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Light- and electron-microscopic investigation of the optic nerve fiber layer in the river lamprey (Lampetra fluviatilis)
LIGHT- AND ELECTRON-MICROSCOPIC INVESTIGATION OF THE OPTIC NERVE FIBER LAYER IN THE RIVER LAMPREY (LA.WE7-RA FLC b’ZATZLIS) K.u HOL~IBERG Department
of Zoology.
Universit!
of Stockholm.
Sweden
IO Januar_i~ 19’51
(Rrceiterl
Abstract-The optic nerve fibers are organized into discreet fascicles which course from the periphery of the retina to the optic disc along the kitreal margin of the inner nuclear layer. Thus. in contrast to gnathostome chordates. the optic fiber laker in lampreys is located sclerally to the inner synaptic layer. A hypothesis is put forward which suggests that the fascicle organiz;Gon is a device which enables cell processes from the inner nuclear layer to extend ~irreall~ beyond the optic fiber lajer and thus facilitate the development of the inner qnaptic layer irithout a splitting process of the inner neuroblastic layer.
KC!, I+‘orrl.s--Lajnprrra
juciarilis:
cyclostome:
opric nerve:
fiber layer
INTRODUCTION In gnathostome
chordates. the optic fiber layer is always located vitreally to the inner synaptic layer. Usually the fiber layer is seen between the layer of ordinary ganglion cells and the inner limiting membrane. but ganglion cells may also lie among the optic fibers and, hence, very close to the vitreal surface of the retina. In lampreys, which are agnath chordates. the optic fiber layer in the adult animal is located sclerally to the inner synaptic layer at the border between the inner nuclear layer and the inner synaptic layer. As a consequence of this organization, the inner synaptic layer comprises the region between the inner nuclear layer/optic fiber layer and the inner limiting membrane. This unique location of the optic fiber layer in lampreys was recognized early by several authors (e.g. Kohl, 1892; Diicker. 1924; Franz, 1932). However. in the earlier works. the retina of lampreys was, without exception, presented in transverse sections. In some illustrations. the optic fiber layer may be seen as a continuous layer along the vitreal border of the inner nuclear layer (e.g. Kohl. 1892). In other cases, either the layer is indicated with some fibers which have been traced for a few micrometers (e.g. Walls. 1942, Fig. 162a) or it is not shown at all. The optic fiber layer is readily seen in transverse sections of the retina through the optic disc, but within a few micrometers from the disc the layer can be traced only in favorable transverse sections and for relatively short distances. Further out towards the periphery of the retina, the layer is very rarely seen. These observations suggested that the possibility to show ihe optic fiber layer in a transverse section was obviously dependent on the plane of section and on the distance from the optic disc. Therefore. in the present investigation, retinas were analysed in tangential sections. MATERIAL ASD METHODS This investigation is based on nine retinas different adult river lampreys (hmperra obtained from the river Daliilven. Sweden. \.R.
IY’IO--0
from
nine
j?uciarilis)
Five eyes were disected. cornea. iris and lens removed. and the e)e cup fixed b! immersion in a solution containing 4 parts 5:” potasium bichromate. 4 parts IO”, formaldehyde and I part acetic acid (Kolmer’s solutionRomeis. 19-1s). The tissues were fixed for I2 hr at room temperature. dehydrated in graded ethanol series and embedded in paraffin. The sections were stained with Water B;J~ and Thiazin Red according to Hartwig (1967). Electron
microscop)
Four eles were disected. cornea. iris and lens removed. The retinas were fixed for I hr in a cold solution of 2:; osmium tetroxide buffered at pH 7.4-7.5 with 0.07 &l s-collidine buffer (3 retinas) (Bennett and Luft. 1959) or with 2.59; sodium bicarbonate in 0.1 N hydrochloric acid fl retina) (Wood and Luft. 1965). The retinas were dehydrated in graded ethanol series and embedded in Epon. Sections were contrasted for about 20 min with uranyl acetate in water followed by 2 min with lead citrate and examined with a Philips EM 301 electron microscope.
OBSERVATIONS
The most common appearance of the retina in transverse sections is seen in the left part of Fig. I: the inner nuclear layer adjoins directly on the inner synaptic layer. which extends to the vitreous body. and there is no sign of an optic fiber layer. In favorable transverse sections near the optic disc. part of an optic fiber bundle could occasionally be seen at the border between the inner nuclear layer and the inner synaptic layer (Fig. I). Further away from the optic disc. towards the periphery of the retina. the bundles were observed very rarely. In tangential sections. the bundles were readily seen as discreet fascicles at the inner margin of the inner nuclear layer (Fig. 2). The fascicles seem to branch occasionally. but most often they course separated from each other by cell bodies of the inner nuclear layer. Although the fascicles usually border to the neuropile of the inner synaptic layer (Fig. 2, inset). they may occasionally course partly within the inner synaptic layer close to the vitreal margin of the inner
1313
151.4
Ii4J
HOttfBERG
nuclear layer The fasncles uer2 tracrd m tangential at first contiguous uith the inner neurobiastic layer sections from the periphery of the retina to the optic The ganglion cells are subsequentI> separated from disc v.herc they mrrge into th2 optic nerv2 (Fig. 3). the inner neurobldstic layer by th2 developing inner Large cei1 bodies. vrhich clearly outrange neighsynaptic layer. The Inner portron of the margma! bourtng cells in size. were occasionally found among layer. adjacent to the vitreous hod!. wifI gradualiy the mncrmost ceil bodies of the inner nuclear layer form the optic fiber layer m the adult ammal. Thus. ~Ftg. 4) In a feu cases it was possible to see perikarya m the retma of adult gnuthostome chordates the ordiof such cells gve off processes which entered into nary ganglion cells and the optic fiber layer are neighbourmg fasciclrs (Fig. 5). These large cells have located vitrerdly to the inner synaptic layer. a voluminous perikaryon (Figs 4-6) rich m mitochonAccording to Kohl (189’) and Studnii-ka (1911). the drra and abundant granular endoplasmic reticulum optic fiber layer m I l-63 mm lampre) larvae lammouith numerous ribosomes. Clusters and beaded coetes) IS contiguous rvtth the innsr nuclear layer and strands of free ribosomes are also found in the adjathe Inner l~rn~t~ngmembrane. .&n Inner synaptic layer cent cytoplasm The frequent) of these large cells IS has apparently not developed durtng thesr stages: the fairly low. perhaps a few per cent of the total number retmal organization resembles the neuroblastic stag2 of cell bodies in the innermost cell rows of the inner in gnathostome chordates. The opnc fiber layer is nuclear layer. subsequentlv separated from the inner limiting memIn transverse sections through the retma, scattered brane by a fibrous layer (Kohl. 1893: “Granulosa inc2lf bodies were seen in the innsr synaptic layer as ternn”: Evfoiejko. 191’: “die mnere Grenzschicht”. far in as at the vitreal surface of the retina (Figs i Dbckrr. 19X: ‘-die mnere Netzfasserschicht”: Ketbel. and 4). Houever. a sparse layer of relatively small 1917. ‘*dieinnere Grenzschicht”) tihich remains conticell bodies uas found in tangential sections approxiguous uith the Inner nuclear layer Jlso during later mately I5 itrn from the vitreous surface of the retina. larval stages and in the adult animal Keibzl (1927) These cell bodies in th2 inner synaptic layer never considered this layer. ivhich apparentI> IS the inner rzach the CIZZof the large cells at the vitreal margm synaptic layer. to be a gfral cell layer. uhereas Kohl of the Inner nuclear layer. (1892) and McZqko (1912) stated that tts meantng The fasctcles were identified in i him sectrons itas unknown Thus. in contrast to the later developst‘lined tvith Toluidinr Blue (e.g. Fig. 5) and further ment m gnathostomc chordatcs. the optic fiber layer on in sections for electron microscopy (Figs 6 and in lampreys never seems to be sep;irated from the 7). The nerve fibers m a fascicle vary considerably inner nuclear layer. It is located sclerally to the inner m size. ranging approxtmately between 0.5 and 5 pm s\naptic !a)er. Houever. the observations reported by in diamrter. Their cytoplasm contains evenly distri- ‘Kohi (1892). Studnrfka (1913). ?vloiejko (1911). buted n2~Irofilaments. scattered m~tochondrla and Dticker (1931) and Kcibe! (1917) are rither based on relativeIt, few stages or not detatled enough to psrmlt smooth membranes of endoplasmic reticulum. The tibers are ensheathed by glia! cells without any mye- a conceivable explanation to the unique iocation of lination of the fibers. the optic tibrr laqer and the inner s!naptic layer. The inner synaptic layer of the adult river lamprey The entire thickness of the inner synaptic layer was IS a aell-developed and complex neuropile (Holmanalysed on montages of electron micrographs and berg. 1975). There are bipolar ason terminals with by scanning numerous sections with the electron typical ribbon synapses arranged in characteristic microscope. Special attention was given to the region adjacent to the vitreous body. i.e. the region which dyads. The post-synaptic processes at the dyads may have or may lack synaptic tesrcles. i.e. they corresponds to the optic fiber layer in gnathostome chordates. These analyses did not reveal any adare presumably amacrine cell processzs or ganglion ditional optic fiber bundles. cell dzndrites. Reciprocal synapses back onto the bipolar ceil terminals have been observed as uel! as serial synapses between amacrine cell processes. Tvvo layers of catecho!am~ne-containing fibers The optic fiber layer in the river lamprey is occur in the inner synaptic layer. A third fiber layer is found in the inner nuclear layer around the mnerorganized into discreet fascrcles which course from most perikarya. among which catecholamme and inthe periphery of the retina to the optic disc as spokes doleamine contajning neurons are located (Ehinger, in a wheel. This organization explains why the optic Holmberg and Ohman. 1977). It 1s questionable if fiber layer seldom or never is observed in transverse such a complex neuroprle would dzrelop accordmg sections. unless th2se pass near or through the optic to the gnathostome pattrm. It implies that the innrr disc, During the early development of the retina in synaptic layer develops scleratly to the ganghon cell gnathostome chordates (see. for example Arey. 1965; nuclei and the optic fiber layer. and then. at a later stage. IS relocated vttreally to the optic fiber layer. Rodieck. 1973). the mterior layer of the optic cup Instead, the fascicle organization of the optic fiber differentiates mto a primitive neuroepithelium and layer may be a device which enablrs amacrine, gangvitreally into a cell-free marginal zone. The neuroelion and especially bipolar cells to extend their propithelium gives rrse to the outer and the inner neuroblastic layers separated by the outer portion of the cesses vitreally beyond the optic fiber layer. thus facilitating the development of the Inner synaptic layer marginal layer. The cells of the outer neuroblastic wtthout a splitting process of a neuroblastic layer. layer differentiate into photoreceptor, horizontal and It should be possible to test this hypothesis by a bipolar cells, those of the inner neuroblastic layer into amacrine. glial (Miiller) and ganglion cells. Thus, the detailed examination of as many as possible conxcuganglion cells and the developing optic fiber Iayer are tive larval stages.
Fig. I. Transverse section through the retina showing part of an optic fiber bundle or fascicle (F) at the border between the inner nuclear layer (INL) and the inner synaptic layer (ISL). Scattered cell bodies (arrows) occur in the inner synaptic layer. H: horizontal cells; V: vitreous body. Scale: 20 pm. Fig. 2. Tangential section showing fascicles IF) of ceil bodies of the inner nuclear layer. H: horizontal sectioned fascicies (F) in which optic nerve fibers of presumably a giant ganglion cell: ISL:
optic nerve fibers surrounded b>- the innermost cells. Scale: 20 pm. Inset shows two transversally varying diameter are seen. G: a large cell body. inner synaptic layer. Scale: 10pm.
Fip. 3. Fasciclrs
of optic nerve fibers (F) which enter into ISL: inner synaptic la)er: V: xitrcous
Fig. 4. .J, Irtrg~ csll of the inner ntxiear rhs rrrins. tf: Fig. 5. The Ia?zr ilNL).
the obliquely hod, Scale:
sectioned Xl gm.
optic
nerve
(04).
hod! (G). presumably a giant ganglion crll. is seen among the imxrmost csfis la!er (CC Fig. 61. A cell bed\ (zr:ow) is also seen near the vitreous surface of horizontal czlls,: ISL: inner synaptic laler: V: ritrcous hod\. Scale: _W Lgrn.
*~~undecib\_ ceil boci~ of ii grinr ganglion cell (G) is seen SL..< A process 1;1rrow~ from the gan_eiiort ceil enters into a fascisls ISL: inner qnapfic ia!cr. Scale: X !im.
crlls of IFI
the Inner nuclesr of optk rw\s fiber<.
1317
Fig. 6. Electron micrograph showing transversally sectioned optic nerve fibers (OF) in a fascicle. The large cell body (G) at the upper right. corresponding to the one shown in Fig. 4 (G). is presumably a giant ganglion cell. Scale: 1 pm.
Fig. 7. Obliqusl~ sectioned optiu txrve iibcrs {OFI in ;L t’asc~cfe. Purr oi a crll body at :?c ;ttreai margin of the inner nucle:ar la\cr is seen at the upper right ol the electron micrograpk kz~lr: I
iim.
Optic
nerve fiber layer
In this connection it may be relevant to recall that a similar organization is found in the retina of the hagfishes Eptatretus burgeri. E. cirrhatus. E. stouti and Paramyxine atami (Hoimberg. 1971; Femholm and Holmberg 1975), which also are agnath chordates. In these species. there is no distinct ganglion cell layer. but the inner fibrous layer. which presumably is an inner synaptic layer. comprises the region between the inner nuclear layer and the inner limiting membrane adjacent to the vitreous body. The precise location of the ordinary ganglion cells is still uncertain. A general opinion is that they are located in the inner nuclear layer, presumably among the innermost cells of the layer (e.g. Franz. 1932; Walls. 1942; Kleerekoper, 1972). According to Diicker (1924) and Rochon-Duvingneaud (1943) the ganglion cells are situated in the inner synaptic layer, mainly within the inner half of the layer (cf. p. 1314) and adjacent to the inner limiting membrane. The large cell bodies. which in the present investigation were seen to send processes into fascicles. are undoubtedly ganglion cells. The overall low frequency of these ceil bodies indicates, however, that they comprise no more than a few per cent of the innermost cell bodies of the inner nuclear layer. The large size and the low frequency suggest that they are giant ganglion cells. Similarly located giant ganglion cells were more recently described in the retina of the smooth dogfish. Mustelus canis (Stell and Witkovsky, 1973) and of a hammerhead shark. Sphyrna lewini (Anctil and Ah. 1974). In these cases. the cells are called displaced ganglion cells. since the perikarya are located near the outer margin of the inner synaptic layer. In view of the proposed hypothesis concerning the development of the inner synaptic layer in lampreys. the giant ganglion cells in the river lamprey are rather ordinary giant ganglion cells, because their perikarya lie adjacent to the optic fiber layer and presumably among ordinary ganglion cells. Displaced amacrine and ganglion cells have been found in the inner synaptic layer in all classes of gnathostome chordates. Since catecholamine and indoleamine accumulating neurons have been identified close to the vitreous body in the adult river lamprey (Ehinger et al., 1977). it is reasonable to assume that these cell bodies are displaced amacrine cells. On the other hand, no fluorescent perikarya were found further out in the inner synaptic layer, which suggests that the cell bodies within the inner synaptic layer of the river lamprey are displaced amacrine cells with other transmitter substances and/or displaced ganglion cells (cf. p. 1314). In most gnathostome chordates. the optic fibers form into bundles which are not separated into discreet fascicles. Regardless of the plane of section. the optic fiber layer displays a homogenous. fibrous appearance. However, the occurrence of optic nerve fibers located as fascicles in the ordinary ganglion cell layer and the absence of a separate layer of optic nerve fibers was reported by Munk (1964, 1968) in Amia. Lepisosteus (order Holostei), Calamoichthys (order Brachiopterygei) and Protopterus (order Dip noi). Munk suggested that the location of the optic fibers as fascicles in the ordinary ganglion ceil layer is probably a phylogenetically old characteristic, which has also been preserved in a few deep-sea
1319
in lamprey
teleosts of the order Isospondyli (Munk, 1966). The occurrence of discreet fascicles in the river lamprey. probably in the ordinary ganglion cell layer. seems to support Munk’s suggestion. Furthermore. the unique location of the optic fiber layer in lampreys may be another fundamental difference between gnathostome and agnath chordates. Acknowledgements-This investigation was supported by grants from the Swedish Natural Science Research Council (B 2124-037. B 2935-003 004). the Harald & Greta Jeansson Foundation. and the Helge Ax : son Johnson Foundation.
REFERE\CES Anctil M. and Ali M. A. (1974) Giant ganglion cells m the retina of the hammerhead shark (Sph_rrno Irwiru). Vision Rrs. 14. 903-904. Arey L. B. (1965) Dereloprnental Anatorn.r. A Textbook and Laboratory Manual of Embryology. Saunders. Philadelphia. Bennett H. S. and Luft J. H. (1959) s-collidine as a basis for buffering fixatives. J. biophrs. . brochem. C ~rol. 6. 113-114. Diicker M. (1924) ijber die Augen der Zyklostomen. Jeno Z. Nururwiss. 60. 471-528.
Ehinger B.. Holmberg K. and Ghman P.
(1977) Aminergic and indoleamine accumulating neurons in the retina of the river lamprey (Lnmprrru j7uciurilis). .~cra soot. (Srockh.) 58. 117-123. Fernholr.: B. and Holmberg K. (1975) The eyes in three genera of hagfish (Eprarreru\. Pararn_r’trrtr and .Mysmet-a case of degenerative evolution. li>ron Rex. 15. 253-259 Franz V. (1932) Auge und Akkommodation van Pzrrorri.rrott (La/rlperru)pu~iuri/is L. Zoo/. Jb. (Zoo/.) 52. 118-I 78. Hartwig H.-G. (1967) Eine neue Methode l%r farberischen Differenzierung von Stlbchen und Zapfen. Z. antis. .L/lkrosk. 68. 235-240. Holmberg K. (1971) The hagfish retina: electron microscopic study comparmg receptor and epitheltal cells in the Pacific hagfish. Pohsrorrenla stouri. with those m the Atlantic haghsh. Myxiw gluriuosa. 2. Zrllforxch. 121. 249-269. Holmberg K. (1978) Electron microscopic analysis of the inner synaptic layer in the river lamprey (Lomperra fluciatihs). Acra :ool. (Stockh.) 59(2) (in press). Keibel F. (1927) Beitrage zur Anatomie. zur Entwicklungsgeschichte und zur Stammesgeschichte der Sehorgane der Cyklostomen. Z. mikr.-anat. forsch. 12. 39i-lS6. Kleerekoper H. (1972) The sense organs. In T/N, Bto/og_i o/ Lumpreys (edited by Hardisty H. W. and Potter I. C.). Vol. 2. pp. 373-404. Academic Press. London. Kohl C. (1892) Rudimentare Wirbelthieraugen. Zoo/ogica
(Srurrg.) 13. 48-51. Moriejko B. (19121 1st das Cyclostomenause- primitiv oder degeneriert? Anat. AK. 42; 612-620. Munk 0. (1964) The eve of Calumoichrhvs calubaricus Smith. 1865 (Polypteridae. Pisces) compared with the eye of other fishes. Vidensk. iMeddr dansk nuritrh. Form. 127. 113-126. Munk
0. (1966) Ocular anatomy
of some deep-sea teleosts.
Dana Rep. 70. l-62. 0. (1968) The eyes of Anna and Lepisosreus (Pisces. Holostei) compared with the brachiopterygian and teleostean eyes. Vidensk. &feddr dunsk nuturh. Foren. 131. 109-127. . Rochon-Duvigneaud A. (1943) Les Yeux er la ITsion des Vertebres. Masson & Cie, Paris. Munk
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KM HOLMBERG
Rodteck R. W (1973) The Verrrhrarr Rerina. Prmuples of Srrucrurr and Funcrion. pp. 338-343. Freeman. San Francisco. Romeis B. (1948) Mikroskopische Technk. \42348. Letbnitz. Munchen. Stell W: K. and Witkovsky P. (1973) Retmal structure in the smooth dogfish. Musrelus cants: general description and light microscopy of giant ganglion cells. J. camp. Yrurol. 148. l-32. Studnifka F. K. (1912) L’eber dte Entwtcklung und dte
Bedeutung der Seitenaugen von Ammocoetes. .-lnur ._(IIz. 41. 561-578. Walls G L. (1932) The trrrrhrare Eye and frs .A&prw Radiarron. pp. 559-560. Cranbrook lnstttute of Scrence. Bloomfield Hills. Repnnted as facsimile edition by Hafner Pubhshmg Co.. New York (1967). Wood R. L. and Luft J. H. (1965) The influence of buffer systems on fixation with osmium tetroxide. J Clarasrrucr. Rex. 12. 22-45.
of Zoology.
Universit!
of Stockholm.
Sweden
IO Januar_i~ 19’51
(Rrceiterl
Abstract-The optic nerve fibers are organized into discreet fascicles which course from the periphery of the retina to the optic disc along the kitreal margin of the inner nuclear layer. Thus. in contrast to gnathostome chordates. the optic fiber laker in lampreys is located sclerally to the inner synaptic layer. A hypothesis is put forward which suggests that the fascicle organiz;Gon is a device which enables cell processes from the inner nuclear layer to extend ~irreall~ beyond the optic fiber lajer and thus facilitate the development of the inner qnaptic layer irithout a splitting process of the inner neuroblastic layer.
KC!, I+‘orrl.s--Lajnprrra
juciarilis:
cyclostome:
opric nerve:
fiber layer
INTRODUCTION In gnathostome
chordates. the optic fiber layer is always located vitreally to the inner synaptic layer. Usually the fiber layer is seen between the layer of ordinary ganglion cells and the inner limiting membrane. but ganglion cells may also lie among the optic fibers and, hence, very close to the vitreal surface of the retina. In lampreys, which are agnath chordates. the optic fiber layer in the adult animal is located sclerally to the inner synaptic layer at the border between the inner nuclear layer and the inner synaptic layer. As a consequence of this organization, the inner synaptic layer comprises the region between the inner nuclear layer/optic fiber layer and the inner limiting membrane. This unique location of the optic fiber layer in lampreys was recognized early by several authors (e.g. Kohl, 1892; Diicker. 1924; Franz, 1932). However. in the earlier works. the retina of lampreys was, without exception, presented in transverse sections. In some illustrations. the optic fiber layer may be seen as a continuous layer along the vitreal border of the inner nuclear layer (e.g. Kohl. 1892). In other cases, either the layer is indicated with some fibers which have been traced for a few micrometers (e.g. Walls. 1942, Fig. 162a) or it is not shown at all. The optic fiber layer is readily seen in transverse sections of the retina through the optic disc, but within a few micrometers from the disc the layer can be traced only in favorable transverse sections and for relatively short distances. Further out towards the periphery of the retina, the layer is very rarely seen. These observations suggested that the possibility to show ihe optic fiber layer in a transverse section was obviously dependent on the plane of section and on the distance from the optic disc. Therefore. in the present investigation, retinas were analysed in tangential sections. MATERIAL ASD METHODS This investigation is based on nine retinas different adult river lampreys (hmperra obtained from the river Daliilven. Sweden. \.R.
IY’IO--0
from
nine
j?uciarilis)
Five eyes were disected. cornea. iris and lens removed. and the e)e cup fixed b! immersion in a solution containing 4 parts 5:” potasium bichromate. 4 parts IO”, formaldehyde and I part acetic acid (Kolmer’s solutionRomeis. 19-1s). The tissues were fixed for I2 hr at room temperature. dehydrated in graded ethanol series and embedded in paraffin. The sections were stained with Water B;J~ and Thiazin Red according to Hartwig (1967). Electron
microscop)
Four eles were disected. cornea. iris and lens removed. The retinas were fixed for I hr in a cold solution of 2:; osmium tetroxide buffered at pH 7.4-7.5 with 0.07 &l s-collidine buffer (3 retinas) (Bennett and Luft. 1959) or with 2.59; sodium bicarbonate in 0.1 N hydrochloric acid fl retina) (Wood and Luft. 1965). The retinas were dehydrated in graded ethanol series and embedded in Epon. Sections were contrasted for about 20 min with uranyl acetate in water followed by 2 min with lead citrate and examined with a Philips EM 301 electron microscope.
OBSERVATIONS
The most common appearance of the retina in transverse sections is seen in the left part of Fig. I: the inner nuclear layer adjoins directly on the inner synaptic layer. which extends to the vitreous body. and there is no sign of an optic fiber layer. In favorable transverse sections near the optic disc. part of an optic fiber bundle could occasionally be seen at the border between the inner nuclear layer and the inner synaptic layer (Fig. I). Further away from the optic disc. towards the periphery of the retina. the bundles were observed very rarely. In tangential sections. the bundles were readily seen as discreet fascicles at the inner margin of the inner nuclear layer (Fig. 2). The fascicles seem to branch occasionally. but most often they course separated from each other by cell bodies of the inner nuclear layer. Although the fascicles usually border to the neuropile of the inner synaptic layer (Fig. 2, inset). they may occasionally course partly within the inner synaptic layer close to the vitreal margin of the inner
1313
151.4
Ii4J
HOttfBERG
nuclear layer The fasncles uer2 tracrd m tangential at first contiguous uith the inner neurobiastic layer sections from the periphery of the retina to the optic The ganglion cells are subsequentI> separated from disc v.herc they mrrge into th2 optic nerv2 (Fig. 3). the inner neurobldstic layer by th2 developing inner Large cei1 bodies. vrhich clearly outrange neighsynaptic layer. The Inner portron of the margma! bourtng cells in size. were occasionally found among layer. adjacent to the vitreous hod!. wifI gradualiy the mncrmost ceil bodies of the inner nuclear layer form the optic fiber layer m the adult ammal. Thus. ~Ftg. 4) In a feu cases it was possible to see perikarya m the retma of adult gnuthostome chordates the ordiof such cells gve off processes which entered into nary ganglion cells and the optic fiber layer are neighbourmg fasciclrs (Fig. 5). These large cells have located vitrerdly to the inner synaptic layer. a voluminous perikaryon (Figs 4-6) rich m mitochonAccording to Kohl (189’) and Studnii-ka (1911). the drra and abundant granular endoplasmic reticulum optic fiber layer m I l-63 mm lampre) larvae lammouith numerous ribosomes. Clusters and beaded coetes) IS contiguous rvtth the innsr nuclear layer and strands of free ribosomes are also found in the adjathe Inner l~rn~t~ngmembrane. .&n Inner synaptic layer cent cytoplasm The frequent) of these large cells IS has apparently not developed durtng thesr stages: the fairly low. perhaps a few per cent of the total number retmal organization resembles the neuroblastic stag2 of cell bodies in the innermost cell rows of the inner in gnathostome chordates. The opnc fiber layer is nuclear layer. subsequentlv separated from the inner limiting memIn transverse sections through the retma, scattered brane by a fibrous layer (Kohl. 1893: “Granulosa inc2lf bodies were seen in the innsr synaptic layer as ternn”: Evfoiejko. 191’: “die mnere Grenzschicht”. far in as at the vitreal surface of the retina (Figs i Dbckrr. 19X: ‘-die mnere Netzfasserschicht”: Ketbel. and 4). Houever. a sparse layer of relatively small 1917. ‘*dieinnere Grenzschicht”) tihich remains conticell bodies uas found in tangential sections approxiguous uith the Inner nuclear layer Jlso during later mately I5 itrn from the vitreous surface of the retina. larval stages and in the adult animal Keibzl (1927) These cell bodies in th2 inner synaptic layer never considered this layer. ivhich apparentI> IS the inner rzach the CIZZof the large cells at the vitreal margm synaptic layer. to be a gfral cell layer. uhereas Kohl of the Inner nuclear layer. (1892) and McZqko (1912) stated that tts meantng The fasctcles were identified in i him sectrons itas unknown Thus. in contrast to the later developst‘lined tvith Toluidinr Blue (e.g. Fig. 5) and further ment m gnathostomc chordatcs. the optic fiber layer on in sections for electron microscopy (Figs 6 and in lampreys never seems to be sep;irated from the 7). The nerve fibers m a fascicle vary considerably inner nuclear layer. It is located sclerally to the inner m size. ranging approxtmately between 0.5 and 5 pm s\naptic !a)er. Houever. the observations reported by in diamrter. Their cytoplasm contains evenly distri- ‘Kohi (1892). Studnrfka (1913). ?vloiejko (1911). buted n2~Irofilaments. scattered m~tochondrla and Dticker (1931) and Kcibe! (1917) are rither based on relativeIt, few stages or not detatled enough to psrmlt smooth membranes of endoplasmic reticulum. The tibers are ensheathed by glia! cells without any mye- a conceivable explanation to the unique iocation of lination of the fibers. the optic tibrr laqer and the inner s!naptic layer. The inner synaptic layer of the adult river lamprey The entire thickness of the inner synaptic layer was IS a aell-developed and complex neuropile (Holmanalysed on montages of electron micrographs and berg. 1975). There are bipolar ason terminals with by scanning numerous sections with the electron typical ribbon synapses arranged in characteristic microscope. Special attention was given to the region adjacent to the vitreous body. i.e. the region which dyads. The post-synaptic processes at the dyads may have or may lack synaptic tesrcles. i.e. they corresponds to the optic fiber layer in gnathostome chordates. These analyses did not reveal any adare presumably amacrine cell processzs or ganglion ditional optic fiber bundles. cell dzndrites. Reciprocal synapses back onto the bipolar ceil terminals have been observed as uel! as serial synapses between amacrine cell processes. Tvvo layers of catecho!am~ne-containing fibers The optic fiber layer in the river lamprey is occur in the inner synaptic layer. A third fiber layer is found in the inner nuclear layer around the mnerorganized into discreet fascrcles which course from most perikarya. among which catecholamme and inthe periphery of the retina to the optic disc as spokes doleamine contajning neurons are located (Ehinger, in a wheel. This organization explains why the optic Holmberg and Ohman. 1977). It 1s questionable if fiber layer seldom or never is observed in transverse such a complex neuroprle would dzrelop accordmg sections. unless th2se pass near or through the optic to the gnathostome pattrm. It implies that the innrr disc, During the early development of the retina in synaptic layer develops scleratly to the ganghon cell gnathostome chordates (see. for example Arey. 1965; nuclei and the optic fiber layer. and then. at a later stage. IS relocated vttreally to the optic fiber layer. Rodieck. 1973). the mterior layer of the optic cup Instead, the fascicle organization of the optic fiber differentiates mto a primitive neuroepithelium and layer may be a device which enablrs amacrine, gangvitreally into a cell-free marginal zone. The neuroelion and especially bipolar cells to extend their propithelium gives rrse to the outer and the inner neuroblastic layers separated by the outer portion of the cesses vitreally beyond the optic fiber layer. thus facilitating the development of the Inner synaptic layer marginal layer. The cells of the outer neuroblastic wtthout a splitting process of a neuroblastic layer. layer differentiate into photoreceptor, horizontal and It should be possible to test this hypothesis by a bipolar cells, those of the inner neuroblastic layer into amacrine. glial (Miiller) and ganglion cells. Thus, the detailed examination of as many as possible conxcuganglion cells and the developing optic fiber Iayer are tive larval stages.
Fig. I. Transverse section through the retina showing part of an optic fiber bundle or fascicle (F) at the border between the inner nuclear layer (INL) and the inner synaptic layer (ISL). Scattered cell bodies (arrows) occur in the inner synaptic layer. H: horizontal cells; V: vitreous body. Scale: 20 pm. Fig. 2. Tangential section showing fascicles IF) of ceil bodies of the inner nuclear layer. H: horizontal sectioned fascicies (F) in which optic nerve fibers of presumably a giant ganglion cell: ISL:
optic nerve fibers surrounded b>- the innermost cells. Scale: 20 pm. Inset shows two transversally varying diameter are seen. G: a large cell body. inner synaptic layer. Scale: 10pm.
Fip. 3. Fasciclrs
of optic nerve fibers (F) which enter into ISL: inner synaptic la)er: V: xitrcous
Fig. 4. .J, Irtrg~ csll of the inner ntxiear rhs rrrins. tf: Fig. 5. The Ia?zr ilNL).
the obliquely hod, Scale:
sectioned Xl gm.
optic
nerve
(04).
hod! (G). presumably a giant ganglion crll. is seen among the imxrmost csfis la!er (CC Fig. 61. A cell bed\ (zr:ow) is also seen near the vitreous surface of horizontal czlls,: ISL: inner synaptic laler: V: ritrcous hod\. Scale: _W Lgrn.
*~~undecib\_ ceil boci~ of ii grinr ganglion cell (G) is seen SL..< A process 1;1rrow~ from the gan_eiiort ceil enters into a fascisls ISL: inner qnapfic ia!cr. Scale: X !im.
crlls of IFI
the Inner nuclesr of optk rw\s fiber<.
1317
Fig. 6. Electron micrograph showing transversally sectioned optic nerve fibers (OF) in a fascicle. The large cell body (G) at the upper right. corresponding to the one shown in Fig. 4 (G). is presumably a giant ganglion cell. Scale: 1 pm.
Fig. 7. Obliqusl~ sectioned optiu txrve iibcrs {OFI in ;L t’asc~cfe. Purr oi a crll body at :?c ;ttreai margin of the inner nucle:ar la\cr is seen at the upper right ol the electron micrograpk kz~lr: I
iim.
Optic
nerve fiber layer
In this connection it may be relevant to recall that a similar organization is found in the retina of the hagfishes Eptatretus burgeri. E. cirrhatus. E. stouti and Paramyxine atami (Hoimberg. 1971; Femholm and Holmberg 1975), which also are agnath chordates. In these species. there is no distinct ganglion cell layer. but the inner fibrous layer. which presumably is an inner synaptic layer. comprises the region between the inner nuclear layer and the inner limiting membrane adjacent to the vitreous body. The precise location of the ordinary ganglion cells is still uncertain. A general opinion is that they are located in the inner nuclear layer, presumably among the innermost cells of the layer (e.g. Franz. 1932; Walls. 1942; Kleerekoper, 1972). According to Diicker (1924) and Rochon-Duvingneaud (1943) the ganglion cells are situated in the inner synaptic layer, mainly within the inner half of the layer (cf. p. 1314) and adjacent to the inner limiting membrane. The large cell bodies. which in the present investigation were seen to send processes into fascicles. are undoubtedly ganglion cells. The overall low frequency of these ceil bodies indicates, however, that they comprise no more than a few per cent of the innermost cell bodies of the inner nuclear layer. The large size and the low frequency suggest that they are giant ganglion cells. Similarly located giant ganglion cells were more recently described in the retina of the smooth dogfish. Mustelus canis (Stell and Witkovsky, 1973) and of a hammerhead shark. Sphyrna lewini (Anctil and Ah. 1974). In these cases. the cells are called displaced ganglion cells. since the perikarya are located near the outer margin of the inner synaptic layer. In view of the proposed hypothesis concerning the development of the inner synaptic layer in lampreys. the giant ganglion cells in the river lamprey are rather ordinary giant ganglion cells, because their perikarya lie adjacent to the optic fiber layer and presumably among ordinary ganglion cells. Displaced amacrine and ganglion cells have been found in the inner synaptic layer in all classes of gnathostome chordates. Since catecholamine and indoleamine accumulating neurons have been identified close to the vitreous body in the adult river lamprey (Ehinger et al., 1977). it is reasonable to assume that these cell bodies are displaced amacrine cells. On the other hand, no fluorescent perikarya were found further out in the inner synaptic layer, which suggests that the cell bodies within the inner synaptic layer of the river lamprey are displaced amacrine cells with other transmitter substances and/or displaced ganglion cells (cf. p. 1314). In most gnathostome chordates. the optic fibers form into bundles which are not separated into discreet fascicles. Regardless of the plane of section. the optic fiber layer displays a homogenous. fibrous appearance. However, the occurrence of optic nerve fibers located as fascicles in the ordinary ganglion cell layer and the absence of a separate layer of optic nerve fibers was reported by Munk (1964, 1968) in Amia. Lepisosteus (order Holostei), Calamoichthys (order Brachiopterygei) and Protopterus (order Dip noi). Munk suggested that the location of the optic fibers as fascicles in the ordinary ganglion ceil layer is probably a phylogenetically old characteristic, which has also been preserved in a few deep-sea
1319
in lamprey
teleosts of the order Isospondyli (Munk, 1966). The occurrence of discreet fascicles in the river lamprey. probably in the ordinary ganglion cell layer. seems to support Munk’s suggestion. Furthermore. the unique location of the optic fiber layer in lampreys may be another fundamental difference between gnathostome and agnath chordates. Acknowledgements-This investigation was supported by grants from the Swedish Natural Science Research Council (B 2124-037. B 2935-003 004). the Harald & Greta Jeansson Foundation. and the Helge Ax : son Johnson Foundation.
REFERE\CES Anctil M. and Ali M. A. (1974) Giant ganglion cells m the retina of the hammerhead shark (Sph_rrno Irwiru). Vision Rrs. 14. 903-904. Arey L. B. (1965) Dereloprnental Anatorn.r. A Textbook and Laboratory Manual of Embryology. Saunders. Philadelphia. Bennett H. S. and Luft J. H. (1959) s-collidine as a basis for buffering fixatives. J. biophrs. . brochem. C ~rol. 6. 113-114. Diicker M. (1924) ijber die Augen der Zyklostomen. Jeno Z. Nururwiss. 60. 471-528.
Ehinger B.. Holmberg K. and Ghman P.
(1977) Aminergic and indoleamine accumulating neurons in the retina of the river lamprey (Lnmprrru j7uciurilis). .~cra soot. (Srockh.) 58. 117-123. Fernholr.: B. and Holmberg K. (1975) The eyes in three genera of hagfish (Eprarreru\. Pararn_r’trrtr and .Mysmet-a case of degenerative evolution. li>ron Rex. 15. 253-259 Franz V. (1932) Auge und Akkommodation van Pzrrorri.rrott (La/rlperru)pu~iuri/is L. Zoo/. Jb. (Zoo/.) 52. 118-I 78. Hartwig H.-G. (1967) Eine neue Methode l%r farberischen Differenzierung von Stlbchen und Zapfen. Z. antis. .L/lkrosk. 68. 235-240. Holmberg K. (1971) The hagfish retina: electron microscopic study comparmg receptor and epitheltal cells in the Pacific hagfish. Pohsrorrenla stouri. with those m the Atlantic haghsh. Myxiw gluriuosa. 2. Zrllforxch. 121. 249-269. Holmberg K. (1978) Electron microscopic analysis of the inner synaptic layer in the river lamprey (Lomperra fluciatihs). Acra :ool. (Stockh.) 59(2) (in press). Keibel F. (1927) Beitrage zur Anatomie. zur Entwicklungsgeschichte und zur Stammesgeschichte der Sehorgane der Cyklostomen. Z. mikr.-anat. forsch. 12. 39i-lS6. Kleerekoper H. (1972) The sense organs. In T/N, Bto/og_i o/ Lumpreys (edited by Hardisty H. W. and Potter I. C.). Vol. 2. pp. 373-404. Academic Press. London. Kohl C. (1892) Rudimentare Wirbelthieraugen. Zoo/ogica
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0. (1966) Ocular anatomy
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Dana Rep. 70. l-62. 0. (1968) The eyes of Anna and Lepisosreus (Pisces. Holostei) compared with the brachiopterygian and teleostean eyes. Vidensk. &feddr dunsk nuturh. Foren. 131. 109-127. . Rochon-Duvigneaud A. (1943) Les Yeux er la ITsion des Vertebres. Masson & Cie, Paris. Munk
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KM HOLMBERG
Rodteck R. W (1973) The Verrrhrarr Rerina. Prmuples of Srrucrurr and Funcrion. pp. 338-343. Freeman. San Francisco. Romeis B. (1948) Mikroskopische Technk. \42348. Letbnitz. Munchen. Stell W: K. and Witkovsky P. (1973) Retmal structure in the smooth dogfish. Musrelus cants: general description and light microscopy of giant ganglion cells. J. camp. Yrurol. 148. l-32. Studnifka F. K. (1912) L’eber dte Entwtcklung und dte
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