- Email: [email protected]
Appearance of glutamate-like immunoreactivity during retinal regeneration in the adult newt
Brain Research 785 Ž1998. 171–177
Short communication
Appearance of glutamate-like immunoreactivity during retinal regeneration in the adult newt Chikafumi Chiba
)
UniÕersity of Tsukuba, Institute of Biological Sciences, Tsukuba, Ibaraki 305, Japan Accepted 18 November 1997
Abstract The appearance of endogenous glutamate during retinal regeneration in the newt was examined by immunohistochemistry. Glutamate-like immunoreactivity ŽGlu-LI. first appeared in prospective ganglion cells along the vitreal margin of retinas that were about six cells thick, in prospective photoreceptors immediately before segregation of retinal plexiform layers and then in prospective bipolar cells immediately after the initial appearance of thin plexiform layers. In retinas nearing complete regeneration, Muller cells showed ¨ immunoreactivity. The appearance of glutamatergic phenotypes during retinal regeneration seemed to follow the order of cell differentiation wT. Saito, Y. Kaneko, F. Maruo, M. Niino, Y. Sakaki, Study of the regenerating newt retina by electrophysiology and immunohistochemistry Žbipolar- and cone-specific antigen localization., J. Exp. Zool. 270 Ž1994. 491–500x. However, changes in the amount of endogenous glutamate during retinal regeneration were more complex. On the one hand, Glu-LI at the prospective ganglion cell layer temporarily increased during the initial period of segregation of the inner plexiform layer. On the other hand, immunoreactivity in the photoreceptor layer declined during segregation of the outer plexiform layer. The transient expression of immunoreactivity may represent a function of glutamate in events such as cell survival or neurite extension during retinal regeneration. q 1998 Elsevier Science B.V. Keywords: Newt; Retina; Regeneration; Glutamate; Immunohistochemistry
Certain species of urodele amphibia, such as newts and salamanders, have a capacity, even in adulthood, to regenerate retinas following complete removal of the original tissue w9,26x. It is widely accepted that the primary origin of the regenerated retina is the retinal pigment epithelial ŽRPE. cell w9,10,18x. RPE cells undergo depigmentation and proliferation following retinal ablation, producing the various retinal neurons and glia which eventually form a new functional retina. This process of regeneration has been the subject of many morphological studies w3,9,10,18,23,26x, making the urodelian retinal regeneration system an ideal model for understanding the cytogenesis and the genesis of neural circuitry in the central nervous system ŽCNS.. An amino acid, L-glutamate, is the major excitatory
) Corresponding author. Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan. Fax: q81-0298-53-6614; E-mail: [email protected]
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 4 2 7 - 3
neurotransmitter in the vertebrate CNS w13,17x. In the retina, glutamate appears to mediate synaptic transmission from photoreceptors to bipolar cells and from bipolars to ganglion cells as part of the primary visual pathway w6,12,13,27x. Advances in developmental neurobiology suggest that this substance can also participate in various events in neuronal development w11,14x. At mature stages, high levels of extracellular glutamate appear to be toxic to retinal neurons w25,28x. In contrast, glutamate is likely to act as a neurotrophic agent during embryonic development. For example, glutamate can promote survival and growth of retinal neurons w19x, and affect the dendritic stratification w2x or synaptic organization w1,5x. In a previous electrophysiological study, we used the newt retinal regeneration system to demonstrate that the sensitivity of spiking cells Žpossibly amacrine and ganglion cells. to glutamate appears before synaptogenesis, and tends to increase temporarily during the formation of the inner synaptic layer w4x. However, it was not determined whether glutamate participates directly in retinal regenera-
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tion. The present study is designed to examine this question by using immunohistochemistry to investigate the appearance and maturation of glutamatergic phenotypes during newt retinal regeneration. Adult newts Ž Cynops pyrrohgaster . were anesthetized, and the neural retinas were surgically removed as previously described w8x. The animals were maintained in a moist chamber Žabout 228C. and sacrificed on selected post-operative Žp.o.. days Ž15–45 days.. The regenerating retinas were divided into four groups Žearly, intermediate, late-I and late-II. following Chiba and Saito w4x. In the present experiment, the early, intermediate, late-I and late-II stages corresponded to 15–20, 24–26, 28–30, and more than 35 days p.o., respectively. For immunohistochemical study, eye cups from normal and operated animals were dipped in turn into the following three ice-cold fixatives: Ž1. 0.1% glutaraldehyde–4% paraformaldehyde mixture in 0.1 M phosphate buffer ŽPB; pH 7.4. for 15 min; Ž2. 4% paraformaldehyde in PB for 15 min; and Ž3. 4% paraformaldehyde in a 0.1 M sodium bicarbonate buffer ŽPh 10.4. for 9 h. The eye cups were transferred to 30% sucrose in PB, left overnight at 48C and then cryosectioned at a thickness of about 20 m m. Tissue sections were rinsed in 0.02 M phosphate buffered saline ŽPBS; Ph 7.4., 1% Triton X-100 in PBS, and PBS for 15 min each at 48C. A rabbit glutamate antiserum ŽChemicon. diluted in 1.5% normal goat serum– 0.3% Triton X-100 mixture in PBS Ž1:250. was then placed over the section for 48 h at 48C. After the antiserum was washed out, the second antibody, FITC-conjugated goat anti-rabbit IgG ŽSigma., was applied to the sections at a dilution of 1:160 in PBS for 3 h at room temperature. The specificity of the antiserum has been described previously w12x. To control for non-specific binding of the antiserum to the newt retinal tissues, other sections were treated in the same way with non-immune rabbit serum ŽSigma.. No staining was detected in either normal or regenerating control retinas, and thus the observed immunoreactivity likely results from a reaction between the antibody and the tissue glutamate. Fig. 1 shows the glutamate-like immunoreactivity ŽGluLI. in the normal retina. The outer ŽOPL. and the inner plexiform layers ŽIPL. were stained intensely ŽFig. 1A.. Photoreceptors were weakly stained. Relatively high Glu-LI was observed in the outer half of the inner nuclear layer ŽINL., with apparent staining in both horizontal and bipolar cells. In some sections, immunoreactive bipolar cells having a Landolt Club Ždouble arrow in Fig. 1B. were clearly visible. In the inner half of the INL, only a small number of cells within the amacrine cell layer were stained Žsingle arrow in Fig. 1B.. These immunoreactive cells may represent displaced ganglion cells. Glu-LI was also observed in the ganglion cell layer ŽGCL. ŽFig. 1C.. Nerve fibers entering the optic disk Žod. were stained intensely ŽFig. 1D., indicating that many of immunoreactive cells in the GCL were ganglion cells. In the GCL, however, there
were a small number of somata with no immunoreactivity Žarrows in Fig. 1C., which could be displaced amacrine cells. Vertically extending processes in the GCL were also stained intensely Žarrow heads in Fig. 1A.. Judging from their morphology, these appeared to be projections of Muller glial cells which expand as ‘end feet’ to form the ¨ inner limiting membrane. Glu-LI was examined in regenerating retinas at different days after operation. At 15 to 20 days p.o. Ži.e., the early stage., regenerating retinas were one to two cells thick and consisted of cells with either pigmented or non-pigmented cytoplasm. At this stage, retinas were not immunoreactive to glutamate Ždata not shown.. At 24 to 26 days p.o. Ži.e., the intermediate stage., the regenerating retinas were several cells thick but lacked plexiform layers. Fig. 2A,B show sample sections of this stage. Regenerating retinas of about four-cell thickness did not show immunoreactivity ŽFig. 2A.. Immunoreactive cells first appeared along the vitreal margin of the regenerating retinas that had reached a thickness of about six cells ŽFig. 2B.. Single or clustered somata were clearly stained Žtwo of them are indicated by arrows.. Fluorescence was also observed in the extracellular space surrounding the immunoreactive somata. At 27 days p.o., single or clustered prospective photoreceptors had appeared at the site along the scleral margin of the regenerating retinas. Fig. 2C shows a sample section in which only a small number of individual photoreceptors had appeared. In this section, photoreceptors were faintly visible under the fluorescent microscope because there was no immunoreactivity, but the number of immunoreactive somata at the vitreal side had increased. Within the outer half of this region, single or clustered somata with no staining were recognizable Žarrow heads.. Their position presumably indicates that these cells were amacrine cells w3x. Fig. 2D shows another section taken at the same day that seemed slightly advanced, with photoreceptors lined up along the scleral margin, forming the outer nuclear layer ŽONL.. In this section, photoreceptors showed a detectable level of immunoreactivity Žtwo of them are indicated by arrows.; these cells were conical and had extended a single short process inwardly Žarrow head.. The number of immunoreactive cells at the vitreal side of the regenerating retinas seemed constant. On the other hand, the number of cells with no staining at the site of the prospective amacrine cell layer had increased. Within the following few days Ži.e., the late-I stage., two plexiform layers appeared in the regenerating retinas. Fig. 3A shows a sample section of this stage. Glu-LI appeared along the segregating plexiform layers, and increased immunoreactivity was observed at the site of the prospective GCL. However, staining in the ONL declined at this stage and showed weak labelling characteristic of the intact original tissue. Vertically extending processes seemed to be stained lightly Žarrow.. Fig. 3B shows another section at the same stage with a higher magnifica-
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Fig. 1. Glutamate-like immunoreactivity in the normal retina. ŽA. The outer ŽOPL. and inner plexiform layers ŽIPL. were stained intensely. The outer nuclear layer ŽONL. was weakly stained. The outer half of the inner nuclear layer ŽINL. and the ganglion cell layer ŽGCL. were clearly labelled. Vertically extending processes in the GCL were also stained Žarrow heads.. ŽB. An intensely immunoreactive bipolar cell extending a Landolt Club Ždouble arrow. was distinguishable. A soma along the amacrine cell layer was also stained Žarrow.. ŽC. Most somata in the GCL were immunoreactive. However, a small number of somata along the proximal margin of the IPL were unlabelled Žarrows.. ŽD. Optic nerve fibers entering the optic disc Žod. were stained intensely. Bar: ŽA. 60 m m; ŽB, C. 30 m m; ŽD. 80 m m.
tion. The process, which extended into the immature IPL Žarrow., originated from an oval soma located at the most outer part of the prospective INL Žarrow head.. By 34 days p.o., the appearance of the regenerating retinas was identical to that of the original retinas in most but not all aspects. Fig. 3C shows a sample section. Immunoreactivity at the GCL was decreased from the higher levels seen at earlier stages and was similar to the original tissues. However, immunoreactivity of the plexiform layers was still lower than seen in control retinas. Processes that extended across the retina were weakly
stained Žarrows.. These were apparently apical and basal processes of Muller ¨ cells. Fig. 3D shows another section of this stage. The Muller cells could be distinguished from ¨ the background by their relatively high immunoreactivity. The somata of the Muller cells were located in the inner ¨ two-fifths of the INL Žarrow head. and had extended single processes in both inner and outer directions Žarrows.; one process ran across the IPL and terminated at the vitreal margin of the GCL. At more than 35 days p.o., retinal regeneration appeared to be complete; this stage was defined as late-II. Fig. 3E shows a sample section at 45 days
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Fig. 2. Glutamate-like immunoreactivity in regenerating retinas before appearance of plexiform layers. ŽA, B. Sample sections of the intermediate retina. At 24 days p.o., the Glu-LI was not detected ŽA.. At 26 days p.o. ŽB., the Glu-LI appeared at somata along the vitreal margin Žarrows.. Extracellular space around the labelled cells was also stained. ŽC, D. Sample sections of retinas between the intermediate and the late stages Ž27 days p.o... In one section ŽC., where a small number of prospective photoreceptors appeared singly at site of the scleral margin Žthe cells are hardly visible because of no immunoreactivity., only somata within a 2-to-4 cell layer at the vitreal side were stained. Somata with no labelling were distinguishable within the outer half of this region Žarrow heads.. In the other section ŽD., a line of photoreceptors along the scleral margin showed the Glu-LI. Two of them are indicated by arrows. The cells were conical and had extended a single short process Žarrow head.. Immunoreactive somata were also present at a few vitreal cell layers. Bar: 40 m m.
p.o. Immunoreactivity of the plexiform layers had increased and the overall pattern and intensity of Glu-LI throughout the regenerated retina was identical to that of the original tissue. The present study suggests that photoreceptors, bipolars and ganglion cells in the newt retina are glutamatergic,
corroborating other studies of vertebrate retina w6,12,13,27x. Furthermore, horizontal cells also seemed to be labelled by the glutamate antiserum. However, at least some, if not all, of these horizontal cells are probably GABAergic w3x. The glutamate in horizontal cells may serve as a precursor for the production of GABA rather than acting directly as a
Fig. 3. Glutamate-like immunoreactivity in regenerating retinas during and after the segregation of plexiform layers. ŽA, B. Sample sections of retinas at the late-I stage. In ŽA., the segregating OPL and IPL showed the Glu-LI. Somata within the prospective GCL showed increased immunoreactivity. Vertically extending processes within the INL were lightly labelled Žarrow.. In ŽB., oval soma Žarrow head. extending a single process Žarrow. into the IPL were lightly labelled. ŽA. 29 days p.o.; ŽB. 28 days p.o. ŽC, D. Sample sections of retinas between the late-I and II stages. The appearance of the retina was almost identical to the normal retina. In ŽC., vertically extending processes across the retina were lightly stained Žarrows.. In ŽD., a Muller cell having a ¨ soma which was located at the inner two-fifths of the INL Žarrow head. and had extended single processes in both inner and outer directions Žarrows. was stained clearly. ŽC. 33 days p.o.; ŽD. 34 days p.o. ŽE. A sample section of retinas at the late-II stage Ž45 days p.o... The Glu-LI was substantially the same as that of the normal retina. Bar: ŽA, C, E. 40 m m; ŽB, D. 30 m m.
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neurotransmitter w12x. Muller cells were also labelled, pos¨ sibly representing their avid uptake and metabolism of glutamate w21,24x. During retinal regeneration, Glu-LI first appeared at somata Žprobably ganglion cells. along the vitreal margin of regenerating retinas that were about six cells thick Žintermediate stage.. Since previous electron microscopic studies show that the optic nerve structure has already appeared in regenerating retinas of several cells’ thickness w9x, fluorescence of the extracellular space surrounding the labelled cells might represent the axonal processes of ganglion cells. Immunoreactivity subsequently appeared at the prospective ONL immediately before the segregation of the plexiform layers Žbetween the intermediate and late stages.. The labelled cells were conical and had extended a single short process inwardly; these are probably cones. The occurrence of similar cone shaped cells during retinal regeneration has been described in other papers w23x. Immediately after the appearance of thin plexiform layers Žlate-I stage., immunoreactivity was observed in oval somata located along the outer most prospective INL. Each cell had extended a single labelled process up to the developing IPL. Such morphological characteristics appeared to be identical to those of PKC-immunoreactive ‘ON’-shaped bipolar cells, whose appearance at the same stage has also been described w18,23x. These observations suggest that the appearance of glutamatergic phenotypes during retinal regeneration follows the order of cell differentiation Ži.e., ganglion cells ™ photoreceptors™ bipolar cells. proposed by Saito et al. w23x. On the other hand, changes in the amount of endogenous glutamate during retinal regeneration were complex. Glu-LI at the prospective GCL rose excessively during the initial phase of IPL segregation, and later decreased to near the level of the original tissue. Furthermore, the prospective ONL, which had expressed immunoreactivity immediately before the appearance of the plexiform layers, declined to near the level of control tissue as these layers segregated. Such a transient increase in neurotransmitters has been reported not only in retinal regeneration but also in its development w3,15,20x. It has been suggested that the early neurotransmitters have a trophic role for immature neurons w11,14,16,19,20,22x. If released non-synaptically, glutamate may affect survival and neurite extension of releasing neurons themselves or surrounding neurons w11,14,22x. Bodnarenko et al. w2x have reported that the injection of an agonist for a glutamate receptor subtype ŽAPB receptors. on ON-bipolar cells into developing cat eyes modulates dendritic stratification of ganglion cells, and suggested that glutamate-mediated afferent activity regulates appropriate synaptic connections within the retina. During newt retinal regeneration, the IPL seemed to increase in thickness gradually following the appearance of Glu-LI in bipolar cells. This may suggest that activity-dependent regulation, mediated by glutamate, participates in a process
of synaptogenesis within the IPL in regeneration, as well. In fact, as we have reported previously w4x, spiking retinal cells Žamacrine and ganglion cells. tend to increase in responsiveness to glutamate while the IPL is increasing in thickness. An immunocytochemical marker of Muller cells, glu¨ tamine synthetase Žan enzyme that converts glutamate into glutamine. appears at a time when retinal regeneration is complete w18x. Correspondingly, Glu-LI in Muller cells ¨ appeared at a later regeneration stage. This suggests that the uptake and metabolism of glutamate in Muller cells ¨ increases later in regeneration. Haberecht and Redburn w7x have reported that, in the rabbit retina, increased uptake activity of Muller cells is a major determinant of extracel¨ lular glutamate levels in the first postnatal week. They have suggested that Muller cells might regulate levels of ¨ extracellular glutamate in retinal generation which could participate in a variety of events in neuronal development. This might also be true in retinal regeneration. In conclusion, our present observations suggest the possibility that glutamate could play a variety of roles in neuronal development during newt retinal regeneration. However, it is necessary to design further experiments to determine how glutamate actually functions in retinal regeneration. Our data, along with similar observations in developing retinas, provide a background for future studies to determine the functional roles of glutamate during normal maturation and regeneration.
Acknowledgements We are grateful to Dr. Takehiko Saito ŽUniversity of Tsukuba. for his valuable suggestions on this work and to Dr. Dianna A. Johnson ŽUniversity of Tennessee at Memphis. for her critical reading of the manuscript and valuable comments. We thank Mr. Richard Gabbert for correcting grammatical errors in the initial drafts. This work was supported by grants from the University of Tsukuba Research Project and from the Ministry of Education of Japan.
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C. Chibar Brain Research 785 (1998) 171–177 w5x H.T. Cline, M. Constantine-Paton, NMDA receptor antagonists disrupt the retinotectal map, Neuron 3 Ž1989. 413–426. w6x B. Ehinger, O.P. Ottersen, J. Storm-Mathisen, J.E. Dowling, Bipolar cells in the turtle retina are strongly immunoreactive for glutamate, Proc. Natl. Acad. Sci. U.S.A. 85 Ž1988. 8321–8325. w7x M.F. Haberecht, D.A. Redburn, High levels of extracellular glutamate are present in retina during neonatal development, Neurochem. Res. 21 Ž1996. 285–291. w8x Y. Kaneko, T. Saito, Appearance and maturation of voltage-dependent conductances in solitary spiking cells during retinal regeneration in the adult newt, J. Comp. Physiol. A 170 Ž1992. 411–425. w9x J.R. Keefe, An analysis of urodelian retinal regeneration: I. Studies of the cellular source of retinal regeneration in Notophthalmus Õiridescens utilizing 3 H-thymidine and colchicine, J. Exp. Zool. 184 Ž1973. 185–206. w10x L.R. Klein, P.R. MacLeish, T.N. Wiesel, Immunolabelling by a newt retinal pigment epithelium antibody during retinal development and regeneration, J. Comp. Neurol. 293 Ž1990. 331–339. w11x S.A. Lipton, S.B. Kater, Neurotransmitter regulation of neuronal outgrowth, plasticity and survival, TINS 12 Ž1989. 265–270. w12x R.E. Marc, W.-L.S. Liu, M. Kalloniatis, S.F. Raiguel, E.V. Haesendonck, Patterns of glutamate immunoreactivity in the goldfish retina, J. Neurosci. 10 Ž1990. 4006–4034. w13x S.C. Massey, Cell types using glutamate as a neurotransmitter in the vertebrate retina, in: N.N. Osborne, G.J. Chader ŽEds.., Progress in Retinal Research, Vol. 9, Pergamon, Oxford, 1990, pp. 399–425. w14x J.W. McDonald, M.V. Johnston, Physiological and pathophysiological roles of excitatory amino acids during central nervous system development, Brain Res. Rev. 1 Ž1990. 446–455. w15x E.K. Messersmith, D.A. Redburn, g-Aminobutyric acid immunoreactivity in multiple cell types of the developing rabbit retina, Vis. Neurosci. 8 Ž1992. 201–211. w16x E.K. Messersmith, D.A. Redburn, The role of GABA during development of the outer retina in the rabbit, Neurochem. Res. 18 Ž1993. 463–470. w17x D.T. Monaghan, R.J. Bridges, C.W. Cotman, The excitatory amino
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acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol. 29 Ž1989. 365–402. K. Negishi, S. Shinagawa, M. Ushijima, Y. Kaneko, T. Saito, An immunohistochemical study of regenerating newt retinas, Dev. Brain Res. 68 Ž1992. 255–264. K.A. Nichol, M.W. Schulz, M.R. Bennett, Nitric oxide-mediated death of cultured neonatal retinal ganglion cells: neuroprotective properties of glutamate and chondroitin sulfate proteoglycan, Brain Res. 697 Ž1995. 1–16. D.V. Pow, D.K. Crook, R.O.L. Wong, Early appearance and transient expression of putative amino acid neurotransmitters and related molecules in the developing rabbit retina: an immunocytochemical study, Vis. Neurosci. 11 Ž1994. 1115–1134. D.V. Pow, S.R. Robinson, Glutamate in some retinal neurons is derived solely from glia, Neuroscience 60 Ž1994. 355–366. M.B. Robinson, J.T. Coyle, Glutamate and related acidic excitatory neurotransmitters: from basic science to clinical application, FASEB 15 Ž1987. 41–70. T. Saito, Y. Kaneko, F. Maruo, M. Niino, Y. Sakaki, Study of the regenerating newt retina by electrophysiology and immunohistochemistry Žbipolar- and cone-specific antigen localization., J. Exp. Zool. 270 Ž1994. 491–500. P.V. Sarthy, D.M.-K. Lam, Biochemical studies of isolated glial Muller ¨ cells from the turtle retina, J. Cell. Biol. 78 Ž1978. 675–684. R. Siliprandi, R. Canella, G. Carmignoto, N. Schiavo, A. Zanellato, R. Zanoni, G. Vantini, NMDA induced neurotoxicity in the adult rat retina, Vis. Neurosci. 8 Ž1992. 567–773. L.S. Stone, The role of retinal pigment cells in regenerating neural retinae of adult salamander eyes, J. Exp. Zool. 113 Ž1950. 9–32. M. Tachibana, T. Okada, Release of endogenous excitatory amino acids from ON-type bipolar cells isolated from the goldfish retina, J. Neurosci. 11 Ž1991. 2199–2208. G.D. Zeevalk, W.J. Nicklas, Chemically-induced hypoglycemia and anoxia: relationship to glutamate receptor mediated toxicity in the retina, J. Pharmacol. Exp. Ther. 253 Ž1990. 1285–1292.
Short communication
Appearance of glutamate-like immunoreactivity during retinal regeneration in the adult newt Chikafumi Chiba
)
UniÕersity of Tsukuba, Institute of Biological Sciences, Tsukuba, Ibaraki 305, Japan Accepted 18 November 1997
Abstract The appearance of endogenous glutamate during retinal regeneration in the newt was examined by immunohistochemistry. Glutamate-like immunoreactivity ŽGlu-LI. first appeared in prospective ganglion cells along the vitreal margin of retinas that were about six cells thick, in prospective photoreceptors immediately before segregation of retinal plexiform layers and then in prospective bipolar cells immediately after the initial appearance of thin plexiform layers. In retinas nearing complete regeneration, Muller cells showed ¨ immunoreactivity. The appearance of glutamatergic phenotypes during retinal regeneration seemed to follow the order of cell differentiation wT. Saito, Y. Kaneko, F. Maruo, M. Niino, Y. Sakaki, Study of the regenerating newt retina by electrophysiology and immunohistochemistry Žbipolar- and cone-specific antigen localization., J. Exp. Zool. 270 Ž1994. 491–500x. However, changes in the amount of endogenous glutamate during retinal regeneration were more complex. On the one hand, Glu-LI at the prospective ganglion cell layer temporarily increased during the initial period of segregation of the inner plexiform layer. On the other hand, immunoreactivity in the photoreceptor layer declined during segregation of the outer plexiform layer. The transient expression of immunoreactivity may represent a function of glutamate in events such as cell survival or neurite extension during retinal regeneration. q 1998 Elsevier Science B.V. Keywords: Newt; Retina; Regeneration; Glutamate; Immunohistochemistry
Certain species of urodele amphibia, such as newts and salamanders, have a capacity, even in adulthood, to regenerate retinas following complete removal of the original tissue w9,26x. It is widely accepted that the primary origin of the regenerated retina is the retinal pigment epithelial ŽRPE. cell w9,10,18x. RPE cells undergo depigmentation and proliferation following retinal ablation, producing the various retinal neurons and glia which eventually form a new functional retina. This process of regeneration has been the subject of many morphological studies w3,9,10,18,23,26x, making the urodelian retinal regeneration system an ideal model for understanding the cytogenesis and the genesis of neural circuitry in the central nervous system ŽCNS.. An amino acid, L-glutamate, is the major excitatory
) Corresponding author. Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan. Fax: q81-0298-53-6614; E-mail: [email protected]
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 4 2 7 - 3
neurotransmitter in the vertebrate CNS w13,17x. In the retina, glutamate appears to mediate synaptic transmission from photoreceptors to bipolar cells and from bipolars to ganglion cells as part of the primary visual pathway w6,12,13,27x. Advances in developmental neurobiology suggest that this substance can also participate in various events in neuronal development w11,14x. At mature stages, high levels of extracellular glutamate appear to be toxic to retinal neurons w25,28x. In contrast, glutamate is likely to act as a neurotrophic agent during embryonic development. For example, glutamate can promote survival and growth of retinal neurons w19x, and affect the dendritic stratification w2x or synaptic organization w1,5x. In a previous electrophysiological study, we used the newt retinal regeneration system to demonstrate that the sensitivity of spiking cells Žpossibly amacrine and ganglion cells. to glutamate appears before synaptogenesis, and tends to increase temporarily during the formation of the inner synaptic layer w4x. However, it was not determined whether glutamate participates directly in retinal regenera-
172
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tion. The present study is designed to examine this question by using immunohistochemistry to investigate the appearance and maturation of glutamatergic phenotypes during newt retinal regeneration. Adult newts Ž Cynops pyrrohgaster . were anesthetized, and the neural retinas were surgically removed as previously described w8x. The animals were maintained in a moist chamber Žabout 228C. and sacrificed on selected post-operative Žp.o.. days Ž15–45 days.. The regenerating retinas were divided into four groups Žearly, intermediate, late-I and late-II. following Chiba and Saito w4x. In the present experiment, the early, intermediate, late-I and late-II stages corresponded to 15–20, 24–26, 28–30, and more than 35 days p.o., respectively. For immunohistochemical study, eye cups from normal and operated animals were dipped in turn into the following three ice-cold fixatives: Ž1. 0.1% glutaraldehyde–4% paraformaldehyde mixture in 0.1 M phosphate buffer ŽPB; pH 7.4. for 15 min; Ž2. 4% paraformaldehyde in PB for 15 min; and Ž3. 4% paraformaldehyde in a 0.1 M sodium bicarbonate buffer ŽPh 10.4. for 9 h. The eye cups were transferred to 30% sucrose in PB, left overnight at 48C and then cryosectioned at a thickness of about 20 m m. Tissue sections were rinsed in 0.02 M phosphate buffered saline ŽPBS; Ph 7.4., 1% Triton X-100 in PBS, and PBS for 15 min each at 48C. A rabbit glutamate antiserum ŽChemicon. diluted in 1.5% normal goat serum– 0.3% Triton X-100 mixture in PBS Ž1:250. was then placed over the section for 48 h at 48C. After the antiserum was washed out, the second antibody, FITC-conjugated goat anti-rabbit IgG ŽSigma., was applied to the sections at a dilution of 1:160 in PBS for 3 h at room temperature. The specificity of the antiserum has been described previously w12x. To control for non-specific binding of the antiserum to the newt retinal tissues, other sections were treated in the same way with non-immune rabbit serum ŽSigma.. No staining was detected in either normal or regenerating control retinas, and thus the observed immunoreactivity likely results from a reaction between the antibody and the tissue glutamate. Fig. 1 shows the glutamate-like immunoreactivity ŽGluLI. in the normal retina. The outer ŽOPL. and the inner plexiform layers ŽIPL. were stained intensely ŽFig. 1A.. Photoreceptors were weakly stained. Relatively high Glu-LI was observed in the outer half of the inner nuclear layer ŽINL., with apparent staining in both horizontal and bipolar cells. In some sections, immunoreactive bipolar cells having a Landolt Club Ždouble arrow in Fig. 1B. were clearly visible. In the inner half of the INL, only a small number of cells within the amacrine cell layer were stained Žsingle arrow in Fig. 1B.. These immunoreactive cells may represent displaced ganglion cells. Glu-LI was also observed in the ganglion cell layer ŽGCL. ŽFig. 1C.. Nerve fibers entering the optic disk Žod. were stained intensely ŽFig. 1D., indicating that many of immunoreactive cells in the GCL were ganglion cells. In the GCL, however, there
were a small number of somata with no immunoreactivity Žarrows in Fig. 1C., which could be displaced amacrine cells. Vertically extending processes in the GCL were also stained intensely Žarrow heads in Fig. 1A.. Judging from their morphology, these appeared to be projections of Muller glial cells which expand as ‘end feet’ to form the ¨ inner limiting membrane. Glu-LI was examined in regenerating retinas at different days after operation. At 15 to 20 days p.o. Ži.e., the early stage., regenerating retinas were one to two cells thick and consisted of cells with either pigmented or non-pigmented cytoplasm. At this stage, retinas were not immunoreactive to glutamate Ždata not shown.. At 24 to 26 days p.o. Ži.e., the intermediate stage., the regenerating retinas were several cells thick but lacked plexiform layers. Fig. 2A,B show sample sections of this stage. Regenerating retinas of about four-cell thickness did not show immunoreactivity ŽFig. 2A.. Immunoreactive cells first appeared along the vitreal margin of the regenerating retinas that had reached a thickness of about six cells ŽFig. 2B.. Single or clustered somata were clearly stained Žtwo of them are indicated by arrows.. Fluorescence was also observed in the extracellular space surrounding the immunoreactive somata. At 27 days p.o., single or clustered prospective photoreceptors had appeared at the site along the scleral margin of the regenerating retinas. Fig. 2C shows a sample section in which only a small number of individual photoreceptors had appeared. In this section, photoreceptors were faintly visible under the fluorescent microscope because there was no immunoreactivity, but the number of immunoreactive somata at the vitreal side had increased. Within the outer half of this region, single or clustered somata with no staining were recognizable Žarrow heads.. Their position presumably indicates that these cells were amacrine cells w3x. Fig. 2D shows another section taken at the same day that seemed slightly advanced, with photoreceptors lined up along the scleral margin, forming the outer nuclear layer ŽONL.. In this section, photoreceptors showed a detectable level of immunoreactivity Žtwo of them are indicated by arrows.; these cells were conical and had extended a single short process inwardly Žarrow head.. The number of immunoreactive cells at the vitreal side of the regenerating retinas seemed constant. On the other hand, the number of cells with no staining at the site of the prospective amacrine cell layer had increased. Within the following few days Ži.e., the late-I stage., two plexiform layers appeared in the regenerating retinas. Fig. 3A shows a sample section of this stage. Glu-LI appeared along the segregating plexiform layers, and increased immunoreactivity was observed at the site of the prospective GCL. However, staining in the ONL declined at this stage and showed weak labelling characteristic of the intact original tissue. Vertically extending processes seemed to be stained lightly Žarrow.. Fig. 3B shows another section at the same stage with a higher magnifica-
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Fig. 1. Glutamate-like immunoreactivity in the normal retina. ŽA. The outer ŽOPL. and inner plexiform layers ŽIPL. were stained intensely. The outer nuclear layer ŽONL. was weakly stained. The outer half of the inner nuclear layer ŽINL. and the ganglion cell layer ŽGCL. were clearly labelled. Vertically extending processes in the GCL were also stained Žarrow heads.. ŽB. An intensely immunoreactive bipolar cell extending a Landolt Club Ždouble arrow. was distinguishable. A soma along the amacrine cell layer was also stained Žarrow.. ŽC. Most somata in the GCL were immunoreactive. However, a small number of somata along the proximal margin of the IPL were unlabelled Žarrows.. ŽD. Optic nerve fibers entering the optic disc Žod. were stained intensely. Bar: ŽA. 60 m m; ŽB, C. 30 m m; ŽD. 80 m m.
tion. The process, which extended into the immature IPL Žarrow., originated from an oval soma located at the most outer part of the prospective INL Žarrow head.. By 34 days p.o., the appearance of the regenerating retinas was identical to that of the original retinas in most but not all aspects. Fig. 3C shows a sample section. Immunoreactivity at the GCL was decreased from the higher levels seen at earlier stages and was similar to the original tissues. However, immunoreactivity of the plexiform layers was still lower than seen in control retinas. Processes that extended across the retina were weakly
stained Žarrows.. These were apparently apical and basal processes of Muller ¨ cells. Fig. 3D shows another section of this stage. The Muller cells could be distinguished from ¨ the background by their relatively high immunoreactivity. The somata of the Muller cells were located in the inner ¨ two-fifths of the INL Žarrow head. and had extended single processes in both inner and outer directions Žarrows.; one process ran across the IPL and terminated at the vitreal margin of the GCL. At more than 35 days p.o., retinal regeneration appeared to be complete; this stage was defined as late-II. Fig. 3E shows a sample section at 45 days
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Fig. 2. Glutamate-like immunoreactivity in regenerating retinas before appearance of plexiform layers. ŽA, B. Sample sections of the intermediate retina. At 24 days p.o., the Glu-LI was not detected ŽA.. At 26 days p.o. ŽB., the Glu-LI appeared at somata along the vitreal margin Žarrows.. Extracellular space around the labelled cells was also stained. ŽC, D. Sample sections of retinas between the intermediate and the late stages Ž27 days p.o... In one section ŽC., where a small number of prospective photoreceptors appeared singly at site of the scleral margin Žthe cells are hardly visible because of no immunoreactivity., only somata within a 2-to-4 cell layer at the vitreal side were stained. Somata with no labelling were distinguishable within the outer half of this region Žarrow heads.. In the other section ŽD., a line of photoreceptors along the scleral margin showed the Glu-LI. Two of them are indicated by arrows. The cells were conical and had extended a single short process Žarrow head.. Immunoreactive somata were also present at a few vitreal cell layers. Bar: 40 m m.
p.o. Immunoreactivity of the plexiform layers had increased and the overall pattern and intensity of Glu-LI throughout the regenerated retina was identical to that of the original tissue. The present study suggests that photoreceptors, bipolars and ganglion cells in the newt retina are glutamatergic,
corroborating other studies of vertebrate retina w6,12,13,27x. Furthermore, horizontal cells also seemed to be labelled by the glutamate antiserum. However, at least some, if not all, of these horizontal cells are probably GABAergic w3x. The glutamate in horizontal cells may serve as a precursor for the production of GABA rather than acting directly as a
Fig. 3. Glutamate-like immunoreactivity in regenerating retinas during and after the segregation of plexiform layers. ŽA, B. Sample sections of retinas at the late-I stage. In ŽA., the segregating OPL and IPL showed the Glu-LI. Somata within the prospective GCL showed increased immunoreactivity. Vertically extending processes within the INL were lightly labelled Žarrow.. In ŽB., oval soma Žarrow head. extending a single process Žarrow. into the IPL were lightly labelled. ŽA. 29 days p.o.; ŽB. 28 days p.o. ŽC, D. Sample sections of retinas between the late-I and II stages. The appearance of the retina was almost identical to the normal retina. In ŽC., vertically extending processes across the retina were lightly stained Žarrows.. In ŽD., a Muller cell having a ¨ soma which was located at the inner two-fifths of the INL Žarrow head. and had extended single processes in both inner and outer directions Žarrows. was stained clearly. ŽC. 33 days p.o.; ŽD. 34 days p.o. ŽE. A sample section of retinas at the late-II stage Ž45 days p.o... The Glu-LI was substantially the same as that of the normal retina. Bar: ŽA, C, E. 40 m m; ŽB, D. 30 m m.
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neurotransmitter w12x. Muller cells were also labelled, pos¨ sibly representing their avid uptake and metabolism of glutamate w21,24x. During retinal regeneration, Glu-LI first appeared at somata Žprobably ganglion cells. along the vitreal margin of regenerating retinas that were about six cells thick Žintermediate stage.. Since previous electron microscopic studies show that the optic nerve structure has already appeared in regenerating retinas of several cells’ thickness w9x, fluorescence of the extracellular space surrounding the labelled cells might represent the axonal processes of ganglion cells. Immunoreactivity subsequently appeared at the prospective ONL immediately before the segregation of the plexiform layers Žbetween the intermediate and late stages.. The labelled cells were conical and had extended a single short process inwardly; these are probably cones. The occurrence of similar cone shaped cells during retinal regeneration has been described in other papers w23x. Immediately after the appearance of thin plexiform layers Žlate-I stage., immunoreactivity was observed in oval somata located along the outer most prospective INL. Each cell had extended a single labelled process up to the developing IPL. Such morphological characteristics appeared to be identical to those of PKC-immunoreactive ‘ON’-shaped bipolar cells, whose appearance at the same stage has also been described w18,23x. These observations suggest that the appearance of glutamatergic phenotypes during retinal regeneration follows the order of cell differentiation Ži.e., ganglion cells ™ photoreceptors™ bipolar cells. proposed by Saito et al. w23x. On the other hand, changes in the amount of endogenous glutamate during retinal regeneration were complex. Glu-LI at the prospective GCL rose excessively during the initial phase of IPL segregation, and later decreased to near the level of the original tissue. Furthermore, the prospective ONL, which had expressed immunoreactivity immediately before the appearance of the plexiform layers, declined to near the level of control tissue as these layers segregated. Such a transient increase in neurotransmitters has been reported not only in retinal regeneration but also in its development w3,15,20x. It has been suggested that the early neurotransmitters have a trophic role for immature neurons w11,14,16,19,20,22x. If released non-synaptically, glutamate may affect survival and neurite extension of releasing neurons themselves or surrounding neurons w11,14,22x. Bodnarenko et al. w2x have reported that the injection of an agonist for a glutamate receptor subtype ŽAPB receptors. on ON-bipolar cells into developing cat eyes modulates dendritic stratification of ganglion cells, and suggested that glutamate-mediated afferent activity regulates appropriate synaptic connections within the retina. During newt retinal regeneration, the IPL seemed to increase in thickness gradually following the appearance of Glu-LI in bipolar cells. This may suggest that activity-dependent regulation, mediated by glutamate, participates in a process
of synaptogenesis within the IPL in regeneration, as well. In fact, as we have reported previously w4x, spiking retinal cells Žamacrine and ganglion cells. tend to increase in responsiveness to glutamate while the IPL is increasing in thickness. An immunocytochemical marker of Muller cells, glu¨ tamine synthetase Žan enzyme that converts glutamate into glutamine. appears at a time when retinal regeneration is complete w18x. Correspondingly, Glu-LI in Muller cells ¨ appeared at a later regeneration stage. This suggests that the uptake and metabolism of glutamate in Muller cells ¨ increases later in regeneration. Haberecht and Redburn w7x have reported that, in the rabbit retina, increased uptake activity of Muller cells is a major determinant of extracel¨ lular glutamate levels in the first postnatal week. They have suggested that Muller cells might regulate levels of ¨ extracellular glutamate in retinal generation which could participate in a variety of events in neuronal development. This might also be true in retinal regeneration. In conclusion, our present observations suggest the possibility that glutamate could play a variety of roles in neuronal development during newt retinal regeneration. However, it is necessary to design further experiments to determine how glutamate actually functions in retinal regeneration. Our data, along with similar observations in developing retinas, provide a background for future studies to determine the functional roles of glutamate during normal maturation and regeneration.
Acknowledgements We are grateful to Dr. Takehiko Saito ŽUniversity of Tsukuba. for his valuable suggestions on this work and to Dr. Dianna A. Johnson ŽUniversity of Tennessee at Memphis. for her critical reading of the manuscript and valuable comments. We thank Mr. Richard Gabbert for correcting grammatical errors in the initial drafts. This work was supported by grants from the University of Tsukuba Research Project and from the Ministry of Education of Japan.
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