RESUMEN
Successful axonal regeneration is a complex process determined by both axonal environment and endogenous neural capability of the regenerating axons in the central and the peripheral nervous systems. Numerous external inhibitory factors inhibit axonal regeneration after injury. In response, neurons express various regeneration-associated genes to overcome this inhibition and increase the intrinsic growth capacity. In the present study, we show that the brain-expressed X-linked (Bex1) protein was over-expressed as a result of peripheral axonal damage. Bex1 antagonized the axon outgrowth inhibitory effect of myelin-associated glycoprotein. The involvement of Bex1 in axon regeneration was further confirmed in vivo. We have demonstrated that Bex1 knock-out mice showed lower capability for regeneration after peripheral nerve injury than wild-type animals. Wild-type mice could recover from sciatic nerve injury much faster than Bex1 knock-out mice. Our findings suggest that Bex1 could be considered as regeneration-associated gene.
Asunto(s)
Axones/fisiología , Regeneración Nerviosa/fisiología , Proteínas del Tejido Nervioso/fisiología , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas Motoras/fisiología , Glicoproteína Asociada a Mielina/antagonistas & inhibidores , Glicoproteína Asociada a Mielina/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Regulación hacia Arriba/fisiologíaRESUMEN
Carnosine-like peptides (carnosine-LP) are a family of histidine derivatives that are present in the nervous system of various species and that exhibit antioxidant, anti-matrix-metalloproteinase, anti-excitotoxic, and free-radical scavenging properties. They are also neuroprotective in animal models of cerebral ischemia. Although the function of carnosine-LP is largely unknown, the hypothesis has been advanced that they play a role in the developing nervous system. Since the zebrafish is an excellent vertebrate model for studying development and disease, we have examined the distribution pattern of carnosine-LP in the adult and developing zebrafish. In the adult, immunoreactivity for carnosine-LP is specifically concentrated in sensory neurons and non-sensory cells of the olfactory epithelium, the olfactory nerve, and the olfactory bulb. Robust staining has also been observed in the retinal outer nuclear layer and the corneal epithelium. Developmental studies have revealed immunostaining for carnosine-LP as early as 18 h, 24 h, and 7 days post-fertilization in, respectively, the olfactory, corneal, and retinal primordia. These data suggest that carnosine-LP are involved in olfactory and visual function. We have also investigated the effects of chronic (7 days) exposure to carnosine on embryonic development and show that 0.01 microM to 10 mM concentrations of carnosine do not elicit significant deleterious effects. Conversely, treatment with 100 mM carnosine results in developmental delay and compromised larval survival. These results indicate that, at lower concentrations, exogenously administered carnosine can be used to explore the role of carnosine in development and developmental disorders of the nervous system.
Asunto(s)
Carnosina/análogos & derivados , Carnosina/administración & dosificación , Embrión no Mamífero/efectos de los fármacos , Sistema Nervioso/metabolismo , Pez Cebra/metabolismo , Animales , Anserina/biosíntesis , Carnosina/biosíntesis , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Ojo/citología , Ojo/embriología , Ojo/metabolismo , Inmunohistoquímica , Sistema Nervioso/citología , Sistema Nervioso/embriología , Bulbo Olfatorio/citología , Bulbo Olfatorio/embriología , Bulbo Olfatorio/metabolismo , Mucosa Olfatoria/citología , Mucosa Olfatoria/embriología , Mucosa Olfatoria/metabolismo , Nervio Olfatorio/citología , Nervio Olfatorio/embriología , Nervio Olfatorio/metabolismo , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The olfactory system is an outstanding model for understanding activity-dependent neuronal plasticity in mammals. Olfactory sensory neurons (OSNs) in the periphery project onto mitral/tufted cells in the olfactory bulb (OB) and these mitral/tufted cells in turn project to piriform cortex (PC). Numerous studies have examined changes in OB after a permanent OSN ablation, but little is known about "trans-transsynaptic" changes taking place in the PC. Permanent zinc sulfate lesion of the olfactory epithelium resulted in a selective loss of the NMDA receptor NR2B protein and mRNA expression in pyramidal cells in layer IIb of PC after 2-7 d. Regulatory elements affected by NR2B signaling, namely the phosphorylation of CREB, were also downregulated only in layer IIb neurons. These changes could be caused by OSN axon loss in the zinc sulfate lesion, or to a reduced activity. To test this hypothesis, we performed both permanent and reversible naris occlusion, which blocks odorant access to the nasal cavities and OSN activity. The expression of NR2B and phospho-CREB were downregulated 5 d after occlusion and this reduction was fully restored 10 d after reopening of the naris. Subsequently, we identified the subset of pyramidal cells in layer IIb that are especially sensitive to the loss of odor-evoked activity using double retrograde tracers. In summary, the present study provides an initial characterization of the molecular mechanisms associated with odor stimulation on second order neuronal plasticity and phenotype in the olfactory system.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Vías Olfatorias/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Olfato/fisiología , Sinapsis/metabolismo , Animales , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Ácido Glutámico/metabolismo , Masculino , Ratones , Cavidad Nasal/lesiones , Cavidad Nasal/cirugía , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Neurotoxinas/efectos adversos , Odorantes , Bulbo Olfatorio/metabolismo , Vías Olfatorias/efectos de los fármacos , Neuronas Receptoras Olfatorias/efectos de los fármacos , Neuronas Receptoras Olfatorias/metabolismo , Fosforilación/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Privación Sensorial/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Sulfato de Zinc/efectos adversosRESUMEN
Sodium/calcium (Na(+)/Ca(2+)) exchangers are membrane transport systems that regulate Ca(2+)-homeostasis in many eukaryotic cells. In olfactory and vomeronasal sensory neurons ligand-induced olfactory signal transduction is associated with influx and elevation of intracellular Ca(2+), [Ca(2+)](i). While much effort has been devoted to the characterization of Ca(2+)-related excitation and adaptation events of olfactory chemosensory neurons (OSNs), much less is known about mechanisms that return [Ca(2+)](i) to the resting state. To identify proteins participating in the poststimulus Ca(2+)-clearance of mouse OSNs, we analyzed the expression of three potassium (K(+))-independent (NCX1, 2, 3) and three K(+)-dependent (NCKX1, 2, 3) Na(+)/Ca(2+) exchangers. In situ hybridization showed that mRNAs of all six Na(+)/Ca(2+) exchangers coexist in neurons of the olfactory and vomeronasal systems, and that some are already detectable in the embryo. Of these, NCX1 and NCKX1 represent the most and least abundant mRNAs, respectively. Moreover, immunohistochemistry revealed that the NCX1, 2, and 3 proteins are expressed in nearly all neurons of the olfactory epithelium, the vomeronasal organ, the septal organ of Masera, and the Grueneberg ganglion. These three exchanger proteins display different expression profiles in dendrites, knobs, and plasma membranes of OSNs and in sustentacular cells. Furthermore, we show that NCX1 mRNA in rat olfactory mucosa is expressed as 8 alternative splice variants. This is the first comprehensive analysis of Na(+)/Ca(2+) exchanger expression in the mammalian olfactory system. Our results suggest that Ca(2+)-extrusion by OSNs utilizes multiple different Na(+)/Ca(2+) exchangers and that different subtypes are targeted to different subcellular compartments.
Asunto(s)
Neuronas Aferentes/metabolismo , Vías Olfatorias/metabolismo , Olfato/fisiología , Intercambiador de Sodio-Calcio/metabolismo , Órgano Vomeronasal/metabolismo , Factores de Edad , Animales , Calcio/metabolismo , Femenino , Masculino , Ratones , Neuronas Aferentes/citología , Mucosa Olfatoria/citología , Mucosa Olfatoria/metabolismo , Vías Olfatorias/citología , Isoformas de Proteínas/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Intercambiador de Sodio-Calcio/clasificación , Intercambiador de Sodio-Calcio/genética , Órgano Vomeronasal/citologíaRESUMEN
Beta-tubulin genes code for very similar proteins, sharing extensive identity in amino acid sequence within and across species, each of which manifests characteristic patterns of cell and tissue expression. While searching for olfactory specific mRNAs in the channel catfish (Ictalurus punctatus), we isolated a novel beta-tubulin cDNA. In the putative ORF, 1298 nucleotides were 80-88% identical to cloned cDNAs from zebrafish to human for beta-tubulin isotype IVb. This ORF is interrupted by an insert of 111 nucleotides located between the regions corresponding to exons 2 and 3 in other species. This insert lacks similarity to any sequence in the NCBI databases. We showed that this novel cDNA fragment hybridizes specifically to catfish olfactory epithelium mRNA on Northern analysis. Here we demonstrate by in situ analysis of catfish olfactory epithelium that the expression of this mRNA is spatially restricted to the outer two-thirds of each olfactory lamella where olfactory receptor neurons reside. These results suggest that this nucleotide sequence is the result of incomplete RNA transcript processing. The growing awareness of the regulatory roles played by RNAs transcribed from intronic regions of genes suggests that this observation may have relevance to regulation of gene expression in olfactory tissue during development and axon targeting.
Asunto(s)
Ictaluridae/genética , Intrones , Mucosa Olfatoria/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , ARN Mensajero/metabolismo , Tubulina (Proteína)/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , ADN Complementario/metabolismo , Expresión Génica , Ictaluridae/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Mucosa Olfatoria/citología , ARN Mensajero/genética , Alineación de Secuencia , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMEN
PURPOSE: Few proteins are known to be selectively expressed in retinal ganglion cells (RGCs), the neurons directly affected by optic nerve stroke and glaucoma. In addition, subsets of RGCs are reported to project to various CNS areas via the retinohypothalamic pathway in rodents and primates. Many of these areas exhibit immunoreactivity for the brain-expressed X-linked (Bex) proteins Bex1 and Bex2. This prompted us to evaluate expression of these proteins in retina. METHODS: We utilized rats and transgenic mice, coupled with a new rodent model of isolated optic nerve stroke (rodent anterior ischemic optic neuropathy, rAION). An anti-Bex1 antibody was reacted to retinal tissue extracts. To evaluate short term effects of rAION on RGC Bex expression, a double transgenic mouse strain was employed expressing cyan fluorescent protein (CFP) under control of the Thy-1 protein promotor, and beta-galactosidase (lacZ) under control of the immediate early stress c-fos gene promotor. Positive identification of rat RGCs was performed by retrograde fluorogold labeling via stereotactic CNS injection. Retinas were analyzed using both diaminobenzidine (DAB)-linked immunochemistry and confocal microscopy. RESULTS: Bex immunoreactivity is present at high levels in the retina. Bex1 and Bex2 are selectively expressed in RGCs and differentially expressed in a subset of large RGC neurons. Bex signal levels are lower in small RGC neurons, which preferentially express high levels of the transcription factor Brn3b. Post-stroke, Bex accumulates in the RGC cytoplasm, consistent with the optic nerve edema produced by clinical AION. CONCLUSIONS: Bex immunoreactivity can be used to evaluate, ex vivo, the distribution of RGC cell bodies and their axons in the retina and rAION effects on RGC axonal loss. Thus, Bex can be utilized to evaluate both long- and short-term effects of optic nerve stroke and may play a significant role in regulating RGC functions in both the axonal and cell body components of RGC neurons.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuropatía Óptica Isquémica/metabolismo , Retina/metabolismo , Animales , Axones/metabolismo , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/farmacocinética , Técnicas Inmunológicas , Ratones , Ratones Transgénicos/genética , Ratas , Células Ganglionares de la Retina/metabolismo , Coloración y Etiquetado , Estilbamidinas , Distribución Tisular , Factor de Transcripción Brn-3B/metabolismoRESUMEN
Bex proteins are expressed from a family of "brain expressed X-linked genes" that are closely linked on the X-chromosome. Bex1 and 2 have been characterized as interacting partners of the olfactory marker protein (OMP). Here we report the distribution of Bex1 and Bex2 mRNAs in several brain regions and the development and characterization of an antibody to mouse Bex1 protein that cross-reacts with Bex2 (but not Bex3), and its use to determine the cellular distribution of Bex proteins in the murine brain. The specificity of the antiserum was characterized by immunoprecipitation and Western blots of tissue and transfected cell extracts and by immunocytochemical analyses of cells transfected with either Bex1 or Bex2. Antibodies preabsorbed with Bex2 still recognize Bex1, while blocking with Bex1 eliminates all immunoreactivity to both Bex1 and Bex2. Bex immunoreactivity (ir) was primarily localized to neuronal cells within several regions of the brain, including the olfactory epithelium, bulb, peri/paraventricular nuclei, suprachiasmatic nucleus, arcuate nucleus, median eminence, lateral hypothalamic area, thalamus, hippocampus, and cerebellum. RT-PCR and in situ hybridization demonstrated the presence of Bex mRNA in several of these regions. Double-label immunocytochemistry indicates that Bex-ir is colocalized with OMP in mature olfactory receptor neurons (ORNs) and in the OMP-positive subpopulation of neurons in hypothalamus. This is the first anatomical mapping of Bex proteins in the mouse brain and their colocalization with OMP in ORNs and hypothalamus.
Asunto(s)
Encéfalo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Vías Olfatorias/metabolismo , Factores de Edad , Secuencia de Aminoácidos , Animales , Encéfalo/citología , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Ratones , Proteínas del Tejido Nervioso/genética , Proteína Marcadora Olfativa , Mucosa Olfatoria/citología , Mucosa Olfatoria/metabolismo , Vías Olfatorias/citología , Neuronas Receptoras Olfatorias/metabolismo , Especificidad de Órganos , ARN Mensajero/análisis , Alineación de SecuenciaRESUMEN
Olfactory marker protein (OMP) is a ubiquitous, cytoplasmic protein found in mature olfactory receptor neurons of all vertebrates. Electrophysiological and behavioral studies demonstrate that it is a modulator of the olfactory signal transduction pathway. Here, we demonstrate that the solution structure of OMP, as determined by NMR studies, is a single globular domain protein comprised of eight beta-strands forming two beta-sheets oriented orthogonally to one another, thus exhibiting a "beta-clam" or "beta-sandwich" fold: beta-sheet 1 is comprised of beta3-beta8-beta1-beta2 and beta-sheet 2 contains beta6-beta5-beta4-beta7. Insertions include two, long alpha-helices located on opposite sides of the beta-clam and three flexible loops. The juxtaposition of beta-strands beta6-beta5-beta4-beta7-beta2-beta1-beta8-beta3 forms a continuously curved surface and encloses one side of the beta-clam. The "cleft" formed by the two beta-sheets is opposite to the closed end of the beta-clam. Using a peptide titration series, we have identified this cleft as the binding surface for a peptide derived from the Bex1 protein. The highly conserved Omega-loop structure adjacent to the Bex1 peptide-binding surface found in OMP may be the site of additional OMP-protein interactions related to its role in modulating olfactory signal transduction. Thus, the interaction between the OMP and Bex1 proteins could facilitate the interaction between OMP and other components of the olfactory signaling pathway.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Proteína Marcadora Olfativa , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor EphB2 , Alineación de Secuencia , Termodinámica , VolumetríaRESUMEN
Gamma-aminobutyric acid (GABA) has a dual role as an inhibitory neurotransmitter in the adult central nervous system (CNS) and as a signaling molecule exerting largely excitatory actions during development. The rate-limiting step of GABA synthesis is catalyzed by two glutamic acid decarboxylase isoforms GAD65 and GAD67 coexpressed in the GABAergic neurons of the CNS. Here we report that the two GADs show virtually nonoverlapping expression patterns consistent with distinct roles in the developing peripheral olfactory system. GAD65 is expressed exclusively in undifferentiated neuronal progenitors confined to the proliferative zones of the sensory vomeronasal and olfactory epithelia In contrast GAD67 is expressed in a subregion of the nonsensory epithelium/vomeronasal organ epithelium containing the putative Gonadotropin-releasing hormone (GnRH) progenitors and GnRH neurons migrating from this region through the frontonasal mesenchyme into the basal forebrain. Only GAD67+, but not GAD65+ cells accumulate detectable GABA. We further demonstrate that GAD67 and its embryonic splice variant embryonic GAD (EGAD) concomitant with GnRH are dynamically regulated during GnRH neuronal migration in vivo and in two immortalized cell lines representing migratory (GN11) and postmigratory (GT1-7) stage GnRH neurons, respectively. Analysis of GAD65/67 single and double knock-out embryos revealed that the two GADs play complementary (inhibitory) roles in GnRH migration ultimately modulating the speed and/or direction of GnRH migration. Our results also suggest that GAD65 and GAD67/EGAD characterized by distinct subcellular localization and kinetics have disparate functions during olfactory system development mediating proliferative and migratory responses putatively through specific subcellular GABA pools.
Asunto(s)
Glutamato Descarboxilasa/genética , Hormona Liberadora de Gonadotropina/metabolismo , Neuronas/citología , Vías Olfatorias/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Línea Celular , Movimiento Celular/genética , Epitelio/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glutamato Descarboxilasa/deficiencia , Ratones , Ratones Noqueados , Mucosa Olfatoria/citología , Vías Olfatorias/embriología , Transducción de Señal/genéticaRESUMEN
The bone morphogenetic proteins (BMPs) play fundamental roles during the organization of the central nervous system. The presence of these proteins has also been demonstrated in regions of the adult brain that are characterized by neural plasticity. In this study, we examined the expression of BMP4, 6, and 7 mRNAs and proteins in the murine olfactory system. The olfactory system is a useful model for studying cell proliferation and neural differentiation because both of these processes persist throughout life in the olfactory epithelium (OE) and olfactory bulb (OB). Our results demonstrate a differential expression of BMP4, 6, and 7 in the embryonic, postnatal, and adult olfactory system. In particular, BMP4 and BMP7 showed similar immunostaining patterns, being expressed in the olfactory region from the earliest stages studied (embryonic day 15.5) to adulthood. During development BMPs were expressed in the OE, olfactory bulb nerve layer, glomerular layer (GL), mitral cell layer (MCL), and subventricular zone. During the first postnatal week of life, BMP4 and 7 immunoreactivity (-ir) was particularly evident in the GL, MCL, and in the subependymal layer (SEL), which originates postnatally from the subventricular zone. In adults, BMP4 and 7 immunostaining was present in the GL and SEL. Within the SEL, BMP4 and 7 proteins were expressed primarily in association with the astrocytic glial compartment. BMP6-ir was always found in mature olfactory receptor neurons and their axonal projections to the OB. In summary, these data support the hypothesis that BMPs play a role in the morphogenesis of the olfactory system during development and in its plasticity during adulthood.
Asunto(s)
Astrocitos/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular/genética , Ratones Endogámicos/embriología , Neuronas/metabolismo , Vías Olfatorias/embriología , Células Madre/metabolismo , Factor de Crecimiento Transformador beta , Animales , Animales Recién Nacidos , Astrocitos/citología , Proteína Morfogenética Ósea 4 , Proteína Morfogenética Ósea 6 , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/genética , Femenino , Feto , Regulación del Desarrollo de la Expresión Génica/fisiología , Masculino , Ratones , Ratones Endogámicos/crecimiento & desarrollo , Ratones Endogámicos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/genética , Neuronas/citología , Bulbo Olfatorio/citología , Bulbo Olfatorio/embriología , Bulbo Olfatorio/crecimiento & desarrollo , Proteína Marcadora Olfativa , Mucosa Olfatoria/citología , Mucosa Olfatoria/embriología , Mucosa Olfatoria/crecimiento & desarrollo , Nervio Olfatorio/citología , Nervio Olfatorio/embriología , Nervio Olfatorio/crecimiento & desarrollo , Vías Olfatorias/citología , Vías Olfatorias/crecimiento & desarrollo , Embarazo , ARN Mensajero/metabolismo , Células Madre/citologíaRESUMEN
Mice from which the olfactory marker protein (OMP) gene has been deleted demonstrate a number of neurophysiologic and behavioral defects that suggest OMP is an important component in olfactory signal transduction and is critically involved in odor processing. Recently, the potential pleiotropic effects of gene deletion were addressed by adenoviral vector-mediated rescue of the neurophysiologic defects, in vivo. As a complement to this study, the authors used a recombinant adenoviral vector to transiently introduce OMP into olfactory sensory neurons of adult OMP-null mice and, using psychophysical methods, demonstrated the resulting reacquisition of behavioral function subsequent to gene replacement. The rescue of the OMP-null behavioral phenotype further supports the hypothesis that OMP is an important component in olfactory signal amplification and/or transduction processing.
Asunto(s)
Conducta Animal/fisiología , Proteínas del Tejido Nervioso/fisiología , Odorantes , Fenotipo , Umbral Sensorial/fisiología , Adenoviridae/genética , Animales , Condicionamiento Operante , Aprendizaje Discriminativo , Relación Dosis-Respuesta a Droga , Estesioneuroblastoma Olfatorio/metabolismo , Estesioneuroblastoma Olfatorio/virología , Vectores Genéticos/metabolismo , Vectores Genéticos/farmacología , Proteínas Fluorescentes Verdes , Humanos , Inmunohistoquímica/métodos , Infecciones , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteína Marcadora Olfativa , Neuronas Receptoras Olfatorias/metabolismo , Distribución Aleatoria , Umbral Sensorial/efectos de los fármacos , TransfecciónRESUMEN
BACKGROUND: The main olfactory epithelium (MOE) is a complex organ containing several functionally distinct subpopulations of sensory neurons. One such subpopulation is distinguished by its expression of the guanylyl cyclase GC-D. The axons of GC-D-expressing (GC-D+) neurons innervate 9-15 "necklace" glomeruli encircling the caudal main olfactory bulb (MOB). Chemosensory stimuli for GC-D+ neurons include two natriuretic peptides, uroguanylin and guanylin, and CO2. However, the biologically-relevant source of these chemostimuli is unclear: uroguanylin is both excreted in urine, a rich source of olfactory stimuli for rodents, and expressed in human nasal epithelium; CO2 is present in both inspired and expired air. FINDINGS: To determine whether the principal source of chemostimuli for GC-D+ neurons is external or internal to the nose, we assessed the consequences of removing external chemostimuli for afferent activity to the necklace glomeruli. To do so, we performed unilateral naris occlusions in Gucy2d-Mapt-lacZ +/- mice [which express a beta-galactosidase (beta-gal) reporter specifically in GC-D+ neurons] followed by immunohistochemistry for beta-gal and a glomerular marker of afferent activity, tyrosine hydroxylase (TH). We observed a dramatic decrease in TH immunostaining, consistent with reduced or absent afferent activity, in both necklace and non-necklace glomeruli ipsilateral to the occluded naris. CONCLUSION: Like other MOB glomeruli, necklace glomeruli exhibit a large decrease in afferent activity upon removal of external stimuli. Thus, we conclude that activity in GC-D+ neurons, which specifically innervate necklace glomeruli, is not dependent on internal stimuli. Instead, GC-D+ neurons, like other OSNs in the MOE, primarily sense the external world.
RESUMEN
BACKGROUND: The role of olfactory marker protein (OMP), a hallmark of mature olfactory sensory neurons (OSNs), has been poorly understood since its discovery. The electrophysiological and behavioral phenotypes of OMP knockout mice indicated that OMP influences olfactory signal transduction. However, the mechanism by which this occurs remained unknown. PRINCIPAL FINDINGS: We used intact olfactory epithelium obtained from WT and OMP(-/-) mice to monitor the Ca(2+) dynamics induced by the activation of cyclic nucleotide-gated channels, voltage-operated Ca(2+) channels, or Ca(2+) stores in single dendritic knobs of OSNs. Our data suggested that OMP could act to modulate the Ca(2+)-homeostasis in these neurons by influencing the activity of the plasma membrane Na(+)/Ca(2+)-exchanger (NCX). Immunohistochemistry verifies colocalization of NCX1 and OMP in the cilia and knobs of OSNs. To test the role of NCX activity, we compared the kinetics of Ca(2+) elevation by stimulating the reverse mode of NCX in both WT and OMP(-/-) mice. The resulting Ca(2+) responses indicate that OMP facilitates NCX activity and allows rapid Ca(2+) extrusion from OSN knobs. To address the mechanism by which OMP influences NCX activity in OSNs we studied protein-peptide interactions in real-time using surface plasmon resonance technology. We demonstrate the direct interaction of the XIP regulatory-peptide of NCX with calmodulin (CaM). CONCLUSIONS: Since CaM also binds to the Bex protein, an interacting protein partner of OMP, these observations strongly suggest that OMP can influence CaM efficacy and thus alters NCX activity by a series of protein-protein interactions.
Asunto(s)
Calcio/metabolismo , Calmodulina/fisiología , Proteína Marcadora Olfativa/fisiología , Neuronas Receptoras Olfatorias/metabolismo , Intercambiador de Sodio-Calcio/fisiología , Animales , Calmodulina/genética , Electrofisiología/métodos , Cinética , Ratones , Ratones Noqueados , Modelos Biológicos , Proteína Marcadora Olfativa/genética , Fenotipo , Mapeo de Interacción de Proteínas , Transducción de Señal , Intercambiador de Sodio-Calcio/genética , Resonancia por Plasmón de SuperficieRESUMEN
Altered distribution of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR1 has been linked to stimulation-dependent changes in synaptic efficacy, including long-term potentiation and depression. The main olfactory bulb (OB) remains plastic throughout life; how GluR1 may be involved in this plasticity is unknown. We have previously shown that neonatal naris occlusion reduces numbers of interneuron cell bodies that are immunoreactive for GluR1 in the external plexiform layer (EPL) of the adult mouse OB. Here, we show that immunoreactivity of mouse EPL interneurons for GluR1 is also dramatically reduced following olfactory deafferentation in adulthood. We further show that expression of glutamic acid decarboxylase (GAD) 65, 1 of 2 GAD isoforms expressed by adult gamma-aminobutyric acidergic interneurons, is reduced, but to a much smaller extent, and that in double-labeled cells, immunoreactivity for the Ca(2+)-binding protein parvalbumin (PV) is also reduced. In addition, GluR1 expression is reduced in presumptive tufted cells and interneurons that are negative for GAD65 and PV. Consistent with previous reports, sensory deafferentation resulted in little neuronal degeneration in the adult EPL, indicating that these differences were not likely due to death of EPL neurons. Together, these results suggest that olfactory input regulates expression of the GluR1 AMPA receptor subunit by tufted cells that may in turn regulate GluR1 expression by interneurons within the OB EPL.
Asunto(s)
Interneuronas/metabolismo , Bulbo Olfatorio/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Receptores AMPA/metabolismo , Olfato/fisiología , Sulfato de Zinc/farmacología , Animales , Recuento de Células , Glutamato Descarboxilasa/metabolismo , Ratones , Ratones Transgénicos , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/efectos de los fármacosRESUMEN
Olfactory marker protein (OMP), a phylogenetically conserved protein, is highly, and almost exclusively, expressed in vertebrate olfactory receptor neurons (ORNs). Although OMP is widely used as a marker for ORNs, its function has remained largely elusive. Here we used suction-pipette recordings from isolated ORNs of OMP(-/-) mice to investigate its role in olfactory transduction. Vertebrate olfactory transduction is initiated when odourants bind to receptor proteins to activate an adenylyl cyclase via a G protein-coupled signalling pathway. This leads to an increase in cAMP and the opening of a cyclic nucleotide-gated (CNG), non-selective cation channel which depolarizes the cells. Ca(2+) influx through the CNG channel in turn activates a Ca(2+)-activated Cl(-) channel, causing a Cl(-) efflux and further depolarization. In the absence of OMP, the time-to-transient-peak of the response, the latency to first spike, and the response termination were slowed 2- to 8-fold, indicating its role in regulating olfactory response kinetics and termination. This phenotype persisted in OMP(-/-) ORNs even in low external Ca(2+) solution chosen to prevent Cl(-) channel activation, suggesting OMP acts upstream of Cl(-) channel activation. Furthermore, the response kinetics in cilia are virtually indistinguishable between OMP(-/-) and wild-type ORNs when intracellular cAMP level was elevated by the phospho-diesterase inhibitor, IBMX, suggesting OMP acts upstream of cAMP production. Together, our results suggest a role for OMP in regulating the kinetics and termination of olfactory responses, implicating a novel mechanism for fast and robust response termination to ensure the temporal resolution of the odour stimulus. These observations also help explain the deficits in odour detection threshold and odour quality discrimination seen in the OMP(-/-) mice.
Asunto(s)
Cilios/fisiología , AMP Cíclico/fisiología , Odorantes , Proteína Marcadora Olfativa/fisiología , Neuronas Receptoras Olfatorias/fisiología , Adaptación Fisiológica/fisiología , Animales , Ciclohexanoles/farmacología , Electrofisiología , Eucaliptol , Femenino , Cinética , Ratones , Ratones Noqueados , Monoterpenos/farmacología , Proteína Marcadora Olfativa/deficiencia , Proteína Marcadora Olfativa/genética , Inhibidores de Fosfodiesterasa/farmacología , Soluciones , Estimulación QuímicaRESUMEN
Bex1 and Calmodulin (CaM) are upregulated during skeletal muscle regeneration. We confirm this finding and demonstrate the novel finding that they interact in a calcium-dependent manner. To study the role of Bex1 and its interaction with CaM in skeletal muscle regeneration, we generated Bex1 knock out (Bex1-KO) mice. These mice appeared to develop normally and are fertile, but displayed a functional deficit in exercise performance compared to wild type (WT) mice. After intramuscular injection of cardiotoxin, which causes extensive and reproducible myotrauma followed by recovery, regenerating muscles of Bex1-KO mice exhibited elevated and prolonged cell proliferation, as well as delayed cell differentiation, compared to WT mice. Thus, our results provide the first evidence that Bex1-KO mice show altered muscle regeneration, and allow us to propose that the interaction of Bex1 with Ca(2+)/CaM may be involved in skeletal muscle regeneration.
Asunto(s)
Fatiga Muscular/fisiología , Músculo Esquelético/fisiología , Proteínas del Tejido Nervioso/fisiología , Esfuerzo Físico/fisiología , Regeneración/fisiología , Animales , Masculino , Ratones , Ratones NoqueadosRESUMEN
Interest in manipulating gene expression in olfactory sensory neurons (OSNs) has led to the use of adenoviruses (AdV) as gene delivery vectors. OSNs are the first order neurons in the olfactory system and the initial site of odor detection. They are highly susceptible to adenovirus infection although the mechanism is poorly understood. The Coxsackie-Adenovirus receptor (CAR) and members of the integrin family have been implicated in the process of AdV infection in various systems. Multiple serotypes of AdV efficiently bind to the CAR, leading to entry and infection of the host cell by a mechanism that can also involve integrins. Cell lines that do not express CAR are relatively resistant, but not completely immune to AdV infection, suggesting that other mechanisms participate in mediating AdV attachment and entry. Using in situ hybridization and western blot analyses, we show that OSNs and olfactory bulbs (OB) of mice express abundant CAR mRNA at embryonic and neonatal stages, with progressive diminution during postnatal development. By contrast to the olfactory epithelium (OE), CAR mRNA is still present in the adult mouse OB. Furthermore, despite a similar postnatal decline, CAR protein expression in the OE and OB of mice continues into adulthood. Our results suggest that the robust AdV infection observed in the postnatal olfactory system is mediated by CAR and that expression of even small amounts of CAR protein as seen in the adult rodent, permits efficient AdV infection and entry. CAR is an immunoglobulin domain-containing protein that bears homology to cell-adhesion molecules suggesting the possibility that it may participate in organization of the developing olfactory system.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Bulbo Olfatorio/crecimiento & desarrollo , Bulbo Olfatorio/fisiología , Mucosa Olfatoria/crecimiento & desarrollo , Mucosa Olfatoria/fisiología , Receptores Virales/genética , Infecciones por Adenoviridae/fisiopatología , Factores de Edad , Animales , Secuencia de Bases , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Femenino , Hibridación in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Bulbo Olfatorio/embriología , Mucosa Olfatoria/embriología , Embarazo , ARN Mensajero/análisis , Receptores Virales/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Nuclear magnetic resonance (NMR) (15)N relaxation measurements of the olfactory marker protein (OMP) including longitudinal relaxation (T(1)), transverse relaxation (T(2)), and (15)N-{(1)H} NOE data were collected at low protein concentrations (Asunto(s)
Proteínas del Tejido Nervioso/química
, Termodinámica
, Animales
, Anisotropía
, Modelos Moleculares
, Proteínas del Tejido Nervioso/fisiología
, Isótopos de Nitrógeno
, Resonancia Magnética Nuclear Biomolecular/métodos
, Proteína Marcadora Olfativa
, Conformación Proteica
, Estructura Secundaria de Proteína
, Ratas
, Transducción de Señal/fisiología
, Soluciones
RESUMEN
Mice pre-trained in an olfactometer were tested daily on odor detection and discrimination tasks after irrigation of their olfactory epithelium in each naris with 50 microl of 5% zinc sulfate or saline. Anterograde transport of a wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) conjugate from the epithelium to the olfactory bulb was used to assess anatomical connectivity in these and in mice that were used only for histological analyses. One day after treatment, saline controls performed at high levels of accuracy in detecting vapor from solutions of 5-0.01% ethyl acetate and in an odor discrimination task but most ZnSO4-treated mice performed at chance for 5-30 days before showing recovery. Although dense WGA-HRP reaction product was found in the accessory olfactory bulb, there was little or no evidence for axonal transport to glomeruli of the main olfactory bulb in the first 4-8 days after treatment. These results demonstrate that intranasal application of ZnSO4 to mice produces a brief but essentially total disruption of functional connections from the olfactory epithelium to the main olfactory bulb and a corresponding transient anosmia.
Asunto(s)
Trastornos del Olfato/inducido químicamente , Trastornos del Olfato/fisiopatología , Bulbo Olfatorio/anatomía & histología , Bulbo Olfatorio/efectos de los fármacos , Nervio Olfatorio/anatomía & histología , Nervio Olfatorio/efectos de los fármacos , Olfato/efectos de los fármacos , Sulfato de Zinc/administración & dosificación , Sulfato de Zinc/farmacología , Acetatos , Administración Intranasal , Animales , Transporte Axonal/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Femenino , Ratones , Bulbo Olfatorio/fisiología , Bulbo Olfatorio/fisiopatología , Nervio Olfatorio/fisiología , Nervio Olfatorio/fisiopatología , Olfato/fisiología , Factores de TiempoRESUMEN
Olfactory marker protein (OMP) expression is a hallmark of mature vertebrate olfactory receptor neurons (ORNs). Evidence for OMP function derives from altered behavioral and electrophysiological activities of OMP-KO mice. The molecular basis for the altered phenotype following the deletion of OMP is still unclear. Recent structural studies predict the involvement of OMP in protein-protein interaction. Here we report the identification of an OMP partner, Bex2, by phage-display screening of an olfactory mucosal cDNA-library. In situ hybridization demonstrates cellular co-localization of OMP mRNA with mRNAs for Bex1, Bex2, and Bex3 in ORNs of olfactory tissue of the mouse. The OMP/Bex interaction has been confirmed by demonstrating the chemical cross-linking of recombinant rat OMP with a synthetic peptide derived from the Bex amino acid sequence. The subcellular localization of Bex and OMP proteins was evaluated in transfected HEK293 cells. Bex is visualized in the nucleus and cytoplasm. Following co-transfection we observed the unexpected presence of some OMP in the nucleus along with Bex. Together, these data argue convincingly that we have identified Bex as an OMP partner whose further characterization will provide insight to the role of OMP and to the mechanism of the OMP/Bex interaction in ORN differentiation and function.