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Biol Signals 1992;1:46-56
Q.X. Guo S.Z. Yang C. L. Liang D. Tsang L.S. Jen
Tyrosine-HydroxylaseImmunoreactive Neurons in Retinal Transplants in the Rat
KeyWords Catecholaminergic neurons Tyrosine hydroxylase Retinal transplant Rat
G.Q.X., Y.S.Z. and L.C.L. are visiting research fellows on leave from Neuroscicnces Research Institute, Guangzhou Medical College, Guangzhou, The Sun Yat-sen University of Medical Sciences, Guangzhou, and Institute of Biophysics, Beijing, China, respectively.
Abstract Embryonic rat retinae were transplanted to the brains of new born rats, and the distribution of catecholaminergic neurons in the retinal tissue was studied 1-2 months after transplanta tion, using the tyrosine hydroxylase (TH) immunohistochemi cal method. The results showed that distinct TH-positive cells were identified in all retinal transplants examined. The so mata of the majority of these TH-immunoreactive cells were located along the inner margin of the inner nuclear layer in the retinal transplants; the processes of these cells were distributed mainly in the outer portion of the inner plexiform layer. This pattern is comparable to that observed in retinae of normal and host rats, suggesting that the organization of the catechol aminergic neurons in the transplant is largely similar to that in the normal retina. However, a reduction of the immunoreactivity in the plexiform layers and subpopulations of TH-positive cells with somatic diameter smaller than 8 pm or larger than 18 pm was observed in most of the retinal transplants studied. This implies that the organization of the catecholam inergic system in the transplant may not be as intact as in the normal retina.
Received: February 20, 1991 Accepted: March 12,1991
L.S. Jen, PhD Department of Anatomy The Chinese University of Hong Kong Shatin, NT (Hong Kong)
©1992 S. Karger AG, Basel 1016-0922/92/ 0011-004652.75/0
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Departments of Anatomy and Biochemistry'. The Chinese University of Hong Kong. Shatin. NT. Hong Kong
Intracranial retinal transplantation is one of the useful models for studying the survival and maturation of transplanted central neu rons. as well as for investigating specific con nections and interactions between the trans plants and the host brain [1-15]. The advan tages of this approach are that the retinal transplants require no prelabeling, and that the ganglion cells in the transplants can sur vive and are capable of establishing func tional connections with specific visual targets of the host brain [3, 4, 7-9, 11, 14, 15]. How ever, most of the previous studies focused on interactions between the transplanted retinae and visual centers of the host brain, and rela tively little information is available concern ing the structural and functional organization of interneurons in intracranially transplanted retinae. Recently, we have demonstrated that in retinal tissue transplanted to the brainstem of host rats the two subpopulations of cholin ergic neurons were distributed in a bilaminated fashion with their somata located in the inner nuclear and ganglion cell layers, respec tively [6], a pattern which is essentially the same as that observed in the normal retina [6, 11, 17-19]. As the conventional and dis placed cholinergic neurons which had their somata distributing on the two sides of the inner plexiform layer (IPL) in the normal retina are believed to be involved in mediat ing the on- and off-responses of the retinal ganglion cells [20-23], it has been suggested that corresponding cholinergic neurons in the retinal transplant also provide similar inputs to the ganglion cells and play a significant role in the on- and off-visual channels. This is sup ported by results obtained from electrophysiological recording experiments showing that the characteristic on-off responses of central visual neurons can be driven by intracranially transplanted retinae [1, 15]. In view of this,
we decided to investigate further the organi zation of the catecholaminergic system in the retinal transplant by examining the distribu tion pattern of the enzyme tyrosine hydroxy lase (TH), which has been commonly used as a neuronal marker in the retina [18, 24-29]. The present study is intended to provide some useful information concerning the struc tural and functional organization of trans planted catecholaminergic neurons, which in turn provides insight into factors determining the survival and maintenance of neurons in the mammalian retina [3-6, 9-14. 30]. Materials and Methods The animals and experimental procedures used were basically the same as those described in previous studies [5, 6, 11]. In brief, retinae obtained from embryonic day 14 Sprague-Dawley (SP) rats were dis sected in Ham's F10 medium (Gibco) on ice and then injected, using a glass pipette, into the left side of the brainstem of newborn rats of the same strain under ether anesthesia. The gestational period of SP rats is around 22 days. For comparison, embryonic retinae were also introduced to the cortex or ventricle of the host brain in some of the littermates. In addition, a small number of animals which received no retinal transplants served as normal controls. In the present experiments, the right eye of all recipient rats was also enucleated at the time of transplantation, a procedure known to enhance the survival of ganglion cells and promote ingrowth of optic axons to the denervated visual targets [1-6]. After transplantation, the recipi ent rats were returned to their mothers and raised until 1-2 months old before further experimentation. The animals were then anesthetized deeply by intraperitoneal injection of chloral hydrate (7%, 0.5ml/100g body weight), and were perfused transcardially with physiological saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The eyes of all experimental rats and brains of rats with retinal trans plants were dissected in the same fixative. The dorsal pole of the eyeballs was marked by a cut for orientation purpose, and the eyeballs were sectioned horizontally so that the nasal, central and temporal parts of the retina could be included in some of the sections. The brains and eyes were sectioned at a thickness of 2050 pm on a cryostat or vibrotome, and the sections
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Introduction
Results In the retinae of normal and host rats, THimmunoreactive (TH-I) cells were observed primarily in two laminar zones in the inner retinal layers. The majority of the TH-I somata, as reported in previous studies [ 18, 24-26, 33], were located in the inner nuclear layer (INL) though some were also found consis tently in the ganglion cell layer (GCL). As shown in figure 1, the TH-I somata in INL were usually widely separated and rarely found adjacent to each other, and the number of somata encountered in all sections exam ined was scarce. Nevertheless, the TH-I so mata could be divided at least into two major populations on the basis of their somatic size. One population had somatic diameters rang ing from 10 to 24 pm and were moderately to darkly stained (large arrows in fig. 1a-c, 2a-c) and this corresponds to the class l TH-I cells described previously by other investigators [ 18,24,25,28]. The somata of these cells were distributed primarily in the inner parts of the INL, though a small number of them were found to occupy or extend into the outer half of the INL (fig. la, d, 2a). TH-I somata of comparable sizes were also encountered in the
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GCL, but the number was much reduced as compared with that in the INL. and appeared invariably as solitary cells (fig. 2d). The sec ond population of TH-I cells observed in the retina had somata which varied from approxi mately 5 to 10 pm in diameter, and distrib uted mainly along the inner margin of the INL. This population of small TH-I cells is similar to the class 2 TH-I cells reported in the rat retina [18,24,25,28]. The somata of these cells, perhaps because of their small sizes, were generally round in shape and appeared to be lighter in immunostaining (fig. 1). Another interesting feature concerning the TH-I somata in the retina is that there were relatively more large TH-I somata encoun tered in the temporal (fig. la, b) than in the nasal (fig. lc) part of the retina, and a gradual decrease in somatic sizes towards the nasal part of the retina was consistently detected. This finding supports a nasotemporal differ ence in TH-I somata in the rat retina, as noted previously by Mitrofanis et a!. [ 18, 24], While the TH-I somata were mainly ob served in the inner parts of the INL and GCL, TH-positive cellular processes were found to be distributed primarily in the outermost onethirds of the IPL, and in the outer plexiform layer (OPL). The TH-I processes in the IPL appeared usually as a fairly distinct TH-posi tive band which could be visualized readily even at low magnification. However, varia tion in the density, extent and intensity of immunostaining of TH-I processes in the IPL was commonly observed not only between different retinae but in different parts of the same retinae as well (fig. la, c, 2a-c). For ex ample, the difference in immunostaining be tween the temporal (fig. la) and nasal (fig. lc) parts of the retina observed in figure 1 was not representative because the immunostaining in other regions of the nasal retina could be as darkly stained as the part of the temporal reti na. The immunoreactive processes in the
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were collected on subslides in phosphate buffer. The brain and retinal sections were washed in phosphatebuttered saline which contains 1% Triton 2C-100 and 10% fetal serum for 1 h, and then incubated in a medium containing a mouse monoclonal anti-TH anti body (Incstar, Stillwater, Minnesota, Minn., USA) for 24 h before they were processed by the avidin-biotin complex method, as described in previous studies [6, 18. 24], Control sections were incubated in a solution with the primary' antibody replaced by phosphate buff er, mouse or rat serum. Some of the sections were also counterstained with cresyl violet for examining the cytoarchitectonies of the normal or transplanted reti nae. A total of 18 retinae obtained from 8 normal and 10 host rats, and 25 retinal transplants which were bet ter immunohistochemicallv stained were selected and included in this study.
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Fig. 1. Photomicrographs showing the TH immunoreactivity in a normal retina at differ ent magnifications, a -c Low-power views of the temporal (a, b) and nasal (c) parts in the periphery of the retina. Note that the TH-I somata can be divided clearly into two categories, with large (large arrows) and small (small arrows) somata located primarily in the inner parts of the INL. Very' few large TH-I somata were encountered in the far nasal part of the retina as shown in c. d, e High-power view showing details of the TH-I profiles in the temporal and nasal parts of the retina, respectively. Moderately immunostained processes were distributed mainly in the outer parts of IPL and OPL (open stars). Closed stars at the bottom of the photomicrographs indicate the ganglion cell layer. Scale bars: 50 pm.
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Fig. 2. Photomicrographs showing some of the large TH-1 cells in the 1NL (a-c) and gan glion cell layer (d) in a normal retina. Note the different shapes of the somata, and that the extent and intensity of immunostaining varied considerably in different regions of the same retina. The open stars indicate the OPL. e shows TH-I cells in a portion of a retinal trans plant. The broken line indicates the interface between the transplanted retinal tissue (Tp) and lens (Ln). Arrows indicate the somata of TH-I cells located in regions corresponding to the INL. Scale bars: 50 pm.
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Fig. 3. Photomicrographs showing three different retinal transplants in which immunoreactive processes were located in the IPL. The thick arrows in c indicate the outermost parts of the IPL. The thin arrows indicate TH-I somata distributed along the inner margin of the INL. Arrow heads indicate the GCL. The dotted line in a indicates the interface between the host (H) and transplanted retinal tissue (Tp). Scale bars: 50 pm.
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Fig. 4. Photomicrographs showing two retinal transplants in which no immunorcactive processes in organized form could be identified in regions corresponding to the 1PL. The dendrites of the TH-positive cells (arrows) arc prominently immunostained, however. Note that the majority of the TH-1 somata are medium-sized and few cells with large or small somata can be visualized. Scale bars: 50 pm.
thin band of immunoreactive processes was readily recognized in regions corresponding to the outermost part of the IPL but not in the OPL. In some of the transplants, however, no immunoreactive processes in well-organized form were easily observed even in the IPL, though the dendrites of the TH-I cells were prominently immunostained. The lack of or ganized immunostained processes in the plexiform layers in these transplants was not due to plane of sectioning, because many sec tions covering a large portion of the retinal tis sues were examined. One typical observation is illustrated in figures 2eand 4. In addition, it is noteworthy to point out that the reduc tion or absence of organized distribution pat tern of TH-I cellular processes in the plexi form layers did not seem to bear any relation ship with the location of the retinal trans plant, as similar patterns were observed in retinae transplanted to the cortex or mid brain.
Discussion The most important finding in this study is that a well-organized population of TH-I cells was identified in intracranially transplanted retinae, and the distribution pattern of these cells was comparable to that observed in the normal or host retina. Since TH, a rate-limit ing enzyme in the biosynthesis of catechol amines has been used as a marker of catecholaminergic neurons, our results suggest that most precursor cells of catecholaminergic neurons in embryonic retina transplanted to the foreign brain can survive, continue to dif ferentiate, and relate visual signal even in a completely abnormal environment. As it has been shown that the catecholaminergic neu rons in the normal retina represent mainly the conventional amacrine cells with their somata located in INL, the results obtained in this
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OPL were usually but not necessarily lighter in staining and less extensive as compared with those in the IPL. Nevertheless, no imraunoreactive processes could be traced to those TH-I somata which were located mainly in the inner parts of the INL. As reported in previous studies [5, 6, 11, 12], the transplanted retinae are usually folded or in rosette form, though occasionally organized in the form of continuous sheets (fig. 2, 3,4). However, all cellular and plexiform layers observed in normal retinae were identified in the retinal transplant despite a considerable variation in the form of organi zation and a general reduction in size and thickness of the retinal tissue. But distinct populations of TH-I cells were identified in all retinal transplants examined. By comparing adjacent sections which were processed for Nissl’s stain and TH immunohistochemistry, it was obvious that the TH-I somata were located primarily in the inner parts of the INL, regardless of the form of the trans planted tissues. As in the normal or host reti na, a small number of TH-I somata were also located in the GCL, but the density observed in the transplant appeared to be slightly in creased; this increase could be due to a more compact arrangement of the transplanted reti nal tissue. Although the TH-I cells in retinal transplants could also be divided into two subpopulations on the basis of their somatic sizes, the large majority of the immunoreactive somata fell into the range of 8-18 pm in diameter (fig. 2e, 3,4), and few cells with so mata smaller than 8 or larger than 18 pm were detected. By contrast, the density, extent and inten sity of immunostained cellular processes in the retinal transplant were more variable or substantially reduced (fig. 2e, 3, 4) as com pared with those of the normal or host retina. In most of the transplants in which a contin uous sheet of retinal tissue was observed, a
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activity in the plexiform layers in the trans plant. The observation of a less prominent band of immunoreactive processes in the outer parts of the IPL and the absence of immunostaining in the OPL of retinal trans plants indicate a relatively loose organization of the processes of TH-I cells in the trans planted retina. Whether these reflect struc tural defects and functional deficiencies of the retinal transplant is not altogether clear and needs to be confirmed by further ultrastruc tural and physiological studies. However, the absence of detectable and patterned immuno reactive profiles in the OPL in the transplant suggests that some of the TH-I cells which would normally send their processes to the OPL failed to do so under the present experi mental conditions. As it is generally accepted that cells which extend processes to the OPL are interplexiform cells [24,-26], the finding in this study implies that the catecholaminer gic interplexiform cells failed to develop or differentiate in the retinal transplant. Similarly, the reduction of a population of TH-I cells with very small and large sornata in the transplanted tissue suggests that some of catecholaminergic interneurons did not sur vive or differentiate after transplantation. Since it has been postulated that many of the catecholaminergic neurons with small sornata are adrenergic [41], it seems reasonable to conclude that the great majority of the adren ergic cells failed to survive or develop in the transplant and the TH-I cells observed are mainly dopaminergic neurons. Moreover, the fact that most of the TH-I cells in the retinal transplant are of medium size suggests further that there is a iack of a clear nasotemporal gra dient of the TH-I cells. Taken together, the results obtained in this study showing some defects in the arrangement and development of the catecholaminergic system in the intra cranial retinal transplant are in contrast with our previous observations of a near perfect
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study suggest further that those TH-I cells observed in the retinal transplant were most likely catecholaminergic amacrine cells. The fact that these cells had their sornata located in the inner parts of the INL and ramified, at least in some of the cases studied, to the adja cent IPL implying that these cells interact, as demonstrated in previous studies in the mam malian retina [24-27, 29, 34, 35], with other noncatecholaminergic or All rod amacrine cells which in turn synapse with the rod bipo lar cells [36-39], and are therefore involved in the rod pathways [35, 37, 40]. The observation of TH-I cells in all retinal transplants also indicates that the catechol aminergic neurons in transplanted retinal tis sues can survive and mature regardless of whether they were transplanted to a region near the central visual centers or not. This is supported by results of a recent study showing that TH-I interneurons can differentiate in developing retinal tissue following intraocular transplantation [30]. These findings, together with the evidence indicating that an appar ently well-organized cholinergic neuronal net work in similarly transplanted retinal tissue [6], suggest strongly that interneurons in the retinal transplant can be maintained and ma ture when grafted to a completely foreign or ectopic site in the host brain. Furthermore, the maintenance of these interneurons seems to be independent of the ganglion cells be cause it is known that most if not all of the retinal ganglion cells would degenerate within 5 weeks or so after transplantation of the retina to a region which is far away from their normal targets [9, 14]. While the overall pattern of immunostaining and distribution of TH-I cells and their processes in the transplant are similar to those observed in the normal retina, several charac teristic features of the TH-I cells in the normal retina were missing. One of the most obvious differences is the reduction of the immunore-
organization pattern of cholinergic neurons and their processes in the transplanted retina [6], and suggesting that the catecholaminergic system is more susceptible than the choliner gic system to changes in the microenviron ment or experimental manipulations. On the other hand, our results showing a lack of a clear nasotemporal gradient of TH-I cells with relatively large somata coincides well with results of a recent study which demonstrated that there was an absence of topography of the retinotectal projection of the retinal trans plant [2]. Thus, a general conclusion of these findings is that most of the interneurons can survive and differentiate following intracra nial transplantation, but the more refined pat tern of distribution, development and matu
ration of certain special features of the cate cholaminergic neurons can be affected con siderably. The issues as to how and when the presumably predetermined programs during development can be altered by various factors or experimental conditions are currently be ing investigated by comparing the time courses and distribution patterns of different neuronal populations in the transplanted and normal retina during development.
Acknowledgements This study was supported by UPGC grant 221400030 and NSFC grant 39070464.
References 7 Klassen H, Lund RD: Retinal transplants can drive a pupillary reflex in host rat brains. Proc Natl Acad Sei USA 1987,84:69586960. 8 Klassen H, Lund RD: Retinal graft-induced pupillary responses in rats: Restoration of a reflex function in the mature mammalian brain. J Neurosci 1990; 10:578587. 9 Lund RD, Hankin MH, Sefton AJ. Perry VH: Conditions for optic axon outgrowth. Brain Behav Evol 1988;31:218-226. 10 Lund RD. Rao K, Kunz HW. Gill III TJ: Instability of neural xeno grafts placed in neonatal rat brains. Transplantation 1988;46:216-223. 11 McLoon SC. Lund RD: Specific projections of retina transplanted to rat brain. Exp Brain Res 1980: 40:273-282. 12 Perry VH, Lund RD, McLoon SC: Ganglion cells in retinae trans planted to newborn rats. J Comp Neurol 1985;231:353-363.
13 Radel JD. Hankin MH. Lund RD: Proximity as a factor in the inner vation of host brain regions by reti nal transplants. J Comp Neurol 1990:300:211-229. 14 Sefton AJ. Perry VH. Lund RD: The influence of target regions on the outgrowth and survival of gan glion cells in retinae transplanted to the cerebral cortex. Dev Brain Res 1987;33:145-149. 15 Simons DJ, Lund RD: Fetal reti nae transplanted over tecta of neo natal rats respond to light and evoke patterned neuronal dis charges in the host superior collicu lus. Dev Brain Res 1985:21:156— 159. 16 Famiglietti Jr EV: 'Starbursl’ amacrinc cells and cholinergic neurons: Mirror-symmetric ON and OF amacrine cells of rabbit retina. Brain Res 1983;261:138-144. 17 Famiglietti EV, Kolb H: A bistratified amacrine cell and synaptic circuitry in the inner plexiform layer of the retina. Brain Res 1975: 84:293-300.
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1 Crâner SL. Radel JD, Jen LS, Lund RD: Light evoked conical activity produced by illumination of intra cranial retinal transplants: Experi mental studies in rals. Exp Neurol 1989;104:93-100. 2 Galli L. Rao K, Lund RD: Trans planted rat retinae do not project in a topographic fashion on the host tectum. Exp Brain Res 1989:74: 427-430. 3 Hankin MH. Lund RD: Specific target-directed axonal outgrowth from transplanted embryonic rodent reti nae intoneonatal rat superiorcolliculus. Brain Res 1987;408:344-348. 4 Hankin MH. Lund RD: Induction of target-directed optic axon out growth: Effect of retinae trans planted to anophthalmic mice. Dev Biol 1990:38:136-146. 5 Jen LS. Chau RMW. Tsang D: Cy tochrome oxidase activity in reti nas transplanted to the brainstem in rats. Neurosci Lett 1989:105: 275-280. 6 Jen LS, Tsang D. Chau RMW. Shcn WZ: the cholinergic system in retinal transplants in rats. Brain Res 1990;523:156-160.
27 Tork I. Stine J: Morphology of catecholamine-containing amacrine cells in the cats’s retina, as seen in retinal wholemounts. Brain Res 1979;169:261-273. 28 Vcrsaux-Botteri C, Martin-Martinelli E, Nguycn-LeGros J. Geffard M. Vigny A. Denoroy L: Regional specialization of the rat retina: Catecholaminergic-containing amacrine cell characterization and distribu tion. J Comp Neurol 1986:243:422— 433. 29 Voigt T. Wassle H: Dopaminergic innervation of AH amacrine cells in mammalian retina. J Neurosci 1987;7:4115-4128. 30 Aramant R, Seiler M. Ehinger B, Bergstrom A, Adolph AR, Turner JE: Neuronal markers in rat retinal grafts. Dev Brain Res 1990:53:4961. 31 Chan SO. Chow KL. Jen LS: Postna tal development of the ipsilaterally projecting retinal ganglion cells in normal rats and rats with neonatal lesions. Dev Brain Res 1989:49: 265-274. 32 Jen LS, Chan SO, Chau RWM: Pres ervation of the entire population of normally transient ipsilaterally pro jecting retinal ganglion cells by neo natal lesions in the rat. Exp Brain Res 1990;80:205-208. 33 Ehinger B: Ncurotransmitter sys tems in the retina. Retina 1983:2: 305-321.
34 Dacey DM: Dopamine-accumulat ing retinal neurons revealed by in vitro fluorescence display a unique morphology. Science 1988:240: 1196-1198. 35 Pourcho RG: Dopaminergic ama crine cells in the cat retina. Brain Res 1982;252:101-109. 36 Kolb H: The inem plexiform layer in the retina of the cat; electron microscropic observations. J Neurocylol 1979;8:295-329. 37 Kolb H. Famiglietti EV: Rod and cone pathways in the inner plexi form layer of the cat retina. Science 1974;186:47-49. 38 Kolb H. Nelson R, Mariani AP: Amacrine cells, bipolars and gan glion cells of the cat retina: A Golgi study. Vision Res 1981;21:10811114. 39 Nelson R, Kolb H. Famiglietti EV, Gouras P: Neural responses in the rod and cone systems of the cat reti na: Intracellular records and procion stains. Invest Ophthalmol 1976;15:946-953. 40 Hamasaki DL Trattler WB. Hajek AS: Light on depresses and light off enhances the release of dopamine from the cat’s retina. Neurosci Lett 1986:68:112-116. 41 Park DH. Teitelman G, Evingcr MJ. Woo JI, Ruggiero DA. Al bert VR, Baetge EE, Pickel VM, Rees DJ, Joh TH: Phenylethanolamine N - methyltransferase-containing neurons in rat retina: Imrnunohistochemistry and molecular bi ology. J Neurosci 1986;6:11081113.
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18 MitrofanisJ, Maslim J, Stone J: Catecholaminergicand cholinergic neu rons in the developing retina of the rat. J Comp Neurol 1988:276:343359. 19 Voigt T: Cholinergic amacrine cells in the rat retina. J Comp Neurol 1986:248:19-35. 20 Bloomfield SA. Miller RF: A func tional organization of ON and OFF pathways in the rabbit retina. Neu roscience 1986;6:1-13. 21 Nelson R. Famiglietti EV. Kolb H: Intracellular staining reveals differ ent levels of stratification for ONand OFF-center ganglion cells in cat retina. J Neurophysiol 1978;41: 472-483. 22 Schmidt M. Wassle H. Humphrey M: Action and localization of acetyl choline in the cat retina. J Neuro physiol 1987;58:997-1015. 23 Tauchi M. Masland RH: The shape and arrangement of the cholinergic neurones in the rabbit retina. Proc R Soc Lond (Biol) 1984;223:101-109. 24 Mitrofanis J. Vigny A. Stone J: Dis tribution of catecholaminergic cells in the retina of the rat, guinea pig, cat, and rabbit: Independence from ganglion cell distribution. J Comp Neurol 1988:267:1-14. 25 Nguycn-LeGros J, Vigny A, Gay M: Postnatal development of TH-like immunoreactive interplexiform cells in the rat retina. Exp Eye Res 1983;37:23-32. 26 Oyster CW, Takahashi ES, Cillffo M, Brecha NC: Morphology and dis tribution of TH-like immunoreac tive neurones in the cat retina Proc Natl Acad Sci USA 1985,82:63356339.
I G I N A L
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Biol Signals 1992;1:46-56
Q.X. Guo S.Z. Yang C. L. Liang D. Tsang L.S. Jen
Tyrosine-HydroxylaseImmunoreactive Neurons in Retinal Transplants in the Rat
KeyWords Catecholaminergic neurons Tyrosine hydroxylase Retinal transplant Rat
G.Q.X., Y.S.Z. and L.C.L. are visiting research fellows on leave from Neuroscicnces Research Institute, Guangzhou Medical College, Guangzhou, The Sun Yat-sen University of Medical Sciences, Guangzhou, and Institute of Biophysics, Beijing, China, respectively.
Abstract Embryonic rat retinae were transplanted to the brains of new born rats, and the distribution of catecholaminergic neurons in the retinal tissue was studied 1-2 months after transplanta tion, using the tyrosine hydroxylase (TH) immunohistochemi cal method. The results showed that distinct TH-positive cells were identified in all retinal transplants examined. The so mata of the majority of these TH-immunoreactive cells were located along the inner margin of the inner nuclear layer in the retinal transplants; the processes of these cells were distributed mainly in the outer portion of the inner plexiform layer. This pattern is comparable to that observed in retinae of normal and host rats, suggesting that the organization of the catechol aminergic neurons in the transplant is largely similar to that in the normal retina. However, a reduction of the immunoreactivity in the plexiform layers and subpopulations of TH-positive cells with somatic diameter smaller than 8 pm or larger than 18 pm was observed in most of the retinal transplants studied. This implies that the organization of the catecholam inergic system in the transplant may not be as intact as in the normal retina.
Received: February 20, 1991 Accepted: March 12,1991
L.S. Jen, PhD Department of Anatomy The Chinese University of Hong Kong Shatin, NT (Hong Kong)
©1992 S. Karger AG, Basel 1016-0922/92/ 0011-004652.75/0
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Departments of Anatomy and Biochemistry'. The Chinese University of Hong Kong. Shatin. NT. Hong Kong
Intracranial retinal transplantation is one of the useful models for studying the survival and maturation of transplanted central neu rons. as well as for investigating specific con nections and interactions between the trans plants and the host brain [1-15]. The advan tages of this approach are that the retinal transplants require no prelabeling, and that the ganglion cells in the transplants can sur vive and are capable of establishing func tional connections with specific visual targets of the host brain [3, 4, 7-9, 11, 14, 15]. How ever, most of the previous studies focused on interactions between the transplanted retinae and visual centers of the host brain, and rela tively little information is available concern ing the structural and functional organization of interneurons in intracranially transplanted retinae. Recently, we have demonstrated that in retinal tissue transplanted to the brainstem of host rats the two subpopulations of cholin ergic neurons were distributed in a bilaminated fashion with their somata located in the inner nuclear and ganglion cell layers, respec tively [6], a pattern which is essentially the same as that observed in the normal retina [6, 11, 17-19]. As the conventional and dis placed cholinergic neurons which had their somata distributing on the two sides of the inner plexiform layer (IPL) in the normal retina are believed to be involved in mediat ing the on- and off-responses of the retinal ganglion cells [20-23], it has been suggested that corresponding cholinergic neurons in the retinal transplant also provide similar inputs to the ganglion cells and play a significant role in the on- and off-visual channels. This is sup ported by results obtained from electrophysiological recording experiments showing that the characteristic on-off responses of central visual neurons can be driven by intracranially transplanted retinae [1, 15]. In view of this,
we decided to investigate further the organi zation of the catecholaminergic system in the retinal transplant by examining the distribu tion pattern of the enzyme tyrosine hydroxy lase (TH), which has been commonly used as a neuronal marker in the retina [18, 24-29]. The present study is intended to provide some useful information concerning the struc tural and functional organization of trans planted catecholaminergic neurons, which in turn provides insight into factors determining the survival and maintenance of neurons in the mammalian retina [3-6, 9-14. 30]. Materials and Methods The animals and experimental procedures used were basically the same as those described in previous studies [5, 6, 11]. In brief, retinae obtained from embryonic day 14 Sprague-Dawley (SP) rats were dis sected in Ham's F10 medium (Gibco) on ice and then injected, using a glass pipette, into the left side of the brainstem of newborn rats of the same strain under ether anesthesia. The gestational period of SP rats is around 22 days. For comparison, embryonic retinae were also introduced to the cortex or ventricle of the host brain in some of the littermates. In addition, a small number of animals which received no retinal transplants served as normal controls. In the present experiments, the right eye of all recipient rats was also enucleated at the time of transplantation, a procedure known to enhance the survival of ganglion cells and promote ingrowth of optic axons to the denervated visual targets [1-6]. After transplantation, the recipi ent rats were returned to their mothers and raised until 1-2 months old before further experimentation. The animals were then anesthetized deeply by intraperitoneal injection of chloral hydrate (7%, 0.5ml/100g body weight), and were perfused transcardially with physiological saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The eyes of all experimental rats and brains of rats with retinal trans plants were dissected in the same fixative. The dorsal pole of the eyeballs was marked by a cut for orientation purpose, and the eyeballs were sectioned horizontally so that the nasal, central and temporal parts of the retina could be included in some of the sections. The brains and eyes were sectioned at a thickness of 2050 pm on a cryostat or vibrotome, and the sections
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Introduction
Results In the retinae of normal and host rats, THimmunoreactive (TH-I) cells were observed primarily in two laminar zones in the inner retinal layers. The majority of the TH-I somata, as reported in previous studies [ 18, 24-26, 33], were located in the inner nuclear layer (INL) though some were also found consis tently in the ganglion cell layer (GCL). As shown in figure 1, the TH-I somata in INL were usually widely separated and rarely found adjacent to each other, and the number of somata encountered in all sections exam ined was scarce. Nevertheless, the TH-I so mata could be divided at least into two major populations on the basis of their somatic size. One population had somatic diameters rang ing from 10 to 24 pm and were moderately to darkly stained (large arrows in fig. 1a-c, 2a-c) and this corresponds to the class l TH-I cells described previously by other investigators [ 18,24,25,28]. The somata of these cells were distributed primarily in the inner parts of the INL, though a small number of them were found to occupy or extend into the outer half of the INL (fig. la, d, 2a). TH-I somata of comparable sizes were also encountered in the
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GCL, but the number was much reduced as compared with that in the INL. and appeared invariably as solitary cells (fig. 2d). The sec ond population of TH-I cells observed in the retina had somata which varied from approxi mately 5 to 10 pm in diameter, and distrib uted mainly along the inner margin of the INL. This population of small TH-I cells is similar to the class 2 TH-I cells reported in the rat retina [18,24,25,28]. The somata of these cells, perhaps because of their small sizes, were generally round in shape and appeared to be lighter in immunostaining (fig. 1). Another interesting feature concerning the TH-I somata in the retina is that there were relatively more large TH-I somata encoun tered in the temporal (fig. la, b) than in the nasal (fig. lc) part of the retina, and a gradual decrease in somatic sizes towards the nasal part of the retina was consistently detected. This finding supports a nasotemporal differ ence in TH-I somata in the rat retina, as noted previously by Mitrofanis et a!. [ 18, 24], While the TH-I somata were mainly ob served in the inner parts of the INL and GCL, TH-positive cellular processes were found to be distributed primarily in the outermost onethirds of the IPL, and in the outer plexiform layer (OPL). The TH-I processes in the IPL appeared usually as a fairly distinct TH-posi tive band which could be visualized readily even at low magnification. However, varia tion in the density, extent and intensity of immunostaining of TH-I processes in the IPL was commonly observed not only between different retinae but in different parts of the same retinae as well (fig. la, c, 2a-c). For ex ample, the difference in immunostaining be tween the temporal (fig. la) and nasal (fig. lc) parts of the retina observed in figure 1 was not representative because the immunostaining in other regions of the nasal retina could be as darkly stained as the part of the temporal reti na. The immunoreactive processes in the
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were collected on subslides in phosphate buffer. The brain and retinal sections were washed in phosphatebuttered saline which contains 1% Triton 2C-100 and 10% fetal serum for 1 h, and then incubated in a medium containing a mouse monoclonal anti-TH anti body (Incstar, Stillwater, Minnesota, Minn., USA) for 24 h before they were processed by the avidin-biotin complex method, as described in previous studies [6, 18. 24], Control sections were incubated in a solution with the primary' antibody replaced by phosphate buff er, mouse or rat serum. Some of the sections were also counterstained with cresyl violet for examining the cytoarchitectonies of the normal or transplanted reti nae. A total of 18 retinae obtained from 8 normal and 10 host rats, and 25 retinal transplants which were bet ter immunohistochemicallv stained were selected and included in this study.
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Fig. 1. Photomicrographs showing the TH immunoreactivity in a normal retina at differ ent magnifications, a -c Low-power views of the temporal (a, b) and nasal (c) parts in the periphery of the retina. Note that the TH-I somata can be divided clearly into two categories, with large (large arrows) and small (small arrows) somata located primarily in the inner parts of the INL. Very' few large TH-I somata were encountered in the far nasal part of the retina as shown in c. d, e High-power view showing details of the TH-I profiles in the temporal and nasal parts of the retina, respectively. Moderately immunostained processes were distributed mainly in the outer parts of IPL and OPL (open stars). Closed stars at the bottom of the photomicrographs indicate the ganglion cell layer. Scale bars: 50 pm.
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Fig. 2. Photomicrographs showing some of the large TH-1 cells in the 1NL (a-c) and gan glion cell layer (d) in a normal retina. Note the different shapes of the somata, and that the extent and intensity of immunostaining varied considerably in different regions of the same retina. The open stars indicate the OPL. e shows TH-I cells in a portion of a retinal trans plant. The broken line indicates the interface between the transplanted retinal tissue (Tp) and lens (Ln). Arrows indicate the somata of TH-I cells located in regions corresponding to the INL. Scale bars: 50 pm.
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Fig. 3. Photomicrographs showing three different retinal transplants in which immunoreactive processes were located in the IPL. The thick arrows in c indicate the outermost parts of the IPL. The thin arrows indicate TH-I somata distributed along the inner margin of the INL. Arrow heads indicate the GCL. The dotted line in a indicates the interface between the host (H) and transplanted retinal tissue (Tp). Scale bars: 50 pm.
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Fig. 4. Photomicrographs showing two retinal transplants in which no immunorcactive processes in organized form could be identified in regions corresponding to the 1PL. The dendrites of the TH-positive cells (arrows) arc prominently immunostained, however. Note that the majority of the TH-1 somata are medium-sized and few cells with large or small somata can be visualized. Scale bars: 50 pm.
thin band of immunoreactive processes was readily recognized in regions corresponding to the outermost part of the IPL but not in the OPL. In some of the transplants, however, no immunoreactive processes in well-organized form were easily observed even in the IPL, though the dendrites of the TH-I cells were prominently immunostained. The lack of or ganized immunostained processes in the plexiform layers in these transplants was not due to plane of sectioning, because many sec tions covering a large portion of the retinal tis sues were examined. One typical observation is illustrated in figures 2eand 4. In addition, it is noteworthy to point out that the reduc tion or absence of organized distribution pat tern of TH-I cellular processes in the plexi form layers did not seem to bear any relation ship with the location of the retinal trans plant, as similar patterns were observed in retinae transplanted to the cortex or mid brain.
Discussion The most important finding in this study is that a well-organized population of TH-I cells was identified in intracranially transplanted retinae, and the distribution pattern of these cells was comparable to that observed in the normal or host retina. Since TH, a rate-limit ing enzyme in the biosynthesis of catechol amines has been used as a marker of catecholaminergic neurons, our results suggest that most precursor cells of catecholaminergic neurons in embryonic retina transplanted to the foreign brain can survive, continue to dif ferentiate, and relate visual signal even in a completely abnormal environment. As it has been shown that the catecholaminergic neu rons in the normal retina represent mainly the conventional amacrine cells with their somata located in INL, the results obtained in this
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OPL were usually but not necessarily lighter in staining and less extensive as compared with those in the IPL. Nevertheless, no imraunoreactive processes could be traced to those TH-I somata which were located mainly in the inner parts of the INL. As reported in previous studies [5, 6, 11, 12], the transplanted retinae are usually folded or in rosette form, though occasionally organized in the form of continuous sheets (fig. 2, 3,4). However, all cellular and plexiform layers observed in normal retinae were identified in the retinal transplant despite a considerable variation in the form of organi zation and a general reduction in size and thickness of the retinal tissue. But distinct populations of TH-I cells were identified in all retinal transplants examined. By comparing adjacent sections which were processed for Nissl’s stain and TH immunohistochemistry, it was obvious that the TH-I somata were located primarily in the inner parts of the INL, regardless of the form of the trans planted tissues. As in the normal or host reti na, a small number of TH-I somata were also located in the GCL, but the density observed in the transplant appeared to be slightly in creased; this increase could be due to a more compact arrangement of the transplanted reti nal tissue. Although the TH-I cells in retinal transplants could also be divided into two subpopulations on the basis of their somatic sizes, the large majority of the immunoreactive somata fell into the range of 8-18 pm in diameter (fig. 2e, 3,4), and few cells with so mata smaller than 8 or larger than 18 pm were detected. By contrast, the density, extent and inten sity of immunostained cellular processes in the retinal transplant were more variable or substantially reduced (fig. 2e, 3, 4) as com pared with those of the normal or host retina. In most of the transplants in which a contin uous sheet of retinal tissue was observed, a
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activity in the plexiform layers in the trans plant. The observation of a less prominent band of immunoreactive processes in the outer parts of the IPL and the absence of immunostaining in the OPL of retinal trans plants indicate a relatively loose organization of the processes of TH-I cells in the trans planted retina. Whether these reflect struc tural defects and functional deficiencies of the retinal transplant is not altogether clear and needs to be confirmed by further ultrastruc tural and physiological studies. However, the absence of detectable and patterned immuno reactive profiles in the OPL in the transplant suggests that some of the TH-I cells which would normally send their processes to the OPL failed to do so under the present experi mental conditions. As it is generally accepted that cells which extend processes to the OPL are interplexiform cells [24,-26], the finding in this study implies that the catecholaminer gic interplexiform cells failed to develop or differentiate in the retinal transplant. Similarly, the reduction of a population of TH-I cells with very small and large sornata in the transplanted tissue suggests that some of catecholaminergic interneurons did not sur vive or differentiate after transplantation. Since it has been postulated that many of the catecholaminergic neurons with small sornata are adrenergic [41], it seems reasonable to conclude that the great majority of the adren ergic cells failed to survive or develop in the transplant and the TH-I cells observed are mainly dopaminergic neurons. Moreover, the fact that most of the TH-I cells in the retinal transplant are of medium size suggests further that there is a iack of a clear nasotemporal gra dient of the TH-I cells. Taken together, the results obtained in this study showing some defects in the arrangement and development of the catecholaminergic system in the intra cranial retinal transplant are in contrast with our previous observations of a near perfect
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study suggest further that those TH-I cells observed in the retinal transplant were most likely catecholaminergic amacrine cells. The fact that these cells had their sornata located in the inner parts of the INL and ramified, at least in some of the cases studied, to the adja cent IPL implying that these cells interact, as demonstrated in previous studies in the mam malian retina [24-27, 29, 34, 35], with other noncatecholaminergic or All rod amacrine cells which in turn synapse with the rod bipo lar cells [36-39], and are therefore involved in the rod pathways [35, 37, 40]. The observation of TH-I cells in all retinal transplants also indicates that the catechol aminergic neurons in transplanted retinal tis sues can survive and mature regardless of whether they were transplanted to a region near the central visual centers or not. This is supported by results of a recent study showing that TH-I interneurons can differentiate in developing retinal tissue following intraocular transplantation [30]. These findings, together with the evidence indicating that an appar ently well-organized cholinergic neuronal net work in similarly transplanted retinal tissue [6], suggest strongly that interneurons in the retinal transplant can be maintained and ma ture when grafted to a completely foreign or ectopic site in the host brain. Furthermore, the maintenance of these interneurons seems to be independent of the ganglion cells be cause it is known that most if not all of the retinal ganglion cells would degenerate within 5 weeks or so after transplantation of the retina to a region which is far away from their normal targets [9, 14]. While the overall pattern of immunostaining and distribution of TH-I cells and their processes in the transplant are similar to those observed in the normal retina, several charac teristic features of the TH-I cells in the normal retina were missing. One of the most obvious differences is the reduction of the immunore-
organization pattern of cholinergic neurons and their processes in the transplanted retina [6], and suggesting that the catecholaminergic system is more susceptible than the choliner gic system to changes in the microenviron ment or experimental manipulations. On the other hand, our results showing a lack of a clear nasotemporal gradient of TH-I cells with relatively large somata coincides well with results of a recent study which demonstrated that there was an absence of topography of the retinotectal projection of the retinal trans plant [2]. Thus, a general conclusion of these findings is that most of the interneurons can survive and differentiate following intracra nial transplantation, but the more refined pat tern of distribution, development and matu
ration of certain special features of the cate cholaminergic neurons can be affected con siderably. The issues as to how and when the presumably predetermined programs during development can be altered by various factors or experimental conditions are currently be ing investigated by comparing the time courses and distribution patterns of different neuronal populations in the transplanted and normal retina during development.
Acknowledgements This study was supported by UPGC grant 221400030 and NSFC grant 39070464.
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