Jörgen Fex

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.

Glutamic acid decarboxylase immunoreactivity of olivocochlear neurons in the organ of Corti of guinea pig and rat

The distribution of glutamic acid decarboxylase (GAD)-like immunoreactivity in the organ of Corti of guinea pig and rat was studied under the light microscope. Indirect immunohistochemical techniques were used. Cochleae were first incubated with a specific antiserum to rat brain GAD and then stained through an avidin-biotin-horseradish peroxidase (HRP) procedure. GAD-like immunoreactivity was visualized as staining with HRP reaction product. Surface preparations were prepared from the immunoreacted cochleae. GAD-like immunoreactivity was found in the inner spiral bundle, tunnel spiral bundle, tunnel crossing fibers, outer hair cell synaptic regions and outer spiral bundles. Little staining was seen in the basal turn. Most of the immunoreactivity was seen in the third and lower fourth turn of the guinea pig cochlea, but even there many efferent fibers and endings were unstained. It is concluded that GAD-like immunoreactivity is present in a subpopulation of cochlear efferents that contains elements from both the medial and the lateral olivocochlear system. Future studies are needed to determine whether this subpopulation is GABA-ergic (i.e. uses gamma-aminobutyric acid as a neurotransmitter) and/or cholinergic.

GABA visualized by immunocytochemistry in the guinea pig cochlea in axons and endings of efferent neurons

Antiserum raised against GABA coupled with glutaraldehyde to bovine serum albumin was applied to the guinea pig cochlea. Immunoreactivity was visualized as horseradish peroxidase reaction product in surface preparations of the organ of Corti using immunocytochemical techniques. Bright-field, differential interference contrast and video-enhanced contrast light microscopy were used. GABA-like immunoreactivity was found in axons and endings of efferent neurons in all turns of the cochlear spiral, but predominantly in the third turn and first half of the fourth turn. In these apical turns, immunoreactivity was seen in the efferent components: inner spiral bundle, tunnel spiral bundle, tunnel-crossing fibers, large nerve endings synapsing on outer hair cell bases, nerve endings high up on outer hair cells, nerve endings or varicosities close to outer hair cells, and outer spiral fibers. Some immunoreactive large nerve endings at outer hair cells were found in the apical half of the fourth turn. This study shows that axons and endings of efferent neurons in the organ of Corti of guinea pig contain GABA-like immunoreactivity with a distribution similar to that of GAD-like immunoreactivity as shown in a previous study. In both studies, many efferent nerve axons and endings were unstained, even in regions of maximal density of immunoreactivity in the apical turns. The evidence indicates that a subpopulation of efferent neurons projecting to the organ of Corti is GABAergic and very likely different from the lateral and the medial olivocochlear efferent systems.

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.

Neurotransmitter-related immunocytochemistry of the organ of Corti.

The principles of immunocytochemistry were outlined in 1942 by Coons et al. and in the 1970s immunocytochemistry emerged as a powerful method for identifying structures and tracing pathways in the nervous system. It now plays a fundamental role in the neuroanatomical and histochemical analysis of the central nervous system. The first immunocytochemical studies of the mammalian cochlea were reported in 1980, from three different laboratories. Since then many studies on cochlear immunocytochemistry have been carried out, concerned with questions about neurotransmitter candidates or about structural proteins. This review describes immunoreactivity of enkephalin, choline acetyltransferase (ChAT), glutamate decarboxylase (GAD), gamma-aminobutyric acid (GABA), aspartate aminotransferase (AATase) and glutaminase (GLNase) in the organ of Corti. ChAT is the enzyme that catalyzes the synthesis of acetylcholine (ACh). GAD is the terminal enzyme in the biosynthesis of the inhibitory neurotransmitter GABA. AATase and GLNase are two enzymes involved in the metabolism of the excitatory neurotransmitter candidates aspartate and glutamate. We have much relied on surface preparations of the organ of Corti. We have also used cryostat sectioning of the cochlea, particularly when there was a need to apply a number of different antisera to comparable preparations from one and the same cochlea. We have used immunofluorescence and immunoperoxidase procedures. Immunoperoxidase procedures have given us better signal noise ratio for specific immunoreactivity (in surface preparations) than has immunofluorescence. Occasionally, to achieve maximal resolution of surface preparations in light microscopy studies, we have used enhanced contrast video display. We have found immunoreactivity in efferent fibers in the organ of Corti following the application of antisera to enkephalin, ChAT, GAD, GABA, AATase and GLNase. Most of these different antisera give different distributions of immunoreactivity and other antisera have evoked no immunoreactivity in the organ of Corti. To the best of our knowledge, the cells of origin of efferent axons and terminals in the organ of Corti are located in the brainstem. Originally described as crossed and uncrossed olivocochlear neurons, these efferents have recently been classified into a medial and a lateral system predominantly innervating, respectively, the outer hair cell region and the inner hair cell region. However, our findings on the distribution of GAD- and GABA-like immunoreactivity indicate that there may be more than two different systems of efferents in the organ of Corti, as previously suggested by Schwartz and Ryan (1983).

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.

Compartmentalized vesicular traffic around the hair cell cuticular plate

Through thin-section and freeze-fracture electron microscopy, we identify structural correlates of an intense vesicular traffic in a narrow band of cytoplasm around the cuticular plate of the bullfrog vestibular hair cells. Myriads of coated and uncoated vesicles associated with longitudinally oriented microtubules populate the narrow cytoplasmic region between the cuticular plate and the actin network of the apical junctional belt. If microtubules in the sensory hair cells, like those in axons, are pathways for organelle transport, then the characteristic distribution of microtubules around the cuticular plate represents transport pathways across the apical region of the hair cells. This compartmentalized membrane traffic system appears to support an intense vesicular release and uptake along a band of apical plasma membrane near the cell border. Functions of this transport system may include membrane recycling as well as exocytotic and endocytotic exchange between the hair cell cytoplasm and the endolymphatic compartment.

Cochlear disorder associated with melanocyte anomaly in mice with a transgenic insertional mutation

We have generated eight lines of transgenic mice containing mouse vasopressin-beta-galactosidase fusion constructs. One of these lines, VGA-9, harbors approximately 50 transgene copies at a single chromosomal site. When bred to transgene homozygosity, mice of this line showed a complete loss of skin pigmentation, microphthalmia, and cochlear abnormalities. The vascular stria of the cochlea was thin and deficient in melanin pigment which is normally produced by presumably neural crest-derived melanocytes. The marginal cells of the stria were thin and lacked basal infoldings. Degeneration of outer hair cells was also observed in homozygous mice, but this alteration may be secondary to the strial abnormalities. In contrast to homozygous VGA-9 mice, heterozygous VGA-9 mice were pigmented and appeared to have no anatomical alterations in either eye or cochlea. Since the integrated transgene provides a marker for cloning an endogenous gene necessary for normal pigmentation and proper development of the inner ear, the transgenic line VGA-9 may become valuable for the study of the molecular genetics of inner ear disorders associated with pigment abnormalities in both mice and humans.

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.

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.