Cloning and characterization of alpha9 subunits of the nicotinic acetylcholine receptor expressed by saccular hair cells of the rainbow trout (Oncorhynchus mykiss).

alpha9/alpha10 Subunits are thought to constitute the nicotinic acetylcholine receptors mediating cholinergic efferent modulation of vertebrate hair cells. The present report describes the cloning and sequence analysis of subunits of the alpha9-containing receptor of a hair-cell layer from the saccule of the rainbow trout (Oncorhynchus mykiss). A major alpha9 subunit, termed alpha9-I, displayed typical features of a nicotinic alpha subunit, with total coding sequence of 572 amino acids including a 16 amino-acid signal peptide. It possessed an extended cytoplasmic loop between membrane-spanning regions M3 and M4, compared with mammalian homologs. Transcript for alpha9-I was robustly expressed in the saccular hair cell layer and less prominently in trout olfactory mucosa, spleen, pituitary gland, and liver, as determined by reverse transcription-polymerase chain reaction. alpha9-I cDNA was not detected in trout brain, skeletal muscle, retina, and kidney. The alpha9-I nicotinic receptor protein was immunolocalized, with an affinity-purified antibody directed against a trout alpha9-I epitope, to hair-cell and neural sites in the saccular hair-cell layer. Foci were found at basal and basolateral membrane sites on hair cells as well as on afferent nerve. Receptor clustering was observed in hair cells bordering non-sensory epithelium. Since in higher vertebrates the alpha9 is reported to associate with another nicotinic subunit, alpha10, we examined the possibility of expression of additional nicotinic subunits in trout saccular hair cells. Message for another nicotinic subunit, termed alpha9-II, was found to be expressed in the hair cells, although more difficult to amplify than alpha9-I. In contrast to alpha9-I, alpha9-II was expressed in brain, as well as in olfactory mucosa, less prominently in pituitary gland and liver, but not in spleen, skeletal muscle, retina, or kidney. The cloned alpha9-II had a total coding sequence of 550 amino acids, which included a 17-amino-acid signal peptide, and an extended M3-M4 loop. A third nicotinic subunit message, termed alpha9-III, was PCR-amplified from trout olfactory mucosa where it was strongly expressed. However, message for alpha9-III was not detected in hair cells. Message for alpha9-III was moderately expressed in trout brain, retina, and pituitary gland but not in trout spleen, skeletal muscle, liver, and kidney. Thus, alpha9-I and alpha9-II may together contribute to the formation of the hair-cell nicotinic receptor of teleosts, where no ortholog of alpha10 appears to exist. The current work is, to our knowledge, the first description of alpha9 coding sequences directly from a vertebrate hair cell source. Further, the generality of hair cell expression of subunits for the alpha9-containing nicotinic cholinergic receptor has been extended to fishes, suggesting a similar efferent mechanism across all vertebrate octavolateralis sensory systems.

Vestibular afferent responses to microrotational stimuli.

Intracellular microelectrode recording/labelling techniques were used to investigate vestibular afferent responses in the bullfrog, to very small amplitude (less than 0.5 degree p-p) sinusoidal rotations in the vertical plane over the frequency range of 0.063-4 Hz. The axis of rotation was congruent with the axis of the anterior semicircular canal. Robust responses to peak accelerations as low as 0.031 degree/S2 were obtained from units subsequently traced to either the central portion of the anterior canal crista or the striolar region of the utricle. All of these microrotationally sensitive afferent neurons had irregular resting discharge rates and the majority had transfer ratios (relative to rotational velocity) of 1-40 spikes/s per degree/s. Individual utricular afferent velocity transfer ratios were nearly constant over the frequency range of 0.125-4 Hz. Canal units generally displayed decreasing response transfer ratios as stimulus frequencies increased. These findings indicate that although utricular striolar and central crista afferent velocity transfer ratios to microrotations were very similar, utricular striolar afferent neurons were more faithful sensors of very small amplitude rotational velocity in the vertical plane.

Hair cell tufts and afferent innervation of the bullfrog crista ampullaris.

Within the bullfrog semicircular canal crista, hair cell tuft types were defined and mapped with the aid of scanning electron microscopy. Intracellular recording and Lucifer Yellow labeling techniques were used to study afferent responses and arborization patterns. Dye-filled planar afferent axons had mean distal axonal diameters of 1.6-4.9 microns, highly branched arbors, and contacted 11-24 hair cells. Dye-filled isthmus afferent axons had mean distal axonal diameters of 1.8-7.9 microns, with either small or large field arbors contacting 4-9 or 25-31 hair cells. The estimated mean number of contacts per innervated hair cell was 2.2 for planar and 1.3 for isthmus afferent neurons. Data on evoked afferent responses were available only for isthmus units that were observed to respond to our microrotational stimuli (less than 3 degrees/s peak rotational velocity). Of 21 such afferent neurons, 8 were successfully dye-filled. Within this small sample, high-gain units had large field arbors and lower-gain units had small field arbors. The sensitivity of each afferent neuron was analyzed in terms of noise equivalent input (NEI), the stimulus amplitude for which the afferent response amplitude is just equivalent to the RMS deviation of the instantaneous spike rate. NEI for isthmus units varied from 0.63 to 8.2 degrees/s; the mean was 3.2 degrees/s.

Forward masking properties of neurons in the dorsal cochlear nucleus: possible role in the process of echo suppression.

The majority of single unit studies in the auditory system have been carried out using stimuli whose temporal and spectral contexts are held constant. Relatively little attention has been given to the influence of context on unit response properties. Indeed, auditory nerve fiber responses are known to be context-dependent due to the property of forward masking, a phenomenon by which the response to one sound results in a reduction in the response to a subsequent sound. Forward masking might be expected to be even more influential at central levels of the auditory pathway where the responses are reshaped by additional synaptic interactions. The purpose of the present study was to characterize the forward masking properties of neurons in the dorsal cochlear nucleus (DCN). A tool was developed for measuring the response to a probe tone as a function of delay following a previous tone-burst. The frequency of the probe was held constant at the unit's characteristic frequency while the frequency of the leading tone (masker) was varied. These measures provided a description of neural masking effects in different temporal and spectral contexts. The data yielded two patterns of suppression. In the first pattern (Type A), the suppression of the probe response became evident immediately following offset of the masker; the suppression bandwidth showed a gradual narrowing as the delay between masker and probe was increased. In the second class (Type B), the suppression of the probe response did not become evident until well after offset of the masker; this pattern appeared more circumscribed in that the suppression bandwidth gradually increased as a function of delay up to a maximum then decreased with further increases in delay. The results imply that mechanisms intrinsic to the DCN contribute to further modification and reshaping of the spectral and temporal context of masking effects beyond those seen in the auditory nerve. It is hypothesized that such properties may be specialized for suppressing the response to echoes thus facilitating communication and localization of sound in enclosed spaces.

Risk factors for ototoxicity due to cisplatin.

OBJECTIVE: To determine whether abnormalities in routine blood tests were associated with increased susceptibility to hearing loss induced by cisplatin chemotherapy. DESIGN: Cohort study of patients with head and neck cancer receiving cisplatin chemotherapy who underwent audiometric testing. SETTING: A large, university-based hospital; part of a larger study regarding rehabilitation of patients with cancer. PATIENTS: Forty-two patients with head and neck cancer who agreed to participate underwent at least three serial treatments with high-dose cisplatin therapy. Routine blood tests and audiometric testing were performed before each course of chemotherapy. One hundred eighty audiograms were performed. OUTCOME MEASURES: A deterioration of auditory threshold of 15 dB or more at one frequency or of 10 dB or more at three or more frequencies was considered a significant loss. Only frequencies at and below 4000 Hz were considered. RESULTS: Multiple analysis of variance results indicated that decreased serum albumin level, hemoglobin level, red blood cell count, and hematocrit were associated with an increased likelihood of significant hearing loss during chemotherapy. CONCLUSIONS: Patients in poor general medical condition with low levels of red blood cells or serum proteins are at increased risk for development of hearing loss from cisplatin chemotherapy. We recommend that hearing be tested before chemotherapy begins and after the first course of cisplatin. If there is no significant hearing loss at or below 4000 Hz at that time, then subsequent audiometric testing is required only if symptoms of hearing loss develop.

Myelin-sheath abnormalities in the vestibular nerves of chronically diabetic rats.

Electron microscopic examination of the saccular and utricular nerves of rats with streptozocin-induced diabetes mellitus revealed differences from age-matched control animals, mainly related to myelin sheaths. Nerves from diabetic rats had a high incidence of a variety of osmiophilic inclusion bodies, often associated with disrupted myelin-sheath lamellae, lysosomal bodies, and periaxonal expansions of Schwann cell cytoplasm. The occurrence of osmiophilic inclusion bodies within nerve profiles was correlated with the severity of diabetes as measured by blood glucose levels (p < 0.001, linear-regression ANOVA). Duration of diabetes was not related to incidence of inclusion bodies. No evidence of demyelination/remyelination or neuronal degeneration was observed. An unusual Schwann cell reaction was noted, involving lysosomal digestion of large, pinched-off portions of myelinated nerve fibers of some diabetic rats. Both this Schwann cell reaction and the high incidence of inclusion bodies may be due to increased nonenzymatic glycosylation of myelin proteins in the diabetic animals.

Patterns of hearing loss resulting from cis-platinum therapy.

The purpose of this study was to provide a clinically useful classification of the hearing loss that occurs with chemotherapy using cis-diamminedichloroplatinum (cis-platinum). Serial audiometric testing was performed on 66 patients who underwent cis-platinum chemotherapy (2 to 6 courses, 100 mg/m2). Subsequent hearing losses were classified according to (1) whether hearing loss occurred after the first or second course of chemotherapy, (2) the magnitude of loss, and (3) the frequencies affected. Of 39 patients who could be classified, 21 (54%) had no loss or mild loss, 14 (36%) had early loss, and 4 (10%) had late loss. Different patterns of hearing loss were observed. The observation that different patterns of hearing loss occur implies that different mechanisms of hearing loss exist. Some mechanisms must be more important in different patients. Our data are important with regard to continuation of chemotherapy treatment after hearing loss occurs. Most significant hearing loss (90%) occurred early or not at all. If early hearing loss occurred and treatment was continued, the speech frequencies were eventually affected in 71% of patients. Recognition of the patterns of hearing loss are important considerations when discontinuation of therapy is being considered.

Is cis-platinum vestibulotoxic?

Serial vestibular testing was performed on 34 patients undergoing cis-platinum chemotherapy and 12 age-matched normal subjects. Vestibular testing comprised caloric and low-frequency, sinusoidal rotational testing (0.01 to 0.16 Hz). Test-retest variability in the caloric and rotational vestibular assessment of the normal subjects formed the basis for evaluation of subclinical loss of vestibular function within our patient population. In spite of high-dose cis-platinum chemotherapy (100 mg/m2) with cumulative dosages to 1600 mg, no clear evidence of vestibular toxicity was found.

Morphometric analysis of horizontal canal nerves of chronically diabetic rats.

Horizontal canal nerves of 3-, 6-, 9-, and 12-month-diabetic rats were compared with those of age-matched controls. The myelin sheaths of the horizontal canal nerves in diabetic rats were thinner than those of age-matched controls (mean +/- SD 0.63 +/- 0.04 micron (n = 16) vs. 0.71 +/- 0.05 micron (n = 9); p < 0.0001, one-tailed t test). Regression analysis revealed that myelin sheath thickness did not correlate with severity of diabetes, but myelin thinning did occur as a function of the duration of diabetes (p < 0.05, regression ANOVA). The progression of myelin thinning over time is consistent with the possibility of an accelerated decline in vestibular function with age in diabetic patients. That myelin thinning did not correlate with the severity of diabetes suggests that this thinning is not directly related to the aging effects attributed to nonenzymatic glycosylation of myelin proteins. Multivariate analysis revealed a significant difference between diabetic and control groups when fiber diameter and intrasheath diameter were considered together (p < 0.008, canonical discriminant-function analysis). Diabetic and control groups did not differ significantly in total nerve fiber counts. In the diabetic group, however, nerve fiber counts were higher in animals with higher blood glucose levels (p < 0.02, linear-regression ANOVA; r2 = 0.49). The finding of higher nerve fiber counts in more severely diabetic rats is consistent with an earlier transmission electron microscopic finding of false myelinated nerve fiber profiles in micrographs from more severely diabetic rats. These false profiles are believed to represent phagocytosis-like Schwann cell reactions against their own myelin, triggered by excess myelin glycosylation.

An evaluation of the head-shaking nystagmus test.

Head-shaking nystagmus can be observed in some patients with vestibular lesions. This nystagmus is transient and appears after 15 to 20 cycles of rapid head-shaking. Horizontal head-shaking may be associated with a low-amplitude reversal phase, which follows the primary response. Several previous studies using active head movement have indicated that head-shaking nystagmus is a sensitive indicator of vestibular loss. In this study, the head-shaking nystagmus test using passive head movement was evaluated in 115 patients and 17 control subjects, using three examiners. The data indicate that using passive head movement, the head-shaking nystagmus test is neither sensitive nor specific enough for use as a screening test for vestibular loss.

The "plateau effect" of cis-platinum-induced hearing loss.

Cis-platinum-induced hearing loss has been reported to exhibit a "plateau effect" over the 2 to 8 kHz range. We examined serial audiometry data from 27 patients treated with cis-platinum. Although a plateau at 60 to 70 dB HL often occurred, this hearing loss was clearly exceeded in a number of patients. We identified 14 patients with sufficient cis-platinum-induced sensorineural hearing loss to evaluate a plateau limit to hearing loss over the 4 to 8 kHz range. Half of these 14 patients had hearing losses that exceeded the plateau level. More severe 4 to 8 kHz hearing losses did not correlate with number of treatments, cumulative dosages of cis-platinum, pre-existing hearing loss, radiation therapy, other chemotherapeutic agents, or even hearing losses below 4 kHz. Although cis-platinum therapy can induce a plateau of moderate hearing impairment for some patients over the 4 to 8 kHz range, a significant proportion of patients will experience more severe hearing impairment in this range, even after only one or two courses of cis-platinum.

Morphological evidence of vestibular pathology in long-term experimental diabetes mellitus. II. Connective tissue and neuroepithelial pathology.

The influence of long-term experimental diabetes on the saccule and utricle was investigated with light and electron microscopy. Pathological changes involving the connective tissue cells of the subneuroepithelial connective tissue suggest that these cells may play a potentially important role in diabetic pathology of the inner ear. In diabetic animals there was an increased incidence of secondary lysosomes within the connective tissue cells as well as an accumulation of intracellular lipid droplets that increased with the level of hyperglycemia. Five animals with relatively more severe diabetes also had an extensive accumulation of extracellular matrix. Two of these rats with longer diabetes duration had a small number of degenerating type I hair cells scattered through the saccular neuroepithelium, possibly due to chronic stresses from impaired diffusion of oxygen, nutrients and waste material through the dense extracellular matrix. Utricles of these same animals did not have any degenerating hair cells.

Morphological evidence of vestibular pathology in long-term experimental diabetes mellitus. I. Microvascular changes.

The influence of long-term experimental diabetes on the microvasculature of the saccule and utricle was investigated using quantitative light and electron microscopic techniques. Basal lamina thickening or reduplication, typically seen in diabetic microangiopathy, were not observed. However, morphometric analysis did reveal a statistically significant increase in capillary diameters along with an increased vascularization of both the saccule and utricle. Both of these microvascular abnormalities may have been caused by the hemodynamic alterations known to occur in diabetes. These alterations include decreased deformability of red blood cells and increased blood viscosity. Either of these factors can lead to a greater stress on the capillary wall and possibly to a reduced oxygen delivery to the tissues.

Patterns of low-frequency rotational responses in bilateral caloric weakness patients.

Analysis of low-frequency (0.01 to 0.16 Hz), sinusoidal rotary chair responses of 24 patients with bilateral caloric weakness revealed a wide range of vestibulo-ocular reflex (VOR) impairment. The rotational stimuli presented allowed characterization of the degree of VOR impairment from severe to no impairment even in patients with absent responses to ice water caloric irrigation. Five patterns of responses were described, which helps to explain the lack of correlation often seen between caloric and rotary chair test results, especially when rotational testing does not extend down to at least 0.02 Hz. Bilateral caloric weakness was confirmed in 79% and 71% of patients by test frequencies of 0.01 and 0.02 Hz. Rotational stimuli of 0.04, 0.08, and 0.16 Hz gave notably poorer performance with 54%, 50%, and 29% confirmation.

The high-frequency oscillopsia test.

There is a need to develop bedside tests of the vestibulo-ocular reflex (VOR) that could be used in the clinical situation to screen patients who may be candidates for further evaluation. In 1984 Barber described the oscillopsia test, which compared visual acuity with and without head movement. Barber indicated that head movement should occur at greater than 1 Hz. This study was performed to evaluate the oscillopsia test at higher frequencies (2 to 7 Hertz) in the hope of improving its performance. The sensitivity and specificity of this test were evaluated using three examiners (the authors) and were referenced to clinical electronystagmographic results in 115 patients and 17 control subjects. The oscillopsia test evaluated in this study was highly specific, but not highly sensitive. It did not detect vestibular loss or subjective dizziness in more than 50% of cases. The high frequency oscillopsia test does not appear to be an effective screening test for VOR abnormalities or vestibular loss.

Differential uptake of 3H-thymidine by structures of the pia-arachnoid and cerebral cortex distal to the interrupted middle cerebral artery.

Tritiated thymidine accumulated in pial surface structures 1-10 days after interruption of the right middle cerebral artery (MCA) in the rat. Essentially no radioactive marker was present in the underlying cortical territory of the interrupted MCA other than at the operation site. Control meninges, cortex contralateral to the operated MCA, and structures proximal to the operation site were without appreciable accumulation of 3H-thymidine. Thus, the marked cellular proliferative response to MCA interruption was localized to structures of the pia-arachnoid overlying the cortical territory of the interrupted MCA. The data suggest that after MCA occlusion a cellular proliferative response contributes to the expansion of preexisting pial surface collateral vessels and that the expansion does not extend into the parenchyma.

Efferent neurons and vestibular cross talk in the frog.

A galvanic stimulus (30- to 120-s, 0.3-mA constant current pulse) was used to depolarize the spike-generating region of horizontal and anterior canal afferent neurons. The galvanically induced spike activity from these neurons served as a driving input to the efferent vestibular system in the bullfrog. Efferent-mediated effects were assessed by intracellular recordings of posterior canal afferent spike activity, either ipsilateral or contralateral to the driving stimulus. Ipsilateral to the driving stimulus, efferent-mediated spike rate changes occurred in 62 (39%) of 158 posterior canal afferent neurons. Ipsilateral efferent-mediated effects were overwhelmingly excitatory (92%). Of responding units, 3% were inhibited during stimulus application and 5% showed mixed responses involving 3-20 s of inhibition followed by facilitation. Contralateral to the driving stimulus, efferent-mediated spike rate changes occurred in 18 (23%) of 77 posterior canal afferent neurons. Contralateral efferent-mediated effects were overwhelmingly inhibitory (95%). Only one unit was facilitated during stimulation and no mixed responses to contralateral stimulation were observed. Analysis of the coefficient of variation in interspike intervals (CV) before and during stimulation showed no significant efferent-mediated effects on spike train noise. Comparisons of resting spike rates between units showing efferent-mediated effects and those that did not were in general agreement with previous studies. Responding units had a lower mean spike rate (6.8 +/- 0.70 spikes/s, mean +/- SE) than did nonresponding units (10.7 +/- 0.42 spikes/s, mean +/- SE; P < 0.001; 2-tailed t-test of log-normalized data). Comparison between groups in the regularity of their resting spike rates, as quantified by CV, showed considerable overlap. When responding and nonresponding units with similar resting spike rates were compared, responding units had more irregular resting spike rates than did nonresponding units (P < 0.004; 2-tailed, paired t-test). In most cases (77%) the temporal pattern and general shapes of efferent-mediated responses mirrored the driving input of the galvanically activated afferent neurons. The other 23% of efferent-mediated responses exhibited a marked adaptation of the response. Adapting and nonadapting units were not significantly different in their mean resting spike rates or in the regularity of their resting spike rates.