The rats were sacrificed at W1 and W2 after click stimulation; then, left cochlear sectioning was performed along the paramodiolar axis, followed by hematoxylin/eosin staining. After the aforementioned fixation procedure was executed, the cochleae were decalcified in 10% ethylene-diamine tetra-acetic acid (EDTA) for 4 weeks at 4°C, dehydrated, embedded in paraffin, and sectioned serially (4 μm thick) parallel to the modiolar axis [20 (link)]. The specimens were observed with the spiral ganglion cells counted under a light microscope (Olympus BX51) and photographed (Olympus U-25ND6), and the digital images were saved. After the image collected, we used ImageJ software to encircle the exterior of spiral ganglion cells, then calculate the density of spiral ganglion cells by defining control group as 100%, and then calculate it for other groups proportionally.
U 25nd6
Manufactured by Olympus
The U-25ND6 is a neutral density filter from Olympus. It is designed to reduce the intensity of light entering the optical system, allowing for more precise control of exposure and contrast in microscopy applications.
Automatically generated - may contain errors
2 protocols using u 25nd6
1
Spiral Ganglion Cell Density Analysis
2
Optogenetic Neural Stimulation Protocol
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For iontophoresis of ACh, a sharp glass pipette ($15 MU) was filled with 10 mM acetylcholine chloride (A6625, Sigma) dissolved in external saline. ACh was ejected into the MB calyx by a brief (500 ms) positive current pulse using an iontophoresis unit (Model 260, World Precision Instruments).
Stimulation of optogenetic probes LED/mercury light stimulation Wide-field optical stimulation was achieved by high power LEDs (M470L2 and M590L3 for ReaChR and CsChrimson, respectively, Thorlabs) or filtered light from a mercury lamp (for Arch). An LED (M470L2) with peak output at wavelength of $470 nm was used for both the activation of ReaChR and excitation of Citrine tagged to ReaChR. Because ReaChR is sensitive to a broad spectrum of light (Lin et al., 2013) , blue light was sufficient to make the PNs expressing ReaChR fire at $200 Hz (Figure S3A). Light from an LED or a mercury lamp was collimated and delivered to an upright microscope (BX51Wl, Olympus) equipped with a 40x water-immersion objective lens (NA 0.80). LED light was pulsated at 80 Hz. Neutral-density filters (U-25ND25 or U-25ND6, Olympus) were used to stimulate the cells at lower intensities. All the reported optical intensity of LED light was measured at the back aperture of the objective lens (S120VC sensor, Thorlabs).
Stimulation of optogenetic probes LED/mercury light stimulation Wide-field optical stimulation was achieved by high power LEDs (M470L2 and M590L3 for ReaChR and CsChrimson, respectively, Thorlabs) or filtered light from a mercury lamp (for Arch). An LED (M470L2) with peak output at wavelength of $470 nm was used for both the activation of ReaChR and excitation of Citrine tagged to ReaChR. Because ReaChR is sensitive to a broad spectrum of light (Lin et al., 2013) , blue light was sufficient to make the PNs expressing ReaChR fire at $200 Hz (Figure S3A). Light from an LED or a mercury lamp was collimated and delivered to an upright microscope (BX51Wl, Olympus) equipped with a 40x water-immersion objective lens (NA 0.80). LED light was pulsated at 80 Hz. Neutral-density filters (U-25ND25 or U-25ND6, Olympus) were used to stimulate the cells at lower intensities. All the reported optical intensity of LED light was measured at the back aperture of the objective lens (S120VC sensor, Thorlabs).
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