Immunohistochemical localization of fibronectin-like protein in the inner ear of the developing gerbil and rat.

Immunohistochemistry was used to demonstrate the distribution of fibronectin-like protein within the developing inner ear of two species of altricial rodents: gerbils and rats. While there were temporal differences between the two species, the developmental sequence of immunostaining was virtually identical. Most notably, in rats from embryonic day 18 through day 1 postpartum, and in gerbils from birth through day 4 postpartum, intense, discrete fibronectin-like immunoreactivity was observed in the cochlea immediately beneath the inner and outer hair cells, sites of active auditory nerve fiber growth and nerve-hair cell synaptogenesis at these ages. The results suggest that fibronectin is appropriately positioned spatially and temporally to play a significant role in promoting, guiding and/or maintaining neural innervation within the developing organ of Corti. The temporo-spatial pattern of immunostaining in Schwann cells and auditory (VIIIth cranial) nerve neurons implies that fibronectin also plays a significant role in the early formation of myelin. In non-neural elements of the cochlea, fibronectin is a major structural component within the basilar membrane at all of the developmental stages investigated.     

Postnatal maturation of the organ of Corti in gerbils: Morphology and physiological responses

The organ of Corti, the sensory epithelium of hearing in mammals, matures postnatally in the gerbil. Quantitative analyses of the postnatal development of the organ of Corti, including supporting cells and the basilar membrane, were carried out. The morphological study confirmed that maturation of the sensory cells proceeds with a base-to-apex gradient, with the outer hair cells appearing to mature before the inner hair cells. Maturation of the supporting cells and the basilar membrane commenced first in the middle turn. Expansion of the second row of Deiters' cells began at 6 days after birth in the middle turn, before enlargement of the pillar cell heads at 8 days postnatally. Pillar cell head enlargement continued until 20 days postnatally in the middle turn. The tunnel of Corti and spaces of Nuel appeared first in the middle turn between 8 and 10 days postnatally. The maturation of the basilar membrane involved the thickening of the central hyaline layer and a reduction in the epithelial cells on the tympanic aspect. This process continued until about 20 days after birth. The cochlear microphonic potential, whole nerve action potential, and stimulus frequency otoacoustic emissions were recorded from 12 days after birth onward and related to changes in organ of Corti morphology. The results show that changes in the accessory structures continue throughout the period of onset and development of cochlear responses between 12 and 20 days after birth, and may therefore influence the micromechanical responses of the organ of Corti to acoustic stimuli during this period. J. Comp. Neurol. 386:635–651, 1997. © 1997 Wiley-Liss, Inc.     

Inner hair cell responses to the velocity of basilar membrane motion in the guinea pig

Triangular wave acoustic stimulation at 200 Hz produced the expected square wave cochlear microphonic at the round window membrane and within the scala media. Intracellular recordings from inner hair cells (IHC) of the first cochlear turn showed a combination waveform having both spike impulse and square wave features. The IHC response suggests a sensitivity of these cells to both the displacement and to the velocity of basilar membrane motion.     

Contractile proteins in the hyaline cells of the chicken cochlea.

Hyaline cells are a single layer of epithelial cells found at the inferior edge of the sensory epithelium in the chick cochlea. They rest directly above a specialized region of the basilar membrane at a point where it connects to the fibrocartilaginous skeleton of the cochlear duct. The basal cytoplasm of the hyaline cells contains a bundle of linearly aligned actin filaments that resemble stress fibers in their organization. The actin filaments are anchored in the basal plasma membranes of the cells, which are, in turn, associated with the underlying basal lamina and the extracellular matrix of the basilar membrane. We have used a combination of transmission electron microscopy, differential-interference-contrast and epifluorescence light microscopy, and confocal laser scanning microscopy to study the composition and organization of these actin bundles within the hyaline cells. The bundles are arranged into triangular wedges that are oriented radially across the basilar membrane. Each cell contains one or two actin wedges. Adjacent cells can have them aligned in opposite directions so that in a whole-mount surface preparation they appear as interdigitations. Immunofluorescent staining of the hyaline cells has shown that smooth muscle myosin and alpha-actinin are co-localized to the actin bundles. Smooth muscle myosin is also found throughout the cytoplasm of the cells. The fact that hyaline cells in the chick cochlea are contacted by efferent nerve fibers suggests that these cells may regulate tension on the basilar membrane via the specialized bundle of actin filaments.     

Age-related changes in the distribution of Kv1.1 and Kv3.1 in rat cochlear nuclei

OBJECTIVES: To identify age-related changes in voltage-gated K(+) (Kv) channels that contribute to temporal processing in neurons of the central auditory system, we investigated the distribution of Kv1.1 and Kv3.1 in the auditory brainstem of adult and aged rats. METHODS: Immunohistochemistry was performed in accordance with the free-floating method described earlier. RESULTS: Among the auditory nuclei, only the posterior ventral cochlear nucleus (PVCN) showed age-related changes. Kv1.1 immunoreactivity was increased in the octopus cell bodies, while the staining intensity was significantly decreased in the neuropil. Image analysis demonstrated the specific increase in Kv1.1 immunoreactivity in aged cochlear nucleus neurons although the mean density of the entire selection was significantly decreased. In contrast, the number of Kv1.1-immunoreactive neurons was not significantly different between control and aged groups. The immunoreactivity for Kv3.1 was decreased in the octopus cells and neuropil of aged PVCN, which was confirmed by image analysis. The number of Kv3.1-positive cells was also significantly decreased in aged PVCN. DISCUSSION: This study may provide useful data to compare age-related changes in Kv1.1 and Kv3.1 with known physiological properties of auditory neurons.     

Transgenic BDNF induces nerve fiber regrowth into the auditory epithelium in deaf cochleae

Sensory organs typically use receptor cells and afferent neurons to transduce environmental signals and transmit them to the CNS. When sensory cells are lost, nerves often regress from the sensory area. Therapeutic and regenerative approaches would benefit from the presence of nerve fibers in the tissue. In the hearing system, retraction of afferent innervation may accompany the degeneration of auditory hair cells that is associated with permanent hearing loss. The only therapy currently available for cases with severe or complete loss of hair cells is the cochlear implant auditory prosthesis. To enhance the therapeutic benefits of a cochlear implant, it is necessary to attract nerve fibers back into the cochlear epithelium. Here we show that forced expression of the neurotrophin gene BDNF in epithelial or mesothelial cells that remain in the deaf ear induces robust regrowth of nerve fibers towards the cells that secrete the neurotrophin, and results in re-innervation of the sensory area. The process of neurotrophin-induced neuronal regeneration is accompanied by significant preservation of the spiral ganglion cells. The ability to regrow nerve fibers into the basilar membrane area and protect the auditory nerve will enhance performance of cochlear implants and augment future cell replacement therapies such as stem cell implantation or induced transdifferentiation. This model also provides a general experimental stage for drawing nerve fibers into a tissue devoid of neurons, and studying the interaction between the nerve fibers and the tissue.     

Age-related changes in the distribution of Kv1.1 and Kv3.1 in rat cochlear nuclei

Abstract

Objectives: To identify age-related changes in voltage-gated K⁺ (Kv) channels that contribute to temporal processing in neurons of the central auditory system, we investigated the distribution of Kv1.1 and Kv3.1 in the auditory brainstem of adult and aged rats.

Methods: Immunohistochemistry was performed in accordance with the free-floating method described earlier.

Results: Among the auditory nuclei, only the posterior ventral cochlear nucleus (PVCN) showed age-related changes. Kv1.1 immunoreactivity was increased in the octopus cell bodies, while the staining intensity was significantly decreased in the neuropil. Image analysis demonstrated the specific increase in Kv1.1 immunoreactivity in aged cochlear nucleus neurons although the mean density of the entire selection was significantly decreased. In contrast, the number of Kv1.1-immunoreactive neurons was not significantly different between control and aged groups. The immunoreactivity for Kv3.1 was decreased in the octopus cells and neuropil of aged PVCN, which was confirmed by image analysis. The number of Kv3.1-positive cells was also significantly decreased in aged PVCN.

Discussion: This study may provide useful data to compare age-related changes in Kv1.1 and Kv3.1 with known physiological properties of auditory neurons.     

Immunoreactivity of Calcium-Binding Proteins in the Central Auditory Nervous System of Aged Rats

Objective

While many factors contribute to aging, changes in calcium homeostasis and calcium related neuronal processes are likely to be important. High intracellular calcium is toxic to cells and alterations in calcium homeostasis are associated with changes in calcium-binding proteins, which confine free Ca²⁺. We therefore assayed the expression of the calcium binding proteins calretinin and calbindin in the central auditory nervous system of rats.

Methods

Using antibodies to calretinin and calbindin, we assayed their expression in the cochlear nucleus, superior olivary nucleus, inferior colliculus, medial geniculate body and auditory cortex of young (4 months old) and aged (24 months old) rats.

Results

Calretinin and calbindin staining intensity in neurons of the cochlear nucleus was significantly higher in aged than in young rats (p<0.05) The number and staining intensity of calretinin-positive neurons in the inferior colliculus, and of calbindin-positive neurons in the superior olivary nucleus were greater in aged than in young rats (p<0.05).

Conclusion

These results suggest that auditory processing is altered during aging, which may be due to increased intracellular Ca²⁺ concentration, consequently leading to increased immunoreactivity toward calcium-binding proteins.     

Distribution of glial fibrillary acidic protein (GFAP) in the cochlear nucleus of adult and aged rats

The age-related change in glial fibrillary acidic protein (GFAP) immunoreactivity was analyzed in young (3 months) and old (24 months) adult rat cochlear nuclei (CN). Quantitative analyses show a significant increase with age, in the number of GFAP positive astrocytes and processes in the old adult when compared with the young adult rat. There was also a differential distribution of GFAP immunoreactivity in the young adult CN where it predominates in the granular cell region, whereas in old rats, the GFAP immunoreactivity distribution was homogeneous in all parts of the nucleus. There was no change in the total number of neurons between these two stages in any part of the nucleus except for the antero-ventral CN, where a decrease in neuronal number was observed in the aged rats. The increase in GFAP immunoreactivity was related to an increase of both GFAP positive astrocyte number and processes. The increase of GFAP positive astrocytes may be due either to an alteration of auditory nerve fibers, changing the trophic interactions with post-synaptic cells, or to intrinsic alterations of CN neurons and local circuits reflecting aging of the CN.     

The effect of age and tongue exercise on BDNF and TrkB in the hypoglossal nucleus of rats

Age-associated changes in tongue musculature may contribute to dysphagia. One possible treatment is tongue exercise. Exercise induces synaptic plasticity by increasing neurotrophic factors in spinal cord and limb musculature. However, effects of exercise on neurotrophic factors in the cranial sensorimotor system are unknown. Our purpose was to examine the effects of age and exercise on brain-derived neurotrophic factor (BDNF) and its receptor TrkB in the rat hypoglossal nucleus.Young, middle-aged, and old rats were assigned to exercise or no-exercise control conditions. Exercise animals were trained to perform a tongue press task for 8 weeks. Samples from the hypoglossal nucleus were analyzed for BDNF and TrkB immunoreactivity.Baseline maximum tongue forces were similar in all age groups and increased significantly following exercise. BDNF immunoreactivity did not show a significant decrease with age in control group. However, in the exercise group, BDNF was significantly increased in young animals. TrkB immunoreactivity decreased significantly with age in control group, but did not change with exercise. BDNF and TrkB immunoreactivity levels were positively correlated with exercise in young and middle aged animals, but were negatively or weakly correlated with exercise in old animals and with a lack of exercise in no-exercise controls.Tongue exercise was associated with increased tongue forces in rats at all ages. While increases in BDNF and TrkB levels associated with exercise may play a role in mechanisms contributing to increased tongue forces in young and middle-aged rats, other mechanisms may be involved in increased tongue forces observed in old rats.     

Fos-like immunoreactivity in central auditory neurons of the mouse

Fos-like immunoreactivity was used to study sound-induced activation of neurons in the auditory brainstem. Immunoreactivity was assayed with a polyclonal antibody to Fos. In response to 6-kHz tone bursts, the pattern of staining was a band of immunoreactive neurons positioned at the tonotopically appropriate position within the cochlear nucleus and the inferior colliculus. The band was narrow at low sound pressure levels but wider along the tonotopic axis at higher sound levels. In response to noise bursts, the pattern was broader and often extended throughout the auditory nuclei. Often within this broad pattern were “sub-bands” of immunostained neurons, interspersed with bands of unstained neurons. With increasing sound pressure levels above 35--55 dB, the number of Fos-like immunoreactive neurons increased for the cochlear nucleus, superior olivary complex, and inferior colliculus. In the cochlear nucleus and inferior colliculus, the stained cells were small, and hence their activity would be difficult to sample in electrophysiological studies. In the medial nucleus of the trapezoid body, the stained neurons had larger somata and other characteristics of principal cells. Anesthesia with Nembutal or Avertin, but not with ketamine or urethane, decreased the number of Fos-like immunoreactive neurons in the cochlear nucleus. The different anesthetics produced more variable results in the inferior colliculus. In anesthetized, monaurally stimulated animals, the presence of staining in the contralateral cochlear nucleus indicates that some Fos-like immunoreactivity may be mediated by descending or commissural systems. These observations indicate that Fos assays are useful for studying the pattern of neuronal activation in the auditory system and may also be useful in studying the descending auditory pathways. © 1995 Wiley-Liss, Inc.     

The cochlea in gerbilline rodents

The inner ears of 5 different gerbil species are compared on the basis of cochlear microphonic recordings, serial sections and computerized quantitative reconstructions of the cochleae and their specific morphological structures. The hearing range of most gerbils is below 20 kHz. Some species are extremely sensitive in the frequency range of 1-4 kHz. This special sensitivity is reflected in, among other features, the following cochlear structures and their suggested functions: (1) the rapid width increase of the basilar membrane in the basal portion of the cochlea provides additional space for the representation of lower frequencies at the expense of higher frequencies; (2) the large hyaline mass and the cells of Claudius and Hensen in the medial and apical portions of the cochlea influence the vibratory properties of the cochlear partition, and (3) the specialized structures of the cochlea may be an adaptation to the acoustical environment in arid habitats.     

Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor

The distribution and morphology of glycinergic synapses in the cochlear nucleus were investigated using monoclonal antibodies against the glycine receptor. Glycine receptor immunoreactivity was seen on somas and proximal processes of most cells in all divisions of the cochlear nucleus; distribution of label in neuropil was denser in the dorsal cochlear nucleus and granule cell cap than in the ventral cochlear nucleus. At the ultrastructural level, glycine receptor immunoreactivity was specifically distributed postsynaptically to terminals that contained flattened vesicles in the guinea pig anteroventral cochlear nucleus. These studies show that the immunocytochemical localization of the glycine receptor can provide a means of identifying and characterizing glycinergic synapses throughout the central nervous system.     

Formation of the cochlea in the chicken embryo: sequence of innervation and localization of basal lamina-associated molecules.

The purpose of this study was to determine the temporal and spatial gradients in the innervation of the chicken cochlea, the basilar papilla, as it develops in the early embryo. A series of white Leghorn chick embryos (Hamburger-Hamilton Stage 20-43) were prepared for serial sectioning and stained by Toluidine blue or by antibodies to fibronectin or laminin. Light microscopic observations were made on the first fiber bundles to reach each region of the basilar papilla. There is a distinct temporo-spatial pattern of the ingrowth of fiber bundles to the developing basilar papilla. The primary pattern is not described as any simple linear gradient. Fiber ingrowth begins proximally, shifts to a distal and then to a mid-proximal region. The fiber ingrowth correlates temporally and spatially with disruption of fibronectin and laminin staining of the basal lamina where fiber bundles are penetrating. This pattern may reflect not only the sequence of fiber ingrowth but also the displacement of cells and fibers in the elongating basilar papilla, which grows as a result of a contemporaneous mitotic activity throughout the structure rather than progressing from one end to the other.     

Furosemide alters organ of corti mechanics: evidence for feedback of outer hair cells upon the basilar membrane

A widely held hypothesis of mammalian cochlear function is that the mechanical responses to sound of the basilar membrane depend on transduction by the outer hair cells. We have tested this hypothesis by studying the effect upon basilar membrane vibrations (measured by means of either the M?ssbauer technique or Doppler-shift laser velocimetry) of systemic injection of furosemide, a loop diuretic that decreases transduction currents in hair cells. Furosemide reversibly altered the responses to tones and clicks of the chinchilla basilar membrane, causing response-magnitude reductions that were largest (up to 61 dB, averaging 25-30 dB) at low stimulus intensities at the characteristic frequency (CF) and small or nonexistent at high intensities and at frequencies far removed from CF. Furosemide also induced response-phase lags that were largest at low stimulus intensities (averaging 77 degrees) and were confined to frequencies close to CF. These results constitute the most definitive demonstration to date that mechanical responses of the basilar membrane are dependent on the normal function of the organ of Corti and strongly implicate the outer hair cells as being responsible for the high sensitivity and frequency selectivity of basilar membrane responses. A corollary of these findings is that sensorineural hearing deficits in humans due to outer hair cell loss reflect pathologically diminished vibrations of the basilar membrane.     

Noradrenergic changes and memory loss in aged mice

The present paper addresses the question of whether a decline of central noradrenergic activity is associated with aging and memory loss in mice. Receptor binding techniques were utilized to compare alpha 2-adrenoceptor density in the brains of aged and young mice. Using [3H]rauwolscine, a selective alpha 2-adrenoceptor antagonist, two membrane binding sites were identified which were differentially affected by age. Whereas the density of high-affinity binding sites was unchanged in aged brain as compared to young controls, there was a significant decrease in the number of low-affinity sites. In a separate study, animals were tested for performance on a step-through inhibitory avoidance task, prior to sacrifice and morphological analysis of the brainstem noradrenergic nucleus locus coeruleus (LC). Aged mice exhibited a significant decrease in task retention as compared to young controls; a small, though non-significant, decline was also observed in the numbers of cells within LC. While young mice exhibited low within-group variance, individual aged animals differed greatly in both LC cell number and behavioral performance. Within the aged population, there was a highly significant correlation between the extent of LC cell loss and the degree of memory impairment. These results provide further evidence for an age-related decline in central noradrenergic function which may contribute to an associated memory loss.     

Androgenic regulation of expression of androgen receptor protein in the perineal motoneurons of aged male rats.

Androgen might regulate expression of androgen receptors (AR) in AR-containing motoneurons in young animals. In the present study, it was examined whether expression of AR was also regulated by androgen in aged animals. Twelve male rats were castrated at 26 months of age. Five days following castration, the animals were treated with testosterone propionate (TP; six males) or vehicle (six males) and killed 2 hours later. Six sham-castrated rats served as controls. AR immunoreactivity was examined in motoneurons of the spinal nucleus of the bulbocavernosus (SNB) in these animals by immunohistochemistry using the polyclonal antibody PG21. In control animals, slightly intense AR immunoreactivity was confined to the nuclei of the motoneurons. AR immunoreactivity was completely eliminated in the motoneurons of castrated rats. In castrated, aged animals treated with TP, the intensity of AR immunoreactivity in the nuclei of SNB motoneurons was increased. Plasma levels of testosterone in castrated, aged animals 2 hours following treatment with TP were significantly greater than those in controls. These results suggest that expression of AR in motoneurons of the SNB in aged male rats is up-regulated in response to androgen and that androgen may be, at least in part, involved in the process of aging of the SNB in male rats.     

Cholinergic activation of hippocampal neural stem cells in aged dentate gyrus

Adult hippocampal neurogenesis contributes to the hippocampal circuit's role in cognitive functioning. New neurons are generated from hippocampal neural stem cells (NSCs) throughout life, but their generation is substantially diminished in aged animals due to a decrease in NSC proliferation. Because acetylcholine (ACh) is an important neurotransmitter released in the hippocampus during learning and exercise that is known to decrease with aging, we investigated whether aged NSCs can respond to ACh. In this study, we found that cholinergic stimulation has a positive effect on NSC proliferation in both young adult (8–12 weeks old) and aged mice (>2 years old). In fresh hippocampal slices, we observed a rapid calcium increase in NSCs in the dentate gyrus after muscarinic cholinergic stimulation, in both age groups. Furthermore, we found that the exercise‐induced promotion of aged NSC proliferation was abrogated by the specific lesioning of the septal cholinergic system. In turn, cholinergic activation by either eserine (physostigmine) or donepezil treatment promoted the proliferation of NSCs in aged mice. These results indicate that NSCs respond to cholinergic stimulation by proliferating in aged animals. Physiological and/or pharmacological cholinergic stimulation(s) may ameliorate cognitive decline in aged animals, by supporting adult hippocampal neurogenesis. © 2010 Wiley‐Liss, Inc.     

Fibronectin-like immunoreactivity in the developing cerebral cortex

In the developing cerebral cortex of the mouse, binding of antibodies directed against the extracellular matrix glycoprotein fibronectin occurs with a distinct temporal and spatial pattern. On the 10th embryonic day (E10), when the wall of the telencephalic vesicle is made up of only the proliferating cells of the ventricular zone, antifibronectin (aFN) binding is restricted to the blood vessels and pia-arachnoid. Fibronectin-like immunoreactivity first appears in the neuropil as small points of immunofluorescence among the earliest postmitotic neurons that form the preplate (E11-12). A short time later (E12-13), aFN immunoreactivity becomes more diffuse but continues to be restricted to the preplate. As newly arriving neurons form the cortical plate within the preplate (E13-14), aFN binding is present in the marginal zone above the cortical plate and in the subplate below it. Both the marginal zone and the subplate contain early afferents and the cells that were previously part of the preplate. Binding of aFN is transient; by E18-19 it has diminished to the point where it is no longer detectable except in the blood vessels and pia-arachnoid. The transient appearance of fibronectin-like immunostaining in the zones that contain early cortical afferents suggests that fibronectin plays a role in forming the migratory pathway for the growth cones of these axons. In this role it may be acting in concert with other extracellular matrix components such as hyaluronectin, glycosaminoglycans, and laminin, which have been shown to have similar spatial distributions.(ABSTRACT TRUNCATED AT 250 WORDS)