NORMAL LENS POTENTlAL OF THE RA§NBOW TROUT (SALMD QAIRDNERI) Thesis for the Degree of Phi D. MICHIGAN STATE UNIVERSITY JAMES L. RAE 1968 ABSTRACT NORMAL LENS POTENTIAL OF THE RAINBOW TROUT (Salmo gairdneri) by James L. Rae The rainbow trout lens exhibits a potential differ— ence of about 59 mv between its interior and exterior, the inside being negative with respect to the outside. The same potential was measured at all positions on the lens even though the epithelium is absent on the posterior surface. The thickness of the tissue across which the potential occurs is less, however, for the posterior than for the anterior surface. Both the capsule and epithelium appear to contribute to the maintenance of the potential although the exact con- tribution of each cannot be determined quantitatively. That another part of the lens contributes to the potential also seems likely. Studies made using metabolic inhibitors suggest that at least part of the potential is actively maintained but does not directly depend on either the entire glycolytic pathway or the cytochrome chain. The existence of an adenosine triphosphatase transport system seems very likely. James L. Rae The effect of temperature change on the lens poten— tial also suggests that metabolic activity is important in the maintenance of the potential. Such studies also indicate that the lens of the rainbow trout, in comparison to that of mammals, has some special mechanisms for maintaining transparency at low temperatures. The potential difference depends upon the presence of both Na+ and K+, although an increase in the concentra— tion of K+ in the bathing medium has a much greater effect on the potential than does a reduction of the concentration of Na+. In all cataractous lenses measured, a substantial reduction in the potential was found which indicates that the techniques employed could be of value in the investigation of cataract formation. NORMAL LENS POTENTIAL OF THE RAINBOW TROUT (Salmo gairdneri) By (1. turf r l}, James UK Rae A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 1968 ACKNOWLEDGMENTS The author wishes to expresslfiesappreciation to Dr. J. R. Hoffert for his guidance and support throughout the study. Thanks also are extended to Dr. P. O. Fromm for his aid in the preparation of this dissertation. He also wishes to thank the other members of his guidance committee, Dr. W. L. Frantz, Dr. E. P. Reineke, and Dr. J. B. Scott for their interest and help in the project. Special thanks are extended to Mr. K. Irish. His idea for the motor drive mechanism eas well as invaluable suggestions throughout the project made the completion of the work possible. Also, special thanks to Dr. J. Diana whose moral support was invaluable at a time when the project was stalled because of technical difficulties. In addition, the writer is indebted to the U.S.P.H.S., Division of National Institute of General Medical Sciences, grant GM-llZl for a pre-doctoral traineeship. The research was also supported by grant NB-0u125 from the National Insti- tute of Neurological Diseases and Blindness. ii Dedicated to my grandfather, the late James Goulding,whose financial and moral support and whose interest in learning made my entire education possible. Dedicated also to my wife, Joan, who sacrificed and tolerated a great deal to make the completion of this project a reality. iii TABLE OF INTRODUCTION . . . . . . . . . LITERATURE REVIEW. . . . . . . Microelectrode potentials. Lens potential studies . . Other potential profiles . Cation studies . . . . . . Inhibitors and metabolism. Location . . . . . . . . . MATERIALS AND METHODS. . . . . Experimental animals . . . Electronics. . . . . . . . Instruments an Balancing amplifiers . Electrodes . . . . . . . . Reference electrodes . CONTENTS shielding. . Pulling microelectrodes. . Filling microelectrodes. . Selecting microelectrodes. Manipulator and drive. . . Procedure for potential measurements Removal of lenses. . . Q measuring normal potential Procedure for Procedure for mapping studies. . . . . . Procedure for inhibitor studies. . . . Procedure for ion studies. . . . . . . . Procedure for study of osmotic effects Procedure for determining effect of temperature on the lens potential. . . Procedure for determining electrode characteristics. Procedure for measuring pressure effect. Procedure for measuring electrode distortion . RESULTS 0 o o o o O o 0 Effect of physical factors on microelectrode potential (Tables 1, 2, and 3) . . . . . . Comparison of in-vivo and in-vitro lens potentials iv Page 15 15 17 18 18 19 20 21 22 23 23 2M 25 26 27 28 28 29 29 3O 31 31 35 Effect of repeated microelectrode penetrations on lens potential. Normal potential of rainbow trout lens Relationship between lens potential and electrode tip diameter Measurement of potentials at adjacent regions. Effect of Janus Green on lens potential. . "dead" Comparison of potentials in living vs. lens tissue. Potentials at various sites on lens. Effect of on lens Effect of Stability Effect of potential. Alteration of lens potential during changes in 0 temperature of the bathing medium. Effect of ions on lens potential Effect of alteration of osmolarity of bathing medium on lens potential . DISCUSSION . . . SUMMARY AND CONCLUSIONS. LITERATURE CITED removal of capsule and epithelium potential. capsule damage on potential. of lens potential with time. some metabolic inhibitors on lens 36 36 37 39 39 40 H1 1+1 1+3 H7 '48 NS 52 55 59 74 76 TABLE 1. 10. ll. 12. 13. 1M. 15. 16. 17. 18. LIST OF TABLES Effect of pressure on electrode . . . . . . Distortion against inclined plane . . . . . Effect of electrolyte matrix on electrode . Frog sartorius muscle cell potentials . . . Comparison of in-vivo and in-vitro lens potentials. Repeated penetrations into same site. . . . Normal potential of rainbow trout lens: comparison of right and left eyes . . . . . Adjacent region potentials. . . . . . . . . Potential in dye spot versus potential along Side Of dye SpOto O O O O O O O O O 0 0 O O 0 Potential Potential Effect of potential Effect of of living and fixed lenses. . . . versus position on lens . . . . . removal of capsule and epithelium on the difference of the rainbow trout lens. lenticular capsule damage on the lens potential. . . . . . . . . . . . . . . Stability Effect of potential Effect of Effect of Effect of of lens potential with time . . . inhibitors on the maximum lenticular temperature on rainbow trout potential. ions on lenticular potential. . . osmolarity on potential . . . . vi Page 3l 32 33 35 35 36 37 39 no H1 H2 us us A7 49 50 53 55 LIST OF FIGURES FIGURE Page 1. The effect of electrode tip diameter on the maximum lens potential . . . . . . . . . . . . . . .‘. 38 2. Photomicrographs of normal and decapsulated lenses . . H5 3. The effect of temperature on the maximum potential Of the lens. 0 O O O O O Q 0 O O O O O O O I O O O O O 51 u. The effect of changes in K+'concentration in the bathing medium on the lens potential . . . . . . . . . 5H 5. Photomicrographs of condition of epithelium after exposure of lens to hyper and hyposmotic insult. . . . 58 APPENDICES FIGURES Microelectrode Assembly. . . . . . . . . . . . . . . . . . 82 Physical Dimensions of the Microelectrode. . . . . . . . . 83 Block Diagram of the Circuitry . . . . . . .’. . . . . . . 85 Front End Circuitry for Tip Potential, Electrode Resistance, and Amplifier Drift Determinations . . . . . 86 vii Appendix 1: Appendix 2: Appendix 3: LIST OF APPENDICES The Microelectrode. . . . . Circuitry . . . . . . . . . Solutions . . . . . . . . . viii Page INTRODUCTION It has been observed by Allison (1963) and Hoffert and Fromm (1965) that hatchery-reared lake trout are sus— ceptible to ocular pathology. The foregoing is true for hatchery-reared fish in general (Allison, 1967). This pathology often involves lenticular lesions which are virtually identical to human cataracts. Because of the susceptibility of rainbow trout to cataract formation and because of their availability, they offer an excellent experimental animal for the investiga- tion of physiological changes which occur during the onset of cataracts. In addition, trout eyes are large in compari- son to their body size, and thus even small fish have lenses which are convenient to handle. Very little is known about normal physiology of rainbow trout lenses and since it seems superfluous to look at pathology without knowledge of normal function, this study of the normal physiology of the rainbow trout lens was under- taken. The rainbow trout lens is normally crystal clear which implies that the internal environment of the lens is well controlled. It is known that the ionic composition of the lens is vastly different from that of aqueous humor and the vitreous (Hoffert, 1967; O'Brien and Salit, 1931). These facts being true, one would expect a consistent poten- tial difference to occur across the lens membranes. Altera— tion of this potential then would be an indication of change in normal lens physiology. The membranes of the lens do not offer a convenient or classical preparation with which to investigate and correlate ion movements with potential changes. Obviously the lens membranes cannot be physically isolated into an external side and an internal side. One can alter the ex— ternal environment of the lens, but since the internal lens has little interstitial fluid (Thoft and Kinoshita, 1965b; Huggert, 1959a, 1959b), the internal environment cannot directly be altered or sampled. Any changes which occur in the internal composi- tion of the lens are a result of the lens membranes reacting to external environment changes or to breakdown in normal function of the membranes. Thus, it seems that the logical approach is to monitor, with microelectrodes, the changes in potential difference which might occur as the external environment is altered. The use of microelectrodes would allow the measurements to be made with minimal disruption of the normal function of lens membranes. The answers to the following questions seem per— tinent to a knowledge of normal lens function: 1. Does a potential exist across the lens membranes? 2. If so, what is its magnitude? 3. What layers in the lens are responsible for the potential? A. Is the potential the same at any position on the lens? 5. What is the ionic dependence of the potential? 6. Does the maintenance of the potential require the expenditure of metabolic energy? 7. Does induced clouding of the lens affect the potential and, if so, how? It was the purpose of this study to answer all of the above questions with the hope of ultimately settling upon an approach to investigate the mechanisms whereby the transparency of the lens is maintained and to determine what failures in the mechanisms occur when cataracts are formed. LITERATURE REVIEW Microelectrode#potentials Studies on the potential difference across cell membranes using one internal electrode and one extracellular reference electrode were begun as early as 1939 by Hodgkin and Huxley. The studies of Hodgkin and Huxley (1939, l9u5) using microelectrodes to determine the potential difference across the squid giant axon membrane pioneered the field. Cole and Curtis (19u0, 1942), also investigating the giant squid axon, made valuable contributions to the technique. Because of their inability to make microelectrodes of con— sistent characteristics, both groups experienced low potential difference values and a great deal of variability from one axon to another. Graham.and Gerard (l9u6), because of the diffi— culty in obtaining and preparing squid axons, investigated the striated fibers of the frog sartorius muscle. They were able to consistently make glass capillary electrolyte electrodes with 2 to 5 u tip diameters. The values reported in these studies were 41.0 to 80.9 mv, inside negative, with a mean of 61.8 mv for the membrane potential of 65 fibers—- an improvement over earlier work. In l9u9, Ling and Gerard (19H9a) perfected a tech- nique for pulling microelectrodes of about 1 u tip diameter u which were rigid enough to penetrate frog muscle cells. Their 97.6:5.7 mv, inside negative, values more closely approached the calculated membrane potential. Technology in transmembrane potential measurements did not improve much until Alexander and Nastuk (1953) designed a solenoid-operated electronic microelectrode puller. This in- strument allowed.the consistent production of extremely small electrodes (0.3 to 0.5 u tip diameter). These small elec— trodes eliminated a great deal of the variance in recorded potential values. A brief sampling of the literature on the frog sar- torius muscle transmembrane potentials shows a typical value of about 90 mv, inside negative (Adrian, 1956). That the potential is dependent upon the expenditure of metabolic energy has been shown by Shanes, Abraham, and Brown (19H2) and Ling and Gerard (19u9b). Increasing the potassium ion concentration in the bathing medium resulted in reduction of the potential, nearing zero at high K+ levels; whereas altering the chloride ion level while keeping the potassium ion level constant resulted in almost no change of the potential (Hodgkin and Horowicz, 1959). Ling and Gerard (19H9a) reported that the fiber potentials were independent of muscle length, of pH between five and ten, and were sensitive only to potassium ion of the normal ions in Ringers solution. Two excellent reviews of microelectrode technology and construction are currently available: Kennard, 1958, and Frank and Becker, l96u. Lens potential studies Few studies have been done on the potential differ— ence which exists across the outer membranes of the lens. Constant (1958) measured the oxidation-reduction potential of lenses cultured in the presence of several acid or basic dyes. She found the potential to be more negative in basic dyes. No alteration of the potential occurred when the lenses were placed in the acid dyes studied or in cataractogenic 2-6 dinitrophenolindophenol. Brindley (1956), while investigating the electrical activity of the retina with microelectrodes, quite by acci- dent discovered a potential of about 70 mv, inside negative, across the lens membranes of the frog. Repeating the experi— ment on rabbits, he found a 66 mv potential, inside negative. He found the potential to be the same for the anterior and posterior surface in both frog and rabbit. In the frog u 0 + C 0 potential. Replacement of NaCl by 100 mEq K /l1ter 1n lens, replacement of C1- by 80 produced no change in the Ringers caused the potential to drop to near zero. He con— cluded that the lens is much less permeable to Cl- than to K+ and is about 32 times as permeable to K+ as to Na+. He stated that the structure responsible for developing the potential difference could be either the lens capsule or the cell membranes of lens fibers. Sperelakis and Potts (1959), using microelectrodes, recorded potentials of 23.3 mv (inside negative) across individual bovine lens fiber membranes. Using AnggCl agar-saline wick electrodes they reported a transcapsular potential of 26.2 mv (inside negative). This potential in- creased linearly from about 8 mv to 28 my (Q10=1.93) as the temperature was increased from 17 to 36 C respectively. The potential was decreased by 2-H DNP, Iodoacetate (IAA), EDTA, ultraviolet radiation, and N2 bubbling through the medium. Isosmotic NaCl and Choline C1 had no effect on the potential, whereas isosmotic KCl and KZSOM reduced the negative poten- tial to zero or slightly positive values. Isosmotic Na2SOu reduced the potential to 50% of its control value. They reported a short circuit current of 18 uA/cm2 but did not correlate it with movement of any ion. They concluded that the lens capsule is active in maintaining the potential and that the lens is best considered to be many cells within one giant cell. Other potential profile: Using a technique almost identical to the one reported in this thesis, Kikkawa (l96u) measured the potential profile of the rabbit cornea. The potential was found to be negative inside and increased as the microelectrode penetrated through the epithelium; the maximum value was obtained at an average depth of 53 u. On penetrating the stroma, the potential dropped sharply toward zero but still remained negative. Attempts to extend the measurement of the potential to the interior were unsuccessful, for the microelectrodes broke upon deeper penetration into the stroma. Engbaek and Hoshiko (1957) used the microelectrode technique to determine the potential profile across the frog skin. They found total skin potentials ranging from 73 to 145 mv, the inside positive with respect to ground. The total skin potential was reached in two potential difference jumps, whereas Ottoson SE 31. (1953) reported only one jump which was across the dermoepidermal region. Engbaek and Hoshiko, after comparison of their emf values and thickness measurements,suggested that the first jump is in the epidermis while the second occurs at the junction of the epidermis and corium. Cation studies That a potential difference occurs across the mem- branes of the lens is not surprising in light of the cation differences between the lens and aqueous humor and vitreous body cited earlier. Additional studies have shown that rupture of the lens capsule results in Na+ influx into the lens with simultaneous K+ efflux (Harris and Gehrsitz, 1951). Absence of Ca++ in the incubation medium gave comparable results in the same study. Investigations of rat lenses (Thoft and Kinoshita, 1965a) show that they become generally more permeable in the absence of calcium: Sucrose and mannose enter the lens more readily, whereas the accumulation of K+ or Rb+ is de— creased, presumably due to increased leakage of these materials from the lens. That sodium penetration into the lens is also calcium dependent is suggested by the fact that in the absence of calcium, the sodium space is nearly equal to the water space. Merola, Kern and Kinoshita (1960) also reported a reduction in the ratio of K+/Na+ for calf lenses incubated in calcium deficient media, a result which supported data from Harris, Gehrsitz, and Nordquist (1953) for rabbit lenses. Long-term infection in rats also resulted in reduced phosphate and increased calcium content of lenses (Currie and Kenny, 1967). Treatment with antibiotics resulted in a reduction of Ca++ and an increase in phosphate. The rate of exchange of K+ between the lens and its bathing medium at equilibrium has been determined indepen- dently by Kinsey and McLean (1969) and Thoft and Kinoshita (1965c). Their results show that about 85% of the lens potassium ion is available for exchange but that the internal- external concentration difference is maintained. Inhibitors and metabolism Kinoshita, Kern, and Merola (1961) and Kern, Roosa, and Murray (1962) found that ouabain inhibited the maintenance of normal ionic gradients between the lens and the in-vitro extracellular—like fluid in which it was bathed. 10 Further evidence for the metabolic dependence of the lens cation equilibrium was obtained by Harris and Gruber (1962). They reported that the cation imbalance could not be retained in the presence of iodoacetate or ouabain. It seems likely from their studies that the maintenance of the cation state depends only on anaerobic metabolism. They reported that ATP is probably involved in the lens cation transport and suggested the presence of a Na-K activated adenosine triphosphatase. Their kinetic studies with Na21+ suggest that the lens capsule is the barrier to sodium movement. Hydration of the lens occurred in every situation where cation shift occurred. Studies by Hauschildt, Harris, and Nordquist (1955) show there is no correlation between cation transport and the concentration of organic acid-soluble phosphorous or seven minute—hydrolyzable phosphorous of the lens. Also, no paral- lelism exists between cation movement against a concentration gradient and movement of inorganic phosphorous. It has been shown by Schwartz, Danes, and Leinfelder (1959) that metabolic energy is needed by the bovine lens to maintain its normal hydration state. Reduced temperature, lack of glucose, anoxia, lowered pH, and treatment with cyanide or iodoacetate all caused increases in lens weight due to hydration. The authors suggested that changes in weight occur as a result of the inability of the lens to maintain its ionic balance; however, no ions were measured. They also 11 reasoned that, since hydration occurs under conditions of reduced metabolism, the occurrence of hydration must indicate a metabolic deficiency. Additional supporting evidence for the metabolic dependence of lens cation levels has been presented by Becker and Cotlier (1962). In their study, rabbit lenses which had accumulated Rb86 were incubated in a medium containing suffi- cient nonlabeled rubidium to saturate the carrier. The rate of runout of Rb86 could be increased by reducing the concen— + in the medium or by the addition of iodoacetate, tration of Ca+ fluoride, or cyanide ions. Lowering of the lens temperature by refrigeration or poisoning by iodoacetic acid, fluoride,.or sodium cyanide resulted in increased sodium influx into the lens with simul— taneous K+ efflux (Harris and Gehrsitz, 1951). It was also shown that pyruvate, but not xylose, fructose, or galactose, could replace glucose in the medium without the occurrence of this cation shift. Harris gt El. (1953) showed that in-vitro reversal of refrigeration-induced cation shift occurred when the lens was incubated at 37 C. This return-to-normalcy reversal was greatly aided by the presence of glutamic acid. They concluded that any cation ratio across the lens is a balance between two opposing movement of cations,one against a concentration gra— dient which is due to an active transport mechanism, and the other with a concentration gradient representing a passive movement through normally permeable barriers. 12 Location The location of the barrier to cation movement between the lens and ocular humors is subject to some question. Becker (1962) suggests the location of a transport system in the epithelium.‘ He reported that incubating-lenses with only the anterior or the posterior surface in contact with a medium containing Rb86 resulted in Rb uptake almost entirely through the anterior side. In additional studies, lenses were quick frozen after submersion in an incubating medium containing Rb86. A 6 mm diameter core was trephined from the lens going from the anterior to the posterior surface and was sectioned into ten parallel slices. The most anterior slice containing the epithelium was found to have the highest activity, 1.7 times as high as the most posterior slice and 2.9 times as high as the center slice. After treatment with ouabain,Rb86 uptake was reduced, and the activity in the anterior and posterior slices was nearly equal. Reddy and Kinsey (1963) also confirmed the epithelial location of the transport system. In their study, 3.5 times as much K“2 was found to pass through the anterior side as through the posterior. In the presence of 10"5 M ouabain, the anterior uptake was reduced to the same value as the posterior uptake while the posterior uptake was unchanged. Concomitantly, Na22 entry through the anterior side was in- creased 70% by ouabain, whereas posterior uptake of Na22 was not changed. 13 Bonting, Caravaggio, and Hawkins (1963) found that an enzyme, Na-K activated adenosine triphosphatase, which is closely related to the active transport of sodium and potassium in human erythrocytes, occurred in the epithelium of the lens of the cat, calf, and rabbit in relatively high activities. No significant activity could be detected in anterior and posterior capsule, cortex, and nucleus. It has been reported by Mandel and Klethi (1958) that the ATP level of the mammalian lens is as high as that of the heart, liver, kidney, and brain. The ATP level was found to be highest in the superficial lens cortex including the epithelium. Further studies by Kinsey and Reddy (1965) support the epithelial location of the transport mechanism. Removal of the lens surface membranes was found to alter, in a manner similar to metabolic poisons, the rate of accumulation and exit of all ions studied. Penetration of K“2 and Rb86 was greater across the anterior than posterior surface, a dif— ference which was abolished by metabolic poisons and removal of capsule and epithelium. Penetration of Na22 and Cl36 was greater from the posterior side, and removal of capsule and epithelium increased influx across both surfaces as did iodoacetate and ouabain for Na22 but not C135. Removal of these surface membranes also abolished the effect of inhi- bitors. It was concluded that potassium and Rb+ are actively transported into and sodium out of the lens by carrier systems 1” located in the epithelium and that movement of H20 and Cl— between lens and its in-vitro bathing fluid occurs passively by diffusion. Using the rubidium washout method, Becker and Cotlier (1965) found that enzymatic decapsulation of the rabbit lens, leaving the epithelium intact, did not alter the Rb86 uptake or runout. They again concluded that the transport mechanism is located in the epithelium of the lens. Information which suggests that the capsule is in— volved in the control of ionic movements in and out of the lens was presented by Brindley (1956) and Sperelakis and Potts (1959). These authors found the same potential differ- ence across the anterior and posterior surfaces in the lenses measured. The latter authors even reported a potential dif- ference of about 26 mv across the capsule alone in bovine lenses and concluded that the lens capsule is metabolically active in the maintenance of the potential difference. MATERIALS AND METHODS Experimental animals The rainbow trout (Salmo gairdneri) used in these experiments were obtained from the Michigan Department of Conservation Hatchery at Grayling, Michigan. The fish were transported to the East Lansing campus in a galvanized metal tank lined with a non-toxic paint. The tank, fitted with an agitator to provide aeration, was housed in a polystyrene- lined plywood box to prevent temperature changes of the tank water. In the laboratory, the fish were kept in fiberglass- lined plywood tanks supplied with a continuous flow of de— chlorinated water. Constant aeration was provided by acti- vated charcoal-filtered air lines. Conditions of 13 C and 15 hours light, 9 hours darkness were maintained each day. The animals were transported from the holding facility to the research laboratory in a polyethylene bucket filled with dechlorinated water. Care was taken to use the trout before the temperature of the bucket water changed more than 2 C. Electronics Instruments and shielding.-—Since the measurement of membrane potentials depends upon the use of high impedance microelectrodes, a preamplifier with a high input impedance 15 16 was necessary. The Grass P6-l2 DC Preamplifier (Grass Medical Instruments Co., Quincy, Mass.) with an input impedance of 1011 Q was chosen. The P6-l2 was initially adjusted for operation in the manner suggested by the instruction manual. The instru- ment was left on continuously to allow maximum stability to be obtained. The gain and output level of the amplifier were checked daily, whereas all other adjustments were checked about once per month. It was found that only the output level varied from day to day. All other characteristics of the preamplifier remained constant until some degree of tube malfunction oc- curred. The highly selected paired tubes of the P6-12 high impedance input probe were replaced every four months because after this period of time the tube characteristiCs had drifted far enough apart that proper common Imade discrimination was no longer attainable. Even at optimal amplifier adjustment, drift measured over an hour or more averaged about 20 uv/min. However, minute to minute fluctuation of 200 to 700 uv were found to occur frequently. The Grass RPS 106—B Power Supply accompanying the P6-l2 regulates line voltage to within 1%, and it was found that no measurable fluctuation occurred in any of the output voltages of the power supply as a result of normal line voltage fluctuations. Consequently, a separate line voltage stabi- lizer was unnecessary. However, a Cornell-Dubilier (Allied 17 Radio Corp., Chicago, Ill.) type IF-u power line filter was used at all times. Because high impedance circuitry is prone to elec- trical noise, proper shielding was necessary. All experiments were performed in a Model 81 Radio Frequency Shielded Room (Ray-Proof Corporation, Norwalk, Conn.) which was maintained at absolute ground by way of a cable connection to a copper bar driven 30 feet into the earth. Care was taken to avoid ground loops. It was found that the preamplifier had lower noise voltages when it was shielded, therefore it and all other electronic components were brought into the shielded room. Because of this, the biological preparation required shielding from the electronics. A homemade galvanic chamber was used for this purpose. Since DC measurements were made and changes in poten- tial were slow, a Mosely 1700B 10 inch Servo Potentiometric Strip Chart Recorder (Hewlett Packard, Mosely Division, Pasadena, Calif.) was used instead of the usual oscilloscope. This permitted long—term, continuous recordings to be made. Balancing amplifiers.-—There was a tendency for the high impedance amplifier to drift and for spontaneous baseline shifts to occur when the microelectrode was in the circuit. It was therefore necessary to frequently check the system for baseline changes. This was done by shorting each of the input grids to ground. Any change in the baseline was then due to an alteration in amplifier balance and was not affected 18 by the microelectrode. To allow automatic measurement of this baseline change at consistent intervals, a DC relay and a model CM-u Industrial Timer (Allied Radio Corp., Chicago, Ill.) with a Cel2 gear assembly were placed in the circuit (see Appendix 2) and were adjusted so that the grids were shorted to ground for three seconds every minute. Any shift was corrected by use of the amplifier balance circuit. Shift in baseline due to the microelectrode was' corrected by placing the reference electrode and the micro- electrode in the Ringers bath (see Appendix 2). The ampli- fier was balanced by the procedure discussed in the preceeding paragraph. Any baseline change which occurred when the grids were switched from their shorted mode to operating mode was due to the electrodes. These changes were nulled to zero by use of the electrode balance circuit. Electrodes Reference electrodes.--Reference electrodes used for this study were Sargent (E.H. Sargent Co., Detroit, Mich.) S-30080—17 miniature calomel cells. It was necessary to shorten the leads to about 50% of their initial length to reduce capacitive-coupled noise signals. Also, their pin connectors were replaced by banana plugs to allow them to be plugged directly into the banana plug jacks on the P6-l2 preamplifier input probe. The leads were soldered to the banana plugs to insure good electrical contact. After the above modifications, 19 the entire lead from electrode to banana plug was covered with braided shielding which was then grounded. The electrodes were stored in Ringers solution with their leads shorted together to keep their potential difference as low as possible. Initially the potential differenCe between the electrodes was 2 mv and one year later they had decayed to a difference of only 7 mv. The electrodes were filled with saturated KCl and were frequently checked to be certain their KCl level was not depleted. It was also necessary to periodically wash the KCl deposits out of the bottom of the electrodes, because large deposits increased the time constants of the electrodes to the point where their speed of response was no longer acceptable. Pulling microelectrodes.--The microelectrodes were drawn from 1 mm OD Pyrex capillary tubing by use of an Industrial Science Associates, Incorporated, (Ridgewood, N.Y.) Horizontal Model Ml Micropipette Puller. The puller was adjusted by addition of external resistors so that the total weak pull resistance was 175 Q and total strong pull resistance was A20 0. The duration of weak pull was set at 50% of maximum. The heater was adjusted to give a total pulling cycle of 70 to 90 seconds. These settings resulted 60% of the time in one electrode per pull that had a resistance of 5 to 20 mega and a tip 20 potential of less than 5 mv. The usable electrode was always on the solenoid side of the puller. Tip diameters of these electrodes, as measured using a compound microscope with an ocular micrometer, were 2 to 3 u. Other characteristics of the electrodes are presented in Appendix 1. It was found that these electrodes were strong enough to repeatedly penetrate the tough lens capsule without the electrodes breaking or appreciably changing their electrical characteristics. Filling microelectrodes.——The microelectrodes were filled by using a modification of the Tasaki method (Tasaki, Polley, and Orrego, l95u). The pulled electrodes were moUnted, lu3 at a time, tips up, in a specially designed acrylic plastic holder. The tips—up position was chosen because it allowed more pipettes to be filled at one time with less change of damage to the tips. The electrodes were vigorously boiled under reduced pressure in warm methanol (temperature about 30 to 40 C) for 2 to 5 minutes. At this time, a bubble could be seen near the tip of almost every electrode. Upon returning the electrodes to atmospheric pressure, the bubbles disappeared in 5 to 10 minutes. The pipettes were then carefully dipped in distilled water, after which they were placed in 3 M KCl in acrylic plastic containers at room temperature. In A to 19 days, the electrodes had, by diffusion, completely filled with 314KC1 and were ready for use. They were stored under 21 3 M KCl in the above containers, but after 6 weeks in storage their tip potentials were found to increase beyond the usable point and they were discarded. Selecting electrodes.—-For testing, each electrode was taken from the storage container, dipped in distilled H20 to remove excess KCl, and placed in the microelectrode holder (see Appendix 1). Ringers solution was then added to the holder and the holder attached to the micromanipulator and lowered into the Ringers-filled measuring chamber until the tip of the microelectrode was well immerSed in Ringers solution. At this time both calomel reference electrodes were also placed in the measuring chamber. The amplifier was balanced by the procedure stated earlier. The potential difference of the calomel electrodes was balanced to zero by the application of a counter emf from the electrode balance circuit of the P6-12 preamplifier. One calomel cell was placed in the appropriate position in the top of the microelectrode holder where it was connected to the microelectrode by a Ringers fluid bridge. The amplifier was then balanced again. The potential difference between the reference electrode—microelectrode unit and the other reference electrode was the tip potential. Its value was always negative and was acceptable if it was between 0 and -5 mv. The resistance of the electrode was measured by switching a high resistance decade box (values from 1 to 22 100 mega in 1 mega steps) in series with the electrode and measuring the voltage drop across this added resistance. The resistance of the decade box was adjusted so that 50% of any voltage applied from the P6-l2 preamplifier calibration battery circuit was recorded. At this point, the resistance of the decade box was equal to the resistance of the electrode. Thus, the electrode resistance could be read directly from the decade box. Electrodes with resistance between 5 and 10 megQ were found to be strong enough for repeated penetrations, of low enough resistance to be very stable, and small enough to give consistent and repeatable values. Manipulator and drive The microelectrode was lowered into the lens by means of a Pfeiffer (Beryllium Mfg. Co., Valley Stream, N.Y.) HP MAX ultramicro manipulator, which was mounted on a heavy steel frame. The frame was bolted to a piece of soapstone which in turn was placed on a rubber shock-absorbing material. The manipulator was driven by a rubber belt from a well- shielded reversible Hurst (Allied Radio Corp., Chicago, Ill.) 1 rpm synchronous motor. The motor drive pulley was twice the diameter of the pulley used on the drive shaft of the manipulator, thus the manipulator drive shaft turned at 2 rpm. This speed coupled with the drive adjustments on the manipulator itself allowed penetration and withdrawal rates of from 5 to 100 microns per minute. 23 For many of the experiments, the rubber belt was not used and the manipulator drive was turned by hand. This procedure was satisfactory for any study were knowledge of depth of penetration was not required. Procedure for potential measurements Removal of lenses.—-The fish were decapitated at a level just posterior to the operculi. The eyeball was held, while in the head, with a rat tooth forceps and an incision made at the limbus of the cornea with a sharp-pointed scapel. The cornea was then cut around its entire periphery with iris scissors and gently removed from the eyball. Small curved forceps were then placed under the lens, which was gently lifted out of the eye, carefully washed in Ringers solution to remove excess aqueous humor, and immediately placed in a beaker of fresh Ringers solution at room temperature. This entire procedure resUlted in damage to about one out of every eight lenses. Data from any lens whose potential value was 75% or less of that of the contralateral lens of the same fish was dis- carded. The lenses were held by gravity in a depression in aniacrylrcplastic rectangular bar cemented inside an acrylic plastic chamber. The bar was drilled so that vacuum could be applied from below to hold the lens in position. Vacuum was used only during the electrode withdrawal phase of a few lens potential measurements. 24 Procedure for measuring normalgpotentia1.—-To make a measurement of the normal lens potential, the lens was gently lifted from the beaker’ofRingers solution by use of a small, curved forceps (which could easily be slipped under the lens) and placed in the depression of the measuring chamber which had been prefilled with Ringers solution (200 cc). The Ringers solution was then drawn out through polyethylene tubing connected to a 50 cc syringe until the level was just below the top of the lens, leaving it exposed to air. The electrodes were then switched to the operating mode. Since the microelectrode was raised above the lens and was not in contact with the bath, an open circuit was present between the recording electrode and reference electrode. This resulted in wild voltage fluctuations on the recorder. Lowering the electrode until it just touched the lens removed the fluc- tuations. At this point, a depth reading which represented the upper surface of the lens was taken from the micrometer dial of the micromanipulator. The electrode was then raised 100 u above the surface of the lens and the Ringers solution was replaced so that both reference and recording electrodes made contact with it. The amplifier and electrode were then balanced by the proce- dure discussed earlier. The micromanipulator was then either driven by motor or by hand to lower the microelectrode into the lens. The potential difference between the inside of the lens and the 25 reference electrode in the bath was recorded. During this time a frequent amplifier drift check was made with the timer- relay dircuit. After either hand- or motor-mediated removal of the electrode from the lens, any change in the balance through the electrodes was corrected by use of the electrode balance circuit. If this change was more than :3 mv, the measure— ment was discarded. The resistance of the electrode was also checked after each penetration, and if more than a 10% change in resistance occurred, the measurement was discarded. Procedure for mapping studies.——After removal of the cornea, the anterior surface of the lens was marked with Janus Green dye. The dye was delivered by means of a piece of polyethylene tubing which was attached to a 22 gauge syringe needle. A 1 cc tuberculin syringe worked well to hold and deliver minute amounts of dye. The polyethylene tubing was filled with dye and then was lowered into the eye until it just touched the lens. Usually this resulted in a light blue spot on the mid-anterior portion of the lens. Another spot was similarly placed well above the first and in the direction of the upper suspensory ligament. This procedure resulted in a lens marked both on the anterior surface and on the upper pole. The dye had no effect on the lens potential (see Table 9). The lens was then removed and prepared for measure— ment by the usual technique. The measurements, all with 26 the lenses bathed with Ringers solution, were made first on the side nearest the electrode when the lens was placed in the chamber. Minor position adjustments were made by touching the lens lightly to rotate it. After the initial measurements were made, the lens was rotated 1800 and the potential of the opposite side measured. With the contralateral lens of the same fish, the side opposite that measured first in the previous lens was the first to be measured. For example, if the anterior surface was measured first in the right lens, the posterior surface was measured first in the left lens. Only two positions were measured on any one lens, for it was found that excessive rotation of the lens in the experimental chamber frequently resulted in lens damage and a reduced potential. Procedure for inhibitor studies.--Lenses were handled in the usual manner. All lenses were initially measured in Ringers solution with the electrode being lowered by manually turning the micromanipulator until maximum potential was recorded. After the initial measurement, one lens was placed in a beaker of Ringers solution for every three placed in the experimental solution. The experimental solution was composed of the inhibitor dissolved in Ringers. Subsequent measurements were made at 1 and 2 hours. In each case, the control was measured in Ringers solution, whereas each of the experimentals was measured in the experimental solution. 27 Usually there was no change in the control value over two hours. However, if the electrode broke anywhere in the procedure and had to be replaced, some change in the control value was always noted. All experimental lenses were corrected for this change by multiplying their values by the ratio: P.D. P.D O 'T lens potential at beginning of experiment and where P.D. o P.D.T lens potential at specified time. Procedure for ion studies.--All lenses were measured by the usual technique in Ringers solution. The micromanipu- lator was turned by hand until maximum potential was recorded. One control lens was placed in a beaker of Ringers solution for every three placed in the experimental solution. Ini- tially, the experimental solution was added to the measuring chamber. Each lens was individually placed in the chamber and measurements were made every minute until the same value persisted for three consecutive measurements. At this point, equilibrium was assumed to have been achieved. Each lens was then placed in a beaker of the experimental solution. Measurements were again made when each lens had been in the experimental solution for a total of 1 hour. These values were corrected as before for any potential change in the control lenses. 28 Procedure for study of osmotic effects.--As before, all lenses were measured in Ringers and controls were placed in beakers of Ringers solution for storage. Lenses were soaked in distilled H20 for 10 minutes or in 0.3 M NaCl for 15 minutes. Immediately after this treatment their potentials were measured in Ringers solution. They were then placed in individual beakers of Ringers solution and allowed to recover for 1 hour, at whiCh time their potential was again measured. As before all values were corrected for any change in the controls. Procedure for determining effect of temperature on the lens p9tential.--All lens potentials were measured by the usual technique and the maximum potential recorded. The lenses were then placed in Ringers solution and maintained at 0 C in an ice H20 bath. After 30 minutes at 0 C, each lens was placed in the measuring chamber which was filled with Ringers previously kept at 0 C. During the transfer of the Ringers solution from the ice H 0 bath to the measuring 2 chamber some warming occurred. Consequently, 3 C was the lowest temperature attainable in the chamber. The tempera- ture of the chamber was monitored by means of a YSI (Yellow Springs Instrument Co., Yellow Springs,0hio) Model H23 Thermistor and Model H3TD Telethermometer. As the solution and lens warmed, potential measurements were made at the reported temperatures. 29 For the effect of temperatures above room tempera- ture, the same procedure was followed except that the solution and lens were initially maintained in warm Ringers solution and were measured as they cooled. A few lens potentials were measured continuously as the temperature of the bathing medium was gradually reduced by thermoelectric cooling. The potential in these studies fell smoothly as the temperature was reduced. Procedure for determining electrode characterisfics Procedure for measuring pressure effect.--The entire tip of the microelectrode was placed in the lumen of a Ringers- filled rubber tube attached to a pressure bottle. In order to keep from breaking the microelectrode during its inser- tion into the tube, it was lowered by means of a micromanipu— lator through a 12 gauge syringe needle whose tip was in the lumen of the tube. The syringe needle was then removed. The Ag-AgCl wire electrode was placed in the lumen of the rubber tube by the same technique to prevent damage to the AgCl coating. Both electrodes were then plugged into the amplifier input. The pressure in the pressure bottle was then increased over a range of 0 to 100 mm Hg and the potential recorded after balancing and calibration (see p. 17).- Procedure for measuring electrode distortion.--The electrode was driven at 100 u/min. onto an acrylic plastic 30 bar that was inclined 160 to horizontal and immersed in Ringers solution. This inclination was necessary to insure that the tip of the microelectrode was not occluded by the nonconducting acrylic plastic, for it was found that occlu- sion of the tip by a non-conductor resulted in the genera- tion of large 60 cycle AC potentials. The electrode was observed optically by means of a dissecting microscope and was seen to bend considerably and finally to break. RESULTS Effect of physical factors on micro— electrode potentithTTables l, 2,_and 3) After insertion of both microelectrode and reference electrode into the Ringer-filled rubber hose from the pres— sure bottle, both calomel asymmetry potential and tip potential were reduced to zero by application of a counter- electromotive force from the electrode balance battery. The pressure on the electrode tip was then increased slowly from 0 to 100 mm Hg while the potential was being recorded. Table 1 summarizes the data from these experiments. No potential was induced by a pressure increase at the tip of the microelectrode throughout the pressure range which was utilized. Table l.--Effect of pressure on electrode Electrode Maximum potential number at 100 mm Hg 1 . 0.0 mv 2 +0.2 mv 3 _ 0.0 mv Table 2 gives the results of lowering a microelec— trode onto an inclined plastic bar immersed in Ringers solution. 31 32 H.m+ 0.0: H.3I 0.0+ H.5+ 5 0.0+ 0.0 0.0: 0.0+ H.5+ 0 H.5+ 3.0: 0.0: 0.H+ H.5+ 0 0.0+ H.0n 0.0: N.H+ 0.5+ : 0.0+ 0.Hn 5.0: 0.0+ 0.5+ m 0.5+ 5.0: 0.0: 0.3+ 0.5+ m 0.0+ 0.0 0.0: H.m+ 0.5+ H “>50 A>Ev A>EV A>Ev A>EV ponesc wcwxmmpn mcflxmmpn deflucmvoa HMflPCMpom gap xaumeazmm mooppomam amumm HMflpcmpom whommn mmcmno aflH + >nmeE>mm Hmanmo amapcmvom Edeflxmz Hwanmo mamHm omcwaocw pmcwmwm covaODmflQI|.m magma 33 Observation through a dissecting microscope showed that considerable bending and finally breaking of the microelec- trode occurred. No significant change in potential occurred during the distortion. However, since the tip potential depends on a small microelectrode tip, breaking the tip abolishes the tip potential. The potential which remains after breaking is quite comparable to the initial calomel asymmetry potential. Potential measurements were made on a 10% agar in Ringers immersed in a Ringers reference solution. Ten per- cent Ringer agar was used because its consistency seemed more dense than the lens and thus should distort a microelectrode tip more than the internal lens would. The electrode was lowered at 100 u/min. to a depth of 1,000 u. No significant potential difference resulted from any penetration, as shown in Table 3. Table 3.--Effect of electrolyte matrix on electrode I L Calomel Calomel Calomel asymmetry + Tip asymmetry + Electrode asymmetry tip potential potential tip potential number (mv) (mv) (mv) at 100011(mv) 1 ' +7.1 +3.2 —2.9 +3.2 2 +7.1 +u.1 -3.0 +3.9 3 +7.1 +1.2 —5.9 +1.2 u +7.1 [+2.0 -5.1 +2.0 5 +7.1 +2.2 —u.9 +2.1 34 The amount of distortion, if any, could not be measured opti- cally. If the 10% agar was made up in some solution other than Ringers, e.g. KCl or Na SO”, a potential difference 2 between the agar and the bathing solution could be measured. These data indicate that the electrode was sensitive to the potential which results from ionic differences across a boundary. However, just placing the electrode into an electrolyte mass does not give rise to a potential if the internal electrolyte is the same as that of the bathing solution. It was also found that the electrodes did not reSpond to temperature changes (3 to A3 C) in the bath or to pH changes over the range of pHH to pH9. Application of a calibration signal through the reference electrode into a Ringers bath in which both micro and refer- ence electrode are immersed results in the recording of the same potential difference as when the calibration signal is applied directly to the input grids of the amplifier. Conse- quently, the microelectrode measures potential. The mean and standard error of 28 penetrations into frog sartorius muscle fibers was -59.5:2.2 mv, as shown in Table A. These values correlate well with the -6l.u mv mean values obtained by Graham and Gerard (19H7) on the same tissue with electrodes of about the same (2 to 5 u) tip diameter. 35 Table u.--Frog sartorius muscle cell potentials Number of Potential observations (mv) 28 -59.5:2.2* *Mean 1 standard error Comparison of in-vivo and In-vitro lens potentials The potential difference of four lenses was measured while they were in the eye. The cornea was removed, the refer- ence electrode placed in contact with the aqueous humor, and the microelectrodes (8 u tip diameter) were lowered into the lens_until maximum potential was recorded. The mean and standard error for the potentials was -HH.0:7.5 mv. The lenses were then removed from the eyes and measured as usual in the Ringers—filled chamber. The -u3.718.5 mv value obtained from the in—vitro lenses show that removal of the lens from the eye does not appreciably affect the potential. The values in Table 5 also suggest that the ionic composition of the Ringers solution used in this study is close to that of aqueous humor. Table 5.-—Comparison of in-vivo and in-vitro lens potentials Number of Potential Potential observations in-vivo (mv) in—vitro (mv) A —uu.0:7.5* -u3.7:8.5* *Mean 1 standard error 36 Effect of repeated microelectrode penetrations the lens was‘ same 81 That te in on lenspotential_ each lens (Table 6). the microelectrode does not appreciably damage shown by making repeated penetrations into the Table 6.--Repeated penetrations into same site No. of Probe #1 Probe #2 Probe #3 Probe #u Probe #5 obser- potential potential potential potential potential vations (mv) (mv) (mv) (mv) (mv) 11 -49.0i2.6* —H9.3:2.8* -H8.5:2.7* -H7.3i3.0* ~H8.0:2.9* *Mean 1 standard error Five measurements on each of eleven lenses established that there was approximately a 2% reduction in potential by the fifth penetration, and the reduction is not statistically significant. Up to 22 penetrations have been made on a few lenses within an hour with no more reduction than about 5% by the 22nd penetration. These data suggest that holes which the electrode made were readily sealed by the lens tissue. It is still possible that the electrode makes the lens "leaky" while it is inside, resulting in a lowered potential. Normal potential of ‘ rainbow trout lens The resting potential of lenses of rainbow trout eyes were measured in vitro using microelectrodes. The mean t -59.0:1.l mv. The potential standard error for 52 lenses was 37 was negative inside with respect to the outside bathing solu— tion (Table 7). Table 7.--Normal potential of rainbow trout lens: comparison of right and left eyes Number of Potential observations 2(mv) 52 —59.0il.l* Potential of Potential of right eye (mv) left eye (mv) 26 . -59.3:1.6* 59.nil.6* *Mean 1 standard error The same data were divided to compare right and left lens potentials and the values which resulted were ~59.3tl.6 mv for the right eye and -59.H:l.6 for the left. The fact that these values were almost identical was not surprising and lends credibility to the technique. Relationship between lens poten- tial and electrode tip diameter The data in Fig. 1 indicate that the potential mea- sured is a linear function of the logarithm of the tip diameter in microns. Because of this the potential reported (Table 7) may be of lesser magnitude than that which truly exists in the lens. LENS POTENTIAL (-mv) 80 7O 6O 50 MO 30 2O 10 38 Figure l.~—The effect of electrode tip diameter on the maximum lens potential \+ ,3” 8n no“ I l l l 0.5 1.0 1.5 2.0 LOG OF TIP DIAMETER (u) *Mean.i standard error 39 Measurement of_potentials at adjacent regions To determine the reliability of the measurement tech- nique, potentials were first measured in a given position with 8 to 10 p electrodes, and then the electrode was moved laterally 100 u where another measurement was made. Initial position potentials were -33.5:l.3 mv, whereas adjacent region poten- tials were -33.lil.2 mv. These potentials, shown in Table 8, were not significantly different at the a=0.05 level indicating reliability and repeatability of the technique. Table 8.-—Adjacent region potentials '—-_‘—_—_-_—‘—‘—T'_"‘—'—-""-_——'———_"—_—_—-—_“—_-_————'- Potential at Potential at Number of control position adjacent position observations (mv) (mv) 23 -33.Sil.3* .1-33.1tl.2* *Mean 1 standard error Effect of Janus Green on lensgpotential To determine whether or not the Janus Green marking technique affects the lens potential, 18 lenses were marked by the usual technique. Using 8 to 10 u tip diameter micro— electrodes, a potential difference measurement was made on each lens in the dye spot. The electrode was then moved laterally until it was outside the dye spot and the potential measured again. The results are shown in Table 9. HO Table 9.--Potential in dye spot versus potential along side of dye spot Potential in Potential in Number of dye spot adjacent non-dyed observations (mv) position (mv) 18 -32.5:1.H* -32.1i1.3* *Mean 1 standard error The values obtained by the two measurements are not different from each other and are not different from normal potentials measured on other lenses with 8 to 10 u electrodes. Conse— quently, the dye does not affect the magnitude of the poten— tial. Comparison of potentials in living vs. "dead" lens tissue To determine if the potential is a function of living lens tissue, potentials of freshly removed lenses were measured in Ringers, after which the lenses were placed in Dietrich's fixative (Appendix 3) for 6 hours. The potentials were again measured in Ringers solution. The data in Table 10 shows that after fixation the potential not only decreases but reverses polarity. It was concluded that the negative lens potential depends upon native protein in lens membranes. H1 Table 10.--Potential of living and fixed lenses mP—w— ? “—21-“ Potential in Potential in Number of Ringers before Ringers after Observations fixation (mv) fixation (mv) *Mean 1 standard error Potentials at various sites on lens Potentials were measured at selected positions on the lens by use of 8 p tip diameter microelectrodes. The lens was marked with spots of Janus Green dye to allow proper orien- tation of the lens. Only two positions were measured on any one lens so as to reduce the possibility of manipulation damage. The data in Table 11 indicates that the lens is equi— potential, i.e. the potential is the same regardless of where one measures it. The depth at which the maximum potential occurs is not the same. At 183.0117.H u penetration the maximum posterior potential occurs, whereas 2n1.7:19.3 u penetration is necessary to achieve the maximum anterior potential. Effect of removal of capsule and epithelium on lens potential To investigate the effect of removal of the capsule and epithelium, potentials were first measured on six lenses in the usual manner, and the maximum potential recorded. M2 mocmnmmmao Hmfipcmpoa u .Q.m poahm ppmocmvm H Goose «0.HH04001 «0.HH0.00| 0 A>Ev “>50 Hmwpcmpoa Hmwucmpom maoa guano hOflpmpc< «N.HH:.001 «m.awm.mmu 0 A>EV A>EV HMflpcmuom HMflpcmpoa mHom bozo; maom Loam: 00.0vmvmmo.0 «:.5HH0.00H «0.0HH5.H:N mm AmCOQOHEV Amcomowav .Q.m hoapmpmoa Edawxme .o.m QOflnmpcm Esafixme pom apnea pow 06060 «0.HHNH.00| «5.HHN.NmI mm A>Ev “>80 mcowpm>pmmno HMflpcmyom wapcmpoa mo amnadz soapmvmom howpmpc< i! |I .mcmH co cOflprom mdmhm> Hmwpcmvomll.aa mHAMB U3 Following these measurement the capsule and epithelium were removed and the potentials measured were 80% less than those of intact lenses (Table 12). Table l2.—-Effect of removal of capsule and epithelium on the potential difference of rainbow trout lenses P.D. with P.D. without No. of capsule and capsule and obser— epithelium epithelium P.D. without vations (mv) (mv) _P.D. with 6 -u5.u:3.u* -9.2:.5u* 0.206:.017* p<0.001 *Mean 1 standard error P.D. = potential difference This suggests that the capsule and epithelium are responsible for the majority of the potential. The remaining 20% is presumably a potential difference between interstitial fluid and Ringers or due to some additional action of the lens fiber membranes on ion movement. Periodically, deeper penetration _gave rise to abrupt 15 mv increases in potential which fell to normal after a few more microns penetration. Three or four of these potential "peaks" could be recorded for each penetration of #00 to 500 microns. It is suggested that these potentials represent transmembrane potentials of lens fibers. Effect of capsule damage onppotential In another experiment, the potential of seven lenses was measured in Ringers with 8 u tip diameter microelectrodes. nu Figure 2 A. Photomicrograph of a normal rainbow trout lens (H and E, X 532) B. Photomicrograph of rainbow trout lens with capsule and epithelium removed (H and E, x 532) 95 Figure 2 H6 After a nick was placed in the capsule by use of a sharp pointed scalpel blade, the potential dropped significantly, as shown in Table 13. Table l3.--Effect of lenticular capsule damage on the lens potential No. of P.D. before P.D. after obser- damage damage P.D. after vations (mv) (mv) P.D. before 7 -uu.3:2.9* -2u.0:1.0* 0.5u7:.023* p<0.001 *Mean 1 standard error P.D. potential difference The extent of the lesion did not seem to affect the magnitude of the potential decrease. A minute nick and a well-defined slit produced about the same change. The resulting potential, about 50% of the control, seemed stable and did not decay appreciably with time. In about one of every eight lenses, one of the sus- pensory ligaments would adhere tenaciously to the capsule and would cause a minute rupture of the capsule as the lens was being removed from the eye. These lenses always had a poten- tial of about 50% of that of the intact lens from the opposite eye of the same fish. Since with capsule and epithelium removed the poten- tial dr0ps 80% and with a capsule nick the drop is about 50%, it seems possible that both capsule and epithelium con— tribute to the potential. H7 Stability of lens potential with time To test the stability of the potential, eight lenses were measured in Ringers within 30 minutes after their removal from the eyes. The lenses were then stored in Ringers and subsequent measurements were made every hour for up to 6 hours. The values shown in Table 1H were reported as the ratio of potential at the specified time to the potential at the beginning of the experiment, Timeo. Table lH.--Stability of lens potential with time ~17 Number of P.D. at TimeT observations TimeT P.D. at Timeo 8 1 hour 0.962:0.036* 8 2 hours 0.92510.0H7 8 3 hours 0.958:0.096 8 . H hours 0.938:0.061 8 5 hours 0.939:0.059 8 6 hours 0.9H710.088 *Mean 1 standard error P.D. at Time = potential difference at beginning of exper1ment. After 6 hours the potential had not decayed significantly from the initial value. Since all experiments to be run would require four hours or less, it was concluded that no change in potential difference would occur in untreated lenses over the experimental period. ue Effect of some metabolic inhibitors on lens potential In experiments to determine the effects of inhibitors on the potential, all lenses were first measured in Ringers solution. Control lenses were placed in beakers of Ringers solution. The experimentals were treated with Ringers solu— tion to which inhibitor was added. Measurements were made at l and 2 hours after the start of the treatment. Controls were measured in Ringers and experimentals were measured in the experimental solution. Since the l and 2 hours values were not significantly different from each other, only the two hour values were reported (Table 15). The mean potential of 1H lenses placed in 10’6 M NaCn was found to increase by about 20%. Likewise, ll lenses measured in 10’5M potassium iodoacetate had 1H% higher poten- tials. The stimulatory effect of both of these inhibitors was abolished when they were used together. An adenosine triphosphatase inhibitor, ouabain, caused significant reduction in potential in 6 lenses. The reduc- tion, however, was only 25% of the control value. Alteration of lens potential duringichanges in temperature of the bathing medium To investigate the effect of temperature on the lens potential, measurements were made at temperatures ranging from 3 C to H3 C. Each value was reported as the ratio of the value at the given temperature to the maximum potential obtained H9 mOCmQOMMHU Howpcmpom u .Q.m mpmvaMOUOfi Edflmmmvomm nophm UQMUCMPm H cmw2+ 000.0vm 0:0.0HH05.0 m.mHN.Hmu 5.~H~.00| 0 cflmnmso z mnoa o I o o I. o | o I o I <EV mnmwcwm Ca .Q.m A>EV mCOMvm>pmmno povanwncH Loafinfincw + wnwmcflm mo amnfisz .sca + .wcwm cw .Q.m .wcflm Ca .Q.m ca HMflpcmvom HMflvcmvom MMHSOfipcmH ESEHxME may no mmoufinflncw mo pommmmlu.ma mHDMH 50 from that.lens (Table.16). These values were plotted against the temperature in degrees centigrade (Fig. 3). Table 16.--Effect of temperature on rainbow trout lens potential Number of Fraction of observations Temperature 0C maximum potential 5 3 0.285i0.003* 3 5 0.HH910.019 2 - 6 0.570i0.005 H 7 0.631i0.002 3 10 O.79Hi0.036 3 , 1H O.9H5:0.010 5 18 O.9H2i0.020 5 21 0.91Hi0.026 5 27 ‘ 0.987:0.013 .5 29 0.929:0.0H9 5 35 0.932:0.02H 2 H0 0.990:0.00l 2 H2 0.991:0.001 H H3 0.201:0.020 *Mean 1 standard error The potential was found to level off at about 1H C which is close to the optimum environmental temperature for the rainbow trout. The potential remained almost constant from 51 90996 U9mocmvm H cmmz« AOUV mmDB9wppmm oocmamm moo9pomam 9699mm 039 UH90 .Hmo 9mo9ooom mamm9m >9pwdo9flo .%9HH:O9HQ m00H5 NHamm xooflo onMHmflmmm zawmoz mmt9m 99990 woo9powam oco p99wxll4lllllllllllll nopflzm c300 wwo 9090E a: msoco9£oc>m 86 Front End Circuitry for Tip Potential, Electrode Resistance, and Amplifier Drift Determinations Am909m9mmm wmv mmomomo 039 C9 ossomoz 00919 seesaw Homo xom momomm fl llllllllllllllll _fillllll| llllllll L "n 0009A malwm "u ow No No No _. C(z?‘ __ 00 t O (\5 AX 4:1.kfll H00 Ham ’/;p-———w——Ho Ch !I———~__ /——o —0 0’0" >9mppmm @999m UCD090 >90mmmoo< APPENDIX 3 Solutions 87 88 Composition of Rainbow Trout Ringers Solution NaCl 7.37 gm/liter KCl 0.31 gm/liter CaCl2 0.10 gm/liter MgSOlJr 0.1H gm/liter KHZPOu 0.H6 gm/liter NaHPOu 2.02 gm/liter 5 Osmolarity = 298 milliosmols if CompoSition of Chloride Free Ringers Same as for aboVe Ringers except NaZSOu, K280”, and CaSOu were substituted for their respective chloride salts with care being taken to keep total Na+, K+, and Ca++ levels unchanged. Composition of Dietrich's Fixative‘ Tap Water 150 ml 95% Ethanol 75 ml H0% Formalin 25 m1 Glacial Acetic Acid 5 ml MICHIGAN STATE umveasnv LIBRARIES VIlIIIIIHIWIIWIW||WIWIIIUH"HANNAH 31293 03175 7366