I and Dr. Altschuler pioneered through our collaboration in my Laboratory the use of immunocytochemistry in auditory research for the study of the organ of Corti, of the auditory nerve and of the cochlear nucleus. Two other teams, Dr. Flock et al. and, respectively, Dr. Zenner et al. were the first to use immunocytochemistry to study organelles of the auditory sensory cells.
My presentation at this Meeting of the Japan Society of Ear Research concerns immunocytochemistry as applied to the cochlea as an organ and will be mainly about studies in my Laboratory, complemented with a brief overview of some recent and exciting studies carried out mainly by research teams in Japan. The positive findings of all these different studies are very much focused on the efferent nerve fibers and nerve endings in the cochlea. On the one hand it has been shown that several different neurotransmitter candidates may co-exist in many of the cochlear efferents. On the other hand the findings indicate strongly there may exist sub-populations of cochlear efferents that are chemically different from the two major sub-populations, the so-called medial and lateral olivocochlear efferents.
The described findings are of great interest from the point of view of auditory research, since the efferents may play an important role in modifying the micromechanics of the auditory sensory cells. The findings are also interesting from the point of view of neuroscience in general, since the cochlear efferent neurons originating in the brainstem form exceptionally long and well defined centrifugal tracts within the central nervous system.
The negative side of all these immunocytochemical studies of the cochlea is a disappointing lack of positive evidence towards a definition of the neurotransmitter, or neurotransmitters, of the inner and outer hair cells of the cochlea. Even so, it is practically certain that such positive immunocytochemical evidence will eventually be forth-coming, be important and perhaps be decisive.

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Coenzyme Q (CoQ) has already received a high evaluation in clinical treatment of heart diseases. It has also reported that CoQ is effective for recovery from an acute sudden deafness in our field. However, pharmacokinetics of CoQ in inner ear has not been clarified. The present study focuses on the pharmacokinetics of CoQ using guinea pigs with an acute sensorineural hearing loss which is caused by hypoxic condition. The animals were controlled under the artificial respirater, the blood gas concentration, and the measurements of ABR, ECG, and blood pressure. The ABR gradually went off after the repeated hypoxia. In this occasion, the animals were sacrificed, measured with each of the above tests, and examined the brain and the inner ear by the histological and histochemical methods, SEM and TEM examination.
As the results, CoQ is effective on recovery from the damage of auditory hairs and prevents from the damage of respiratory metabolism of the hair cells which is conducted by the hypoxia.

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On etiology of aminoglycoside ototoxication, basic particulars such as ionic environments, membrane properties, transduction channels and presynaptic events in hair cells should be considered. The ionic environment of the apical surface of hair cells is characterized by a high concentration of potassium and a very low concentration of calcium. This calcium content is necessary for maintaining the gating action of the transduction channels. It seems likely that triphosphoinositide is a candidate for the binding site of calcium which is competed with aminoglycoside. It has been also reported that the aminoglycoside competes with calcium at the basolateral side of hair cells. The aminoglycoside acts in two phases on the basolateral side in which the actions are reversible and irreversible. There is a difference in causing process of aminoglycoside ototoxication between the apical side and the basolateral side.

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It was proved by the toluidine blue method in vitro that heparin and chondroitin sulfate had strong binding ability to aminoglycoside. Reduction of Kanamycin ototoxicity was slightly observed by a simultaneous application of heparin to guinea pigs through electrocochleography and morphological investigation with SDH staining. But the protective effect of gentamicin ototoxicity by chondroitin sulfate was not detected. In order to protect cisplatin (CDDP) ototoxicity, sodium thiosulfate (STS) was used. When STS was administrated early after CDDP injection to the guinea pigs, remarkably reduced CDDP ototoxicity was noted by means of scanning electron microscopy. However, when an application of STS was late, protective effect was not strong. Furthermore, there was a mild protection effect by infusion of STS into the middle ear cavity.

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The protective effect of fosfomycin against ototoxicity induced by aminoglycoside antibiotic and cisplatin and its analog carboplatin was investigated in Fisher rats. Drug-induced toxic side effects of ototoxicity and nephrotoxicity, or myelo-suppression effect due to carboplatin were reduced functionally or histopathologically by the coadministration with fosfomycin. No influence of fosfomycin against the antitumor effect of cisplatin was clarified using ddy-strain mice inoculated with Ehrlich carcinoma cells. Clinically, nephrotoxicity was lower after the combined administration of cisplatin and fosfomycin than after cisplatin alone. The combined administration of fosfomycin and ototoxic drugs has significantly lower toxicities and greater therapeutic efficacy.

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Drug pathways to Corti's organ of guinea pig was investigated under normal and experimental conditions by morphological technique. Under normal condition systemically injected small molecular weight substance which are kanamycin (KM) and lanthanum ion (LA) can get to Corti's organ from early time via capillaries of tympanic lip and basilar membrane. As time goes on (1.5 hs. after administration), KM which comes from perilymphatic space accumulates to Corti's organ.
On the other hand, pre-administered KM very easily can get to Corti's organ from early time via capillary and perilymphatic pathways when furosemide (FM) is added. This alteration aspect of BLB might lead to inner ear damage and inner ear therapy by using drug.

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Isosorbide, one of the osmotic diuretics, is clinically confirmed to be effective for Meniere's disease. In order to evaluate the basic reaction of isosorbide, inner ear pressure, osmotic pressure and morphological change were studied after intravenous administration of 1.6 ml/kg of isosorbide to the guinea pigs with endolymphatic hydrops. The results were as follows. 1) The inner ear pressure, 30 mmH₂O in normal condition, was gradually decreased to 0 mmH₂O in 10-15 minutes, 2) The osmotic pressure of perilymph, 300 osm/L in control, was increased to 335 osm/L in 60 minutes. 3) In 9 cases out of 13, Reisner's membrane especially in the lower turns of the cochlea, showed foldings of the membrane, indicating the decrease of the endolymphatic hydrops. The other 4 cases did not show foldings, although they showed profound hydrops with severe atropy of the endolymphatic duct. From these results, it is considered that isosorbide may react on the endlymphatic duct, not on the stria vascularis.

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After introducing potassium ion through the round window into the perilymphatic space of 40 guinea pigs by means of iontophoresis, physiological and histochemical investigations were performed. About 15 min after the iontophoretic procedure, electronystagmography revealed irritative nystagmus for the first 5 min and then paralytic nystagmus for the following 6 to 24 hr. Irritative nystagmus had irregular large amplitudes and paralytic nystagmus had regular small amplitudes. Histochemical analysis of the vestibular sensory epitheria revealed the increased activity of succinic dehydrogenase during irritative nystagmus and the decreased activity during paralytic nystagmus. On the other hand, electrocochleography revealed the decrease of the action potential without any initial irritative cochlear sign, and the enzyme activity of the cochlear sensory cells was decreased from the beginning.

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Endocochlear potential (EP) and K⁺ activity in the scala media were recorded by means of K⁺ sensitive doublebarrelled microelectrodes in the second turn of the cochlea of the guinea pig during the perilymphatic perfusion of furosemide and ouabain. Furosemide produced a less decrease in K⁺ activity relative to the EP decrease than ouabain. The result suggests that furosemide reduces the EP by different mechanisms from the deterioration of K⁺ secretion potentig resulting from the inhibition of Na⁺-K⁺ ATPase.

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The accepted cause of Meniere's disease is endolymphatic hydrops. Osmotic diuretics were often administrated to the treatment of Meniere's disease. The purpose of this experiense is to know the effects of osmotic diuretics on the endolymphatic pressure and EP in the guinea pigs. The changes of endolymphatic pressure and EP were recorded with a servo-controlled micro-pressure system (MODEL 900, WPI). Intravenous injection of 80% isosorbide (2.0 ml/Kg) caused a intermittent increase in the pressure, and showed gradual decrease, and then gradually recovered. The maximum reduction of pressure was recorded at about 10 minutes after intravenous injection. The EP showed no significant change after intravenous injection of isosorbide. Intravenous injection of 50% glycerol (2.4 ml/Kg) showed the results as isosorbide. The maximum reduction of pressure was recorded at about 30 minutes after intravenous injection of glycerol. Isosorbide, in the same manner as glycerol, produce a pressure reduction in endolymph.

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The two types of vasopressin receptors have been now characterized: these have been termed the V1 and V2 receptors. In order to know which receptor mediates the action of vasopressin on the EP, the effect of V1 antagonist and V2 agonist on the EP was examined in the guinea pig. V1 antagonist failed to block the EP change induced by arginine vasopressin (AVP). V2 agonist produced the reversible EP reduction like AVP. The results suggest that V2 receptor mediate the action of vasopressin on the EP.

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To alter the intracellular pH, ammonium and propionate were perfused by inner ear vascular perfusion technique. On HEPES buffer perfusate intracellular alkarinization with perfusion of ammonium led to a strong decline of EP (-38.2±2.5mV) while intracellular acidification with propionate produced a small positive shift of +3.7±1.2mV. The responses of EP on bicarbonate buffer perfusate exhibited similar patterns but less in magnitude than that on HEPES buffer. These results suggest that EP is dependant upon a intracellular pH (presumably in strial marginal cells), though the magnitude of pH changes during these conditions was not determined.

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Alterations of the inner ear fluids pressure and cochlear microphonics (CM) with increased cerebrospinal fluid (CSF) pressure were studied in the guinea pig. Hydrostatic pressures in endolymph and perilymph of the cochlea were measured by use of a servo-controlled micropipet system. Endolymphatic pressure as well as perilymphatic pressure increased in a linear manner with little or no time lag as the CSF was pressurized over a range of 0 to 50 mmHg. There was no statistical difference in the pressure between endolymph and perilymph. The CM in response to 500 Hz and 4, 000 Hz tone burst was observed before, during and after the CSF pressurization. Inputoutput functions of the CM showed a suppression in both maximum output and sensitivity with CSF pressure greater than 30 mmHg.

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Aspirin is injected to the guinea pigs to understand its effects on the cochlear nerve excitability. For this purpose, the compound AP's evoked by electrical stimulation to the cochlea, were recorded using micropipettes introduced in the cochlear nerve bundle. The latency of compound AP's was found to be increased. In one of our previous studies we observed that the cochlear nerve spontaneous discharge rate also increases following the injection of Aspirin. These two results indicate that Aspirin suppresses the cochlear nerve excitability and the decreased excitability contributes to the impairment of hearing function.

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The effect of calcium antagonist on asphyxia induced spontaneous discharges was examined. As the experimental animal young adult cats which were anestetized with pentobarbital (40 mg/kg), petazocine and diazepam were used. Characteristic frequency, threshold and spontaneous discharge rate of the auditory neuron in the cochlear nucleus was obtained before, during and after asphyxia. As a calcium antagonist diltiazem was selected and injected into the femoral vein. Change of spontaneous discharges, EEG and ECG were obtained and concentration in the blood of diltiazem was also measured. After administration of diltiazem spontaneous discharges were proportionaly decreased in grade of amount of injection from slight to complete. From the present observation the following results were obtained that asphyxia induced spontaneous discharges of the auditory neurons in the cochlear nucleus were suppressed by calcium antagonist and dose related effect was predictable.

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By ³H-thymidine autoradiography, we have previously shown that DNA synthesis occurs in the cochlea of the adult mice at various sites such as stria vascularis, Reissner's membrane, basilar membrane and spiral ligament (Murakami et al. 1986). In this study we traced the labeled cells by prolongation of the survival time after isotope injection up to 14 days.
Labelling index peaked at 3 days after isotope injection and thereafter decreased gradually. In the stria vascularis the labeled intermediate cell seems to move towards apical and basal portions with time progress. The intermediate cell may be a precursor of other cells of the stria vascularis. In the Reissner's membrane grains accumulated in mesotherial cells in the central portion and cells at the edge. The accumulation former cells remains but that in the later cells decreased with time progress. Mitosis was observed only in the spiral ligament cells throughout experiment.

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Na, K-ATPase activity in the lateral cochlear wall of guinea pigs was measured by using the lumiphotometric method. The cochlea was rapid-frozen into the liquid nitrogen and was freeze-dried sufficiently. The specimen was seperated into three parts and then each part was compared with the other two in the degree of relative activity. The partiality of Na, K-ATPase activity was observed and of the three, the stria vascularis had the highest level of activity. The relative activity at the S. L, the S. P and the S. V were estimated at 1.75, 23.2 and 100 respectively in the basal turn and showed a similar tendency in the other two. At the stria vascularis, where the highest turn increased the magnitude of the relative activity, the values of the basal, the second and the third turn to the apex were 100, 121 and 127 respectively. These results suggested that the stria vascularis should function as the critical factor to the production of the endolymph-perilymph in the inner ear accompanied by Na and K ion dynamics as well as the productive site of the endocochlear DC potential.

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Prostaglandin (PG) D₂ is a major PG in the central nervous system. In this study PG synthetase, and enzyme responsible for the biosynthesis of PGD₂ was localized immunocytochemically in the rat cochlea.
PG D synthetase was purified from Wistar rat brain or spleen to apparent homogeneity; monoclonal or polyclonal antibodies were raised in the mouse or rabbit, respectively. Using these antibodies the distribution of the PGD synthetase-like immunoreactivity in the cryosections of the 5 weeks old rat cochlea was examined by avidinbiotin complex techniques.
Immuroreactivity was obtained only when antibody against brain enzyme was use In the organ of Corti, immunoreactivity was distributed mainly in the outer and inner hair cells. In the vascular stria immunoreactivity was found in the marginal cells and basal cells. Immunoreactivity was also observed in the cells of the Reissner's membrane.
It is now clear that brain type PGD synthetase occurs in the rat cochlea. The significance of this finding should be explored.

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This study described the exogeneous and endogeneous synthesis of prostaglandins (PGs) by the cochlea in vitro.
1) After incubation of the whole membraneous cochlea with ³H-arachidonic acid, synhthesis of various kinds of PGs was evidenced. Although PGF_2α was the most abundantly synthesized PG than others, the rate of conversion from arachidonic acid to it was calculated to be only 0.03%. The synthesis of PGD₂, PGE₂, 6ketoF_1α and TXB₂ were also evidensed in this order.
2) The whole membraneous cochlea was incubated at 37°C for 0-15 minutes and PG formed were determined by radioimmunoassay. The main PG produced was PGE₂, and PGD₂, PGF_2α, PGI₂ and TXA₂ were also found in the cochlea. At incubation for 0 minutes without incubation PGs were already formed to considerable level and the maximal level was obtained at 5 minutes incubation. Thereafter the levels of PGD₂ PGE₂ and PGF_2α decreased gradually.
3) The cochlea was dissected into three parts: OC+M (organ of Corti +modiolus), LW (lateral wall) and CN (cochlear nerve), and PGs formed by these tissues was determined. In OC+M and CN main PG was PGE, whereas in LW the amount of PGD₂, PGE₂, PGF_2α and PGI₂ was about same level.
These result revealed that the cochlea tissues can also prodused main five PGs as other organs. Possible involvement of PGs in various cochlear functions was briefly discussed.

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The distribution of the antibacterial enzyme lysozyme-and lactoferrin-secreting cells in the tubotympanum of normal chinchilla was studied immunohistochemically. The middle ear mucosa contained lysozyme-secreting cells and lactoferrin-secreting cells. The former were localized primarily in the columnar epithelium are and the latter primarily in the cuboidal epithelium area of the mucosa. In the eustachian tube, the lysozyme was localized in goblet cells of the mucosal epithelium and mucous cells of the glands, while lactoferrin was localized in serous cells of the glands. Our results indicate that secretory lysozyme and lactoferrin are secreted by different cell types. This finding is consistent with the concept that antibacterial enzymes are an integral part of the normal mucosal defense system in the tubotympanum.

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The guinea pig middle ear mucosa, including that of the eustachian tube, was embedded in Epon or Lowicryl K4M. Thin sections of the mucosa were stained with wheat germ agglutinin (WGA) -gold complex, and examined under electron microscope. WGA-reactive glycoconjugates were primarily localized over the glycocalyx made of fine filaments surrounding microvilli and over mucous blanket overlying cell surfaces. In addition to this apical surface labeling, several intracellular structures such as Golgi apparatus and all three types (electron-lucent (EL), intermediately electron-dense (ID), or electron-dense (ED) type) of secretory granules were heavily labeled. Among the granules EL granules showed stronger labeling than the others. The results presented here indicate that WGA-binding sites are closely associated with the secretory granules and with the luminal surface structures of the middle ear epithelium, suggesting the distribution of glycoconjugates in both secretory products and membrane consituents.

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Guinea pigs were immunized with human serum and albumin of chicken egg. Antigen and antibody were investigated by immunofluorescent method. Antigen for antihuman immunoglobulin (goat) was found widely in the membranous labyrinth, but antibody for albumin of chicken egg was found only in the limbus.
In cases of the endolymphatic hydrops which was made by the endolymphatic sac obliteration, IgG was found widely in the membranous labyrinth including the aqueduct. In cases of acute acoustic trauma of the cochlea, IgG was found only in the endolymphatic sac and on the floating cells in vestibular aqueduct.

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Lateral wall of the rat cochlear duct was fixed by perfusion with 4% paraformaldehyde and embedded in LR-GOLD. The ultrathin sections were incubated with antibody against the holo (Na⁺, K⁺) ATPase purified from rat kidney, subsequently with protein A gold complex and quantitatively analysed. In the stria vascularis, many gold particles were found over the basolateral membrane of the marginal cell and hardly found over the endolymphatic surface. Numerous gold particles were also found over the plasma membrane of the spiral prominence stromal cell and over the intercellular microvilli of the external sulcus cell. These results suggest (Na⁺, K⁺) ATPase on the lateral wall of the cochlear duct plays important roles for maintaining endolymphatic cation gradients and generation of the endocochlear potential.

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The immunohistochemical localization of γ-aminobutyric acid (GABA) -and aspartate-containing neurons was evidenced in the guinea pig vestibular nuclei, using purified antisera to GABA and aspartate. Most GABA-containing neurons had small cell bodies and were scattered unevenly throughout the vestibular nuclei. The largest number of these cells were found in the medial nucleus. GABA-containing terminals were distributed throughout the nuclei. A large number of various sized aspartate-containing cells were present in the nuclei. Giant cells in the lateral nucleus also contained aspartate and were surrounded by GABA-like immunoreactive terminals, suggesting the modulation of aspartate-containing neurons by GABAergic fibers from Purkinje cells.

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Gamma-aminobutyric acid (GABA) -like immunoreactivity in the chick vestibular endorgans was studied by using an antiserum against GABA coupled with glutaraldehyde to bovine serum albumin. GABA-like immunoreactivity was localized in the cytoplasma of the sensory hair cells in both cristae ampullares and maculae. GABA-like immunoreactivity positive cells were found to be evenly distributed throughout the sensory epithelium. There was no difference in staining between type I and type II cells. The results provide immunocytochemical evidence that GABA-like immunoreactivity was localized in sensory cells, and also, the possibility that GABA may be an afferent neurotransmitter in the chick vestibular endorgans.

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The distribution of cholin acethyltranseferase-like immunoreactivities (CATI) and glutamic oxaloacetic transaminase-like immunoreactivities (GOTI) were studied immunohistochemically in the auditory and vestibular system of rat brain. CATI neurons were iocated in the superior olivary nuclear complex, the medial vestibular nucleus and the prepositus hypoglossal nucleus. Both cytosolic (s-) and mitochondrial (m-) GOTI neurons and fibers were unevenly distributed in the all auditory and vestibular nuclei. However, the Iatelar vestibular nucleus contained only m-GOTI neurons. These findings suggested that acethylcholine and glutamate/aspartate play important roles in the central auditory and vestibular system.

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The distribution of cells which showed calcitonin gene-related peptide (CGRP). like immunoreactivity in the superior olivary complex was examined in mature and premature rats by avidin-biotin complex-alkaline phosphatase method. In mature rats immunoreactivity was observed in the dorsomedial part of the lateral superior olive, but not in the medial superior olive. The immunoreactivity was not observed at 2 weeks after birth and appeared at 3 weeks after birth, when cochlear microphonics are known to appear. In conclusion it seems that CGRP is associated with lateral olivocochlear system and have some significance for functional role of its system.

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In order to clarify injury of the inner ear and of the brain after head trauma, we observed morphological changes in guinea pigs after experimentary induced trauma. The relationship between morphological changes and function were studied in some of the animal with the use of auditory brain stem response (ABR). We performed a direct blow to the bulla in 16 guinea pigs and after one week histological changes and ABR were examined. (Nembutal was injected intraperitoneally for anesthesia.)

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The auditory brain stem response can be used as a functional test of brain activity in comatose patients. In these patients, a variety of pathological and physiological conditions is frequently found. In the present paper, the effects of blood pressure, blood respiratory gas level and rectal temperature on ABR in guines pig were investigated. When the mean blood pressure dropped 32.9% from the value of control, there were no change in the record of ABR. Further more, in no case was the ABR affected by high value of PaO₂ on normothermic, hypothermic and after rewarming conditions. The rectal temperature was manipulated from 37°C to 27°C, and the data indicated that a reduction of 1°C in rectal temperature was associated with an increase of 5.9% in the interval between waves I to IV. It should be emphasized the measurement of body temperature requires attention and control in the ABR interpretation.

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Birds and mammals are known to be two phylogenic branches of warm-blooded and have many things in common as to their bilogical mechanisms. For the sake of aviation, birds are supposed to have bette communication system. Comparisons of function and morphology of the inner ear have been made between birds and mammals. The ABR was utilized as an indicator of audition of birds. Male parakeets were used. Daily intra-muscular injections of Kanamycin (100, 150 and 200mg/kg) were given and changes in ABR were observed. Hearing ability of the birds was disturbed in every case as judged from ABR. The ABR was more deeply affected by the size of daily dose rather than by the total dosage. Complete deafness was not brought about in these experiments. Changes in ABR were reversible in experiments of shorter duration.

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Cochlear damage of the Hamster induced by intramuscular injection of KM was studied using ABR and SEM. The following findings were obtained.
1) The latency of wave I was almost no change in ABR.
2) Two cases had the sudden elongation of wave I and the threshold shift.
3) The changes in the organ of Corti were apparent in the outer hair cells of the lower turn. The change in the inner hair cells were slight.
4) The damage of first row was severest of outer hair cells, but in some cases, the damage of second and third row was severer in the middle turn.
5) In the stria vascularis, the absence of microvilli and the protrusion of marginal cells were recognized.

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Kanamycin (KM) was injected in muscle of young and adult rats. Secondary effects on Ganglion cells were detected.
Secondary degeneration occurred after destruction of hair cells which were induced by KM.
The degree of damage depended upon the dose of KM and the time after administration.
The Spiral Ganglion Cells were lost from about 25 days after administration.
The degeneration reached a plateau after 150 days, leaving some cells even after 300 days.
The myelin sheath became loose and as the damage became hard, mitochondria and endoplasmic reticulums showed swollen.
Unmyelinated cells were rich in mitochondria, endoplasmic reticulums, etc...

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The previous biochemical studies suggested that Cepharantin has the potency to stabilize the structure of the cell membrane. The present study was performed to evaluate the preventive effect of Cepharantin on the hair cell damage by Kanamycin (KM) in guinea pigs. KM was injected into 5 animals at 200mg/kg i. m. for 4 weeks and Cepharantin into another 5 ones at 5mg/kg i. p. for 4 weeks immediately before KM injection. Pinna reflex test (20 -0.5 kHz) and supravital histochemical reduction reaction with Nitro-BT indicated that Cepharantin has a relatively effective potency to prevent the damage of the outer and inner hair cells by KM. However, the outer hair cells in the first raw of the spiral organ and in the first turn of the cochlea were prevented insufficiently from the damage.

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Carboplatin (CBDCA, Cis-diammine [1, 1-cyclobutanedicarboxylato] platinum) is synthetic second generation chemotherapeutic platinum analog with greater antitumor activity, but less nephrotoxicity than cisplatin. Few studies on the ototoxicity of CBDCA have been reported. The purpose of this study is to compare ototoxicity of carboplatin and its transferability into inner ear with those of cisplatin. Hair cell damage was determined under a phase contrastmicroscopy with the surface preparation technique and scanning electron microscopy. Cisplatin showed severe damage of outer hair cells of the cochlea, but carboplatin induced no cochlear damage. Carboplatin treated animals showed higher platinum concentration in the serum and perilymph at 1 hour after injection, but lower concentration at 24 hours after injection than cisplatin treated animals.

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Eighteen guinea pigs received infrasound (1, 10, 20Hz) at 120-163dB SPL for one hour. They were sacrificed fourteen days after the exposure. The temporal bones were examined light microscopically. Pathological findings were loss and degeneration of outer hair cells and globus formation on the under surface of the tectorial membrane in the apical turn of the cochlea. These morphological changes were noted in the ears exposed by 20Hz at 163dB SPL. The stria vascularis and the spiral ligament were normal in all ears examined. There were no atrophy of the spiral and vestibular ganglion cells and of the cochlear and vestibular nerves in all experimented animals. The saccule, the utricule and semicircular canals were normal in all sectioned specimens. It is concluded from the present light microscopic observation that one hour exposure of the infrasound below 140dB SPL does not induce any morphological changes in the guinea pig.

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The albino guinea pigs with normal Preyer's reflex were exposed to 2kHz pure tone of 80, 100 and 120dB SPL for 30, 60 and 120 minutes and the effects upon CM and AP were examined. The AP was far more sensitive to intense sound than CM and its threshold sift was proportional to the sound energy given to the animals, indicating the validity of the equal energy principle. The elevation of the AP threshold was the largest at 3kHz in case of 100dB×30 min but the largest elevation moved into 8kHz in 120dB×30min, whereas the CM was the most sensitive at 1kHz in any stimulus condition. The measurements 7 days after the exposure of the sound revealed the recovery of the potential was better in CM than AP. It was concluded that the response to the intense sound was different in CM and AP.

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32-P was most rapidly incorporated phosphatidic acid, phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidy1-4, 5-bisphosphate. This rate of incorporation was uneffected by sound. When the hydrolysis of prelabelled lipids was measured in “chase” experiment, the results indicated a decrease in the relative 32-P content of phosphatidylinositol 4, 5-bisphosphate with intense sound stimulation in the organ of Corti. There were no significant changes in the other phosphoinositides. It was suggested that the sound stimulation increased the turnover of phospholipid, especially the hydrolysis of phosphatidyl-inositide 4, 5-bisphosphate in the organ of Corti.

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The effect of noise exposure on the endocochlear potential (EP), and K⁺, Na⁺, Cl^-, H⁺, HCO^-₃ and Ca²⁺ concentrations in endolymph were investigated using ion-selective microelectrodes. Albino guinea pig was anesthetized and double-barreled ion-selective microelectrode was inserted into scala media of the basal turn through the round window. At the same time, animal was exposed to a pure tone of 2000Hz at 120dB SPL for 30min which was generated with a probe-tube microphone in a closed system. The noise induced a slight increased of the K⁺ concentration and a slight alkalinization, but do little change of the Na⁺, Cl^- and HCO^-₃ concentrations in endolymph. The findings of H⁺ and HCO^-₃ imply the depression of Pco, which suggests to be due to an increase of the blood flow to the cochlea. On the other hand, the Ca²⁺ concentration showed a marked and abrupt elevation by noise exposure. In contrast, no significant change of the Ca²⁺ concentration exhibited in hair cell damaged cochlear. We discuss the mechanism of the noise-induced Ca²⁺ elevation in endolymph and its effect on hearing.

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The ultrastructural observations were performed on neuromasts in lateral line systems of guppies (Poecilia reticulatus var.) under transmission electronmicroscope (H-700) with accelerating voltage of 200kV by complete serial section technique. We described the differences of the morphological quantity and features of the synaptic body (SB) between acoustically stimulated and not stimulated on the sensory hair cells of 39 guppies with noise exposure (65dB of tone burst at 200Hz for 45min) and 30 those without noise exposure were examined. The latter was designated as a control. Although 5 out of 28 (14.3%) non-stimulated sensory cells (SCs) possessed synaptic bodies (SBs) (4 globoid, single discoid), 19 SBs were found in 17 out of 37 (37.8%) stimulated SCs (10 SBs showed globoid shape, 7 were discoid and 2 seemed to lose the electron-density.). So SB might take an active part in neurotransmission and show metabolic change in cytoplasm of SC.

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To know whether the olivocochlear bundle (OCB) has a protective function to acoustic trauma, guinea pigs were exposed to 2kHz pure tone of 120dB SPL with the stimulation or section of OCB and the effects upon AP and CM were analysed. In comparison with the results in control animals, the AP threshold and CM amplitude were significantly less damaged in the OCB stimulated group but there was no remarkably increased sensitivity to acoustic trauma in the OCB sectioned group. The recovery process was faster in the OCB sectioned group than in other two. It was concluded that OCB has some protective role to the cochlea in case of intense sound exposure.

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In order to investigate the state of microcirculation during loud sound exposure, the intracochlear partial oxygen pressure was measured with an oxygen-sensitive microelectrode having a tip of 2 μm in diameter and with a needle type electrode with a 100 μm tip. In the region including the scala vestibuli of the second turn, the scala media and the scala tympani of the first turn, no significant increase or decrease of the PO₂ were recorded. The measurement of the systemic blood pressure was also performed but no significant change was observed during sound exposure.

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It is generally thought that the hypothermic condition reduces the metabolic rate of the tissue of the sensory cells and the energy exhaustion by intense sound is one of the causes of acoustic trauma. To examine the influence of hypothermia upon the acoustic trauma, the albino guinea pigs were exposed to intense sound (2 kHz pure tone at 120 dB SPL for 30 minutes) at normal and reduced body temperature (31, 28, and 25°C) and the effects upon AP and CM were evaluated between two conditions. In hypothermic groups, the CM and AP amplitudes were less reduced and the threshold shift smaller than in the normal group. The results obtained in the present study indicate that hypothermia suppresses energy consumption during intense sound exposure resulting in protection of the cochlea from acoustic injury.

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A total of fifty two guinea pigs has been exposed to a two octave band noise centered at 4 KHz at 92 dB SPL for 30 minutes. These animals were divided into two groups; one is normal (control), the other is cochlear aqueduct-blocked animals. Input-output function of CM was deteriorated in 57% of the control animals, and 50% of the cochlear aqueduct-blocked animals. However, when two groups of the animals were compared in difference between the weighted noise level and the sound level to produce maximum output of CM, the CM was found to be deteriorated in 50% of the cochlear aqueduct-blocked animals, but only 16.6% of the control animals. These findings suggest that patency of the cochlear aqueduct may play an important role in noise-induced hearing loss.

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In 8.0 d embryo as viewed by SEM, the integumentary cells were generally characterized by scattered microvilli and central cilia. The cilia were peculiary devoid of a central pair of fibers as revealed by TEM. The temporal regions of 8.5-9.5 d embryos demonstrated a series of developmental stages of the otic vesicle formation. Concomitantly, microvilli and cilia turned atrophic or vanished completely from the body surface. In the sensory kinocilia of the adult mice, however, we confirmed that the embryonic cilia, lacking the central pair, were preserved.
The sensory epithelium of the inner-ear will be regarded as a part of embryonic epiderm which has survived the ontogeny of the terrestrial vertebrate under the protection of the submerged environment of the closed inner-ear formation.

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The ear region in the 10th gestation day mouse embryo was explanted into the organ culture system and was cultured for 4 days to 10 days. BJGb medium (Fitton-Jackson modification) was used in this study. Fetal bovine serum and penicillin-streptomycin solutions were added to the medium. The temperature in the incubator was kept at 37°C and the atomosphere in incubator was also kept constantly with carbon dioxide in 5%. The explants grew on the Falcon organ culture dish.
Seventy seven of 90 cultured specimens from the mouse embryos were serially sectioned at the thickness of 5 microns, stained with hematoxylin and eosin solution and studied histologically with the light microscope.
The tissues of ear region cultured for 4 or 5 days showed the developing sensory epithelium in the inner ear, cartilaginous otic capsule, acoustico-facial ganglion cells, facial nerve fibers and anlage of ossiles in their histological specimens.
The tissues cultured for 5 days were observed histologically as similar as in the same stage of developing ear region in vivo.

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Ossification process of the otic capsule and long bones of the rats were observed with microradiographical technique. Animals used were non-treated and vitamine A treated rats. The otic capsule and knee joints including femur and tibia were removed. The materials were embedded with polyester resine and grinded by grindstone and frosted glass. The microradiography was performed by soft x-ray using SOFRON SPO-50S and non-decalcified sections were stained with H-E staining. New bone formation takes place on the part of canalicular division of otic capsule but calcification is weak on the 5th day. Otic capsule removed from 30-day-old rats were entirely calcified. Hypervitaminosis A causes the degeneration of cartlage and demineralization of otic capsule removed from young rats.

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Authors showed the inhibitory effect of Cepharanthin, a kind of Alkaloid, on the teratogenicity of maternal hypervitaminosis A in the rat in the previous reports. In the present study, the similar inhibitory effect of Cepharanthin was confirmed in the mouse too. The ddN mother mice were injected intraperitoneally excessive Vitamin A on the 8th day of pregnancy and some of them were injected simultaneously Cepharanthin in the same way. The mouse fetuses from mothers treated with excessive Vitamin A showed external abnormality much more than fetuses from mothers treated with Vitamin A and Cepharanthin. Besides, internal malformations in the ear region observed by means of bone and cartilage staining showed inhibitory effect of Cepharanthin on teratogenicity of excessive Vitamin A as like in the rat.

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The carrassius, fresh water fish, has semicircular canal, utricle and saccule, but has no cochlea. We observed the structure of membranous labyrinth stained by HE.
In this fish, osseous or cartilaginous labyrinthine capsule was not formed. Membranous labyrinth was composed with cartilage, pars superior (semicircular canal and utricle) exposed in the cranial cavity, whereas pars inferior (saccule and lagena) located at the skull base and was immersed in cerebro spinal fluid.
Osseous labyrinth was seen at the part of semicircular canal, and succule and lagena. That is, closed perilymphatic space did not exist in this fish. Endolymphatic duct was not observed in the carrasius in this study.

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Colored guinea pigs were studied in this paper. We used guinea pigs with black coat in the large part of the body, which could be obtained by mating with black animals. Also 3 colored (black, brown and white) guinea pigs, with one external ear colored and other ear uncolored, were used. Membranous labyrinth were dissected from these animals. Surface preparation of stria vascularis and utricular membrane were observed in the unstained specimen and distribution of melanin was compared among these groups. Melanin of these colored guinea pigs was found to distribute in the similar grade. The grade of coat and location of the color did not influence on the quantity of melanin in the inner ear. The distribution and quantity of melanin in the inner ear was found to be independent on the coat color.

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A specific staining of the chochlear hair cell stereocilia was investigated according to Santi's method. The inner and three rows of the outer hair cell stereocilias were well stained by alcian blue and were easily visualized against back ground. The basis of this phenomenon is an affinity between cations of dye and polyanions of sulfated compounds in the stereocilia.
We also examined other cationic dyes such as neutral red and toluidine blue, but those stainabilities for the cochlear tissue were not so good and no staining specificity was recognized. Further investigations about their optimum pH, concentration, and temperature on staining are necessary.
This cochlear hair cell staining will contribute to functional analysis rather than morphological analysis of the stereocilia.

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