RESUMEN

The role of amphiphysin in clathrin-mediated endocytosis of synaptic vesicles is well established. However, it is still uncertain if the protein is also involved in developmental mechanisms, e.g. axon outgrowth and synapse formation. To investigate the developmental changes in the expression of amphiphysin we used the retinotectal system of the chick, a highly ordered and easily accessible primary neuronal pathway. Reverse transcription polymerase chain reaction (RT-PCR) of total RNA from chick retina and tectum revealed first transcripts for amphiphysin, dynamin and synaptotagmin at embryonic day 5 (E5) for both regions. Surprisingly, Western blots of the retina revealed an increase of protein expression for amphiphysin only after E11 in the retina and the tectum. Immunofluorescence for amphiphysin was not detectable before E10 in the developing chick retina, while other presynaptic proteins like synaptotagmin showed already intense signals in the inner and outer plexiform layers. Subsequently, amphiphysin immunoreactivity follows the expression of synaptotagmin and synaptic vesicle protein 2 (SV2) as seen in the retina and the tectum, and exhibits the same staining as the other proteins in the mature chick brain. Ultrastructural data revealed for the first time that amphiphysin is not only limited to conventional synapses but is also abundant in retinal ribbon terminals. Taken together our data reveal that: (i) there is a developmental delay between mRNA transcription and protein expression for key proteins involved in endocytosis; (ii) amphiphysin gets upregulated after synapse formation; and (iii) amphiphysin is present in the synaptic vesicle cycle in retinal ribbon synapses.

Asunto(s)

Proteínas de Unión al Calcio , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Retina/fisiología , Colículos Superiores/fisiología , Envejecimiento , Animales , Embrión de Pollo , Pollos , Dinamina III , Dinaminas , GTP Fosfohidrolasas/genética , Glicoproteínas de Membrana/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Retina/embriología , Retina/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Colículos Superiores/embriología , Colículos Superiores/crecimiento & desarrollo , Sinapsis/fisiología , Sinapsis/ultraestructura , Sinaptotagminas , Transcripción Genética

RESUMEN

Newly synthesized presynaptic integral membrane proteins in neurons are transported in precursor vesicles from the site of protein biosynthesis in the cell body by fast axonal flow to the presynaptic terminal. We followed the path that presynaptic proteins travel on the way to their central targets of the highly ordered primary visual pathway of the chick and analyzed the developmental changes in the expression of synaptic vesicle protein 2 (SV2), synaptotagmin, and syntaxin. Immunofluorescences revealed that: (1) the onset of protein expression in the retinal ganglion cells occurs in a central to peripheral developmental pattern from embryonic day 4 (E4) onward; (2) the proteins were found first in the inner and later in the outer plexiform layer of the retina; and (3) they were redistributed from the photoreceptor inner segments and cell bodies to the terminals in the outer plexiform layer. From E4 onward, immunopositive axons for SV2, synaptotagmin, and syntaxin were found in the optic nerve, disappearing after E9 for SV2 and synaptotagmin. The optic tract was stained for SV2 and synaptotagmin between E7 and E12, for syntaxin until the posthatching period. Finally, immunoreactivities for the investigated proteins were present at the surface of the tectum from E8 onward, when first retinal axons arrived there. The present study revealed that SV2 and synaptotagmin, but not syntaxin, are, expressed in a transient wave that follows the advancement of optic axons and the proteins towards the optic tectum.

Asunto(s)

Embrión de Pollo/metabolismo , Embrión de Pollo/ultraestructura , Proteínas del Tejido Nervioso/metabolismo , Terminales Presinápticos/metabolismo , Retina/embriología , Colículos Superiores/embriología , Animales , Animales Recién Nacidos/anatomía & histología , Animales Recién Nacidos/metabolismo , Axones/ultraestructura , Embrión de Pollo/fisiología , Inmunohistoquímica , Glicoproteínas de Membrana/metabolismo , Quiasma Óptico/embriología , Quiasma Óptico/metabolismo , Quiasma Óptico/ultraestructura , Nervio Óptico/embriología , Nervio Óptico/metabolismo , Nervio Óptico/ultraestructura , Terminales Presinápticos/ultraestructura , Retina/ultraestructura , Colículos Superiores/ultraestructura , Factores de Tiempo , Vías Visuales/embriología , Vías Visuales/metabolismo , Vías Visuales/ultraestructura

RESUMEN

Dynamin I, a GTPase involved in the endocytic cycle of synaptic vesicle membranes, is believed to support axonal outgrowth and/or synaptogenesis. To explore the temporal and spatial patterns of dynamin I distribution in neuronal morphogenesis, we compared the developmental expression of dynamin with the expression of presynaptic membrane proteins such as SV2, synaptotagmin, and syntaxin in the chick primary visual pathway. Western blots of retina and tectum revealed a steady increase of synaptotagmin and syntaxin from embryonic Day 7 (E7) to E11, whereas for the same time frame no detectable increase of dynamin was found. Later stages showed increasing amounts of all tested proteins until the first postnatal week. Immunofluorescence revealed that SV2, synaptotagmin, and syntaxin are present in retinal ganglion cell axons from E4 on. In later stages, the staining pattern in the retina and along the visual pathway paralleled the formation and maturation of axons. In contrast, dynamin is not detectable by immunofluorescence in the developing retina and optic tectum before synapse formation. Our data indicate that, in contrast to the early expression of synaptotagmin, SV2, and syntaxin during axonal growth, dynamin is upregulated after synapse formation, suggesting its function predominantly during and after synaptogenesis but not in axonogenesis.(J Histochem Cytochem 47:1297-1306, 1999)

Asunto(s)

Proteínas de Unión al Calcio , GTP Fosfohidrolasas/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Retina/metabolismo , Colículos Superiores/metabolismo , Envejecimiento , Animales , Western Blotting , Embrión de Pollo , Pollos/crecimiento & desarrollo , Dinamina I , Dinaminas , Técnica del Anticuerpo Fluorescente Indirecta , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Qa-SNARE , Retina/embriología , Retina/crecimiento & desarrollo , Colículos Superiores/embriología , Colículos Superiores/crecimiento & desarrollo , Sinaptotagminas , Factores de Tiempo

RESUMEN

Clathrin-mediated endocytosis involves cycles of assembly and disassembly of clathrin coat components and their accessory proteins. Dephosphorylation of rat brain extract was shown to promote the assembly of dynamin 1, synaptojanin 1, and amphiphysin into complexes that also included clathrin and AP-2. Phosphorylation of dynamin 1 and synaptojanin 1 inhibited their binding to amphiphysin, whereas phosphorylation of amphiphysin inhibited its binding to AP-2 and clathrin. Thus, phosphorylation regulates the association and dissociation cycle of the clathrin-based endocytic machinery, and calcium-dependent dephosphorylation of endocytic proteins could prepare nerve terminals for a burst of endocytosis.

Asunto(s)

Clatrina/metabolismo , Endocitosis , GTP Fosfohidrolasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Subunidades alfa de Complejo de Proteína Adaptadora , Subunidades beta de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Carbazoles/farmacología , Cromatografía de Afinidad , Ciclosporina/farmacología , Dimerización , Dinamina I , Dinaminas , Inhibidores Enzimáticos/farmacología , Alcaloides Indólicos , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Dominios Homologos src

RESUMEN

The GLUT4 glucose transporter continuously recycles between the cell surface and an endosomal compartment in adipocytes. Insulin decreases the rate of GLUT4 endocytosis in addition to increasing its exocytosis. Endocytosis of the transporter is thought to occur at least in part via the clathrin-mediated endocytic system. The protein dynamin is involved in the final stages of clathrin-coated vesicle formation. Here we show that the dynamin II isoform is expressed in 3T3-L1 adipocytes and is present in isolated plasma membrane and low density microsomal fractions. Insulin reduced the levels of dynamin II associated with the plasma membrane by about half, raising the possibility that the hormone may reduce GLUT4 endocytosis by removing dynamin from the cell surface. A fusion protein containing the amphiphysin SH3 domain selectively bound dynamin II from 3T3-L1 adipocyte cell lysates. Microinjection of the fusion protein into these cells inhibited transferrin endocytosis and increased the levels of GLUT4 at the cell surface. Glutathione S-transferase alone, the SH3 domains of spectrin and Crk, and a mutated amphiphysin SH3 domain unable to bind dynamin II did not affect GLUT4 distribution. However, a peptide containing the dynamin II sequence that binds amphiphysin increased the surface presence of GLUT4. Moreover, in cells first treated with insulin to externalize GLUT4, the dynamin peptide, but not an unrelated control peptide, inhibited GLUT4 internalization upon insulin removal. These results suggest that interactions of dynamin II with amphiphysin may play an important role in GLUT4 endocytosis. We hypothesize that insulin may reduce GLUT4 endocytosis by regulating the function of dynamin II at the cell surface, as part of the mechanism to increase glucose uptake.

Asunto(s)

Adipocitos/metabolismo , Membrana Celular/metabolismo , GTP Fosfohidrolasas/metabolismo , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas del Tejido Nervioso/metabolismo , Células 3T3 , Animales , Dinaminas , Endocitosis , Transportador de Glucosa de Tipo 4 , Insulina/metabolismo , Ratones , Dominios Homologos src

Asunto(s)

Vesículas Sinápticas/fisiología , Sinaptosomas/fisiología , Animales , Membrana Celular/fisiología , Clatrina/fisiología , Invaginaciones Cubiertas de la Membrana Celular/fisiología , Dinaminas , Exocitosis/fisiología , GTP Fosfohidrolasas/fisiología , Proteínas de la Membrana/metabolismo , Microtúbulos/fisiología , Modelos Neurológicos , Terminaciones Nerviosas/fisiología , Transmisión Sináptica/fisiología

RESUMEN

Amphiphysin (amphiphysin I), a dominant autoantigen in paraneoplastic Stiff-man syndrome, is a neuronal protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. The yeast homologue of amphiphysin, Rvs167, has pleiotropic functions, including a role in endocytosis and in actin dynamics, suggesting that amphiphysin may also be implicated in the function of the presynaptic actin cytoskeleton. We report here the characterization of a second mammalian amphiphysin gene, amphiphysin II (SH3P9; BIN1), which encodes products primarily expressed in skeletal muscle and brain, as differentially spliced isoforms. In skeletal muscle, amphiphysin II is concentrated around T tubules, while in brain it is concentrated in the cytomatrix beneath the plasmamembrane of axon initial segments and nodes of Ranvier. In both these locations, amphiphysin II is colocalized with splice variants of ankyrin3 (ankyrinG), a component of the actin cytomatrix. In the same regions, the presence of clathrin has been reported. These findings support the hypothesis that, even in mammalian cells, amphiphysin/Rvs family members have a role both in endocytosis and in actin function and suggest that distinct amphiphysin isoforms contribute to define distinct domains of the cortical cytoplasm. Since amphiphysin II (BIN1) was reported to interact with Myc, it may also be implicated in a signaling pathway linking the cortical cytoplasm to nuclear function.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Axones/química , Proteínas Portadoras/análisis , Corteza Cerebral/química , Proteínas Musculares/análisis , Músculo Esquelético/química , Proteínas Nucleares/análisis , Nódulos de Ranvier/química , Proteínas Supresoras de Tumor , Secuencia de Aminoácidos , Animales , Axones/ultraestructura , Secuencia de Bases , Química Encefálica , Células COS , Proteínas Portadoras/genética , Clonación Molecular , Citoplasma/química , ADN Complementario , Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Musculares/genética , Músculo Esquelético/ultraestructura , Proteínas del Tejido Nervioso/química , Proteínas Nucleares/genética , Conejos , Nódulos de Ranvier/ultraestructura , Ratas , Células Tumorales Cultivadas , Dominios Homologos src

RESUMEN

Amphiphysin is an SH3 domain-containing neuronal protein that is highly concentrated in nerve terminals where it interacts via its SH3 domain with dynamin I, a GTPase implicated in synaptic vesicle endocytosis. We show here that the SH3 domain of amphiphysin, but not a mutant SH3 domain, bound with high affinity to a single site in the long proline-rich region of human dynamin I, that this site was distinct from the binding sites for other SH3 domains, and that the mutation of two adjacent amino acids in dynamin I was sufficient to abolish binding. The dynamin I sequence critically required for amphiphysin binding (PSRPNR) fits in the novel SH3 binding consensus identified for the SH3 domain of amphiphysin via a combinatorial peptide library approach: PXRPXR(H)R(H). Our data demonstrate that the long proline-rich stretch present in dynamin I contained multiple SH3 domain binding sites that recognize interacting proteins with high specificity.

Asunto(s)

GTP Fosfohidrolasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Dinamina I , Dinaminas , GTP Fosfohidrolasas/química , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Prolina/metabolismo , Conejos , Análisis de Secuencia , Dominios Homologos src/genética

RESUMEN

The proline-rich COOH-terminal region of dynamin binds various Src homology 3 (SH3) domain-containing proteins, but the physiological role of these interactions is unknown. In living nerve terminals, the function of the interaction with SH3 domains was examined. Amphiphysin contains an SH3 domain and is a major dynamin binding partner at the synapse. Microinjection of amphiphysin's SH3 domain or of a dynamin peptide containing the SH3 binding site inhibited synaptic vesicle endocytosis at the stage of invaginated clathrin-coated pits, which resulted in an activity-dependent distortion of the synaptic architecture and a depression of transmitter release. These findings demonstrate that SH3-mediated interactions are required for dynamin function and support an essential role of clathrin-mediated endocytosis in synaptic vesicle recycling.

Asunto(s)

Endocitosis , GTP Fosfohidrolasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Sitios de Unión , Membrana Celular/ultraestructura , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Dinaminas , Humanos , Lampreas , Microscopía Electrónica , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Prolina/química , Proteínas Recombinantes de Fusión/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Transmisión Sináptica , Vesículas Sinápticas/ultraestructura

RESUMEN

GTP-binding rab proteins, present in synaptic vesicles and endocrine secretory granules, have been shown to be involved in the control of regulated exocytosis. We found rab3 proteins in immunoblots of diverse areas of the mouse central nervous system (spinal cord, olfactory bulb, hippocampus, cerebellum and neocortex). Immunohistochemical observations at light- and electron-microscopical levels in the hippocampus and other areas revealed rab3 proteins in virtually all synaptic fields and terminals of the areas investigated. In the retina, rab3A immunoreactivity was confined to the inner and outer plexiform layers. Ultrastructural examination revealed that rab3A was present in conventional terminals in the inner plexiform layer and in horizontal cell processes of the outer plexiform layer. In contrast ribbon synapses, which play a key role in transferring information from the photoreceptor cells to the central nervous system, were immunonegative. We also tested whether other proteins of the rab3 family are present in ribbon synapses. However, using an antibody recognizing rab3B and rab3C in addition to rab3A, we found no immunoreactivity in these synapses. Interestingly, we observed also no immunoreactivity for synaptosomal-associated protein 25 (SNAP-25) in ribbon synapses, but conventional synapses and horizontal cell processes were heavily stained. Our data show that the known rab3 and SNAP-25 isoforms, which are components of the secretory apparatus of conventional synapses, are absent from ribbon synapses of the retina. Our observations suggest different mechanisms of transmitter exocytosis in conventional and ribbon terminals.

Asunto(s)

Encéfalo/citología , Exocitosis , Proteínas de Unión al GTP/análisis , Proteínas de la Membrana , Proteínas del Tejido Nervioso/análisis , Retina/fisiología , Sinapsis/fisiología , Vesículas Sinápticas/fisiología , Animales , Química Encefálica , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microscopía Inmunoelectrónica , Proteínas del Tejido Nervioso/fisiología , Especificidad de Órganos , Retina/citología , Sinapsis/química , Sinapsis/ultraestructura , Vesículas Sinápticas/química , Proteína 25 Asociada a Sinaptosomas , Proteínas de Unión al GTP rab3

RESUMEN

The expression and subcellular location of glutamate receptor subunits 2&3 was investigated in the developing postnatal cerebellum. Immunoblotting revealed that glutamate receptor subunits 2/3 is expressed in an identical pattern of immunoreactive bands of approximately 108 kDa from postnatal day zero to adult animals. Light microscopy showed that within the cerebellar cortex, GluR 2/3 immunoreactivity was essentially confined to Purkinje neurons. Strong immunostaining could be observed at postnatal days 1-3 within Purkinje cell bodies and primary dendrites. With ongoing development, the cell body and an increasingly elaborate dendritic tree was outlined by immunoreaction product. In adult animals, staining of Purkinje cell dendrites was patchy, and staining intensity of the cell body, in particular, was greatly reduced. Ultrastructural analysis revealed that during early postnatal development, immunoreaction product was localized to the cell membrane, but was not confined to postsynaptic densities. From the second postnatal week, glutamate receptor subunits 2/3 immunoreactivity was largely restricted to postsynaptic densities. These observations reveal a developmentally regulated refinement of the subcellular distribution of defining subunits of the AMPA-type glutamate receptor. The presence of membrane bond receptors prior to the formation of synapses also provides a rationale for the known transmitter-mediated modulation of Purkinje cell dendritogenesis.

Asunto(s)

Corteza Cerebelosa/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/biosíntesis , Células de Purkinje/metabolismo , Receptores AMPA/biosíntesis , Fracciones Subcelulares/química , Factores de Edad , Animales , Corteza Cerebelosa/crecimiento & desarrollo , Corteza Cerebelosa/ultraestructura , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/química , Células de Purkinje/ultraestructura , Receptores AMPA/análisis , Receptores AMPA/química

RESUMEN

Synaptojanin is a nerve terminal protein of relative molecular mass 145,000 which appears to participate with dynamin in synaptic vesicle recycling. The central region of synaptojanin defines it as a member of the inositol-5-phosphatase family, which includes the product of the gene that is defective in the oculocerebrorenal syndrome of Lowe. Synaptojanin has 5-phosphatase activity and its amino-terminal domain is homologous with the yeast protein Sac1 (Rsd1), which is genetically implicated in phospholipid metabolism and in the function of the actin cytoskeleton. The carboxy terminus, which is of different lengths in adult and developing neurons owing to the alternative use of two termination sites, is proline-rich, consistent with the reported interaction of synaptojanin with the SH3 domains of Grb2 (refs 1, 2). Synaptojanin is the only other major brain protein besides dynamin that binds the SH3 domain of amphiphysin, a presynaptic protein with a putative function in endocytosis. Our results suggest a link between phosphoinositide metabolism and synaptic vesicle recycling.

Asunto(s)

Proteínas del Tejido Nervioso/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Terminales Presinápticos/enzimología , Secuencia de Aminoácidos , Animales , Encéfalo/enzimología , Línea Celular , Clonación Molecular , ADN Complementario , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Células PC12 , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Ratas , Homología de Secuencia de Aminoácido , Dominios Homologos src

RESUMEN

The closely related synaptic vesicle membrane proteins synaptophysin and synaptoporin are abundant in the hippocampal formation of the adult rat. But the prenatal hippocampal formation contains only synaptophysin, which is first detected at embryonic day 17 (E17) in perikarya and axons of the pyramidal neurons. At E21 synaptophysin immunoreactivity extends into the apical dendrites of these cells and in newly formed terminals contacting these dendrites. The transient presence of synaptophysin in axons and dendrites suggests a functional involvement of synaptophysin in fibre outgrowth of developing pyramidal neurons. Synaptoporin expression parallels the formation of dentate granule cell synaptic contacts with pyramidal neurons: the amount of hippocampal synaptoporin, determined in immunoblots and by synaptoporin immunostaining of developing mossy fibre terminals; increases during the first postnatal week. Moreover, in the adult, synaptoporin is found exclusively in the mossy fibre terminals present in the hilar region of the dentate gyrus and the regio inferior of the cornu ammonis. In contrast, synaptophysin is present in all synaptic fields of the hippocampal formation, including the mossy fibre terminals, where it colocalizes with synaptoporin in the same boutons. Our data indicate that granule neuron terminals differ from all other terminals of the hippocampal formation by the presence of both synaptoporin and synaptophysin. This difference, observed in the earliest synaptic contacts in the postnatal hippocampus and persisting into adult life, suggests distinct functions of synaptoporin in these nerve terminals.

Asunto(s)

Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Proteínas de la Membrana/biosíntesis , Red Nerviosa/metabolismo , Sinaptofisina/biosíntesis , Animales , Femenino , Hipocampo/ultraestructura , Immunoblotting , Inmunohistoquímica , Fibras Nerviosas/metabolismo , Fibras Nerviosas/ultraestructura , Embarazo , Células Piramidales/metabolismo , Células Piramidales/ultraestructura , Ratas , Ratas Sprague-Dawley

RESUMEN

The expressions of two closely related synaptic vesicle antigens synaptophysin and synaptoporin were examined in the olfactory system of the adult rat and during pre- and postnatal development. In the adult, immunocytochemistry showed that the continuously regenerating olfactory receptor neurons (primary neurons) produce both synaptophysin and synaptoporin which were localized in the cell bodies of the receptor neurons in the olfactory epithelium, their dendrites, axonal processes in the olfactory nerve and their terminals in the olfactory bulb glomeruli. Furthermore, ultrastructural analysis revealed synaptophysin- and synaptoporin-immunoreactivities associated with synaptic vesicles in most olfactory receptor axonal terminals impinging on dendrites of the mitral and tufted neurons (secondary neurons in the olfactory bulb circuitry) in the olfactory glomeruli. In like manner, tufted neurons, granule and periglomerular neurons (interneurons in the olfactory bulb circuitry) express both synaptophysin and synaptoporin. In contrast, mitral neurons expressed only the synaptophysin antigen which was likewise associated with mitral axonal terminals in their target the olfactory cortex. The patterns of synaptophysin and synaptoporin expressions in mitral neurons (synaptophysin only) and tufted neurons (synaptophysin and synaptoporin) were similar in prenatal, postnatal and adult rats as revealed by immunocytochemistry and in situ hybridization. However, the biosynthesis of synaptophysin and synaptoporin by granule and periglomerular neurons, olfactory bulb interneurons, occurred mainly postnatally.

Asunto(s)

Proteínas de la Membrana/biosíntesis , Neuronas Aferentes/metabolismo , Corteza Somatosensorial/metabolismo , Sinaptofisina/biosíntesis , Animales , Femenino , Inmunohistoquímica , Hibridación in Situ , Interneuronas/metabolismo , Interneuronas/ultraestructura , Masculino , Terminaciones Nerviosas/metabolismo , Terminaciones Nerviosas/ultraestructura , Neuronas Aferentes/ultraestructura , Bulbo Olfatorio/metabolismo , Bulbo Olfatorio/ultraestructura , Nervio Olfatorio/metabolismo , Nervio Olfatorio/ultraestructura , Embarazo , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Ratas , Ratas Sprague-Dawley , Corteza Somatosensorial/embriología , Corteza Somatosensorial/ultraestructura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura

RESUMEN

The expression of the synaptic vesicle antigens synaptophysin (SY) and synaptoporin (SO) was studied in the rat striatum, which contains a nearly homogeneous population of GABAergic neurons. In situ hybridization revealed high levels of SY transcripts in the striatal anlage from embryonic day (E) 14 until birth. In contrast, SO hydridization signals were low, and no immunoreactive cell bodies were detected at these stages of development. At E 14, SY-immunoreactivity was restricted to perikarya. In later prenatal stages of development SY-immunoreactivity appeared in puncta (identified as terminals containing immunostained synaptic vesicles), fibers, thick fiber bundles and 'patches'. In postnatal and adult animals, perikarya of striatal neurons exhibited immunoreaction for SO; ultrastructurally SO antigen was found in the Golgi apparatus and in multivesicular bodies. SO-positive boutons were rare in the striatum. In the neuropil, numerous presynaptic terminals positive for SY were observed. Our data indicate that the expression of synaptic vesicle proteins in GABAergic neurons of the striatum is developmentally regulated. Whereas SY is prevalent during embryonic development, SO is the major synaptic vesicle antigen expressed postnatally by striatal neurons which project to the globus pallidus and the substantia nigra. In contrast synapses of striatal afferents (predominantly from cortex, thalamus and substantia nigra) contain SY.

Asunto(s)

Cuerpo Estriado/crecimiento & desarrollo , Proteínas de la Membrana/metabolismo , Sinaptofisina/metabolismo , Animales , Animales Recién Nacidos , Cuerpo Estriado/embriología , Cuerpo Estriado/metabolismo , Desarrollo Embrionario y Fetal , Femenino , Histocitoquímica , Inmunohistoquímica , Hibridación in Situ , Masculino , Neuronas Aferentes/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

Regarding the hippocampal formation and especially the external two thirds of it's dentate molecular layer a lot of possible morphological changes after long-term potentiation (LTP) have been described in literature. The present morphometric-stereological study of vesicles in axo-spino-dendritic synapses of the inner third of the molecular layer was done under the aspect of heterosynaptic influences following LTP. Because of the hierarchical link of the three analytic levels (test-group, animal, synapse), for statistical interpretation we used the analysis of variance with two-way hierarchical classification. Between the 3 groups (passive control, active control, LTP-group) we found no significant differences. Because of the great differences between the vesicles even within a single synapse we subsequently investigated the middle third of the molecular layer, i.e. the terminal area of the stimulated perforant path. No differences between the three groups we found here either. There was no confirmation for the expected greater homogenization of the synapses based on the uniform input. As a result of this study pure morphological studies without selective staining of specific population of synapses are considered inadvisable. Only with the help of selective staining in the area of the synapses possible differences between the groups may be found.

Asunto(s)

Hipocampo/citología , Hipocampo/fisiología , Sinapsis/fisiología , Sinapsis/ultraestructura , Análisis de Varianza , Animales , Axones/fisiología , Axones/ultraestructura , Dendritas/fisiología , Dendritas/ultraestructura , Estimulación Eléctrica , Potenciales Evocados , Hipocampo/ultraestructura , Masculino , Ratas , Ratas Wistar

RESUMEN

Regarding the hippocampal formation and especially the external two thirds of it's dentate molecular layer a lot of possible morphological changes after long-term potentiation (LTP) have been described in literature. The present morphometric-stereological study of axo-spino-dendritic synapses from the inner third of the molecular layer was done under the aspect of heterosynaptic influences following LTP. We were looking for differences in the number of transmission zones, in the total length of the transmission zone and in the qualitative shape of the single transmission membrane. Because of the hierarchical link of the three analytic levels (test-group, animal, synapse), for statistical interpretation we used the analysis of variance with two-way hierarchical classification. We detected large differences between the single synapses but not significant differences between the 3 groups (passive control, active control, LTP-group). Our quantitative studies showed the same results also in the middle third of the molecular layer (Grabs et al. 1991). Pure morphological studies should be done under selective staining of specified population of synapses to differ in stimulated and non-stimulated synapses. Only with the help of selective staining in the area of the synapses possible differences between the groups may be found.

Asunto(s)

Axones/ultraestructura , Dendritas/ultraestructura , Hipocampo/fisiología , Sinapsis/ultraestructura , Animales , Axones/fisiología , Dendritas/fisiología , Estimulación Eléctrica , Hipocampo/ultraestructura , Masculino , Ratas , Ratas Endogámicas , Sinapsis/fisiología , Transmisión Sináptica , Factores de Tiempo