RESUMO
Similar to hair cells in the mammalian cochlear system, mechanosensory neurons in the Central American wandering spider Cupiennius salei are strongly innervated by efferent fibers that originate from neurons whose somata are located in the central nervous system (CNS). In both the mammalian and arachnid systems, efferent fibers have been shown to co-express two or more transmitters; however, our understanding regarding co-transmission and how it affects sensory signal transduction and processing in these systems is only fragmentary. The spider model system is exceptionally suitable for this type of investigation due to the large size and easy accessibility of the sensory and efferent neurons in this system. Thus far, GABA and glutamate have been identified as the main fast-acting transmitters in efferent axons that form synaptic contacts onto sensory neurons in slit sense organs. Ultrastructural investigations suggest an abundance of neuropeptides within these peripheral synapses. In an effort to identify these peptides and conduct functional studies, we have employed immunohistochemistry to investigate whether the neuropeptide proctolin is present in neurons of the leg ganglia and in peripheral leg structures. Here, we demonstrate that ~ 73% of all neurons in the CNS of C. salei show proctolin-like immunoreactivity (proc-LIR) including the leg ganglia. We demonstrate that both strongly and weakly labeled neurons can be distinguished. The majority of proc-LIR neurons show weak labeling intensity and ~ 86.2% co-localize with glutamate. In future experiments, we plan to undertake functional studies to investigate the significance of this co-expression, which has yet to be investigated.
Assuntos
Sistema Nervoso Central/metabolismo , Ácido Glutâmico/metabolismo , Neuropeptídeos/metabolismo , Oligopeptídeos/metabolismo , Células Receptoras Sensoriais/metabolismo , Venenos de Aranha/metabolismo , AnimaisRESUMO
FMRFamide-related proteins have been described in both vertebrate and invertebrate nervous systems and have been suggested to play important roles in a variety of physiological processes. One proposed function is the modulation of signal transduction in mechanosensory neurons and their associated behavioral pathways in the Central American wandering spider Cupiennius salei; however, little is known about the distribution and abundance of FMRFamide-related proteins (FaRPs) within this invertebrate system. We employ immunohistochemistry, Hoechst nuclear stain and confocal microscopy of serial sections to detect, characterize and quantify FMRFamide-like immunoreactive neurons throughout all ganglia of the spider brain and along leg muscle. Within the different ganglia, between 3.4 and 12.6% of neurons showed immunolabeling. Among the immunoreactive cells, weakly and strongly labeled neurons could be distinguished. Between 71.4 and 81.7% of labeled neurons showed weak labeling, with 18.3 to 28.6% displaying strong labeling intensity. Among the weakly labeled neurons were characteristic motor neurons that have previously been shown to express ɣ-aminobutyric acid or glutamate. Ultrastructural investigations of neuromuscular junctions revealed mixed presynaptic vesicle populations including large electron-dense vesicles characteristic of neuropeptides. Double labeling for glutamate and FaRPs indicated that a subpopulation of neurons may co-express both neuroactive compounds. Our findings suggest that FaRPs are expressed throughout all ganglia and that different neurons have different expression levels. We conclude that FaRPs are likely utilized as neuromodulators in roughly 8% of neurons in the spider nervous system and that the main transmitter in a subpopulation of these neurons is likely glutamate.
Assuntos
Encéfalo/metabolismo , FMRFamida/metabolismo , Gânglios dos Invertebrados/metabolismo , Neurônios/metabolismo , Aranhas/metabolismo , Animais , Feminino , Neurotransmissores/metabolismoRESUMO
The spider Cupiennius salei is a well-established model for investigating information processing in arthropod sensory systems. Immunohistochemistry has shown that several neurotransmitters exist in the C. salei nervous system, including GABA, glutamate, histamine, octopamine and FMRFamide, while electrophysiology has found functional roles for some of these transmitters. There is also evidence that acetylcholine (ACh) is present in some C. salei neurons but information about the distribution of cholinergic neurons in spider nervous systems is limited. Here, we identify C. salei genes that encode enzymes essential for cholinergic transmission: choline ACh transferase (ChAT) and vesicular ACh transporter (VAChT). We used in-situ hybridization with an mRNA probe for C. salei ChAT gene to locate somata of cholinergic neurons in the central nervous system and immunohistochemistry with antisera against ChAT and VAChT to locate these proteins in cholinergic neurons. All three markers labeled similar, mostly small neurons, plus a few mid-sized neurons, in most ganglia. In the subesophageal ganglia, labeled neurons are putative efferent, motor or interneurons but the largest motor and interneurons were unlabeled. Groups of anti-ChAT labeled small neurons also connect the optic neuropils in the spider protocerebrum. Differences in individual cell labeling intensities were common, suggesting a range of ACh expression levels. Double-labeling found a subpopulation of anti-VAChT-labeled central and mechanosensory neurons that were also immunoreactive to antiserum against FMRFamide-like peptides. Our findings suggest that ACh is an important neurotransmitter in the C. salei central and peripheral nervous systems.
Assuntos
Neurônios Colinérgicos/citologia , FMRFamida/análise , Células Receptoras Sensoriais/citologia , Aranhas/anatomia & histologia , Aranhas/citologia , Animais , Proteínas de Artrópodes/análise , Colina O-Acetiltransferase/análise , Feminino , Mecanotransdução Celular , Proteínas Vesiculares de Transporte de Acetilcolina/análiseRESUMO
Spider sensory neurons with cell bodies close to various sensory organs are innervated by putative efferent axons from the central nervous system (CNS). Light and electronmicroscopic imaging of immunolabeled neurons has demonstrated that neurotransmitters present at peripheral synapses include γ-aminobutyric acid (GABA), glutamate and octopamine. Moreover, electrophysiological studies show that these neurotransmitters modulate the sensitivity of peripheral sensory neurons. Here, we undertook immunocytochemical investigations to characterize GABA and glutamate-immunoreactive neurons in three-dimensional reconstructions of the spider CNS. We document that both neurotransmitters are abundant in morphologically distinct neurons throughout the CNS. Labeling for the vesicular transporters, VGAT for GABA and VGLUT for glutamate, showed corresponding patterns, supporting the specificity of antibody binding. Whereas some neurons displayed strong immunolabeling, others were only weakly labeled. Double labeling showed that a subpopulation of weakly labeled neurons present in all ganglia expresses both GABA and glutamate. Double labeled, strongly and weakly labeled GABA and glutamate immunoreactive axons were also observed in the periphery along muscle fibers and peripheral sensory neurons. Electron microscopic investigations showed presynaptic profiles of various diameters with mixed vesicle populations innervating muscle tissue as well as sensory neurons. Our findings provide evidence that: (1) sensory neurons and muscle fibers are innervated by morphologically distinct, centrally located GABA- and glutamate immunoreactive neurons; (2) a subpopulation of these neurons may co-release both neurotransmitters; and (3) sensory neurons and muscles are innervated by all of these neurochemically and morphologically distinct types of neurons. The biochemical diversity of presynaptic innervation may contribute to how spiders filter natural stimuli and coordinate appropriate response patterns.
Assuntos
Sistema Nervoso Central/metabolismo , Ácido Glutâmico/metabolismo , Neurônios/metabolismo , Aranhas/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Esôfago/metabolismo , Feminino , Imunofluorescência , Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Gânglios dos Invertebrados/metabolismo , Imageamento Tridimensional , Músculos/metabolismo , Músculos/ultraestrutura , Aranhas/ultraestrutura , Sinapses/metabolismo , Sinapses/ultraestruturaRESUMO
Neurodegenerative diseases are among the main causes of death in the United States, leading to irreversible disintegration of neurons. Despite intense international research efforts, cellular mechanisms that initiate neurodegeneration remain elusive, thus inhibiting the development of effective preventative and early onset medical treatment. To identify underlying cellular mechanisms that initiate neuron degeneration, it is critical to identify histological and cellular hallmarks that can be linked to underlying biochemical processes. Due to the poor tissue preservation of degenerating mammalian brain tissue, our knowledge regarding histopathological hallmarks of early to late degenerative stages is only fragmentary. Here, we introduce a novel model organism to study histological hallmarks of neurodegeneration, the spider Cupiennius salei. We utilized toluidine blue-stained 0.9-µm serial semithin and 50-nm ultrathin sections of young and old spider nervous tissue. Our findings suggest that the initial stages of neurodegeneration in spiders may be triggered by (1) dissociation of neuron- and glia-derived microtubules, and (2) the weakening of microtubule-associated desmosomal junctions that lead to the unraveling of neuron-insulating macroglia, compromising the structural integrity of affected neurons. The involvement of macroglia in the disposal of neuronal debris described here-although different in the proposed transport mechanisms-shows resemblance to the mammalian glymphatic system. We propose that this model system is highly suitable to investigate invertebrate neurodegenerative processes from early onset to scar formation and that this knowledge may be useful for the study of neurodegeneration in mammalian tissue.
Assuntos
Neurônios , Aranhas , Animais , Adesão Celular , Neurônios/metabolismo , Encéfalo , Microtúbulos , Invertebrados , MamíferosRESUMO
Aplysia VAP-33 (VAMP-associated protein) has been previously proposed to be involved in the control of neurotransmitter release. Here, we show that a Drosophila homolog of VAP-33, DVAP-33A, is localized to neuromuscular junctions. Loss of DVAP-33A causes a severe decrease in the number of boutons and a corresponding increase in bouton size. Conversely, presynaptic overexpression of DVAP-33A induces an increase in the number of boutons and a decrease in their size. Gain-of-function experiments show that the presynaptic dose of DVAP-33A tightly modulates the number of synaptic boutons. Our data also indicate that the presynaptic microtubule architecture is severely compromised in DVAP-33A mutants. We propose that a DVAP-33A-mediated interaction between microtubules and presynaptic membrane plays a pivotal role during bouton budding.
Assuntos
Proteínas de Transporte/isolamento & purificação , Proteínas de Drosophila , Drosophila melanogaster/crescimento & desenvolvimento , Dosagem de Genes , Proteínas de Membrana/isolamento & purificação , Microtúbulos/metabolismo , Sistema Nervoso/crescimento & desenvolvimento , Junção Neuromuscular/crescimento & desenvolvimento , Terminações Pré-Sinápticas/metabolismo , Proteínas de Transporte Vesicular , Animais , Transporte Axonal/genética , Proteínas de Transporte/genética , Diferenciação Celular/fisiologia , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Testes Genéticos , Imuno-Histoquímica , Proteínas de Insetos/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Dados de Sequência Molecular , Mutação/genética , Sistema Nervoso/metabolismo , Sistema Nervoso/ultraestrutura , Junção Neuromuscular/metabolismo , Junção Neuromuscular/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/fisiologia , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestrutura , Proteínas Supressoras de Tumor/metabolismoRESUMO
Presynaptic GABAA receptors modulate synaptic transmission in several areas of the CNS but are not known to have this action in the cerebral cortex. We report that GABAA receptor activation reduces hippocampal mossy fibers excitability but has the opposite effect when intracellular Cl- is experimentally elevated. Synaptically released GABA mimics the effect of exogenous agonists. GABAA receptors modulating axonal excitability are tonically active in the absence of evoked GABA release or exogenous agonist application. Presynaptic action potential-dependent Ca2+ transients in individual mossy fiber varicosities exhibit a biphasic dependence on membrane potential and are altered by GABAA receptors. Antibodies against the alpha2 subunit of GABAA receptors stain mossy fibers. Axonal GABAA receptors thus play a potentially important role in tonic and activity-dependent heterosynaptic modulation of information flow to the hippocampus.
Assuntos
Fibras Musgosas Hipocampais/metabolismo , Receptores de GABA-A/metabolismo , Transmissão Sináptica/fisiologia , Animais , Agonistas de Receptores de GABA-A , Cobaias , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Fibras Musgosas Hipocampais/efeitos dos fármacos , Fibras Musgosas Hipocampais/ultraestrutura , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/ultraestrutura , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismoRESUMO
Retrieval of synaptic vesicles from the membrane of neurons is crucial to maintain normal rates of neurotransmitter release. Photoreceptor terminals of the fly's eye release neurotransmitter in a tonic manner. They therefore rely heavily on vesicle regeneration. Null mutations in endophilin (endo) block clathrin-mediated endocytosis at the Drosophila neuromuscular junction, where previous analysis of hypomorphic mutations has suggested a function for Endophilin (Endo) before vesicle fission, during membrane bending. Here, at fly photoreceptor synapses, we show that Endo is localized to synaptic vesicles at sites of endocytosis that are glial invaginations called capitate projections, and that when the photoreceptor synapses lack Endo they are impaired in their ability to release neurotransmitter. Detailed ultrastructural analysis of endo null mutant photoreceptor synapses fails to reveal a defect at early stages of vesicle reformation but, instead, reveals an accumulation of clusters of electron-dense, apparently nonfunctional, late endocytotic vesicles. Using dynamin;endo double-mutant photoreceptors, we provide further evidence that ultimately the function of Endophilin is required late in endocytosis, allowing vesicles to progress through the synaptic vesicle cycle.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Drosophila/fisiologia , Endocitose/fisiologia , Neuroglia/metabolismo , Células Fotorreceptoras de Invertebrados/metabolismo , Animais , Proteínas de Transporte/genética , Clatrina/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Eletrorretinografia , Epistasia Genética , Luz , Atividade Motora/efeitos da radiação , Mutação , Estimulação Luminosa , Células Fotorreceptoras de Invertebrados/efeitos da radiação , Células Fotorreceptoras de Invertebrados/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Sinapses/metabolismo , Sinapses/ultraestrutura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Vesículas Transportadoras/metabolismoRESUMO
Several neurotransmitters, including GABA acting at presynaptic GABA(B) receptors, modulate glutamate release at synapses between hippocampal mossy fibers and CA3 pyramidal neurons. This phenomenon gates excitation of the hippocampus and may therefore prevent limbic seizure propagation. Here we report that status epilepticus, triggered by either perforant path stimulation or pilocarpine administration, was followed 24 hr later by a loss of GABA(B) receptor-mediated heterosynaptic depression among populations of mossy fibers. This was accompanied by a decrease in the sensitivity of mossy fiber transmission to the exogenous GABA(B) receptor agonist baclofen. Autoradiography revealed a reduction in GABA(B) receptor binding in the stratum lucidum after status epilepticus. Failure of GABA(B) receptor-mediated modulation of mossy fiber transmission at mossy fibers may contribute to the development of spontaneous seizures after status epilepticus.
Assuntos
Fibras Musgosas Hipocampais/fisiopatologia , Plasticidade Neuronal , Receptores de GABA-B/fisiologia , Estado Epiléptico/fisiopatologia , Sinapses/fisiologia , Animais , Baclofeno/farmacologia , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores , Agonistas GABAérgicos/farmacologia , Masculino , Fibras Musgosas Hipocampais/química , Fibras Musgosas Hipocampais/efeitos dos fármacos , Inibição Neural , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Estado Epiléptico/metabolismo , Transmissão Sináptica , Ácido gama-Aminobutírico/análise , Ácido gama-Aminobutírico/metabolismoRESUMO
Two types of sensory organs in crustaceans and arachnids, the various mechanoreceptors of spiders and the crustacean muscle receptor organs (MRO), receive extensive efferent synaptic innervation in the periphery. Although the two sensory systems are quite different-the MRO is a muscle stretch receptor while most spider mechanoreceptors are cuticular sensilla-this innervation exhibits marked similarities. Detailed ultrastructural investigations of the synaptic contacts along the mechanosensitive neurons of a spider slit sense organ reveal four important features, all having remarkable resemblances to the synaptic innervation at the MRO: (1) The mechanosensory neurons are accompanied by several fine fibers of central origin, which are presynaptic upon the mechanoreceptors. Efferent control of sensory function has only recently been confirmed electrophysiologically for the peripheral innervation of spider slit sensilla. (2) Different microcircuit configuration types, identified on the basis of the structural organization of their synapses. (3) Synaptic contacts, not only upon the sensory neurons but also between the efferent fibers themselves. (4) Two identified neurotransmitter candidates, GABA and glutamate. Physiological evidence for GABAergic and glutamatergic transmission is incomplete at spider sensilla. Given that the sensory neurons are quite different in their location and origin, these parallels are most likely convergent. Although their significance is only partially understood, mostly from work on the MRO, the close similarities seem to reflect functional constraints on the organization of efferent pathways in the brain and in the periphery.
Assuntos
Crustáceos , Mecanorreceptores , Neurônios Eferentes , Aranhas , Transmissão Sináptica , Animais , Crustáceos/fisiologia , Crustáceos/ultraestrutura , Imuno-Histoquímica , Mecanorreceptores/fisiologia , Mecanorreceptores/ultraestrutura , Microscopia Eletrônica , Neurônios Eferentes/fisiologia , Neurônios Eferentes/ultraestrutura , Aranhas/fisiologia , Aranhas/ultraestrutura , Transmissão Sináptica/fisiologiaRESUMO
The RNA-binding protein Sam68 has been implicated in the signal-dependent processing of pre-mRNA and in the utilization of intron-containing retroviral mRNAs. Sam68 is predominantly nuclear but exhibits remarkable binding affinity for signalling proteins located at the membrane. We have investigated the subcellular distribution of Sam68 in adult rat cortex and hippocampus. Subcellular fractionation showed that the protein was most abundant in nuclei but also was present at a significant level in the cytosol and membrane fractions, including light and synaptic membranes derived from crude synaptosomes. Sam68 extracted from the synaptosomal fraction cosedimented with polysomes on sucrose gradients. In agreement with these findings, immunohistochemical staining indicated that Sam68 was concentrated in neuronal nuclei but was also detectable in the soma and dendrites. Sam68 immunoreactivity examined at the ultrastructural level was found to associate with dendritic microtubules, endoplasmic reticulum, and free polyribosomes, sometimes close to synapses. A combination of immunoprecipitation and RT-PCR directly confirmed that Sam68 was bound to polyadenylated mRNA in cortical lysates. The alphaCaMKII mRNA was identified as one of the coprecipitated transcripts; in contrast, the gephyrin and NR1-1 mRNAs were not coprecipitated, indicating a certain degree of sequence specificity in the association. In electrophoretic mobility shift assays, recombinant GST-Sam68 as well as brain-derived Sam68 bound with high affinity to the alphaCaMKII 3' untranslated region. These results suggest that Sam68 may accompany and, conceivably, regulate mature mRNAs during nuclear export, somatodendritic transport, and translation.
Assuntos
Córtex Cerebral/metabolismo , Dendritos/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Córtex Cerebral/citologia , Hipocampo/citologia , Imuno-Histoquímica , Masculino , Neurônios/ultraestrutura , Polirribossomos/metabolismo , Transporte Proteico/fisiologia , Ratos , Ratos Endogâmicos , Frações Subcelulares/metabolismo , Sinaptossomos/metabolismoRESUMO
Depending on their subunit composition, GABA(A) receptors can be highly sensitive to Zn(2+). Although a pathological role for Zn(2+)-mediated inhibition of GABA(A) receptors has been postulated, no direct evidence exists that endogenous Zn(2+) can modulate GABAergic signaling in the brain. A possible explanation is that Zn(2+) is mainly localized to a subset of glutamatergic synapses. Hippocampal mossy fibers are unusual in that they are glutamatergic but have also been reported to contain GABA and Zn(2+). Here, we show, using combined Timm's method and post-embedding immunogold, that the same mossy fiber varicosities can contain both GABA and Zn(2+). Chelating Zn(2+) with either calcium-saturated EDTA or N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine had no effect on stratum-radiatum-evoked inhibitory postsynaptic currents (IPSCs), but enhanced IPSCs evoked by stimuli designed to recruit dentate granule cells. We also show that IPSCs recorded in CA3 pyramidal neurons in acute hippocampal slices are depressed by exogenous Zn(2+). This depression was of similar amplitude whether the IPSCs were evoked by stimulation in s. radiatum (to recruit local interneurons) or in the s. granulosum of the dentate gyrus (to recruit mossy fibers). These results show for the first time that GABAergic IPSCs can be modulated by endogenous Zn(2+) and are consistent with GABA release at Zn(2+)-containing mossy fiber synapses.