The reinnervation of regenerated hair cells in the basilar papilla of chicks after kanamycin ototoxicity.

When newly hatched chicks were injected with kanamycin on 8 successive days, the hair cells were destroyed completely in the area 0.4 to 0.8 mm from the proximal end of the basilar papilla. At this time, and 1 and 7 days following the completion of 10 days of injections, the nerve fibres in the basilar papilla of chicks show no sign of injury. On the first day following 10 days kanamycin administration the regenerated hair cells obtained not only afferent innervation, but also efferent innervation. At 15 days following drug cessation, afferent innervation of the regenerated hair cells was already similar to the controls, and the thresholds measured at this time were significantly better than those at 1 and 7 days. The chalice efferent terminals did not appear until the 60th day of drug cessation. Efferent innervation of the regenerated hair cells also approached maturity at this time. Compared with the half month after the completion of drug injection, the hearing of birds had no evident increase. It was obvious that afferent innervation of the regenerated hair cells was related more to the recovery of hearing than efferent innervation. The regenerated hair cells beginning reinnervation early and maturing were important factors influencing the recovery of the birds' hearing function after kanamycin ototoxicity.

The alteration of ultrastructure and immunoreactivity of human embryonic organ of Corti tissue culture after exposure to aminoglycoside (neomycin) ototoxicity.

Using electron microscopy and GABA immunohistochemistry we evaluated the effects on human embyronic organ of Corti tissue culture of exposure to the ototoxic aminoglycoside antibiotic neomycin at a dose of 1 mM for 48 to 96 hrs. Neomycin induces the formation of multilamellar myeloid structures. These lesions, found only in the basal coil but both in inner and outer hair cells, were characteristic of the membrane-associated neomycin-induced damage. A large amount of lipofucsin and numerous lipoid vacuoles as well as vesicle-filled mound-like protrusions were also observed after exposure to neomycin. It seems there is no obvious effect on GABAergic innervation.

[Morphologic study of GABA-ergic neurons in dissociated cell culture of spinal cord and dorsal root ganglion].

gamma-Aminobutyric acid (GABA) is an important inhibitive neurotransmitter in central nervous system (CNS). Many studies have been made about the distribution of GABA-ergic neurons in the spinal cord (SC), but little is known about the morphology of GABA-ergic spinal cord neuron (SCN) in culture. Moreover, whether GA-BA-ergic neuron existed in dorsal root ganglion (DRG) or not is still under discussion. Considering together with the fact that the same neuron can synthesize different. kinds of neurotransmitter in different periods of development, we find that it is attractive to study the GABA immunoreactivity of cultured SCNs and DRG cells. The SC and DRG were dissected from 12-14 day old mouse (C57BL/6J) embryo and plated at a density of 1 x 10(6)-2 x 10(6) cells per dish. At 5 day in vitro (DIV), the cells reaggregate and form complicated neurite network. While SCNs varies in the morphology of cell body and neurite, DRG cells of different sizes can be easily discriminated by their round cell bodies and sharply defined nuclei and nucleoli, and their sizes do not undergo major change during the culture period. Immunoreaction was performed by using a polyclonal anti-GABA serum (rabbit) and PAP procedure. Two types of immunoreactive SCNs were observed: 1. SCNs with intensely positive reacted somata, nucleoli, and neurites. 2. SCNs with reactivity shown only on part or whole of neurites and cell membrane while the cell body is negative (to be considered as the result of GABA uptake).(ABSTRACT TRUNCATED AT 250 WORDS)

SEM observation and GABA immunoreactivity of human embryonic organ of Corti tissue culture.

Scanning electronic microscopy (SEM) observation and GABA immunoreactivity of the tissue culture of 6 human embryonic organs of Corti are reported. The normal structural appearance of the hair cells and the facts that there were no bulging of cuticular plates, and cytoplasmic protrusion proved that the hair cells were healthy. After GABA immunostaining, radial bundles, innerspiral bundles, and the ending of inner and outer hair cell regions displayed typical GABA-positive reactivity. The results indicate that the human embryonic organ of Corti is well developed differentiated, and mature at 19 gestational weeks and can continue to grow and differentiate in vitro. The study offers an ideal and important biological model for further research into the human ear.

[The synaptic architecture of propriospinal neurons in cultured mouse spinal cord].

An electron microscopic analysis of the synaptic architecture in propriospinal neurons of cultured fetal mouse spinal cord has been undertaken. The size of the perikarya in the cultured spinal cord represents a range from small- to medium sized neurons, which form many synapses each other. There are many axo-dendritic and axo-somatic synapses in the culture but direct dendro-dendritic apposition is rarely seen. Four morphological types of synaptic boutons, S, F, M and G are classified according to criteria used by previous investigators. The ultrastructural details available suggest that the propriospinal neurons receive synaptic input from propriospinal fibers through simple synapses. It may indicate that their impulses can be controlled only postsynaptically.

Human embryonic organ of Corti in tissue culture and NSE expression.

For the first time ever, the cochlear tissue of the human embryo has been successfully grown in vitro in two cases, and neuro-specific enolase (NSE) immunoreactivity was studied in one of these tissues. The outer hair cells were arranged in three rows, and the inner hair cells in one row and in better order than the outer hair cells. After NSE immunostaining, the outer spiral bundle, tunnel fiber, outer hair cells, inner hair cells, and the spiral ganglion cells showed positive staining. This data suggests that the human embryonic cochlea has nearly reached complete maturation by 16 weeks, and that the tissue of the Corti organ can be differentiated and matured in vitro.

[Morphological study on the role of coated vesicle in the specialization of synaptic membrane in synaptogenesis].

Our object was to characterize the morphological changes of coated vesicles and synaptic membranes during synaptogenesis. Neurons from spinal cords of fetal mice were established as isolated cells in primary culture. After a few days in vitro, the neurons extended their neurites and started their interaction. At timed intervals thereafter, cultures were fixed for electron microscopic observation. Coated vesicles were prominent in the neuronal cytoplasm at the time of synaptogenesis (about 7-10 days in vitro). Similar vesicles were seen in continuity with some cisternae in the Golgi regions and there was an increase in number during the synaptogenic period. Indeed it is not established whether the coated vesicles were exocytotic or pinocytotic in nature, but the cisternae which were in continuity with coated vesicles could be labelled by glucose-6-phosphatase (G6Pase) but not by thiamine pyrophosphatase (TPPase). Such vesicles were also seen in continuity with the neuronal plasmalemma near the closest contact site and contributed their undercoating to pre- and postsynaptic densities. The formation of bilateral membrane specialization was described as being structurally similar to synaptic active zones and appeared to be the first definitive sign of synapseformation. It has been suggested that the synaptic dense material may derive wholly or in part from the exocytic coated vesicles which apparently budding off from endoplasmic reticulum cisternae. This incorporation could provide the mechanism for confining specific characteristics of neuronal membrane to the synaptic region.

[Development of endoplasmic reticulum and its enzymic marker in cultured mouse spinal neurons].

In an attempt to elucidate the relationship between synapse formation and cell development, the morphology and cytochemistry of the endoplasmic reticulum and its enzymic marker, glucose-6-phosphatase (G-6-Pase), in cultured mouse spinal neurons were investigated ultrastructurally. It was found that in the early period of the development, neurons were characterized by scarceness of organelles; only a few of granular or agranular endoplasmic reticulum and mitochondria were seen. The endoplasmic reticulum and nuclear envelope were packed specifically with G-6-Pase resection product but the product was weak. After a period of culture, most of the neurons had well-developed endoplasmic reticulum, Golgi apparatus, mitochondria and microtubules, etc. The Golgi apparatus was relatively large, having some cisternae associated with vesicles. Either concave of convex face of the saccules was labeled by thiamine pyrophosphatase (TPPase) specifically. GERL, labeled by cytidine monophosphatase (CMPase), was also seen close to the inner or outer face of some Golgi apparatus. The endoplasmic reticulum at this stage was distributed throughout the cytoplasm, including that in dendrites; its enzyme marker (G-6-Pase) localized consistently within the lumen of all endoplasmic reticulum, nuclear space and subsurface cisternae, and frequently in the concave saccules of the Golgi apparatus. After a long-term culture, some neurons became "aged". The endoplasmic reticulum cisternae enlarged and G-6-Pase reaction reduced. Along with the neuronal development, especially maturation of the endoplasmic reticulum and its enzymic marker, synapse formation was begun at the neuropile area. The axo-dendritic synapses always occurred between the axonal terminals and dendrites where the endoplasmic reticulum had showed positive G-6-Pase reactions. Considering the fact, it suggests that the appearance and change of these specific enzymes may be related to the maturation of the neurons in vitro, and also related to the synapse formation between neurons.