Ca2+ changes in the hair bundle were monitored with Calcium Green-1, the dextran conjugate (3000 Da) being used to circumvent artifacts caused by dye binding to cytoplasmic structures, which can alter the Ca2+ response (Blatter & Weir, 1990; Gray-Keller & Detwiler, 1994). Epifluorescence illumination was generated with a 100 W mercury lamp powered by a variable current supply (Attoarc, Carl Zeiss, Thornwood, NY, USA). The exciting light passed through a remotely controlled Uniblitz shutter, a KG1 heat filter, a 430 nm long-pass filter to exclude ultraviolet, a 450-490 nm excitation filter, a 510 nm dichroic beam splitter and the × 63 water-immersion objective. The fluorescence emission was long-pass filtered at 520 nm and imaged with an intensified CCD camera (Hamamatsu C2400). During the fluorescence measurements, the Nomarski analyser was removed from the light path. Images were stored on videocassette using a Sony S-VHS tape recorder (SVO 9500MD), and subsequent analysis was performed with the Image-1 software package (Universal Imaging, West Chester, PA, USA). To follow the time course of a fluorescence change, the average intensity was measured in each image over an approximately 5 μm-square region overlying the hair bundle. Since Calcium Green-1 is a single wavelength dye, changes in fluorescence, ΔF, were expressed relative to the background fluorescence, F.
To ensure reproducibility of the measurements, a fixed sequence of events was followed in the experimental protocol. After attaining a whole-cell recording, between 5 and 10 min were allowed for the electrode solution to equilibrate with the intracellular milieu and for the dye fluorescence to stabilize in the hair bundle. During this period, the cell was viewed by transmitted illumination through a 700 nm long-pass filter to avoid bleaching the dye. Patch electrodes used for fluorescence measurements all had uncompensated resistances of less than 10 MΩ to ensure rapid diffusion of dye into the cell. The stimulating probe was advanced onto the hair bundle while delivering small displacement steps until a response was detected with no deflection in the baseline current. Correct positioning of the probe was often difficult to achieve and it was easy to ‘over-push’ the bundle so that it was displaced positive from its resting position. In some experiments, the bundle fluorescence was monitored simultaneously, since it was found to be the most sensitive indicator of the probe's contact with the bundle: over-pushing the bundle resulted in a rise in the background fluorescence. The microscope was focused on the tip of the bundle which in fluorescence could be judged by the location of the stimulating probe (Fig. 3) and the shutter was then closed. To execute an experiment, the shutter was opened just prior to the stimulus, the bundle was displaced by a maximal step of normal duration 225 ms, and the fluorescence change was followed for about 5 s before closing the shutter. In the low-Ca2+ solutions, the step was often lengthened to improve the accuracy of the fluorescence measurement. Fluorescence changes and transducer currents were acquired in a series of endolymph solutions, several minutes elapsing between successive stimuli.
Figure 3. Experimental preparation and fluorescence responses
A,top, papilla surface showing hair bundles in Nomarski optics. The patch pipette, deep to the plane of focus, was introduced through a small hole in the side of the papilla so that the apical surface remained intact. Stimulating probe (arrow) is situated on the hair bundle. Bottom, focus at the top of the bundle in fluorescent illumination, showing Ca2+ signal from hair bundle. Arrow indicates the stimulating probe. Scale bar, 10 μm. B,simultaneous recordings of transducer current (bottom) and hair bundle Ca2+ fluorescence (middle) for a 225 ms bundle deflection (top). Though the transducer current shows some adaptation, its initial amplitude is near maximal. Endolymph, Na+, 2.8 mM Ca2+. Holding potential, -90 mV.
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The increase in fluorescence, ΔF/F, during bundle stimulation provides a measure of the amount of Ca2+ that has entered through the transducer channel. ΔF/F is assumed to be proportional to Ca2+ entry and can therefore be expressed in the following form:
where K is a constant and ICa, the current carried by Ca2+, is integrated over the stimulus duration. The fluorescence change observed with a mixed transducer current carried by Ca2+ and Na+ was calibrated to determine K by replacing Na+ with an impermeable monovalent ion so that Ca2+ became the sole charge carrier. This calibration method has been used to estimate the fractional Ca2+ current in neuronal nicotinic acetylcholine receptors (Vernino, Rogers, Radcliffe & Dani, 1994). The fraction, f, of the total transducer current IT, carried by Ca2+ is defined as:
where INa and ICa are the currents due to Na+ and Ca2+, respectively. The peak change in fluorescence, ΔF, associated with a given transducer current IT was first measured in endolymph containing Na+ and 2.8 mM Ca2+. Tris, which will be shown to be effectively an impermeant ion, was then substituted for Na+ producing a transducer current, ITris, and a fluorescence change ΔFTris. Values in the standard Na+ solution before and after the Tris substitution were usually averaged. The fractional Ca2+ current, f, was then calculated from:
where ΔΦ=ΔF/F, ΔΦTris= (ΔF/F)Tris and the time integrals of the current, ƒIdt, were determined for the duration of the stimulus. To check that the dye was not saturated, several stimulus durations were initially employed to verify that the fluorescence change grew linearly with stimulus duration. In such cases, ΔF/F was proportional to the current integral (Fig. 4). A second estimate of the fractional Ca2+ current, denoted as fc, was obtained from the ratio of the peak currents in Tris and in Na+:
The two estimates f (based on the fluorescence ratio) and fc (based on the current ratio) will differ if Tris is slightly permeable or if the Ca2+ flux through the channel is influenced by the nature of the monovalent ion.