Jorge J. Prieto

Connections of the auditory cortex with the claustrum and the endopiriform nucleus in the cat

We studied the connections of eleven auditory cortical areas with the claustrum and the endopiriform nucleus in the cat, by means of cortical injections of either wheat germ agglutinin conjugated to horseradish peroxidase, or biotinylated dextran amines. Unlike previously accepted reports, all auditory areas have reciprocal connections with the ipsi- and contralateral claustrum, though they differ in strength and/or topography. The areas that send the strongest projections are the intermediate region of the posterior ectosylvian gyrus and the insular cortex, followed by the primary auditory cortex and the dorsal portion of the posterior ectosylvian gyrus. The high degree of convergence of cortical axons in the intermediate region of the claustrum, arising from tonotopic and nontonotopic areas, suggests that claustral neurons are unlikely to be well tuned to the frequency of the acoustic stimulus. Corticoclaustral axons from any given area cover territories largely overlapping with those occupied by the claustrocortical neurons projecting back to the same area. The location of cortically projecting neurons in the claustrum matches the position of the target cortical area in the cerebral hemisphere, both rostrocaudally and dorsoventrally. These findings suggest that the intermediate region of the claustrum integrates inputs from all auditory cortical areas, and then sends the result of such processing back to every auditory cortical field. On the other hand, the endopiriform nucleus, a limbic-related structure thought to play a role in the acquisition of conditioned fear, would process mostly polymodal information, since it only receives projections from the insular and temporal cortices.

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.

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.

Pilocarpine elicits the interdental cell secretory activity of the inner ear

Subcutaneous injection of pilocarpine in guinea pigs resulted in the following ultrastructural changes: 1) the apical cavities of the interdental cells were filled with a substance indistinguishable from the overlying amorphous layer of the TM; 2) a great number of spherical structures appeared over the limbal portion of the tectorial membrane. In TEM photomicrographs these structures displayed the same appearance as the amorphous layer of the TM and were usually continuous to it; 3) the number of holes that decorate the upper surface of the limbal portion of the TM was dramatically increased and it was found that they connect the endolymphatic space to the apical cavities of the interdental cells; 4) there was an increase in the number of the small extracellular vesicles found in the clear spaces of the tectorial membrane. These facts suggest that pilocarpine stimulates the secretion of the interdental cells, confirming the existence of the secretory processes previously described (Prieto et al., 1990). These findings can be related to the turnover of the TM in the adult animal and, perhaps, to the secretion of some organic compound to the endolymph. We postulate that the actions of pilocarpine on the interdental cells are most probably mediated by the activation of muscarinic acetylcholine receptors in these cells.

The effect of hypothyroidism on the development of the glycogen content of organ of Corti's hair cells.

The density of glycogen particles in organ of Cortis sensory cells was measured to determine the effect of congenital hypothyroidism upon the normal development of this energy source. This density in both normal and hypothyroid inner hair cells remains in low values from birth to adulthood. On the other hand, that of normal outer hair cells undergoes a great increase between the 10th and the 20th postnatal days, coinciding with the maturation of both the efferent innervation of these cells and the tuning properties of the auditory receptor. The glycogen stores of the hypothyroid outer hair cells do not show any significant increase from birth to adulthood. This latter fact suggests that the congenital hypothyroidism restrains the development of an important energy source of outer hair cells, most surely disturbing the physiological processes relying on glycogen metabolism.

Subsurface material in outer hair cells

Tannic acid stains a homogenous material inside the outer hair cells of the organ of Corti of the guinea pig. This material is always placed between the plasma membrane and the first layer of subsurface cisterns, but only in those areas along the lateral surface of the outer hair cell lining the spaces of Nuel. The possibility that this material is related to some particular function of outer hair cell lateral face is discussed.

GABAergic organization of the auditory cortex in the mustached bat (Pteronotus p. parnellii)

The structure and distribution of neurons and axon terminals (puncta) immunostained for gamma-aminobutyric acid (GABA) in the parietotemporal neocortex of the mustached bat (Pteronotus p. parnellii) was studied. The types of GABAergic neurons and puncta (putative terminals) were analyzed, and the immunocytochemical patterns were compared to those in cat auditory cortex (AC). The classic map of mustached bat primary auditory cortex (AI) corresponds to a belt of granular six-layered cortex on the temporal convexity. This area encompasses the Doppler-shifted constant frequency 60 kHz domain (DSCF) described in physiological investigations, as well as its flanking, low-frequency, posterior field (AIp) and the anterior high-frequency region (AIa). Many types of GABAergic neurons correspond to those in cat primary AC. However, the bat had a significantly lower proportion of such cells in five of the six layers. The classes of GABAergic neurons in most layers were small, medium-sized, and large multipolar cells, and bipolar and bitufted neurons. Types found in only one or two layers included horizontal cells (layers I and VI) or extraverted multipolar neurons (layer II). Only layer IV had comparable percentages (∼ 26%), suggesting that the GABAergic influence on lemniscal thalamocortical input is conserved phylogenetically. While the cellular basis for GABAergic cortical processing may reflect shared neural circuits and common modes of inhibitory processing, laminar differences could underlie adaptations specific to microchioptera.