mortalities in farmed Australian abalone (Haliotis laevigata and Haliotis rubra).

OBJECTIVE: To investigate an outbreak of sudden severe mortality in farmed abalone from coastal Victoria. RESULTS: The outbreaks occurred almost simultaneously in three farms following abalone movements from the wild and between farms. The initial on farm investigation identified a number of features that when considered together were highly suggestive of an infectious aetiology. In many cases, dead abalone had no significant gross lesions. Others had swollen mouths and some had prolapse and eversion of the radula. Histologically, the lesions centred on the nerves innervating the labial apparatus, primarily the cerebral and buccal ganglia, cerebral commissure and peripheral nerve branches arising from these. Nervous tissue necrosis and haemocyte infiltration were the dominant lesions seen microscopically in affected nerves. CONCLUSIONS: A recent outbreak of mortality in Australian abalone was associated with neurotropic lesions, which have not previously been described in this country. The on farm and between farm pattern of spread of the outbreak, a history of abalone movements linking farms, clinical observation of moribund and dead abalone were all highly suggestive of a virulent infectious agent.

mRNAs encoding the Aplysia homologues of fasciclin-I and beta-thymosin are expressed only in the second phase of nerve injury and are differentially segregated in axons regenerating in vitro and in vivo.

Studies using Aplysia californica have demonstrated that transcription after nerve injury occurs during a rapid, transient first phase and a delayed, prolonged second phase. Although the second phase is especially important for regeneration, the mRNAs produced during this phase have not been identified. We characterized two such mRNAs following axotomy. One encodes a novel fasciclin-I homologue, Aplysia fasciclin-like protein (apFasP), and the other encodes Aplysia beta-thymosin (apbetaT). In addition to mRNA synthesis, proteins required for regeneration must be available at the site of growth, and the transport and local translation of certain extrasomatic mRNAs aids in this process. We found apbetaT and apFasP proteins and mRNA at growth cones in vitro. However, only the mRNA for apbetaT was present in regenerating axons in vivo. This implies that the membrane protein apFasP is supplied by rapid transport from the soma, whereas the soluble apbetaT is synthesized locally.

Seasonal differences and protection by creatine or arginine pretreatment in ischemia of mammalian and molluscan neurons in vitro.

We investigated the dose-response relationship of protection by creatine against ischemic damage, and we asked whether or not such protection may be observed in invertebrate neurons that might provide a simpler experimental model. Rat isolated pyramidal neurons from the CA3 region of hippocampus subjected to ischemia ("in vitro ischemia") showed anoxic depolarization (AD) after 3-7 min of incubation in anoxic medium. Membrane potential (MP) was reduced 25-78% from preanoxic value. Inward current was decreased by an average 49%. Supplementation with creatine protected against these changes, with 1 mM being the minimal effective concentration, 2 mM providing a near-maximal protection, a maximal effect being obtained with 5 mM creatine. No additional protection was provided by up to 20 mM creatine. Isolated giant neurons of Lymnaea stagnalis showed a similar response to in vitro ischemia. However, a clear seasonal dependence of sensitivity of these cells was detected. In cells obtained during summertime (May-August), AD latency ranged from 3 to 10 min; during wintertime (December-March), this response did not occur even after 25-50 min. The addition of creatine to the medium did not cause changes in AD latency, probably because these neurons rely on a phosphoarginine/arginine energy system. However, treatment of the cells, harvested during summertime, with 2 mM arginine did provide clear protection against anoxic-aglycaemic changes. Summing up, besides confirming previous findings on creatine protection in mammalian neurons, we (1) better characterized their dose-response relationship and extended the findings to the CA3 region and to isolated neurons, (2) found that invertebrate neurons are not protected by creatine but by arginine supplementation and (3) reported a novel mechanism of seasonal dependence in sensitivity of in vitro ischemia by invertebrate neurons.

Drosophila Dscam is required for divergent segregation of sister branches and suppresses ectopic bifurcation of axons.

Axon bifurcation results in the formation of sister branches, and divergent segregation of the sister branches is essential for efficient innervation of multiple targets. From a genetic mosaic screen, we find that a lethal mutation in the Drosophila Down syndrome cell adhesion molecule (Dscam) specifically perturbs segregation of axonal branches in the mushroom bodies. Single axon analysis further reveals that Dscam mutant axons generate additional branches, which randomly segregate among the available targets. Moreover, when only one target remains, branching is suppressed in wild-type axons while Dscam mutant axons still form multiple branches at the original bifurcation point. Taken together, we conclude that Dscam controls axon branching and guidance such that a neuron can innervate multiple targets with minimal branching.

Synaptic exocytosis and nervous system development impaired in Caenorhabditis elegans unc-13 mutants.

C. elegans mutants defective in unc-13 exhibited severe behavioral abnormalities including paralyzed locomotion and slow pharyngeal pumping and irregular defecation cycle. Consistent with the phenotypes, the mutants accumulated abnormally high levels of the neurotransmitter acetylcholine and were resistant to acetylcholinesterase inhibitors. The unc-13 gene was expressed in most, if not all, neurons when analyzed by using chimeric constructs consisting of the unc-13 promoter and green fluorescence protein or beta-galactosidase reporter gene. While Ca(2+)-regulated acetylcholine release is lacking, the mutants were still able to release acetylcholine in vivo and in vitro at similar levels to that mediated by the regulated mechanism. Double mutants defective in both unc-13 and other genes involved in synaptic transmission showed the Unc-13 phenotype, rather than other mutant phenotypes, in terms of locomotion as well as of acetylcholine accumulation. Furthermore, electron microscopic reconstruction of the mutant nervous system uncovered that a majority of neurons developed and connected as those in the wild type except for subtle abnormalities including inappropriate connections through gap junctions and morphological alterations of neurons. These results demonstrate that the unc-13 gene product plays an essential role at a late stage in Ca(2+)-regulated synaptic exocytosis. Neurotransmitters released through the Ca(2+)-regulated mechanism are required for, but do not play major roles in the nervous system development. The large amount of Ca(2+)-independent neurotransmitter release observed in the unc-13 mutants suggests that there may be a distinct mechanism from evoked or spontaneous release in neurotransmission.

[Sensory neuronopathy]. / Neuronopatie czuciowe.

The authors present the question of sensory neuronopathy which are disorders affecting intervertebral ganglia. The neuropathological background and clinical symptoms of sensory neuronopathy are emphasised, as well as diagnostic difficulties resulting from a variety of ethiological conditions: toxic, inflammatory, and autoimmunological ones, and from lack of unequivocal clinical criteria enabling a difference diagnosis along with neuropathy and radiculopathy, which in turn requires a broad spectrum of diagnostic tests and prolonged observation of patients. The authors discuss also the clinical outcome, prognosis, and current therapeutic possibilities focusing on intensive immunosuppressive management.

Heptanol exerts epileptiform effects in identified neurons of the buccal ganglia of Helix pomatia.

1-Heptanol (0.2-5.0 mM) known to block electrical contacts was tested under epileptic and non-epileptic conditions in the buccal ganglia of Helix pomatia. Synchronicity of epileptiform activity was not affected. In concentrations below 1 mM, heptanol accelerated epileptiform activity induced by pentylenetetrazol. In concentrations above 1 mM, it evoked epileptiform activity without admixture of an epileptogenic drug. Coupling coefficient was increased and decreased in low and high concentration ranges of heptanol, respectively. The measured decrease of coupling is interpreted as a result of the activation of 'epileptiform' membrane conductances accompanied by decreased length constants of neuronal fibers.

Ultrastructural neuropathologic effects of Taxol on neurons of the freshwater snail Lymnaea stagnalis.

Cerebral ganglia of the freshwater snail Lymnaea stagnalis were incubated in vitro in 10 microM Taxol for 8 and 24 h. Cremophor EL (0.1%) was used as a diluant. The tissue was processed for electron microscopy. Various ultrastructural parameters were assessed quantitatively. Cremophor EL appeared to seriously affect the cell somata of the multipeptidergic caudodorsal cells. In the Cremophor-controls the mean area of Golgi zones, the percentage dense material (neuropeptides) in these zones, the number of large electron dense granules (these are involved in neuropeptide processing) and the mean nuclear heterochromatin clump size, were significantly smaller than in the Ringer-controls, whereas the number of lipid droplets was higher. All these parameters, except for the lipid droplets, were not different in the Cremophor-controls and the Taxol-treated specimens. After 24 h treatment, but not after 8 h, Cremophor EL furthermore induced an increase in the number of axonal microtubules. It is argued that the results might signify activation of the neurons by Cremophor EL. Taxol induced a significant increase in the number of microtubules in axons and cell somata. Furthermore an increase in the number of Golgi zones was observed, suggesting activated neuropeptide synthesis. In all groups immunostaining with antibodies to neuropeptides produced by the caudodorsal cells was normal. Release of neuropeptide (exocytosis) from axon endings was elevated after Taxol treatment, and exceptionally high in specimens cotreated with Taxol and Org 2766 (incubation time 22 h). The effect of Org 2766 and Taxol on the number of microtubules was cumulative.(ABSTRACT TRUNCATED AT 250 WORDS)