Jaime A. Merchan

The effects of kainic acid on the cochlear ganglion of the rat

The effects of locally applied kainic acid on cells and fibers in the rat cochlea were examined in a quantitative and ultrastructural study. Doses of 5 nM per microliter of artificial perilymph destroyed part of the spiral ganglion type I cell population, with no ototoxic effects on cochlear hair cells or supporting cells. Type II cells also appeared unaffected. A quantitative evaluation of the cell loss with the 5 nM dosage showed that 34% of spiral ganglion neurons were lost 10 days after treatment. Doses of 20 nM per microliters and 40 nM per microliters did not result in increasing neuronal loss. This differential toxicity could reflect the presence of a sub-population of spiral ganglion cells with an increased number of KA receptors.

Neuronal Loss in the Spiral Ganglion of Young Rats

A quantitative study of spiral ganglion neurones was performed in rats during postnatal days 4, 5, 6, 30 and 60. There are 25,194 +/- 462 ganglion cells on postnatal day 4, abruptly falling to 18,809 +/- 514 on the 6th postnatal day. This neuronal loss accounts for the 22% of the overall ganglion cell population. The number of neurones remains almost unchanged from the 6th to the 60th postnatal day. This numerical variation in the neuronal population of the spiral ganglion seems to be related to the changes that take place during cochlear synaptogenesis, at the end of the first postnatal week, on the base of the outer hair cells. These changes involve competition among efferent endings approaching the cell and some afferents connected with it at birth, that disappear as a result of such a competition.

Lectin staining of saccharides in the normal and hypothyroid developing organ of Corti

Lectin staining has been used to detect mono- and oligosaccharides in normal and hypothyroid developing organs of Corti in the rat. Eight developmental stages were studied (1, 5, 8, 10, 15, 20, 50 and 60 days after birth). Congenital hypothyroidism was induced by oral administration of propylthyouracil to pregnant rats. Labelling of the tectorial membrane with 3 lectins, Ulex europaeus agglutinin-I (UEA-I), Lens culinaris agglutinin (LCA) and Ricinus communis agglutinin-I (RCA-I) showed no significant differences between normal and hypothyroid animals. Staining with peanut agglutinin (PNA) showed that the hypothyroid adult tectorial membrane (but not the normal one) possesses the disaccharide galactose + N-acetyl galactosamine. Phaseolus vulgaris agglutinin-L (PHA-L) labels the whole tectorial membrane in both groups of animals, but the staining is more intense in the hypothyroid one for a narrow band of oligosaccharide located just between the tectorial membrane and the underlying organ of Kölliker. Both soybean agglutinin (SBA) and succinylated wheat germ agglutinin (WGA) stain the tectorial membrane as well as the cytoplasm of the cells constituting the inner portion of the organ of Kölliker; this latter feature disappears in the normal animals about the 8th postnatal day, but it is abnormally preserved until the 60th postnatal day in the hypothyroid ones. In the adult hypothyroid animals, 3 of the lectins (LCA, PHA-L and WGA) stain extracellular conglomerates located under the synaptic pole of the outer hair cells.

Tannic acid staining of the cell coat of the organ of Corti.

Tannic acid staining of the cell coat of the organ of Corti shows a deep asymmetry between the endo- and the perilymphatic surfaces, the former being 6 times thicker. This fact may be related to a barrier mechanism against potassium-induced cell damage. Horizontal cross-links between stereocilia were heavily stained, but vertical ones were not preserved, thus suggesting they are not glycoconjugates.

Localization of anionic sulfate groups in the tectorial membrane.

Colloidal iron hydroxide (CIH) staining demonstrates the existence of anionic sulfate groups of glycoconjugates associated with several constituents of the tectorial membrane (TM). In the adult animal, labelling in the main body of the TM appears as long, electron-dense patches surrounding type A fibrils which show alternating stained and unstained zones. On the other hand, labelling of the fibrils of the matrix of the TM appears as single, CIH particles with no special arrangement. Some of the structurally distinct regions of the TM are also labelled (limbal zone, Hensens stripe and inner portions of the cover net), while others are not (marginal band and outer portions of the cover net). Staining of type A fibrils in the major TM is already present in newborn animals; while, both the outermost region of the TM closest to the cells of the organ of Kölliker and the minor TM are not labelled. The implications of these distributions of sulfated glycoconjugates for the electrochemical properties of the TM are discussed.

A reliable method for Golgi staining of retina and brain slices

Although the classical Golgi method is a powerful means for structural analysis of the brain, it is generally considered to be an unpredictable technique making anatomists wary of using it. Often, even when successful staining has occurred, deposits of silver chromate crystals on the surface of the tissue obscure examination. This paper describes a simple procedure for Golgi impregnation of retina and brain slices so that good, even staining is obtained and crystal formation is avoided. The most outstanding feature of the method is the consistency of results. This consistency is due to two factors: (1) the accurate determination of the optimal chromation by measuring the rise of pH in the solutions and (2) the uniform penetration of dichromate and silver nitrate to the specimen by using a freely hanging, sandwiching technique. We suggest that the method described here can be applied to other parts of the nervous system and will be a reliable way to identify and better classify new cell types.

Hypothyroidism prevents developmental neuronal loss during auditory organ development.

The deficit of thyroid hormone leads to several structural and physiological modifications in the auditory receptor: the outer hair cells present an immature morphology, abnormal persistence of the afferent dendrites and incomplete development of the efferent terminals. The aim of this work was to perform a quantitative and morphometric study of the spiral ganglion neurons in control and hypothyroid animals. The cochleae from both experimental groups were processed in order to obtain plastic sections. In control animals the size of the neurons increased throughout development and was larger in the basal than in the apical portion of the cochlea. In hypothyroid animals, the cell death that takes place normally during development did not occur, and there was no differentiation into types I and II neurons. The size of the neurons also increased with development in treated animals, but they were smaller than in control animals, and in this case the neurons in the apex were larger than in the base. This study shows that hypothyroidism alters the normal development of the spiral ganglion neurons.

Regional specialization of the cell coat in the hair cells of the organ of Corti

The anionic sulfated groups of the cell coat glycoconjugates were studied in sensory and supporting cells of the organ of Corti with the colloidal iron hydroxide technique of Mowry, modified by Seno et al. [(1985) Histochemistry 82, 307-312]. This technique specifically stains the apical (endolymphatic) surface of both inner and outer hair cells. As this is the area at which the influx of potassium into the sensory cells takes place, the accumulation of negative charges in this location may be a mechanism to concentrate cations (mainly potassium) close to this portion of the hair cell membrane.

Reversible damage to the nerve fibres in the organ of corti after surgical opening of the cochlea in the rat

After minimal opening of the cochlear bony wall, the efferent and the outer spiral fibres showed no changes; inner radial fibres (afferents to inner hair cells) were highly sensitive to this mild trauma, appearing swollen and empty of cytoplasmic content. Available data suggest that this may be due to alterations in the cochlear micromechanical environment, related to the surgical manipulation. The swellings were reversible, although the normal structure had not completely recovered until one month after the manipulation.

Olivocochlear efferent innervation of the organ of corti in hypothyroid rats.

Congenital hypothyroidism induces developmental abnormalities in the auditory receptor, causing deafness due to a poor development of the outer hair cells (OHCs) and a lack of synaptogenesis between these cells and the olivocochlear axons. This efferent innervation is formed by two separate systems: the lateral system, which originates in the lateral superior olive (LSO) and reaches the inner hair cells; and the medial system, which originates in the ventral nucleus of the trapezoid body (VNTB) and innervates the OHCs. A previous study carried out in our laboratory showed that in congenitally hypothyroid animals, the neurons which give rise to the efferent system are normal in number and distribution, although smaller in size. The aim of the present work was to study the efferent fibers in the auditory receptor of hypothyroid animals, by means of stereotaxic injections of biotinylated dextran amine in the nuclei that give rise to the olivocochlear system: LSO and VNTB. In hypothyroid animals, injections in LSO gave rise to lateral olivocochlear fibers lacking their characteristic dense terminal arbors, while injections in the VNTB‐labeled fibers terminating in the spiral bundle region, far from the OHCs with which they normally contact. In the latter case, only a small percentage of labeled fibers reached the OHCs area, giving off only two radial branches maximum. Because the number of neurons which develop into the efferent innervation was normal in hypothyroid animals, we conclude that medial fibers may contact a new target. J. Comp. Neurol. 459:454–467, 2003.