Fm1-43 reveals membrane recycling in adult inner hair cells of the mammalian cochlea - PubMed (original) (raw)

Fm1-43 reveals membrane recycling in adult inner hair cells of the mammalian cochlea

Claudius B Griesinger et al. J Neurosci. 2002.

Abstract

Neural transmission of complex sounds demands fast and sustained rates of synaptic release from the primary cochlear receptors, the inner hair cells (IHCs). The cells therefore require efficient membrane recycling. Using two-photon imaging of the membrane marker FM1-43 in the intact sensory epithelium within the cochlear bone of the adult guinea pig, we show that IHCs possess fast calcium-dependent membrane uptake at their apical pole. FM1-43 did not permeate through the stereocilial mechanotransducer channel because uptake kinetics were neither changed by the blockers dihydrostreptomycin and d-tubocurarine nor by treatment of the apical membrane with BAPTA, known to disrupt mechanotransduction. Moreover, the fluid phase marker Lucifer Yellow produced a similar labeling pattern to FM1-43, consistent with FM1-43 uptake via endocytosis. We estimate the membrane retrieval rate at approximately 0.5% of the surface area of the cell per second. Labeled membrane was rapidly transported to the base of IHCs by kinesin-dependent trafficking and accumulated in structures that resembled synaptic release sites. Using confocal imaging of FM1-43 in excised strips of the organ of Corti, we show that the time constants of fluorescence decay at the basolateral pole of IHCs and apical endocytosis were increased after depolarization of IHCs with 40 mm potassium, a stimulus that triggers calcium influx and increases synaptic release. Blocking calcium channels with either cadmium or nimodipine during depolarization abolished the rate increase of apical endocytosis. We suggest that IHCs use fast calcium-dependent apical endocytosis for activity-associated replenishment of synaptic membrane.

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Figures

Fig. 1.

An in situ preparation of the organ of Corti allows studying apical endocytosis in hair cells.A, The cochlear bone was opened at the helicotrema to expose the organ of Corti (OC) within the fourth turn of the cochlea. The dashed line delineates the edge of the cochlear bone. SV, Stria vascularis. Scale bar, 0.5 mm.B, After removal of Reissner's membrane, three rows of outer hair cells (OHC) and one row of inner hair cells (IHC) can be discerned. C, Signal obtained from the second harmonic generation. The OHCs (arrowheads) and the stereocilia of the IHCs can be seen. This image allowed alignment of the imaging window before application of the dye so that the time course of uptake could be studied in distinct structures of the cells. Scale bars:B, C, 50 μm.

Fig. 2.

FM1-43 is taken up apically by cochlear hair cells. Two-photon images of the organ of Corti and hair cells_in situ_. A, 3-D reconstruction of the organ of Corti showing uptake of FM1-43 from the scala media. Exposure time to FM1-43 20 min, producing labeling of three rows of OHCs and one row of IHCs. No uptake into supporting cells is apparent. Reissner's membrane is seen as a sheet of cells that also took up FM1-43. Scale bar, 40 μm. B, A series of confocal sections (in 1.2 μm intervals) through an IHC showing structures taking up dye: AA, apical aggregate; BA, basal aggregate; ER, perinuclear endoplasmic reticulum. Arrowheads indicate punctate staining (hotspots) around the basolateral pole. Scale bar, 10 μm.C, Progressive uptake and appearance of FM1-43 in basal structures. Scale bar, 10 μm. Hotspots marked by_arrowheads_. D, Time course of fluorescent signal. FM1-43 was applied at t = 0 sec with no washout (solid bar). Average fluorescence values for apex (circles) and base (triangles). The fluorescence increased to a steady state. Data from two adjacent cells are shown as open and closed symbols.E, Time course of fluorescence development in two adjacent cells when FM1-43 was bath applied for 300 sec (solid bar) and then washed out.

Fig. 3.

IHCs exhibit fluid phase uptake at their apical membrane. A, The fluid-phase endocytosis marker Lucifer Yellow applied to scala media was excluded from the stereocilia (arrowheads). Scale bar, 5 μm. B, Same cell as in A, with optical section 4 μm deeper into the cell. Lucifer Yellow was detectable in distinct internal structures which correspond to the apical aggregate seen after FM1-43 labeling.C, _z-_reconstruction of a cell labeled with Lucifer Yellow showing uptake into AA and BA structures.N, Nucleus. Scale bar, 10 μm. D,_z-_reconstruction of a FM1-43-labeled cell demonstrates that structures labeled with Lucifer Yellow correspond to AA and BA aggregates labeled by FM1-43. Scale bar, 10 μm. E, The rate of FM1-43 uptake into the apex was not influenced by pharmacological block of the mechanotransducer channel or destruction of the tip links. Comparison of normalized fluorescence uptake in the cuticular plate region in control cells (n = 6,dotted lines show SDs), in cells whose apical membrane was exposed to 5 m

BAPTA for 5 min before imaging (filled circles, n = 4), or 50 μ

streptomycin (open circles,n = 4) before and during imaging. F, The reticular lamina was not compromised in the _in situ_preparation. Alexa 488 (100 μ

), applied to scala media (SM), was not detected in the perilymphatic space (PL) beneath the reticular lamina. _Arrows_show position of the stereocilia. The signal in the cell originated from cross-talk of an FM1-43 signal in the Alexa 488 channel. Scale bar, 10 μm. G, The same cell showing the FM1-43 labeling pattern. The signal in SM originates from cross talk in the Alexa 488 channel. The labeling pattern with AA and BA aggregates and basolateral hotspots (small arrow) is as found in Figure1. FM1-43 labeled the stereocilial membrane (arrows).H, Combined image.

Fig. 4.

Apical endocytosis is fast. A, A time series of images of three IHCs in a strip preparation of the organ of Corti. The optical section is shown in the schematic (not drawn to scale). In cells 1 and 2, both external and internal structures are visible. In cell 1, the stereocilia (SC) and part of the apical aggregate (AM) are visible. In cell 3, the sections cuts only through the apical aggregate (AA).Boxes indicate the dimension of ROIs for B and C. The application pipette was located 40 μm above the apical membrane.B, Time course of fluorescence intensity of the ROIs depicted in A. FM1-43 is applied for 60 sec starting at_t_ = 10 sec. The SC fluoresced intensely because of their large membrane surface and served as a time marker for the dye arrival. After a delay of ∼20 sec, the apical aggregate of cell 2 showed signal, indicating internalization of FM1-43. The signal of AA is not caused by signal spillover from the stereocilia because the time course of AA is similar to that in area AM of cell 1, located adjacent to the stereocilia. C, Extended representation of the first 80 sec, as indicated by the dashed box in_B_. Identical experiments were performed in the in situ preparation.

Fig. 5.

Apical endocytosis depends on external calcium. Two-photon imaging of IHCs in the in situ preparation show fluorescence from an ROI at the apex (solid circles) and base (open circles) of IHCs. FM1-43 is applied to scala media compartment at t = −50 sec (A) and t = 0 sec (B) and present throughout the rest of the experiment. A, Uptake was greater in perilymph than in endolymph. Increase in Ca2+ indicated below.B, The enhancement was caused by the relative increase of Ca2+ in perilymph. The experiment also shows that apical endocytosis is not an artifact produced by perilymph surrounding the apical membrane.

Fig. 6.

Apex-to-base trafficking of internalized membrane is inhibited by monastrol, a kinesin inhibitor. A, Four consecutive _z-_sections of an IHC in the in situ preparation taken by two-photon imaging.Arrows show particle structures extending from the perinuclear region to the basal aggregate. Scale bar, 10 μm.B, Controls show the normal rise of apical and basal signals during continuous application of FM1-43. Data from six cells were averaged; error bars show SD. C, Averaged fluorescence intensity over time for seven cells that had been treated with 50 μ

monastrol, a kinesin inhibitor, in the bath. Imaging parameters were identical to those in controls. The increase in the basal signal was strongly attenuated. In the steady state the apical signal was increased compared with controls. D, 3-D reconstruction of IHCs shows the hotspots (arrows) at the basal pole remained in the presence of monastrol, although the region between the nucleus and the basal region was largely devoid of fluorescent signal. In contrast, fluorescence intensity of the apex (AA) was increased. E, Single sections through the midnuclear level of control and monastrol-treated IHCs show a strong signal in the apex (AA) and basal aggregate (BA), whereas the basal aggregate was only weakly labeled in a monastrol-treated cell.

Fig. 7.

FM1-43 is taken up through the basolateral membrane. Using IHCs in a strip of organ of Corti, FM1-43 was applied as a 90 sec pulse either from the apical side (A, C) or from the basal (B, D) side of the tissue. Cells were visualized by conventional confocal microscopy. The ROIs for the apical and basal regions are shown in the image sequence (A, B). Applied from the apical side, the signal increased in the apical structures (filled circles) before it appeared in the basal structures (open circles). When applied from the basal side, signal appeared first in the basal compartment but increased rapidly in the apex because of fast apical endocytosis. Scale bars, 20 μm.

Fig. 8.

Comparison of in situ and strip preparations reveals basolateral endocytosis. Uptake rates of IHCs of_in situ_ (A, B) organ of Corti and excised strips (C, D). A, Two-photon image of an IHC in the intact organ of Corti during application of FM1-43 (start,t = 0). Line indicates position of line for line image shown to the right.B, Time series of ROI fluorescence. The rise of the signal in the basal aggregate was delayed relative to that of the apex.C, Confocal image of IHC in strip preparation during 100 sec exposure to dye. The intensity along the line shown is displayed as a pseudocolored line scan on the right.D, Time series of the intensities for the associated ROIs. The arrow indicates simultaneous rise of fluorescence signal in spatially separated structures along the apex-to-base axis of the IHC. E, Comparison of data shown in B and D with apical signals scaled to the same initial slope. Time axis is that of_D_. Solid circles, FM1-43 applied to apical membrane only (from B); open circles, FM1-43 signal (from D) with apical and basolateral membrane accessible to dye. Scale bars: A, C, 10 μm.

Fig. 9.

K+ depolarization accelerates loss of fluorescence from the base of IHCs and increases apical endocytosis. A, Fluorescence signals in IHC before, during, and after depolarization with 40 m

K+. Data are from three consecutive experiments performed in a strip of the organ of Corti. FM1-43 is applied at_t_ = 10 sec for 70 sec first in normal extracellular solution (1), then in depolarizing solution containing 40 m

K+(2), followed by dye application in control solution after washout (3). The fluorescence of each trace was normalized. The decay rate of basolateral fluorescence was accelerated compared with the pre- and post-K+ controls.B–D, K+ depolarization accelerates apical endocytosis. Confocal imaging of IHCs used to measure fluorescence signals at apex (filled circles) and base (open circles). FM1-43 was applied for 60 sec (B) or 90 sec (C, D), as indicated by the stimulus bar. Time course of fluorescence when FM1-43 was applied apically (B, D) and basally (C). The endocytosis rate during potassium stimulation was unchanged (B), decreased (C), or increased (D). In all experiments performed (9 cells), there was a marked increase of apical endocytosis on return to control solution containing 4.6 m

K+ (Fig. 10_C_).

Fig. 10.

Increase of apical endocytosis is sensitive to L-type Ca2+ channel blockers. The experimental design was the same as in Figure 9 but with 100 μ

Cd2+ (A) and 10 μ

nimodipine (B) in the bath during stimulation of the cells with extracellular solution containing 40 m

K+. In both experiments, FM1-43 was applied on the apical surface for 90 sec (solid bar). A, Average fluorescence intensity for three cells. The washout of signal from the basal compartment was slower when cells were bathed in depolarizing solution containing 100 μ

Cd2+. Inclusion of Cd2+ abolished the delayed increase of the apical endocytosis. B, Average fluorescence intensity of seven cells, stimulated with high K+ external solution containing 100 μ

nimodipine. Presence of nimodipine abolished the delayed increase of the apical endocytosis.C, Bar graph showing the increase of apical (solid bars) and basal (open bars) fluorescence signals after K+ depolarization and the reduction of the enhancement to control levels in the presence of Cd2+ and nimodipine during K+-induced depolarization. Peak fluorescence intensity of the FM1-43 signal is normalized to the peak fluorescence of the FM1-43 signal before depolarization.

Fig. 11.

Transepithelial stimulation selectively destains FM1-43-labeled hotspots. A, Diagram showing position of the stimulating pipette. B, Series of images through an inner hair cell labeled with FM1-43. Dye was applied for 30 min before the start of the imaging and 50 μ

monastrol for 20 min before the start. The IHC shows the distinctive FM1-43 labeling pattern with AA and hotspots (white arrowhead). With no transepithelial stimulation (from t = 0–375 sec), hotspot fluorescence did change. With anodal pulses (250 μA at 20 Hz from 375–450 sec), fluorescence in the hotspots decreased (arrow). Fluorescence in the AA was unaffected. Scale bar, 10 μm. C, Decay of fluorescence in hotspots and apex after transepithelial stimulation. Average of eight cells (2 experiments). Stimulation (same parameters as in B) leads to a significant decay in fluorescence in the hotspots within 15 sec after onset of the electrical pulses. AA showed only bleaching caused by repeated scanning. Interval between scans, 15 sec.

Fig. 12.

A simple model of the IHC can explain the observed time course of fluorescent signal and the relative strength of label in the apical receptor compartment _F_aand the basal synaptic compartment _F_b.A, Uptake and transfer between extracellular space and two internal compartments (apical and basal) of the IHC is determined by first order kinetics with six free rate constants, as shown. The external marker (FM1-43), C_a or_C_b is applied at the apex or base, respectively. The rate constants k_xx are reaction rate constants. B, Simulation of the uptake rates when FM1-43 is continuously applied to the apex of an IHC as in Figure 1_D. The dashed lines are a simulation of the experiment of Figure 5_C, in which the transfer rate of dye from apex to base (k_ab) is reduced by inhibiting kinesin, which mediates apex-to-base membrane trafficking. Note that the basal signal is decreased (arrow) and that the slope of the apical signal gets steeper (arrow) because of accumulation of dye in the apical compartment. C, Simulation of the FM1-43 uptake and decay when the IHC is presented with a 400 sec pulse as in Figure1_E. Kinetic parameters chosen for the figure are in the ratio _k_a0:_k_ab:_k_ba:_k_bo= 0.3:1:2:2 and_C_a._k_oa=3_k_a0. The responses scale linearly with the applied marker concentrations _C_a,b

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References

1. Assad JA, Shepard MG, Corey DP. Tip-link integrity and mechanical transduction in vertebrate hair cells. Neuron. 1991;7:985–994. - PubMed
1. Bananis E, Murray JW, Stockert RJ, Satir P, Wolkoff AW. Microtubule and motor-dependent endocytotic vesicle sorting in vitro. Cell Biol. 2000;151:179–186. - PMC - PubMed
1. Bauerfeind R, Huttner WB. Biogenesis of constitutive secretory vesicles, secretory granules and synaptic vesicles. Curr Opin Cell Biol. 1993;5:628–635. - PubMed
1. Betz WJ, Bewick GS. Optical monitoring of transmitter release and synaptic vesicle recycling at the frog neuromuscular junction. J Physiol (Lond) 1993;460:287–309. - PMC - PubMed
1. Bomsel M, Parton R, Kuznetsov SA, Schroer TA, Gruenberg J. Microtubule- and motor-dependent fusion in vitro between apical and basolateral endocytotic vesicles from MDCK cells. Cell. 1990;62:719–731. - PubMed

Fm1-43 reveals membrane recycling in adult inner hair cells of the mammalian cochlea - PubMed (original) (raw)