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1 University of Illinois at Chicago, Chicago, IL
2 Marine Biological Laboratory, Woods Hole, MA
Horizontal cells are second order retinal neurons that receive direct input from photoreceptors and are involved in establishing a number of key features of visual perception. These cells mediate the formation of the inhibitory surround portion of the classic center-surround receptive fields of retinal neurons (1). The center-surround receptive fields are important for enhancing the contrast of visual objects and are also involved in color perception. The molecular mechanisms by which horizontal cells send lateral inhibitory signals to photoreceptors and bipolar cells are still under debate, but protons released from horizontal cells have been hypothesized to alter the flow of visual information within the outer retina (2). Indeed, small changes in extracellular pH can dramatically alter neural signals within the retina, in part because photoreceptor calcium channels are highly sensitive to protons. When protons bind to photoreceptor calcium channels, the voltage activation range of the channels shifts to more depolarized potentials and the overall conductance of the cell to calcium is reduced, which significantly reduces neurotransmitter release (3). Our previous work has shown that glutamate, the neurotransmitter released by photoreceptors onto horizontal cells, modulates the flux of hydrogen ions from skate retinal horizontal cells (4). Glutamate-induced changes in H+ flux depend on the presence of extracellular calcium and likely reflect the activation of plasma membrane calcium/H+ ATPases. These transporters extrude intracellular calcium in exchange for extracellular hydrogen ions, decreasing the concentration of protons at the extracellular face of the horizontal cells (5).
We would like to know whether local changes in calcium cause localized alterations in proton flux from horizontal cells. In the experiments reported here, we sought to develop methods to locally raise intracellular calcium levels in horizontal cells. We explored the use of small optical fibers that can apply focal ultraviolet stimuli to cells loaded with the caged calcium buffer NP-EGTA. This compound contains a UV-sensitive calcium binding site that releases its bound calcium upon absorption of ultraviolet light (6).
Horizontal cells were isolated from skate retinas by enzymatic dissociation, as described by Malchow et al. (7). The dissociated horizontal cells were placed in primary culture, where they were readily identified due to their distinct morphology and large size, about 150 µm. Cells plated on Falcon 3001 35-mm culture dishes were loaded with the cell membrane permeable AM ester forms of NP-EGTA and the calcium-sensitive dye Oregon Green, prepared as follows. NP-EGTA (50 µg) and Oregon Green (50 µg) were dissolved in DMSO with 20% pluronic acid and added to 8 ml of skate Ringer’s solution, yielding final concentrations of 5 µM Oregon Green-AM and 8 µM NP-EGTA-AM. The cultured horizontal cells were then incubated with this solution for 30 min at 14 °C, washed twice with skate Ringer’s solution, and allowed to stand for a minimum of 1 h. During this time, endogenous esterases cleave off the AM portion of the dye and the caged calcium compound, thereby trapping them inside the cell. Because NP-EGTA enters the cell unbound to calcium, it is important to pre-expose the preparation to a brief rise in intracellular calcium. Application of 100 µM glutamate for 20 s permits calcium entry and loading of NP-EGTA compounds trapped within the cell. The cells were then thoroughly washed with fresh Ringer’s solution, and experiments were typically conducted 10–30 min after calcium loading. Imaging of intracellular calcium concentration was performed with a Zeiss Attofluor imaging system.
Multi-mode optical fibers, F-MCB-T, were obtained from the Newport Corp.; they have a core diameter of 100 µm, a cladding diameter of 100 µm, a coating diameter of 140 µm, and a numerical aperture (NA) of 0.22, which gives them a high coupling efficiency. We prepared these fibers as follows. First, the coating and cladding were burned off the fibers with a heated tungsten coil, leaving a 5-cm portion bare. This portion of the fiber was then pulled to a final tip diameter of 1–2 µm with a Sutter P-2000 laser puller. The fiber was inserted into a borosilicate capillary tube previously pulled to a 5-µm tip diameter and placed so that the tip of the optical fiber protruded about 5 µm from the tip of the glass. The fiber was then glued in place with cyanoacrylate glue. Fibers were coupled to another multi-mode fiber, which was coupled to an ultraviolet laser.
Figure 1A shows the ultraviolet light output from one such pulled fiber. In this experiment, the dish was filled with a fluorescein solution, and the fluorescence of the solution, caused by ultraviolet light stimulation, was examined. The fluorescence was cone-shaped, with the region of highest intensity localized at the aperture of the optical fiber. A Narashige hydraulic manipulator was then used to place the fiber in a dish with the tip positioned 5 µm away from the membrane of a horizontal cell. Figure 1B shows the change in Oregon Green fluorescence measured from a single horizontal cell upon the photolytic release of calcium from NP-EGTA-loaded cells. When the cell is stimulated for 5 s with ultraviolet light from the pulled optical fiber, a significant increase in fluorescence is detected, indicative of a rise in intracellular calcium concentration. The stimulus was then repeated two more times, which induced calcium increases that were smaller with each subsequent stimulation. In control experiments, with cells loaded only with Oregon Green, application of ultraviolet stimuli led to increases in measured fluorescence. Note that these increases were about 85% smaller (n = 2) than those on cells containing NP-EGTA. Moreover, the control fluorescence disappeared immediately after the ultraviolet stimulus was removed. In contrast, changes in fluorescence with NP-EGTA-loaded cells persisted for many seconds after the UV stimulus was turned off. Additionally, the size of the fluorescent signal did not decay with subsequent UV stimuli.
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This work was supported by grants from the National Center for Research Resources (P41 RR01395), the National Science Foundation (009-1240), and a Grass Foundation Summer Fellowship.
Literature Cited
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