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Technion-Israel Institute of Technology, Haifa, Israel
1 University of Illinois at Chicago, Chicago, IL
2 Marine Biological Laboratory, Woods Hole, MA
3 Technion-Israel Institute of Technology, Haifa, Israel
The vertebrate retina has been the focus of extensive anatomical, biochemical and physiological research for the past 50 years due, not only to its inherent importance for the process of vision, but also to its excellence as a model of information processing in the central nervous system (1). In general, two different approaches have been adopted by researchers examining retinal physiology. First, electrophysiological recordings of retinal activity (the electroretinogram) or of single cells have been used to analyze information processing; and second, biochemical techniques have been employed to study metabolic pathways within the retina. If these two techniques were combined, we would have the advantage of correlating information processing with metabolism.
The retina of the turtle Pseudemys scripta elegans has long been used as a model preparation for examining retinal function (2,3,4). Here we have developed a new variation of the turtle eyecup preparation, which permits us to examine metabolic parameters, including oxygen consumption and changes in extracellular hydrogen ion concentration, while simultaneously assessing the physiological state of retinal function from the electroretinogram.
Turtles were placed in ice for 20 min and then decapitated and pithed, in accordance with NIH guidelines and as approved by the institutional animal care committee of the MBL. The eyes were enucleated and the anterior portion of the eye was removed. The remaining eyecup was then carefully everted atop a conical mound of wax and fastened to the wax with minuten pins. The wax, with the eyecup attached, was firmly anchored to the bottom of a 140-mm glass petri dish. The Ringer solution contained (in mM): NaCl, 110; KCl, 2.6; CaCl2, 2.0; MgCl2, 2.0; NaHCO3, 22; glucose, 10; it had been bubbled with a mixture of 95% air/5% CO2 to reach a pH of 7.4. A polyethelene tube attached to the cover of the petri dish (but not touching the fluid) was used to supply an environment of 95% air/5% CO2, which maintained the bath solution at a pH of 7.4. The electroretinogram was recorded between two electrodes: an Ag-AgCl wire that had previously been inserted into the wax (resting firmly against the back of the sclera); and an Ag-AgCl reference electrode placed in the bath solution. Oxygen-sensitive or pH-selective microelectrodes were placed within 5–10 µm of the retinal surface; the placement was guided visually through an upright microscope. Oxygen was measured with amperometric microelectrodes held at a constant voltage of -0.6 volts (5). The response of these electrodes was calibrated in Ringer solutions bubbled with air or with 100% nitrogen. Hydrogen ion activity was monitored with voltametric microelectrodes, whose response varied as a function of the log of the hydrogen ion concentration (6). Electrode responses were calibrated using turtle Ringer solutions adjusted to pH-6, 7, and 8. A white LED placed 1.5 inches from the retinal surface was used to generate light stimuli of varying intensity; at its brightest setting, this LED produced a light of 440 lux at the surface of the retina, as measured with an Extech Instruments light meter. The intensity of the light was controlled by varying the voltage supplied to the LED.
Figure 1A illustrates the oxygen gradient obtained from one eyecup as a function of the distance of the electrode from the retinal surface. A reading of approximately 50 pA was measured with the electrode within 10 µm of the retina, a value significantly less than that obtained in the bulk solution. As the electrode was withdrawn from the retina in a stepwise manner, the current reading increased until a maximum value of 145 pA was obtained with the electrode 1.4 mm away from the eyecup. This value corresponds to an oxygen concentration of about 250 µM. From the linear characteristics of the oxygen electrode, we estimated the oxygen concentration in the layer close to the retina in this example to be approximately 40 µM. Thus, the solution in contact with the retinal surface was significantly lower in oxygen content, indicating a high rate of oxygen consumption, as has been observed in other species (7). The turtle eyecup preparation also exhibited a significant pH gradient from the retinal surface to the bulk solution (Fig. 1B). In this example, the surface of the retina was approximately 0.6 pH units more acidic than the bulk solution, indicating retinal production of hydrogen ions, and again replicating similar findings from several other species (8). Note that our results reflect the summed total response of the entire retina.
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In summary, we have demonstrated that metabolite gradients can be readily recorded outside the retina in parallel with the electroretinogram, thus enhancing our assessment of the physiological responses to light. Moreover, these measurements are possible from the vitreal side, using the eyecup preparation, and from the photoreceptor side, using the isolated retina. These preparations, coupled with self-referencing probes, make possible a detailed and comprehensive analysis of the relationships between retinal biochemistry (including production of messenger molecules) and signal processing.
This project was supported by a Gruss Lipper Foundation Fellowship (I.P.), a Grass Foundation Fellowship (G.T.), NSF grant 009-1240 (R.P.M), and NCRR grant P41-RRO1395 (P.J.S.S.). We are grateful to Mr. Richard Sanger for expert electronic assistance during the course of this research.
Literature Cited
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