Neurotrophins and their receptors in rat peripheral trigeminal system during maxillary nerve growth.

We examined the expression of the neurotrophins (NTFs) and their receptor mRNAs in the rat trigeminal ganglion and the first branchial arch before and at the time of maxillary nerve growth. The maxillary nerve appears first at embryonic day (E)10 and reaches the epithelium of the first branchial arch at E12, as revealed by anti-L1 immunohistochemistry. In situ hybridization demonstrates, that at E10-E11, neurotrophin-3 (NT-3) mRNA is expressed mainly in the mesenchyme, but neurotrophin-4 (NT-4) mRNA in the epithelium of the first branchial arch. NGF and brain-derived neurotrophic factor (BDNF) mRNAs start to be expressed in the distal part of the first brachial arch shortly before its innervation by the maxillary nerve. Trigeminal ganglia strongly express the mRNA of trkA at E10 and thereafter. The expression of mRNAs for low-affinity neurotrophin receptor (LANR), trkB, and trkC in trigeminal ganglia is weak at E10, but increases by E11-E12. NT-3, NT-4, and more prominently BDNF, induce neurite outgrowth from explant cultures of the E10 trigeminal ganglia but no neurites are induced by NGF, despite the expression of trkA. By E12, the neuritogenic potency of NGF also appears. The expression of NT-3 and NT-4 and their receptors in the trigeminal system prior to target field innervation suggests that these NTFs have also other functions than being the target-derived trophic factors.

Indispensable role of Mdm2/p53 interaction during the embryonic and postnatal inner ear development.

p53 is a key component of a signaling network that protects cells against various stresses. As excess p53 is detrimental to cells, its levels are tightly controlled by several mechanisms. The E3 ubiquitin ligase Mdm2 is a major negative regulator of p53. The significance of balanced p53 levels in normal tissues, at different stages of lifetime, is poorly understood. We have studied in vivo how the disruption of Mdm2/p53 interaction affects the early-embryonic otic progenitor cells and their descendants, the auditory supporting cells and hair cells. We found that p53 accumulation, as a consequence of Mdm2 abrogation, is lethal to both proliferative progenitors and non-proliferating, differentiating cells. The sensitivity of postmitotic supporting cells to excess p53 decreases along maturation, suggesting that maturation-related mechanisms limit p53's transcriptional activity towards pro-apoptotic factors. We have also investigated in vitro whether p53 restricts supporting cell's regenerative capacity. Unlike in several other regenerative cellular models, p53 inactivation did not alter supporting cell's proliferative quiescence nor transdifferentiation capacity. Altogether, the postmitotic status of developing hair cells and supporting cells does not confer protection against the detrimental effects of p53 upregulation. These findings might be linked to auditory disturbances observed in developmental syndromes with inappropriate p53 upregulation.

Rescue of hearing, auditory hair cells, and neurons by CEP-1347/KT7515, an inhibitor of c-Jun N-terminal kinase activation.

We have studied the mechanisms of auditory hair cell death after insults in vitro and in vivo. We show DNA fragmentation of hair cell nuclei after ototoxic drug and intense noise trauma. By using phospho-specific c-Jun-N-terminal kinase (JNK) and c-Jun antibodies in immunohistochemistry, we show that the JNK pathway, associated with stress, injury, and apoptosis, is activated in hair cells after trauma. CEP-1347, a derivative of the indolocarbazole K252a, is a small molecule that has been shown to attenuate neurodegeneration by blocking the activation of JNK (). Subcutaneously delivered CEP-1347 attenuated noise-induced hearing loss. The protective effect was demonstrated by functional tests, which showed less hearing threshold shift in CEP-1347-treated than in nontreated guinea pigs, and by morphometric methods showing less hair cell death in CEP-1347-treated cochleas. In organotypic cochlear cultures, CEP-1347 prevented neomycin-induced hair cell death. In addition to hair cells, CEP-1347 promoted survival of dissociated cochlear neurons. These results suggest that therapeutic intervention in the JNK signaling cascade, possibly by using CEP-1347, may offer opportunities to treat inner ear injuries.

Cellular retinol-binding protein type I is prominently and differentially expressed in the sensory epithelium of the rat cochlea and vestibular organs.

To understand the possible role of retinoic acid during inner ear development and cellular regeneration, we have examined the expression pattern of two intracellular retinoid-binding proteins, the cellular retinol- and retinoic acid-binding proteins of type I in the developing and mature rat inner ear. Expression of cellular retinol-binding protein type I was seen in the supporting cells of the organ of Corti and vestibular organs as soon as the first signs of differentiation of the adjacent hair cells were seen. In the developing organ of Corti, the expression pattern followed the basal-to-apical coil differentiation gradient. After the 1st postnatal week, detectable expression of cellular retinol-binding protein type I disappeared from the organ of Corti, but persisted in the supporting cells of vestibular organs throughout life. Expression of cellular retinoic acid-binding protein type I was not found in the inner ear sensory epithelia. Cellular retinol-binding protein type I has previously been shown to act as a substrate carrier in the synthesis of retinoic acid from its precursor, retinol. Our data suggest that retinoic acid is synthesized in the developing sensory epithelium of the cochlear and vestibular organs and that a concentration gradient formed by retinoic acid may have a role in differentiation of the cochlear sensory epithelium. Furthermore, retinoic acid may have a role in damage-induced hair cell regeneration in the developing and mature vestibular organs as well as in the developing auditory organ. The absence of cellular retinol-binding protein type I from the supporting cells of the mature organ of Corti may be associated with the inability of this organ to regenerate hair cells after damage.

Histaminelike immunoreactive neurons innervating putative neurohaemal areas and central neuropil in the thoraco-abdominal ganglia of the flies Drosophila and Calliphora.

The fused thoraco-abdominal ganglia of the flies Calliphora vomitoria and Drosophila melanogaster were investigated immunocytochemically with antisera against histamine. In both insect species, 18 histaminelike immunoreactive (HA-IR) neurons were resolved in these ganglia. Six of these neurons have cell bodies in the thoracic neuromeres and 12 in the fused abdominal neuromeres. All cell bodies are situated ventrally. In Calliphora all cell bodies are arranged in a segmental pattern. In Drosophila only the thoracic cell bodies have a segmental arrangement, whereas the abdominal ones are clustered anteriorly close to the last thoracic neuromere. In both species the six thoracic neurons supply processes to the synaptic neuropil in the thoracic neuromeres and to the dorsal neural sheath. The processes in the neural sheath run anteriorly in the lateral portions of the ganglion into the cervical connective. In a few regions laterally arborizing terminals are found in putative neurohaemal areas. These areas were investigated by electron microscopic immunocytochemistry in Calliphora. The HA-IR terminals (containing small granular vesicles) were found outside the "blood-brain barrier" below the acellular basal lamina of the neural sheath. Release of histamine into the circulation is therefore theoretically possible. The central processes of the six thoracic HA-IR neurons may interact synaptically with large numbers of other neurons in the neuropil, and the peripheral varicose fibers from the same HA-IR neurons possibly are neurohaemal release sites. The abdominal HA-IR neurons, in contrast, form extensive arborizations within the synaptic neuropil only. Both thoracic and abdominal neurons have ipsilateral and contralateral branches as well as processes that invade more than one neuromere. A single HA-IR neuron thus invades large volumes of synaptic neuropil. Histamine may be used by neurons of the ventral ganglia both as neurotransmitter (or neuromodulator) and as a circulating neurohormone released from the neural sheath.

Distribution of histamine in the cockroach brain and visual system: an immunocytochemical and biochemical study.

The distribution of histamine-immunoreactivity in the carbodiimide-fixed brain and visual system of the cockroach was revealed immunocytochemically with an antiserum against histamine (HA). Histamine levels were measured with high-pressure liquid chromatography. The results show a widespread distribution of histamine-containing somata and fibers in the brain, particularly in the visual system. The most intense immunolabeling was seen in the retinal photoreceptors and in the first optic ganglion, the lamina, where the short visual fibers make synaptic connections with the monopolar neurons, which also displayed immunofluorescence. Immunoreactive long visual fibers traversed the lamina and outer chiasma, terminating in the distal medulla. Tracts of histamine-immunopositive fibers appeared to link the optic ganglia to the protocerebrum. Prominent histamine-containing neurons were situated in the lateral protocerebrum. Immunolabeled pathways consisting of large-diameter fibers also were seen in the cockroach brain. The central parts of the brain, including the central body, were reached by thick immunoreactive fibers that gave rise to intensely fluorescent varicose processes there. In the mushroom bodies, immunoreactivity was limited to the calyces. The protocerebral bridge was nonreactive. Immunofluorescence was seen also in the antennal lobes, but not in the antennal nerves. The biochemical measurements correlated well with the immunocytochemical data. The retinas and optic lobes, measured together, contained remarkably large amounts of histamine. These results reinforce the hypothesis presented by Hardie ('87) and Elias and Evans ('83) that histamine functions as a neurotransmitter in the photoreceptors of some, if not all, insect species.

A histamine-containing neuronal system in human brain.

A well-organized network of varicose fibers was revealed throughout the frontal and temporal cortex of adult humans with specific antisera against histamine. The densest network of fibers was seen in lamina I, where varicose fibers were seen to run in parallel to the overlying pia mater. Electron microscopic immunohistochemistry revealed histamine-immunostaining in granules in a small number of nerve fibers and varicosities. Hypothalamic samples obtained from autopsy brains of adult humans revealed numerous histamine-immunoreactive nerve cell bodies in the posterior basal hypothalamus in and around the tuberomammillary nucleus. The results suggest that a histaminergic neuronal system reminiscent of that described in rodents is present in human brain.

Histamine-immunoreactive nerve fibers in the rat brain.

A new immunohistochemical method that utilizes carbodiimide as a tissue fixative was applied to study the distribution of histamine-immunoreactive neuronal fibers and terminals in the rat brain. Immunoreactive fibers were observed in almost all major regions of the brain. They were most numerous in the different hypothalamic nuclei. Dense networks of immunoreactive fibers were also seen in the medial septum, nucleus of the diagonal band and ventral tegmental area. A moderate density of fibers was seen throughout the cerebral cortex, in some parts of the olfactory bulb and tubercle, bed nucleus of the stria terminalis, amygdala, basal parts of the hippocampus, inferior and superior colliculi, substantia nigra, lateral and medial parabrachial nucleus, and the nucleus of the solitary tract. Few histamine-immunoreactive fibers were seen in most parts of the caudate putamen, most thalamic nuclei, most pontine and ventral medullary nuclei. Histamine-immunoreactive neuronal cell bodies were found exclusively in the tuberomammillary nucleus, in agreement with previous reports. The results provide evidence for a widespread distribution of histamine-containing nerve fibers and terminals in the rat brain. Although immunohistochemical localization of histamine does not give direct evidence of a functional role of histamine in any brain area, this distribution suggests involvement in functions of the limbic system including the septal nuclei, hypothalamus and amygdala. The relatively dense histamine-immunoreactive fiber networks in the colliculi and dorsal cochlear nucleus indicate that this amine may play a role in visual functions and hearing. The paucity of immunoreactive fibers in the pontine and medullary areas suggests that the caudal projections originating from the tuberomammillary complex are minor ones compared to the major rostral projections. Several fiber projections originating from the tuberomammillary complex could be deduced from serial frontal, sagittal and horizontal sections. They contained fibers that crossed the midline at several levels of the brain. The results provide information on the target areas of the histaminergic neurons and form a basis for the examination of cellular contracts between the histaminergic neurons and other cells.

Neurotrophic factors in the auditory periphery.

Many of the neurotrophic factors promote the survival of developing peripheral sensory neurons, and they might be useful as therapeutic agents in the adult neuronal systems. During development, neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) mRNAs are expressed in the auditory sensory epithelium, which composes the peripheral target field of the cochlear (spiral) neurons. NT-3 mRNA is also expressed in the inner hair cells of the mature organ of Corti. mRNAs encoding their signal-transducing receptors, TrkC and TrkB, respectively, are expressed in the cochlear neurons. In addition to neurotrophins, which seem to have an important role during development, another neurotrophic factor, glial cell-line-derived neurotrophic factor (GDNF), seems to be involved in the maintenance of postnatal auditory neurons. In the present work, the cellular distribution of neurotrophins and GDNF in the developing and adult rat inner ear are compared. The effects of recombinant neurotrophins and GDNF on dissociated cochlear neurons in vitro are also compared. Recently, NT-3 and BDNF were used in vivo as therapeutic agents to protect guinea pig cochlear neurons from aminoglycoside-induced degeneration and GDNF from noise-induced degeneration. These data demonstrate that NT-3, BDNF, and GDNF might be potential candidates for prevention of degeneration of the auditory nerve in man.

Rescue and regrowth of sensory nerves following deafferentation by neurotrophic factors.

Trauma and loss of cochlear inner hair cells causes a series of events that result first in the retraction of the peripheral processes of the auditory nerve, scar formation in the organ of Corti, and over the course of weeks to months (depending on the species) the loss of auditory nerve cell bodies (spiral ganglion cells). Neurotrophic factors play an important role in the mature nervous system as survival factors for maintenance and protection and also can play a role in regrowth. Studies in the cochlea now show that application of exogenous neurotrophic factors can enhance survival of spiral ganglion cells after deafness and induce regrowth of peripheral processes, perhaps by replacing lost endogenous factors. Combinations of factors may be most effective for achieving greatest survival and regrowth. Our studies find that brain-derived neurotrophic factor (BDNF) and glial-line-derived neurotrophic factor (GDNF) are very effective at enhancing spiral ganglion cell survival following deafness from ototoxic drugs or noise. It has also been found that BDNF plus fibroblast growth factor (FGF) is very effective at inducing process regrowth. Electrical stimulation also acts to enhance spiral ganglion cell survival, and the combination of electrical stimulation and neurotrophic factors could prove a most effective intervention.

Histamine-immunoreactive nerve fibers in the mammalian spinal cord.

New sensitive antisera against histamine were used to study the distribution of histamine-immunoreactive nerve fibers in the spinal cord of several mammalian species. Tissues were fixed with carbodiimide by transcardiac perfusion or immersion. A few immunoreactive nerve fibers were found in the cervical spinal cord of the rat in the superficial laminae of the dorsal horn, around the central canal and scattered in the anterior horn. The density of immunoreactive fibers in the cervical spinal cord of the guinea pig and tree shrew was higher, but still low. The densest networks of histamine-immunoreactive fibers were seen in the cervical spinal cord of the pig. The laminar distribution of histamine-immunoreactive fibers was similar in all species. Histamine-immunoreactive fibers were densest in lamina X, followed by laminae I-II. Scattered fibers were also seen in the white matter in the lateral and posterior funiculus in the pig. In the rat and the guinea pig, no histamine-immunoreactive cell bodies were seen in the spinal sensory ganglia. The results suggest that the histamine-immunoreactive nerve fibers in the spinal cord may originate from the brain, probably from the posterior hypothalamus, and the fiber projection is more extensive in higher mammalian species. The role of histamine in the spinal cord is not known, but it may be involved in, e.g., pain sensation.

Distribution of F-actin and fodrin in the hair cells of the guinea pig cochlea as revealed by confocal fluorescence microscopy.

We double-stained paraformaldehyde fixed guinea pig cochleas with rhodaminated phalloidin to detect F-actin and with a monoclonal antibody against non-erythroid spectrin (fodrin). The hair cells were studied in surface specimens of the organ of Corti with confocal fluorescence microscopy. In serial optical sections, phalloidin stained the stereocilia, cuticular plate, and a circumferential ring beneath it in the inner and outer hair cells (IHCs and OHCs). The cytoplasm of the IHCs and the OHCs was unlabelled, but the infracuticular network of the OHCs in the upper turns showed a strong reaction. The lateral plasma membrane was unreactive with phalloidin in the IHCs and OHCs, except in the basal turn, where a moderate reaction, probably representing actin of Deiter's cups, was seen along the lateral walls of the basal pole of the OHCs. Fodrin was similarly seen in the cuticular plate, in a circumferential ring beneath it, and in the infracuticular network of the apical OHCs. The most interesting finding was the fodrin-specific distinct labelling of the lateral cell surface in the OHCs of the basal cochlear turn. This staining diminished towards the apex and was practically absent in the OHCs located above the level of 15 mm from the round window. The lateral cell surface of IHCs showed moderate fodrin labelling in all cochlear turns. This staining was much weaker than that seen in the basal OHCs. Fodrin labelling revealed deformation from the regular cylindrical shape in midportion of the OHC bodies in the basal turn of the cochlea.

Spatiotemporal development of cochlear innervation and hair cell differentiation in the rat.

The apical cytoskeleton of cochlear hair cells is largely comprised of actin microfilaments and actin-associated proteins, of which fodrin is one of the most prominent. We studied the development of this mechanosensory apical portion of cochlear hair cells of the rat by fluorescence microscopy using rhodamine conjugated phalloidin to detect F-actin and an antibody against alpha-fodrin. An antibody against the 160 kDa neurofilament polypeptide was used for tracing nerve fibers. The first sign of differentiation of the mechanosensory region, actin-containing stereocilia, was observed on the 19th gestational day in the inner hair cells of the basal coil. The appearance of expression of cytoskeletal actin in the cochlear hair cells proceeded gradientally from basal to apical coil and from inner to outer hair cells. Corresponding maturation sequences were observed in the development of fodrin immunoreactivity in the cuticular plates, but the first evidence of this reactivity was found one day later than the appearance of stereocilia in the hair cells at the same location. Also the penetration of neurofilament-positive neurites into the sensory epithelium followed the same kind of longitudinal and radial maturation gradients throughout the cochlea. Fibers were revealed beneath the sensory cells shortly before the first appearance of differentiation of their mechanosensory region. The results suggest that ingrowing nerve fibers may influence the timing of the apical cytoskeleton differentiation in cochlear hair cells or that both these processes could be controlled by the same external signals that are gradientally expressed throughout the cochlea.

Nonerythroid spectrin (fodrin) is a prominent component of the cochlear hair cells.

We studied the distribution of nonerythroid spectrin, fodrin, in surface preparations and cryosections of the cochlear hair cells as well as isolated hair cells of the guinea pig by using a monoclonal antibody (Mab) reacting with Mr 240 kD alpha-fodrin polypeptide. The Mab gave a strong reaction with the cuticular plate of both the inner and outer hair cells (IHCs and OHCs). Stereocilia were nonreactive and only a weak cell surface reaction was seen in the supporting cells. In the outer hair cells the upper turns of the cochlea, fodrin was observed in a cytoplasmic spiralling structure extending from the cuticular plate towards the cell nucleus. Some labelling was also seen along the cell surface membrane and in the synaptic region. The results suggest that fodrin may be an important constituent in the active processes of hair cells such as cell motility and/or signal transduction.

Coordinated expression and function of neurotrophins and their receptors in the rat inner ear during target innervation.

We show that trkB and trkC mRNAs, encoding the high-affinity receptor tyrosine kinases for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), respectively, as well as low-affinity nerve growth factor receptor (p75LNGFR) mRNA are expressed in the cochleovestibular ganglion (CVG) before and during innervation of the target fields. Correspondingly, from preinnervation stages onward, BDNF and NT-3, but neither nerve growth factor (NGF) nor neurotrophin-4 (NT-4) mRNAs are expressed in the sensory epithelium of the otic vesicle, the peripheral target field of CVG neurons. No neurotrophin transcripts were detected by in situ hybridization in the medullary central targets. In explant cultures, neuritogenesis from both the cochlear and vestibular part of the CVG was promoted by BDNF, while NT-3 evoked neurites mainly from the cochlear neurons. Also NT-4 stimulated neurite outgrowth from the CVG in vitro. In dissociated neuron-enriched cultures, NT-3 and BDNF promoted survival of overlapping subsets of CVG neurons and, correspondingly, results from in situ hybridization showed that both trkC and trkB mRNAs were expressed in most neurons of this ganglion. The negligible effect of NGF seen in the bioassays agrees well with the expression of only a few trkA transcripts, encoding the high-affinity receptor for NGF, in the CVG. Based on the spatiotemporal expression patterns and biological effects in vitro, peripherally-synthesized BDNF and NT-3 regulate the survival of CVG neurons as well as the establishment of neuron-target cell contacts in the early-developing inner ear. In addition, the expression of trkB mRNA, more specifically its truncated form, and trkC as well as p75LNGFR mRNAs in distinct non-neuronal structures indicates novel roles for these molecules during development.

Expression patterns of neurotrophin and their receptor mRNAs in the rat inner ear.

In situ hybridization was used to study the expression of mRNAs of nerve growth factor (NGF), brain-derived neutrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-5 (NT-5) and the components of their high-affinity receptors in the early postnatal and adult rat inner ears. NGF or NT-5 transcripts were not detected in the inner ear neuroepithelium or in the innervating neurons. NT-3 mRNA was intensely expressed over the one-week-old and adult inner hair cells (IHCs) but in the outer hair cells (OHCs) and vestibular maculae only during the early postnatal period. BDNF mRNA was expressed in the IHCs and OHCs of the early postnatal cochlea but not in the adult organ of Corti. High levels of BDNF transcripts were observed in the sensory epithelia of all vestibular end organs. mRNAs of low affinity NGF receptor, trkB and trkC, but not of trk, were expressed in the spiral and vestibular ganglia. In addition, the non-catalytic form of trkB mRNA localized to the sensory epithelia of maculae utriculi and sacculi. The present results show that of the neurotrophins examined, NT-3 is the predominant neurotrophin in the adult organ of Corti and BDNF is that in vestibular organs. The expression patterns of NT-3 and BDNF mRNAs suggest that these neurotrophins may participate in the maintenance of mature cochleovestibular neurons and they may be involved in the survival response of injured neurons.

Guinea pig auditory neurons are protected by glial cell line-derived growth factor from degeneration after noise trauma.

For patients with profound hearing loss, cochlear implants have become the treatment of choice. These devices provide auditory information through direct electrical stimulation of the auditory nerve. Prosthesis function depends on survival and electrical excitability of the cochlear neurons. Degeneration of the auditory nerve occurs after lesions of its peripheral target field (organ of Corti), specifically, including loss of inner hair cells (IHCs). There is now evidence that local treatment of the cochlea with neurotrophins may enhance survival of auditory neurons after aminoglycoside-induced deafness. Glial cell line-derived neurotrophic factor (GDNF) has recently been shown to be an important survival factor in other regions of the nervous system. By in situ hybridization, we now show that IHCs of the neonatal and mature rat cochlea synthesize GDNF and that GDNF-receptor alpha, but not c-Ret, is expressed in the rat spiral ganglion. We also show that GDNF is a potent survival-promoting factor for rat cochlear neurons in vitro. Finally, we examined GDNF efficacy to enhance cochlear-nerve survival after IHC lesions in vivo. We found that chronic intracochlear infusion of GDNF greatly enhances survival of guinea pig cochlear neurons after noise-induced IHC lesions. Our results demonstrate that GDNF is likely to be an endogeneous survival factor in the normal mammalian cochlea and it could have application as a pharmacological treatment to prevent secondary auditory nerve degeneration following organ of Corti damage.

Immunohistochemical demonstration of neuroactive substances in the inner ear of rat and guinea pig.

The presence and localization of different neuropeptides and other putative neurotransmitters or -modulators were examined by immunohistochemistry in the cochleovestibular end organs and in neurons innervating them in rats and guinea pigs. In the organ of Corti neural elements beneath inner hair cells showed immunoreactivity for enkephalin (ENK), calcitonin gene-related peptide (CGRP), L-glutamate decarboxylase (GAD), substance P (SP) and tyrosine hydroxylase (TH). Nerve chalices of type I vestibular hair cells contained SP and GAD, but not consistently. SP was only occasionally observed in neuronal cell bodies of the 8th cranial nerve but fine fibers with different neuroactive substances were seen in the nerve trunk in the following relative numbers: TH greater than SP greater than CGRP greater than ENK. The present data demonstrate the presence of several different neuroactive substances in the rat and guinea pig inner ear suggesting a multiplicity of neurotransmitters or -modulators in this system.

Hyperbaric oxygen therapy seems to enhance recovery from acute acoustic trauma.

CONCLUSION: The average recovery of hearing and cessation of tinnitus was significantly better after hyperbaric oxygen therapy (HBOT) than after normobaric oxygen therapy (NBOT). HBOT can be valuable adjuvant therapy for patients with acute acoustic trauma (AAT). OBJECTIVES: AAT was one of the early indications for the use of HBOT. The rationale of administering oxygen to patients with AAT is based on experimental studies showing that noise exposure results in cochlear hypoxia, which could be compensated by HBOT. The aim of this study was to investigate the efficacy of HBOT in patients with AAT. PATIENTS AND METHODS: We compared the recovery from hearing impairment and tinnitus in 60 ears treated with HBOT with 60 ears treated with NBOT. The HBOT was given daily for 1-8 days. There were no significant differences in clinical or audiological data between HBOT and NBOT groups. RESULTS: The average recovery of hearing both at high and speech frequencies was significantly better and tinnitus persisted less commonly after the HBOT than after the NBOT. Normal hearing at the end of the follow-up period was regained in 42 ears in the HBOT group and in 24 ears in the NBOT group (p<0.01).

Distribution of taurine in the rat cerebellum and insect brain: application of a new antiserum against carbodiimide-conjugated taurine.

The production, specificity and application of an antiserum against taurine conjugated to succinylated ovalbumin by means of 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide is reported. The antiserum was produced in rabbits. The carbodiimide was used also as a tissue fixative. The development of the antibody titre was followed by dot-blot tests on nitrocellulose filters using different amino acid conjugates and with immunohistochemical reaction in the rat and insect brain. Blocking controls were also used. Taurine antiserum, sufficiently specific and sensitive, developed after the fourth booster injection, after which the antiserum was characterized. In the insect brain, intense taurine-like immunoreactivity was observed in the photoreceptors, in the Kenyon cells and the neuropile of the mushroom bodies, in the lower part of the central body and in the antennal lobes. In the rat carebellum, intense taurine-like immunoreactivity was seen in the Purkinje cells. Immunoreaction was seen also in small cells most probably corresponding to the basket cells. The use of the carbodiimide in the production of antisera against taurine provides a parallel method for comparison of the distribution of taurine-like immunoreactivity obtained with antisera made against conjugates prepared with aldehydes.