Three-dimensional cultured ampullae from rats as a screening tool for vestibulotoxicity: Proof of concept using styrene.

Numerous ototoxic drugs, such as some antibiotics and chemotherapeutics, are both cochleotoxic and vestibulotoxic (causing hearing loss and vestibular disorders). However, the impact of some industrial cochleotoxic compounds on the vestibular receptor, if any, remains unknown. As in vivo studies are long and expensive, there is considerable need for predictive and cost-effective in vitro models to test ototoxicity. Here, we present an organotypic model of cultured ampullae harvested from rat neonates. When cultured in a gelatinous matrix, ampulla explants form an enclosed compartment that progressively fills with a high-potassium (K+) endolymph-like fluid. Morphological analyses confirmed the presence of a number of cell types, sensory epithelium, secretory cells, and canalar cells. Treatments with inhibitors of potassium transporters demonstrated that the potassium homeostasis mechanisms were functional. To assess the potential of this model to reveal the toxic effects of chemicals, explants were exposed for either 2 or 72 h to styrene at a range of concentrations (0.5-1 mM). In the 2-h exposure condition, K+ concentration was significantly reduced, but ATP levels remained stable, and no histological damage was visible. After 72 h exposure, variations in K+ concentration were associated with histological damage and decreased ATP levels. This in vitro 3D neonatal rat ampulla model therefore represents a reliable and rapid means to assess the toxic properties of industrial compounds on this vestibular tissue, and can be used to investigate the specific underlying mechanisms.

An in vitro model to assess the peripheral vestibulotoxicity of aromatic solvents.

Epidemiological and experimental studies indicate that a number of aromatic solvents widely used in the industry can affect hearing and balance following chronic exposure. Animal studies demonstrated that long-term exposure to aromatic solvents directly damages the auditory receptor within the inner ear: the cochlea. However, no information is available on their effect on the vestibular receptor, which shares many structural features with the cochlea and is also localized in inner ear. The aim of this study was to use an in vitro approach to assess and compare the vestibular toxicity of different aromatic solvents (toluene, ethylbenzene, styrene and ortho-, meta-, para-xylene), all of which have well known cochleotoxic properties. We used a three-dimensional culture model of rat utricles ("cysts") with preserved functional sensory and secretory epithelia, and containing a potassium-rich (K+) endolymph-like fluid for this study. Variations in K+ concentrations in this model were considered as biomarkers of toxicity of the substances tested. After 72 h exposure, o-xylene, ethylbenzene and styrene decreased the K+ concentration by 78 %, 37 % and 28 %, respectively. O- xylene and styrene both caused histopathological alterations in secretory and sensory epithelial areas after 72 h exposure, whereas no anomalies were observed in ethylbenzene-exposed samples. These in vitro results suggest that some widely used aromatic solvents might have vestibulotoxic properties (o-xylene, styrene and ethylbenzene), whereas others may not (p-xylene, m-xylene, toluene). Our results also indicate that variations in endolymphatic K+ concentration may be a more sensitive marker of vestibular toxicity than histopathological events. Finally, this study suggests that cochleotoxic solvents might not be necessarily vestibulotoxic, and vice versa.

Styrene alters potassium endolymphatic concentration in a model of cultured utricle explants.

Despite well-documented neurotoxic and ototoxic properties, styrene remains commonly used in industry. Its effects on the cochlea have been extensively studied in animals, and epidemiological and animal evidence indicates an impact on balance. However, its influence on the peripheral vestibular receptor has yet to be investigated. Here, we assessed the vestibulotoxicity of styrene using an in vitro model, consisting of three-dimensional cultured newborn rat utricles filled with a high­potassium (K+) endolymph-like fluid, called "cysts". K+ entry in the cyst ("influx") and its exit ("efflux") are controlled by secretory cells and hair cells, respectively. The vestibular epithelium's functionality is thus linked to K+ concentration, measured using a microelectrode. Known inhibitors of K+ efflux and influx validated the model. Cysts were subsequently exposed to styrene (0.25; 0.5; 0.75 and 1 mM) for 2 h or 72 h. The decrease in K+ concentration measured after both exposure durations was dose-dependent, and significant from 0.75 mM styrene. Vacuoles were visible in the cytoplasm of epithelial cells from 0.5 mM after 2 h and from 0.25 mM after 72 h. The results presented here are the first evidence that styrene may deregulate K+ homeostasis in the endolymphatic space, thereby altering the functionality of the vestibular receptor.

Effects of subchronic exposure to styrene on the extracellular and tissue levels of dopamine, serotonin and their metabolites in rat brain.

At present, there is controversy over the neurotoxic potential of styrene. Several epidemiological and clinical studies have shown that styrene exposure causes alterations of central nervous system functions in humans. Neurotransmitters have been implicated in the pathogenesis of styrene neurotoxicity in rodents. Several studies carried out on postmortem brain tissue suggest that styrene may alter dopaminergic neurotransmission in rabbit or rat brain. Moreover, in vitro studies suggest that both styrene and styrene oxide inhibit the uptake of dopamine (DA) in purified synaptic vesicles prepared from rat brain striata. To date, biochemical studies on animals have explored global tissue levels of neurotransmitters with sub-acute exposures to styrene. However, extracellular levels of neurotransmitters are more closely related to behaviour than are global tissue levels. The present study determined changes in the extracellular concentrations of DA, serotonin (5-HT) and their acid metabolites, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA), in striatal dialysates from freely moving adult male rats after exposure to 750 and 1,000 ppm styrene, 6 h per day, 5 days per week for 4 weeks. We also determined the concentrations of DA, 5-HT and their acid metabolites in striatum, nucleus accumbens and prefrontal cortex obtained postmortem from similarly exposed rats. Exposure to 1,000 ppm of styrene caused a significant decrease in extracellular acid metabolite concentrations. Tissue levels of acid metabolites were also decreased to a lesser extent. The effects were observed 72 h after discontinuing exposure but had vanished 17 days later. There was no change in DA or 5-HT concentrations either in the dialysates or tissues. Exposure to 750 ppm styrene caused no changes in the concentrations of DA, 5-HT and their acid metabolites either in the dialysates or tissues. The possibility that the effect of styrene is mediated by monoamine oxidase (MAO) inhibition is discussed.

Repeating triplets of spikes and oscillations in the mitral cell discharges of freely breathing rats.

The olfactory bulb responses to odours display evident temporal organization, both in the form of high-frequency oscillations and precisely replicating triplets of spikes. In this study, the frequency of replicating triplets in a sample of 118 individual responses from 45 cells was compared with that in simulations of non-homogeneous Poisson processes, constructed from the experimental post-stimulus time histograms (PSTHs). In a large majority of the records, replicating triplets (to a precision of 0.5 ms) are found to be more numerous in the physiological records; in some of them, they are approximately 10 times more abundant. An excess of precisely replicating triplets is also found in records where no oscillations are apparent in the autocorrelograms. Triplet replication thus seems a more robust phenomenon than transient oscillation. Not unlike fast oscillations observed in other preparations, replicating triplets produced by a given mitral cell are generally observed only during a restricted period of time of the respiratory cycle (at least in the case of the responses under olfactory stimulation). No relation was found, however, between the nature and strength of the olfactory stimulus and the frequency of replicating patterns. In the absence of olfactory stimulation, some mitral cell discharges also contain more replicating triplets than the non-homogeneous Poisson simulations. Thus, replicating triplets in single-cell discharges seem to play only an indirect role in the coding of olfactory information at the mitral cell output level.

Short-lasting exposure to one odour decreases general reactivity in the olfactory bulb of adult rats.

We investigated in adult rats whether a relatively short exposure to a novel odour can lead to changes in reactivity of olfactory bulb principal neurons. Naive rats were exposed to isoamyl acetate for 20 min per day either for 6 consecutive days or for a single 20-min exposure. Control group was non-exposed. Under anaesthesia, responsiveness of each recorded single mitral/tufted cell was tested towards isoamyl acetate and four other odours. Results show that the proportion of responding cells in the exposed groups decreased drastically when compared to controls. In the two experimental groups recorded 24 h following the last exposure, mitral/tufted cells show a significant decrease in the number of excitatory responses. In parallel, the number of non-responsive cells increased by at least a fourfold factor. This decrease in reactivity was not selective towards the odour used during the exposure but concerned any of the five test-odours presented during recordings. Finally, this lower responsiveness was long lasting as it was still observed 10 days after the end of the last exposure. This preliminary study points out the importance of even limited sensory experience in neural representation of odours.

Olfactory bulb output cell temporal response patterns to increasing odor concentrations in freely breathing rats.

This study compares the single-unit responses of 74 mitral/tufted cells recorded in freely breathing rats to step increases of the intensity of five odorants from 2 x 10(-4) to 10(-1) of saturated vapor pressure. It reveals a stability of the responses of these olfactory bulb output cells. Olfactory stimulation has frequently been shown to produce a strong patterning of mitral/tufted cell discharges highly correlated with respiration. In this study, cells were generally found to show the same response type to two consecutive concentrations, and only a few cells switched their response from excitation to suppression or vice versa. Their firing peak and/or trough occupied the same position in a high proportion of respiratory cycles recorded during a stimulation, and they remained significantly time-locked to the same respiratory epoch for the next higher concentration. Increasing odor concentration did not cause the mean firing frequency of individual cells during a peak to change appreciably between successive or extreme concentrations. By contrast, it tended to shift their maximum frequency during this peak towards an earlier respiratory cycle after stimulation onset. These results are compared with data reported in other electrophysiological studies and with results given by olfactory bulb models before being discussed for their implications in odor coding.

Recording the slow potentials evoked by odors in the olfactory mucosa of awake animals.

The aim of this report is to expose several improvements which are essential for obtaining good recordings from the basal side of the olfactory mucosa of awake rats with Ag-AgCl electrodes implanted through holes drilled in the roof of the nasal bone. In a first step, we present how this minimally invasive method was developed and validated in anesthetized rats. We insist particularly on several important points such as the size and form of the electrode tip, the careful deposit of silver chloride on this tip or the location of the implanting site. Then we demonstrate that the recorded signals have the characteristics of an electro-olfactogram (EOG), i.e that they have a local origin, that they change with odors and concentrations, and that they do not appear during pure air delivery, nor after ipsilateral nostril closure. Lastly we show that this method was successfully utilized in awake rats. In provided data demonstrating the rhythmicity of EOGs in freely-breathing animals and allowed us to study their relationships with respiration.