Morphology of labeled efferent fibers in the guinea pig cochlea

Efferent axons to the guinea pig cochlea were labeled by extracellular injections of horseradish peroxidase into the intraganglionic spiral bundle within the spiral ganglion. The terminal fibers formed by these axons were classified according to their patterns of termination within the basal turn of the cochlea. A class of terminal fibers designated "autonomic" forms a highly branched plexus in the osseous spiral lamina but does not enter the organ of Corti. The termination of single autonomics includes blood vessels as well as areas of the osseous spiral lamina not adjacent to blood vessels. Two major classes of efferent axons from the olivocochlear bundle enter the cochlea by way of the vestibulocochlear anastomosis and terminate either in areas near inner hair cells (IHC efferents) or onto outer hair cells (OHC efferents). The IHC efferents have thin axons throughout their course within the cochlea and can be divided into two subclasses. The most numerous subclass of IHC efferents (unidirectional) enters the inner spiral bundle and turns to spiral in only one direction for less than 1 mm and then forms a discrete termination including many en passant and terminal swellings that are within both the inner and tunnel spiral bundles. A less common subclass of IHC efferents (bidirectional) bifurcates upon entry into the inner spiral bundle to send branches both apically and basally. These terminal fibers take spiral courses that are greater than 1 mm in extent, often course in the tunnel spiral bundle for a large portion of the spiral, and form terminals throughout their extended spiral course. None of the IHC efferent fibers send branches to cross the tunnel to innervate the outer hair cells. A second major class of olivocochlear fibers, OHC efferent fibers, forms large boutons on the outer hair cells, and although they sometimes spiral beneath the IHCs for some length, they do not give off terminals to this region. The OHC efferent axons are thick and myelinated as they enter the cochlea, and they branch near the spiral ganglion to form several terminal fibers. Some of these terminal fibers are thin as they travel from the intraganglionic spiral bundle across the osseous spiral lamina to the organ of Corti, whereas others are thick and obviously myelinated as far peripheral as the habenula.(ABSTRACT TRUNCATED AT 400 WORDS)     

A study of cochlear innervation patterns in cats and rats with the Golgi method and Nomarski optics

Cochlear innervation patterns were studied in infant cats and rats with the rapid Golgi method. Examination of thick serial sections and surface preparations with the differential interference contrast microscope (Nomarski optics) allowed direct visualization of individually impregnated spiral ganglion cells, complete with their peripheral processes and endings in the organ of Corti. Individually impregnated efferent fibers could be recognized as heavily varicose axons that project radially to endings beneath inner and outer hair cells after taking a tangential course in the intraganglionic spiral bundle. It was often possible to visualize unimpregnated hair cells in contact with the impregnated endings of both types of fibers. There are at least two types of spiral ganglion cells in the cochlea of the infant cat and rat. One type innervates only inner hair cells by means of radial fibers. These ganglion cells constitute the overwhelming majority of ganglion cells impregnated in our preparations, and each cell typically innervates two inner hair cells. Hence, these ganglion cells establish nearly “point-to-point” connections between the auditory nerve and the organ of Corti. The other type of ganglion cell innervates outer hair cells by means of long spiral fibers; each cell typically innervates many outer hair cells through the numerous angular enlargements and short end branches of its spiral fiber. In addition, a few of these spiral fibers also send branches to inner hair cells by means of short collaterals; it remains to be seen if such fibers also occur in mature cochleas. Efferent fibers have been traced to inner and outer hair cell regions. The simplest pattern is formed by fine beaded axons with only a few branches ending mainly beneath inner hair cells. More complex patterns are formed by larger axons with many branches ending beneath inner or outer hair cells. Many efferent fibers send branches to both inner and outer hair cells. Electrophysiological studies so far have not demonstrated different populations of units that clearly correspond to the spiral and radial fibers. Therefore, the physiological differences between inner and outer hair cell innervation remain undefined.     

The combined effects of trkB and trkC mutations on the innervation of the inner ear

Previous research has demonstrated that only the two neurotrophins and their cognate receptors are necessary for the support of the inner ear innervation. However, detailed analyses of patterns of innervation in various combinations of neurotrophin receptor mutants are lacking. We provide here such an analysis of the distribution of afferent and efferent fibers to the ear in various combinations of neurotrophin receptor mutants using the lipophilic tracer DiI. In the vestibular system, trkC^+/− heterozygosity aggravates the trkB^−/− mutation effect and causes almost complete loss of vestibular neurons. In the cochlea innervation, various mutations are each characterized by specific topological absence of spiral neurons in Rosenthals canal of the cochlea. trkC^−/− mutation alone or in combination with trkB^+/− heterozygosity causes absence of all basal turn spiral neurons and afferent fibers extend from the middle turn to the basal turn along inner hair cells with little or no contribution to outer hair cells. Both types of basal turn spiral neurons appear to develop and project via radial fibers to inner and, more sparingly, outer hair cells. Simple trkB^−/− mutations show a reduction of fibers to outer hair cells in the apex and, less obvious, in the basal turn. Basal turn spiral neurons may be the only neurons present at birth in the cochlea of a trkB^−/− mutant mouse combined with trkC^+/− heterozygosity. In addition, the trkB^−/− mutation combined with trkC^+/− heterozygosity has a patchy and variable loss of middle turn spiral neurons in mice of different litters. Comparisons of patterns of innervation of afferent and efferent fibers show a striking similarity of absence of fibers to topologically corresponding areas. For example, in trkC^−/− mutants afferents reach the basal turn, spiraling along the cochlea, rather than through radial fibers and efferent fibers follow the same pathway rather than emanating from intraganglionic spiral fibers. The data presented suggest that there are regional specific effects with some bias towards a specific spiral ganglion type : trkC is essential for support of basal turn spiral neurons whereas trkB appears to be more important for middle and apical turn spiral neurons.     

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.     

Innervation of supporting cells in the apical turns of the guinea pig cochlea is from type II afferent fibers

The outer supporting cells in the apical turns of the guinea pig cochlea receive a dense innervation. Our previous study (Fechner et al. [1998] J. Comp. Neurol. 400:299-300) suggested that this innervation of the Deiters' and Hensen's supporting cells was not derived from efferent fibers of the olivocochlear bundle, but its origin has not been further specified. To test the hypothesis that the innervation was afferent in origin, we traced apical afferent fibers that were retrogradely labeled by extracellular injections of horseradish peroxidase. Labeled afferent fibers were of two types: type I fibers contacted inner hair cells, whereas type II fibers crossed the tunnel and contacted outer hair cells. Significantly, most of the type II fibers also formed branches to the outer supporting cells. Although a few olivocochlear efferent fibers formed such branches, counts indicated that the overwhelming majority of the branches were produced by type II afferent fibers. These branches were not produced by basal type II fibers. Apical type II fibers also differed from basal fibers by having shorter lengths, spiraling both apically and basally, and contacting all three rows of outer hair cells. These innervation differences suggest differences in the ways that information from outer hair cells is processed in the apex versus the base of the cochlea.     

Synaptic arrangements between inner hair cells and tunnel fibers in the mouse cochlea

Hair cells, the sensory cells of the organ of Corti, receive afferent innervation from the spiral ganglion neurons and efferent innervation from the superior olivary complex. The inner and outer hair cells are innervated by distinctive fiber systems. Our electron microscopical studies demonstrate, however, that inner hair cells, in addition to their own innervation, are also synaptically engaged with the fibers destined specifically to innervate outer hair cells, within both the afferent and efferent innervation. Serial sections of the afferent tunnel fibers (destined to innervate outer hair cells) in the apical turn demonstrate that, while crossing toward the tunnel of Corti, they receive en passant synapses from inner hair cells. Each inner hair cell (in a series of five in the apical turn) was innervated by two tunnel fibers, one on each side. We show here for the first time that, in the adult, the afferent tunnel fibers receive a ribbon synapse from inner hair cells and form reciprocal contacts on their spines. Vesiculated efferent fibers from the inner pillar bundle (which carries the innervation to outer hair cells) form triadic synapses with inner hair cells and their synaptic afferent dendrites; the vesiculated terminals of the lateral olivocochlear fibers from the inner spiral bundle synapse extensively on the afferent tunnel fibers, forming triadic synapses with both afferent tunnel fibers and their synaptic inner hair cells. This intense synaptic activity involving inner hair cells and both afferent and efferent tunnel fibers, at their crossroad, implies functional connections between both inner and outer hair cells in the process of hearing.     

Unmyelinated axons of the auditory nerve in cats.

This paper describes some central terminations of type II spiral ganglion neurons as labeled by extracellular injections of horseradish peroxidase (HRP) into the auditory nerve of cats. After histological processing with diaminobenzidine, both thick (2-4 microns) and thin (0.5 microns) fibers of the auditory nerve were stained. Whenever traced, thick fibers always originated from type I spiral ganglion neurons and thin fibers always from type II ganglion neurons. Because the labeling of type II axons faded as fibers projected into the cochlear nucleus, this report is limited to regions of the ventral cochlear nucleus near the auditory nerve root. The central axons of type II neurons are unmyelinated, have simple yet variable branching patterns in the cochlear nucleus, and form both en passant and terminal swellings. Under the light microscope, most swellings are located in the neuropil but they are also found in the vicinity of cell bodies, nodes of Ranvier of type I axons, and blood vessels. Eighteen en passant swellings in the neuropil were located by light microscopy and resectioned for electron microscopy; two of these swellings exhibited ultrastructural features characteristic of chemical synapses. The data indicate that inputs from outer hair cells might be able to influence auditory processing in the cochlear nucleus through type II primary neurons.     

Collaterals from lateral and medial olivocochlear efferent neurons innervate different regions of the cochlear nucleus and adjacent brainstem.

Two populations of superior olivary neurons which project to different sensory cell regions in the cochlea also give off collateral projections to the ventral cochlear nucleus (VCN) and adjacent brainstem. To determine whether these VCN projections also have different targets they were characterized by selective retrograde amino acid transport. Retrograde transport of 3H-d-aspartate (D-ASP) selectively labeled the unmyelinated fibers and neurons of the lateral olivocochlear (OC) system including a dense collateral projection to the central VCN. Retrograde transport of 3H-nipecotic acid (NIP) labeled the myelinated fibers and neurons of the medial OC system, including collateral projections to the peripheral VCN, subpeduncular granule cells, and nucleus Y. Medial and lateral OC efferent collaterals thus innervate different regions of the CN. Lateral system collaterals overlap extensively with Type I spiral ganglion cell afferent input. They are well positioned to play a role in modulating afferent input to the central auditory system, as is the primary projection of these efferents to the cochlea. The medial system collaterals project near the recently described afferent projections of Type II spiral ganglion cells. The medial system collaterals may therefore be related to the function of outer hair cells, as the medial system primary axons appear to be in the cochlea.     

Immunocytochemical Localization of a High-affinity Glutamate-Aspartate Transporter, GLAST, in the Rat and Guinea-pig Cochlea

Glutamate transporters play an important role in the reuptake of glutamate after its release from glutamatergic synapses. Four such transporters have so far been cloned from the rat brain. One, the glutamate-aspartate transporter GLAST, has been detected in the mammalian cochlea, in which the principal afferent synapse of the auditory nerve, between the inner hair cells and neurites of type I spiral ganglion neurons, has been suggested to be glutamatergic. The distribution of GLAST was therefore investigated to provide clues to the handling of glutamate in the cochlea. This was studied using light and electron microscopic immunocytochemistry in rats and guinea pigs with antibodies raised against synthetic peptides based on the sequence for GLAST. Significant immunoreactivity was found in the myelin sheath formed by satellite cells surrounding the type I spiral ganglion neurons, and along the plasma membranes of supporting cells around the inner hair cells; other cells in both locations were only weakly labelled, if at all. The absence of substantial numbers of synapses in the spiral ganglion suggests that GLAST is unlikely to be associated with the uptake of synaptic glutamate after release in this region. lmmunoreactivity associated with the inner hair cells is consistent with the utilization of glutamate at the afferent synapse.     

NADPH-diaphorase histochemistry reveals an autonomic-like innervation in the postnatal hamster cochlea.

Previous studies used nicotinamide adenine diphosphate (NADPH)-diaphorase histochemistry as an indicator of nitric oxide synthase (NOS) expression in the adult mammalian cochlea. In this study, we investigated the early postnatal expression of diaphorase activity in the hamster cochlea. Two types of extrinsic fibers were intensely labeled as early as postnatal day 3 (P3) in the portion of the cochlear nerve that innervates the base of the modiolus. By P10, these fibers had reached the spiral ganglion and were projecting toward the organ of Corti. The perivascular type of fiber did not project into the organ of Corti; however, the nonperivascular type could be traced among the supporting cells below the outer hair cells. Spiral ganglion cell somata were also labeled as early as P3. The onset of diaphorase expression in the spiral ganglion cells corresponds to a critical period of synaptogenesis for these sensorineural cells. If NADPH-diaphorase activity is an indicator of NOS, then our results suggest that NO may play a role during postnatal cochlear development.     

Immunohistochemical localization of adrenergic receptors in the rat organ of corti and spiral ganglion

Alpha(1)-, beta(1)-, and beta(2)-adrenergic receptors (ARs), which mediate responses to adrenergic input, have been immunohistochemically identified within the organ of Corti and spiral ganglion with polyclonal antibodies of established specificity. Alpha(1)-AR was immunolocalized to sites overlapping supranuclear regions of inner hair cells as well as to nerve fibers approaching the base of inner hair cells, most evident in the basal cochlear turn. A similar preponderance across cochlear turns for alpha(1)-AR in afferent cell bodies in the spiral ganglion pointed to type I afferent dendrites as a possible neural source of alpha(1)-AR beneath the inner hair cell. Foci of immunoreactivity for alpha(1)-AR, putatively neural, were found overlapping supranuclear and basal sites of outer hair cells for all turns. Beta(1)- and beta(2)-ARs were immunolocalized to sites overlapping apical and basal poles of the inner and outer hair cells, putatively neural in part, with immunoreactive nerve fibers observed passing through the habenula perforata. Beta(1)- and beta(2)-ARs were also detected in the cell bodies of Deiters' and Hensen's cells. Within the spiral ganglion, beta(1)- and beta(2)-ARs were immunolocalized to afferent cell bodies, with highest expression in the basal cochlear turn, constituting one possible neural source of receptors within the organ of Corti, specifically on type I afferent dendrites. Beta(1)- and beta(2)-ARs in Hensen's and Deiters' cells would couple to Galphas, known to be present specifically in the supporting cells. Overall, adrenergic modulation of neural/supporting cell function within the organ of Corti represents a newly considered mechanism for modifying afferent signaling.     

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.     

The development of vestibulocochlear efferents and cochlear afferents in mice

We have reinvestigated the embryonic development of the vestibulocochlear system in mice using anterograde and retrograde tracing techniques. Our studies reveal that rhombomeres 4 and 5 include five motor neuron populations. One of these, the abducens nucleus, will not be dealt with here. Rhombomere 4 gives rise to three of the remaining populations: the facial branchial motor neurons; the vestibular efferents; and the cochlear efferents. The migration of the facial branchial motor neurons away from the otic efferents is completed by 13.5 days post coitum (dpc). Subsequently the otic efferents separate into the vestibular and cochlear efferents, and complete their migration by 14.5 dpc. In addition to their common origin, all three populations have perikarya that migrate via translocation through secondary processes, form a continuous column upon completion of their migrations, and form axonal tracts that run in the internal facial germ. Some otic efferent axons travel with the facial branchial motor nerve from the internal facial genu and exit the brain with that nerve. These data suggest that facial branchial motor neurons and otic efferents are derived from a common precursor population and use similar cues for pathway recognition within the brain. In contrast, rhombomere 5 gives rise to the fourth population to be considered here, the superior salivatory nucleus, a visceral motor neuron group. Other differences between this group and those derived from rhombomere 4 include perikaryal migration as a result of translocation first through primary processes and only then through secondary processes, a final location lateral to the branchial motor/otic efferent column, and axonal tracts that are completely segregated from those of the facial branchial and otic efferents throughout their course inside the brain. Analysis of the peripheral distribution of the cochlear efferents and afferents show that efferents reach the spiral ganglion at 12.5 dpc when postmitotic ganglion cells are migrating away from the cochlear anlage. The efferents begin to form the intraganglionic spiral bundle by 14.5 dpc and the inner spiral bundle by 16.5 dpc in the basal turn. They have extensive collaterals among supporting cells of the greater epithelial ridge from 16.5 dpc onwards. Afferents and efferents in the basal turn of the cochlea extend through all three rows of outer hair cells by 18.5 dpc. Selective labeling of afferent fibers at 20.5 dpc (postnatal day 1) shows that although some afferents are still in early developmental stages, some type II spiral ganglion cells already extend for long distances along the outer hair cells, and some type I spiral ganglion cells end on a single inner hair cell. These data support previous evidence that in mice the early outgrowth of afferent and efferent fibers is essentially achieved by birth.     

Developmental regulation of neuron-specific P2X3 receptor expression in the rat cochlea

ATP-gated ion channels assembled from P2X3 receptor (P2X3R) subunits contribute to neurotransmission and neurotrophic signaling, associated with neurite development and synaptogenesis, particularly in peripheral sensory neurons. Here, P2X3R expression was characterized in the rat cochlea from embryonic day 16 (E16) to adult (P49-56), using RT-PCR and immunohistochemistry. P2X3R mRNA was strongly expressed in the cochlea prior to birth, declined to a minimal level at P14, and was absent in adult tissue. P2X3R protein expression was confined to spiral ganglion neurons (SGN) within Rosenthal's canal of the cochlea. At E16, immunolabeling was detected in the SGN neurites, but not the distal neurite projection within the developing sensory epithelium (greater epithelial ridge). From E18, the immunolabeling was observed in the peripheral neurites innervating the inner hair cells but was reduced by P6. However, from P2-8, immunolabeling of the SGN neurites extended to include the outer spiral bundle fiber tract beneath the outer hair cells. This labeling of type II SGN afferent fiber declined after P8. By P14, all synaptic terminal immunolabeling in the organ of Corti was absent, and SGN cell body labeling was minimal. In adult cochlear tissue, P2X3R immunolabeling was not detected. Noise exposure did not induce P2X3R expression in the adult cochlea. These data indicate that ATP-gated ion channels incorporating P2X3R subunit expression are specifically targeted to the afferent terminals just prior to the onset of hearing, and likely contribute to the neurotrophic signaling which establishes functional auditory neurotransmission.     

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.     

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.     

Ultrastructural evaluation of calcitonin gene-related peptide immunoreactivity in the human cochlea and vestibular endorgans

Calcitonin gene-related peptide (CGRP) is a neuropeptide widely distributed in the peripheral and central nervous system. Demonstrated in the efferent systems of the mammalian cochlea and vestibule, immunoreactive patterns of CGRP may vary by species. There is, however, no information in the literature investigating CGRP localization in the human cochlea. In the present study, the ultrastructural localization of CGRP immunoreactivity was evaluated in the human inner ear with immunoelectron microscopy. It was found that, in human cochlea, CGRP immunoreactivity was located in unmyelinated nerve fibres of the spiral lamina, inner spiral fibres beneath inner hair cells, tunnel spiral fibres, tunnel crossing fibres and outer radial fibres. In endorgans of human vestibule, CGRP immunoreactivity was located in vesiculated nerve fibres and bouton-type nerve terminals which were seen to contact afferent nerve chalices surrounding type I sensory cells and afferent nerve fibres, or to form an en passant contact with afferent dendrites. CGRP immunoreactivity appeared to be confined to efferent systems in all cases. This study presents evidence that CGRP could serve a role in neurotransmission or neuroregulation in both cochlear and vestibular efferent systems of human.     

Peripherin-like immunoreactivity in type II spiral ganglion cell body and projections

Peripherin, an intermediate filament protein, is present in neuronal subpopulations of both peripheral and central nervous systems. The distribution of peripherin was studied in the adult rat cochlea using immunohistochemistry on whole mount material, in cryostat sections and sections of plastic embedded tissue. In the spiral ganglion, peripherin labeling was restricted to the perikarya of a subpopulation of neurons and their peripheral and central processes. Peripherin positive neurons had the following features: (i) they have a large eccentric nucleus, they were often found in a cluster of 2 or 3 cells, (ii) they were often located near the intraganglionic spiral bundle fibers, (iii) they represented roughly 8% of the whole ganglion population and (iv) on the average they had smaller perikarya than non-immunoreactive cells. Immunostaining on semithin plastic sections revealed positive reactivity on Type II ganglion cells, while Type I neurons were negative. Double labeling using peripherin and three neurofilament (NF) subunit antibodies confirmed the presence of both markers within the same spiral ganglion cell type. Type II neurons have been previously documented as the only subpopulation of the spiral ganglion that presents a strong positive NF immunoreactivity within their perikarya. In the organ of Corti, peripherin-positive fibers formed bundles that course beneath the outer hair cells and send branches that end as boutons contacting the outer hair cells. All these characteristics suggest that peripherin-positive cells are Type II neurons, and that peripherin constitutes a reliable marker for this spiral ganglion subpopulation, as well as their peripheral and central processes.     

Tenascin-C in the cochlea of the developing mouse

Tenascin-C is a glycoprotein of the extracellular matrix that acts in vitro as both a permissive and a nonpermissive substrate for neurite growth. We analyzed, by immunocytochemistry, the distribution of tenascin-C along neural growth pathways in the developing mouse cochlea. In the spiral lamina, tenascin-C coexists in a region where nerve bundles arborize. In the organ of Corti, tenascin-C lines the neural pathways along pillar and Deiters' cells before and during the time of nerve fiber ingrowth. By embryonic day 16, tenascin-C is abundant on the pillar side of the inner hair cell but does not accumulate on the modiolar side until about birth, a time after the arrival of afferent fibers. The synaptic zones beneath outer hair cells are strongly labeled during the time when early events in afferent synaptogenesis are progressing but not during the time of efferent synaptogenesis. At the age when most neural growth ceases, tenascin-C immunoreactivity disappears. Faint tenascin-C immunolabeling of normal hair cells, strong tenascin immunolabeling in pathological hair cells of Bronx waltzer (bv/bv) mice, and staining for beta-galactosidase, whose gene replaces tenascin in a "knockout" mouse, indicate that hair cells supply at least part of the tenascin-C. The changing composition of the extracellular matrix in the synaptic region during afferent and efferent synaptogenesis is consistent with a role for tenascin in synaptogenesis. The presence of tenascin-C along the growth routes of nerve fibers, particularly toward the outer hair cells, raises the possibility that growth cone interactions with tenascin-C helps to guide nerve fibers in the cochlea.     

Topographic organization of the central projections of the spiral ganglion in cats

The morphological organization of inputs from restricted sectors of the cat cochlear spiral ganglion into the cochlear nucleus was studied by making focal extracellular injections of horseradish peroxidase (HRP) into the spiral ganglion. Injections resulted in Golgi-like labeling of a small cluster of spiral ganglion cells and their peripheral and central axons. Large injections involved most of the cells within Rosenthal's canal in sectors of the spiral ganglion innervating greater than or equal to 1 mm of the basilar membrane and resulted in narrow, complete laminae of labeled axons and preterminal fields within each cochlear nucleus subdivision. The positions of these bands were consistent with the "isofrequency laminae" appropriate for the frequencies represented at the injection sites, with high frequency laminae situated more dorsally, and lower frequencies progressively more ventral. A discrete projection to the small cell cap area was observed that was discontinuous with the main projection laminae in the ventral cochlear nuclei (VCN). In the dorsal cochlear nucleus, projecting fibers and terminals were excluded from the molecular cell layer. No labeled fibers entered the granule cell areas. In contrast to larger injections, very small HRP deposits labeled only part of an isofrequency lamina. Specifically, injections restricted to the scala tympani aspect of the spiral ganglion labeled only the lateral part of VCN isofrequency laminae, whereas injections limited to the scala vestibuli aspect of the ganglion labeled the medial aspect of the isofrequency planes. Thus these data indicate a previously unrecognized topographic representation of the vertical dimension of the spiral ganglion across VCN isofrequency laminae. Some possible functional implications of this projection organization are discussed.