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.

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.

Effects of aging on potassium homeostasis and the endocochlear potential in the gerbil cochlea.

Previous work has shown that the endocochlear potential (EP) decreases with age in the gerbil. Concomitant with the EP decrease is an age-related loss of activity of Na,K-ATPase in the lateral wall and stria vascularis. We hypothesized that the EP decrease is associated with a similar decrease in the endolymphatic potassium concentration [Ke+]. This hypothesis was tested using double-barrelled, K(+)-selective electrodes introduced into scala media through the round window in young and quiet-aged gerbils. Results show that the means (+/- S.D.) of the [Ke+] in young and aged gerbils were not significantly different (178.2 +/- 14.2 mM and 171.2 +/- 34.4 mM, respectively), although the intersubject variability was much greater in the aged animals than in the young. These values of [Ke+] are slightly higher than those found for other mammals and may reflect the higher plasma osmolarity found in the gerbil. The concentration of perilymphatic potassium [Kp+] in scala tympani at the round window was also similar for the young and aged groups (3.57 +/- 1.17 mM and 4.18 +/- 2.03 mM, respectively). On the other hand, mean EP values in the young and aged gerbils were 92.0 +/- 5.7 mV and 64.8 +/- 15.8 mV, respectively and were statistically different (P < 0.001). Overall, EP and [Ke+] showed little correlation (R2 = 0.23), except that when [Ke+] fell below 150 mM, the EP was always less than 60 mV. An analysis of the chemical potential for Ke+ with respect to Kp+ shows that it was similar for young and aged gerbils (overall mean of 103.1 +/- 13.7 mV) and remained constant with respect to the EP, in spite of an overall electrochemical potential of Ke+ that varied from 120 to 210 mV. Thus, the system maintains Ke+ homeostasis at the expense of the EP, even when the EP is on the verge of collapse.

Spontaneous rates, thresholds and tuning of auditory-nerve fibers in the gerbil: comparisons to cat data.

Characteristics of 245 auditory nerve fibers in eleven Mongolian gerbils are described in terms of spontaneous rates, thresholds, and tuning curves. The animals were reared in a low-noise environment and had similar hearing thresholds across frequency. Tuning curves were obtained with an algorithm developed to characterize the tuning of auditory fibers in cat, thereby allowing direct comparisons to published data from cat. The results demonstrate that basic similarities exist between gerbil and cat data, although some minor differences are also apparent. Tuning curve bandwidths, as measured 10 and 40 dB above the thresholds at the characteristic frequency (CF), follow trends found in cat data. Like cat, auditory nerve fibers in the gerbil have a range of spontaneous rates. In individual gerbils, fibers associated with low spontaneous rates have higher thresholds than do fibers of similar CF with high rates. Five of the eleven gerbils showed profiles of spontaneous rate across frequency reminiscent of those obtained from quiet-raised young cats. The profiles of the remaining gerbils tended to be compressed to a smaller range of spontaneous rates for characteristic frequencies above about five kHz, much like older cats with unknown noise histories. The cause of the spontaneous compression is not obvious. The correspondence between cat and gerbil with regard to spontaneous rate and CF threshold implies the presence of fundamental mechanisms that are common to mammalian auditory systems.

Boundaries of two-tone rate suppression of cochlear-nerve activity.

Two-tone rate suppression was examined in the responses of single cochlear-nerve fibers in Mongolian gerbils. The iso-rate tracking algorithm developed by Kiang and Moxon (Kiang, N.Y.-S. and Moxon, E.C. (1974): J. Acoust Soc. Am. 55, 620-630) for obtaining tuning curves was modified to track iso-rate suppression boundaries as a function of frequency with the excitor tone fixed at the characteristic frequency (CF) of the fiber. Lower threshold boundaries of the areas of suppression flanking the tuning curve above and below CF were outlined for fibers over a large CF range. It was found that the boundaries of rate suppression obtained below CF were very stable in their absolute positions on the intensity-frequency plane. This stability was evident both as a function of fiber CF (0.6-15 kHz) and as a function of the shape of the tuning curve at a given CF. In other words, the suppression boundary obtained below CF was largely independent of the tuning curve. In a second series of experiments tuning curves were taken in the presence of a fixed tone placed in the suppression area located above the fiber CF. The fixed tone by itself was not excitatory. These tuning curves were compared to tuning curves obtained with a single tone. It was found that frequencies around the fiber CF were most affected (suppressed) by the presence of the second tone, and that the low-frequency tail of the tuning curve tended to shift toward the boundary of the suppression area below CF. Because this suppression boundary lies below the threshold of the normal tail of the tuning curve for many mid- and high-CF fibers, these fibers often became hypersensitive at low frequencies in the presence of the second tone above CF.

Compound action potential input/output functions in young and quiet-aged gerbils.

Auditory-nerve compound action potentials (CAP) and cochlear microphonic (CM) potentials were measured with round window electrodes in two sets of quiet-reared gerbils: young (N = 9 ears, 4-7 months) and aged (N = 11 ears, 35-37 months). CAP thresholds, measured at probe frequencies from 0.5 to 25.6 kHz, are plotted as audibility curves. Input/output (I/O) functions were derived from CAP and CM amplitude measurements at six frequencies. When compared to young controls, CAP audibility curves from aged animals all show some degree of threshold shift, ranging from minimal to severe, as well as increased variability. Our data suggest that some of the variability in the aged-animal audibility curves can be attributed to variations in individual genetic factors. Maximum CAP amplitudes for the aged animals average significantly less than those of the young controls at all frequencies tested. Young control I/O functions are generally steeper than those of the aged gerbils. Differences in the CM amplitudes of the young and aged gerbils are not as clear cut as the differences in the CAP. Possible mechanisms explaining the decrease in amplitudes and slopes of the CAP I/O functions in aged animals include changes in numbers or thresholds of primary ganglion cells, or a decrease in synchrony in discharges of auditory-nerve fibers.

Lateral wall Na,K-ATPase and endocochlear potentials decline with age in quiet-reared gerbils.

Changes in the integrity of cochlear ion transport systems with age were examined in gerbils raised for 5-38 months in a quiet environment. Ion transport function was assessed by light microscopic immunohistochemical staining for the enzyme, Na,K-ATPase and by measurement of the endocochlear potential (EP). Small foci of strial atrophy accompanied by loss of immunostaining for Na,K-ATPase were observed in the stria vascularis of the apical and basal turns as early as 5 months of age. Cochleas from 29-38 month-old gerbils showed a loss of immunostaining for Na,K-ATPase in the stria in most of the apical turn with the degeneration extending well into the middle turn in many of the oldest ears. The extent of strial atrophy and loss of immunoreactive Na,K-ATPase in the basal turn varied considerably among the oldest cochleas. Populations of lateral wall fibrocytes (type II fibrocytes) normally rich in Na,K-ATPase exhibited a corresponding decrease in enzyme content in regions of advanced strial atrophy. The volume of immunostained stria vascularis correlated well with the magnitude of the resting EP. The results demonstrate that lateral wall ion transport systems in the gerbil cochlea degenerate as a function of age. The findings also provide good evidence for a functional relationship between the stria vascularis and the Na,K-ATPase-rich type II fibrocytes in generating and maintaining the EP.

Rate/level functions of auditory-nerve fibers in young and quiet-aged gerbils.

Steady-state rate/level functions of single auditory-nerve fibers to characteristic frequency (CF) tone bursts were measured in quiet-aged (35-37 months) and young control (4-7 months) gerbils. Rate/level functions of aged gerbils are different from those of young controls in that the thresholds are shifted to higher sound levels, but otherwise the shapes of the aged and young rate/level functions are similar. Specifically, there is little difference in the slope of the dynamic range portion of the rate/level functions when comparing aged gerbils to young controls. This is in contrast to whole-nerve input/output (I/O) functions of aged gerbils, which exhibit slopes that are less steep than those of the young controls (Hellstrom and Schmiedt, 1990b). Thus, it is likely that the deterioration of the CAP I/O function in aged animals is not due to a deterioration of rate/level functions in single units, but rather to other factors such as spiral ganglion cell degeneration or a loss of synchrony.

Stimulated acoustic emissions in the ear canal of the gerbil.

Ear-canal sound pressure and cochlear potentials were monitored inthe anesthetized gerbil to study the origins of acoustic emissions produced by transient and continuous stimuli. No evidence was found of any delayed emissions (echoes) originating within the cochlea in the acoustic or cochlear microphonic (CM) waveforms. However, strong acoustic and CM distortion products occurred when two primary tones of moderate levels were presented to the ear; the site of origin of these products was traced to the cochlea. Further, the levels of distortion followed a complex time course after anoxia, often becoming stronger after animal death for up to one hour, then decaying to the noise floor of the system. The disappearance of the distortion products was coincident with elimination of both the negative endocochlear potential and the CM response to a fundamental tone.

Method for tracing single fibers in the organ of Corti with horseradish peroxidase.

A method based on the axonal transport of horseradish peroxidase (HRP) has been developed to trace single fibers in the organ of Corti. HRP was injected into the auditory nerve or cochlear nucleus of the adult chinchilla, and the entire cochlea was subsequently incubated for reaction product. Of most significance was the Golgi-like labelling of single radial afferents innervating the inner hair cells. Heretofore, radial afferents have been difficult to trace with more conventional staining techniques, especially in adult animals. Outer spiral fibers, presumably afferent, were also labelled in one of the cochleas.

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.

A comparison of brainstem, whole-nerve AP and single-fiber tuning curves in the gerbil: normative data.

Tuning curves were obtained from brainstem responses (BSRs) and whole-nerve action potential (AP) responses of gerbils using a forward masking procedure. These are compared with single-fiber tuning curves of the gerbil. The broadness of the tips, tip-to-tail ratios and the slopes of the high frequency sides of the BSR tuning curves are similar to those of the AP curves for probe frequencies ranging from 1 to 8 kHz. Also, the BSR and AP curves share a number of characteristics with single-fiber tuning curves. These results suggest that, like the AP, components of the BSR can be used to measure frequency selectivity in the periphery of an intact auditory system.

Brainstem, whole-nerve AP and single-fiber suppression in the gerbil: normative data.

Suppression of gerbil brainstem responses (BSRs) and whole-nerve AP responses was studied by means of a forward masking procedure in which a tone-burst probe was preceded by a narrow-band masker. The effectiveness of the masker in reducing the brainstem response to the probe can be diminished by presenting a tone burst simultaneously with the masker. By varying the frequency and intensity of the third stimulus, BSR suppression areas can be determined. These flank the tails and high-frequency sides of BSR tuning curves in a manner similar to the suppression areas of AP tuning curves. The shapes and sizes of the BSR and AP suppression areas vary greatly across probe frequencies and animals. However, the lower boundaries of suppression areas associated with the tails of the tuning curves occur at similar absolute levels regardless of probe frequency or tuning curve shape. The BSR and AP suppression areas are in some respects similar to areas of two-tone rate suppression in single auditory nerve fibers of the gerbil.

Age-related changes in auditory potentials of Mongolian gerbil.

The Mongolian gerbil is being evaluated as an animal model of age-related hearing loss (presbyacusis). Part of this evaluation involves estimating auditory thresholds from evoked potentials arising from the auditory nerve and brainstem. The gerbils are born and reared in an environment where the ambient noise level is less than 40 dBA. Some animals are followed longitudinally (8, 19, 23.5 and 36 months), others are studied at 6-8 months (controls), or at 36 months (cross-sectional). Physiological responses are obtained with the animals anesthetized with ketamine and xylazine and transdermal electrodes attached to the head. Auditory signals are tone pips with center frequencies from 1 to 16 kHz in octave steps. Signal levels are varied from 10 to 80 dB SPL in 10 dB steps. For animals (N = 48) in the age range of 6-8 months, mean auditory thresholds were about 20 dB SPL between 2.0 and 8.0 kHz, 25 dB at 16 kHz and 30 dB at 1.0 kHz. By age 22-24 months (N = 15) thresholds had increased by about 10 dB at nearly all frequencies. By age 36 months (N = 37 ears, 32 animals) threshold increases were about 30-35 dB at 8 and 16 kHz, were 25 dB at 4 kHz and 2 kHz, and were 19 dB at 1 kHz. These hearing losses in 36-month gerbil are qualitatively similar to human data for 60-65-year-old males and 70-year-old females. Individual differences in hearing loss were large with the range exceeding 65 dB. While some animals (26/37) had a high-frequency sloping loss, others (11/37) had a bimodal audiometric shape where the hearing loss was smallest at 4 kHz and increased by at least 10 dB at adjacent frequencies.

Tuning and suppression in auditory nerve fibers of aged gerbils raised in quiet or noise.

Mongolian gerbils were reared either in quiet or in a continuous noise field (85 dBA, 500-4000 Hz). The gerbils began the noise exposure at 8 months of age and, after the exposure, spent the remainder of their lives in the quiet vivarium with the quiet-aged group. The duration of the noise exposure was between 365 and 724 days. At the terminal experiment the ages of the animals varied between 24 and 43 months, with a mean age of about 36 months, an age representing the average life span of a gerbil in our colony. During the terminal experiment, tuning curves and boundaries of two-tone rate suppression were obtained from single fibers in the auditory nerve. Threshold shifts occurred in both groups of animals. The shift was largely confined to the tip of the tuning curve; i.e., the region around the characteristic frequency (CF) of the fiber. The CF shifts effectively reduced the tip-to-tail ratios of the tuning curves. Two-tone suppression areas above and below CF were present for all fibers in the quiet-aged animals, but were often absent for fibers in the noise-aged group. The presence of suppression was largely independent of fiber thresholds in both groups of animals. Indeed, fibers were found with clearly-defined suppression boundaries above and below CF despite threshold shifts of up to 60 dB. Moreover, in the noise-aged group suppression below CF was sometimes found without concomitant suppression above CF and vice versa, suggesting an independence between the two suppression areas. For fibers with CFs within the bandwidth of the noise, two-tone suppression above CF was always absent, even though suppression below CF was sometimes present. In sum, two-tone suppression was near normal in ears aged in quiet despite relatively large threshold shifts at the fiber CF. However, suppression, especially that above CF, was vulnerable to the effects of chronic noise. Taken with the results of other studies, our data suggest that the micromechanics of the cochlea are largely responsible for two-tone suppression, especially that above CF, and that different mechanisms may underlie suppression above and below CF.

Age-related decreases in endocochlear potential are associated with vascular abnormalities in the stria vascularis.

The density and diameter of strial capillaries were assessed in whole-mount preparations of the cochlear lateral wall from 18 gerbils aged in quiet for at least 36 months. Following morphometric analysis, histopathologic changes in selected regions of the lateral wall were examined by light and transmission electron microscopy. Alterations in strial vasculature were compared with the endocochlear potential (EP) measurements from the same ear. Vascular degeneration occurred in a segmental fashion in that regions of atrophic capillaries were found throughout the cochlea but primarily in the apical and lower basal turns and in the hook. The amount of stria with normal capillaries varied greatly among the aged ears, ranging from 19 to 87%. The resting EP also varied markedly, ranging from 23 to 83 mV. Little correlation was found between vascular alterations and the corresponding EP value from individual cochlear turns. However, significant correlations were found between the total strial area with normal vasculature and both the mean EP value and that recorded at either the round window or first turn in that ear.

Development of cochlear potentials in the neonatal gerbil.

The onset and maturation of hearing was examined in separate groups of sibling and nonsibling neonatal Mongolian gerbils (Meriones unguiculatus). Auditory nerve compound action potentials (CAP) and cochlear microphonics (CM) were measured at the round window, and the endocochlear potential (EP) was recorded at three different locations in pups aged 13 to 30 days after birth (DAB) and in 90 day-old animals. Maturational trends for the three potentials were similar to those previously reported for gerbil neonates. However, CAP thresholds continued to decrease, and CM and CAP input/output functions and EP continued to increase beyond 30 days of age, a time at which many investigators have considered hearing in the gerbil to be mature. The EP developed simultaneously throughout the cochlea and approached 80 mV by 20 DAB. CAP thresholds showed a highly correlated log-linear relationship with EP in groups of nonlittermates and in siblings studied at different ages. In contrast, maximum CAP and CM amplitudes increased with increasing EP, but did not show significant growth until the EP exceeded 70 mV.

Frequency selectivity of the middle latency response.

A forward masking paradigm was used to assess the frequency selectivity of the middle latency response (MLR). Tuning curves of the MLR were obtained in unanesthetized gerbils. Changes in the amplitudes of MLR waves A, B, and C with latency values of 10 to 13 ms, 14 to 17 ms, and 20 to 25 ms, respectively, were analyzed as a function of masker frequency and intensity. Tuning curves of the MLR were also compared to tuning curves of the auditory brainstem response (ABR), which was recorded simultaneously with the MLR. The MLR and ABR differed in their response to forward masking. The MLR was reduced in amplitude or eliminated by masker stimuli that had minimal or no effect on the ABR. Forward masking often caused variable and non-monotonic changes in the amplitude of the MLR. Tuning curves of the MLR indicate that the MLR is less frequency selective than the ABR. The MLR is an electrophysiological measure of auditory function central to the auditory brainstem. Therefore, it may provide information concerning central components of normal and pathological auditory function. However, because of the variability of MLR amplitudes with forward masking, tuning curves of the MLR are difficult to obtain and are not efficient for routine measurements of frequency selectivity.

Age-related decreases in endocochlear potential are associated with vascular abnormalities in the stria vascularis.

The density and diameter of strial capillaries were assessed in whole-mount preparations of the cochlear lateral wall from 18 gerbils aged in quiet for at least 36 months. Following morphometric analysis, histopathologic changes in selected regions of the lateral wall were examined by light and transmission electron microscopy. Alterations in strial vasculature were compared with the endocochlear potential (EP) measurements from the same ear. Vascular degeneration occurred in a segmental fashion in that regions of atrophic capillaries were found throughout the cochlea but primarily in the apical and lower basal turns and in the hook. The amount of stria with normal capillaries varied greatly among the aged ears, ranging from 19 to 87%. The resting EP also varied markedly, ranging from 23 to 83 mV. Little correlation was found between vascular alterations and the corresponding EP value from individual cochlear turns. However, significant correlations were found between the total strial area with normal vasculature and both the mean EP value and that recorded at either the round window or first turn in that ear.