Signal and morphological changes in the endolymph of patients with vestibular schwannoma on non-contrast 3D FLAIR at 3 Tesla.

BACKGROUND: Non-contrast FLAIR revealed increased signal within the inner ear in patients with vestibular schwannoma, which is generally assumed to occur in the perilymph; however, the majority of previous studies did not differentiate between the endolymph and perilymph. Therefore, endolymph signal changes have not yet been investigated in detail. The purpose of the present study was three-fold: (1) to assess perilymph signal changes in patients with vestibular schwannoma on heavily T2-weighted (T2W) 3D FLAIR, also termed positive perilymphatic images (PPI), (2) to evaluate signal and morphological changes in the endolymph on PPI, and (3) to establish whether vertigo correlates with the signal intensity ratios (SIR) of the vestibular perilymph or vestibular endolymphatic hydrops. METHODS: Forty-two patients with unilateral vestibular schwannoma were retrospectively recruited. We semi-quantitatively and qualitatively evaluated the perilymph signal intensity on the affected and unaffected sides. We also quantitatively examined the signal intensity of the vestibular perilymph and assessed the relationship between vertigo and the SIR of the vestibular perilymph on the affected side. We semi-quantitatively or qualitatively evaluated the endolymph, and investigated whether vestibular hydrops correlated with vertigo. RESULTS: The perilymph on the affected side showed abnormal signal more frequently (signal intensity grade: overall mean 1.45 vs. 0.02; comparison of signal intensity: overall mean 36 vs. 0 cases) and in more parts (the entire inner ear vs. the basal turn of the cochlea and vestibule) than that on the unaffected side. No significant difference was observed in the SIR of the vestibular perilymph with and without vertigo (5.54 vs. 5.51, p = 0.18). The endolymph of the vestibule and semicircular canals showed the following characteristic features: no visualization (n = 4), signal change (n = 1), or vestibular hydrops (n = 10). A correlation was not observed between vestibular hydrops and vertigo (p = 1.000). CONCLUSIONS: PPI may provide useful information on signal and morphological changes in the endolymph of patients with vestibular schwannoma. Further research is warranted to clarify the relationship between vertigo and the MR features of the inner ear.

Evolution of Endolymph Secretion and Endolymphatic Potential Generation in the Vertebrate Inner Ear.

The ear of extant vertebrates reflects multiple independent evolutionary trajectories. Examples include the middle ear or the unique specializations of the mammalian cochlea. Another striking difference between vertebrate inner ears concerns the differences in the magnitude of the endolymphatic potential. This differs both between the vestibular and auditory part of the inner ear as well as between the auditory periphery in different vertebrates. Here we provide a comparison of the cellular and molecular mechanisms in different endorgans across vertebrates. We begin with the lateral line and vestibular systems, as they likely represent plesiomorphic conditions, then review the situation in different vertebrate auditory endorgans. All three systems harbor hair cells bathed in a high (K+) environment. Superficial lateral line neuromasts are bathed in an electrogenically maintained high (K+) microenvironment provided by the complex gelatinous cupula. This is associated with a positive endocupular potential. Whether this is a special or a universal feature of lateral line and possibly vestibular cupulae remains to be discovered. The vestibular system represents a closed system with an endolymph that is characterized by an enhanced (K+) relative to the perilymph. Yet only in land vertebrates does (K+) exceed (Na+). The endolymphatic potential ranges from +1 to +11 mV, albeit we note intriguing reports of substantially higher potentials of up to +70 mV in the cupula of ampullae of the semicircular canals. Similarly, in the auditory system, a high (K+) is observed. However, in contrast to the vestibular system, the positive endolymphatic potential varies more substantially between vertebrates, ranging from near zero mV to approximately +100 mV. The tissues generating endolymph in the inner ear show considerable differences in cell types and location. So-called dark cells and the possibly homologous ionocytes in fish appear to be the common elements, but there is always at least one additional cell type present. To inspire research in this field, we propose a classification for these cell types and discuss potential evolutionary relationships. Their molecular repertoire is largely unknown and provides further fertile ground for future investigation. Finally, we propose that the ultimate selective pressure for an increased endolymphatic potential, as observed in mammals and to a lesser extent in birds, is specifically to maintain the AC component of the hair-cell receptor potential at high frequencies. In summary, we identify intriguing questions for future directions of research into the molecular and cellular basis of the endolymph in the different compartments of the inner ear. The answers will provide important insights into evolutionary and developmental processes in a sensory organ essential to many species, including humans.

The enigmatic root cell - emerging roles contributing to fluid homeostasis within the cochlear outer sulcus.

Despite their curious morphology prompting numerous hypotheses of their normal function, the root cells lining the cochlear outer sulcus have long evaded physiological characterization. A growing body of evidence now suggests that they regulate the solute content of the endolymph and/or the perilymph, and may be essential in safe-guarding the global homeostasis of the cochlea. Immuno-labeling experiments have demonstrated polarized expression of key ion transport proteins, and recent electrophysiological recordings have identified specific membrane conductances. These studies have painted a clearer picture of how this unusual cell type may contribute to the maintenance of sound transduction, and how they may be central to pathological processes associated with various forms of hearing loss. This article is part of a Special Issue entitled "Annual Reviews 2013".

Induced endolymphatic flow from the endolymphatic sac to the cochlea in Ménière's disease.

OBJECTIVE: The aim of the present study was to verify whether drugs injected into the endolymphatic sac (ES) can reach the cochlea and possibly treat inner ear disorders. STUDY DESIGN: Prospective cohort study. SETTING: Tertiary referral center, Otolaryngology Department, University of Verona. SUBJECTS AND METHODS: Patients with Ménière's disease (MD) who were candidates for ES decompression were selected. Nineteen subjects received dexamethasone (DEX) via injection into the ES. To objectively define whether substances administered into the ES could reach the cochlea, we added gadolinium (GD) in three patients. All subjects had intraoperative electrocorticogram recordings and an audiologic follow-up. The three subjects who underwent injection of the DEX-GD solution were followed-up with magnetic resonance imaging. The audiological data are presented during a follow-up period of 12 months. RESULTS: Intraoperative electrocochleography recordings revealed no changes in two patients and summating potentials and compound action potential latency and wave-form modifications in all the other subjects. GD distribution was observed from 48 hours to one week after ES injection into the cochlea of the three subjects injected with DEX-GD. GD-related enhancement of inner ear structures lasted more than two weeks in all subjects. Pure tone average results showed hearing improvement of at least 20 dB HL in 42 percent of patients (8 of 19) at the 12-month follow-up. Statistically significant differences emerged between the mean pure tone average of the ES procedure subjects at one and 12 months after surgery (P = 0.0096). CONCLUSION: This novel approach might reveal new prospects for treating viral, metabolic, autoimmune, and genetic disorders of the cochlea.

Calcium balance and mechanotransduction in rat cochlear hair cells.

Auditory transduction occurs by opening of Ca(2+)-permeable mechanotransducer (MT) channels in hair cell stereociliary bundles. Ca(2+) clearance from bundles was followed in rat outer hair cells (OHCs) using fast imaging of fluorescent indicators. Bundle deflection caused a rapid rise in Ca(2+) that decayed after the stimulus, with a time constant of about 50 ms. The time constant was increased by blocking Ca(2+) uptake into the subcuticular plate mitochondria or by inhibiting the hair bundle plasma membrane Ca(2+) ATPase (PMCA) pump. Such manipulations raised intracellular Ca(2+) and desensitized the MT channels. Measurement of the electrogenic PMCA pump current, which saturated at 18 pA with increasing Ca(2+) loads, indicated a maximum Ca(2+) extrusion rate of 3.7 fmol x s(-1). The amplitude of the Ca(2+) transient decreased in proportion to the Ca(2+) concentration bathing the bundle and in artificial endolymph (160 mM K(+), 20 microM Ca(2+)), Ca(2+) carried 0.2% of the MT current. Nevertheless, MT currents in endolymph displayed fast adaptation with a submillisecond time constant. In endolymph, roughly 40% of the MT current was activated at rest when using 1 mM intracellular BAPTA compared with 12% with 1 mM EGTA, which enabled estimation of the in vivo Ca(2+) load as 3 pA at rest. The results were reproduced by a model of hair bundle Ca(2+) diffusion, showing that the measured PMCA pump density could handle Ca(2+) loads incurred from resting and maximal MT currents in endolymph. The model also indicated the endogenous mobile buffer was equivalent to 1 mM BAPTA.

Inward-rectifier chloride currents in Reissner's membrane epithelial cells.

Sensory transduction in the cochlea depends on regulated ion secretion and absorption. Results of whole-organ experiments suggested that Reissner's membrane may play a role in the control of luminal Cl(-). We tested for the presence of Cl(-) transport pathways in isolated mouse Reissner's membrane using whole-cell patch clamp recording and gene transcript analyses using RT-PCR. The current-voltage (I-V) relationship in the presence of symmetrical NMDG-Cl was strongly inward-rectifying at negative voltages, with a small outward current at positive voltages. The inward-rectifying component of the I-V curve had several properties similar to those of the ClC-2 Cl(-) channel. It was stimulated by extracellular acidity and inhibited by extracellular Cd2+, Zn2+ and intracellular ClC-2 antibody. Channel transcripts expressed include ClC-2, Slc26a7 and ClC-Ka, but not Cftr, ClC-1, ClCa1, ClCa2, ClCa3, ClCa4, Slc26a9, ClC-Kb, Best1, Best2, Best3 or the beta-subunit of ClC-K, barttin. ClC-2 is the only molecularly-identified channel present that is a strong inward rectifier. This study is the first report of conductive Cl(-) transport in epithelial cells of Reissner's membrane and is consistent with an important role in endolymph anion homeostasis.

Stria vascularis and vestibular dark cells: characterisation of main structures responsible for inner-ear homeostasis, and their pathophysiological relations.

The regulation of inner-ear fluid homeostasis, with its parameters volume, concentration, osmolarity and pressure, is the basis for adequate response to stimulation. Many structures are involved in the complex process of inner-ear homeostasis. The stria vascularis and vestibular dark cells are the two main structures responsible for endolymph secretion, and possess many similarities. The characteristics of these structures are the basis for regulation of inner-ear homeostasis, while impaired function is related to various diseases. Their distinct morphology and function are described, and related to current knowledge of associated inner-ear diseases. Further research on the distinct function and regulation of these structures is necessary in order to develop future clinical interventions.

Fluid dynamics vascular theory of brain and inner-ear function in traumatic brain injury: a translational hypothesis for diagnosis and treatment.

It is hypothesized that in all traumatic brain injury (TBI) patients with a clinical history of closed or penetrating head injury, the initial head trauma is associated with a vibratory sensation and noise exposure, with resultant alteration in vascular supply to the structures and contents of the fluid compartments of brain and ear (i.e., the fluid dynamics vascular theory of brain-inner-ear function [FDVTBE]). The primary etiology-head trauma-results in an initial fluctuation, interference, or interaction in the normal fluid dynamics between brain and labyrinth of the inner ear, with a resultant clinical diversity of complaints varying in time of onset and severity. Normal function of the brain and ear is a reflection of a normal state of homeostasis between the fluid compartments in the brain of cerebrospinal fluid and perilymph-endolymph in the labyrinth of the ear. The normal homeostasis in the structures and contents between the two fluid compartment systems--intracerebral and intralabyrinthine--is controlled by mechanisms involved in the maintenance of normal pressures, water and electrolyte content, and neurotransmitter activities. The initial pathophysiology (a reflection of an alteration in the vascular supply to the brain-ear) is hypothesized to be an initial acute inflammatory response, persistence of which results in ischemia and an irreversible alteration in the involved neural substrates of brain-ear. Clinically, a chronic multisymptom complex becomes manifest. The multisymptom complex, individual for each TBI patient regardless of the diagnostic TBI category (i.e., mild, moderate, or severe), initially reflects processes of inflammation and ischemia which, in brain, result in brain volume loss identified as neurodegeneration and hydrocephalus ex vacuo or an alteration in cerebrospinal fluid production (i.e., pseudotumor cerebri) and, in ear, secondary endolymphatic hydrops with associated cochleovestibular complaints of hearing loss, tinnitus, vertigo, ear blockage, and hyperacusis. The FDVTBE integrates and translates a neurovascular hypothesis for Alzheimer's disease to TBI. This study presents an FDVTBE hypothesis of TBI to explain the clinical association of head trauma (TBI) and central nervous system neurodegeneration with multisensory complaints, highlighted by and focusing on cochleovestibular complaints. A clinical case report, previously published for demonstration of the cerebrovascular medical significance of a particular type of tinnitus, and evidence-based basic science and clinical medicine are cited to provide objective evidence in support and demonstration of the FDVTBE.

Clinical experience with symptomatic superior canal dehiscence in a single neurotologic practice.

OBJECTIVE: To discuss the range of signs and symptoms of patients with superior canal dehiscence syndrome identified in a single neurotologic practice. STUDY DESIGN: Retrospective chart review of 35 patients diagnosed with superior canal dehiscence from April 2000 to June 2007. RESULTS: All patients had evidence of superior canal dehiscence on computed tomography. These 35 patients represented 0.56% of new patients seen over a five-year period. The incidence of clinical symptoms and signs is compared to other published series. The mean vestibular evoked myogenic potential thresholds of affected ears are significantly lower than the mean vestibular evoked myogenic potential thresholds of unaffected ears. To date, 5 of 35 patients have undergone transmastoid occlusion of the superior semicircular canal with significant improvement in clinical symptoms in 4 of 5 cases. CONCLUSION: Not all patients with a diagnosis of superior canal dehiscence syndrome will have classic symptoms and signs. A high index of suspicion with careful clinical examination and properly performed ancillary testing is required to confirm this diagnosis.

Molecular mechanisms of epithelial cell-specific expression and regulation of the human anion exchanger (pendrin) gene.

Pendrin, a Cl(-)/anion exchanger encoded by the gene PDS, is highly expressed in the kidney, thyroid, and inner ear epithelia and is essential for bicarbonate secretion, iodide accumulation, and endolymph ion balance, respectively. This study aimed to define promoter regulatory elements essential for renal, thyroid, and inner ear epithelial cell-specific expression of human PDS (hPDS) and to explore the effect of ambient pH and aldosterone on hPDS promoter activity. Endogenous pendrin mRNA and protein were detected in renal HEK293, thyroid LA2, and inner ear VOT36 epithelial cell lines, but not in the fibroblast cell line, NIH3T3. A 4.2-kb hPDS 5'-flanking DNA sequence and consecutive 5'-deletion products were cloned into luciferase reporter vectors and transiently transfected into the above cell lines. Distinct differences in expression/activity of deduced positive/negative regulatory elements within the hPDS promoter between HEK293, LA2, and VOT36 cells were demonstrated, with only basal activity in NIH3T3 cells. Acidic pH (7.0-7.1) decreased and alkaline pH (7.6-7.7) increased hPDS promoter activity in transfected HEK293 and VOT36, but not in LA2 cells. Aldosterone (10(-8) M) reduced hPDS promoter activity in HEK293 but had no effect in LA2 and VOT36 cells. These pH and aldosterone-induced effects on the hPDS promoter occurred within 96-bp and 89-bp regions, respectively, which likely contain distinct response elements to these modulators. Acidic pH and aldosterone decreased, and alkaline pH increased, endogenous pendrin mRNA level in HEK293 cells. In conclusion, pendrin-mediated HCO3(-) secretion in the renal tubule and anion transport in the endolymph may be regulated transcriptionally by systemic pH and aldosterone.

A new animal model for Ménière's disease.

CONCLUSION: A new murine model for the study of Ménière's disease has been developed by treatment with both lipopolysaccharide (LPS) and aldosterone. Induction of vestibular dysfunction in the hydropic animal model may entail additional stress such as reduced inner ear blood flow, and sudden acute changes in endolymph volume and/or pressure. OBJECTIVE: The purpose of this study was to develop a more suitable animal model, showing closer resemblance to the pathophysiological process in Ménière's disease. MATERIALS AND METHODS: Adult CBA/J mice were treated by intratympanic injection of LPS, intraperitoneal injection of aldosterone, or injection of both LPS and aldosterone. Morphological analyses were performed in the cochlea and endolymphatic sac. RESULTS: All experimental animals showed mild to moderate endolymphatic hydrops. Those treated with both LPS and aldosterone showed reversible vestibular dysfunction after the intratympanic injection of epinephrine.

Soluble megalin is accumulated in the lumen of the rat endolymphatic sac.

The endolymphatic sac (ES) is believed to play an important role in maintaining homeostasis in the inner ear by the absorption and endocytosis of endolymph. Megalin is a 600-kDa multiligand endocytic receptor expressed in certain types of absorptive epithelia including kidney proximal tubules. We analyzed the immunoreactivity for megalin in rat ES by immunofluorescence, immunogold electron microscopy, and immunoblotting. With immunostaining, the luminal substances of the ES were strongly stained for megalin. Megalin was also localized in luminal macrophage-like cells and both types of epithelial cell (mitochondria-rich cells and ribosome-rich cells). In these cells, the megalin was localized in the lumen of endosomes, but was not membrane associated. This localization pattern indicates that the megalin in these cells is not a membrane receptor, but merely one of the constituents that are endocytosed from the lumen of the ES. Immunoblotting indicated that the megalin in the ES is a 210-kDa molecule lacking a cytoplasmic domain. This suggests that the megalin in the ES may be a soluble form, different from the 600-kDa membrane-bound receptor expressed in kidneys. Taken together, it is likely that the megalin in the ES lumen is a soluble component and may be endocytosed by the ES epithelial cells. Furthermore, we found that the tectorial membrane, an acellular structure in the cochlea, gave a strong megalin immunoreaction. Since the cochlea is connected to the ES, the megalin may be transported alone or with the components of the tectorial membrane from the cochlea to the ES lumen through longitudinal flow.

Intralabyrinthine fluid dynamics: Meniere disease.

PURPOSE OF REVIEW: Meniere disease has long been postulated to be a disorder of intralabyrinthine fluid dynamics. RECENT FINDINGS: More recent developments in this field indicate that the control of fluid movement may be at a cellular level and that hormonal influence may be important. SUMMARY: The control of fluid and ion movements through aquaporins and gap junctions in the cell membranes are creating new perspectives in the mechanism of development of endolymphatic hydrops as well as potential methods for treatment. Intralabyrinthine fluid dynamics also play a role in the ability to locally deliver drugs to the inner ear through the middle ear.

Purinergic modulation of cochlear partition resistance and its effect on the endocochlear potential in the Guinea pig.

Introduction of adenosine 5'-triphosphate (ATP) into the endolymphatic compartment of the guinea-pig cochlea decreases the endocochlear potential (EP). To determine if this is due to an ATP-induced change in compartment resistance, the cochlear partition resistance (CoPR) was measured using constant current injections into scala media before, during, and after microinjection of ATP into the same compartment. The CoPR (mean = 3.13 +/- 0.13 kOmega) decreased with ATP in a dose-dependent manner (25.1 +/- 3.0% decrease in relation to baseline values) and this was linearly correlated ( R(2) = 0.91) to the magnitude of the ATP-induced decline in EP (41.6 +/- 7.0% decline in relation to the baseline). Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, a P2X receptor antagonist) injected prior to ATP application blocked this ATP-induced reduction in EP and CoPR. This indicates that ATP-gated ion channels (P2X receptors) provide a latent shunt capable of regulating the majority of the electrical potential across the luminal surface of the sensory hair cells, which is necessary for sound transduction. The results suggest a novel sound transduction regulatory mechanism, which, via extracellular ATP, has the capability of adjusting hearing sensitivity.

Sodium- and potassium-activated ATPase in the mammalian vestibular system.

Autoradiographic and cytochemical procedures were employed to determine the cellular distribution of the Na,K-ATPase enzyme in the mammalian vestibular system. A light-microscope survey of vestibular tissues incubated with [(3)H]ouabain shows high densities of ouabain binding sites within the dark cell epithelium (DC) of the ampullae of the semi-circular canals, and to a lesser extent, the DC of the utricular macula. A moderate number of binding sites was found in nerve fibers penetrating the connective tissue beneath the sensory epithelium (SE) of the ampullae and the maculae. A small number of binding sites is distributed in the deep portion of the SE, both in the ampullae and in the maculae. These latter binding sites seem to be associated with nerve terminals and receptor cells. At the ultrastructural level, the vestibular dark cells exhibit extensive basolateral membrane infolding, a morphological hallmark of cells engaged in trans-epithelial ion transport. The cytochemical reaction product is K(+)-dependent, ouabain inhibitable, and is restricted to the basolateral membrane extensions, with little or no product on the luminal membrane. The extent of membrane infolding in dark cells of the utricle is less pronounced than that of the ampullar dark cells and the intensity of the cytochemical reaction appears to correlate with the extent of membrane infolding. The results support the widely held hypothesis that the vestibular dark cells play a role in endolymph production. They also suggest that the vestibular sensory epithelia may be a site of ion exchange.

H+,K+-ATPase is not involved in endolymph pH homeostasis.

In endolymph, the calculated electrochemical gradients suggest the presence of active transport of H+ and K+. The presence of H+,K+-ATPase, an active transporter that mediates the exchange of intracellular H+ for extracellular K+, was investigated using reverse transcription-polymerase chain reaction on rat microdissected inner ear tissues. Distal colon and kidney were used as positive controls. The two H+,K+-ATPase colonic or gastric isoforms were not detected in the rat cochlea and endolymphatic sac. This result suggests that H+,K+-ATPase is not involved in H+ and K+ endolymph homeostasis.

Developmental aspects of the rat endolymphatic sac and functional implications.

The purpose of this study was to determine specific characteristics of endolymphatic sac (ES) cells of the developing rat that are considered to be involved in endolymph homeostasis. Because intermediate filament proteins (IFPs) are regarded as markers of cell differentiation and basal lamina proteins (BLPs) are essential in cell<=>matrix interactions, we determined the presence of IFPs [cytokeratins (CKs) and vimentin] and BLPs [collagen IV, heparan sulphate proteoglycan (HSPG) and laminin] at different developmental stages before and after birth. In addition, we studied the expression of two enzymes of oxidative metabolism: cytochrome oxidase and succinate dehydrogenase. The presence of CKs 8, 18 and 19 in all epithelial cells of the ES during the embryonic stage is characteristic of simple (glandular) epithelial cells. Interestingly, a distinct population of these cells shows additional expression of CK 7, which is a feature of secretory cells. These CK 7-positive cells also contain a high concentration of oxidative enzymes and are rich in mitochondria, indicating that they are light cells. It is suggested that light cells possess specific energy-requiring transport capabilities. Loss of CK 19 expression in the distal part and in a large region of the intermediate part of the ES implies that these cells do not differentiate any further and acquire the capacity to proliferate. Furthermore, prominent co-expression of vimentin with the CKs in the distal part of the ES may confer viscoelastic properties on this epithelium. This may facilitate expansion and thus enable cushioning of pressure fluctuations. Finally, the early prominent occurrence of HSPG in the basal lamina of the ES enables transport of ions. In this light our recent observations of early functioning NaK-ATPases in certain ES cells are interesting.

Role of the new imaging modalities in the investigation of Meniere's disease.

The incidence of nonvisualization of the vestibular aqueduct and the endolymphatic duct-endolymphatic sac (ED-ES) complex as well as the type of periaqueductal pneumatization were evaluated in 23 patients with definite Meniere's disease (MD) by high-resolution CT and by MRI. Fifty subjects with no previous history of any ear disease were used as a control group. High-resolution CT results disclosed that in the control group the percentage of nonvisualized vestibular aqueduct (3.4%) was statistically significantly lower than in the MD group, when either the diseased ear (27.8%) or the nondiseased ear of the same group (22.2%) was examined. In addition, no differences were observed between the diseased and nondiseased ears of the patients with MD. The periaqueductal pneumatization was also found to be statistically significantly lower in the MD group. As regards the MRI examination, the results of the analysis showed that the ED-ES complex was visualized more frequently in the ears of the control subjects (64.1%) than in the diseased ears of the patients with Meniere's disease (39.1%). This difference had a marginal statistical significance (P approximately 0.05). We discussed the results in relation to possible underlying pathophysiologic mechanisms involving the flow of endolymph toward the ES during the different stages of the disease.

Signs of endolymphatic hydrops after perilymphatic perfusion of the guinea pig cochlea with cholera toxin; a pharmacological model of acute endolymphatic hydrops.

There are indications that endolymph homeostasis is controlled by intracellular cAMP levels in cells surrounding the scala media. Cholera toxin is a potent stimulator of adenylate cyclase, i.e. it increases cAMP levels. We hypothesized that perilymphatic perfusion of cholera toxin might increase endolymph volume by stimulating adenylate cyclase activity, providing us with a pharmacological model of acute endolymphatic hydrops (EH). Guinea pig cochleas were perfused with artificial perilymph (15 min), with or without cholera toxin (10 microg/ml). The endocochlear potential (EP) was measured during and after perfusion. The summating potential (SP), evoked by 2, 4 and 8 kHz tone bursts, was measured via an apically placed electrode 0, 1, 2, 3 and 4 h after perfusion. Thereafter, the cochleas were fixed to enable measurement of the length of Reissner's membrane, reflecting EH. After perfusion the EP increased significantly over time in the cholera toxin group as compared to the controls. Also, the SP increased gradually at all frequencies in the cholera toxin group. Comparison within animals showed that the increase in SP became significant after 2 h at 4 kHz, after 3 h at 2 kHz and after 4 h at 8 kHz. In the control group the SP did not change significantly. The compound action potential (CAP) amplitude decreased monotonically over time at all frequencies in both the cholera toxin group and the control group, but it decreased faster in the cholera toxin group. Also, the cochlear microphonics amplitude decreased over time at all frequencies in both groups, but the decrease was significant only in the cholera toxin group after 3 h at 2 and 4 kHz. Quantification of the length of Reissner's membrane showed a small but insignificant enlargement in the cholera toxin treated animals compared to controls. These results are in accord with our view that EH is accompanied by an increase in SP and a decrease in CAP. Our results partially confirm previous results of Feldman and Brusilow (Proc. Natl. Acad. Sci. USA (1973) 73, 1761-1764). New aspects in relation to that study are the significantly increased EP and SP. In the classical EH model, based on obstruction of the absorptive function of the endolymphatic sac, increased SPs are accompanied by decreased EPs. In this cholera toxin model of EH, it is unlikely that the endolymphatic sac is involved. Apparently, EH can be based on mechanisms located in the cochlea itself as opposed to mechanisms located in the endolymphatic sac.