RESUMEN

We investigated the microscopic structure of transverse sections of the oculomotor, trochlear and abducens nerves of Arabian foals using stereological methods. Bilateral nerve pairs from 2-month-old female Arabian foals were analyzed. The tissues were embedded in plastic blocks, then 1 µm thick sections were cut and stained with osmium tetroxide and methylene blue-azure II. Stereology was performed using light microscopy. Morphometry showed that the right and left pairs of nerves were similar. The transverse sectional areas of the oculomotor, trochlear and abducens nerves were 1.93 ± 0.19 mm2, 0.32 ± 0.06 mm2 and 0.70 ± 0.08 mm2, respectively. The oculomotor nerve exhibited a significantly greater number of myelinated axons (16755 ± 1279) and trochlear (2656 ± 494) and the abducens nerves (4468 ± 447). The ratio of the axon diameter to myelinated nerve fiber diameter was 0.58, 0.55 and 0.55 for the oculomotor, trochlear and abducens nerves, respectively. Of the three nerves studied, the abducens nerve exhibited the greatest nerve fiber area, myelin area, nerve and axon diameters, and myelin thickness. The ratio of small myelinated nerve fibers was greatest in the oculomotor nerve.

Asunto(s)

Nervio Abducens/metabolismo , Axones/metabolismo , Vaina de Mielina/metabolismo , Fibras Nerviosas Mielínicas/metabolismo , Nervio Oculomotor/metabolismo , Animales , Femenino , Caballos , Microscopía/métodos

RESUMEN

Interaction of the axon guidance receptor Neuropilin-1 (Npn-1) with its repulsive ligand Semaphorin 3A (Sema3A) is crucial for guidance decisions, fasciculation, timing of growth and axon-axon interactions of sensory and motor projections in the embryonic limb. At cranial levels, Npn-1 is expressed in motor neurons and sensory ganglia and loss of Sema3A-Npn-1 signaling leads to defasciculation of the superficial projections to the head and neck. The molecular mechanisms that govern the initial fasciculation and growth of the purely motor projections of the hypoglossal and abducens nerves in general, and the role of Npn-1 during these events in particular are, however, not well understood. We show here that selective removal of Npn-1 from somatic motor neurons impairs initial fasciculation and assembly of hypoglossal rootlets and leads to reduced numbers of abducens and hypoglossal fibers. Ablation of Npn-1 specifically from cranial neural crest and placodally derived sensory tissues recapitulates the distal defasciculation of mixed sensory-motor nerves of trigeminal, facial, glossopharyngeal and vagal projections, which was observed in Npn-1(-/-) and Npn-1(Sema-) mutants. Surprisingly, the assembly and fasciculation of the purely motor hypoglossal nerve are also impaired and the number of Schwann cells migrating along the defasciculated axonal projections is reduced. These findings are corroborated by partial genetic elimination of cranial neural crest and embryonic placodes, where loss of Schwann cell precursors leads to aberrant growth patterns of the hypoglossal nerve. Interestingly, rostral turning of hypoglossal axons is not perturbed in any of the investigated genotypes. Thus, initial hypoglossal nerve assembly and fasciculation, but not later guidance decisions depend on Npn-1 expression and axon-Schwann cell interactions.

Asunto(s)

Movimiento Celular , Nervios Craneales/metabolismo , Fasciculación/metabolismo , Neuropilina-1/metabolismo , Células de Schwann/metabolismo , Nervio Abducens/embriología , Nervio Abducens/metabolismo , Animales , Axones/metabolismo , Nervios Craneales/embriología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Fasciculación/genética , Femenino , Nervio Hipogloso/embriología , Nervio Hipogloso/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Neuronas Motoras/metabolismo , Mutación , Cresta Neural/embriología , Cresta Neural/metabolismo , Neuropilina-1/genética , Factores de Transcripción SOXE/metabolismo , Semaforina-3A/genética , Semaforina-3A/metabolismo , Células Receptoras Sensoriales/metabolismo , Transducción de Señal

RESUMEN

Previous studies using an in vitro model of eyeblink classical conditioning in turtles suggest that increased numbers of synaptic AMPARs supports the acquisition and expression of conditioned responses (CRs). Brain-derived neurotrophic factor (BDNF) and its associated receptor tyrosine kinase, TrkB, is also required for acquisition of CRs. Bath application of BDNF alone induces synaptic delivery of GluR1- and GluR4-containing AMPARs that is blocked by coapplication of the receptor tyrosine kinase inhibitor K252a. The molecular mechanisms involved in BDNF-induced AMPAR trafficking remain largely unknown. The aim of this study was to determine whether BDNF-induced synaptic AMPAR incorporation utilizes similar cellular mechanisms as AMPAR trafficking that occurs during in vitro classical conditioning. Using pharmacological blockade and confocal imaging, the results show that synaptic delivery of GluR1 subunits during conditioning or BDNF application does not require activity of NMDARs but is mediated by extracellular signal-regulated kinase (ERK). In contrast, synaptic delivery of GluR4-containing AMPARs during both conditioning and BDNF application is NMDAR- as well as ERK-dependent. These findings indicate that BDNF application mimics AMPAR trafficking observed during conditioning by activation of some of the same intracellular signaling pathways and suggest that BDNF is a key signal transduction element in postsynaptic events that mediate conditioning.

Asunto(s)

Factor Neurotrófico Derivado del Encéfalo/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Tortugas/metabolismo , Nervio Abducens/metabolismo , Animales , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/farmacología , Condicionamiento Psicológico/fisiología , Flavonoides/farmacología , Expresión Génica/efectos de los fármacos , Neuronas Motoras/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Sinaptofisina/metabolismo

RESUMEN

Premotor inhibitory neurons responsible for the decrease in the firing discharge during fast or slow eye movements selectively target the cell bodies and the dendrites of abducens motoneurons. Gamma-aminobutyric acid (GABA) and glycine, the main inhibitory synaptic neurotransmitters in the central nervous system, act via glycine and GABAA receptors, assembled from various types of subunits, which determine the kinetics of the currents mediated. Therefore, our hypothesis was that the expression of the inhibitory receptors on the somatic and the dendritic compartments, involved in different functions, may differ. In this study, we compared the subcellular patterns of expression of the main GABAA receptor subunits (GABAARalpha1, alpha2, alpha3, alpha5), glycine receptors (GlyRalpha1), and gephyrin in the somatic and dendritic compartments of rat abducens motoneurons, using double or triple immunocytochemical experiments with confocal microscopy. Significant differences exist in the patterns of organization and the synaptic expression of the GlyR and GABAAR subunits in the cell bodies and dendrites of abducens motoneurons. In the somata, only the GABAARalpha1 subunit was expressed, whereas both GABAARalpha1 and GABAARalpha3 were present in the dendrites. The GlyRalpha1 to GABAARalpha1 density ratio was reversed in the somatic and dendritic compartments (0.9 vs. 2.3). A quantitative electron microscopy study showed that the modes whereby gephyrin reaches its postsynaptic inhibitory synaptic target differ between the somata and the dendrites. Therefore, our results support the idea that a structure-function adaptation occurs at the single-neuron level.

Asunto(s)

Nervio Abducens/metabolismo , Neuronas Motoras/metabolismo , Receptores de GABA-A/metabolismo , Receptores de Glicina/metabolismo , Nervio Abducens/citología , Animales , Transporte Biológico , Dendritas/metabolismo , Espacio Intracelular/metabolismo , Masculino , Ratas , Ratas Wistar , Distribución Tisular

RESUMEN

Many studies have shown that the nucleus prepositus hypoglossi (PH) participates with the vestibular nuclear complex, the cerebellum and the oculomotor nuclei in the control of eye movements. We have looked at the neurochemical organization of PH in the cat and monkey using a recently developed antibody, 8B3, that recognizes a chondroitin sulfate proteoglycan. In the cat, immunoreactivity to 8B3 labels a set of cells in PH. On frontal sections, these cells form a cluster that is seen over the entire anterior-posterior (A-P) extent of PH, but the number of cells in the cluster changes with A-P level. Earlier studies have identified an A-P cell column in PH of the cat whose neurons synthesize nitric oxide. We have used both single- and double-label protocols to investigate the relation between the two cell groups. Single-label studies show spatial overlap but that the cells immunoreactive to nitric oxide synthase (nNOS) are more numerous than cells immunoreactive to 8B3. Double-label studies show that all cells immunoreactive to 8B3 were also immunoreactive to nNOS, but, as suggested by the single-label data, there are many nNOS-immunoreactive cells not immunoreactive to 8B3. Populations of 8B3 and nNOS-immunoreactive cells are also found in PH of squirrel and macaque monkeys. The results suggest that nNOS-immunoreactive cells in PH may consist of two functionally different populations.

Asunto(s)

Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Bulbo Raquídeo/metabolismo , Neuronas/metabolismo , Neuronas Nitrérgicas/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Óxido Nítrico/metabolismo , Nervio Abducens/citología , Nervio Abducens/metabolismo , Animales , Química Encefálica/fisiología , Gatos , Cerebelo/citología , Cerebelo/metabolismo , Movimientos Oculares/fisiología , Inmunohistoquímica , Macaca , Bulbo Raquídeo/citología , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Neuronas/citología , Neuronas Nitrérgicas/citología , Músculos Oculomotores/inervación , Saimiri , Especificidad de la Especie

RESUMEN

Immunohistochemical study was performed to examine if calcium-binding proteins are involved in the degeneration of motor neurons in the brain stems and the spinal cords of transgenic mice carrying a G93A mutant human SOD1 gene. Specimens from age-matched non-transgenic wild-type mice served as controls. In the spinal cord of the controls, the density of parvalbumin-immunoreactive neurons was highest in the large anterior horn neurons and lower in the posterior horn neurons in the spinal cord. On the other hand, calbindin D-28k immunoreactivity was much less apparent than that observed with parvalbumin antisera. Rexed's lamina II was densely immunostained for calbindin D-28k, whereas, in the anterior horn, calbindin-D-28k-positive small neurons were barely dispersed in a scattered pattern. In transgenic mice, parvalbumin-positive anterior horn neurons were severely reduced, even at the presymptomatic stage, whereas calbindin-positive neurons were largely preserved. At the symptomatic stage, both parvalbumin and calbindin D-28k immunoreactivity markedly diminished or disappeared in the anterior horn. Immunoblotting analysis revealed a significant reduction of immunoreactivity to parvalbumin antibody in transgenic mice compared with the controls. In the brain stem, parvalbumin-positive oculomotor and abducens neurons and the calbindin D-28k-positive sixth nucleus were well-preserved in transgenic mice as well as in the controls. Thus, the diffuse and severe loss of parvalbumin immunoreactivity of large motor neurons even at early stages in SOD1-transgenic mice and the absence of calbindin D-28k immunoreactivity of normal large motor neurons suggest that these calcium-binding proteins may contribute to selective vulnerability and an early loss of function of large motor neurons in this SOD1-transgenic mouse model.

Asunto(s)

Esclerosis Amiotrófica Lateral/metabolismo , Tronco Encefálico/metabolismo , Neuronas Motoras/metabolismo , Parvalbúminas/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Médula Espinal/metabolismo , Superóxido Dismutasa/genética , Nervio Abducens/metabolismo , Nervio Abducens/patología , Nervio Abducens/fisiopatología , Esclerosis Amiotrófica Lateral/genética , Animales , Tronco Encefálico/patología , Tronco Encefálico/fisiopatología , Calbindinas , Tamaño de la Célula , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación hacia Abajo/genética , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/patología , Mutación/genética , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Nervio Oculomotor/metabolismo , Nervio Oculomotor/patología , Nervio Oculomotor/fisiopatología , Médula Espinal/patología , Médula Espinal/fisiopatología , Transfección , Regulación hacia Arriba/genética

RESUMEN

The authors reviewed clinical profiles and laboratory findings for 100 cases of abducens nerve paresis without impairment of the other cranial nerves, limb weakness, and ataxia throughout the clinical course. Review of the medical records of 9300 patients referred to our neuoroimmunological laboratory for serum anti-ganglioside antibody testing. Information was obtained from each primary physician on symptoms of preceding infection; initial symptoms; neurological signs during the illness; the clinical course; treatment provided; and outcome. Isolated abducens nerve paresis was present in 100 patients and bilateral paresis in 29. Tentative diagnoses made by the primary physicians on request of anti-ganglioside antibody testing were abducens nerve palsy (n = 68), Fisher syndrome (n = 14), acute ophthalmoparesis without ataxia (n = 14). Symptoms of infection anteceded in 63. Tendon reflexes were absent or decreased in 27. Distal paresthesias were experienced by seven. Serum anti-GQ1b antibody was positive in 25. These findings suggest that some cases of isolated abducens nerve palsy can be categorized as a regional variant of Guillain-Barré syndrome or mild form of Fisher syndrome.

Asunto(s)

Enfermedades del Nervio Abducens/diagnóstico , Enfermedades del Nervio Abducens/etiología , Autoanticuerpos/sangre , Gangliósidos/inmunología , Síndrome de Guillain-Barré/complicaciones , Síndrome de Guillain-Barré/diagnóstico , Nervio Abducens/inmunología , Nervio Abducens/metabolismo , Nervio Abducens/fisiopatología , Enfermedades del Nervio Abducens/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Autoanticuerpos/inmunología , Niño , Preescolar , Diagnóstico Diferencial , Femenino , Síndrome de Guillain-Barré/sangre , Humanos , Inmunoglobulinas Intravenosas/uso terapéutico , Lactante , Infecciones/complicaciones , Infecciones/inmunología , Masculino , Persona de Mediana Edad , Síndrome de Miller Fisher/sangre , Síndrome de Miller Fisher/complicaciones , Síndrome de Miller Fisher/diagnóstico , Plasmaféresis , Valor Predictivo de las Pruebas , Reflejo Anormal/inmunología , Esteroides/uso terapéutico

RESUMEN

The question whether general tetanus arises from the independent sum of multiple local tetani or results from the actions of the transynaptic tetanus neurotoxin (TeNT) in higher brain centres remains unresolved. Despite the blood-borne dissemination of TeNT from an infected wound, the access to the central nervous system is probably prevented by the blood-brain barrier. However, several long-term sequelae (e.g. autonomic dysfunction, seizures, myoclonus, and sleep disturbances) present after the subsidence of muscle spasms might be indicative of central actions that occur farther away from lower motoneurons. Subsequently, the obvious entry route is the peripheral neurons followed by the transynaptic passage to the brain. We aimed at describing the pathophysiological correlates of TeNT translocation using the oculomotor system as a comprehensive model of cell connectivity and neuronal firing properties. In this study, we report that injection of TeNT into the medial rectus muscle of one eye resulted in bilateral gaze palsy attributed to firing alterations found in the contralaterally projecting abducens internuclear neurons. Functional alterations in the abducens-to-oculomotor internuclear pathway resembled in part the classically described TeNT disinhibition. We confirmed the transynaptic targeted action of TeNT by analysing vesicle-associated membrane protein2 (VAMP2) immunoreactivity (the SNARE protein cleaved by TeNT). VAMP2 immunoreactivity decreased by 94.4% in the oculomotor nucleus (the first synaptic relay) and by 62.1% presynaptic to abducens neurons (the second synaptic relay). These results are the first demonstration of physiological changes in chains of connected neurons that are best explained by the transynaptic action of TeNT on premotor neurons as shown with VAMP2 immunoreactivity which serves as an indicator of TeNT activity.

Asunto(s)

Metaloendopeptidasas/toxicidad , Oftalmoplejía/inducido químicamente , Toxina Tetánica/toxicidad , Nervio Abducens/efectos de los fármacos , Nervio Abducens/metabolismo , Nervio Abducens/fisiopatología , Animales , Biomarcadores/metabolismo , Gatos , Movimientos Oculares/efectos de los fármacos , Femenino , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/farmacocinética , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Conducción Nerviosa/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Vías Nerviosas/fisiopatología , Músculos Oculomotores/efectos de los fármacos , Músculos Oculomotores/metabolismo , Músculos Oculomotores/fisiopatología , Nervio Oculomotor/efectos de los fármacos , Nervio Oculomotor/metabolismo , Nervio Oculomotor/fisiopatología , Oftalmoplejía/metabolismo , Oftalmoplejía/fisiopatología , Proteínas R-SNARE , Toxina Tetánica/farmacocinética

RESUMEN

Nerve transection induces complex changes in gene regulation and expression that can have profound phenotypic effects on the fate of axotomized neurons. The transcription factors c-Jun and ATF-2 (activating transcription factor-2) are components of a regulatory network that mediates survival, regeneration, and apoptosis following axotomy in rodents. The activation and function of c-Jun and ATF-2 after nerve injury have not been examined in primates. Using a novel model of cranial nerve injury in baboons, we have examined the temporality of c-Jun activation (phosphorylation) in cranial nerve (CN) III and CN VI neurons and ATF-2 activation in CN VI neurons at 2, 4, and 9 days post-injury by immunohistochemistry. Furthermore, we have addressed whether the activation of these factors is associated with apoptosis by the TUNEL assay. We report that activated c-Jun is present in CN III and CN VI neurons ipsilateral to axotomy at 2, 4, and 9 days post-injury, but not in neurons contralateral to injury. Additionally, CN VI neurons ipsilateral to injury at 4 and 9 days contain activated ATF-2. Furthermore, no evidence of TUNEL reactivity was observed in either nucleus, regardless of laterality, at any of the examined time points. These findings suggest that activation of both c-Jun and ATF-2 does not mediate apoptosis in axotomized primate CN III and CN VI neurons at time points up to 9 days. This report serves as a basic inquiry into the neuronal response to cranial nerve injury in primates.

Asunto(s)

Apoptosis/fisiología , Traumatismos del Nervio Craneal/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Neuronas Motoras/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Degeneración Retrógrada/metabolismo , Factores de Transcripción/metabolismo , Nervio Abducens/citología , Nervio Abducens/metabolismo , Traumatismo del Nervio Abducente/metabolismo , Traumatismo del Nervio Abducente/fisiopatología , Factor de Transcripción Activador 2 , Animales , Axotomía , Tronco Encefálico/metabolismo , Tronco Encefálico/patología , Traumatismos del Nervio Craneal/patología , Traumatismos del Nervio Craneal/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Lateralidad Funcional/fisiología , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Neuronas Motoras/patología , Nervio Oculomotor/citología , Nervio Oculomotor/metabolismo , Traumatismos del Nervio Oculomotor , Papio anubis , Fosforilación , Degeneración Retrógrada/patología , Degeneración Retrógrada/fisiopatología , Factores de Tiempo , Activación Transcripcional/fisiología

RESUMEN

The synaptic delivery of GluR4-containing AMPA receptors during in vitro classical conditioning of a neural correlate of an eyeblink response was examined by fluorescence imaging of punctate staining for glutamate receptor subunits and the presynaptic marker synaptophysin. There was a significant increase in GluR4-containing AMPA receptors to synaptic sites after conditioning as determined by colocalization of GluR4 subunit puncta with synaptophysin. Moreover, the trafficking of these receptor subunits requires NMDA receptor activation as it was blocked by D,L-2-amino-5-phosphonovaleric acid (AP-5). In contrast, colocalization of NR1 subunits with synaptophysin was stable regardless of whether the preparations had undergone conditioning or had been treated by AP-5. The enhanced colocalization of GluR4 and synaptophysin was accompanied by an increase in both the total number and size of puncta for both proteins, suggesting greater synthesis and aggregation during conditioning. Western blot analysis confirmed upregulation of synaptophysin and GluR4 following conditioning. These data support the hypothesis that GluR4-containing AMPA receptors are delivered to synaptic sites during conditioning. Further, they suggest coordinate presynaptic and postsynaptic modifications during in vitro classical conditioning.

Asunto(s)

Condicionamiento Clásico/fisiología , Receptores AMPA/metabolismo , Sinapsis/metabolismo , 2-Amino-5-fosfonovalerato/farmacología , Nervio Abducens/citología , Nervio Abducens/metabolismo , Animales , Parpadeo/fisiología , Western Blotting , Antagonistas de Aminoácidos Excitadores/farmacología , Inmunohistoquímica , Técnicas In Vitro , Neuronas Motoras/metabolismo , Fenómenos Fisiológicos del Sistema Nervioso , Receptores AMPA/antagonistas & inhibidores , Sinaptofisina/metabolismo , Distribución Tisular , Tortugas

RESUMEN

In the present study, an optimized Transmission Electron Microscopy Color Imaging (TEMCI) procedure was used to map and quantify the pathways involved in the trafficking and subcellular targeting of gephyrin in identified abducens motoneurons. Gephyrin is a scaffolding protein, which plays a crucial role in the clustering of the GABA(A) and glycine receptors to the cytoskeleton. TEMCI associated several accurate tools: (i) nanogold immunodetection of gephyrin in motoneurons identified on the basis of their immunoreactivity to Choline Acetyl Transferase, (ii) low magnification color scale coding of gephyrin densities on series of ultrathin sections of motoneurons, which gave a map of the cytoplasmic distribution of the protein, (iii) statistical analysis of the subcellular distribution of the immunolabeling. The color map of gephyrin densities in the cell bodies reflected the distribution of inhibitory synapses over the membrane. The TEMCI analysis of motoneurons with various patterns of synaptic covering made it possible to visualize for the first time the cytoplasmic transport pathway of gephyrin towards its target at synaptic contact. A high magnification quantitative analysis, including the study of 109 inhibitory synapses, showed that most gephyrin-associated immunogold particles (67%) were located in the subsynaptic regions facing the active zones, and the second most densely occupied regions were the perisynaptic regions (19.5% of immunogold particles). A consistent proportion of the gephyrin (11.5%), significantly higher than densities present in the rest of the cytoplasm (2%), was detected in the extrasynaptic submembrane region.

Asunto(s)

Nervio Abducens/metabolismo , Proteínas Portadoras/metabolismo , Citoplasma/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas Motoras/metabolismo , Puente/metabolismo , Nervio Abducens/ultraestructura , Animales , Mapeo Encefálico/instrumentación , Mapeo Encefálico/métodos , Inmunohistoquímica , Masculino , Microscopía Electrónica/instrumentación , Microscopía Electrónica/métodos , Neuronas Motoras/ultraestructura , Inhibición Neural/fisiología , Puente/ultraestructura , Transporte de Proteínas/fisiología , Ratas , Ratas Wistar , Agregación de Receptores/fisiología , Receptores de GABA-A/metabolismo , Receptores de Glicina/metabolismo , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestructura , Transmisión Sináptica/fisiología

RESUMEN

The pattern of innervation of the extraocular muscles is highly conserved across higher vertebrate species and mediates sophisticated visuomotor processes. Defects in oculomotor development often lead to strabismus, a misalignment of the eyes that can cause partial blindness. Although it has been intensively studied from a clinical perspective, relatively little is known about how the system develops embryonically. We have therefore mapped the development of the oculomotor nerve (OMN) in chick embryos by using confocal microscopy. We show that OMN development follows a series of stereotyped steps that are tightly regulated in space and time. The OMN initially grows past three of its targets to innervate its distal target, the ventral oblique muscle, only later forming branches to the more proximal muscles. We have also investigated spatiotemporal aspects of the unusual contralateral migration of a subpopulation of oculomotor neurons by using molecular markers and have found the semaphorin axon guidance molecules and their receptors, the neuropilins, to be expressed in discrete subnuclei during this migration. Finally, we have created an embryological model of Duane retraction syndrome (DRS), a form of strabismus in which the OMN is believed to innervate aberrantly the lateral rectus, the normal target of the abducens nerve. By ablating rhombomeres 5 and 6 and hence the abducens, we have mimicked a proposed oculomotor deficit occurring in DRS. We find that the absence of the abducens nerve is not sufficient to produce this inappropriate innervation, so other factors are required to explain DRS.

Asunto(s)

Axones/fisiología , Proteínas de la Membrana , Neuronas/metabolismo , Nervio Oculomotor/embriología , Nervio Abducens/embriología , Nervio Abducens/metabolismo , Animales , Proteínas Portadoras/metabolismo , Embrión de Pollo , Proteínas del Citoesqueleto , Modelos Animales de Enfermedad , Síndrome de Retracción de Duane/metabolismo , Síndrome de Retracción de Duane/fisiopatología , Glicoproteínas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Proteínas con Homeodominio LIM , Microscopía Confocal/métodos , Miosinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Neurofilamentos/metabolismo , Neuronas/fisiología , Neuropilina-1/metabolismo , Neuropilina-2/metabolismo , Nervio Oculomotor/citología , ARN Mensajero/metabolismo , Semaforina-3A/metabolismo , Semaforinas , Factores de Transcripción , Nervio Troclear/embriología , Nervio Troclear/metabolismo

RESUMEN

In the developing hindbrain, the functional loss of individual Hox genes has revealed some of their roles in specifying rhombomere (r) identity. However, it is unclear how Hox genes act in concert to confer the unique identity to multiple rhombomeres. Moreover, it remains to be elucidated how these genes interact with other transcriptional programs to specify distinct neuronal lineages within each rhombomere. We demonstrate that in r5, the combined mutation of Hoxa3 and Hoxb3 result in a loss of Pax6- and Olig2-expressing progenitors that give rise to somatic motoneurons of the abducens nucleus. In r6, the absence of any combination of the Hox3 paralogous genes results in ectopic expression of the r4-specific determinant Hoxb1. This ectopic expression in turn results in the differentiation of r4-like facial branchiomotoneurons within this rhombomere. These studies reveal that members of the Hox1 and Hox3 paralogous groups participate in a 'Hox code' that is necessary for coordinating both suppression and activation mechanisms that ensure distinction between the multiple rhombomeres in the developing hindbrain.

Asunto(s)

Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Neuronas Motoras/fisiología , Rombencéfalo/embriología , Proteínas de Xenopus/genética , Nervio Abducens/citología , Nervio Abducens/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Tipificación del Cuerpo , Diferenciación Celular/fisiología , Linaje de la Célula , Movimiento Celular/fisiología , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Proteínas del Ojo , Proteínas de Homeodominio/metabolismo , Ratones , Morfogénesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Proteínas Represoras , Rombencéfalo/citología , Proteínas de Xenopus/metabolismo

RESUMEN

Previous work showed that in vitro abducens eyeblink classical conditioning of turtle brain stem-cerebellum preparations involved NMDA-mediated mechanisms and redistribution of GluR4-containing AMPA receptors in the abducens motor nuclei. Since conditioning can be obtained in brain stem preparations without the cerebellum, we examined whether similar mechanisms were involved during conditioning of the brain stem alone. The results showed that conditioning could not be induced in the presence of the NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP-5) and that abducens nerve conditioned responses, once initiated in normal saline, were significantly attenuated in the presence of AP-5. The effects of AP-5 did not generally depress physiological responsiveness of preparations because some abducens nerve reflexes were not significantly reduced by the compound. GluR4-containing AMPA receptors in the abducens motor nuclei were significantly upregulated and positively correlated with the levels of conditioning similar to that of preparations having an intact cerebellum. Furthermore, increased GluR4 subunits after brain stem conditioning was confirmed by Western blot analysis. These results suggest that NMDA receptor-mediated mechanisms and GluR4 upregulation may mediate in vitro abducens eyeblink classical conditioning and that these mechanisms reside in the brain stem eyeblink circuitry.

Asunto(s)

Tronco Encefálico/metabolismo , Condicionamiento Palpebral/fisiología , Receptores AMPA/biosíntesis , Receptores de N-Metil-D-Aspartato/fisiología , Tortugas/metabolismo , Nervio Abducens/efectos de los fármacos , Nervio Abducens/metabolismo , Animales , Tronco Encefálico/efectos de los fármacos , Cerebelo/efectos de los fármacos , Cerebelo/metabolismo , Condicionamiento Palpebral/efectos de los fármacos , Técnicas In Vitro , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/biosíntesis , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología

RESUMEN

The loss of afferent synaptic boutons is a prominent alteration induced by axotomy on adult central neurons. In this work we attempted to prove whether synapse loss could be reverted by reconnection with a new target. We severed the medial longitudinal fascicle of adult cats and then transplanted embryonic cerebellar primordia at the lesion site immediately after lesion. As previously shown, the transected axons from abducens internuclear neurons penetrate and reinnervate the graft [J Comp Neurol 444 (2002) 324]. By immunocytochemistry and electron microscopy we studied the synaptology of abducens internuclear neurons under three conditions: control, axotomy and transplant (2 months of survival time). Semithin sections of the abducens nucleus were immunostained against calretinin, to identify abducens internuclear neurons, and either synaptophysin (SF), to label synaptic terminals, or glial fibrillary acidic protein (GFAP) to detect the astrocytic reaction. Optical and linear density of SF and GFAP immunostaining were measured. Data revealed a significant decrease in the density of SF-labeled terminals with a parallel increase in GFAP-immunoreactive elements after axotomy. On the contrary, in the transplant group, the density of SF-labeled terminals was found similar to control, and the astrocytic reaction induced by lesion was significantly reduced. At the ultrastructural level, synaptic coverage and linear density of boutons were measured around the somata of abducens internuclear neurons. Whereas a significant reduction in both parameters was found after axotomy, cells of the transplant group received a normal density of synaptic endings. The ratio between F- and S-type boutons was found similar in the three groups. Therefore, these findings indicate that the grafting of a new target can prevent the loss of afferent synaptic boutons produced by the axotomy.

Asunto(s)

Trasplante de Tejido Encefálico/métodos , Interneuronas/metabolismo , Regeneración Nerviosa/fisiología , Terminales Presinápticos/metabolismo , Degeneración Retrógrada/prevención & control , Degeneración Retrógrada/terapia , Trasplante de Células Madre/métodos , Nervio Abducens/metabolismo , Nervio Abducens/ultraestructura , Animales , Axotomía , Calbindina 2 , Gatos , Tamaño de la Célula/fisiología , Extensiones de la Superficie Celular/fisiología , Extensiones de la Superficie Celular/ultraestructura , Cerebelo/embriología , Cerebelo/trasplante , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/fisiopatología , Gliosis/prevención & control , Gliosis/terapia , Inmunohistoquímica , Interneuronas/ultraestructura , Mesencéfalo/fisiología , Mesencéfalo/ultraestructura , Microscopía Electrónica , Vías Nerviosas/lesiones , Vías Nerviosas/cirugía , Nervio Oculomotor/fisiología , Nervio Oculomotor/ultraestructura , Puente/metabolismo , Puente/ultraestructura , Terminales Presinápticos/ultraestructura , Degeneración Retrógrada/fisiopatología , Proteína G de Unión al Calcio S100/metabolismo , Sinaptofisina/metabolismo

RESUMEN

A relationship between motoneuron activity and calcitonin gene-related peptide (CGRP) expression was previously suggested based on indirect inferences. We show here a positive correlation between CGRP immunoreactivity and firing activity in an experimental model that used tetanus neurotoxin (TeNT) to alter basal firing levels. A low dose (0.5 ng/kg) of TeNT injected in the lateral rectus muscle raised the basal firing rate of ipsilateral abducens motoneurons, estimated as the firing rate at straight-ahead gaze (F(0)); the firing rate returned to control values after 2 weeks. In contrast, a high dose (5 ng/kg) of TeNT decreased basal firing, which recovered slowly over a 7-week period. Expression of CGRP immunoreactivity by abducens motoneurons, preferentially related to betaCGRP gene expression, was analyzed during these periods of altered firing activity. The number of CGRP-immunofluorescent abducens motoneurons increased to approximately 120% by 7 days after low-dose TeNT, to include all available motoneurons in the nucleus. In addition, the average CGRP immunofluorescence optical density inside motoneurons almost doubled after 4 days and returned toward control values in the following 2 weeks. In contrast, a high-dose injection of TeNT reduced the number of CGRP-immunofluorescent motoneurons to 5.4% of control 7 days post injection, and the number returned to 77.6% after 42 days. CGRP immunofluorescence intensity inside motoneurons was also reduced. Regression analysis of F(0) values with either the number of CGRP-immunolabeled motoneurons, their average immunofluorescence intensity, or both factors combined resulted in positive correlations with regression coefficients of 0.87 or higher. Therefore, CGRP expression and firing activity in abducens motoneurons are positively correlated.

Asunto(s)

Nervio Abducens/metabolismo , Potenciales de Acción/fisiología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Gatos/metabolismo , Neuronas Motoras/metabolismo , Regeneración Nerviosa/fisiología , Puente/metabolismo , Regulación hacia Arriba/fisiología , Nervio Abducens/citología , Nervio Abducens/efectos de los fármacos , Acetilcolina/metabolismo , Animales , Gatos/anatomía & histología , Colina O-Acetiltransferasa/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Técnica del Anticuerpo Fluorescente , Interneuronas/citología , Interneuronas/metabolismo , Neuronas Motoras/citología , Neuronas Motoras/efectos de los fármacos , Músculos Oculomotores/inervación , Puente/citología , Puente/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Toxina Tetánica/farmacología , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

Nitric oxide (NO) production by specific neurons in the prepositus hypoglossi (PH) nucleus is necessary for the correct performance of eye movements in alert cats. In an attempt to characterize the morphological substrate of this NO function, the distribution of nitrergic neurons and NO-responding neurons has been investigated in different brainstem structures related to eye movements. Nitrergic neurons were stained by either immunohistochemistry for NO synthase I or histochemistry for reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase. The NO targets were identified by cyclic guanosine monophosphate (cGMP) immunohistochemistry in animals treated with a NO donor immediately before fixation of the brain. Connectivity between cells of the NO-cGMP pathway was analyzed by injections of the retrograde tracers horseradish peroxidase or fast blue in different structures. The motor nuclei commanding extraocular muscles did not contain elements of the NO-cGMP pathway, except for some scattered nitrergic neurons in the most caudal part of the abducens nucleus. The PH nucleus contained the largest number of nitrergic cell bodies and a rich neuropil, distributed in two groups in medial and lateral positions in the caudal part, and one central group in the rostral part of the nucleus. An abundant cGMP positive neuropil was the only NO-sensitive element in the PH nucleus, where no cGMP-producing neuronal cell bodies were observed. The opposite disposition was found in the marginal zone between the PH and the medial vestibular nuclei, with a large number of NO-sensitive cGMP-producing neurons and almost no nitrergic cells. Both nitrergic and NO-sensitive cell bodies were found in the medial and inferior vestibular nuclei and in the superior colliculus, whereas the lateral geniculate nucleus contained nitrergic neuropil and a large number of NO-sensitive cell bodies. Some of the cGMP-positive neurons in the marginal zone and medial vestibular nucleus projected to the PH nucleus, predominantly to the ipsilateral side. These morphological findings may help to explain the mechanism of action of NO in the oculomotor system.

Asunto(s)

Tronco Encefálico/citología , Gatos/anatomía & histología , Movimientos Oculares/fisiología , Red Nerviosa/citología , Neuronas/citología , Óxido Nítrico/metabolismo , Músculos Oculomotores/inervación , Nervio Abducens/citología , Nervio Abducens/metabolismo , Animales , Transporte Axonal/efectos de los fármacos , Transporte Axonal/fisiología , Tronco Encefálico/metabolismo , Gatos/metabolismo , GMP Cíclico/metabolismo , Femenino , Peroxidasa de Rábano Silvestre/farmacocinética , Inmunohistoquímica , Masculino , NADPH Deshidrogenasa , Red Nerviosa/fisiología , Neuronas/fisiología , Músculos Oculomotores/fisiología , Nervio Oculomotor/citología , Nervio Oculomotor/metabolismo , Desempeño Psicomotor/fisiología , Transmisión Sináptica/fisiología , Nervio Troclear/citología , Nervio Troclear/metabolismo , Núcleos Vestibulares/citología , Núcleos Vestibulares/fisiología

RESUMEN

Spinal and cranial motoneurons express alpha- and beta-calcitonin gene-related peptide (CGRP) mRNAs constitutively at variable ratios, and these two mRNAs are differentially regulated following axotomy in spinal, facial, and hypoglossal motoneurons. The purpose of this study was to investigate the change in CGRP mRNA expression following nerve injury in oculomotor, trochlear, abducens, and trigeminal motor nuclei in which beta-CGRP mRNA is predominantly expressed under normal conditions. Using male Sprague-Dawley rats, either the left eyeball and the orbital contents including the bulbar muscles were removed, or the left masseter nerve was ligated and transected. The rats were allowed to survive for 1, 3, 7, 14, 28, 56 days following these procedures. The levels of mRNAs for alpha- and beta-CGRP and growth-associated protein (GAP)-43 were analyzed by in situ hybridization histochemistry using 35S-labeled oligonucleotide probes. Following nerve injury, the expression of alpha-CGRP mRNA rapidly increased on the directly-injured side in all of these nuclei. Thereafter, it gradually decreased and returned to about the control level at postoperative day 56 within oculomotor, trochlear, and abducens motoneurons, but it sustained at a high level within trigeminal motoneurons. The expression of beta-CGRP was quite variable among these nuclei, and significant changes were also seen on the side contralateral to the directly-injured side. These data indicate that the up-regulation of alpha-CGRP mRNA may be a common response of cranial motor neurons following axotomy even if the constitutive expression of beta-CGRP mRNA exceeds that of alpha-CGRP mRNA in these neurons.

Asunto(s)

Péptido Relacionado con Gen de Calcitonina/genética , Neuronas Motoras/metabolismo , ARN Mensajero/biosíntesis , Nervio Abducens/citología , Nervio Abducens/metabolismo , Análisis de Varianza , Animales , Axotomía , Proteína GAP-43/genética , Masculino , Nervio Oculomotor/citología , Nervio Oculomotor/metabolismo , Ratas , Ratas Sprague-Dawley , Núcleo Espinal del Trigémino/citología , Núcleo Espinal del Trigémino/metabolismo , Nervio Troclear/citología , Nervio Troclear/metabolismo

RESUMEN

In this study, we investigated the effects of long-term synaptic blockade on postsynaptic receptor clustering at central inhibitory glycinergic synapses. High doses of botulinum neurotoxin type A injected in the lateral rectus muscle completely abolishes inhibitory postsynaptic potentials onto abducens motoneurons within 2 days postinjection, and transmission remains blocked for at least 2 months. Using this model, we analyzed the expression of gephyrin, a glycine receptor clustering protein, on the membrane of motoneuron somata after botulinum neurotoxin type A injection in their target muscle. Immunofluorescence or electron microscopy immunohistochemistry revealed gephyrin-immunoreactive clusters (most < 0.5 microm in diameter) densely covering the surface of control abducens motoneurons. Ultrastructurally, presynaptic terminals containing flattened synaptic vesicles (F terminals) were found associated with multiple gephyrin-immunoreactive postsynaptic densities (average 1.24 gephyrin clusters/F+ profile). No significant changes in gephyrin-immunoreactive clusters were observed at 5 days postinjection, but we found significant reductions (25-40%) in the density of gephyrin clusters 19 and 35 days postinjection. Hence, the physiological alterations reported in this model precede structural changes on postsynaptic receptor cluster density. The decrease in gephyrin-immunoreactive clusters was paralleled by reductions in synaptic covering (F+ terminals per 100 microm of membrane). Presumed inactive F+ terminals that remained attached to the motoneuron surface displayed normal gephyrin-immunoreactive clusters; however, the pre- and postsynaptic membranes in between synaptic active zones frequently appeared separated by enlarged extracellular spaces. We concluded that postsynaptic receptor cluster dissolution seemed more directly related to terminal retraction than to inactivity alone.

Asunto(s)

Nervio Abducens/efectos de los fármacos , Toxinas Botulínicas Tipo A/farmacología , Proteínas Portadoras/biosíntesis , Gatos/metabolismo , Proteínas de la Membrana/biosíntesis , Neuronas Motoras/efectos de los fármacos , Proteínas del Tejido Nervioso/biosíntesis , Nervio Abducens/citología , Nervio Abducens/metabolismo , Análisis de Varianza , Animales , Péptido Relacionado con Gen de Calcitonina/análisis , Gatos/anatomía & histología , Peroxidasa de Rábano Silvestre , Microscopía Electrónica , Neuronas Motoras/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Receptores de Glicina/fisiología , Sinapsis/efectos de los fármacos

RESUMEN

The present study investigated the effect of seventh and eight cranial nerve lesions on the prominence of calcitonin gene-related peptide in the hypoglossal (XII), facial (VII), abducens (VI), and oculomotor (III) cranial nerve nuclei. Guinea pigs were anesthetized and subjected to unilateral cochlear removal, vestibular end organ ablation, and seventh nerve transection. After a survival period ranging from 4 h to 5 days, each animal was anesthetized and perfused intracardially. Frozen sections were collected through the brainstem and stained immunohistochemically for calcitonin gene-related peptide using a polyclonal antibody with the Vectastain ABC kit and protocol. Positive cells were counted in each nucleus bilaterally and analyzed for side to side differences. Nuclei XII and III showed no significant difference in the numbers of cells staining positively for calcitonin gene-related peptide between the ipsilateral and the contralateral sides to the lesion. However, nuclei VII and VI showed elevated numbers ipsilateral to the lesion on some days, but not all. For VII, there was no significant difference before 24 h, but there were significant differences 1-5 days after the lesion. Similarly, in VI, there was no difference before 24 h, but differences were significant beginning with day 1 and continuing through day 3, and finally disappearing by day 4. Changes in the numbers of CGRP positive cells in VII measurable 24 h after the lesion and continuing for at least 5 days afterward indicate a central nervous system retrograde response to peripheral motor nerve injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Tronco Encefálico/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Traumatismos del Nervio Facial , Traumatismos del Nervio Vestibulococlear , Nervio Abducens/metabolismo , Animales , Densitometría , Nervio Facial/metabolismo , Femenino , Cobayas , Nervio Hipogloso/metabolismo , Inmunohistoquímica , Masculino , Plasticidad Neuronal/fisiología , Nervio Vestibulococlear/metabolismo