RESUMO
The distribution of 125I-wheat germ agglutinin (WGA) transported by axons of chick retinal ganglion cells to layer d of the optic tectum was studied by electron microscopic autoradiography. We found that 52% of the radioactivity was located in axons and axon terminals in the contralateral optic tectum 22 h after intravitreal injection of affinity-purified 125I-WGA. Axons comprised 43% of the volume of layer d. Dendrites, glial cells, and neuron cell bodies contained 20%, 17%, and 3% of the label, whereas these structures comprised 24%, 21%, and 2% of the tissue volume, respectively. We also measured the distances between the autoradiographic silver grains and the plasma membranes of these profiles, and compared observed distributions of grains to theoretical distributions computed for band-shaped sources at various distances from the plasma membranes. This analysis revealed that the radioactive source within axons was distributed in a band of cytoplasm extending in from the plasma membrane a distance of 63 nm. Because WGA is known to bind to specific membrane glycoconjugates, we infer that at least some glycoconjugates may be concentrated within an annular region of cytoplasm just beneath the axonal plasma membrane after axoplasmic transport from the neuron cell body.
Assuntos
Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Colículos Superiores/fisiologia , Vias Visuais/fisiologia , Animais , Transporte Axonal , Axônios/metabolismo , Galinhas , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Lectinas/metabolismo , Microscopia Eletrônica , Colículos Superiores/citologia , Aglutininas do Germe de TrigoRESUMO
When horseradish peroxidase is injected into the optic tectum of a chick, axons of ganglion cells transport it centripetally to their cell bodies in the retina at a rate of about 72 millimeters per day. After intraocular injections in the young chick, the peroxidase is transported centripetally along efferent axons, and is concentrated in cell bodies within the isthmo-optic nucleus. This retrograde movement of protein from axon terminal to cell body suggests a possible mechanism by which neurons respond to their target areas.
Assuntos
Axônios/metabolismo , Nervo Óptico/metabolismo , Animais , Galinhas , Olho , Injeções , Peroxidases/administração & dosagem , Peroxidases/metabolismo , Retina/metabolismo , Fatores de TempoRESUMO
Many membranous organelles and protein complexes are normally transported anterograde within axons to the presynaptic terminal, and details of the motors, adaptors and cargoes have received significant attention. Much less is known about the transport in neurons of non-membrane bound particles, such as mRNAs and their associated proteins. We propose that herpes simplex virus type 1 (HSV) can be used to study the detailed mechanisms regulating long distance transport of particles in axons. A critical step in the transmission of HSV from one infected neuron to the next is the polarized anterograde axonal transport of viral DNA from the host infected nerve cell body to the axon terminal. Using the in vivo mouse retinal ganglion cell model infected with wild type virus or a mutant strain that lacks the protein Us9, we found that Us9 protein was necessary for long distance anterograde axonal transport of viral nucleocapsid (DNA surrounded by capsid proteins), but unnecessary for transport of virus envelope. Thus, we conclude that nucleocapsid can be transported independently down axons via a Us9-dependent mechanism.
Assuntos
Transporte Axonal/fisiologia , Proteínas do Capsídeo/metabolismo , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/fisiologia , Animais , Western Blotting , Proliferação de Células/efeitos dos fármacos , DNA/biossíntese , DNA/genética , Eletroforese em Gel de Poliacrilamida , Olho , Glicoproteínas/metabolismo , Herpesvirus Humano 1/genética , Imuno-Histoquímica , Injeções , Peptídeos e Proteínas de Sinalização Intracelular , Lipoproteínas/genética , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Terminações Pré-Sinápticas/metabolismo , Células Ganglionares da Retina/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Inoculation of the scarified cornea with herpes simplex virus (type 1) leads to herpetic infection of trigeminal ganglion cells. A recent study of the susceptibility of ganglion cells revealed that there may be at least four populations of trigeminal ganglion cells that are infectable by herpes. Two classes were identified by their neuropeptide content: Substance P or calcitonin gene-related peptide. One class was identified by its affinity for a monoclonal antibody, SSEA-3. The fourth class was recognized by its common affinity for both the monoclonal antibody LD2 and for the lectin Bandeiraea simplicifolia isolectin. However, there has been no direct evidence of which types are infected directly as a result of retrograde transport from the corneal site and which may be infected by cell-to-cell spread. The aim of this study was to determine which classes of neurons, which are known to become infected with HSV after ocular inoculation, supply corneal innervation. We have identified four classes of trigeminal ganglion neurons that supply axons to the central cornea of the mouse, on the basis of their ability to transport Fluoro-Gold retrograde from axons in the central corneal epithelium and stroma. About 40% of the neurons that innervate the cornea contain Substance P or calcitonin gene-related peptide; about 60% of the neurons that innervate the cornea react with the monoclonal antibody SSEA-3. About 36% of all neurons in the whole ophthalmic division react with the LD2 or Bandeiraea simplicifolia isolectin, and Fluoro-Gold labels only 2% of them.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Córnea/inervação , Ceratite Herpética/microbiologia , Neurônios/ultraestrutura , Nervo Oftálmico/anatomia & histologia , Simplexvirus/isolamento & purificação , Gânglio Trigeminal/citologia , Animais , Anticorpos Monoclonais , Biomarcadores , Peptídeo Relacionado com Gene de Calcitonina/análise , Córnea/microbiologia , Vias Eferentes/anatomia & histologia , Feminino , Ceratite Herpética/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/microbiologia , Nervo Oftálmico/microbiologia , Simplexvirus/fisiologia , Substância P/análise , Gânglio Trigeminal/microbiologiaRESUMO
We have assessed the effects of 15 pigmentation mutations on the development of retinal ganglion cell projections in mice in two ways: (1) by analyzing the pattern of innervation of the ipsilateral lateral geniculate nucleus as mapped in autoradiograms of brains of animals killed 12 days after intravitreal injection of 3H-proline into one eye and (2) by determining the ratio of axonally transported radioactive protein in the contralateral and ipsilateral optic tracts after similar intravitreal injections. Analysis of the ratio of transported protein in the two optic tracts provides a new and useful assay of the degree of decussation in experimental animals. The effects of the mutations on eye pigmentation, whole eye melanin content and relative tyrosinase activity also were examined. The degree of ipsilateral innervation generally correlates with the degree of pigmentation of the retinal pigment epithelium and with tyrosinase activity. However, discrepancies have been found in ch and ce mutants. In these animals the pigment epithelium is well pigmented, and the area of ipsilateral innervation in the lateral geniculate nucleus is extensive, despite a high ratio of label in contralateral to ipsilateral optic tracts and low tyrosinase activity. Furthermore, mice heterozygous for the c2J allele have pigmentation and optic projections that are normal even though tyrosinase is reduced to 40% of normal. The few anomalous results suggest that alternative or additional factors may control optic axon projections.
Assuntos
Genótipo , Retina/análise , Vias Visuais/anatomia & histologia , Alelos , Animais , Corpos Geniculados/anatomia & histologia , Cor de Cabelo , Melaninas/análise , Camundongos , Monofenol Mono-Oxigenase/análise , Mutação , Neurônios/fisiologia , Retina/anatomia & histologia , Retina/citologia , Pigmentos da RetinaRESUMO
In pigmented RCS rats with inherited retinal dystrophy, most photoreceptor cells disappear between postnatal days 20 and 100. We have examined the time course of the degeneration of photoreceptor nuclei and synapses and determined whether transneuronal changes occur in the inner nuclear layer (INL), inner plexiform layer (IPL), and retinal ganglion cells following loss of photoreceptor cells in these animals. Electron microscopic photomontages of the entire thickness of the IPL of dystrophic (RCS-p+) and control (RCS-rdy+ p+) rats 334 to 515 days old were prepared, and synapses were counted and identified as either conventional (amacrine) or ribbon (bipolar) types. Neither the incidence of synapses in the IPL nor the ratio of conventional to ribbon synapses differed in the dystrophic and control retinas. Ganglion cell diameter, perimeter, area, and density were measured from drawings of wholemount preparations of dystrophic and control rats 105 days and older. Diameter, perimeter, area and number of ganglion cells were not significantly different in the two genotypes. Anterograde axonal transport was measured by studying the displacement of labeled material as it traveled along ganglion cell axons and accumulated in the superior colliculus. The normal and dystrophic rats showed no significant difference in (1) the rates of rapidly moving components (approximately 110-180 mm/day) and slowly moving components (1.7-2.5 mm/day) or (2) the amount of radioactive material transported to the superior colliculus. The absence of transneuronal changes in retinal ganglion cells of RCS rats contrasts with results obtained earlier in rd mice (Graftstein et al., '72). Unlike the RCS rat, retinal degeneration in rd mice occurs before the maturation of the retina. We hypothesize that the ganglion cells may be more affected by loss of input early in development, and, therefore, ganglion cells of retinal dystrophic rats are less affected despite little or no synaptic input for several months. Furthermore, any reduction in the electrical activity of retinal ganglion cells that might follow loss of photoreceptor cells does not result in a significantly decreased rate of axonal transport.
Assuntos
Neurônios/fisiologia , Retina/fisiopatologia , Degeneração Retiniana/fisiopatologia , Sinapses/fisiologia , Envelhecimento , Animais , Transporte Axonal , Microscopia Eletrônica , Neurônios/ultraestrutura , Ratos , Ratos Mutantes , Retina/crescimento & desenvolvimento , Retina/ultraestrutura , Degeneração Retiniana/genética , Degeneração Retiniana/patologiaRESUMO
Herpes simplex virus displays tropism for neurons and other polarized epithelial cells. We have grown human retinal pigment epithelial cells in culture to study potential mechanisms whereby herpes simplex virus (type I) is transported from the plasma membrane of the cell to the nucleus. The cells were highly polarized as determined by a variety of criteria. They were tightly coupled by junctional complexes, as determined by electron microscopy, immunofluorescent staining of tight junctions and measurements of transepithelial electrical resistances > 200 omega cm2. Immunofluorescence and confocal microscopy were used to visualize microtubule orientation. The microtubules were arranged (i) in a single apical cilium, (ii) in a meshwork beneath the apical membrane and (iii) in longitudinally arranged bundles near the lateral membranes and nucleus. The latter microtubules were primarily oriented with their plus ends directed toward the basal surface of the cells. We infected retinal pigment epithelial cells at the apical surface with virus and assayed the uptake and transport of virus to the nucleus by quantitative immunoblot and immunocytochemical staining for the viral immediate early gene product, infected cell protein 4. The antigen first appeared in retinal pigment epithelial cells 2 h after infection. Treatment of the cells with 33 microM nocodazole, a microtubule-destabilizing drug, delayed the appearance of the viral antigen by 1 h. The effect of nocodazole treatment on microtubule integrity was confirmed by immunofluorescent staining and immunoblots of tubulin. Both cytoplasmic dynein and the ubiquitous form of kinesin were identified in the cells using immunoblots. These novel data indicate that human retinal pigment epithelial cells, like neurons, are susceptible to infection by herpes simplex virus and that the centripetal transport of virus to the nucleus in both cell types is facilitated by microtubules. The orientation of microtubules in retinal pigment epithelial cells suggests that the transport of herpes simplex virus from the apical surface is mediated by a microtubule-activated motor enzyme, possibly kinesin.
Assuntos
Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/virologia , Simplexvirus/metabolismo , Transporte Biológico/fisiologia , Western Blotting , Polaridade Celular/fisiologia , Células Cultivadas/metabolismo , Células Cultivadas/virologia , Dineínas/fisiologia , Humanos , Cinesinas/fisiologia , Microscopia Confocal , Microscopia Eletrônica , Microtúbulos/química , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Epitélio Pigmentado Ocular/ultraestrutura , Simplexvirus/ultraestruturaRESUMO
In order to determine whether a possible barrier exists to diffusion of tracer into the optic nerve during development and to provide a basis for later studies of retrograde axonal transport in embryonic nerves, we studied the diffusion of horseradish peroxidase (HRP) into the nerve after vitreal injections in chicks ranging in age from embryonic day 6 to 3 days after hatching. We found that HRP may reach the periaxonal spaces of the retrobulbar optic nerve after vitreal injection, vitreal injection into the opposite eye, or vascular injection. These and other observations suggest that vitreally injected HRP may reach the periaxonal spaces of the optic nerve by at least two routes: (1) by the obvious diffusion of marker from the vitreal surface into the optic nerve head and (2) by vascular leakage from fenestrated capillaries of the choriocapillaris into the pericapillary spaces of these and other capillaries that feed into the optic nerve parenchyma. There is a breakdown in the blood-brain barrier to HRP in the optic nerve head of the chick at embryonic day 13. The development of the breakdown depends at least in part on the maturation of vasculature in the nerve and the establishment of anastomotic branches between these vessels and those of the choriocapillaris. Our results further suggest that the limited diffusion of HRP into the retrobulbar nerve of fetal and newly hatched chicks is a function of uptake of tracer by glial cells within the nerve. Investigators of axonal transport who use this visual pathway as a model should be reminded of the potential artifact involved in this access of vascularly circulating label into the region of the lamina cribrosa.
Assuntos
Transporte Axonal , Peroxidase do Rábano Silvestre/farmacologia , Nervo Óptico/efeitos dos fármacos , Peroxidases/farmacologia , Animais , Barreira Hematoencefálica , Permeabilidade Capilar , Embrião de Galinha , Galinhas , Injeções , Nervo Óptico/fisiologia , Permeabilidade , Corpo VítreoRESUMO
Several distinct populations of sensory neurons in the ophthalmic region of the mouse trigeminal ganglion have been identified by their reactivity to antibodies raised against substance P (SP), calcitonin gene-related peptide (CGRP), cell-surface glycoconjugates SSEA3 and LD2, and the plant lectin, Bandeiraea simplicifolia lectin 1, isolectin 4 (BSIL4). Thirty-six percent of the neurons in the ophthalmic portion of the mouse trigeminal ganglion express CGRP and 17%, SP. All neurons that express SP also express CGRP. Forty percent of the neurons in the ophthalmic region of the ganglion are recognized by monoclonal antisera to SSEA3, and 66% of this population also express the neuropeptides SP or CGRP. The neuronal population recognized by BSIL4 is identical to the population with the LD2 epitope. This population of cells (BSIL4/LD2) does not express the SSEA3 glycoconjugate and is largely nonpeptidergic. All four populations of sensory neurons (SP, CGRP, SSEA3, and LD2/BSIL4) can be infected by herpes simplex virus (HSV). However, the relative proportion of SSEA3- and LD2/BSIL4-labeled cells that were infected productively with HSV was much less than expected based on the relative size of the populations of these neurons in the ophthalmic region of the ganglion.
Assuntos
Infecções por Herpesviridae/patologia , Simplexvirus , Gânglio Trigeminal/patologia , Animais , Anticorpos Monoclonais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Infecções Oculares Virais , Feminino , Imunofluorescência , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/microbiologia , Ceratite Herpética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/citologia , Substância P/metabolismo , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/microbiologiaRESUMO
The relationship between the dose of Herpes simplex virus type 1 (HSV) inoculated in the cornea and the amount of actively replicating virus recovered from mouse trigeminal ganglion cells 5 d after corneal scratch and inoculation was investigated with a tissue culture plaque assay. A dose response curve of productive viral replication was obtained. The estimated dose of HSV that produces half-maximal recovery of virus within the ganglion was 9.15 x 10(3) plaque forming units per eye, and the maximal amount of HSV recovered was 1.34 x 10(4) pfu per ganglion. This definition of infectivity as a function of dose will be useful for studying the effects of potential inhibitors of the binding, uptake, and transport of HSV by productively or latently infected trigeminal neurons.
Assuntos
Herpes Simples/microbiologia , Ceratite Herpética/microbiologia , Gânglio Trigeminal/microbiologia , Animais , Córnea/inervação , Córnea/microbiologia , Doenças dos Nervos Cranianos/microbiologia , Modelos Animais de Doenças , Feminino , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Simplexvirus/fisiologia , Replicação ViralRESUMO
The molecular mechanisms specifying patterns of gene expression in the vertebrate brain, which in turn determine the developmental fates of specific neurons, are yet to be clearly defined. Individual members of a recently identified family of transcriptional regulatory proteins, the GATA factors, are required for the differentiation of certain hematopoietic cell lineages. We show here that two of the members of this gene family, GATA-2 and GATA-3, are expressed within discrete cell populations of the chicken optic tectum during embryogenesis, and that they have highly restricted patterns of expression in the developing chicken brain. Furthermore, the induction of GATA factor expression within specific cell layers parallels the well established spatial (rostral to caudal) and temporal pattern of optic tectum development. The observation that both the timing of appearance and the localization of expression of GATA-2 and GATA-3 are correlated with optic tectum development suggest that these transcription factors may be associated with the initiation of gene transcription required for the determination of specific neuronal fates within visual areas of the vertebrate brain.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Colículos Superiores/metabolismo , Transativadores/biossíntese , Fatores de Transcrição/biossíntese , Animais , Sequência de Bases , Embrião de Galinha , Galinhas , Proteínas de Ligação a DNA/genética , Fator de Transcrição GATA2 , Fator de Transcrição GATA3 , Regulação da Expressão Gênica , Dados de Sequência Molecular , Morfogênese , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase , Colículos Superiores/embriologia , Colículos Superiores/crescimento & desenvolvimento , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
We have studied the lateral rectus muscles and neuromuscular junctions (NMJs) of abducens motoneurons in wobbler (wr/wr) mutant mice from 26 to 58 days of age. The muscles of wr/wr weighed about 70% of the weight of littermate controls and were composed of fiber types comparable to those of controls, as assayed by succinate dehydrogenase activity. The most obvious difference between wr/wr and control NMJs was a reduction in the length of the postjunctional membrane of wr/wr mice. The mutant muscle endplate membrane was only about 70% (6.58 micron) the length of control muscle regions (9.44 micron). There were no obvious differences at the light microscopic level in the distribution of acetylcholine (ACh) receptors at junctional regions or staining of acetylcholinesterase, as assayed with alpha-bungarotoxin binding or enzyme histochemistry. Indirect immunocytochemical studies using antibodies directed against the subunits of the ACh receptor failed to indicate an abnormal presence of immature receptors clustered at the NMJs of wr/wr mice. Our findings suggest that the formation or maintenance of normal postjunctional folds and the differentiation of receptors at the junctions are under independent control during development. Furthermore, the wobbler mutation may affect muscle cell differentiation as well as neuronal differentiation. This mutant mouse should prove a useful model for study of postjunctional fold formation and function.
Assuntos
Envelhecimento/metabolismo , Camundongos Mutantes Neurológicos/anatomia & histologia , Músculos/ultraestrutura , Junção Neuromuscular/ultraestrutura , Receptores Colinérgicos/metabolismo , Animais , Contagem de Células , Histocitoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos/crescimento & desenvolvimento , Microscopia Eletrônica , Desenvolvimento Muscular , Músculos/metabolismo , Junção Neuromuscular/metabolismo , FenótipoRESUMO
The phenomenon of retrograde intraaxonal transport of extracellular markers introduced at the level of the axon terminal has been suggested as a possible mechanism of communication between the axon terminal and the neuron cell body. We tested the hypothesis that communication after axotomy might consist of a change in the rate of uptake or of transport of material by injured neurons. Small lesions were made with a needle in one retinal quadrant of chicks and immediately afterwards horseradish peroxidase (HRP) was injected into the vitreous body of the eye. The amount of HRP accumulated by some of the neurons of the isthmo-optic nucleus (ION) which project to the damaged area was clearly different from that of nearby cells which project to the non-damaged portions of the retina. The uninjured cells accumulated enzyme marker beginning at 3.5 h after injection. The injured neurons did not accumulate significant amounts of HRP until between 4 and 6 h after injection. Between 6.75 h and 18 h the injured cells in the ION accumulated greater amounts of HRP than cells in other regions, but by 24 h the cells of the ION in the region of injury contained distinctly less label. This pattern of enzyme accumulation was confirmed by counts of the number of HRP-positive granules within cells of chicks fixed 4, 11.75, 12.25, 27.6 and 72 h after injury. In another series of experiments, the axon terminals of the ION were first exposed to HRP, and 1 h later some of the axons were damaged with a needle. In these cases, there was no difference between the injured and control neurons in the time of first appearance of labeled cells in the ION within the first 4 h after injection of HRP. These findings suggest that injury initially results in a decrease in the uptake of the marker rather than a decrease in the rate of retrograde transport. The amount of marker found in the injured neurons later is greater than that found in the control neurons. This subsequent difference may represent an increase in the rate of uptake, transport, or both or a decrease in the rate of degradation of HRP within the cell body as a response to injury of the axon.
Assuntos
Transporte Axonal , Neurônios/metabolismo , Peroxidases/metabolismo , Retina/lesões , Animais , Galinhas/metabolismo , Traumatismos Oculares/metabolismo , Histocitoquímica , Retina/metabolismo , Fatores de Tempo , Vias Visuais/metabolismoRESUMO
We have investigated the rate of anterograde axonal transport of affinity-purified [125I]wheat germ agglutinin (WGA) in chick retinal ganglion cells. By 2 lines of evidence, we found the rate to be 22-44 mm/day. This rate is slower than that reported for many other endogenously synthesized proteins or for horseradish peroxidase, the only other exogenously supplied protein studied as an anterograde transported marker. This difference in rate of transport of [125I]WGA may reflect a novel pathway important in membrane recycling.
Assuntos
Transporte Axonal , Lectinas/metabolismo , Retina/metabolismo , Colículos Superiores/metabolismo , Animais , Galinhas , Neurônios/metabolismo , Vias Visuais/metabolismo , Aglutininas do Germe de TrigoRESUMO
Following intravitreal injection, affinity purified, iodinated wheat germ agglutinin ([125I]WGA) is taken up by chick retinal ganglion cells and transported in an anterograde direction to nerve terminals in the optic tectum. The accumulation of axonally transported label in the tectum may be measured quantitatively. Using such an approach, we find that co-injection of [125I]WGA with an excess of unlabeled WGA reduces the amount of axonally transported labeled lectin. Since co-injection of comparable levels of soybean agglutinin or Ulex Europeanus-I fails to reduce tectal labeling to a similar extent, and since native WGA at the same concentration does not appear to be toxic to retinal ganglion cells, these results support the hypothesis that the uptake and subsequent anterograde axonal transport of WGA by these cells is a selective process, dependent on a limited number of extra- or intracellular binding sites.
Assuntos
Galinhas/metabolismo , Dissacarídeos , Lectinas/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Transporte Axonal , Permeabilidade da Membrana Celular , Glucanos/metabolismo , Masculino , Aglutininas do Germe de TrigoRESUMO
We have studied the subcellular localization of peroxidase-labeled organelles after anterograde axonal transport by chick retinal ganglion cells that had been exposed 23-25 h earlier to wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). After intravitreal injection of WGA-HRP, we found in the optic tectum that 82% of labeled organelles were located within axons and axon terminals. The organelles included: tubules and cisternae of the smooth endoplasmic reticulum, hypolemmal cisternae, vesicles, dense bodies and multivesticular bodies. We also measured the distances between the centers of the labeled organelles and the plasma membrane of these profiles. The density of organelles (number of organelles/micron 2) was plotted as a function of distance from the plasma membrane. Irrespective of the dose of lectin-peroxidase injected, labeled organelles were most densely concentrated in a 30 nm wide annular zone centered 75 nm in from the plasma membrane. In axon terminals the labeled organelles were most concentrated 75-90 nm in from the plasma membrane. Assuming that the peroxidase label indicates the presence of WGA-HRP, we conclude that after anterograde axonal transport the lectin accumulates in lysosomal organelles and elements of the smooth endoplasmic reticulum. Therefore, in contrast to the more restricted localization of [125I]WGA as inferred from electron microscopic autoradiography after uptake and transport by the same cell type, WGA-HRP-labeled organelles are found more diffusely within the axoplasm, particularly in axon terminals. Furthermore, peroxidase-labeled organelles in dendritic, glial or neuronal cell bodies in the tectum were seen less frequently than expected based on evidence of frequent transfer to second cells after intravitreal injections of [125I]WGA. Thus, we infer that at these concentrations WGA labeled with HRP may not be transferred intercellularly as efficiently as even lower concentrations of iodinated WGA are apparently transferred.
Assuntos
Transporte Axonal , Axônios/ultraestrutura , Peroxidase do Rábano Silvestre/metabolismo , Lectinas/metabolismo , Peroxidases/metabolismo , Retina/ultraestrutura , Células Ganglionares da Retina/ultraestrutura , Animais , Galinhas , Microscopia Eletrônica , Organoides/ultraestrutura , Colículos Superiores/anatomia & histologia , Sinapses/ultraestrutura , Aglutininas do Germe de TrigoRESUMO
The involvement of the axonal smooth endoplasmic reticulum as a channel for the retrograde axonal transport of horseradish peroxidase (HRP) has been tested by analysing serial sections of 52 HRP-positive organelles in chick optic nerves. The enzyme marker was injected in the posterior, contralateral optic tectum 10 h before fixation of young chicks. The two optic nerves, retinas and optic tecta were incubated for electron microscopic demonstration of HRP. Thin sections of the retinas and tecta and serial thin sections of the optic nerves were studied in some cases with the aid of a goniometer. Of the 52 organelles, 42% had a tubular shape, 46% were oval and 12% were multivesicular bodies. None of the organelles was found to have continuities with other membranous structures, including tubules or cisternae of the smooth endoplasmic reticulum. In 10 cases, the smooth endoplasmic reticulum was followed in serial sections over a length of up to 4 micrometer. In every case, the reticulum appeared to form a continuous system although some tubular extensions apparently ended blindly near other organelles. In neither the 10 series of serial sections nor in any other individual micrographs did any recognizable profile of the smooth endoplasmic reticulum contain HRP. Measurements of the thickness of the membranes of HRP-containing organelles, of the smooth endoplasmic reticulum and of plasmalemma were made, since these membranes have been distinguished on the basis of their thickness in other cells. The plasmalemma in the axons was about 20% thicker than that of the smooth endoplasmic reticulum, and about 9% thicker than that of HRP-labeled organelles. The membrane of the smooth endoplasmic reticulum and HRP-organelles could also be distinguished by this means. It is concluded that in chick retinal ganglion cell axons, HRP is not transported in a retrograde direction via a continuous channel of smooth endoplasmic reticulum.
Assuntos
Transporte Axonal , Retículo Endoplasmático/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Nervo Óptico/metabolismo , Peroxidases/metabolismo , Animais , Galinhas , Endocitose , Microscopia Eletrônica , Neurônios/metabolismo , Retina/metabolismo , Córtex Visual/metabolismo , Vias Visuais/metabolismoRESUMO
Following corneal inoculation with herpes simplex virus (Type 1) (HSV), virus spreads to the CNS by axonal transport in the central branches of trigeminal ganglion cell neurons. Although this mode of viral entry to the CNS is rare for humans, it appears to be the principal route of entry into the CNS in animal models of herpetic corneal disease. In this study, the corneas of BALB/c mice were unilaterally inoculated with HSV, and the distribution of HSV-immunoreactive label was studied to identify the central branches of the axons of infected trigeminal ganglion cells. Virus was first noted in the brainstem trigeminal complex 4 days after corneal inoculation, when HSV-labeled afferents were found throughout the course of the descending tract of V as well as in interstitial neurons in the tract. By 5 days labeled neurons were also found not only in the n. caudalis and portions of the n. interpolaris of the trigeminal complex but also in laminae I-IV of the dorsal horn of the upper cervical levels of the spinal cord. No immunoreactivity was seen in other regions of the complex, including the n. oralis or the main sensory n. of V. By 6 days, however, the infection had spread to the main sensory division of V.
Assuntos
Encéfalo/microbiologia , Herpes Simples/fisiopatologia , Gânglio Trigeminal/microbiologia , Animais , Encéfalo/patologia , Feminino , Herpes Simples/patologia , Camundongos , Camundongos Endogâmicos BALB C , Gânglio Trigeminal/patologiaRESUMO
Fluoro-Gold has been used previously to identify those trigeminal ganglion cells that innervate the central cornea. To examine the effects of Fluoro-Gold treatment on infection and spread of HSV in vivo, we measured the number of plaque forming units recovered from trigeminal ganglia 3 or 5 days after corneal scratch and inoculation with Fluoro-Gold and HSV. Treatment with Fluoro-Gold reduced the amount of virus recovered after retrograde transport 63% at 3 days and 28% at 5 days after inoculation. When we examined trigeminal ganglion sections from animals treated with HSV and Fluoro-Gold, we found the number of neurons double labeled with antibodies that recognize HSV and Fluoro-Gold was only 13% of all Fluoro-Gold labeled neurons. This was significantly fewer cells that we had anticipated, on the basis of double labeling experiments with wheat germ agglutinin combined with Fluoro-Gold. The effects of varying doses of the retrograde tracer, Fluoro-Gold on Herpes simplex virus (type 1) (HSV) infectivity were also assayed in vitro using a standard viral plaque assay. At 1 x 10(-3) mg/ml Fluoro-Gold there was no effect on the number of plaque forming units. At 5 x 10(-1) mg/ml the number of plaques was reduced about 67%. We conclude that Fluoro-Gold interferes with productive HSV infection in vivo and in vitro after retrograde transport of HSV by neurons.
Assuntos
Antivirais/farmacologia , Corantes Fluorescentes/farmacologia , Simplexvirus/efeitos dos fármacos , Estilbamidinas , Animais , Antivirais/uso terapêutico , Corantes Fluorescentes/uso terapêutico , Herpes Simples/tratamento farmacológico , Células Vero/microbiologia , Ensaio de Placa ViralRESUMO
The 'wobbler' mutant mouse can be recognized at about 4 weeks of age by its tremor and atrophy of forelimb muscles. In addition to degeneration of spinal motoneurons, especially in cervical spinal cord, selected bulbar motoneurons have also been reported to degenerate in the mutant. We examined a cranial motor nucleus and found a 31% loss of abducens motoneurons in 4-5-week-old wobbler mice as compared to age-matched control mice.