RESUMEN
The quantitative stereological method, the optical fractionator, was used for determining the total number of neurons and the total number of neurons immunostained with parvalbumin, calbindin-D28k (calbindin), and calretinin in the dorsal and posteroventral cochlear nucleus (DCN and PVCN) in CBA/CaJ (CBA) mice during aging (1-39 months old). CBA mice have only a modest sensorineural pathology late in life. An age-related decrease of the total number of neurons was demonstrated in the DCN (r=-0.54, P<0.03), while the total number of neurons in the PVCN did not show any significant age-related differences (r=0.16, P=0.57). In the DCN 5.5% of neurons were parvalbumin positive in the very old (30-39 months) mice, vs. 2.2% in the 1 month old mice. In the DCN 3% of the neurons were calbindin immunopositive in the 30-39 months mice compared to 1.9% in the 1 month old group. In the PVCN, 20% of the neurons in the very old mice were parvalbumin immunopositive, compared to 12% in the young mice. Calbindin did not show any significant age-related differences in the PVCN. The total number of calretinin immunopositive neurons both in the DCN and PVCN did not show any significant change with increasing age. In conclusion, the total neuronal number in the DCN and PVCN was age-related and region-specific. While the neuronal number in the DCN and PVCN was decreased or unchanged, respectively, the calcium binding protein positive neuronal number showed a graded increase during aging in a region-specific and protein-specific manner.
Asunto(s)
Envejecimiento/metabolismo , Proteínas de Unión al Calcio/metabolismo , Núcleo Coclear/citología , Núcleo Coclear/metabolismo , Neuronas/citología , Neuronas/metabolismo , Animales , Calbindina 1 , Calbindina 2 , Calbindinas , Recuento de Células , Femenino , Masculino , Ratones , Ratones Endogámicos CBA , Parvalbúminas/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Distribución TisularRESUMEN
How germinal center (GC) B cells diversify their rearranged immunoglobulin genes by somatic hypermutation is unknown. However, the GC-specific activation-induced cytidine deaminase has been identified as a key factor controlling two central GC-specific events: somatic hypermutation and class-switch recombination of immunoglobulin genes. This factor may function as a catalytic subunit of an RNA-editing complex or, more directly, on DNA as a deoxy-cytidine deaminase in the hypermutation domain and class-switch region. Deamination of deoxy-cytidines on both strands may result in staggered DNA double-strand breaks (DSBs) that, in the hypermutation domain, become processed by member(s) of newly identified error-prone DNA polymerases. Direct evidence for DSBs in hot-spots of hypermutating immunoglobulin genes has been provided, implicating DSBs as reaction intermediates of an error-prone DSB-repair pathway acting specifically in GC B cells. These recent findings are key to the identification of the hypermutation mechanism.
Asunto(s)
Linfocitos B/metabolismo , Genes de Inmunoglobulinas/genética , Mutación/genética , Animales , Linfocitos B/enzimología , Linfocitos B/inmunología , Disparidad de Par Base/genética , Citidina Desaminasa/metabolismo , Daño del ADN/genética , Reparación del ADN/genética , Humanos , Transcripción Genética/genéticaRESUMEN
The generation of a diverse antigen receptor repertoire is fundamental for the functionality of the adaptive immune system. While the V(D)J recombination process that generates the primary antigen receptor repertoire is understood in great detail, it is still unclear by which mechanism immunoglobulin (Ig) genes are further diversified by somatic hypermutation. Using mouse strains that carry a non-functional, pre-defined V(H)D(H)J(H) gene segment in their IgH locus we demonstrate DNA double-strand breaks (DSBs) in and around V(H)D(H)J(H) in B cells undergoing somatic hypermutation. The generation of these DSBs depends on transcriptional activity, and their distribution along the V(H)D(H)J(H) segment parallels that of point mutations in the hypermutation domain. Furthermore, similar to hot spots of somatic hypermutation, 50-60% of all DSBs occur preferentially at RGYW motifs. DSBs may transiently dissociate the Ig promoter from the intronic enhancer to block further transcription and to initiate an error-prone non-homologous DSB repair pathway. In accord with this model large deletions are frequently produced, along with point mutations, in a V(H)D(H)J(H) segment inserted together with its promoter into the IgH locus in inverted orientation. Our data suggest that DSBs are reaction intermediates of the mechanism underlying somatic hypermutation.
Asunto(s)
Daño del ADN/genética , Inmunoglobulinas/genética , Mutación , Animales , Linfocitos B , Secuencia de Bases , Inversión Cromosómica , Mapeo Cromosómico , ADN , Región Variable de Inmunoglobulina , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Eliminación de SecuenciaRESUMEN
How rearranged immunoglobulin (Ig) genes are further diversified by somatic hypermutation is unknown. Using VDJ passenger Ig heavy chain (IgH) knockin mouse strains, we now demonstrate a high frequency of DNA double-strand breaks (DSBs) in the targeted VDJ passenger gene of germinal center (GC) B cells. These DSBs parallel the distribution of mutations in the targeted hypermutation domain and are found preferentially at RGYW motifs, the intrinsic hot spots of somatic hypermutation. The introduction of DSBs appears to depend on transcriptional activity. Thus, secondary diversification of rearranged V gene segments relates to an error-prone nonhomologous DSB repair system acting in B cells of the GC.
Asunto(s)
Daño del ADN , Genes de Inmunoglobulinas , Mutación , Animales , Secuencia de Bases , Ratones , Datos de Secuencia MolecularRESUMEN
The cartwheel cell is the most numerous inhibitory interneuron of the dorsal cochlear nucleus (DCN). It is expected to be an important determinant of DCN function. To assess the contribution of the cartwheel cell, we examined the discharge characteristics of DCN neurons and behavioral measures in the Purkinje cell degeneration (pcd) mice, which lack cartwheel cells, and compared them to those of the control mice. Distortion product otoacoustic emissions and auditory brainstem-evoked response thresholds were similar between the two groups. Extracellularly recorded DCN single units in ketamine/xylazine-anesthetized mice were classified according to post-stimulus time histogram (PSTH) and excitatory-inhibitory response area (EI-area) schemes. PSTHs recorded in mouse DCN included chopper, pauser/buildup, onset, inhibited and nondescript types. EI-areas recorded included Types I, II, III, I/III, IV and V. There were no significant differences in the proportions of various unit types between the pcd and control mice. The pcd units had slightly lower thresholds to characteristic frequency tones; however, they had spontaneous rates, thresholds to noise, and maximum driven rates to noise that were similar to those of the control units. Pcd mice had smaller startle amplitudes, but startle latency, prepulse inhibition/augmentation and facilitation by a background tone were comparable between the two groups. From these results, we conclude that DCN function in response to relatively simple acoustic stimuli is minimally affected by the absence of the cartwheel cells. Future studies employing more complex and/or multimodal stimuli should help assess the role of the cartwheel cells.
Asunto(s)
Núcleo Coclear/fisiología , Degeneración Nerviosa/fisiopatología , Células de Purkinje/fisiología , Reflejo de Sobresalto/fisiología , Estimulación Acústica , Animales , Conducta Animal/fisiología , Núcleo Coclear/citología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Valores de ReferenciaRESUMEN
Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.
Asunto(s)
Genoma , Mapeo Físico de Cromosoma , Pez Cebra/genética , Animales , Mapeo Cromosómico , Electroforesis en Gel de Agar , Etiquetas de Secuencia Expresada , Marcadores Genéticos , Escala de Lod , Modelos Genéticos , Polimorfismo Genético , Lugares Marcados de Secuencia , Programas InformáticosRESUMEN
To develop the BALB/c mouse strain as an animal model for the study of progressive sensorineural hearing loss, mice ranging in age from young adult through middle age were studied. Auditory brainstem response thresholds, histopathology [cytocochleograms for hair cells, the packing density of spiral ganglion cells (SGCs), the number of neurons and overall size of the anterior ventral cochlear nucleus (AVCN)], and behavioral paradigms (prepulse inhibition, fear-potentiated startle) were compared with previous data from C57BL/6J (C57) and DBA/2J (DBA) mouse strains. Progressive high frequency hearing loss in BALB/c mice was generally more rapid than C57 and slower than DBA (e.g. mean thresholds for 16 kHz: 10-month-old BALB/c mice = 71 dB SPL; 55-day-old DBA mice = 79 dB SPL; 12-month-old C57 mice = 50 dB SPL). Like the other strains, BALB/c exhibited a progressive loss of hair cells and SGCs that was most severe in the cochlear base and least severe in the middle turns; however, BALB/c mice had relatively more SGC loss in the apex. Unlike C57 and DBA, no loss of neurons was observed in the AVCN following cochlear pathology (although AVCN volume was reduced). Like the other strains, successful fear conditioning was obtained with a 12 kHz conditioned stimulus. Prepulse inhibition showed that middle and low frequency tones (4-12 kHz) became more salient as high frequency hearing declined. Similar results had been previously obtained with C57 and DBA mice and were interpreted as reflecting hearing-loss-induced plasticity in the central auditory system.
Asunto(s)
Umbral Auditivo/fisiología , Pérdida Auditiva Sensorineural/etiología , Estimulación Acústica , Factores de Edad , Animales , Recuento de Células , Núcleo Coclear/patología , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Femenino , Células Ciliadas Auditivas/patología , Pérdida Auditiva Sensorineural/fisiopatología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Plasticidad Neuronal/fisiología , Neuronas/patología , Especificidad de la Especie , Ganglio Espiral de la Cóclea/patologíaRESUMEN
Glycinergic neurons in the cochlear nucleus (CN) of C57BL/6J (C57) and CBA/CaJ (CBA) mice were studied by using immunocytochemical and receptor-binding techniques. Adult C57 mice exhibit progressive cochlear pathology as they age, whereas aging CBA mice retain good hearing. In the CN of old C57 mice (18 months) with severe hearing loss, the number of glycine-immunoreactive neurons decreased significantly. The number (Bmax) of strychnine-sensitive glycine receptors (GlyR) decreased significantly in the dorsal CN of old C57 mice. Significant effects were not observed in the CN of middle-aged C57 mice (with less-severe hearing loss) or in very old CBA mice (which do not exhibit severe hearing loss). The data suggest that the combination of severe hearing loss and old age results in deficits in one or more inhibitory glycinergic circuits in the CN.
Asunto(s)
Núcleo Coclear/metabolismo , Glicina/metabolismo , Ratones Endogámicos C57BL/metabolismo , Ratones Endogámicos CBA/metabolismo , Receptores de Glicina/metabolismo , Envejecimiento/metabolismo , Animales , Enfermedades Cocleares/genética , Enfermedades Cocleares/metabolismo , Enfermedades Cocleares/patología , Núcleo Coclear/patología , Femenino , Trastornos de la Audición/genética , Trastornos de la Audición/metabolismo , Trastornos de la Audición/patología , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL/genéticaRESUMEN
Morphological measurements were made on histological sections of the anteroventral cochlear nucleus (AVCN) in mice of the DBA/2J and C57BL/6J strains to determine the effects of sensorineural cochlear pathology on the number, packing density, and size of neurons and on AVCN volume. Both strains possess alleles that cause progressive cochlear pathology initially affecting the organ of Corti: in DBA mice, hearing loss is evident at 4 weeks of age and progresses rapidly; in C57 mice, hearing loss begins after 2 months of age and progresses more slowly. In both strains AVCN volume decreased, some loss of neurons occurred, and these changes paralleled the progression of peripheral hearing loss. Central changes were rapid in DBA mice, but the ultimate magnitude of the changes in 1-year-old mice did not differ between strains. Both strains differed from well-hearing CBA/J mice which exhibited no changes in the AVCN measures. The findings indicate that pathology of the organ of Corti in adult mice results in degenerative changes in the cochlear nucleus. The data also support earlier findings indicating that, if cochlear pathology does not begin prior to young adulthood, the age of onset and duration of sensorineural impairment have little effect on the ultimate magnitude of central effects.
Asunto(s)
Núcleo Coclear/citología , Sordera/fisiopatología , Neuronas Aferentes/fisiología , Factores de Edad , Animales , Recuento de Células , Tamaño de la Célula , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Degeneración Nerviosa/fisiología , Neuronas Aferentes/citologíaRESUMEN
The effects of chronic cochlear impairment on morphological features of the adult cochlear nucleus (CN) were assessed in CBA/J mice in which severe sensorineural damage had been induced by exposure to intense noise. Sections from various CN subdivisions, stained for Nissl substance and fibers, were quantitatively evaluated in four groups of noise-exposed mice that differed with regard to the age at noise exposure (2, 6, or 11 months), age at the time the CN was evaluated (6, 11, or 24 months), and the duration (chronicity) of sensorineural impairment (4, 5, 13, or 18 months). Like-aged, non-exposed CBA mice were used as controls, so the effects of peripheral damage and aging could be compared. Cochlear damage produced significant changes in CN subdivisions thought to receive the heaviest input from cochlear afferents (anteroventral CN, octopus cell area, dorsal CN layer III). These changes included a reduction of neuropil volume, reductions in neuron size, and increases in neuronal packing density that were complementary to reduced volume in these subdivisions. Effects on neuron number were minimal in all subdivisions. Central changes in noise-exposed mice were absent or diminished in DCN layers I and II, which receive relatively less input from primary fibers. The age at onset and chronicity of damage had little to do with the severity of central effects of cochlear damage. The effects of cochlear damage were not additive with age-related changes seen in the old controls.
Asunto(s)
Núcleo Coclear/patología , Pérdida Auditiva Provocada por Ruido/patología , Factores de Edad , Animales , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico , Pérdida Auditiva Provocada por Ruido/etiología , Pérdida Auditiva Provocada por Ruido/fisiopatología , Ratones , Ratones Endogámicos CBA , Órgano Espiral/patología , Ganglio Espiral de la Cóclea/patologíaRESUMEN
Basic anatomical features were evaluated in the inferior colliculus (IC) of C57BL/6J and CBA/J mice across the adult life span (1.5 to 30 months of age). C57BL/6J mice exhibit progressive age-related cochlear pathology and become severely hearing-impaired during the second year of life; CBA/J mice exhibit little hearing loss as they age. Age had little effect on the size of the IC, the size of IC neurons, or the packing density of IC neurons and there was no evidence of age-related neuron loss. However, old CBA/J mice developed numerous spongiform lesions throughout the brainstem. The absence of morphological changes in the IC of hearing-impaired C57BL/6J mice supports the hypothesis that features such as the size of neurons, survival of neurons, and volume of the neuropil are not affected by chronic sensorineural pathology in central auditory nuclei (e.g., as the IC) that do not receive direct input from primary afferent fibers. The data from both strains taken together indicate that certain basic anatomical properties of the mouse IC persist in the face of aging.
Asunto(s)
Envejecimiento/fisiología , Colículos Inferiores/anatomía & histología , Animales , Vías Auditivas/anatomía & histología , Vías Auditivas/ultraestructura , Tamaño de la Célula/fisiología , Cóclea/anatomía & histología , Colículos Inferiores/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Neuronas/ultraestructura , Especificidad de la EspecieRESUMEN
The morphology of the dorsal cochlear nucleus (DCN) was evaluated across the life span in inbred C57BL/6J (C57) and CBA/J (CBA) mice using 5 age groups (young adult to very old). C57 mice exhibit progressive cochlear sensorineural pathology and hearing loss during middle age; CBA mice have only modest sensorineural pathology late in life. DCN layers I, II, and III were evaluated histologically with serial sections stained for Nissl and fibers. DCN volume decreased with age in C57 mice, but the change began earliest and was most pronounced in layer III. In CBA mice, volume increased during the first year of life and decreased only in the oldest mice. All major DCN cell types were found in both strains at all ages. There was an age-related decrease in the mean size of neurons in C57 mice that was first observed in layer III. In CBA mice, only a nonsignificant trend toward smaller neurons was observed in the oldest mice. An age-related decline in the number of neurons in layer III (but not in layers I and II) occurred in C57 mice. Aged CBA mice exhibited no significant loss of DCN neurons. Thus, age-related changes in the DCN were much more pronounced in C57 mice than in CBA mice, and the changes in C57 mice were most pronounced in layer III. Because layer III receives most of the DCN's primary auditory input, it would be directly affected by age-related hearing loss and degeneration of spiral ganglion cells in C57 mice. This suggests that the age-related changes observed in DCN layer III of C57 mice are affected by progressive peripheral degenerative changes; when peripheral loss is minimal (CBA mice), less substantial age-related changes are observed.
Asunto(s)
Envejecimiento/fisiología , Nervio Coclear/anatomía & histología , Ratones Endogámicos C57BL/crecimiento & desarrollo , Ratones Endogámicos CBA/anatomía & histología , Neuronas/citología , Animales , Percepción Auditiva , Recuento de Células , Nervio Coclear/citología , Nervio Coclear/crecimiento & desarrollo , Ratones , Neuronas/fisiologíaRESUMEN
The influence of aging and age-related cochlear impairment on the ventral cochlear nucleus was evaluated by measuring morphological properties of the octopus cell area (OCA) in five age groups of inbred C57BL/6J and CBA/J mice (young adult to very old). The former strain demonstrates progressive cochlear sensorineural pathology and hearing loss during middle age; the latter has only modest sensorineural pathology late in life. Histological sections of the OCA were evaluated with serial sections and several strains for neurons, glia, and fibers, and Golgi impregnations were also used. Aging was associated with a decrease in volume of the OCA, a loss of neurons, slight decrease in neuron size, increased packing density of glial cells, and changes in dendrites ranging from minor to total loss of primary branches. The greatest changes occurred in extreme old age, beyond the median lifespan. Age-related changes were not exacerbated by sensorineural pathology in aging C57BL/6J mice. Individual octopus cells varied greatly in the extent of age-related abnormality.