- Email: [email protected]
Dopaminergic cell differentiation in the developing chick retina
Bruin Rrsrart~h
Bufietin.
Vol. 10, pp. 97-102. 1983. Printed in the U.S.A
Dopaminergic Cell D~e~~ntiation in the Developing Chick Retina MASASUKE
ARAKI,
TOSHIHIRO
MAEDA”
AND HIROSHI
KI~URA*
Department of Anatomy, Jichi Medical School, Tochigi 329-04 Japan and *Department of Anatomy, Shiga University of Medical Science Qtsu, Shiga 520-21 Japan Received
8 September
1982
ARAKI, M., T. MAEDA AND H. KIMURA. Dopaminergic cell differentiation in developing chick retina. BRAIN RES BULL lo(l) 97-102, 1983.-The distribution and morphology of dopaminergic (DA) neurons in the chick retina was studied in the course of development. Fluorescent DA cells were first detected on the 13th day of incubation. They were always found in positions two or three cell rows externally from the junction between the inner plexiform layer (IPL) and inner nuclear layer (INL). On the 14th day, DA cells were found in the innermost row of the INL. Subsequently their processes extended not only bilaterally along the IPL-INL junction but also vertically into the IPL. As a result, three fiber layers were formed as laminae I, 3 and 5 in the IPL. fn the newly-hatched chick retina, a number of growth cone-like fluorescent structures with fine spikes were seen at the IPL-INL junction, indicating that DA fibers were still growing and elongating at least at hatching. On the 4th postnatal day, the ramification of dendritic processes was very prominent and they often showed a spiral configuration. Cell differentiation
Amacrine cells
Chick
Development
method established by Falck catecholamine-containing neurons have been demonstrated in the retina of various species [6-91. The catecholamine fluorescence in the retina is mostly due to dopamine (DA), and other catecholamines such as noradrenahne and adrenaline seem to play a minor role or are even absent 113,231. In the avian retina DA cells are mostly found in the innermost part of the inner nuclear layer (INL), which contains the cell bodies of the amacrine neurons whose processes extend exclusively into the inner plexiform layer (IPL) [7,16]. Such dopaminergic processes form a dense network at the outermost border of the IPL, but distribute sparsely in the middle and innermost border of the IPL. Currently we have applied a new sensitive method for catecholamine, being modified from the Faglu method [ 121to study the differentiation of DA cells in the developing chick retina aiming to clarify the process whereby these DA cells make a well developed fiber-arborization. USING
(It
ul.
the histofluorescence
[IO],
METHOD
Fertile eggs were incubated in a dark incubator at 37.5”C to 38.o”C with 90% humidity. Under this condition hatching occurred on the 20th or 21st day. After hatching, chicks were raised under a cycled light condition with a lighting schedule from 9 a.m. to 7 p.m. The number of animals used were from two to five at each stage. Eye balls were enucleated immediately after decapitation, and were dissected into anterior and posterior halves with razor blades. The posterior halves were fixed overnight at 4’C by immersing in a fixative containing 4% paraformaldehyde, 0.5% glutaraldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.3) with 20% sucrose. Then 20 pm sections were cut with a freezing microtome and were washed twice with a solution
Copyright
o 1983 ANKHO
International
Dopaminergic
Retina
containing 4% paraformaidehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) with 20% sucrose to remove picric acid. Without washing, these sections were mounted on a glass slide, dried by warm air at 45°C to 50°C for 20 min and cover-slipped with paraffin oil. Finally the specimens were examined under a fluorescent microscope equipped with an epi-illumination system (Olympus BR-RFL). The filters used were BG-3/1F-405 for emission and Y-475 for absorption. RESULTS
During the development of the chick retina, fluorescent DA cells were first detected on the 13th day of incubation. They were always located in positions two or three cell rows externally from the junction between the inner plexiform layer (IPL) and inner nuclear layer (INL) (Fig. 1). These fluorescent cells were situated obliquely among nonfluorescent amacrine ceils, and had oblong somata. Their processes, often single with each neuron, extended toward the IPL but were yet within the INL. On the 14th day, DA cells were found in the innermost row of the INL, immediately adjacent to the IPL (Fig. 2). Such a cell had somewhat round soma whose processes, usually one or two, were oriented horizontally in the IPL. On the 15th day of incubation, DA ceils displayed a similar cytological feature and intensity of fluorescence to those found in the retina of the 14th day (Fig. 3). In rare instances, these DA cells were also found in layers two or three cell rows apart from the junction between the IPL and INL. On the 17th day, DA cells appeared to increase in both the intensity of fluorescence and size of perikaryon (Fig. 4). In addition, their cellular processes extended not only bilaterally along the IPL-INL junction but also vertically into the
Inc .-0361-9230/83/010097-06$03.00/O
98
IPL, such a feature was more prominent on the 19th day. The latter processes formed both the inner (Iarn~~a 5) and middle fiber layer (lamina 3) in the IPL (Fig. 5). At this stage. a number of fluorescent dots, probably of cross-sectioned fragments of DA fibers, were frequently seen in the outer fiber layer (I~mina I) (Fig. SC and D). In chicks immediately after hatching, a number of owth cone-like fluorescent structures with fine spikes we found at the IPL-INL junction (Fig. 6). These st~ctur~s were app~ently o~~inated from DA cell bodies situated in the vicinity by a connection with line DA fibers. The appearance of DA cell somata was essentially identical with that of IY-day retina. The number of DA cells seemed to be nearly const~t during the retinal development, at least after the 13th day of incubation. On the 4th day after hatching, the outer fiber layer (lamina I) at the IPL-INL junction showed a well developed tiber network, and here DA cells extended their processes with many branches to the outer or middle fiber layers (Fig. 7A). These DA neurons appeared to be multist~t~ed amacrine cells as described by Ramon y Cajal [20]. Such belching processes climbing towards the ganglion cell layer often showed a spiral con~~ratjon (Fig. 7 B-D). In ement with reports of Ehinger [7], the density of DA fibers in the outer fiber layer was much higher than that in the inner and middle layer. These events are summarized in an illustration (Fig. 8).
Using our improved method for catecholamine fluorescent histochemistry, we were able to describe in detail the morphological alterations of dopaminergic amacrine cells of the chick retina during development . The onset of dopamine synthesis was observed on the 13th day of i~cuba~~n, one day earlier than that reported previously by Hauschild and Laties [ 161.It is notewo~hy that the fn-stly detects DA cells in the present study occurred two or three cell rows deep in the INL, not in the first row as they reported. This phenomenon appeared to simply depend on sensitivities for detecting DA with the two different techniques. Indeed, in our materials on the following 14th day almost all DA cells were arranged immediately adjacent to the IPL. This fact clearly indicates that a ce~~n type of amacrine cells have synthesized catecholamine. most probably dopamine. before they migrate to attain their final position. This observation is incidentally consistent with a report by Hashimoto cv ul. [IS]. who desc~b~ the differentjation of central ~at~hol~min~containing neurons of the rat embryo.
ARAKI.
MAEDA
AND KIMUKA
The present observation proves that the differ~nt~~ti~~ of DA fibers occurs after hatchin The ~urner~~usfluorescent growth cones with fine spikes und at hatching may imply that DA fibers are still growing and elongating after hatching to form a fiber network which distributes the whole retina a\ seen on the 4th ~s~n~~~ day. The ~~rni~cati~~nof d~ndriti~ processes was also much more pr~~min~nt in the 4th day retina than in the retina of newly hatched chicks. It is interesting that such processes often ~xbibited spiral fe~t~r~s. although the significance of the configuration is unknown There is an interest argument with the present ftnding. Based on developmental studies of synapse formation in the IPL [5,22], it has been shown that the density of s~~~ps~s in the IPL at hatching was almost the ~~~n~e as found in tbe adult chicken retina. Therefore, they concluded that the fiber mat. uration has mostly completed at the time of hatchi is the case, the reason why we could detect the density of DA fibers on the 4th day may be partially expl~ined as follows: dopamine has been synthesized :n the ~~karyon at hatchi~, but is not yet tr~nspot~ed to the distal portion of fibers. and additional four days are r~qui~d for the tr~ns~~~a~on. In addition a lj~~t condition may play some roles in the fiber maturation of DA cells in the chick retina. Nichols r*t rrl. [ 191reported that intensity of dopamine fluorescence increa in the light-adapted retinae of the rabbit and guinea p n corn ‘son with the dark-adapted invest ~~)ndition. Da Prada ed the effect of Ii&t on DA synthesis in retina of several specks and showed that DA synthesis was enhanced consider~bi~ by light, while it ~a\ inhibited in the dark. It is likely. t~~r~for~. that an accelera tion of DA synthesis by light after hatching may contribute to visualization of distal fibers of DA ceil processes. Despite of the morphological h~rnoio~~. amacrine cells of the avian retina can be regarded as ;I beterogen~~~l~popul~tion in the sense of their content of different biological active sub~t~ces. Hisfochemicall~. they are known tc) contain acetylcholine 121,y”aminobuty~c acid [ 181.somat~statin [3]. substance P [ 171, glucagon (211, serotonin 11I]. and acetvlcholinesterase [I]. The position of the amacrine perikaryon in the INL and the processes in lamina~ of the IPI. seems 10 be ch~acter;stic to the substances it cont~ns 121j. For example, as also noted in our rest&. d~~erenti~t~d DA cell bodies always exist in the innerm~)st row of the INL and form a dense network in lamina I and less extensive onec in laminae 3 and S [23]. Such a specific relation between the topography and bioactive constituent ofamacrine cells would possibly have another correlation with the pa~icular pattern of synapse fo~~tion~
FACING PAGE FIG. 1. Retina from 13&y embryo. Fluorescent cell bodies are found two or three ceU rows externally from the Inner plexiform layer (~PL~-inner nuclear layer (INL) junction. Each soma issue a sin& short process towards the IPL. Retina from 14-dav embryo d INL. One or two pries $1 extend ~~zo~la~i~ m the The fluorescent cell bodies are l~~~zed at the bound jes and processes ~~owhe~ds) is almost the same IPL. (x%0) FIG. 3, Retina from ~~d~y embryo. The ce of the ~u~~sceot celf as seen in the 14-day retina. (xS81))FIG. 4. Retina ham I?-day embryo. The intensity of ffuorese~nce seems to incrust. In addition to lateral fibers in the ~nermost layer of the IPL (indicated by ~Kowbead in Fig. 4A and double avower in Fig. 4C1, Rv~~t~c~~Rber towards the inner fiber layer occurs. In Fig. 4C, a fine fluorescent lateral fiber in the inner fiber layer is seen (arrowhead). t x 580~
DOPAMINERGIC
CELL IN RETINA
99
100
AKAM,
MAEDA
AND KIMUKA
DOPAMINERGIC
CELL IN RETINA
,
101
INL
OPL
FIG. 8. Summary of the DA cell differentiation in the developing chick retina. Fluorescence is first detected on the 13th day in the cells located two or three cell rows externally in the INL. On the 14th day, such cells migrate to the first row of the INL, where they first issue fibers horizontally. At 17th day, the intensity of fluorescence increases. Vertical processes are more apparent, which forms the outer and middle fiber layer in the subsequent development. After hatching, vertical processes make several branches. and forms many spiral processes. The outer fiber layer shows much denser fluorescence than the inner and middle fiber layers. NF: nerve fiber layer, G: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer. FACING PAGE FIG. 5. Retina from 1Pday embryo. Processes extending vertically into the IPL formed both the inner and middle fiber layer. Fluorescent lateral fiber layers (Fig. 5C) are more prominent than the retina of 17-day embryo. In Fig. 5D, the fluorescent cell body issues both a meandering vertical fiber and a lateral fiber in the outer fiber layer. (SA-C x580, SD x 1452) FIG. 6. Retina from newly hatched chick. At the IPL-INL junction growth cone-like structures with fine spikes are frequently observed (Fig. 6A and 6B). A number of fibers extend laterally to the outer fiber layer at the proximal segment of the soma (Fig. 6C). f x 1452) FIG. 7. Retina from 4-day chick. In Fig. 7A, three fluorescent fiber layers are indicated by arrowheads. The density of DA fibers in the outer fiber layer is much higher than that in the inner and middle layer. In Fig. 7B, a thick vertical process extends in the IPL and branches into two spiral processes. Figs. 7C and 7D show the same cell at different focuses. The cell body seems to issue at least four spiral processes. (7A x288, 7B-D x580)
1. Araki, M., C. Ide and T. Saito. Ultrastructural localization of acetylcholinesterase activity in the developing chick retina. Acta Histochem Cytochrm 15: 242-255, 1982. 2. Baughman, R. W. and C. R. Bader. Biochemical characterization and cellular localization of the cholinergic system in the chicken retina. Bruin Res 138: 469-485, 1977. 3. Buckerfield, M., J. Oliber and I. W. Chubb. Somatostatin-like immunoreactivity in amacrine cells of the chicken retina. Neurnscience 6: 689-695, 1981. 4. Da Prada, M. Dopamine content and synthesis in retina and N. Accumbens Septi: Pharmacological and light-induced modification. In: Advances in Biochemical Psychopharmacology, vol. 16, edited by E. Costa and G. L. Gessa. New York: Raven Press, 1977. 5. Daniels, M. P. and Z. Vogel. Localization of cu-Bungarotoxin binding sites in synapses of the developing chick retina. Bruin Res 201: 45-56, 1980. 6. Ehinger, B. Adrenergic retinal neurons. Z Zellforsch 71: 146 152. 1966. 7. Ehinger, B. Adrenergic nerves in the avian eye and ciliary ganglion. Z Zeilfnrsch 82: 577-588, 1967.
8. Ehinger, B. Biogenic monoamines as transmitters in the retina. In: Transmitters in the Visaal Process, edited by S. L. Bonting. Oxford, NY: Pergamon Press, 1976, pp. 145-163. 9. Ehinger, B. and I. Flor&r. Indoleamine-accumulating neurons in the retina of rabbit, cat and goldfish. Ceil Tissue Res 175: 37-48, 1976. IO. Falck, B., N.-A. Hiharp. G. Thieme and A. Tarp. Fluorescence of catecholamines and related compounds condensed with formaldehyde. J Histochem Cytochem 10: 348-354, 1962. Il. Floren, I. Indoleamine accumulating neurons in the retina of chicken and pigeon. A comparison with dopaminergic neurons. Acta Ophthalmol (Copenh) 57: 198-210, 1979. 12. Fumess, J. B., M. Costa and A. L. Wilson. Water stable ~uorophores, produced by reaction with aldehyde solutions, for the histochemical localization of catechol and indolethylamines. Histochemistry 52: 159-170, 1977. 13. Haggendal, J. and T. Malmfors. Evidence of dopaminecontaining neurons in the retina of rabbits. Acta Physiol Stand 59: 295-296, 1963. 14. H@endal, J. and T. Malmfors. Identification and cellular localization of the catecholamines in the retina and the choroid of the rabbit. Acta Physiol Scund 64: 58-66, 1965.
ARAKI, M&DA
IO2
15. Hashimoto,
H., H. Kimura and T. Maeda. Histofluorescent method for visualization of central monoamine neurons in preand neo-natal rat by glyoxylic acid. Acta Histochem Cytochem lo: 140, 1976. 16. Hauschild, D. and A. M. Laties. An indoleamine-containing cell in chick retina. Invest Ophthalmol 12: 537-540, 1973. 17. Karten, H. J. and N. Brecha. Localization of substance P immunoreactivity in amacriw cells of the retina. Narrrrr 283: 87-88,
1980.
18. Marshall, J. and M. J. Voaden. An autoradiographic study of the cells accumulating (3H)-y-aminobutyric acid in the isolated retinas of oiaeons and chickens. Invest ODhtha/mol 13: 602-607. 1974.
. -
AND KINURA
19. Nichols, C. W., D. Tocobowitz
20. 21.
22.
and M. Hottenstein. The influence of light and dark on the catecholamine cantent of the retina and choroid. Invest Ophthalmot 6: 642-646, 1967. Ramon y Cajal, S. Histologir du SyctEme Nervcux de I’Hommo ct des VertPbr&s. Paris: A. Maloine, 1911. Tonqvist, K., I. Lo&n, R. H&anson and F. Sundter. Peptidecontaining neurons in the chicken retina. Exp Eyc~Res 33: 55-64, 1981. Vogel, Z. and M. Nirenberg. Localization of acetylchotine receptors during synaptogenesis in retina. Proc Nat! Aiad Sci USA
23.
73: 1806-1810,
1976.
Wassenaar, J. S. The neuronal dopaminergic system of the retina. in: The NeurobiokogJ of Dopamine. edited by A. S. Horn, J. Korf and B. H. t. Westerink. New York: Academic Press. 1979.
Bufietin.
Vol. 10, pp. 97-102. 1983. Printed in the U.S.A
Dopaminergic Cell D~e~~ntiation in the Developing Chick Retina MASASUKE
ARAKI,
TOSHIHIRO
MAEDA”
AND HIROSHI
KI~URA*
Department of Anatomy, Jichi Medical School, Tochigi 329-04 Japan and *Department of Anatomy, Shiga University of Medical Science Qtsu, Shiga 520-21 Japan Received
8 September
1982
ARAKI, M., T. MAEDA AND H. KIMURA. Dopaminergic cell differentiation in developing chick retina. BRAIN RES BULL lo(l) 97-102, 1983.-The distribution and morphology of dopaminergic (DA) neurons in the chick retina was studied in the course of development. Fluorescent DA cells were first detected on the 13th day of incubation. They were always found in positions two or three cell rows externally from the junction between the inner plexiform layer (IPL) and inner nuclear layer (INL). On the 14th day, DA cells were found in the innermost row of the INL. Subsequently their processes extended not only bilaterally along the IPL-INL junction but also vertically into the IPL. As a result, three fiber layers were formed as laminae I, 3 and 5 in the IPL. fn the newly-hatched chick retina, a number of growth cone-like fluorescent structures with fine spikes were seen at the IPL-INL junction, indicating that DA fibers were still growing and elongating at least at hatching. On the 4th postnatal day, the ramification of dendritic processes was very prominent and they often showed a spiral configuration. Cell differentiation
Amacrine cells
Chick
Development
method established by Falck catecholamine-containing neurons have been demonstrated in the retina of various species [6-91. The catecholamine fluorescence in the retina is mostly due to dopamine (DA), and other catecholamines such as noradrenahne and adrenaline seem to play a minor role or are even absent 113,231. In the avian retina DA cells are mostly found in the innermost part of the inner nuclear layer (INL), which contains the cell bodies of the amacrine neurons whose processes extend exclusively into the inner plexiform layer (IPL) [7,16]. Such dopaminergic processes form a dense network at the outermost border of the IPL, but distribute sparsely in the middle and innermost border of the IPL. Currently we have applied a new sensitive method for catecholamine, being modified from the Faglu method [ 121to study the differentiation of DA cells in the developing chick retina aiming to clarify the process whereby these DA cells make a well developed fiber-arborization. USING
(It
ul.
the histofluorescence
[IO],
METHOD
Fertile eggs were incubated in a dark incubator at 37.5”C to 38.o”C with 90% humidity. Under this condition hatching occurred on the 20th or 21st day. After hatching, chicks were raised under a cycled light condition with a lighting schedule from 9 a.m. to 7 p.m. The number of animals used were from two to five at each stage. Eye balls were enucleated immediately after decapitation, and were dissected into anterior and posterior halves with razor blades. The posterior halves were fixed overnight at 4’C by immersing in a fixative containing 4% paraformaldehyde, 0.5% glutaraldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.3) with 20% sucrose. Then 20 pm sections were cut with a freezing microtome and were washed twice with a solution
Copyright
o 1983 ANKHO
International
Dopaminergic
Retina
containing 4% paraformaidehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) with 20% sucrose to remove picric acid. Without washing, these sections were mounted on a glass slide, dried by warm air at 45°C to 50°C for 20 min and cover-slipped with paraffin oil. Finally the specimens were examined under a fluorescent microscope equipped with an epi-illumination system (Olympus BR-RFL). The filters used were BG-3/1F-405 for emission and Y-475 for absorption. RESULTS
During the development of the chick retina, fluorescent DA cells were first detected on the 13th day of incubation. They were always located in positions two or three cell rows externally from the junction between the inner plexiform layer (IPL) and inner nuclear layer (INL) (Fig. 1). These fluorescent cells were situated obliquely among nonfluorescent amacrine ceils, and had oblong somata. Their processes, often single with each neuron, extended toward the IPL but were yet within the INL. On the 14th day, DA cells were found in the innermost row of the INL, immediately adjacent to the IPL (Fig. 2). Such a cell had somewhat round soma whose processes, usually one or two, were oriented horizontally in the IPL. On the 15th day of incubation, DA ceils displayed a similar cytological feature and intensity of fluorescence to those found in the retina of the 14th day (Fig. 3). In rare instances, these DA cells were also found in layers two or three cell rows apart from the junction between the IPL and INL. On the 17th day, DA cells appeared to increase in both the intensity of fluorescence and size of perikaryon (Fig. 4). In addition, their cellular processes extended not only bilaterally along the IPL-INL junction but also vertically into the
Inc .-0361-9230/83/010097-06$03.00/O
98
IPL, such a feature was more prominent on the 19th day. The latter processes formed both the inner (Iarn~~a 5) and middle fiber layer (lamina 3) in the IPL (Fig. 5). At this stage. a number of fluorescent dots, probably of cross-sectioned fragments of DA fibers, were frequently seen in the outer fiber layer (I~mina I) (Fig. SC and D). In chicks immediately after hatching, a number of owth cone-like fluorescent structures with fine spikes we found at the IPL-INL junction (Fig. 6). These st~ctur~s were app~ently o~~inated from DA cell bodies situated in the vicinity by a connection with line DA fibers. The appearance of DA cell somata was essentially identical with that of IY-day retina. The number of DA cells seemed to be nearly const~t during the retinal development, at least after the 13th day of incubation. On the 4th day after hatching, the outer fiber layer (lamina I) at the IPL-INL junction showed a well developed tiber network, and here DA cells extended their processes with many branches to the outer or middle fiber layers (Fig. 7A). These DA neurons appeared to be multist~t~ed amacrine cells as described by Ramon y Cajal [20]. Such belching processes climbing towards the ganglion cell layer often showed a spiral con~~ratjon (Fig. 7 B-D). In ement with reports of Ehinger [7], the density of DA fibers in the outer fiber layer was much higher than that in the inner and middle layer. These events are summarized in an illustration (Fig. 8).
Using our improved method for catecholamine fluorescent histochemistry, we were able to describe in detail the morphological alterations of dopaminergic amacrine cells of the chick retina during development . The onset of dopamine synthesis was observed on the 13th day of i~cuba~~n, one day earlier than that reported previously by Hauschild and Laties [ 161.It is notewo~hy that the fn-stly detects DA cells in the present study occurred two or three cell rows deep in the INL, not in the first row as they reported. This phenomenon appeared to simply depend on sensitivities for detecting DA with the two different techniques. Indeed, in our materials on the following 14th day almost all DA cells were arranged immediately adjacent to the IPL. This fact clearly indicates that a ce~~n type of amacrine cells have synthesized catecholamine. most probably dopamine. before they migrate to attain their final position. This observation is incidentally consistent with a report by Hashimoto cv ul. [IS]. who desc~b~ the differentjation of central ~at~hol~min~containing neurons of the rat embryo.
ARAKI.
MAEDA
AND KIMUKA
The present observation proves that the differ~nt~~ti~~ of DA fibers occurs after hatchin The ~urner~~usfluorescent growth cones with fine spikes und at hatching may imply that DA fibers are still growing and elongating after hatching to form a fiber network which distributes the whole retina a\ seen on the 4th ~s~n~~~ day. The ~~rni~cati~~nof d~ndriti~ processes was also much more pr~~min~nt in the 4th day retina than in the retina of newly hatched chicks. It is interesting that such processes often ~xbibited spiral fe~t~r~s. although the significance of the configuration is unknown There is an interest argument with the present ftnding. Based on developmental studies of synapse formation in the IPL [5,22], it has been shown that the density of s~~~ps~s in the IPL at hatching was almost the ~~~n~e as found in tbe adult chicken retina. Therefore, they concluded that the fiber mat. uration has mostly completed at the time of hatchi is the case, the reason why we could detect the density of DA fibers on the 4th day may be partially expl~ined as follows: dopamine has been synthesized :n the ~~karyon at hatchi~, but is not yet tr~nspot~ed to the distal portion of fibers. and additional four days are r~qui~d for the tr~ns~~~a~on. In addition a lj~~t condition may play some roles in the fiber maturation of DA cells in the chick retina. Nichols r*t rrl. [ 191reported that intensity of dopamine fluorescence increa in the light-adapted retinae of the rabbit and guinea p n corn ‘son with the dark-adapted invest ~~)ndition. Da Prada ed the effect of Ii&t on DA synthesis in retina of several specks and showed that DA synthesis was enhanced consider~bi~ by light, while it ~a\ inhibited in the dark. It is likely. t~~r~for~. that an accelera tion of DA synthesis by light after hatching may contribute to visualization of distal fibers of DA ceil processes. Despite of the morphological h~rnoio~~. amacrine cells of the avian retina can be regarded as ;I beterogen~~~l~popul~tion in the sense of their content of different biological active sub~t~ces. Hisfochemicall~. they are known tc) contain acetylcholine 121,y”aminobuty~c acid [ 181.somat~statin [3]. substance P [ 171, glucagon (211, serotonin 11I]. and acetvlcholinesterase [I]. The position of the amacrine perikaryon in the INL and the processes in lamina~ of the IPI. seems 10 be ch~acter;stic to the substances it cont~ns 121j. For example, as also noted in our rest&. d~~erenti~t~d DA cell bodies always exist in the innerm~)st row of the INL and form a dense network in lamina I and less extensive onec in laminae 3 and S [23]. Such a specific relation between the topography and bioactive constituent ofamacrine cells would possibly have another correlation with the pa~icular pattern of synapse fo~~tion~
FACING PAGE FIG. 1. Retina from 13&y embryo. Fluorescent cell bodies are found two or three ceU rows externally from the Inner plexiform layer (~PL~-inner nuclear layer (INL) junction. Each soma issue a sin& short process towards the IPL. Retina from 14-dav embryo d INL. One or two pries $1 extend ~~zo~la~i~ m the The fluorescent cell bodies are l~~~zed at the bound jes and processes ~~owhe~ds) is almost the same IPL. (x%0) FIG. 3, Retina from ~~d~y embryo. The ce of the ~u~~sceot celf as seen in the 14-day retina. (xS81))FIG. 4. Retina ham I?-day embryo. The intensity of ffuorese~nce seems to incrust. In addition to lateral fibers in the ~nermost layer of the IPL (indicated by ~Kowbead in Fig. 4A and double avower in Fig. 4C1, Rv~~t~c~~Rber towards the inner fiber layer occurs. In Fig. 4C, a fine fluorescent lateral fiber in the inner fiber layer is seen (arrowhead). t x 580~
DOPAMINERGIC
CELL IN RETINA
99
100
AKAM,
MAEDA
AND KIMUKA
DOPAMINERGIC
CELL IN RETINA
,
101
INL
OPL
FIG. 8. Summary of the DA cell differentiation in the developing chick retina. Fluorescence is first detected on the 13th day in the cells located two or three cell rows externally in the INL. On the 14th day, such cells migrate to the first row of the INL, where they first issue fibers horizontally. At 17th day, the intensity of fluorescence increases. Vertical processes are more apparent, which forms the outer and middle fiber layer in the subsequent development. After hatching, vertical processes make several branches. and forms many spiral processes. The outer fiber layer shows much denser fluorescence than the inner and middle fiber layers. NF: nerve fiber layer, G: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer. FACING PAGE FIG. 5. Retina from 1Pday embryo. Processes extending vertically into the IPL formed both the inner and middle fiber layer. Fluorescent lateral fiber layers (Fig. 5C) are more prominent than the retina of 17-day embryo. In Fig. 5D, the fluorescent cell body issues both a meandering vertical fiber and a lateral fiber in the outer fiber layer. (SA-C x580, SD x 1452) FIG. 6. Retina from newly hatched chick. At the IPL-INL junction growth cone-like structures with fine spikes are frequently observed (Fig. 6A and 6B). A number of fibers extend laterally to the outer fiber layer at the proximal segment of the soma (Fig. 6C). f x 1452) FIG. 7. Retina from 4-day chick. In Fig. 7A, three fluorescent fiber layers are indicated by arrowheads. The density of DA fibers in the outer fiber layer is much higher than that in the inner and middle layer. In Fig. 7B, a thick vertical process extends in the IPL and branches into two spiral processes. Figs. 7C and 7D show the same cell at different focuses. The cell body seems to issue at least four spiral processes. (7A x288, 7B-D x580)
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