H. istochemical 1 " 0 and chemical 7 ' 8 evidence

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1 Dopamine: A retinal neurotransmitter II. Autoradiographic localization of H3-dopamine in the retina Steven G. Kramer, Albert M. Potts, and Yvonne Mangnall Retinal uptake of tritiated dopamine was induced in cats either by preretinal perfusion or by carotid infusion as previously described. After glutaraldehyde fixation and Epon embedding, light-microscopic autoradiographs of the retina were prepared. Following preretinal perfusion, specific localization of activity was demonstrated in nerve fibers at the junction of the inner nuclear and inner plexiform layers, in occasional cell bodies in the same region, and in some cell bodies of the ganglion cell layer. This distribution corresponded exactly with that of known dopamine-containing neurons of the retina. Following carotid infusion, very little localization of activity was found in the retina, suggesting a significant blood-retinal barrier for dopamine. Key words: autoradiography, dopamine, catecholamines, neurotransmission, chemical transmission, dopaminergic, retina, amacrine cell, dopaminergic junctional cell H. istochemical 1 " 0 and chemical 7 ' 8 evidence has suggested the existence of dopaminergic neurons at the junction of the retinal inner nuclear and inner plexiform layers. In order that dopamine be identified as the actual chemical transmitter of these neurons, the first study 9 in From the Department of Ophthalmology, The University of Chicago, Chicago, 111. Supported in part by United States Public Health Service Research Grants Nos. EY and EY-00416, by United States Public Health Service Special Fellowship No. 1 FO3 EY , and by Research to Prevent Blindness, Inc. Presented in part at the Spring National Meeting of the Association for Research in Vision and Ophthalmology, April, Manuscript submitted June 7, 1971; manuscript accepted June 17, Reprint requests to: Steven G. Kramer, M.D., Department of Ophthalmology, 950 East 59th Street, Chicago, this series demonstrated retinal uptake and light-stimulated release of tritiated dopamine in vivo. The material released from the retina was chromatographically identified as dopamine. In dark-adapted animals, the rate of release and the amount of dopamine released per light flash increased disproportionately with increasing flash frequency. In light-adapted animals, high rates of dopamine release were found regardless of flash frequency. The previous experiments 9 showed tritiated dopamine uptake and release by measuring radioactivity levels of perfusion fluid bathing the surface of the retina. The present paper demonstrates that the H3- dopamine extracted from the perfusion fluid is specifically localized within the retina: By autoradiography the study shows that exogenously administered H3-dopamine is concentrated and stored in retinal neurons in the same location as those known to contain endogenous dopamine. 1 " 0 There is

2 618 Kramer, Potts, and Mangnall Investigative Ophthalmology August 1971 PRE-RETINAL PERFUSION STROBE TELE - THERMOMETER INFLOW PUMP RESERVOIR INFLOW ~ PERFUSION, BATH \ \ f^-\ a bl "OUTFLOW / "^MICRO- MANIPU- LATOR OPEN EYE Fig. 1. Schematic diagram of preietinal perfusion perparation. 0 little or no localization of radioactivity elsewhere in the retina, and the material remains stored despite a washout phase of the experiment. The conclusion is drawn that the light-stimulated release of dopamine from the retina is a specific phenomenon of the known retinal dopamine-containing neurons at the junction of the inner nuclear and inner plexiform layers. Methods Routes of H3-dopamine administration. Preretinal perfusion. In order to demonstrate and measure uptake and light-stimulated release of H3-dopamine from retinal neurons in vivo, an experimental preparation in the cat eye called preretinal perfusion was developed (Fig, I). 9 The exposed retinal surface was perfused with an isotonic solution containing H3-dopamine, and the radioactivity of precise aliquots of the perfusate was monitored as a function of time during perfusion. A drop in radioactivity level of the effluent perfusate was considered indicative of uptake of H3-dopamine from the perfusate. Subsequent continuation of perfusion, with the same molar concentration of nonradioactive dopamine, allowed measurement of a steady state, background 1 level of radioactivity in the effluent perfusate. During this continued perfusion with nonradioactive dopamine, the retina was stimulated by a flashing light. A resultant increase in radioactivity above background in the effluent perfusate was considered indicative of release of H3-dopamine induced by the light stimulus. This perfusion experiment was carried out in Table I. Phases of preretinal perfusion Phase (minutes) 1. Uptake (0 to 20) 2. Washout (21 to 30) 3. Release (31 to 60) Perfusion with*': H3-dopamiue Nonradioactive dopamine in same molar concentration Nonradioactive dopamine in same molar concentration, plus light stimulation *Rate of perfusiotl = ^l/min. three phases which are summarized in Table I. During the first or "uptake phase," the perfusion fluid consisted of 20 ml. of isotonic saline containing 40 microcuries of H3-dopamine, with a specific activity of 3,600 millicuries per millimole, and 100 United States Pharmacopeia (USP) units of sodium heparin. At the end of this phase, fluid in the entire system was exhanged for isotonic saline containing nonradioactive dopamine and heparin in the same molar concentrations as those which existed in the original fluid. The fluid within the eye itself was exchanged three times using a Pasteur pipette. Perfusion was then continued for an additional ten minutes during the second or "washout phase." During the third or "release without changing the perfusion fluid, but a flashphase," perfusion was continued for 30 minutes ing light stimulus was added. For the autoradiographic studies reported here, the experiment was concluded at the end of the "washout phase" (Table I) by removing all fluid from the eye with a Pasteur pipette and replacing it with two per cent glutaraldehyde, buffered with 0.1M phosphate buffer at ph 7.4. The open eye was then enucleated, and fixation was continued

3 Volume 10 Number 8 Dopamine: A retinal neurotransmitter 619 in cold 2 per cent buffered glutaraldehyde for two hours. Carotid infusion. Retinal uptake and storage of tritiated dopamine was also investigated by infusion of the radioactive material for 30 minutes into the right carotid artery of cats as previously described. 9 ' 12 For the present study, the eyes were rapidly enucleated and opened by removal of a superior calotte with a razor blade. The open eyes were fixed for two hours in cold two per cent glutaraldehyde, buffered with 0.1M phosphate buffer at ph 7.4. Autoradiography. Small pieces of sclerachoroid-retina from the fixed eyes were cut and washed in cold phosphate buffer for 15 minutes. The tissues were postfixed in two per cent osmium tetroxide in cold buffer for three hours and were dehydrated and embedded in Epon. One-micron sections were cut, heated, and cooled until they adhered to cleaned, glass slides. Under Safelight conditions (Wratten No. 2), the slides were dipped in Uford K-5 nuclear emulsion, dried, and stored in black plastic slide boxes containing 50 to 100 mg. of dried silica gel. The emulsion overlying the sections was photographically developed at one-week intervals over a six-week period and at one-month intervals thereafter: In glass slide-racks in total darkness and at 70 C, the slides were sequentially dipped in Kodak D-19 developer for two minutes, distilled water for 20 seconds, and Edwal Quick-fix photographic fixer for 30 seconds.* They were washed in three changes of distilled water for a total of five minutes, and the sections were stained through the emulsion for 30 seconds with a mixture of toluidine blue and pyronin B. When the emulsion was too heavily stained, destaining was done with ten per cent ethyl alcohol or two per cent polyvinyl alcohol. Coverslips were applied with Preservaslide mounting medium (Matheson, Coleman, and Bell), and photomicrographs were taken with a Zeiss photomicroscope. f Control slides from animals not subjected to catecholamine perfusion or infusion were prepared and treated identically. Such controls for each tissue accompanied experimental tissues during each step of exposure and development. Results Autoradiographs of the retina following the "washout phase" (Table I) of preretinal perfusion with H3-dopamine revealed a striking band of radioactivity "This procedure was found least likely to produce folds in the retinal sections. {Courtesy of Dr. Nicholas Vick. precisely at the junction of the inner nuclear and inner plexiform layers (Fig. 2). Discrete activity was apparent in this location with as little as one day of exposure of the nuclear emulsion to the section. The emulsion overlying the sections tended to obscure detail and to induce an excessively pink color to the over-all section. Fig. 3 shows a section from the same experimental eye as those shown in the other figures, but in this case no nuclear emulsion was applied. It demonstrates satisfactory tissue fixation with clear anatomic detail and lack of the general blur induced by the emulsion. The junctional area (arrow) of radioactivity localization is shown at similar power for comparison in Fig. 4. Diffuse activity is present to greatest extent in the inner plexiform layer, next in the inner nuclear layer, and least in the outer plexiform and outer nuclear layers. Fig. 5, from an area of dense activity, shows a single cell from the junctional region which heavily concentrates the radioactive label. Arrows indicate the outline of this cell body. Occasional single cells with specific localization of radioactivity were seen in the ganglion cell layer. Fig. 6 demonstrates such a cell with almost no surrounding background activity. Neuronal processes containing label could not be seen associated with these cells. Retinal autoradiographs following carotid infusion of H3-catecholamines showed very little discrete localization of activity even with as much as three months of exposure of the emulsion. This was in contradistinction to the choroid in the same sections in which specific neuronal localization of radioactivity was readily seen. 13 Discussion Accurate high-resolution autoradiography with labeled catecholamines is hindered because of the high water solubility of these compounds. 14 The danger of extracting the label from the tissue or at least of moving the label is significant. An

4 620 Kramer, Potts, and Mangnall Investigative Ophthalmology August 1971 Fig. 2. Autoradiograph of cat retina following washout phase of preretinal perfusion with H3- dopamine, three-week exposure. Note band of silver grains at junction of inner nuclear and inner plexiform layers (arrow). IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer. (Toluidine blue-pyronin B, Ilford K-5 emulsion. Original magnification xl60.) effective approach to this problem involving freeze-drying for fixation has been described by Stumpf. 15 On the other hand, Marks and co-workers 16 have shown satisfactory autoradiographic localization of tritiated norepinephrine following potassium dichromate fixation and paraffin embedding, and Wolfe and associates 17 and Lenn ls have shown the feasibility of electron microscopic autoradiography of labeled catecholamines using osmium tetroxide fixation. In the present study we found very satisfactory localization of tritiated dopamine using glutaraldehyde fixation and postfixation with osmium tetroxide. Any compounds containing free amino groups which are even loosely bound in tissues are likely to be precipitated to proteins in situ when exposed to glutaraldehyde. 19 In our preretinal perfusion experiment, most free dopamine was probably removed in the "washout phase." Dopamine which had been accumulated by retinal neurons, however, was precipitated in place by the glutaraldehyde, and the autoradiographs were successful. The details of the synaptic anatomy of these dopamine-containing neurons can be studied by electron microscopic autoradiography 20 and such a study is in progress. As shown in Figs. 2 to 6, autoradiographic localization of H3-dopamine was very discrete and corresponded exactly to the distribution of neurons which are known to contain endogenous dopamine. The band of localized activity at the junction of the inner nuclear and inner plexiform layers (Figs. 2 and 4) corresponds to a fluorescent band seen by the histochemical technique of Falck and Owman 21 in a wide variety of species. 1 " 0 The single cell which is heavily laden with activity in Fig. 5 probably corresponds to similar single fluorescent cells seen within the band of fluorescent fibers at the junction of the inner nuclear and inner plexiform layers. 1 " 0 Such cells are similar to amacrine cells, but in most species they comprise only about ten per cent of these cells. 1 ' 3 Recently, Ehinger and Falck G have proposed the term "adrenergic junctional cells" to describe these. The occasional cell in the ganglion cell layer which concentrates tritiated dopamine following preretinal perfusion (Fig. 6) probably corresponds to the "alloganglion cells" described by Ehinger. 1 In virtually

5 Volume 10 Number 8 Dopamine: A retinal neurotransmitter 621 Figs. 3 and 4. Fig. 3, Section of same retina as shown in other figures without overlying nuclear emulsion. Arrow indicates junctional area. IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer. (Toluidine bluepyronin B. Original magnification x400.) Fig. 4, High-power autoradiograph showing band of activity at junction of inner nuclear and inner plexiform layers as in Fig. 2, three-week exposure. IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer. (Toluidine blue-pyronin B, Ilford K-5 emulsion. Original magnification x400.) all species, occasional cells in the ganglion cell layer fluoresce for the presence of catecholamines. Connections of these have not been observed, and perhaps they represent ectopic cells from the junctional region. In any case, their endogenous catecholamine content suggests a capacity for exogenous catecholamine uptake and storage, 10 ' 11? 18 ' " and this is supported by the autoradiographic findings exemplified by Fig. 6. Finally, the relatively dense though diffuse activity of the inner plexiform region (Fig. 4) suggests the possibility of specific uptake and storage in this region. Catecholamine-containing fibers in this region have been called "eremite" fibers by Ehinger, 1 and they may be responsible for the activity observed. The specific localization of radioactivity following the washout phase of the preretinal perfusion experiment lends credibility to the uptake and release measurements presented in the first paper of this series. 0 The major portion of the radioactivity present at the conclusion of the uptake and washout phases is found specifically in that region of the retina known to contain large numbers of dopamine-containing neurons. The subsequent light-stimulated release of H3-dopamine from the retina, which was demonstrated in the previous paper, 0 can have occurred only as a result of activity of these neurons. The inference is clear that the preretinal perfusion experiment has allowed, first, effective radioactive labeling of a releasable pool of

6 622 Kramer, Potts, and Mangnall Investigative Ophthalmology August 1971 Figs. 5 and 6. Fig. 5, High-power autoradiograph showing labeled cell (arrows) within densely labeled band of activity at junction of inner nuclear and inner plexiform layers, four-week exposure. This is probably the dopaminergic functional cell (see text and Fig. 7). (Toluidine blue-pyronin B, Ilford K-5 emulsion. Original magnification x500.) Fig. 6, High-power autoradiograph showing labeled cell (arrow) within ganglion cell layer, one-week exposure. (Toluidine blue-pyronin B, Ilford K-5 emulsion. Original magnification x400.) dopamine in specific retinal nurons and, second, measurement of release of that dopamine induced by light. Carotid infusion, on the other hand, is relatively ineffective in inducing specific uptake of dopamine into retinal neurons. This is consistent with a significant bloodretinal barrier for dopamine, and on the basis of the known blood-brain barrier for this compound, 23 this is not unexpected. The first paper in this series 9 suggested that several criteria necessary for identifying dopamine as a retinal neurotransmitter had been met. The present study supports this contention because it demonstrates the anatomic basis for the uptake and release phenomena observed. It seems reasonably clear that the cells Ehinger and Falck 0 have called adrenergic junctional cells are actually dopaminergic junctional cells with rather specific characteristics (Fig. 7): They are similar in morphology and location to retinal amacrine cells, though in number they comprise about ten per cent of these in most species. 2 ' 3 On chemical and histochemical grounds, 7> s they contain significant amounts of dopamine. They are capable of accumulating and storing dopamine and of releasing it as a consequence of light stimulation of the retina. 9 They have wide-ranging, horizontally oriented fibers which are among the longest of any retinal cells j 1 " 0 and they probably have inhibitory effects (Fig. 7, Region I). They may have multiple synaptic interactions with a variety of retinal cell types (Region II), 26 and they may have small vertically oriented fibers ex-

7 Volume 10 Number 8 Dopamine: A retinal neurotransmitter 623 Fig. 7. Schematic conception of the dopaminergic junctional cell (D]C) (see text and Fig. 5). H = horizontal cell, B = bipolar cells, A = amacrine cells, G = ganglion cell. Regions I, II, and /// (see text). tending back through the inner nuclear layer toward the horizontal cells (Region III). 5 ' Recently Boycott and co-workers 26 have reported amacrine-like cells, especially in the cat retina, which send distinct fibers vertically toward the outer plexiform layer. These comprise approximately ten per cent of the cat amacrine cells, the same number which tend to stain for the presence of dopamine. In addition in many species, fluorescent fibers indicating the presence of dopamine have been seen extending from junctional cells toward the outer plexiform layers. 0 ' 6 > 2C It is now clear that the dopaminergic junctional cell accumulates, stores, and releases a potent biogenic amine, and its anatomy suggests that it may have wideranging and important effects. It is hoped that further study of this intriguing cell may lead ultimately to pharmacologic manipulation of its physiologic actions. REFERENCES 1. Ehinger, B.: Distribution of adrenergic nerves in the eye and some related' structures in the cat, Acta Physiol. Scand. 66: 123, Laties, A. M., and Jacobowitz, D.: A comparative study of the autonomic innervation of the eye in monkey, cat, and rabbit, Anat. Rec. 156; 383, Laties, A. M,, and Jacobowitz, D.: Histochemical studies of monoamine-containing cells in the monkey retina, J. Histochem, Cytochem. 14: 823, Sano, Y., Yoshikawa, H., and Konishi, M.: Fluorescence microscopic observations on the dog retina, Arch. Histol. Jap. 30: 75, Ehinger, B., Falck, B., and Laties, A. M.: Adrenergic neurons in teletost retina, Z. Zellforsch. Mikrosk. Anat. 97: 285, Ehinger, B., and Falck, B.: Morphological and pharmaco-histochemical characteristics of adrenergic retinal neurons of some mammals, Albrecht von Graefes, Arch. Klin. Ophthalmol. 178: 295, Haggendal, J., and Malmfors, T.: Identification and cellular localization of the catecholamines in the retina and the choroid of the rabbit, Acta Physiol. Scand. 64: 58, Nichols, C. W., Jacobowitz, D., and Hottenstein, M.: The influence of light and dark on the catecholamine content of the retina and choroid, INVEST. OPHTHALMOL. 6: 642, Kramer, S. G.: Dopamine: A retinal neurotransmitter. I. Retinal uptake, storage, and light-stimulated release of H3-dopamine in vivo, INVEST. OPHTHALMOL. 10: 438, Kramer, S. G., and Potts, A. M.: Iris uptake of catecholamines in experimental Homer's syndrome, Am. J. Ophthalmol. 67: 705, Kramer, S. G., and Potts, A. M.; Intraocular pressure and ciliary body norepinephrine uptake in experimental Homer's syndrome, Am. J. Ophthalmol. 68: 1076, Kramer, S. G., and Potts, A. M.: Catecholamine metabolite formation in the iris and ciliary body, Am. J. Ophthalmol. In press. 13. Kramer, S. G., Potts, A. M., and Mangnall, Y.: Autoradiographic localization of catecholamines in the uveal tract. I. A light microscopic study, Am. J. Ophthalmol. In press. 14. Rogers, A. W.: Techniques of autoradiography, Amsterdam, 1967, Elsevier Publishing Company, p Stumpf, W. E.: High-resolution autoradiography of diffusable substances, Science 161: 1262, Marks, B. H., Samorajski, T., and Webster, E. J.: Radioautographic localization of norepinephrine-h3 in the tissues of mice, J. Pharmacol. Exp. Ther. 138: 376, Wolfe, D. E., Porter, L. T., Richardson, K. C, and Axelrod, J.: Localizing tritiated norepinephrine in sympathetic neurons by electron microscopic autoradiography, Science 138:440, Lenn, N. J.: Localization of uptake of tritiated norepinephrine by rat brain in vivo and in vitro using electron microscopic autoradiography, Am. J. Anat. 120: 377, Peters, T., Jr., and Ashley, C. A.: An artefact in radioautography due to binding of free arnino acids to tissues, J. Cell Biol. 33: 54, 1967.

8 624 Kramer, Potts, and Mangnall Investigatioe Ophthalmology August Caro, L. G., vantubergen, R. P., and Kolb, J. A.: High-resolution autoradiography. I. Methods, J. Cell Biol. 15: 173, Falck, B., and Owman, C: A detailed methodological description of the fluorescence method for the cellular demonstration of biogenic monoamines, Acta Univ. Lundensis, Sectio II, No. 7, Whitby, L. G., Axelrod, J., and Weil-Malherbe, H.: The fate of H 3 -norepinephrine in animals, J. Pharmacol. Exp. Ther. 132: 193, Fuxe, K., and Hillarp, N. A.: Uptake of L-dopa and noradrenaline by central catecholamine neurons, Life Sci. 3: 1403, Straschill, M., and Perwein, J.: The inhibition of retinal ganglion cells by catecholamines and gamma-aminobutyric acid, Pfluegers Arch. 312: 45, Ames, A., and Pollen, D. A.: Neurotransmission in central nervous tissue A study of isolated rabbit retina, J. Neurophysiol. 32: 424, Boycott, B., Dowling, J., Fisher, S., Kolb, H., and Laties, A.: A new amacrine cell in the vertebrate retina, Presented at the Spring National Meeting of the Association for Research in Vision and Ophthalmology, April 30, 1971.