High-resolution structure of hair-cell tip links.

Transduction-channel gating by hair cells apparently requires a gating spring, an elastic element that transmits force to the channels. To determine whether the gating spring is the tip link, a filament interconnecting two stereocilia along the axis of mechanical sensitivity, we examined the tip link's structure at high resolution by using rapid-freeze, deep-etch electron microscopy. We found that the tip link is a right-handed, coiled double filament that usually forks into two branches before contacting a taller stereocilium; at the other end, several short filaments extend to the tip link from the shorter stereocilium. The structure of the tip link suggests that it is either a helical polymer or a braided pair of filamentous macromolecules and is thus likely to be relatively stiff and inextensible. Such behavior is incompatible with the measured elasticity of the gating spring, suggesting that the gating spring instead lies in series with the helical segment of the tip link.

Structural basis for mechanical transduction in the frog vestibular sensory apparatus: I. The otolithic membrane.

The mechanical coupling of the otoliths to the hair cell sensory stereocilia at the surface of the vestibular sensory epithelium is mediated by two layers of extracellular matrix, each one with a specific role in the mechanical transduction process. The first is a rigid layer in direct contact with the otolithic mass and is known as the otolithic membrane or gelatin membrane. This structure consists of a dense, randomly cross linked filament network that uniformly distributes the force of inertia of the non-uniform otolithic mass to all stereocilia bundles. The second layer formed by a columnar organization of filaments secures the otolithic membrane above the surface of the epithelium. The long columnar filaments are organized in parallel to the stereocilia bundles and are anchored to the apical surface of the supporting cells. The zonula adherens at the apical region of each supporting cell displays a thick polygonal bundle of actin filaments forming at the surface of the epithelium a transcellular honeycomb organization that provides mechanical ground support for the columnar filament layer. The dominant aspect of this columnar filament layer indicates that it may also have an important role in attenuating the force of inertia of the large otolithic mass during acceleration, screening stresses that would be directed to an effective bending of the stereocilia bundles.

CGRP-like immunoreactivity in the guinea pig organ of Corti: a light and electron microscopy study.

We examined calcitonin-gene related peptide (CGRP)-like immunoreactivity in the guinea pig organ of Corti at both the light and electron microscope level using immunofluorescence and immunoperoxidase techniques. We observed strong CGRP-like immunoreactivity in the inner spiral bundle and tunnel spiral bundle in all turns at both the light and electron microscope level. CGRP immunostaining was localized exclusively in vesiculated efferent fibers. The majority of these fibers in the inner spiral bundle were immunoreactive. We observed immunostained synaptic junctions between two efferent fibers and between efferent varicosities and afferent dendrites. In the region of the outer hair cells (OHC), we found CGRP-like immunoreactivity in the efferent endings at the base of OHCs and at the level of the OHC nucleus. At the base of OHCs, CGRP reactive endings in the first, second and more rarely the third row. CGRP immunoreactivity was localized in large dense core vesicles. In summary, our data demonstrate that the neuropeptide, CGRP, was found in both the lateral and medial efferent systems. Secondly, the immunoreactivity was localized in large dense core vesicles.

Glycine receptor immunoreactivity in the ventral cochlear nucleus of the guinea pig.

Glycine appears to be a major inhibitory neurotransmitter in the cochlear nucleus. In order to determine more precisely the distribution of glycinergic synapses, we have studied the immunocytochemical distribution of the glycine postsynaptic receptor. Two monoclonal antibodies were used, Gly Rec Ab 2, which recognizes the 48kD polypeptide and Gly Rec Ab 7, which primarily recognizes the 93kD subunit of the glycine receptor complex. At the light microscopic level, glycine receptor immunoreactivity was found throughout the ventral cochlear nucleus with a punctuate distribution often found outlining large cell bodies. Indistinguishable patterns of staining were obtained with the two antibodies. Ultrastructural localization was done with Gly Rec Ab 7 because immunoreactivity remained after fixation with glutaraldehyde containing solutions. At the ultrastructural level, immunoreactivity was concentrated at postsynaptic sites on dendrites and cell bodies. In the anteroventral cochlear nucleus, neurons identified as spherical cells contained numerous inmunoreactive synapses on their cell bodies, whereas most immunoreactive synapses on stellate cells were on their proximal dendrites. In the posteroventral cochlear nucleus, neurons identified as octopus cells were immunoreactive on their cell bodies and proximal dendrites. In the granule cell layer, immunoreactivity was found only in the neuropile. Throughout the ventral cochlear nucleus, glycine receptor immunoreactivity was found postsynaptic to terminals containing flattened synaptic vesicles as well as those containing oval/pleomorphic synaptic vesicles.

Development of aspartate aminotransferase and glutaminase immunoreactivity in the rat auditory nerve.

Aspartate aminotransferase and glutaminase immunoreactive labeling of the auditory nerve has previously been reported. In the present study, the development of these immunoreactivities was examined in the auditory nerve of the rat, at ages ranging from 17 days gestation to four postnatal weeks. Cells and processes were examined in the cochlea, and fibers and terminals in the cochlear nucleus. In the cochlea, immunoreactive labeling with antisera to both enzymes was first seen at 20 gestational days, in spiral ganglion cells. It was not until two postnatal weeks, however, that this immunoreactive labeling was first seen in primary afferent terminals around spherical cells in the anteroventral cochlear nucleus. This correlates with the establishment of mature synaptic connections and function.

Ultrastructural localization of GABA-immunoreactive terminals in the anteroventral cochlear nucleus of the guinea pig.

The immunocytochemical distribution of gamma-aminobutyric acid (GABA) was studied by electron microscopy in the anteroventral cochlear nucleus (AVCN) of the guinea pig using affinity-purified antibodies made against GABA conjugated to bovine serum albumin. Our observations confirm that spherical cells are the predominant cell type in the guinea pig AVCN and receive numerous axosomatic contacts (Schwartz and Gulley, (1978) J. Anat. 153, 489-508). Stellate cells receive few axosomatic contacts. Electron microscopic immunocytochemistry shows that GABA immunoreactivity is present in synaptic terminals in the AVCN. Of the several classes of presynaptic terminals present in the AVCN as characterized by vesicle type (large round; oval/pleomorphic; flat; small round) only those containing oval/pleomorphic vesicles were GABA-immunoreactive. However, GABA immunoreactivity may not be present in all these terminals because some oval/pleomorphic terminals are unlabelled. Immunoreactive terminals are widespread in the AVCN; they are abundant on spherical cell bodies, rarely seen on stellate cell bodies and are also found scattered throughout the neuropile.

Immunocytochemical localization of GABA in the cochlear nucleus of the guinea pig.

The immunocytochemical distribution of gamma-aminobutyric acid (GABA) was determined in the cochlear nucleus of the guinea pig using affinity-purified antibodies made against GABA conjugated to bovine serum albumin. Light microscopic immunocytochemistry shows immunoreactive puncta, which appear to be GABA-positive presynaptic terminals, distributed throughout the cochlear nucleus. In the ventral cochlear nucleus, these puncta are often found around unlabeled neuronal cell bodies. While occasional labeled small cells are found in the ventral cochlear nucleus, most GABA-immunoreactive cell bodies are present in the superficial layers of the dorsal cochlear nucleus. Based on size and shape, immunoreactive cells in the dorsal cochlear nucleus are divided into 3 classes: medium round cells with diameters averaging 16 microns, small round cells with average diameters of 9 microns and small flattened cells with major and minor diameters averaging 11 and 6 microns, respectively. Labeled fusiform and granule cells are not seen. A similar distribution of label was seen using antibodies against glutamic acid decarboxylase. Electron microscopic immunocytochemistry of the anteroventral cochlear nucleus shows GABA immunoreactive boutons containing oval/pleomorphic synaptic vesicles on cell bodies and dendrites. Other major classes of terminals, including those with small round, large round and flattened synaptic vesicles are unlabeled.

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

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

Immunocytochemical localization of choline acetyltransferase-like immunoreactivity in the guinea pig cochlea.

The immunocytochemical localization of the enzyme choline acetyltransferase (ChAT) was examined in the guinea pig organ of Corti to determine if both lateral and medial systems of efferents would show immunoreactive labeling for this specific enzyme marker of cholinergic neurons. Cochleae were also examined after lesion of efferents to determine if ChAT-like immunoreactivity is confined to efferents. ChAT-like immunoreactivity was seen in the inner spiral bundle, tunnel spiral bundle and by the bases of inner hair cells corresponding to the lateral system of efferents. ChAT-like immunoreactivity was also seen in crossing fibers and puncta at the bases and by the nuclei of outer hair cells corresponding to the medial system of efferents. With the use of video enhanced contrast microscopy more than 9 ChAT-like immunoreactive puncta at the bases of outer hair cells could be resolved. In cochleae examined 6 weeks after ipsilateral lesion of efferents, no ChAT-like immunoreactivity was observed. These results add strong evidence that acetylcholine is a transmitter of both the medial and lateral systems of efferents.

Glutaminase-like immunoreactivity in the organ of Corti of guinea pig.

The distribution of glutaminase (GLNase)-like immunoreactivity (IR) in the normal and surgically de-efferented organ of Corti of guinea pig was studied. Primary antisera were against phosphate-dependent GLNase from rat kidney. Indirect immunocytochemical techniques were used; IR was visualized in cryostat sections through immunofluorescence, and through immunofluorescence or with horseradish peroxidase reaction product in surface preparations. Standard microscopy and video-enhanced light microscopy with asymmetric illumination contrast were used. GLNase-like IR was found at inner hair cells (IHCs) in the normal and in the de-efferented organ of Corti, in the tunnel spiral bundle, in tunnel-crossing fibers, in endings high up on outer hair cells (OHCs), in outer spiral bundles, in puncta close to OHCs, and in large, efferent endings at OHC bases. There was no GLNase-like IR at OHCs in the de-efferented organ of Corti. It is concluded that GLNase-like IR is present in auditory nerve dendrites at IHCs and in olivocochlear efferents of the medial system, and that future studies are needed to determine whether also the lateral system of olivocochlear efferents contains GLNase-like IR. A diagram is included depicting the relation between OHCs and efferent nerve endings along the cochlear spiral, showing that in the apicalmost 3/4 turn of the spiral OHCs have no efferent endings.

Enkephalin-like immunoreactivity in the guinea pig organ of Corti: ultrastructural and lesion studies.

Enkephalin-like immunoreactivity (ELI) was examined in a light and electron microscopic study of the normal guinea pig cochlea and of cochlea de-efferented through evulsion of the vestibular nerve. Antiserum to methionine enkephalin, 164, which gives immunoreactive labeling of only the lateral system of efferents, and antiserum 163, which gives immunoreactive labeling of lateral and medial efferents, were used. In de-efferented cochleae no immunoreactive labeling was seen with either antiserum, confirming that in the organ of Corti ELI is confined to efferents. At the ultrastructural level antiserum 163 but not 164 showed ELI in efferent terminals at the base of outer hair cells. ELI with 164 was seen in efferents ending on outer hair cells at the level of the nucleus. Medially, ELI was seen with both antisera in the inner and tunnel spiral bundles. Efferent terminals containing ELI were seen apposing afferent dendrites, other efferents and the inner hair cell. However, only rarely could synaptic contacts be unambiguously identified and then only with afferent dendrites.

Colocalization of enkephalin-like and choline acetyltransferase-like immunoreactivities in olivocochlear neurons of the guinea pig.

The guinea pig lateral superior olive was examined immunocytochemically using antisera against enkephalin and choline acetyltransferase sequentially on the same sections. A colocalization of choline acetyltransferase-like and enkephalin-like immunoreactivities was found in cells of the lateral superior olive that give rise to the lateral system of olivocochlear efferents. Only choline acetyltransferase-like immunoreactivity was observed in the group of olivary nuclei that give rise to the medial group of olivocochlear fibers.

Immunocytochemical localization of glutaminase-like immunoreactivity in the auditory nerve.

The immunocytochemical localization of glutaminase, which we have proposed as a marker for excitatory amino acid neurotransmitters was determined in the guinea pig auditory nerve. Glutaminase-like immunoreactivity was seen in auditory nerve terminals in the cochlear nucleus and in the cell bodies of the auditory nerve in the cochlea. This staining was seen in type I and not type II spiral ganglion cells. Glutaminase-like immunoreactivity was also observed in granule cells in the cochlear nucleus.

Localization of enkephalin-like immunoreactivity in acetylcholinesterase-positive cells in the guinea-pig lateral superior olivary complex that project to the cochlea.

Olivocochlear fibers have been demonstrated to have acetylcholinesterase-positive staining both in brainstem and cochlea. Olivocochlear fibres in the cochlea have also been determined to contain enkephalin-like immunoreactivity. In this study, we first determined the source of olivocochlear fibers in the guinea-pig using horseradish peroxidase and wheat germ agglutinin in retrograde transport studies. These cells were then examined for enkephalin-like immunoreactivity followed by acetylcholinesterase staining on the same sections to determine which cells and fibers showed staining for both. It was found that cells in the guinea-pig lateral superior olive that project to the cochlea have both enkephalin-like immunoreactivity staining and acetylcholinesterase-positive staining. Cells in other areas giving rise to olivocochlear fibers showed only acetylcholinesterase staining. These results suggest that there is co-localization of enkephalin and acetylcholine in a population of olivocochlear cells and fibers.

Postnatal development of spiral ganglion cells in the rat.

The postnatal development of the spiral ganglion in the albino rat was studied using light and electron microscopy. The morphological characteristics distinguishing type 1 from type 2 spiral ganglion cells were defined, and the critical period for distinguishing the two types of neurons was identified. At birth, the spiral ganglion consists of a homogeneous population of small, densely packed, spherical cells that have large cytoplasmic-to-nuclear ratios. During the first postnatal week, the cells mature slowly. At this period the myelin sheath around the cell bodies generally consists of only a few layers of loose myelin. Long glial fingers extend around the cell processes and soma, particularly the filopodial extensions of the somatic membrane. Type 2 spiral ganglion cells can be distinguished at postnatal day 8. Viewed with the phase-contrast microscope these cells are smaller and have more darkly staining nuclei and more lightly staining cytoplasm than the type 1 cells. The most characteristic ultrastructural features of the type 2 neurons are the densely packed neurofilaments in the cytoplasm and lack of compact myelin around the cell soma. By day 14, spiral ganglion cells are morphologically mature, although the myelin sheath continues to thicken. The results are discussed in relation to the electrophysiological development of the auditory system and the morphological maturation of the organ of Corti.

Aspartate aminotransferase immunoreactivity in cochlea of guinea pig.

The distribution of aspartate aminotransferase-like immunoreactivity in the cochlea of the guinea pig was studied at the light microscopy level. Indirect immunofluorescence histochemistry using antisera against cytoplasmic aspartate aminotransferase prepared from pig heart was applied to surface preparations of the organ of Corti and cryostat sections of the cochlea. In the modiolus, immunofluorescence was localized to spiral ganglion cells and myelinated fibers of the auditory nerve and intraganglionic spiral bundles. In the organ of Corti, immunofluorescence was seen in upper tunnel crossing fibers and at the base of outer hair cells, following a distribution similar to that of the efferent innervation of the outer hair cells. Weak immunofluorescence was seen in the inner spiral bundle and tunnel spiral bundle, but was not present in all preparations. Immunofluorescence was not seen in inner hair cells, nor at the base of inner hair cells, and may have been absent from outer hair cells. It is concluded that spiral ganglion cells and myelinated auditory nerve axons contain aspartate aminotransferase-like immunoreactivity such immunoreactivity has previously been determined in auditory nerve endings inthe cochlear nucleus. Olivocochlear neurons that innervate outer hair cells also contain such immunoreactivity while other cochlear efferents contain little or none.

Immunocytochemical localization of enkephalin-like immunoreactivity in the retina of the guinea pig.

The distribution of enkephalin-like immunoreactivity in the retina of the guinea pig was studied. Indirect immunofluorescence techniques were used on retinae with and without colchicine pretreatment. In retinae not receiving colchicine pretreatment, enkephalin-like immunoreactivity was seen in fibers in the inner plexiform layer, predominantly in laminae 1, 3, and 5. In colchicine-pretreated retinae, enkephalin immunofluorescent cell bodies were seen in the inner margin of the inner nuclear layer in addition to the immunoreactive fibers. These cells showed morphological characteristics of amacrine cells. No enkephalin-like immunofluorescence was seen in the optic nerve, ganglion cell layer, or outer nuclear or plexiform layers. These findings of enkephalin-like immunoreactive cells and fibers in a mammalian retina add to the findings in nonmammalian retinae and suggest that the enkephalins play a role in primary sensory systems of mammalian and nonmammalian species.