Article Figures & Data
Figures
- Figure 1.
FM1-43 rapidly fills the cytoplasm of auditory and vestibular hair cells, entering from the tops and progressing to the bases of cells. A, Confocal time series of FM1-43 labeling in mouse cochlear outer hair cells. Dye spread through the cell within 60 sec of application. Labeling begins with the hair bundles, three rows of which can be seen (arrowheads). Fluorescence then fills the cytoplasm of the outer hair cells (OHC) from the top down, but is excluded from the nucleus (N). Four cell bodies from the first row of OHCs are shown. B, Confocal images from a time series of FM1-43 labeling of mouse utricular hair cells, before FM1-43(a), during a pulse of FM1-43 (b), and during destaining (c). Regions for quantification of fluorescence in the medium (1), hair bundle (2), cell apex (3), and cell base (4) in C are indicated. C, Time course of fluorescence intensity in the regions indicated during two pulses of FM1-43. At the top (1)is the fluorescence of the medium (expanded vertical axis)to indicate the timing of the FM1-43 pulses. Times of the micrographs in B are indicated. During FM1-43 application, the fluorescence signal in the bundle rapidly saturated (2); after washout, the bundle signal declined as fluorescence appeared in apical (3), then basal (4) cytoplasm. D, Model of fluorescence expected if FM1-43 partitions into and out of the hair bundle plasmalemma and also enters through the tips of stereocilia and diffuses throughout the cytoplasm; regions as in C (see Materials and Methods). E, Schematic of dye exclusion from hair cell apical surfaces by inverting and sealing a saccule onto coverglass. F, Fluorescence micrograph of the apical surface of a saccule positioned as in E and incubated for 10min. The plasma membrane was labeled, but the cytoplasm was unlabeled. G, Fluorescence micrograph of a frog saccule unsealed from a coverglass and incubated for 5 min, showing normal labeling of hair cells by FM1-43.
- Figure 2.
FM1-43 rapidly enters hair cells through a nonendocytotic pathway. A, Spacefilling models of FM1-43 and FM3-25. B, Confocal section of hair cells after a 15 min application of FM3-25 at 22°C; focal plane just below the cuticular plates. The plasma membrane was labeled, but dye was not observed in cytoplasm. C, Hair cells after a 5 min application of FM1-43 at 22°C. Cytoplasm was brightly labeled. D, After a 60 min application of FM3-25 at 22°C. Fluorescence is present in the cytoplasm. E, After a 5 min application of FM1-43 at 4°C. Labeling was similar to but less bright than that at room temperature. F, After a 60 min application of FM3-25 at 4°C. Cytoplasmic labeling was blocked. Inset, A plane of focus at a the apical surface of a cell. Bright labeling is restricted to a ring around the cuticular plate, the site of apical endocytosis.
- Figure 3.
FM1-43 enters bullfrog hair cells through their hair bundles. A, Confocal image of phalloidin labeling, at the level of the apical surface. After being blotted with a nitrocellulose strip, a line of hair cells is missing bundles (arrowheads), although their cuticular plates are still intact (fainter disks). B, Confocal image of FM1-43 labeling, at a level just below the cuticular plates. C, Color overlay of A and B. Hair cells lacking bundles have much less FM1-43 labeling than adjacent cells with bundles. D–I, A series of images from a time series during which FM1-43 was focally applied to the tip of the hair bundle in a narrow stream, with the experimental arrangement in J. E, Outflow pipette lowered so the stream just grazes the tallest stereocilia. F, Stream lowered further to contact the top part of the bundle. Fluorescence appeared in the cytoplasm below the cuticular plate. G, Stream raised above the bundle. The bundle began to destain. H, The bundle completely destained, but fluorescence is retained in the cytoplasm. I, Stream lowered to graze the tallest stereocilia. Only the tallest stereocilia became fluorescent, showing the extent of the dye stream.J, Schematic diagram of outflow and inflow pipettes. Regions for quantification of fluorescence within the bundle (red) and apical cell body (blue) are indicated. K, Quantification of fluorescence during and after FM1-43 application in the bundle (red) and cell body (blue), with the times of the images D–I indicated.
- Figure 4.
FM1-43 can enter hair cells through the mechanotransduction channel. A, Normal loading of frog saccular hair cells incubated in FM1-43. B, Hair cells incubated in 1 mm Gd3+ before and during FM1-43 loading. Inset, Region of B intensified to show faint labeling of the plasma membrane. Gd3+ blocked internalization of FM1-43. C, Quantification of Gd3+ block of FM1-43 loading and restoration of loading after Gd3+ washout, reduction in loading by depolarization to approximately −25 mV with 45 mm external K+, and by pretreatment with 5 mM BAPTA to cut tip links (mean±SEM). D, Quantification of FM1-43 loading into frog saccular hair cells in saline with no added Ca2+, 0.1, 1.0 or 10 mM Ca2+. Entry was significantly reduced only by 10 mm Ca2+ (mean ± SEM). E, Schematic diagram of the stimulus used to open or close transduction channels. Because of the change in bundle orientation at the striola, a fluid jet aimed across the macula closes transduction channels in hair cells on the left side of the striola and opens channels on the right side. F, A frog saccule after FM1-43 was blown across the saccule with the fluid jet for 2 min. The saccule and the pipette are outlined in white and the approximate location of the striola in yellow. Cells within the dye stream on the right (positively deflected) side of the striola internalized FM1-43, whereas most cells on the left (negative) side did not. G, DIC view of the boxed region in G at the plane of the hair bundles. Arrows mark the orientation of 68 hair bundles; a yellow line demarcates the striola. Three hair cells on the left with their bundles oriented toward the right are circled. H, Fluorescence micrograph of the field in H, taken at the level of the cell bodies. Hair cells with their bundles pushed negatively remained unlabeled, whereas hair cells with their hair bundles pushed positively were brightly labeled with FM1-43.
- Figure 5.
FM1-43 permeates through the capsaicin receptor channel TRPV1 and the ATP receptor channel P2X2. A, HEK cells transfected with TRPV1 and exposed to capsaicin (0.5 μm) with FM1-43 (10 μm) for 3 min. B, Mock transfected cells exposed to capsaicin and FM1-43. C, TRPV1-transfected HEK cells exposed to FM1-43 at 47°C for 3 min. D, TRPV1-transfected HEK cells exposed to capsaicin and FM1-43, along with the TRPV1 antagonist Ruthenium Red (10 μm). E, HEK cells transfected with P2X2 and exposed to 20 μm ATP with 10 μm FM1-43 for 3 min. F, P2X2-transfected HEK cells exposed to ATP and FM1-43, along with the P2X antagonist PPADS (100 μm). Experiments were done at room temperature unless noted.
- Figure 6.
The fixable FM1-43 analog AM1-43 labels many sensory structures after subcutaneous injection into juvenile mice. A–C, Cochlear hair cells at increasing magnification. Both inner and outer hair cells were brightly labeled, but other cells in the cochlea were not. D, Hair cells in the utricular macula. E, Exterior of the head of an injected neonatal mouse. All hair follicles, from snout to tail, were labeled, with the vibrissal follicles most intensely labeled. F, The apical half of a single vibrissal follicle. Both Merkel cells and fibers innervating the Merkel cells were labeled. G, Merkel cells within a touch dome, just below the surface of back skin. H, Spiral fiber in a spindle organ within dissected muscle. I, Eye, showing labeling of corneal nociceptive fibers. J, Nociceptive fibers from the nasal epithelium. K, Cross-section of kidney, with cortical labeling. L, Nerve endings within the soft palate. M, Labeling in the geschmacksstreifen in the hard palate. N, Tongue, with individual taste buds brightly labeled. O, Fungiform papillae in tongue. The fibers innervating the taste bud were labeled, but not the taste receptor cells. P, Circumvallate papillae in tongue. Taste receptor cells themselves were labeled.
- Figure 7.
AM1-43 labels primary sensory neurons after systemic injection into juvenile mice. A, Brain and initial segment of the spinal cord of a P15 mouse, injected 24 hr before killing. The labels indicate the location of the labeled sensory ganglia. Bottom view; rostral at left. B, Dorsal root ganglia throughout the spinal cord label with FM1-43; top view. C, Label of neurons within a single dorsal root ganglion; confocal section. Most neurons have accumulated dye, but some are more brightly fluorescent than others. D, Sagittal section of brain from an injected mouse; rostral to right. Within the brain, the only brightly labeled neurons were those of the mesence-phalic nucleus of the trigeminal(MesV, arrow), located just rostral and ventral of the cerebellum (Ce). The choroid plexus (CP) had lighter fluorescence, and all blood vessels within the brain were faintly fluorescent. E, Labeled neurons of MesV in a coronal section of brain. Inset, A single neuron in MesV with the cell body and process labeled with FM1-43; label is throughout cytoplasm.
- Figure 8.
AM1-43 labeling of sensory neurons requires dye at sensory endings. A, AM1-43 fluorescence in whisker follicles (arrowheads) 24 hr after injection of dye into individual follicles. B, The trigeminal ganglion ipsilateral to the injected follicles. A subset of neurons in the nasal portion of the ganglion (top) was labeled with AM1-43. C, Fluorescence in a trigeminal ganglion 24 hr after subcutaneous injection of AM1-43; confocal section. D, Fluorescence in the opposite trigeminal ganglion 24 hr after subcutaneous injection of AM1-43 preceded by ligation of the ipsilateral infraorbital branch of the trigeminal nerve. E, F, Whole-mount view of control and ligated trigeminal ganglia. Ligation blocked labeling of nasal neurons innervating face (F, top) but not posterior neurons (bottom). G, H, Fluorescence in whole mounts of trigeminal ganglia 48 hr after subcutaneous injection of AM1-43, and 24 hr after unilateral ligation of the infraorbital nerve. Most neurons within both the unligated and ligated ganglia are labeled.
