RESUMO
Ascidians are interesting neurobiological models because of their evolutionary position as a sister-group of vertebrates and the high regenerative capacity of their central nervous system (CNS). We investigated the degeneration and regeneration of the cerebral ganglion complex of the ascidian Styela plicata following injection of the niacinamide antagonist 3-acetylpyridine (3AP), described as targeting the CNS of several vertebrates. For the analysis and establishment of a new model in ascidians, the ganglion complex was dissected and prepared for transmission electron microscopy (TEM), routine light microscopy (LM), immunohistochemistry and Western blotting, 1 or 10 days after injection of 3AP. The siphon stimulation test (SST) was used to quantify the functional response. One day after the injection of 3AP, CNS degeneration and recruitment of a non-neural cell type to the site of injury was observed by both TEM and LM. Furthermore, weaker immunohistochemical reactions for astrocytic glial fibrillary acidic protein (GFAP) and neuronal ßIII-tubulin were observed. In contrast, the expression of caspase-3, a protein involved in the apoptotic pathway, and the glycoprotein CD34, a marker for hematopoietic stem cells, increased. Ten days after the injection of 3AP, the expression of markers tended toward the original condition. The SST revealed attenuation and subsequent recovery of the reflexes from 1 to 10 days after 3AP. Therefore, we have developed a new method to study ascidian neural degeneration and regeneration, and identified the decreased expression of GFAP and recruitment of blood stem cells to the damaged ganglion as reasons for the success of neuroregeneration in ascidians.
Assuntos
Gânglios dos Invertebrados/fisiopatologia , Regeneração Nervosa/fisiologia , Urocordados/fisiologia , Animais , Antígenos CD34/metabolismo , Células Sanguíneas/fisiologia , Western Blotting , Caspase 3/metabolismo , Gânglios dos Invertebrados/ultraestrutura , Proteína Glial Fibrilar Ácida/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Modelos Animais , Degeneração Neural , Neuroglia/fisiologia , Neuroglia/ultraestrutura , Piridinas , Tubulina (Proteína)/metabolismo , Urocordados/ultraestruturaRESUMO
A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing.
Assuntos
Sistema Nervoso Central/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Loligo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sistema Nervoso Central/ultraestrutura , Gânglios dos Invertebrados/metabolismo , Gânglios dos Invertebrados/ultraestrutura , Ribonucleoproteínas Nucleares Heterogêneas/química , Ribonucleoproteínas Nucleares Heterogêneas/isolamento & purificação , Loligo/ultraestrutura , Peso Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/isolamento & purificação , Lobo Óptico de Animais não Mamíferos/metabolismo , Lobo Óptico de Animais não Mamíferos/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/isolamento & purificação , Ribonucleoproteínas Citoplasmáticas Pequenas/genética , Ribonucleoproteínas Citoplasmáticas Pequenas/metabolismo , Sinaptossomos/metabolismo , Sinaptossomos/ultraestruturaRESUMO
The aim of this study was to investigate the ultrastructure of the interface zone between the nervous tissue and the connective vascular sheath that surround the central ganglia of the terrestrial snail of Megalobulimus abbreviatus and test its permeability using lanthanum as an electron dense tracer. To this purpose, ganglia from a group of snails were fixed by immersion in a 2% colloidal lanthanum solution, and a second group of animals was injected in the foot with either a 2%, 10% or 20% lanthanum nitrate solution and then sacrificed 2 or 24 h after injection. Ganglia from both groups were processed for transmission electron microscopy. The vascular endothelium, connective tissue and basal lamina of variable thickness that ensheathe the nervous tissue and glial cells of the nervous tissue constitute the interface zone between the haemolymph and the neurones. The injected lanthanum reached the connective tissue of the perineural capsule; however, it did not permeate into the nervous tissue because the basal lamina interposed between both tissues interrupted this passage. Moreover, the ganglia fixed with colloidal lanthanum showed electron dense precipitates between the glial processes in the area adjacent to the basal lamina. It can be concluded from these findings that, of the different components of the haemolymph-neuronal interface, only the basal lamina, between the perineural capsule and the nervous tissue, limits the traffic of substances to and from the central nervous system of this snail.
Assuntos
Gânglios dos Invertebrados/metabolismo , Gânglios dos Invertebrados/ultraestrutura , Hemolinfa/metabolismo , Caramujos/metabolismo , Caramujos/ultraestrutura , Animais , Lantânio , Permeabilidade , Especificidade da EspécieRESUMO
Ribosomes and polyribosomes were detected by immuno-electron microscopy in the giant axon and small axons of the squid using a polyclonal antibody against rat brain ribosomes. The ribosomal fraction used as antigen was purified by ultracentrifugation on a sucrose density gradient and shown to contain ribosomal RNAs and native ribosomes. The polyclonal antibody raised in rabbits reacted with at least ten proteins on immunoblots of purified rat brain ribosomes as well as with a set of multiple ribosomal proteins prepared from the squid giant fiber lobe. Immunoreactions were performed on cryostat sections of the stellate nerve cut at a distance of more than 3 cm from the stellate ganglion, using pre-embedding techniques. Ribosomes and polyribosomes were identified within the giant axon and small axons using electron microscopic methods, following binding of peroxidase-conjugated anti-rabbit IgG secondary antibody. Polysomes were more frequently localized in peripheral axoplasm, including the cortical layer of the giant axon, and were generally associated with unidentified cytoskeletal filaments or with dense matrix material. The immunochemical demonstration of ribosomes and polyribosomes in the giant axon and small axons of the squid confirms similar observations in the squid and the goldfish obtained with the method of electron spectroscopic imaging, and strongly supports the view that a local system of protein synthesis is present in axons. The immunochemical method here described offers an alternative tool for the selective identification of ribosomes, and is likely to prove of value in the analyses of other axonal systems.