Juan Represa

Temporal pattern of nerve growth factor (NGF) binding in vivo and the in vitro effects of NGF on cultures of developing auditory and vestibular neurons.

NGF binding patterns reflect the presence of receptors for this growth factor. High specific binding of 125I 2.5 S-NGF was observed for the 11 gestation day (gd) statoacoustic ganglion (SAG) with lower levels recorded for both 14 gd acoustic ganglion (AG) and vestibular ganglion (VG) samples. Fourteen day AG cells were more than twice as active for binding NGF when compared to VG samples of the same gestational age. Both whole ganglion explants and dissociated cell cultures were grown in chemically defined medium for short term culture to assay changes in neurite outgrowth and survival of neurons in response to the addition of exogenous 2.5 S-NGF. The most vigorous neurite outgrowth and neuronal survival responses were produced by 11 gd SAG samples treated with NGF. Acoustic ganglion specimens of both 11 gd and 14 gd embryos were much more responsive to the neurotrophic effects of NGF when compared to the responses of their VG counterparts. There was a correlation between NGF binding ability and in vitro responsiveness to exogenous NGF. We hypothesize based on the results of this study that NGF (and/or a member of the NGF family of growth factors) is involved in the control of developmentally regulated neuronal cell death of SAG neurons and may play a role in the innervation of developing inner ear sensory structures.

Inositol phospho-oligosaccharide stimulates cell proliferation in the early developing inner ear

The ability of an inositol phospho-oligosaccharide (POS) to mimic the mitogenic effects of nerve growth factor (NGF) and insulin on the early development of the inner ear was investigated. POS (10 microM) stimulated the incorporation of [3H]thymidine into the cochleovestibular ganglion by 3.9-fold. NGF (50 ng/ml) stimulation was 4.7-fold. POS and NGF showed no additivity. Cells induced to proliferate by POS overlapped with those expressing NGF receptors. POS, like insulin, potentiated the mitogenic effect of bombesin on the otic vesicle epithelium. DNA synthesis in the presence of bombesin (100 nM) plus POS (10 microM) was increased by 6.4-fold. POS stimulation was not additive with insulin. The results suggest that POS may play a role in growth factor regulation of cell proliferation during embryonic development.

Neurotrophin and Trk neurotrophin receptors in the inner ear of Salmo salar and Salmo trutta

Neurotrophins (NTs) and their signal transducing Trk receptors play a critical role in the development and maintenance of specific neuronal populations in the nervous system of higher vertebrates. They are responsible for the innervation of the inner ear cochlear and vestibular sensory epithelia. Neurotrophins and Trks are also present in teleosts but their distribution in the inner ear is unknown. Thus, in the present study, we used Western‐blot analysis and immunohistochemistry to investigate the expression and cell localization of both NTs and Trk receptors in the inner ear of alevins of Salmo salar and Salmo trutta. Western‐blot analysis revealed the occurrence of brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3), but not nerve growth factor (NGF), as well as all three Trk receptors, i.e. TrkA, TrkB and TrkC, the estimated molecular weights of which were similar to those expected for mammals. Specific immunoreactivity for neurotrophins was detected mainly in the sensory epithelia. In particular, BDNF immunoreactivity was found in the maculae of the utricle and saccule, whereas NT‐3 immunoreactivity was present in the sensory epithelium of the cristae ampullaris. As a rule the sensory epithelia of the inner ear lacked immunoreactivity for Trks, thus excluding possible mechanisms of autocrinia and/or paracrinia. By contrast, overlapping subpopulations of neurons in the statoacoustic ganglion expressed TrkA (about 15%), TrkB (about 65%) and TrkC (about 45%). The present results demonstrate that, as in mammals and birds, the inner ear of teleosts expresses the components of the neurotrophin–Trk system, but their roles remain to be elucidated.

The thymus of the hairless rhino-j (hr/hr-j) mice

The hairless (hr) gene is expressed in a large number of tissues, primarily the skin, and a mutation in the hr gene is responsible for the typical cutaneous phenotype of hairless mice. Mutant hr mouse strains show immune defects involving especially T cells and macrophages, as well as an age‐related immunodeficiency and an accelerated atrophy of the thymus. These data suggest that the hr mutation causes a defect of this organ, although hr transcripts have not been detected in fetal or adult mice thymus. The present study analyses the thymus of young (3 mo) and adult (9 mo) homozygous hr‐rh‐j mice (a strain of hairless mice) by means of structural techniques and immunohistochemistry to selectively identify thymic epithelial cells, dendritic cells, and macrophages. There were structural alterations in the thymus of both young and adult rh‐rh‐j mice, which were more severe in older animals. These alterations consisted of relative cortical atrophy, enlargement of blood vessels, proliferation of perivascular connective tissue, and the appearance of cysts. hr‐rh‐j mice also showed a decrease in the number of epithelial and dendritic cells, and macrophages. Taken together, present results strongly suggest degeneration and accelerated age‐dependent regression of the thymus in hr‐rh‐j mice, which could explain at least in part the immune defects reported in hairless mouse strains.

Changes in the cerebellar cortex of hairless Rhino-J mice (hr-rh-j)

A mutation in the hr gene is responsible for typical epithelium phenotype in hairless mice. As this gene is expressed at high levels not only in the skin but also in the brain, the aim of the study was to clarify its role in the central nervous system. We have analyzed by morphological and immunocytochemical methods (calbindin D-28k, phosphorylated and 200 kDa neurofilament protein) the cerebellum of a mutated mouse strain, the hairless (hr-rh-j) type carrying the homozygous hr gene rhino mutation. The cerebellar cortex was studied in young (3 months) and adult (9 months) wild type and mutated mice. No major structural change was found in any of the groups and neuronal density or neuronal arrangement were similar in mutated animals to their age-matched controls. Nevertheless there were changes in shape and size of the Purkinje neurons in the old mutated animals respect to their normal littermates, while the molecular and the granule cell layers were apparently invariable. Calbindin (CB) immunohistochemistry revealed a significant decrease in the expression of this protein in the Purkinje cells of the aged mutated mice. Immunohistochemistry for a neurofilament protein (NFP) showed a reduction of staining in all the cerebellar cortex layers in the older animals, which was much more evident in the (hr-rh-j) mutated mice. These results suggest that hr gene is involved in the structural maintenance of the mature cerebellar cortex, rather than in the development. Our findings may also be consistent with an accelerated aging of the central nervous system in rh-rh-j mice.

Expression of the cytoskeletal protein MAP5 and its regulation by neurotrophin 3 (NT3) in the inner ear sensory neurons

Abstractu2002Microtubule-associated proteins (MAPs) are essential cytoskeletal components during development for neurogenesis and neuronal plasticity. Inner ear innervation is accomplished by cochleovestibular ganglion (CVG) neurons in a highly specific, well-defined pattern, which is regulated by neurotrophic factors belonging to the neurotrophin family. The inner ear offers a suitable model for studying the expression of MAPs and assessing their role in neurotrophin-induced effects that are required for neuron-target innervation. The present study was undertaken to analyze the expression and localization of MAP5 isoforms during development of CVG neurons in vivo and in vitro; as well as the regulation of MAP5 by neurotrophin-3 (NT3) in cell culture. MAP5 expression in the inner ear of chick embryos and postnatal specimens was monitored using immunoblots and immunohistochemistry on frozen sections. MAP5 was highly expressed during the early stages of CVG development, at embryonic day (E)4, being located in both neuronal perikarya and neurites. Expression was maintained during the neurite outgrowth phase (E6–E12), when strong MAP5 immunostaining was observed at the same cellular locations. MAP5 expression decreased suddenly at E14, after the establishment of specific connections between the CVG neurons and their targets, the sensory epithelium of the inner ear. In cultured CVG neurons addition of NT3 led to increased MAP5 expression and produced neurite outgrowth. Both effects are differentially regulated in parallel by low (0.5 ng/ml) and high (5 ng/ml) NT3 concentrations. Present results suggest that MAP5 may be involved in neurotrophin-induced microtubule bundling during neurite outgrowth of auditory neurons.

Reduction of glial fibrillary acidic protein-immunoreactive astrocytes in some brain areas of old hairless rhino-j mice (hr-rh-j).

Mutations in the hairless (hr) gene of mice result in hair follicle and other epithelial defects. The hr gene is expressed at high levels in the brain where it probably participates in the survival and maintenance of some neuronal populations, but whether it also supports glial populations of the central nervous system has been not investigated. To clarify this, quantitative immunohistochemistry for astrocytes (glial fibrillary acidic protein (GFAP)) and microglial cells (CD11b macrophage antigen) was used in the brain of a mutant mouse strain, the hairless (hr-rh-j) type, which carries the homozygous hr gene rhino mutation. The glial cell density was assessed in the cerebral cortex, hippocampus, striatum, hypothalamus and cerebellum of young (3 months) and old (9 months) hr-rh-j mice. No significant differences were found between young wild-type and hr-rh-j mice. The density of GFAP immunoreactive astrocytes normally increased as a function of age, but in older hr-rh-j mice there was a severe reduction (P<0.01) in the striatum, hypothalamus, and hippocampus. Conversely, the microglial cells were insensible to aging or to hr-rh-j mutation. These results suggest that the hr gene is involved in the maintenance of the GFAP immunoreactive cells in some cerebral areas. Nevertheless, because these animals do not show any neurological signs, the functional significance of the present findings remains to be established.

Protein kinase C activation is required during the early development of the inner ear in culture

SummaryThe role of protein kinase C (PKC). during the early development of the inner ear was investigated using organ culture techniques. Otocysts isolated from chick embryos were made quiescent by culturing in the absence of serum for 24 h. The normal process of development could be reactivated by restoration of serum and other growth factors. Addition of phorbol ester (TPA) or synthetic diacylglycerol (OAG) to serum-free medium was also effective in reactivating development and stimulation of DNA synthesis was 41% and 52% of that of serum, respectively. Insulin potentiated the effects of TPA and OAG but had no effect when present alone. Morphogenesis and the associated cell proliferation stimulated by either serum or PKC activation were both inhibited by sphingosine, an in vitro inhibitor of PKC. Inhibition by sphingosine was dose-dependent with a half-maximal inhibitory concentration of about 10 μM. The results suggest that PKC activation is an essential step in controlling proliferative growth during early stages of the development of the inner ear.

Morphogenesis and Genetics of Inner Ear Development and Malformation

Publisher Summary This chapter reviews the morphogenesis of the membranous and bony labyrinths of the inner ear and the genetics of inner ear development. The inner ear develops from cephalic surface ectoderm that forms the otic placode. Each otic placode thickens, and then invaginates to form the otic vesicle, the epithelial primordia from which the vestibular and cochlear regions of the membranous labyrinth develop. The otic capsule appears initially at 6 weeks of human embryonic development as a condensation of mesodermal mesenchyme around the developing otocyst. By 8 weeks of gestation, the condensed mesenchyme has formed a fully chondrified otic capsule that serves as a template for the subsequent formation of the endochondral bony labyrinth. Patterning of the inner ear into prospective vestibular and auditory sensory areas is associated with restriction of gene expression domains during the early stages of otic development. A final consideration in the genetic patterning of the inner ear is the role of the sequential expression of growth factor genes and in specifying the development of both the otic capsule and the perilymphatic spaces, which act to protect and nourish the membranous labyrinth, ultimately forming a fully chondrified otic capsule, with its perilymphatic spaces, which serves as the template for subsequent formation of the bony labyrinth, scala vestibuli, and scala tympani of the adult inner ear, and temporal bone.