Hair cell innervation by spiral ganglion neurons in the mouse

Horseradish peroxidase (HRP) was injected extracellularly into the auditory nerve of adult mice so that the enzyme could infuse individual spiral ganglion neurons. Forty-two well-stained neurons were reconstructed through serial sections from their cell bodies to peripheral terminations in the organ of Corti with the aid of a light microscope and drawing tube. No neuron was observed to innervate both inner and outer hair cells (IHCs and OHCs). Previous observations from neonatal mammals that reported that IHCs and OHCs were innervated by the same neuron are thus presumed to describe a transient developmental phenomenon. Two populations of spiral ganglion neurons were determined on the basis of the differences in receptor innervation. The type I neurons innervated exclusively IHCs by way of thick (1-2 microns) radial fibers, whereas the type II neurons innervated only OHCs by way of thin (approximately 0.5 micron) outer spiral fibers. Certain features of the peripheral process in the vicinity of the cell body were highly correlated with fiber type. This pattern of separate innervation of IHCs and OHCs by type I and type II neurons, respectively, may represent the general plan of afferent organization for the adult mammalian cochlea.     

Longitudinal gradients of KCNQ4 expression in spiral ganglion and cochlear hair cells correlate with progressive hearing loss in DFNA2

Mutations in the human KCNQ4 gene were recently found by Kubisch et al. [Cell 96 (1999) 437-446] to cause a non-syndromic, autosomal dominant, progressive hearing loss, DFNA2. The mouse Kcnq4 orthologue was previously localized to the outer hair cells (OHCs) of the inner ear, suggesting the pathophysiological effects were due to dysfunctional OHCs. Yet, OHC dysfunction does not provide a plausible explanation for the progressive nature of the frequency specific hearing loss. We have re-examined and extended the expression analyses of KCNQ4 in the murine inner ear using RT-PCR and whole mount in situ hybridization. Our results confirmed that the rat KCNQ4 orthologue is expressed in both inner and outer hair cells. Reciprocal longitudinal gradients were found in inner hair cells (IHCs) and OHCs. The strongest expression of KCNQ4 in IHCc was in the base of the cochlea and in the apex for OHCs. Similar to the IHCs, a basal to apical gradient was present in the spiral sensory neurons. IHCs mediate hearing via their afferent sensory neurons, whereas OHCs function as active cochlear amplifiers. The complete absence of OHCs leads only to severe sensitivity reduction, but not complete hearing loss. Our data suggest that the primary defect leading to initial high frequency loss and subsequent progressive hearing loss for all frequencies may be due to spiral ganglion and/or IHC dysfunction, rather than an OHC aberration.     

Cochlear innervation in the developing rat: an immunocytochemical study of neurofilament and spectrin proteins

We have studied the innervation of the developing cochlea by immunocytochemical staining of the cytoskeletal proteins, neurofilament (NF), and spectrin (brain spectrin and erythrocyte spectrin). NF immunoreactivity was seen in spiral ganglion cell bodies and their processes and in fibers of the intraganglionic spiral bundle (IGSB) on gestational day 16. NF immunoreactivity with monoclonal antibodies to NF160 and NF68 was present beneath both inner hair cells (the IHC) and outer hair cells (OHCs) on gestational day 20. NF200 immunostaining was located only in the IGSB and in fibers reaching the IHC. The first NF200 immunoreactivity beneath the OHCs was seen in the basal turn at birth. NF labelling began to decrease on postnatal day 9 and its intensity became more like that of the adult. Brain spectrin immunostaining was first seen in the IGSB of the basal turn on gestational day 18. It reached the fibers between the spiral ganglion and the IHC on gestational day 20. Brain spectrin immunoreactivity was first seen beneath the OHCs in the basal turn at birth. It reached all the OHCs of the cochlea by postnatal day 4, and began to decrease 9 days after birth. Erythrocyte spectrin immunostaining was first observed during the second postnatal week, when it labelled spiral ganglion cells. The distribution of NF200 and brain spectrin immunoreactivity suggested that efferent innervation of OHCs is present at birth in the rat, and confirms previous studies showing the early efferent innervation of the OHCs of the mouse and the rat at birth, and the time lag between the appearance of the two spectrin isoforms during development.     

Time course of efferent fiber and spiral ganglion cell degeneration following complete hair cell loss in the chinchilla

Ethacrynic acid (EA) is known to interact with aminoglycoside antibiotics such as gentamicin (GM). In the chinchilla, co-administration of GM and EA can produce hair cell lesions ranging from a small loss of outer hair cells (OHCs) in the base of the cochlea to complete destruction of all hair cells, depending on dosing parameters. Although hair cell loss has been characterized, little is known about the fate of efferent fibers or spiral ganglion neurons (SGNs) in this model. To study the time course of efferent fiber and SGN loss, chinchillas were injected with GM (125 mg/kg IM) followed immediately by EA (40 mg/kg IV). Estimates of efferent fiber loss and density changes were made after 3 days or 1, 2, 3, or 4 weeks of survival. Estimates of SGN loss and density changes were made after 15 days or 1, 2, 4, or 6 months of survival. Cochlear function was rapidly abolished and all cochlear hair cells were missing within 24 h after treatment. Inner hair cells (IHCs) in the middle turn of the cochlea died earlier than cells in the apex or base, and OHCs in Rows 1 and 2 died earlier than OHCs in Row 3. Degeneration of efferent nerve fibers began 3-7 days post-injection, versus 15-30 days for SGNs, and the loss of efferent fibers was essentially complete within 1 month, versus 2-4 months for SGNs. The rapid time course of efferent fiber and SGN loss in the chinchilla may make it a practical model for studying mechanisms of neural loss and survival in the mammalian inner ear.     

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: Evidence for a waiting period

Reconstructions of the efferent innervation of the hamster (Mesocricetus auratus) cochlea were done during postnatal development. Efferent neurons were labeled via injections of biocytin and horseradish peroxidase into the crossed olivocochlear (OC) bundles using an in vitro brainstem technique. Such injections retrogradely labeled cell bodies in ventral periolivary regions of the superior olive consistent with their being medial OC neurons. Anterogradely labeled axons were traced to the cochlea, where they terminated on or below inner hair cells (IHCs) prior to postnatal day 5 (P5). After P5, labeled axons terminated on IHCs and outer hair cells (OHCs) and after P10, the majority of labeled axons terminated on the OHCs. In the electron microscope, small labeled terminals containing densely packed synaptic vesicles were found both adjacent to IHCs (axosomatic) as well as apposed to afferent and efferent fibers below IHCs prior to P5. By P10, large labeled terminals were axosomatic to OHCs and no longer found on IHCs. Consistent with previous reports, these data suggest that medial OC axons form part of an early primary innervation on and below IHCs before terminating on OHCs. This raises the possibility that OC neurons demonstrate a period of waiting below an intermediate target similar to that described in the development of thalamocortical projections. © 1996 Wiley-Liss, Inc.     

Identification and ultrastructural localization of a calretinin-like calcium-binding protein (protein 10) in the guinea pig and rat inner ear

Calretinin has been identified as a brain specific calcium-binding protein which appears as a prominent protein in the cochlear nucleus. We identified and localized calretinin in the guinea pig and rat inner ear using polyclonal antibodies. Immunoblot analyses of guinea pig and rat auditory nerve homogenates revealed an immunoreactive band migrating with the same molecular weight as the purified protein, atM_r = 29k. Immunocytochemistry was carried out at the light and electron microscope levels. In the guinea pig cochlea, inner hair cells, Deiters' cells, Hensen's cells and interdental cells of the spiral limbus were stained. Most of the cochlear ganglion cells were immunostained. In the guinea pig vestibular organs, the staining was exclusively neuronal and localized in large nerve fibers and nerve calices of the apex of the cristae. Only some vestibular ganglion cells were stained. In the rat cochlea, inner hair cells and most of the ganglion neurons were immunoreactive. In the rat vestibule, large nerve fibers and calices were stained as were some type II hairs cells. Only some vestibular ganglion cells were reactive. Electron microscopic observations of immunostained guinea pig cochlea and vestibule showed that the staining was cytosolic. In addition, specific sub-localization was also found in the apical portion of the nerve calices in association with microvesicles. These results describe the discrete localization of calretinin in the cochlea and in the vestibular receptors and suggest a function associated with biochemical regulations at the level of microvesicles in vestibular afferent neurons.     

Developmental segregation in the efferent projections to auditory hair cells in the gerbil

The auditory receptor epithelium of mammals receives efferent innervation from neurons within and surrounding the superior olivary complex of the brainstem (Warr [1975] J. Comp. Neurol. 161:159-181). Disruption of this pathway during early postnatal life, when olivocochlear axons are forming their final connections with auditory hair cells and nerve fibers, can lead to profound and permanent hearing impairments (Walsh et al. [1998] J. Neurosci. 18:3859-3869). Identification of the possible causes for this deterioration in auditory function requires a better understanding of the normal developmental interactions that occur between efferent axons and their target cells within the cochlea. To provide such information, we labeled developing efferent fibers at a constant location within the gerbil cochlea by using the fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI). The terminal arbors of these neurons were then reconstructed by using digital confocal microscopy. By postnatal day (P) 2, the efferent arbors associated with inner hair cells (IHCs) and outer hair cells (OHCs) displayed distinctly different morphologies closely resembling those described for adult animals (Brown [1987] J. Comp. Neurol. 260:605-619). Unlike their mature counterparts, however, P2 efferent axons frequently branched to contact both types of auditory hair cells. Unexpectedly, between P4 and P6, both IHC and OHC efferent axons produced additional branches that crossed the tunnel of Corti to invade the OHC zone. By P8, all of these supernumerary connections were eliminated, yielding completely segregated efferent pathways to IHCs and OHCs.     

Inner hair cells are not required for survival of spiral ganglion neurons in the adult cochlea

Studies of sensorineural hearing loss have long suggested that survival of spiral ganglion neurons (SGNs) depends on trophic support provided by their peripheral targets, the inner hair cells (IHCs): following ototoxic drugs or acoustic overexposure, IHC death is rapid whereas SGN degeneration is always delayed. However, recent noise-trauma studies show that SGNs can die even when hair cells survive, and transgenic mouse models show that supporting cell dysfunction can cause SGN degeneration in the absence of IHC pathology. To reexamine this issue, we studied a model of IHC loss that does not involve noise or ototoxic drugs. Mice lacking the gene for the high-affinity thiamine transporter (Slc19a2) have normal cochlear structure and function when fed a regular (thiamine-rich) diet. However, dietary thiamine restriction causes widespread, rapid (within 10 d) loss of IHCs. Using this model, we show that SGNs can survive for months after IHC loss, indicating that (1) IHCs are not necessary for neuronal survival, (2) neuronal loss in the other hearing loss models is likely due to effects of the trauma on the sensory neurons or other inner ear cells, and (3) that other cells, most likely supporting cells of the organ of Corti, are the main source of SGN survival factors. These results overturn a long-standing dogma in the study of sensorineural hearing loss and highlight the importance of cochlear supporting cells in neuronal survival in the adult inner ear.     

Spatiotemporal expression of TRPM4 in the mouse cochlea

The present study was conducted to elucidate the presence of the transient receptor potential cation channel subfamily M member 4, TRPM4, in the mouse inner ear. TRPM4 immunoreactivity (IR) was found in the cell body of inner hair cells (IHCs) in the organ of Corti in the apical side of marginal cells of the stria vascularis, in the apical portion of the dark cells of the vestibule, and in a subset of the type II neurons in the spiral ganglion. Subsequently, changes in the distribution and expression of TRPM4 in the inner ear during embryonic and postnatal developments were also evaluated. Immunohistochemical localization demonstrated that the emergence of the TRPM4‐IR in IHCs occurs shortly before the onset of hearing, whereas that in the marginal cells happens earlier, at the time of birth, coinciding with the onset of endolymph formation. Furthermore, semiquantitative real‐time PCR assay showed that expressions of TRPM4 in the organ of Corti and in the stria vascularis increased dramatically at the onset of hearing. Because TRPM4 is a Ca²⁺‐activated monovalent‐selective cation channel, these findings imply that TRPM4 contributes to potassium ion transport, essential for the signal transduction in IHCs and the formation of endolymph by marginal cells. © 2014 Wiley Periodicals, Inc.     

Developmental expression of Ca(v)1.3 (alpha1d) calcium channels in the mouse inner ear

Voltage-gated calcium channels are important for neurotransmission at the level of inner hair cells (IHCs) and outer hair cells (OHCs). These channels open when mechanical stimulation depolarises the hair cell membrane and the resulting calcium influx triggers neurotransmitter release. Voltage-gated calcium channels expressed in hair cells are known to be of the L-type with a predominance of the Ca(v)1.3 subunit. The present study describes the developmental expression of the Ca(v)1.3 protein in the cochlea and the vestibular system using immunohistochemical technique. In the adult organ of Corti (OC), Ca(v)1.3 was localized in both sensory and non-sensory cells with a more intense expression in IHCs and Deiters cells when compared to OHCs. In both hair cell types, immunoreactivity was observed in the apical pole, basolateral membrane and at the basal pole (synaptic zone). Similar results were obtained in the vestibular organs. During development, Ca(v)1.3 immunoreactivity was observed in the cochlea as early as embryonic day 15, with expression increasing at birth. At these early stages of cochlear development, Ca(v)1.3 was expressed in all cell types surrounding the scala media. In the OC, the labeling was observed in IHCs, OHCs and supporting cells. The Ca(v)1.3 expression reached an adult-like pattern by the end of the second postnatal week. The present findings suggested that, in addition to their implication in hair cells synaptic transmission, Ca(v)1.3 calcium channels also play an important role in vesicle recycling and transport, as suggested by their extrasynaptic location at the apical pole of the hair cells. The Ca(v)1.3 channels in Deiters cells could participate in active calcium-induced changes in micromechanics of these supporting cells. An early expression during development suggested that these calcium channels are in addition important in the development of the cochlear and vestibular sensory epithelium.     

Alpha-9 nicotinic acetylcholine receptor immunoreactivity in the rodent vestibular labyrinth

Vestibular tissues (cristae ampullares, macular otolithic organs, and Scarpa's ganglia) in chinchilla, rat, and guinea pig were examined for immunoreactivity to the alpha9 nicotinic acetylcholine receptor (nAChR) subunit. The alpha9 antibody was generated against a conserved peptide present in the intracellular loop of the predicted protein sequence of the guinea pig alpha9 nAChR subunit. In the vestibular periphery, staining was observed in calyces around type I hair cells, at the synaptic pole of type II hair cells, and in varying levels in Scarpa's ganglion cells. Ganglion cells were also triply labeled to detect alpha9, calretinin, and peripherin. Calretinin labels calyx-only afferents. Peripherin labels bouton-only afferents. Dimorphic afferents, which have both calyx and bouton endings, are not labeled by calretinin or peripherin. In these experiments, alpha9 was expressed in both calyx and dimorphic afferents. A subpopulation of small ganglion cells did not contain the alpha9 nAChR but did stain for peripherin. We surmise that these are bouton-only afferents. Bouton (regularly discharging) afferents also show efferent responses, although they are qualitatively different from those in irregularly discharging (calyx and dimorphic) afferents, much slower and longer lasting. Thus, regular afferents are probably more affected via a muscarinic cholinergic or a peptidergic mechanism, with a much smaller superimposed fast nicotinic-type response. This latter response could be due to one of the other nicotinic receptors that have been described in studies from other laboratories.     

Immunohistochemical localization of neurocalcin in the rat inner ear

Localization in the rat inner ear of neurocalcin, a three EF-hand calcium-binding protein, was examined immunohistochemically. Neurocalcin-like immunoreactivity was restricted to neurons in neuroepithelial receptor organs, while hair cells and supporting cells showed no such immunoreactivity. In the organ of Corti, both afferent and efferent nerve terminals, which formed synapses on both inner and outer hair cells, showed distinct immunoreactions. Spiral ganglion neurons and cochlear nerves were immunopositive. In the cristae ampullaris, macula utriculi and macula sacculi, afferent nerve terminals forming nerve calices or terminal boutons were strongly immunopositive. Efferent nerve terminals making synapses either on nerve calices or on hair cells showed an intense immunoreactivity. Vestibular ganglion neurons were strongly immunopositive. In electron microscopy, immunoreaction products were diffuse in the cytoplasm of ganglion neurons and nerve terminals. Neurocalcin-like immunoreactivity occurred in association with microtubules, outer mitochondrial membranes, synaptic vesicles and synaptic membranes. It is thus likely that neurocalcin is involved in neural functions in each type of afferent and efferent transmission in the inner ear.     

Immunohistochemical localization of adenosine 5'-triphosphate-gated ion channel P2X(2) receptor subunits in adult and developing rat cochlea

Substantial in vitro and in vivo data support a role for extracellular adenosine 5;-triphosphate (ATP) and associated P2 receptors in cochlear function. However, the precise spatiotemporal distribution of the involved receptor protein(s) has not been determined. By using a specific antiserum and immunoperoxidase labeling, the tissue distribution of the P2X(2) subunit of the ATP-gated ion channel was investigated. Here, we describe the first extensive immunohistochemical mapping of P2X(2) receptor subunits in the adult and developing rat cochlea. In the adult, immunoreactivity was observed in most cells bordering on the endolymphatic compartment (scala media), particularly in the supporting cells. Hair cells were not immunostained by the P2X(2) antiserum, except for outer hair cell stereocilia. In addition, weak immunolabeling was observed in some spiral ganglion neurons. P2X(2) receptor subunit protein expression during labyrinthine ontogeny was detected first on embryonic day 19 in the spiral ganglion and in associated nerve fibers extending to the inner hair cells. Immunostaining also was observed underneath outer hair cells, and, by postnatal day 6 (P6), intense immunolabeling was seen in the synaptic regions of both types of hair cell. Supporting cells of the sensory epithelium were labeled at P0. This labeling became most prominent from the onset of cochlear function (P8-P12). Conversely, expression in the vascular stria declined from this time. By P21, the pattern of immunolabeling was similar to that found in the adult. The localization and timing of P2X(2) immunoreactivity suggest involvement of extracellular ATP and associated ATP-gated ion channels in important physiological events, such as inner ear ontogeny, sound transduction, cochlear micromechanics, electrochemical homeostasis, and auditory neurotransmission.     

Outer Hair Cell Glutamate Signaling through Type II Spiral Ganglion Afferents Activates Neurons in the Cochlear Nucleus in Response to Nondamaging Sounds

Cochlear outer hair cells (OHCs) are known to uniquely participate in auditory processing through their electromotility, and like inner hair cells, are also capable of releasing vesicular glutamate onto spiral ganglion (SG) neurons: in this case, onto the sparse Type II SG neurons. However, unlike glutamate signaling at the inner hair cell-Type I SG neuron synapse, which is robust across a wide spectrum of sound intensities, glutamate signaling at the OHC-Type II SG neuron synapse is weaker and has been hypothesized to occur only at intense, possibly damaging sound levels. Here, we tested the ability of the OHC-Type II SG pathway to signal to the brain in response to moderate, nondamaging sound (80 dB SPL) as well as to intense sound (115 dB SPL). First, we determined the VGluTs associated with OHC signaling and then confirmed the loss of glutamatergic synaptic transmission from OHCs to Type II SG neurons in KO mice using dendritic patch-clamp recordings. Next, we generated genetic mouse lines in which vesicular glutamate release occurs selectively from OHCs, and then assessed c-Fos expression in the cochlear nucleus in response to sound. From these analyses, we show, for the first time, that glutamatergic signaling at the OHC-Type II SG neuron synapse is capable of activating cochlear nucleus neurons, even at moderate sound levels.SIGNIFICANCE STATEMENT Evidence suggests that cochlear outer hair cells (OHCs) release glutamate onto Type II spiral ganglion neurons only when exposed to loud sound, and that Type II neurons are activated by tissue damage. Knowing whether moderate level sound, without tissue damage, activates this pathway has functional implications for this fundamental auditory pathway. We first determined that OHCs rely largely on VGluT3 for synaptic glutamate release. We then used a genetically modified mouse line in which OHCs, but not inner hair cells, release vesicular glutamate to demonstrate that moderate sound exposure activates cochlear nucleus neurons via the OHC-Type II spiral ganglion pathway. Together, these data indicate that glutamate signaling at the OHC-Type II afferent synapse participates in auditory function at moderate sound levels.     

Response characteristics of mammalian cochlear hair cells

Intracellular recordings were made from the low frequency region (third turn) of the guinea pig cochlea. Response characteristics are compared to gross potentials obtained from the organ of Corti fluid space. Inner hair cells (IHCs) possess relatively low (median, -32 mV) initial membrane potentials, whereas that of outer hair cells (OHCs) is higher (median, -53.5 mV). In response to tone burst stimuli, both cell types produce a combination of AC and DC responses. The latter are depolarizing for IHCs but may be of either polarity for OHCs. In terms of their AC responses, IHCs are about 12 dB more sensitive than OHCs. At low sound levels these cells are more linear than high frequency hair cells (Russell, I. J., and P. M. Sellick (1978) J. Physiol. (Lond.) 284: 261-290), judging from the relation between AC and DC response components. At high sound levels pronounced response saturation is seen. The overall tuning properties of the two hair cell types are rather similar, even though IHCs exhibit low frequency velocity dependence, whereas OHCs are displacement sensitive and the cell membrane time constant is larger for IHCs. In order to fit IHC experimental data it is necessary to assume the presence of an underdamped complex pole above the best frequency. The electrical behavior of the OHC does not disqualify it as a conveyor of auditory information to the central nervous system, even though its primary function may be that of a mechanical effector (evidence summarized by Dallos, P. (1985) in Contemporary Sensory Neurobiology, Alan R. Liss, Inc., New York, pp. 207-230).     

Vagal and spinal afferent innervation of the rat esophagus: A combined retrograde tracing and immunocytochemical study with special emphasis on calcium-binding proteins

Vagal afferent neurons contain a variety of neurochemical markers and neuroactive substances, most of which are present also in dorsal root ganglion cells. To test for the suitability of the calcium-binding protein calretinin as a specific marker for vagal afferent fibers in the periphery, immunocytochemistry for this protein was combined with retrograde tracing. Nerve fibers in the rat esophagus, as well as vagal and spinal sensory neurons innervating the esophagus, were investigated for co-localization of calretinin with calbindin, calcitonin gene-related peptide, and NADPH diaphorase. The results indicated that calretinin immunocytochemistry demonstrates neuronal structures known as vagal afferent from other studies, in particular intraganglionic laminar endings. A few enteric neurons whose distribution was unrelated to intraganglionic laminar endings also stained for calretinin. Strikingly, calretinin immunoreactivity was absent from spinal afferent neurons innervating the rat esophagus. In intraganglionic laminar endings and nodose ganglion cells calretinin was highly co-localized with calbindin but not with calcitonin gene-related peptide. On the other hand, calbindin was also found in spinal afferents to the esophagus where it was co-localized with calcitonin gene-related peptide. Vagal afferent neurons innervating the esophagus were never positive for NADPH diaphorase. Thus, calretinin appears to be a more specific marker for vagal afferent structures in the esophagus than calbindin, which is expressed by both vagal and spinal sensory neurons. Calretinin immunocytochemistry may be utilized as a valuable tool for investigations of subpopulations of vagal afferents in certain viscera. J. Comp. Neurol. 398:289–307, 1998. © 1998 Wiley-Liss, Inc.     

Neuron-specific enolase during the development of the organ of Corti

NSE immunoreactivity has been studied in the organ of Corti of the developing mouse from birth to 21 days. NSE immunohistochemical stain is observed in spiral ganglion cells, in nerve fibers and in nerve endings of inner and outer hair cells, and in both populations of sensory cells. Spiral ganglion cells in lower and central parts of the ganglion stain for NSE at birth, but all nerve cells are stained by day 4. Radial and spiral fibers and the endings on inner hair cells stain at birth, but the nerve endings on outer hair cells develop NSE between days 3 and 6. The inner and outer hair cells are NSE-positive at day 2 but the NSE immunoreactivity in the outer hair cells decreases at the end of the second week until the cells become negative. The NSE stain in the neuronal pathways of the inner and outer hair cell regions increases for about 19 days, showing a predominant accumulation in neuronal endings. The data suggest that the development of NSE expression in the organ of Corti reflects the nascence and maturation of the synaptic contacts. Spiral neurons, their fibers and endings as well as inner and outer hair cells express NSE in the isolated organ of Corti in culture. Variability of stain among the different cell populations indicates a role of local factors in the regulation of NSE expression.     

Characterization of different neuron populations in mouse statoacoustic ganglion cultures

Statoacoustic ganglion (SAG) cells were grown in primary culture and the appearance of different neuronal phenotypes was investigated. Analysis criteria were shape, size and staining for the immunocytochemical markers: neurofilament proteins (NF-200 kDa), neuron-specific enolase (NSE), calretinin, a calcium-binding protein and substance P, a neurotransmitter. Cultures were prepared from dissociated SAG cells of 13 gestation-day-old mouse embryos. Neurons were identified and counted after 7 days in vitro. At this stage, neurons were organized in small clusters forming an extensive network of neurites grown on a layer of fibroblasts and glia. Most neurons identified by NF or NSE immunoreactivity showed a typical adult-like bipolar profile. The diameters of the neurons were between 5.62 and 17.00 μm and displayed a normal distribution (mean: 10.6 μm). Two distinct subpopulations were identified by the expression of calretinin and substance P. Calretinin-immunoreactive neurons were large and very rare and had a mean diameter of 11.3 μm; the distribution of substance P was more extensive than that of calretinin and identified a population of small neurons with a mean diameter of 8.9 μm. The distributions of these two markers in SAG cultures were consistent with in vivo results. In conclusion, dissociated SAG cell cultures appear to be a suitable model for analyzing the development of the immunocytochemical and functional characteristics of the neurons of this inner ear ganglio.     

Immunochemical localization of calretinin in the retina of the turbot (Psetta maxima) during development

The expression of the calcium-binding protein calretinin was analysed by immunohistochemistry techniques in the retina of turbot (Psetta maxima) from embryonic to juvenile stages. Calretinin immunoreactivity was first detected in retinae from newly hatched larvae, in which the anlage of the inner plexiform layer and a subset of amacrine and ganglion cells displayed a faint immunolabelling. First appearance of photoreceptors during larval life coincided with an increase in the intensity of the labelling. During subsequent larval development, the expression of calretinin affected distinctive retinal components. The inner plexiform layer, optic fiber layer, and a population of amacrine and ganglion cells were invariably labelled. Occasional bipolar cells were labelled at the end of the larval period. By metamorphosis, calretinin is sequentially expressed in horizontal cells, and bipolar immunoreactive cells become numerous. The pattern of calretinin immunoreactivity of the inner plexiform layer changes from the larval to juvenile period. In all cases, calretinin immunoreactivity exhibited variations between the peripheral retina, which contains the most recently differentiated retinal components, and the remainder of the differentiated retina. Our results suggest that the progressive expression of calretinin in the turbot retina appears associated with some degree of neuronal differentiation. Once the definitive pattern of calretinin immunoreactivity is established in the turbot retina, both similarities and differences with the calretinin location in the retina of other vertebrates can be demonstrated.