Inhibition of the calcium- and voltage-dependent big conductance potassium channel ameliorates cisplatin-induced apoptosis in spiral ligament fibrocytes of the cochlea.

The role of calcium- and voltage-dependent big conductance potassium channels in regulating apoptosis was investigated in cultured type I spiral ligament fibrocytes. Incubation of type I spiral ligament fibrocytes derived from gerbil cochlea with cisplatin induced dose- and time-dependent apoptosis as demonstrated by annexin V conjugated to fluorescein isothiocyanate/prodidium iodide assays. The average voltage activation threshold of whole cell current was sharply shifted to -40 mV in the cisplatin-treated cells as compared with a value of 40 mV in control cells. The average whole-cell current of cisplatin-treated cells induced by a depolarization voltage step from -80 to -10 mV was increased significantly to 1.2+/-0.4 nA as compared with 0.08+/-0.1 nA in control cells. Coincubation with tetraethylammonium and cisplatin retained the whole cell current in the normal range (0.12+/-0.2 nA). The increment of cisplatin-induced whole-cell current was inhibited (97+/-5%) by a specific calcium- and voltage-dependent big conductance potassium channel blocker iberiotoxin. Consistent with this, co-incubation with tetraethylammonium significantly attenuated cisplatin-induced apoptosis in type I spiral ligament fibrocytes by more than 50%. We conclude that the activation of BK channels is an early event associated with cisplatin-induced apoptosis in type I spiral ligament fibrocytes. These findings also point to the calcium- and voltage-dependent big conductance potassium channels as a potential pharmacological target for manipulating cisplatin ototoxicity.

Ouabain induces apoptotic cell death in type I spiral ganglion neurons, but not type II neurons.

Application of ouabain to the intact round-window (RW) membrane of the gerbil cochlea induces apoptosis in most spiral ganglion neurons (SGNs), leaving a few neurons intact (Schmiedt et al. 2002). Here, physiological measures and immunostaining were used to examine the process of SGN degeneration at 3, 6, 12, and 24 h, 4 days, and 1 and 5 months after ouabain treatment. The few remaining neurons surviving up to 5 months after ouabain treatment were immunoreactive for peripherin, a type II neuron marker. Peripherin-positive cell counts indicate that about 7% of the SGNs in the gerbil cochlea are type II neurons, and these neurons survive intact after ouabain treatment. Ouabain exposure had little effect on the outer hair cell and lateral wall systems, even after a 5 month loss of auditory-nerve function. The cellular locations of cytochrome c, poly (ADP-ribose) polymerase (PARP), and activated caspase 3 were examined in control and ouabain-treated cochleas. A redistribution of cytochrome c in peripherin-negative (type I) neurons was observed at 3 h after ouabain exposure. Degraded PARP and activated caspase 3 were also detected in peripherin-negative SGNs at 6 and 24 h after treatment, respectively. These results suggest that the redistribution of cytochrome c is an early event during apoptosis in type I SGNs and that activation of PARP and caspase 3 are associated with apoptosis in these cells. Calcineurin and NF-kappaB are two important signaling pathways that may modulate cell survival in the central nervous system. Here, we found that calcineurin and NF-kappaB selectively labeled type II neurons. It is speculated that the high levels of calcineurin and NF-kappaB in type II SGNs, as compared with type I SGNs, may play protective roles in enhancing the survival of type II neurons exposed to ouabain.

Effects of chronic furosemide treatment and age on cell division in the adult gerbil inner ear.

Atrophy of the stria vascularis and spiral ligament and an associated decrease in the endocochlear potential (EP) are significant factors in age-related hearing loss (presbyacusis). To model this EP decrease, furosemide was delivered into the round-window niche of young adult gerbils by osmotic pump for seven days, chronically reducing the EP by 30-40 mV. Compound action potential (CAP) thresholds were correspondingly reduced by 30-40 dB SPL at high frequencies. Two weeks after withdrawal of furosemide, the treated ears showed an EP recovery of up to 20-30 mV along with a similar recovery of CAP thresholds. The influence of cell division on furosemide-induced and age-related decline of the EP was examined using a mitotic tracer, bromodeoxyuridine (BrdU). Cell proliferation was examined in three groups: young control, furosemide-treated, and aged cochleas. Sections immunostained for BrdU were bleached with H2O2 to eliminate ambiguities with melanin pigment in the inner ear. Cell types positively labeled for BrdU in all three groups included Schwann cells in Rosenthal's canal; glial cells in the osseous spiral lamina; fibrocytes in the limbus, sacculus, and spiral ligament (SL); epithelial cells in Reissner's and round-window membranes; intermediate cells in the stria vascularis; and vascular endothelial cells. Quantitative analysis showed that the mean number of BrdU-positive (BrdU+) intermediate cells in the stria did not differ significantly among the three groups. In contrast, there was a significant increase of BrdU + fibrocytes in the SL of furosemide-treated animals as compared to the young control group. Moreover, there was a significant decrease in labeled fibrocytes in the aged versus the young ears, particularly among the type II and type IV subtypes. The results suggest that the increased fibrocyte turnover in the SL after furosemide treatment may be related to the recovery of EP and CAP thresholds, supporting the hypothesis that fibrocyte proliferation may be essential for maintaining the EP and cochlear function in normal and damaged cochleas. Moreover, the decreased turnover of SL fibrocytes with age may be a contributing factor underlying the lateral wall pathology and consequent EP loss that often accompanies presbyacusis.

A voltage- and Ca2+-dependent big conductance K channel in cochlear spiral ligament fibrocytes.

Evidence is accruing that spiral ligament fibrocytes (SLFs) play an important role in cochlear K(+) homeostasis, but little direct physiological data is available to support this concept. Here we report the presence and characterization of a voltage- and Ca(2+)-dependent big-conductance K (BK) channel in type I SLFs cultured from the gerbil cochlea. A single-channel conductance of 298+/-5.6 pS (n=28) was measured under symmetrical K(+). Membrane potentials for half-maximal open probability (P(o)) were -67, -45 and 85 mV with cytosolic free-Ca(2+) levels of 0.7 mM, 10 microM and 1 microM, respectively (n=8-14). The Hill coefficient for Ca(2+) affinity was 1.9 at a membrane potential of 60 mV (n=6). The BK channel showed very low activity (P(o)=0.0019, n=5) under normal physiological conditions, suggesting a low resting intracellular free [Ca(2+)]. Pharmacological results fit well with the profile of classic BK channels. The estimated half-maximal inhibitory concentration and Hill coefficient for tetraethylammonium were 0.086+/-0.021 mM and 0.99, respectively (n=4-9). In whole cell recordings, the voltage-activated outward K current was inhibited 85.7+/-4.5% (n=6) by 0.1 microM iberiotoxin. A steady-state kinetic model with two open and two closed stages best described the BK gating process (tau(o1) 0.23+/-0.08 ms, tau(o2) 1.40+/-0.32 ms; tau(c1) 0.26+/-0.09 ms, tau(c2) 3.10+/-1.2 ms; n=11). RT-PCR analyses revealed a splice variant of the BK channel alpha subunit in cultured type I SLFs and freshly isolated spiral ligament tissues. The BK channel is likely to play a major role in regulating the membrane potential of type I SLFs, which may in turn influence K(+) recycling dynamics in the mammalian cochlea.

Endocochlear potentials and compound action potential recovery: functions in the C57BL/6J mouse.

The C57BL/6J mouse suffers from cochlear degeneration beginning at an early age and has been used as a model of age-related hearing loss (presbyacusis). Here, the endocochlear potential (EP) and compound action potential (CAP) responses were determined in one-, four-, 12- and 24-month-old C57BL/6J mice. CAP measures included thresholds to tone pips, input/output (I/O) functions, and recovery functions to conditioning tones. EP values among the four age groups did not differ significantly (P>0.05) in either the basal or apical turns. CAP thresholds were increased significantly by 10 to 30 dB in the four-month group compared to the one-month controls at 11.3, 16, 20, and 22.6 kHz. CAP I/O functions were shallower in the four-month group compared to controls at all frequencies. In the 12- and 24-month-old mice, CAP responses were absent, despite normal EP values in these animals. Recovery functions after conditioning tones were obtained at 8, 16, 20 and 22.6 kHz; the functions had fast and slow components at all frequencies tested in both the one- and four-month-old groups. The corresponding recovery curves were identical for both age groups, even with significant threshold shifts in the older group. The two component recovery curves provide the first physiological evidence that different spontaneous rate (SR) classes of auditory neurons exist in the C57BL/6J mouse. Moreover, the unchanged recovery functions in the older group suggest that there was no loss of activity of the low-SR fiber population with age under conditions where the EP remains stable, in contrast to the gerbil model of presbyacusis where there is a loss of low-SR fiber activity and EP does decline with age.

Ouabain application to the round window of the gerbil cochlea: a model of auditory neuropathy and apoptosis.

The physiological and morphological changes resulting from acute and chronic infusion of ouabain onto the intact round-window (RW) membrane were examined in the gerbil cochlea. Osmotic pumps fitted with cannulas allowed chronic (0.5-8 days) infusions of ouabain. Acute and short-term applications of ouabain (1-24 h) induced an increase in auditory-nerve compound action potential (CAP) thresholds at high frequencies with lower frequencies unaffected. The resulting threshold shifts were basically all (no response) or none (normal thresholds), with a sharp demarcation between high and low frequencies. Survival times of 2 days or greater after ouabain exposure resulted in complete auditory neuropathy with no CAP response present at any frequency. Distortion product otoacoustic emissions (DPOAEs) and the endocochlear potential (EP) were largely unaffected by the ouabain indicating normal function of the outer hair cells (OHC) and stria vascularis. One to 3 days after short-term applications, apoptosis was evident among the spiral ganglion neurons assessed both morphologically and with TdT-mediated dUTP-biotin nick end labeling (TUNEL). With 4-8 day survival times, most spiral ganglion cells were absent; however, a few cell bodies remained intact in many ganglia profiles. These surviving neurons had many of the characteristics of type II afferents. Our working hypothesis is that the ouabain induces a spreading depression among the type I ganglion cells by blocking the Na,K-ATPase pump. Because of the constant spike activity of these cells, the ouabain rapidly alters potassium concentrations within ([K+]i) and external to ([K+]o) the ganglion cells, thereby initiating an apoptotic cascade.

Auditory nerve fibers in young and quiet-aged gerbils: morphometric correlations with endocochlear potential.

The number, size and distribution of myelinated nerve fibers were analyzed in the osseous spiral lamina (OSL) of young and old gerbils raised in a quiet environment. Because decreased endocochlear potentials (EPs) play a significant role in age-related hearing loss in the gerbil, we correlated morphometric and topographical data for nerve fibers with EP measurements in the same ear. Fibers were analyzed at the 2 and 10 kHz locations. The number of fibers at the 2 kHz location ranged from 12 to 47% greater than at the 10 kHz place in both young and aged specimens. No significant correlation was found between the number of fibers and the EP. Nerve fibers in gerbil tend to be distributed vertically by size within the OSL [Slepecky et al. (2000) Hear. Res. 144, 124-134], a result also found in cats and guinea pigs. Smaller fibers are more often found towards the scala vestibuli side of the OSL, whereas larger fibers are concentrated towards the scala tympani. The present data confirmed this distribution in young gerbils; however, in aged ears the distribution often became more uniform. Moreover, fiber distribution and ganglion cell size were highly correlated with EP. As EP declined, the fiber size distribution in the OSL became more uniform and the mean cross-sectional area of spiral ganglion cells and fiber diameter decreased. Thus, for whatever reason, certain indices of auditory nerve fiber morphometrics appear to be associated with the EP.

Developmental expression of monocarboxylate transporter in the gerbil inner ear.

The expression of H+-monocarboxylate cotransporters (MCTs) that facilitate cell uptake of lactate, pyruvate and other monocarboxylates was investigated in the adult and postnatally developing gerbil inner ear. In the mature cochlea, immunoreactive MCT1 was present in marginal cells of the stria vascularis and in type II, suprastrial and limbal fibrocytes. In the adult vestibular system, dark cells and a subpopulation of fibrocytes immediately underlying maculae and cristae stained strongly for MCT1. Satellite cells surrounding mature spiral and vestibular ganglia neurons also expressed MCT1. MCT1 immunoreactivity was present at birth in marginal and dark cells, at 8 days after birth in fibrocytes and at 12 days after birth in satellite cells, and coincided precisely with the developmental expression of Na,K-ATPase in these sites. The coexpression of MCT1 and Na,K-ATPase in these cell types points to MCT1 as an important source of energy to drive inner ear Na,K-ATPase activity. In the adult inner ear, MCT2 was detectable only in tectal cells of the cochlea and supporting cells of the crista ampullaris. Immunostaining was first observed at 16 days after birth in tectal and at 20 days after birth in supporting cells, and at the same time immunoreactive aquaporin 4 appeared in these cells. The coexpression of MCT2 and aquaporin 4 suggests a possible role for MCT2 in regulating transcellular water movement. Because MCT2 facilitates the transport of acidic intermediates, its biological significance also could relate to modulation of cell pH and volume. Maintenance of the inner ear's unique ion and fluid gradients is essential to normal hearing and balance and requires the expenditure of large amounts of energy. The cellular distribution of MCT1 and MCT2 points to their participation in generating these electrochemical gradients and their potential involvement in sensory deficits associated with various inner ear disorders.

Potassium recycling pathways in the human cochlea.

OBJECTIVES/HYPOTHESIS: Potential pathways for recycling potassium (K+) used in the maintenance of inner ear electrochemical gradients have been elucidated in animal models. However, little is known about K+ transport in the human cochlea. This study was designed to characterize putative K+ recycling pathways in the human ear and to determine whether observations from animal models can be extrapolated to humans. STUDY DESIGN: A prospective laboratory study using an immunohistochemical approach to analyze the distribution of key ion transport mediators in the human cochlea. METHODS: Human temporal bones were fixed in situ within 1 to 6 hours of death and subsequently harvested at autopsy. Decalcification was accomplished with the aid of microwaving. Immunohistochemical staining was then performed to define the presence and cell type-specific distribution of Na,K-ATPase, sodium-potassium-chloride cotransporter (NKCC), and carbonic anhydrase (CA) in the inner ear. RESULTS: Staining patterns visualized in the human cochlea closely paralleled those seen in other species. Anti-Na,K-ATPase stained strongly the basolateral plasma membrane of strial marginal cells and nerve endings underlying hair cells. This antibody also localized Na,K-ATPase to type II, type IV, and type V fibrocytes in the spiral ligament and in limbal fibrocytes. NKCC was present in the basolateral membrane of strial marginal cells as well as in type II, type V, and limbal fibrocytes. Immunoreactive carbonic anhydrase was present in type I and type III fibrocytes and in epithelial cells lining Reissner's membrane and the spiral prominence. CONCLUSIONS: The distribution of several major ion transport proteins in the human cochlea is similar but not identical to that described in various rodent models. These results support the presence of a complex system for recycling and regulating K+ homeostasis in the human cochlea, similar to that described in other mammalian species.

Steroid hormones modulate expression of cytochrome P450 enzymes in male hamster reproductive tract and leiomyosarcomas.

Syrian hamsters treated with estrogen and androgen for 8 months develop leiomyosarcomas in the vas deferens. Metabolism of estrogen by cytochrome P450s (CYPs) produces catechols and reactive oxygen species, and may contribute to tumor formation. To examine this issue, male hamsters were treated with 17 beta-estradiol (E2), testosterone propionate (TP) or both hormones. Reproductive tract tissues from control and treated animals were immunostained with antibodies specific for four CYP enzymes (1A1, 1A2, 1B1 and 3A1/2). Immunoreactive CYP1A1 was not found in the reproductive tract of control or treated animals. In untreated hamsters, CYP1A2 was detected only in principal cells of the caput epididymis. TP alone had no effect, but treatment with E2 induced expression of CYP1A2 in columnar epithelial cells throughout the epididymis and lining of the vas deferens. Treatment with E2 + TP blocked the induction of CYP1A2 seen in surface epithelial cells treated with E2 alone, but not the constitutive expression of this enzyme. Instead, simultaneous exposure to both hormones induced CYP1A2 in basal cells of the epididymis and vas deferens. CYP3A1/2 was not detected in the reproductive tract of control or TP-treated males, but immunostaining was induced in the inner layer of vas deferens smooth muscle by E2, and in all smooth muscle layers by dual hormone treatment. In controls, CYP1B1 was present in smooth muscle lining the epididymis and surrounding the vas deferens and dual hormone treatment increased staining intensity for CYP1B1 in these cells. Immunoreactive CYP1A2 was not detectable in leiomyosarcomas but the enzyme was present in both columnar and basal cells of the vas deferens epithelium adjacent to the tumors. In contrast, tumor cells showed heterogeneous expression of both CYP1B1 and CYP3A1/2. The relationships between hormone treatment, differential CYP expression and tumor formation strengthen our hypothesis that metabolism of estrogen is an important element in this model of hormonal carcinogenesis.

Microwave decalcification of human temporal bones.

OBJECTIVES/HYPOTHESIS: Morphological and immunohistochemical studies of human temporal bones are challenging as a result of difficulties in obtaining reliably fixed specimens and the lengthy time required for decalcification, typically 4 to 7 months. A novel method of processing human temporal bones using a microwave oven to accelerate decalcification is described. This procedure provides a rapid means of decalcifying temporal bones with good preservation of tissue morphology and antigenicity. METHODS: Human temporal bone specimens obtained at autopsy (n = 12, from specimens aged 43-91 y) were fixed within 6.5 hours of death by transtympanic perilymphatic perfusion of the inner ear. Decalcification was carried out using ethylenediaminetetra-acetic acid (EDTA) in a microwave oven and required only 3 to 6 weeks. Specimens were then dehydrated, embedded in paraffin, sectioned, and mounted on slides for morphological and immunohistochemical evaluation. RESULTS: Microscopic examination revealed no obvious artifacts attributable to the microwave decalcification process. The quality of morphological preservation was largely dependent on the postmortem fixation interval and adequacy of perilymphatic perfusion. Immunohistochemical analysis demonstrated strong positive staining for the enzyme Na,K-ATPase, an integral membrane protein. CONCLUSIONS: This study demonstrates that microwave decalcification provides an efficient and reliable means of processing human temporal bones for histological and histochemical examination. Decalcification time is significantly reduced with no apparent adverse effects on structural preservation or antigenicity.

Immunohistochemical localization of phospholipase C isozymes in mature and developing gerbil cochlea.

The possibility that phospholipase C contributes to intracellular signaling in the cochlea was investigated by immunostaining for eight different isoforms of the enzyme. In the mature gerbil cochlea, expression of the isozymes varied widely among different cell types. The phospholipase C-beta1 isoform was detected in inner and outer hair cells, and spiral ganglion neurons where it may participate in regulating Ca(2+) flux. The beta3 isozyme was expressed in epithelial cells thought to mediate lateral and medial circulation of potassium. The beta2 isozyme was present in border, inner phalangeal and Hensen cells, the stria vascularis, and suprastrial and supralimbal fibrocytes where it also may be involved in regulating ion transport activities. The phospholipase C-gamma isozymes were expressed in supporting cells, the stria vascularis, and certain fibrocytes where they possibly participate in activating tyrosine kinase and modulating ion conductances. The delta2 isoform was found in pillar, outer sulcus and strial marginal cells as well as spiral ganglion neurons and their radial processes. Documentation of changes in the expression pattern of phospholipase C isoforms during postnatal development and knowledge of their distribution in several positive control tissues provided further data for speculation about the biologic significance of the cochlear reactivity. The results demonstrate a wide diversity of isozyme distribution in the cochlea and suggest that the enzymes affect activities of various cochlear cell types in different ways.

Ultrastructural changes in the spiral limbus associated with carboplatin-induced ablation of inner hair cells.

Four months after the selective ablation of inner hair cells by carboplatin, the interdental cell epithelium exhibited dilated intercellular spaces and cytosolic vacuoles not seen in controls. In addition, the wide, often electron-lucent phalanges observed in the interdental cells of the normal chinchilla collapsed into a dense stratum that projected enlarged polypoid profiles into the limbal zone of the tectorial membrane. Carboplatin treatment also resulted in the restructuring of the tectorial membrane overlying the limbus. Changes in this membrane included a variable accumulation of the basal matrix, the rearrangement of intermediate lucent spaces, and the disappearance of a superimposed filamentous mesh. These three strata are, under normal conditions, apparently involved in events underlying tectorial membrane renewal. The post-carboplatin changes in the interdental cells and tectorial membrane occurred exclusively in the proposed medial pathway for K+ diffusion from inner hair cells and presumably resulted from a reduced flow of ions and fluid secondary to the ablation of these cells.

Canalicular reticulum in vestibular hair cells.

A membrane limited system referred to as canalicular reticulum (CR) has been demonstrated in the apical cytosol of the cochlea's inner and outer hair cells. Similarities between cochlear and vestibular hair cells prompted investigation of the presence of CR in hair cells of the gerbil vestibular labyrinth. A method of fixation with glutaraldehyde followed by an osmium-ferrocyanide mixture demonstrated abundant CR in the apex of both type I and type II hair cells. The CR was closely associated with numerous Golgi zones in the apex of the vestibular hair cells, indicating its genesis from Golgi cisternae. Also preserved in upper cytosol were discrete complexes of mitochondria with granular reticulum. These complexes offered a possible site for generating the membrane in Golgi zones and CR. Single and parallel cisternae of granular reticulum were observed in the basal half of the hair cells together with numerous synaptic-like vesicles. These cisternae with their terminal blebbing and accompanying canaliculi were interpreted as novel structures mediating synaptic vesicle genesis in vestibular hair cells in a manner comparable to that postulated for cochlear inner hair cells.

Structural evidence for ion transport and tectorial membrane maintenance in the gerbil limbus.

Cells medial to the tunnel of Corti were examined to assess fine structural features relevant to their proposed role in cochlear K(+) homeostasis. A dense network of canaliculi referred to as canalicular reticulum (CR) resided in the foot body of inner pillar cells, where it bordered and could resorb ions released from inner radial and spiral nerves. Lateral interdental cells (IDCs) formed columns which connected the inner sulcus epithelium with the base of the tectorial membrane's (TM) middle zone. A spout-like neck in cells at the top of lateral IDC columns housed a dense concentration of CR which resembled that characteristic of ion transporting epithelia and appeared to be located here for transporting ions and fluid toward the TM. Clustered IDCs in the center of the limbus connected underlying limbal stroma with the TM's limbal zone and appeared capable of transporting ions from stroma to TM. Abundant CR in limbal stellate fibrocytes evidenced their capacity to transport ions and fluid, presumably from inner sulcus epithelium toward central IDCs. The most medial IDCs possibly function as the terminus of an ion cycling path from scala vestibuli to endolymph. Light fibrocytes situated between supralimbal fibrocytes and medial IDCs appeared to serve as a link in this pathway. The limbal zone of the TM overlying central IDCs consisted of three distinct regions which offered a structural basis for transformation of an amorphous matrix supplied by central IDCs into the protofibrils of the membrane's middle zone.

Distribution of IkappaB proteins in gastric mucosa and other organs of mouse and gerbil.

The NF-kappaB/IkappaB complex is a major transcription regulator of inflammatory and immune responses. Helicobacter pylori infection causes chronic inflammation in gastric mucosa by inducing dissociation of the inhibitory IkappaB protein from the complex with a resulting increased expression of interleukin (IL)-8. To clarify which of several known IkappaB proteins could be involved in this inflammatory response, we undertook immunohistochemical examination of normal mouse stomach as well as other murine tissues for comparison, using polyclonal antibodies specific for alpha-, beta-, gamma-, and in-isoforms of IkappaB. The results showed strong immunoreactivity for the alpha-isoform in parietal cells and for the beta-isoform in pit cells of the stomach, along with the presence of these proteins in various other sites. Comparative staining revealed a similar but not identical distribution of IkappaB proteins in the Mongolian gerbil, a rodent model for H. pylori infection. The findings suggest that the alpha- and beta-isoforms are dominant IkappaB proteins in gastric parietal and foveolar cells, respectively, and point to a role for these transcription regulators in modulating pathological responses in stomach and other organs. (J Histochem Cytochem 48:191-199, 2000)

Temporary ovarian inactivity in elephants: relationship to status and time outside.

The captive elephant population in North America is in reproductive decline and, without importation from the wild, may cease to be viable within the next several decades. The estrous cycle of three captive, reproductive-age African elephants was monitored for 3 years by measuring serum progesterone concentrations. Each elephant experienced one or more episodes of extended low progesterone (>12 weeks), analogous to supposed terminal cessation of estrous cyclicity or 'flatlining' that has been described in some captive Asian and African elephants. Other studies have reported lengthy non-luteal (follicular) phases that indicate extended episodes of ovarian inactivity; however, this phenomenon has not been examined in detail. In this study, total duration of temporary ovarian inactivity or acyclicity followed a social rank pattern, with the most subordinate female having the longest and the dominant female the shortest duration. During periods of acyclicity, the number of hours the elephants spent outside was significantly less than during non-luteal or luteal phases of the cycle. Except in one instance, behavioral data recorded by elephant keepers during their interactions with the elephants showed no change in handling during periods of ovarian inactivity. Further study is necessary to distinguish the causative agent for temporary cessation of estrous cyclicity. Understanding this phenomenon is imperative for the future reproductive viability of captive elephant populations.

Ablation of inner hair cells by carboplatin alters cells in the medial K(+) flow route and disrupts tectorial membrane.

The thesis that K(+) effluxing from inner hair cells (IHCs) cycles medially back to endolymph through inner sulcus and interdental cells (IDCs) was tested by comparing control chinchilla cochleas with those in which IHCs were selectively destroyed by carboplatin. By light microscopy inner sulcus cells appeared tall and nearly empty in control ears, but 4 months after the carboplatin treatment many showed vacuolization and shrinkage. Inner pillar cells also consistently developed abnormal vacuoles after carboplatin treatment. Control cochleas exhibited lateral columns and central clusters of IDCs which at their apex possessed expanded presumably hydrated phalanges. Four months after carboplatin, the IDC epithelium enclosed empty looking spaces and the apical phalangeal compartment collapsed into a thin, apparently dehydrated layer. This alteration was accompanied by changes in the tectorial membrane (TM) whereby the membrane's limbal zone thickened progressively to form a tall hollow mound in advanced lesions. The clear spaces in the epithelium and collapse of the phalanges are thought to reflect diminished flow of ions and fluid through IDCs. The accumulation of limbal TM supports the premise that IDCs secrete macromolecules for TM turnover as well as ions and fluid for promoting lateral migration of its precursor constituents. Occurring after ablation of IHCs by carboplatin, the changes in inner pillar, inner sulcus and IDCs and limbal TM can be viewed as a secondary effect of the interrupted ion efflux from IHCs and as further evidence that this effluent follows a medial route.

Mutation of the Na-K-Cl co-transporter gene Slc12a2 results in deafness in mice.

Hearing impairment is a common human condition, but we know little about the molecular basis of cochlear function. Shaker-with-syndactylism (sy) is a classic deaf mouse mutant and we show here that a second allele, sy(ns), is associated with abnormal production of endolymph, the fluid bathing sensory hair cells. Using a positional candidate approach, we demonstrate that mutations in the gene encoding the basolateral Na-K-Cl co-transporter Slc12a2 (Nkcc1, mBSC2) cause the deafness observed in sy and sy(ns) mice. This finding provides the molecular basis of another link in the chain of K+recycling in the cochlea, a process essential for normal cochlear function.

Novel membranous structures in apical and basal compartments of inner hair cells.

Postfixation with a ferrocyanide-osmium tetroxide solution preserved a dense network of canaliculi extending from the apical to the upper lateral plasma membrane in cochlear inner hair cells (IHCs). Numerous Golgi bodies intermingled with this apical canalicular reticulum (CR). Osmium-ferrocyanide treatment also disclosed several previously unreported structures below the IHC nucleus. The first consisted of stacks of six or eight and sets of three parallel cisternae of rough endoplasmic reticulum spanning between clustered mitochondria. Some parallel cisternae ended with segmentation where they contacted mitochondria, and others terminated by transforming into blebs or continuing into canaliculi. A second feature was comprised of a complex of segmented cisternae and branching canaliculi with clustered mitochondria. Branching minicanaliculi with associated vesicles neighbored the complexes. A fourth entity consisted of synaptic-like vesicles that largely filled the subnuclear cytosol and congregated at synapses. An additional infranuclear structure was composed of slender canaliculi that collected near or streamed to plasmalemma, often next to a synapse. A paradoxical absence of rough endoplasmic reticulum above and Golgi zones below the nucleus provided evidence of atypical mechanisms for generating the membrane in CR and forming synaptic vesicles. The observations offer the view that IHCs are compartmentalized into an apical mechanoreceptor half and a basal half that affects neurotransmission. The apical CR provided a possible structural basis for sequestering the K+ known to influx apically and for directing its diffusion to the site of known efflux across the lateral plasmalemma. The codistribution of parallel cisternae, canalicular-mitochondrial complexes, and synaptic-like vesicles, all of which are unique to IHCs, implicated the cisternae and complexes in the genesis of the vesicles.