RESUMEN
The auditory cortex is the source of descending connections providing contextual feedback for auditory signal processing at almost all levels of the lemniscal auditory pathway. Such feedback is essential for cognitive processing. It is likely that corticofugal pathways are degraded with aging, becoming important players in age-related hearing loss and, by extension, in cognitive decline. We are testing the hypothesis that surface, epidural stimulation of the auditory cortex during aging may regulate the activity of corticofugal pathways, resulting in modulation of central and peripheral traits of auditory aging. Increased auditory thresholds during ongoing age-related hearing loss in the rat are attenuated after two weeks of epidural stimulation with direct current applied to the surface of the auditory cortex for two weeks in alternate days (Fernández del Campo et al., 2024). Here we report that the same cortical electrical stimulation protocol induces structural and cytochemical changes in the aging cochlea and auditory brainstem, which may underlie recovery of age-degraded auditory sensitivity. Specifically, we found that in 18 month-old rats after two weeks of cortical electrical stimulation there is, relative to age-matched non-stimulated rats: a) a larger number of choline acetyltransferase immunoreactive neuronal cell body profiles in the ventral nucleus of the trapezoid body, originating the medial olivocochlear system.; b) a reduction of age-related dystrophic changes in the stria vascularis; c) diminished immunoreactivity for the pro-inflammatory cytokine TNFα in the stria vascularis and spiral ligament. d) diminished immunoreactivity for Iba1 and changes in the morphology of Iba1 immunoreactive cells in the lateral wall, suggesting reduced activation of macrophage/microglia; d) Increased immunoreactivity levels for calretinin in spiral ganglion neurons, suggesting excitability modulation by corticofugal stimulation. Altogether, these findings support that non-invasive neuromodulation of the auditory cortex during aging preserves the cochlear efferent system and ameliorates cochlear aging traits, including stria vascularis dystrophy, dysregulated inflammation and altered excitability in primary auditory neurons.
Asunto(s)
Envejecimiento , Corteza Auditiva , Vías Auditivas , Cóclea , Estimulación Eléctrica , Presbiacusia , Animales , Masculino , Factores de Edad , Envejecimiento/patología , Envejecimiento/metabolismo , Corteza Auditiva/metabolismo , Corteza Auditiva/fisiopatología , Vías Auditivas/fisiopatología , Vías Auditivas/metabolismo , Umbral Auditivo , Proteínas de Unión al Calcio , Colina O-Acetiltransferasa/metabolismo , Cóclea/inervación , Cóclea/metabolismo , Cóclea/fisiopatología , Cóclea/patología , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico , Audición , Proteínas de Microfilamentos , Microglía/metabolismo , Microglía/patología , Neuronas Eferentes/metabolismo , Núcleo Olivar/metabolismo , Presbiacusia/fisiopatología , Presbiacusia/metabolismo , Presbiacusia/patología , Ratas Wistar , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cross-modal reorganization in the auditory and visual cortices has been reported after hearing and visual deficits mostly during the developmental period, possibly underlying sensory compensation mechanisms. However, there are very few data on the existence or nature and timeline of such reorganization events during sensory deficits in adulthood. In this study, we assessed long-term changes in activity-dependent immediate early genes c-Fos and Arc/Arg3.1 in auditory and neighboring visual cortical areas after bilateral deafness in young adult rats. Specifically, we analyzed qualitatively and quantitatively c-Fos and Arc/Arg3.1 immunoreactivity at 15 and 90 days after cochlea removal. We report extensive, global loss of c-Fos and Arc/Arg3.1 immunoreactive neurons in the auditory cortex 15 days after permanent auditory deprivation in adult rats, which is partly reversed 90 days after deafness. Simultaneously, the number and labeling intensity of c-Fos- and Arc/Arg3.1-immunoreactive neurons progressively increase in neighboring visual cortical areas from 2 weeks after deafness and these changes stabilize three months after inducing the cochlear lesion. These findings support plastic, compensatory, long-term changes in activity in the auditory and visual cortices after auditory deprivation in the adult rats. Further studies may clarify whether those changes result in perceptual potentiation of visual drives on auditory regions of the adult cortex.
Asunto(s)
Corteza Auditiva/metabolismo , Proteínas del Citoesqueleto/metabolismo , Regulación de la Expresión Génica/fisiología , Pérdida Auditiva/patología , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Corteza Visual/metabolismo , Animales , Vías Auditivas/metabolismo , Cóclea/lesiones , Cóclea/metabolismo , Cóclea/patología , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos , Pérdida Auditiva/metabolismo , Masculino , Ratas , Ratas Wistar , Ganglio Espiral de la Cóclea/patologíaRESUMEN
Insulin-like growth factor 1 (IGF-1) is a neurotrophic protein that plays a crucial role in modulating neuronal function and synaptic plasticity in the adult brain. Mice lacking the Igf1 gene exhibit profound deafness and multiple anomalies in the inner ear and spiral ganglion. An issue that remains unknown is whether, in addition to these peripheral abnormalities, IGF-1 deficiency also results in structural changes along the central auditory pathway that may contribute to an imbalance between excitation and inhibition, which might be reflected in abnormal auditory brainstem responses (ABR). To assess such a possibility, we evaluated the morphological and physiological alterations in the cochlear nucleus complex of the adult mouse. The expression and distribution of the vesicular glutamate transporter 1 (VGluT1) and the vesicular inhibitory transporter (VGAT), which were used as specific markers for labeling excitatory and inhibitory terminals, and the involvement of the activity-dependent myocyte enhancer factor 2 (MEF2) transcription factors in regulating excitatory synapses were assessed in a 4-month-old mouse model of IGF-1 deficiency and neurosensorial deafness (Igf1 (-/-) homozygous null mice). The results demonstrate decreases in the cochlear nucleus area and cell size along with cell loss in the cochlear nuclei of the deficient mouse. Additionally, our results demonstrate that there is upregulation of VGluT1, but not VGAT, immunostaining and downregulation of MEF2 transcription factors together with increased wave II amplitude in the ABR recording. Our observations provide evidence of an abnormal neuronal cytoarchitecture in the cochlear nuclei of Igf1 (-/-) null mice and suggest that the increased efficacy of glutamatergic synapses might be mediated by MEF2 transcription factors.
Asunto(s)
Núcleo Coclear/metabolismo , Factor I del Crecimiento Similar a la Insulina/deficiencia , Factores de Transcripción MEF2/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Animales , Atrofia , Vías Auditivas , Sistema Nervioso Central/metabolismo , Núcleo Coclear/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Plasticidad Neuronal , Neuronas/metabolismo , Sinapsis/metabolismo , Regulación hacia ArribaRESUMEN
Spherical cells in the anteroventral division of the cochlear nucleus, which relay excitatory inputs from the auditory nerve, also receive both GABAergic and glycinergic inhibitory synapses. Inhibition mediated by GABA and glycine fulfils essential roles in the processing abilities of these and other auditory neurons. However, the developmental program leading to a mature complement of GABAergic and glycinergic synapses and microcircuits is largely unknown. Because of their relatively simple geometry, spherical cells provide an excellent model for unraveling basic developmental patterns of inhibitory synaptogenesis. Using a combination of high resolution immunocytochemical methods, we report that, in the rat, synapses containing GABA or glycine are deployed on spherical cell bodies over a time period extending well beyond hearing onset. Such postnatal developmental recruitment of inhibitory endings is progressive, although there are two distinct leaps in their numbers. The first occurs by the end of the first postnatal week, prior to hearing onset, and the second, during the third postnatal week, after hearing onset. This pattern suggests that adjustments in inhibition could be driven by acoustic experience. While GABAergic and glycinergic endings are maturing and growing in number and size, their neurotransmitter content also appears to be developmentally regulated. Quantitative ultrastructural immunocytochemistry with colloidal gold suggests that GABA and glycine accumulation in synaptic endings follows a staggered pattern, with labeling stabilizing at adult levels by postnatal day 21. This may account for adjustments in synaptic efficacy and strength.
Asunto(s)
Núcleo Coclear/citología , Inhibición Neural/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Factores de Edad , Animales , Animales Recién Nacidos , Regulación del Desarrollo de la Expresión Génica/fisiología , Glicina/metabolismo , Masculino , Microscopía Inmunoelectrónica/métodos , Neuronas/metabolismo , Neuronas/ultraestructura , Ratas , Ratas Wistar , Sinapsis/metabolismo , Sinapsis/ultraestructura , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The regulation of neurotransmitter receptors during synapse formation has been studied extensively at the neuromuscular junction, but little is known about the development of excitatory neurotransmitter receptors during synaptogenesis in central synapses. In this study we show qualitatively and quantitatively that a receptor undergoes changes in localisation on the surface of rat Purkinje cells during development in association with its excitatory synapses. The presence of mGluR1alpha at parallel and climbing fibre synapses on developing Purkinje cells was studied using high-resolution immunoelectron microscopy. Immunoreactivity for mGluR1alpha was detected from embryonic day 18 in Purkinje cells, and showed dramatic changes in its localisation with age. At early postnatal ages (P0 and P3), mGluR1alpha was found both in somata and stem dendrites but was not usually associated with synaptic contacts. At P7, mGluR1alpha became concentrated in somatic spines associated with climbing fibres and in the growing dendritic arborisation even before innervation by parallel fibres. During the second and third postnatal week, when spines and parallel fibre synapses were generated, mGluR1alpha became progressively concentrated in the molecular layer, particularly in the synaptic specialisations. As a result, during the fourth postnatal week, the pattern and level of mGluR1alpha expression became similar to the adult and mGluR1alpha appeared in high density in perisynaptic sites. Our results indicate that mGluR1alpha is present in the developing Purkinje cells prior to their innervation by climbing and parallel fibres and demonstrate that this receptor undergoes a dynamic and specific regulation during postnatal development in association with the establishment of synaptic inputs to Purkinje cell.
Asunto(s)
Diferenciación Celular/fisiología , Corteza Cerebelosa/embriología , Corteza Cerebelosa/crecimiento & desarrollo , Terminales Presinápticos/metabolismo , Células de Purkinje/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Transmisión Sináptica/fisiología , Vías Aferentes/embriología , Vías Aferentes/crecimiento & desarrollo , Vías Aferentes/metabolismo , Envejecimiento/fisiología , Animales , Animales Recién Nacidos , Compartimento Celular/fisiología , Corteza Cerebelosa/metabolismo , Feto , Ácido Glutámico/metabolismo , Inmunohistoquímica , Masculino , Microscopía Electrónica , Núcleo Olivar/metabolismo , Núcleo Olivar/ultraestructura , Terminales Presinápticos/ultraestructura , Células de Purkinje/ultraestructura , Ratas , Ratas WistarRESUMEN
Inhibition by GABA is important for auditory processing, but any adaptations of the ionotropic type A receptors are unknown. Here we describe, using in situ hybridization, the subunit expression patterns of GABA(A) receptors in the rat cochlear nucleus, superior olivary complex, and dorsal and ventral nuclei of the lateral lemniscus. All neurons express the beta3 and gamma2L subunit messenger RNAs, but use different alpha subunits. In the dorsal cochlear nucleus, fusiform (pyramidal) and giant cells express alpha1, alpha3, beta3 and gamma2L. Dorsal cochlear nucleus interneurons, particularly vertical or tuberculoventral cells and cartwheel cells, express alpha3, beta3 and gamma2L. In the ventral cochlear nucleus, octopus cells express alpha1, beta3, gamma2L and delta. Spherical cells express alpha1, alpha3, alpha5, beta3 and gamma2L. In the superior olivary complex, the expression profile is alpha3, alpha5, beta3 and gamma2L. Both dorsal and ventral cochlear nucleus granule cells express alpha1, alpha6, beta3 and gamma2L; unlike their cerebellar granule cell counterparts, they do not express beta2, gamma2S or the delta subunit genes. The delta subunit's absence from cochlear nucleus granule cells may mean that tonic inhibition mediated by extrasynaptic GABA(A) receptors is less important for this cell type. In both the dorsal and ventral nuclei of the lateral lemniscus, alpha1, beta3 and gamma2L are the main subunit messenger RNAs; the ventral nucleus also expresses the delta subunit. We have mapped, using in situ hybridization, the subunit expression patterns of the GABA(A) receptor in the auditory brainstem nuclei. In contrast to many brain regions, the beta2 subunit gene and gamma2S splice forms are not highly expressed in auditory brainstem nuclei. GABA(A) receptors containing beta3 and gamma2L may be particularly well suited to auditory processing, possibly because of the unique phosphorylation profile of this subunit combination.
Asunto(s)
Vías Auditivas/fisiología , Tronco Encefálico/fisiología , Receptores de GABA-A/genética , Transcripción Genética , Animales , Núcleo Coclear/fisiología , Masculino , Neuronas/fisiología , Núcleo Olivar/fisiología , Orgánulos/fisiología , Subunidades de Proteína , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptores de GABA-A/análisisRESUMEN
Voltage-dependent ion channels have specific patterns of distribution along the neuronal plasma membrane of dendrites, cell bodies and axons, which need to be unravelled in order to understand their contribution to neuronal excitability and firing patterns. We have investigated the subcellular compartmentalization of Kv1.4, a transient, fast-inactivating potassium channel, in fusiform cells and related interneurons of the rat dorsal cochlear nucleus. A polyclonal antibody which binds to a region near the N-terminus domain of a Kv1.4 channel was raised in rabbits. Using a high-resolution combination of immunocytochemical methods, Kv1.4 was localized mainly in the apical dendritic trunks and cell bodies of fusiform cells, as well as in dendrites and cell bodies of interneurons of the dorsal cochlear nucleus, likely cartwheel cells. Quantitative immunogold immunocytochemistry revealed a pronounced distal to proximal gradient in the dendrosomatic distribution of Kv1. 4. In plasma membrane localizations, Kv1.4 was preferentially present in dendritic spines, either in the spine neck or in perisynaptic locations, always away from the postsynaptic density. These findings indicate that Kv1.4 is largely distributed in dendritic compartments of fusiform and cartwheel cells of the dorsal cochlear nucleus. Its preferential localization in dendritic spines, where granule cell axons make powerful excitatory synapses, suggests a role for this voltage-dependent ion channel in the regulation of dendritic excitability and excitatory inputs.
Asunto(s)
Núcleo Coclear/citología , Dendritas/ultraestructura , Neuronas/citología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/análisis , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Western Blotting , Retículo Endoplásmico Rugoso/ultraestructura , Epítopos/química , Inmunohistoquímica , Canal de Potasio Kv1.4 , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Neuronas/ultraestructura , Canales de Potasio/química , Canales de Potasio/inmunología , Conejos , Ratas , Ratas Wistar , Sensibilidad y Especificidad , Sinapsis/ultraestructuraRESUMEN
In order to identify cytochemical traits relevant to understanding excitatory neurotransmission in brainstem auditory nuclei, we have analyzed in the dorsal cochlear nucleus the synaptic distribution of aspartate aminotransferase, glutamate, and vesicular zinc, three molecules probably involved in different steps of excitatory glutamatergic signaling. High levels of glutamate immunolabeling were found in three classes of synaptic endings in the dorsal cochlear nucleus, as determined by quantitation of immunogold labeling. The first type included auditory nerve endings, the second were granule cell endings in the molecular layer, and the third very large endings, better described as "mossy." This finding points to a neurotransmitter role for glutamate in at least three synaptic populations in the dorsal cochlear nucleus. The same three types of endings enriched in glutamate immunoreactivity also contained histochemically detectable levels of aspartate aminotransferase activity, suggesting that this enzyme may be involved in the synaptic handling of glutamate in excitatory endings in the dorsal cochlear nucleus. There was also extrasynaptic localization of the enzyme. Zinc ions were localized exclusively in granule cell endings, as determined by a Danscher-selenite method, suggesting that this ion is involved in the operation of granule cell synapses in the dorsal cochlear nucleus.
Asunto(s)
Aspartato Aminotransferasas/análisis , Núcleo Coclear/química , Ácido Glutámico/análisis , Ratas Wistar/fisiología , Zinc/análisis , Animales , Vías Auditivas/química , Vías Auditivas/citología , Núcleo Coclear/citología , Núcleo Coclear/enzimología , Femenino , Microscopía Inmunoelectrónica , Neuronas Aferentes/química , Neuronas Aferentes/enzimología , Neuronas Aferentes/ultraestructura , Terminales Presinápticos/química , Ratas , Transducción de Señal/fisiología , Vesículas Sinápticas/químicaRESUMEN
Adrenomedullary chromaffin cells express at least two subtypes of acetylcholine nicotinic receptors, which differ in their sensitivity to the snake toxin alpha-bungarotoxin. One subtype is involved in the activation step of the catecholamine secretion process and is not blocked by the toxin. The other is alpha-bungarotoxin-sensitive, and its functional role has not yet been defined. The alpha7 subunit is a component of this subtype. Autoradiography of bovine adrenal gland slices with alpha-bungarotoxin indicates that these receptors are restricted to medullary areas adjacent to the adrenal cortex and colocalize with the enzyme phenylethanolamine N-methyl transferase (PNMT), which confers the adrenergic phenotype to chromaffin cells. Transcripts corresponding to the alpha7 subunit also are localized exclusively to adrenergic cells. To identify possible transcriptional regulatory elements of the alpha7 subunit gene involved in the restricted expression of nicotinic receptors, we isolated and characterized its 5' flanking region, revealing putative binding sites for the immediate early gene transcription factor Egr-1, which is known to activate PNMT expression. In reporter gene transfection experiments, Egr-1 increased alpha7 promoter activity by up to sevenfold. Activation was abolished when the most promoter-proximal of the Egr-1 sites was mutated, whereas modification of a close upstream site produced a partial decrease of the Egr-1 response. Because Egr-1 was found to be expressed exclusively in adrenergic cells, we suggest that this transcription factor may be part of a common mechanism involved in the induction of the adrenergic phenotype and the differential expression of alpha-bungarotoxin-sensitive nicotinic receptors in the adrenal gland.
Asunto(s)
Bungarotoxinas/farmacología , Células Cromafines/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas Inmediatas-Precoces , Neuronas/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Sistema Nervioso Simpático/metabolismo , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Sitios de Unión , Bungarotoxinas/metabolismo , Gatos , Bovinos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz , Isomerismo , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Ratas , Sistema Nervioso Simpático/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
Neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells play a primary role in triggering catecholamine secretion. In the present study, their constituent subunits were characterized. In addition to the alpha 3 subunit, which we have previously cloned, the presence of alpha 5 and beta 4 but not of beta 2 subunits was detected by reverse transcription-PCR analysis of mRNA from adrenal medulla. In situ hybridization indicated that alpha 3, alpha 5, and beta 4 subunits are coexpressed in all chromaffin cells. The primary structure of alpha 5 and beta 4 subunits was determined and functional receptors were obtained upon coinjection of subunit cRNAs into Xenopus oocytes. In contrast to other beta 4-containing nicotinic receptors, the ones formed by the bovine beta 4 subunit are insensitive to the agonist cytisine. Finally, we characterized the intergenic region of alpha 3 and alpha 5 subunits, which together with the beta 4 subunit, form a gene cluster in rats and chickens. RNase assays and the existence of overlapping cDNAs indicate that, in the bovine genome, the alpha 3 and alpha 5 genes overlap at their 3' ends. This fact is probably due to inefficient transcription termination, as a result of weak polyadenylation signals.
Asunto(s)
Células Cromafines/química , Receptores Nicotínicos/genética , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , ADN Complementario/genética , Femenino , Regulación de la Expresión Génica/fisiología , Biblioteca de Genes , Genoma , Intrones/genética , Datos de Secuencia Molecular , Neuronas/química , Oocitos/fisiología , ARN Mensajero/análisis , Receptores Nicotínicos/química , Homología de Secuencia de Aminoácido , Transcripción Genética/fisiología , XenopusRESUMEN
Several lines of evidence suggest that alpha-bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors may play a developmental role by modulating plasticity in neuronal circuits. The alpha 7 subunit, a main component of these receptors, is expressed in most regions of the brain, including the cerebellum, where it is present almost exclusively in Purkinje cells and deep cerebellar nuclei. Purkinje cells constitute the only efferent pathway of the cerebellum and their development involves complex interactions, which have been extensively studied. They therefore provide a potentially useful model for analysis of development plasticity which could be influenced by alpha 7 neuronal nicotinic receptors. In the present study a previously characterized monoclonal antibody (mAb 307) has been used to determine the temporal pattern of expression of the alpha 7 subunit in the developing rat cerebellum. No detectable alpha 7 immunoreactivity is found between P0 and P2. Between P3 and P5, however, the Purkinje cell layer shows moderate immunolabeling. alpha 7 expression in this layer increases rapidly between P8 and P15. This increase in alpha 7 staining, which overlaps in time with important developmental and synaptogenic events, is not uniform throughout the cerebellar cortex. Thus, between P3 and P5 all Purkinje cells are weakly labeled, while at later stages (P8-P15) immunolabeling becomes more intense, but at the same time, disappears from Purkinje cells in rostral lobules. In addition, a very well defined pattern for discontinuous or columnar labeling is detected in regions of the Purkinje cell layer where alpha 7 subunits were being expressed. Finally, at P20, alpha 7 subunit labeling is found again in all Purkinje cells, although with lower intensity. These results suggest that alpha 7 receptor expression is developmentally regulated, with a time course that parallels the final differentiation of Purkinje cells. In addition, the heterogeneous spatial distribution of alpha 7-containing nicotinic receptors indicates that, during cerebellar maturation, these cells may receive different signals that modulate receptor gene expression in a very specific way.
Asunto(s)
Cerebelo/crecimiento & desarrollo , Células de Purkinje/química , Receptores Nicotínicos/análisis , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Diferenciación Celular/fisiología , Cerebelo/química , Cerebelo/citología , Pollos , Femenino , Inmunohistoquímica , Ratones , Embarazo , Células de Purkinje/citología , Ratas , Ratas Wistar , Receptores Nicotínicos/química , Receptores Nicotínicos/inmunologíaRESUMEN
Electron microscopic postembedding immunocytochemistry was used to analyze and assess the synaptic distribution of glycine (GLY) and gamma-amino butyric acid (GABA) immunoreactivities in the guinea pig cochlear nucleus (CN). Three classes of endings were identified containing immunolabeling for glycine, GABA, or both glycine and GABA (GLY/GABA). All classes were similar in that the terminals contained pleomorphic vesicles and formed symmetric synapses with their postsynaptic targets. A fourth class, which labeled with neither antibody, contained round vesicles and formed asymmetric synapses. Glycine endings predominated in the ventral CN, while GLY/GABA endings were prevalent in the dorsal CN. GABA endings were the least common and smallest in size. Glycine, GLY/GABA, and GABA endings differed in their proportions and patterns of distribution on the different classes of projection neurons in the CN, including spherical bushy, type I stellate/multipolar, and octopus cells in the ventral CN and fusiform cells in the dorsal CN. The vast majority of anatomically-defined, putative inhibitory endings contain GLY, GABA, or both, suggesting that most of the inhibition in the cochlear nucleus is mediated by these three cytochemically and, probably, functionally distinct classes of endings. The results of this study also suggest that a large proportion of the GABA available for inhibition in the CN coexists in terminals with glycine.
Asunto(s)
Núcleo Coclear/ultraestructura , Glicina/análisis , Inhibición Neural/fisiología , Terminales Presinápticos/química , Ácido gamma-Aminobutírico/análisis , Animales , Núcleo Coclear/química , Cobayas , InmunohistoquímicaRESUMEN
Postembedding immunocytochemistry was used to compare the distribution of GABA and glycine immunoreactive labelling in the cochlear nucleus, in particular the number of immunolabeled synaptic boutons apposing the cell body profiles of three major neuronal types. The proportions and absolute numbers of glycine immunoreactive puncta were greatest on fusiform cell body profiles. Glycine immunoreactive puncta also predominated on spherical cell body profiles, although GABA immunoreactive boutons were more abundant than on fusiform cells. Octopus cell body profiles were apposed by the fewest immunoreactive puncta. Puncta colabeled for glycine and GABA were frequently observed on all three cell types. These findings suggest that each major cell type possesses a distinct pattern of glycinergic and GABAergic input, with glycinergic input predominating. Since both tuberculoventral and cartwheel neurons were immunolabeled and are known to project intrinsically, it is clear that a large percentage of glycinergic and GABAergic input to cochlear nucleus neurons originates from intrinsic sources.
Asunto(s)
Corteza Auditiva/metabolismo , Núcleo Coclear/metabolismo , Glicina/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Cobayas , InmunohistoquímicaRESUMEN
A postembedding silver intensification procedure for immunogold on ultrathin sections has been used to help in the study of localization and co-localization of glycine and GABA in synaptic terminals and cell bodies in the cochlear nucleus of the auditory pathway. Intensification take place in a single step after the immunogold procedure. Intensification times vary from 2 to 6 minutes. This allows visualization of silver enhanced gold particles at low electron microscopic magnifications, which greatly facilitates the analysis of patterns of distribution of putative inhibitory ending immunolabelled for glycine, GABA or both glycine and GABA.
Asunto(s)
Núcleo Coclear/ultraestructura , Glicina/metabolismo , Neuronas/metabolismo , Tinción con Nitrato de Plata/métodos , Ácido gamma-Aminobutírico/metabolismo , Animales , Cobayas , InmunohistoquímicaRESUMEN
Previous molecular cloning studies have revealed that alpha-bungarotoxin binding proteins present in the brain are members of the neuronal nicotinic acetylcholine receptor gene family. The alpha 7 subunit is structurally related to the agonist binding subunits present in the central and peripheral nervous systems and, when expressed in Xenopus oocytes, forms functional channels blockable by alpha-bungarotoxin. In the present study, three different monoclonal antibodies raised against the alpha 7 subunit were used to map its distribution throughout the central nervous system of the rat. Immunohistochemical localization revealed that the alpha 7 subunit is expressed in most regions of the brain, being, overall, well correlated with previous "in situ" localization of alpha 7 transcripts and alpha-bungarotoxin autoradiographic binding studies. Particularly strong immunoreactivity was observed in several sensory and motor nuclei of the brainstem as well as the red nucleus. At the cellular level, alpha 7 immunostaining was usually found both in somata and dendrites, whereas axonal and terminal labeling was not observed. The widespread distribution of the alpha 7 subunit polypeptide is consistent with immunoprecipitation data demonstrating that it is a component of the predominant subtype of brain alpha-bungarotoxin-sensitive nicotinic receptors.
Asunto(s)
Sistema Nervioso Central/química , Fragmentos de Péptidos/análisis , Receptores Nicotínicos/análisis , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Tronco Encefálico/química , Cerebelo/química , Reacciones Cruzadas , Femenino , Técnicas para Inmunoenzimas , Datos de Secuencia Molecular , Prosencéfalo/química , Ratas , Ratas Wistar , Médula Espinal/químicaRESUMEN
We compared the distribution of GABAA and GABAB binding sites in the cochlear nucleus using quantitative receptor autoradiography with [3H]GABA. To visualize GABAA binding sites, GABAB binding sites were blocked with +/- baclofen. To visualize GABAB binding sites, isoguvacine was used to block GABAA binding sites. GABAA binding sites predominated over GABAB, although there were marked regional differences in the distribution of binding. In the ventral cochlear nucleus, GABAA and GABAB binding sites were concentrated in the peripheral granule cell cap, with low binding levels in the central region. In the dorsal cochlear nucleus, binding was concentrated in the superficial (fusiform and molecular) layers, with a distinct laminar pattern. GABAA binding sites predominated in the fusiform cell layer. The molecular layer contained the highest level of GABAB binding sites in the entire cochlear nucleus. These results suggest that GABAergic inhibition in the cochlear nucleus is mediated both by GABAA and GABAB receptors, particularly in the dorsal cochlear nucleus. However, low levels of binding in areas such as the magnocellular regions of the ventral cochlear nucleus, known to contain abundant GABAergic synapses, suggest heterogeneity of GABA receptors in this auditory nucleus.
Asunto(s)
Núcleo Coclear/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Animales , Autorradiografía , Baclofeno/farmacología , Corteza Cerebelosa/efectos de los fármacos , Corteza Cerebelosa/metabolismo , Núcleo Coclear/efectos de los fármacos , Antagonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-B , Cobayas , Ácidos Isonicotínicos/farmacologíaRESUMEN
The goal of this study was to correlate synaptic ultrastructure with transmitter specificity and function in the lateral superior olive (LSO), a nucleus that is thought to play a major role in sound localization. This was accomplished by means of postembedding immunogold immunocytochemistry. Four classes of synaptic terminals were identified in the LSO. They were distinguishable from one another both morphologically and on the basis of their different patterns of immunolabeling for glutamate, glycine, and gamma-aminobutyric acid (GABA). The highest level of glutamate immunoreactivity was found in terminals that contained round vesicles (R) and formed synaptic contacts with asymmetric synaptic junctions. Round-vesicle terminals predominated on small caliber dendrites by a ratio of at least 2:1 over the other classes combined. The thinnest dendrites were typically contacted by R terminals only. The ratio of R terminals to the other types decreased as the caliber of the dendritic profiles they apposed increased so that on the soma, R terminals were outnumbered by at least 2:1 by the other types. Terminals containing flattened vesicles (F) exhibited intense immunoreactivity for both glycine and glutamate, although the glutamate immunolabeling was not as high as that in the R terminals. Flattened-vesicle terminals formed symmetric synaptic contacts with their targets and their distribution was the reverse of that described for R terminals; i.e., they were most abundant on LSO perikarya and fewest on small caliber dendrites. Two terminal types, both containing pleomorphic vesicles and forming symmetric synaptic junctions, were found in far fewer numbers. One group contained large pleomorphic vesicles (LP) and was immunoreactive for both glycine and GABA. The other group contained small pleomorphic vesicles (SP) along with a few dense-core vesicles and labeled for GABA only. The LP terminals were preferentially distributed on somata and large-caliber dendrites, while the SP terminals most often contacted smaller dendrites. Previous work suggests that a large percentage of the R terminals arise from spherical cells in the ipsilateral cochlear nucleus and are excitatory in action. This pathway may use glutamate as a transmitter. Many of the F terminals are thought to originate from the ipsilateral medial nucleus of the trapezoid body and appear to be the inhibitory (glycinergic) terminals from a pathway that originates from the contralateral ear. The origins and functions of LP and SP terminals are unknown, but a few possibilities are discussed along with the significance of cocontainment of neuroactive substances in specific terminal types.
Asunto(s)
Cobayas/metabolismo , Terminaciones Nerviosas/química , Neurotransmisores/análisis , Núcleo Olivar/química , Localización de Sonidos/fisiología , Sinapsis/química , Animales , Glutamatos/análisis , Ácido Glutámico , Glicina/análisis , Inmunohistoquímica , Neuronas/química , Núcleo Olivar/citología , Ácido gamma-Aminobutírico/análisisRESUMEN
Post-embedding immunocytochemical techniques were used to assess distribution of gamma-aminobutyric acid (GABA) in the guinea pig cristae ampullaris. GABA-like immunoreactivity (GABA-LIR) was found in the cytoplasm of both type I (HCI) and type II hair cells (HCII), in the afferent calyx (AC) contacting HCI and some myelinated fibers in the subjacent stroma. HCI and its calyceal contacts showed variation in GABA-LIR, suggesting different populations in HCI and AC. These results support a putative afferent neurotransmitter role of GABA in HC and a possible degradation site of GABA in AC.
Asunto(s)
Vestíbulo del Laberinto/química , Ácido gamma-Aminobutírico/análisis , Animales , Femenino , Cobayas , Técnicas para Inmunoenzimas , Masculino , Vestíbulo del Laberinto/ultraestructuraRESUMEN
Immunocytochemistry with a monoclonal antibody against the GABAA/benzodiazepine receptor showed labeled axo-dendritic synapses in the anteroventral cochlear nucleus. In the dorsal cochlear nucleus, label was seen apposing both axo-somatic and axo-dendritic terminals. The results suggest a heterogeneous distribution of GABA receptors, together with a possible segregation of receptor subtypes between somata and dendrites in certain neurons. The presence of cytoplasmic labeling in some neurons might reflect a higher receptor turnover rate in these neurons.