Catecholamine-independent transient expression of tyrosine hydroxylase in primary auditory neurons is coincident with the onset of hearing in the rat cochlea

During the last stages of neuronal maturation, tyrosine hydroxylase is transiently expressed in the absence of the other catecholamine-synthesizing enzymes. We show here that it is expressed in rat spiral ganglion neurons between postnatal days 8 and 20, with a peak of expression at postnatal day 12. These tyrosine hydroxylase-immunoreactive neurons did not display aromatic amino acid decarboxylase- or dopamine-beta-hydroxylase-immunoreactivities, ruling out the possibilities of dopamine or noradrenaline synthesis. They also did not display peripherin- or intense neurofilament 200-kDa-immunoreactivities, two indicators of type II primary auditory neurons. Tyrosine hydroxylase-immunoreactive dendrites were seen in synaptic contact with the inner hair cells and expressed the GluR2 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, further confirming the type I nature of the neurons transiently expressing the enzyme. The end of the tyrosine hydroxylase expression was not due to cell death because the immunoreactive neurons did not show TUNEL-labelled nuclei. Finally, all the type I neurons expressed the tyrosine hydroxylase mRNA at postnatal day 12, suggesting that the expression of the enzyme is a maturational step common to all these neurons and that the expression of the protein is not synchronized. Because the period of transient expression of tyrosine hydroxylase in type I neurons parallels the periods of maturation of evoked exocytosis in inner hair cells and of appearance and maturation of the cochlear potentials, we propose that the expression of the enzyme indicates the onset of hearing in individual type I primary auditory neurons. This enzyme expression could rely on a Ca2+ activation of its encoding gene subsequent to a sudden and massive Ca2+ entry through voltage-activated Ca2+ channels.     

Presynaptic bodies in outer hair cells of the chinchilla organ of Corti

Some, but not all synapses of chinchilla outer hair cells with terminals of the spiral afferent nerve fibers have presynaptic bodies which are often surrounded by a halo of synaptic vesicles. The variety of appearance of presynaptic bodies and vesicles may result from the conditions of fixation or may represent differences in functional state.     

Phosphorylation of cysteine string protein in the brain: developmental, regional and synaptic specificity

Protein phosphorylation modulates regulated exocytosis in most cells, including neurons. Cysteine string protein (CSP) has been implicated in this process because its phosphorylation on Ser10 alters its interactions with syntaxin and synaptotagmin, and because the effect of CSP overexpression on exocytosis kinetics in chromaffin cells requires phosphorylatable Ser10. To characterize CSP phosphorylation in the brain, we raised phosphospecific antibodies to Ser10. Western blotting revealed that the proportion of phosphorylated CSP (P-CSP) varies between distinct brain regions and also exhibits developmental regulation, with P-CSP highest early in development. Immunohistochemical analysis of the cerebellar cortex revealed a novel pool of P-CSP that did not colocalize with synaptic vesicle markers during early development. Strikingly, in the adult cerebellar granular layer P-CSP was highly enriched in a subset of glutamatergic synapses but undetectable in neighbouring GABA-ergic synapses. In view of the functional consequences of CSP phosphorylation, such differences could contribute to the synapse-specific regulation of neurotransmitter release.     

Selective reduction in synaptic proteins involved in vesicle docking and signalling at synapses in the ataxic mutant mouse stargazer

The spontaneous recessive mutant mouse stargazer has a specific and pronounced deficit in brain-derived neurotrophic factor (BDNF) mRNA expression in the cerebellum. Cerebellar granule cells, in particular, show a selective and near-total loss of BDNF. The mutation involves a defect in the calcium channel subunit Cacng2. This severely reduces expression of stargazin. A stargazin-induced failure in BDNF expression is thought to underlie the cerebellar ataxia with which the mutant presents. BDNF is known to regulate plasticity at cerebellar synapses. However, relatively little is known about the mechanism involved. We previously demonstrated that the stargazer mutation affects the phenotype of cerebellar glutamatergic neurons. Stargazer neurons have less glutamate and proportionally fewer docked vesicles at presynaptic sites than controls. In the current study, we investigate the mechanism underlying BDNF-induced synaptic changes by analyzing alterations in synaptic signalling proteins in the stargazer cerebellum. Expression levels of synaptic proteins were evaluated by measuring relative density of immunogold label over granule cell terminals in ultrathin sections from ataxic stargazer mutants compared with matched nonataxic littermates. We show that there is a selective and marked depletion in the levels of vesicle-associated proteins (synaptobrevin, synaptophysin, synaptotagmin, and Rab3a) but not of plasma membrane-associated protein (SNAP-25) in the terminals of the BDNF-deficient granule cells. Changes are restricted to the cerebellum; levels in the hippocampus are unaltered. These data suggest that the BDNF deficits in the cerebellum of stargazer affect synaptic vesicle docking by selectively altering synaptic-protein distribution and abundance.     

Ryanodine receptors and BK channels act as a presynaptic depressor of neurotransmission in cochlear inner hair cells

Ryanodine receptors (RyRs) are known to contribute to the regulation of free cytosolic calcium concentration. This family of intracellular calcium channels plays a significant role in calcium-induced-calcium-release (CICR), and have been implicated in calcium-dependent processes requiring exquisite spatio-temporal regulation. In order to characterize the importance of these intracellular calcium channels in cochlear physiology, we perfused the guinea pig cochlea with antagonistic concentrations of ryanodine. The distortion products of the cochlear microphonic and the compound action potential of the auditory nerve were reversibly inhibited by ryanodine (IC(50)=27.3 microm, Hill coefficient=1.9), indicating an action at the cochlear amplifier. Single auditory nerve fibre recordings showed that ryanodine slightly increased spontaneous firing rates by 22%, suggesting an excitatory effect of ryanodine. This paradoxical effect could be explained by an inhibitory action of ryanodine on presynaptic BK channels of inner hair cells (IHC). Indeed, perfusing iberiotoxin also increased the spontaneous firing activity of the auditory nerve fibres. Furthermore, whole-cell patch-clamp recordings demonstrated that ryanodine inhibits BK currents at the IHC level. Conversely, immunohistochemistry demonstrated a strong expression of RyR in IHCs and, more particularly, below the cuticular plate where membranous BK channels are highly expressed. Overall, the study demonstrated a key role for RyR and CICR in signal transduction at the IHCs. We therefore propose that coupled RyR--BK channels act to suppress the fast neurotransmission in IHCs.     

Reciprocal synapses between inner hair cell spines and afferent dendrites in the organ of corti of the mouse

We provide, for the first time, ultrastructural evidence for the differentiation of reciprocal synapses between afferent dendrites of spiral ganglion neurons and inner hair cells. Cochlear synaptogenesis of inner hair cells in the mouse occurs in two phases: before and after the onset of hearing at 9-10 postnatal (PN) days. In the first phase, inner hair cells acquire afferent innervation (1-5 PN). Reciprocal synapses form around 9-10 PN on spinous processes emitted by inner hair cells into the dendritic terminals, predominantly in conjunction with ribbon afferent synapses. During the second phase, which lasts up to 14 PN, synaptogenesis is led by the olivocochlear fibers of the lateral bundle, which induce the formation of compound and spinous synapses. The afferent dendrites themselves also develop recurrent presynaptic spines or form mounds of synaptic vesicles apposed directly across inner hair cell ribbon synapses. Thus, in the adult 2-month mouse, afferent dendrites of spiral ganglion neurons are not only postsynaptic but also presynaptic to inner hair cells, providing a synaptic loop for an immediate feedback response. Reciprocal synapses, together with triadic, converging, and serial synapses, are an integral part of the afferent ribbon synapse complex. We define the neuronal circuitry of the inner hair cell and propose that these minicircuits form synaptic trains that provide the neurological basis for local cochlear encoding of the initial acoustic signals.     

Neuron-specific enolase-like immunoreactivity in inner hair cells but not outer hair cells in the guinea pig organ of Corti

Neuron-specific enolase (NSE) has been localized only in neurons and cells with characteristics of neurons. The immunocytochemical localization of NSE was examined in guinea pig cochleae to determine if hair cells, which have some neuronal characteristics, would show NSE-like immunoreactive labeling. NSE-like immunoreactivity was seen in inner hair cells but not in outer hair cells. This is the first report of NSE-like immunoreactivity in a receptor cell. NSE-like immunoreactivity was also seen in efferent fibers and terminals and in both type I and type II spiral ganglion cells. The finding of NSE-like immunoreactivity in inner but not outer cells adds to the number of differences found between them and may be related to differences in function and action.     

Light and electron microscopic analysis of synaptic development in Macaca monkey retina as detected by immunocytochemical labeling for the synaptic vesicle protein,SV2

The development of synapses has been followed in Macaca monkey fetal and infant retina using immunocytochemical labeling for the transmembrane synaptic vesicle glycoprotein, SV2. Electron microscopy (EM) was used to verify the presence of morphological synapses at selected ages. EM immunocytochemical labeling in adult retina showed that all synaptic types contained SV2 in inner (IPL) and outer (OPL) plexiform layers. In fetal retina, SV2 expression and the appearance of morphological synapses were closely related in time, demonstrating that SV2 is a reliable marker for synaptogenesis. SV2 expression appears along a foveal to peripheral gradient. Both SV2 and synapses appear in the foveal IPL at Fd50–55, and reach the retinal edge by Fd90–103. Cone ribbon synapses and SV2 labeling are not present in the foveal OPL until Fd60. Photoreceptors in the far periphery contain SV2 by Fd119–125. This pattern demonstrates an “inner to outer” direction of synaptogenesis. Cones show SV2 labeling before rods at the same retinal eccentricity. In the cone-dominated fovea, SV2 labeling and bipolar cell ribbon-containing terminals are present at Fd55 when amacrine cell conventional terminals are very scarce, indicating that bipolar synapses precede amacrine synapses in monkey foveal IPL. SV2 labeling and bipolar terminals appear first in the outer IPL which contains “OFF” ganglion and bipolar processes in the adult, suggesting that “OFF” midget bipolar cells may form the first synapses. Both SV2 immunocytochemical labeling and EM morphology find that monkey retina follows a generalized inner before outer, and cone before rod synaptic developmental pattern, similar to that in other mammals. The cone-dominated fovea initiates synaptogenesis, and shows a different synaptic sequence from rod-dominated peripheral retina. © 1994 Wiley-Liss, Inc.     

Postnatal development of the hamster coclea. I. Growth of hair cells and the organ of Corti

A morphometric analysis of the developing organ of Corti and its component hair cells was carried out in an age-graded series of Syrian golden hamsters with the aid of scanning electron microscopy. The purpose was to establish a quantitative framework that would provide insight into the rules and principles by which the mammalian cochlea attains its adult proportions. This study examined postnatal development at two day intervals from birth to 22 days after birth. Our analysis included measures of cochlear length and hair cell numbers as well as measures of hair cell sizes in each of five sectors along the cochlear spiral. Our results demonstrate several principles of cochlear development: (1) The full two and one-fourths turns seen in the adult cochlea are already present at birth, but the cochlea continues to elongate for the next 10–12 days. (2) Development of hair cells in the apex generally lags behind that in the base. Whereas the stereocilia and apical margins of hair cells are clearly defined in the basal turn, they become well defined in the apex only postnatally. (3) Growth in cochlear length occurs mainly by increases in cell size rather than in cell numbers; although hair cells do increase in numbers during the first 4 days of cochlear growth, this increase involves addition of hair cells only to preexisting regions of the cochlear apex. Moreover, the full complement of hair cells is established 6 days before the full size of the cochlea is attained; in contrast, hair cell growth occurs at all positions along the cochlear spiral and spans the entire period of cochlear elongation. (4) The period of hair cell growth exceeds the period of organ of Corti growth and appears to be possible by decreases in intercellular spacing, primarily in the apical region of the cochlea; inner and outer hair cell growth was complete between 16 and 18 days after birth. (5) Inner and outer hair cell neighbors remain virtually constant at different ages indicating that the spatial relationships between the two hair cell populations is preserved as the cochlea grows. (6) Comparison with previous developmental studies of auditory function in the hamster reveals that the age of 16 days after birth, when hair cells attain their mature sizes, coincides with the onset of brainstem auditory evoked responses. Growth of hair cell somas alone, however, cannot explain either the subsequent maturation of evoked potential thresholds or changes in frequency representation in the developing cochlea. © Wiley-Liss, Inc.     

Multi-locus genome-wide association analysis supports the role of glutamatergic synaptic transmission in the etiology of major depressive disorder

Major depressive disorder (MDD) is a common psychiatric illness characterized by low mood and loss of interest in pleasurable activities. Despite years of effort, recent genome-wide association studies (GWAS) have identified few susceptibility variants or genes that are robustly associated with MDD. Standard single-SNP (single nucleotide polymorphism)-based GWAS analysis typically has limited power to deal with the extensive heterogeneity and substantial polygenic contribution of individually weak genetic effects underlying the pathogenesis of MDD. Here, we report an alternative, gene-set-based association analysis of MDD in an effort to identify groups of biologically related genetic variants that are involved in the same molecular function or cellular processes and exhibit a significant level of aggregated association with MDD. In particular, we used a text-mining-based data analysis to prioritize candidate gene sets implicated in MDD and conducted a multi-locus association analysis to look for enriched signals of nominally associated MDD susceptibility loci within each of the gene sets. Our primary analysis is based on the meta-analysis of three large MDD GWAS data sets (total N=4346 cases and 4430 controls). After correction for multiple testing, we found that genes involved in glutamatergic synaptic neurotransmission were significantly associated with MDD (set-based association P=6.9 × 10⁻⁴). This result is consistent with previous studies that support a role of the glutamatergic system in synaptic plasticity and MDD and support the potential utility of targeting glutamatergic neurotransmission in the treatment of MDD.     

Differential expression of PKC beta II in the rat organ of Corti

To investigate a possible involvement of protein kinase C (PKC) in cochlear efferent neurotransmission, we studied the expression of the calcium-dependent PKC beta II isoform in the rat organ of Corti at different postnatal ages using immunofluorescence and immunoelectron microscopy. We found evidence of PKC beta II as early as postnatal day (PND) 5 in efferent axons running in the inner spiral bundle and in Hensen cells. At PND 8, we also found PKC beta II in efferents targeting outer hair cells (OHCs), and a slight detection at the synaptic pole in the first row of the basal and middle cochlear turns. At PND 12, PKC beta II expression declined in the efferent fibres contacting OHCs, whereas expression was concentrated at the postsynaptic membrane, from the basal and middle turns. The adult-like pattern of PKC beta II distribution was observed at PND 20. Throughout the cochlea, we found PKC beta II expression in the Hensen cells, non-sensory cells involved in potassium re-cycling, and lateral efferent terminals of the inner spiral bundle. In addition, we observed expression in OHCs at the postsynaptic membrane facing the endings of the medial efferent system, with the exception of some OHCs located in the most apical region of the cochlea. These data therefore suggest an involvement of PKC beta II in both cochlear efferent neurotransmission and ion homeostasis. Among other functions, PKC beta II could play a role in the efferent control of OHC activity.     

SNARE protein expression in synaptic terminals and astrocytes in the adult hippocampus: a comparative analysis

Several evidences suggest that astrocytes release small transmitter molecules, peptides, and protein factors via regulated exocytosis, implying that they function as specialized neurosecretory cells. However, very little is known about the molecular and functional properties of regulated secretion in astrocytes in the adult brain. Establishing these properties is central to the understanding of the communication mode(s) of these cells and their role(s) in the control of synaptic functions and of cerebral blood flow. In this study, we have set-up a high-resolution confocal microscopy approach to distinguish protein expression in astrocytic structures and neighboring synaptic terminals in adult brain tissue. This approach was applied to investigate the expression pattern of core SNARE proteins for vesicle fusion in the dentate gyrus and CA1 regions of the mouse hippocampus. Our comparative analysis shows that astrocytes abundantly express, in their cell body and main processes, all three protein partners necessary to form an operational SNARE complex but not in the same isoforms expressed in neighbouring synaptic terminals. Thus, SNAP25 and VAMP2 are absent from astrocytic processes and typically concentrated in terminals, while SNAP23 and VAMP3 have the opposite expression pattern. Syntaxin 1 is present in both synaptic terminals and astrocytes. These data support the view that astrocytes in the adult hippocampus can communicate via regulated exocytosis and also indicates that astrocytic exocytosis may differ in its properties from action potential-dependent exocytosis at neuronal synapses, as it relies on a distinctive set of SNARE proteins.     

Quantitative analysis of ribbons,vesicles, and cisterns at the cat inner hair cell synapse: Correlations with spontaneous rate

Cochlear hair cells form ribbon synapses with terminals of the cochlear nerve. To test the hypothesis that one function of the ribbon is to create synaptic vesicles from the cisternal structures that are abundant at the base of hair cells, we analyzed the distribution of vesicles and cisterns around ribbons from serial sections of inner hair cells in the cat, and compared data from low and high spontaneous rate (SR) synapses. Consistent with the hypothesis, we identified a “sphere of influence” of 350 nm around the ribbon, with fewer cisterns and many more synaptic vesicles. Although high‐ and low‐SR ribbons tended to be longer and thinner than high‐SR ribbons, the total volume of the two ribbon types was similar. There were almost as many vesicles docked at the active zone as attached to the ribbon. The major SR‐related difference was that low‐SR ribbons had more synaptic vesicles intimately associated with them. Our data suggest a trend in which low‐SR synapses had more vesicles attached to the ribbon (51.3 vs. 42.8), more docked between the ribbon and the membrane (12 vs. 8.2), more docked at the active zone (56.9 vs. 44.2), and more vesicles within the “sphere of influence” (218 vs. 166). These data suggest that the structural differences between high‐ and low‐SR synapses may be more a consequence, than a determinant, of the physiological differences. J. Comp. Neurol. J. Comp. Neurol. 521:3260–3271, 2013. © 2013 Wiley Periodicals, Inc.     

Localization of the presynaptic cytomatrix protein Piccolo at ribbon and conventional synapses in the rat retina: comparison with Bassoon

In recent years significant progress has been made in the elucidation of the molecular assembly of the postsynaptic density at synapses, whereas little is known as yet about the components of the presynaptic active zone. Piccolo and Bassoon, two structurally related presynaptic cytomatrix proteins, are highly concentrated at the active zones of both excitatory and inhibitory synapses in rat brain. In this study we used immunocytochemistry to examine the cellular and ultrastructural localization of Piccolo at synapses in the rat retina and compared it with that of Bassoon. Both proteins showed strong punctate immunofluorescence in the outer and the inner plexiform layers of the retina. They were found presynaptically at glutamatergic ribbon synapses and at conventional GABAergic and glycinergic synapses. Although the two proteins were coexpressed at all photoreceptor ribbon synapses and at some conventional amacrine cell synapses, at bipolar cell ribbon synapses only Piccolo was present. Our data demonstrate similarities but also differences in the molecular composition of the presynaptic apparatuses of the synapses in the retina, differences that may account for the functional differences observed between the ribbon and the conventional amacrine cell synapses and between the photoreceptor and the bipolar cell ribbon synapses in the retina.     

Contribution of bone marrow hematopoietic stem cells to adult mouse inner ear: mesenchymal cells and fibrocytes

Bone marrow (BM)-derived stem cells have shown plasticity with a capacity to differentiate into a variety of specialized cells. To test the hypothesis that some cells in the inner ear are derived from BM, we transplanted either isolated whole BM cells or clonally expanded hematopoietic stem cells (HSCs) prepared from transgenic mice expressing enhanced green fluorescent protein (EGFP) into irradiated adult mice. Isolated GFP(+) BM cells were also transplanted into conditioned newborn mice derived from pregnant mice injected with busulfan (which ablates HSCs in the newborns). Quantification of GFP(+) cells was performed 3-20 months after transplant. GFP(+) cells were found in the inner ear with all transplant conditions. They were most abundant within the spiral ligament but were also found in other locations normally occupied by fibrocytes and mesenchymal cells. No GFP(+) neurons or hair cells were observed in inner ears of transplanted mice. Dual immunofluorescence assays demonstrated that most of the GFP(+) cells were negative for CD45, a macrophage and hematopoietic cell marker. A portion of the GFP(+) cells in the spiral ligament expressed immunoreactive Na, K-ATPase, or the Na-K-Cl transporter (NKCC), proteins used as markers for specialized ion transport fibrocytes. Phenotypic studies indicated that the GFP(+) cells did not arise from fusion of donor cells with endogenous cells. This study provides the first evidence for the origin of inner ear cells from BM and more specifically from HSCs. The results suggest that mesenchymal cells, including fibrocytes in the adult inner ear, may be derived continuously from HSCs.     

Differential expression of synaptic vesicle proteins after repeated electroconvulsive seizures in rat frontal cortex and hippocampus

Electroconvulsive therapy (ECT) remains the treatment of choice for patients with severe or drug-resistant depressive disorders, yet the mechanism behind its efficacy and the effect on neurotransmission is essentially unknown. As synaptic vesicle proteins (SVPs) are required for vesicle fusion and neurotransmitter release, we have examined the effect of single and repeated electroconvulsive seizures (ECS), an animal model of ECT, on the expression of 14 SVPs in the rat frontal cortex and the hippocampus using quantitative real-time polymerase chain reaction (real-time qPCR). Only in the frontal cortex, the mRNA level of synapsin II was significantly upregulated after repeated ECS. In contrast, the mRNA levels of 6 of the 14 SVPs were significantly regulated in the hippocampus after ECS. We found that SNAP29 was upregulated and synaptotagmin III was downregulated after one single ECS in the hippocampus. Furthermore, SNAP29, synapsin I, synapsin III, VAMP2, and VAMP5 were significantly upregulated, whereas synaptotagmin III was significantly downregulated after repeated ECS in the hippocampus. We suggest that these genes are highly important in the long-term therapeutic effect of ECS, and thus it can be hypothesized that the SVPs are involved in the pathophysiology of depression.     

Low density of membrane particles in auditory hair cells of lizards and birds suggests an absence of somatic motility

Hair cells are the mechanoreceptive cells of the vertebrate lateral line and inner ear. In addition to their sensory function, hair cells display motility and thus themselves generate mechanical energy, which is thought to enhance sensitivity. Two principal cellular mechanism are known that can mediate hair-cell motility in vitro. One of these is based on voltage-dependent changes of an intramembrane protein and has so far been demonstrated only in outer hair cells of the mammalian cochlea. Correlated with this, the cell membranes of outer hair cells carry an extreme density of embedded particles, as revealed by freeze fracturing. The present study explored the possibility of membrane-based motility in hair cells of nonmammals, by determining their density of intramembrane particles. Replicas of freeze-fractured membrane were prepared from auditory hair cells of a lizard, the Tokay gecko, and a bird, the barn owl. These species were chosen because of independent evidence for active cochlear mechanics, in the form of spontaneous otoacoustic emissions. For quantitative comparison, mammalian inner and outer hair cells, as well as vestibular hair, cells were reevaluated. Lizard and bird hair cells displayed median densities of 2,360 and 1,880 intramembrane particles/microm2, respectively. This was not significantly different from the densities in vestibular and mammalian inner hair cells; however, it was about half the density in of mammalian outer hair cells. This suggests that nonmammalian hair cells do not possess high densities of motor protein in their membranes and are thus unlikely to be capable of somatic motility.     

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.     

Influence of neurotrophins on the synaptogenesis of inner hair cells in the deaf Bronx waltzer (bv) mouse organ of Corti in culture

The Bronx waltzer (bv) deaf mouse is characterized by massive degeneration of the primary auditory receptors, the inner hair cells, which occurs during the time of expected afferent synaptogenesis. The process is associated with degeneration and protracted division of the normally postmitotic afferent spiral ganglion neurons. To investigate the potential role of neurotrophins in the afferent synaptogenesis of inner hair cells, we exposed bv newborn cochleas in organotypic culture to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF), and also to gamma aminobutyric acid (GABA), for up to 8 days. The study was done using light and electron microscopy. Only about 20% of the inner hair cells survived in culture, regardless of the treatment, similar to the number in the intact mutant in our colony. Depending on the exogenous treatment, this population consisted of either innervated ultrastructurally normal cells or denervated dedifferentiated cells wrapped-in lieu of nerve endings-by the supporting inner phalangeal and border cells. In the control and GABA cultures, inner hair cells were mostly denervated. BDNF and NT-3 alone or combined increased synaptogenesis and hair cell survival only during the first 3 days (by about 10%); however, the cells became denervated by 8 postnatal (PN). Only NGF induced stable innervation and differentiation of neurosensory relationships, including supernumerary innervation characteristic of the intact bv. Denervation among the remaining 20% of inner hair cells induced a reactive wrapping by inner phalangeal and border cells which evidently extended inner hair cell survival. Immunocytochemical studies of these reactive supporting cells were done in the intact (8 PN) mutant cochlea. The supporting cells that provide sustenance to the denervated inner hair cells displayed strong BDNF (and possibly NT-3) immunoreactivity. Subsequently, we revealed the presence of all three neurotrophins in the inner hair cell region of the developing (1-8 PN) cochlea of the normal ICR mouse. The inner hair cells expressed all three neurotrophins; BDNF prevailed in the inner phalangeal cells, NT-3 in the pillar cells and inner phalangeal cells, and NGF in the pillar cells. In conclusion: initially, the 80% loss of inner hair cells is apparently caused by their failed afferent synaptogenesis. Exogenous neurotrophins influence synaptogenesis in the bv in culture, but NGF alone is successful in promoting stable neurosensory relationships. The presence of neurotrophins in supporting cells in the normal and degenerating cochlea indicates their role in the sustenance of inner hair cells.     

Light-evoked synaptic activity of retinal ganglion and amacrine cells is regulated in developing mouse retina

Recent studies have shown a continued maturation of visual responsiveness and synaptic activity of retina after eye opening, including the size of receptive fields of retinal ganglion cells (RGCs), light-evoked synaptic output of RGCs, bipolar cell spontaneous synaptic inputs to RGCs, and the synaptic connections between RGCs and ON and OFF bipolar cells. Light deprivation retarded some of these age-dependent changes. However, many other functional and morphological features of RGCs are not sensitive to visual experience. To determine whether light-evoked synaptic responses of RGCs undergo developmental change, we directly examined the light-evoked synaptic inputs from ON and OFF synaptic pathways to RGCs in developing retinas, and found that both light-evoked excitatory and inhibitory synaptic currents decreased, but not increased, with age. We also examined the light-evoked synaptic inputs from ON and OFF synaptic pathways to amacrine cells in developing retinas and found that the light-evoked synaptic input of amacrine cells is also downregulated in developing mouse retina. Different from the developmental changes of RGC spontaneous synaptic activity, dark rearing has little effect on the developmental changes of light-evoked synaptic activity of both RGCs and amacrine cells. Therefore, we concluded that the synaptic mechanisms mediating spontaneous and light-evoked synaptic activity of RGCs and amacrine cells are likely to be different.