Displaced ganglion cells in the chick retina

Neuroscience Research, 6 (1989) 329-339

329

Elsevier Scientific Publishers Ireland Ltd. NSR 00273

Displaced ganglion cells in the chick retina Francisco A. Prada 1, Carola E. Chmielewski 1, Manuel E. D o r a d o 1, Carmen Prada 2 and Jos6 Maria G6nis-G~lvez 1 I lnstituto de Biologia del Desarrollo, Facultad de Medicina, Universidad de Sevilla and 2 Departamento de Fisiologia, Facultad de Medicina, Unioersidad Complutense, Madrid (Spain)

(Received 25 May 1988; Revised version received 6 October, 1988; Accepted 14 November 1988) Key words: Displaced ganglion cells; Silver methods; Electron microscopy; Topographical distribution; Retina;

Chick

SUMMARY New morphological and cytological data on the displaced ganglion cells (DGCs) in the chick retina are presented. Analysis of the topographic distribution, cellular number, dendritic field, perikaryon size and ultrastructural characteristics are included. The DGCs were found predominantly in the peripheral retina. The sizes of the DGCs, 18-42 #m, observed either by Normarsky's interferential contrast or by silver impregnation techniques, spanned the size range of the other retinal neurons. The results support the hypothesis that DGCs, in the chick retina, may constitute a specific morphofunctional system, and therefore they might not be considered as neurons that fail to attain the normal location of ganglion cells during the developmental process of migration.

INTRODUCTION

The early morphological description of the displaced ganglion cells (DGCs) by Dogiel 6 was enlarged mainly by Ram6n y Cajal 22 and Polyak 20. In the avian retina, the bodies of these cells are located along the inner border of the inner nuclear layer (INL). T h e a x o n traverses the i n n e r p l e x i f o r m layer ( I P L ) a n d the g a n g l i o n cell l a y e r ( G C L ) going into the o p t i c nerve fiber layer ( O N F L ) . T h e i r d e n d r i t e s h a v e b e e n s h o w n to b r a n c h o u t within the first s t r a t a o f the IPL. In the m a m m a l i a n r e t i n a the d i s t r i b u t i o n a n d p r o j e c t i o n s of the D G C s v a r y c o n s i d e r a b l y with the species. T w o m o r p h o l o g i c a l t y p e s o f D G C s , of s m a l l a n d large cellular b o d i e s are a c k n o w l e d g e d 3,4,9,10,25. H o w e v e r , in the a v i a n r e t i n a there are s o m e c o n t r o v e r sies. W h i l e K a r t e n et al. 17 c o n s i d e r that there is o n l y o n e p o p u l a t i o n o f D G C s p r o j e c t i n g to the b a s a l o p t i c r o o t nucleus" ( B O R n ) in the c o n t r a l a t e r a l h e m i s p h e r e , G r a n d a l et al. 13 a n d H e a t o n et al. 14 suggest the existence of a n o t h e r p o p u l a t i o n of s m a l l D G C s p r o j e c t i n g to the tectum. T h e u l t r a s t r u c t u r e o f the D G C s is n o t well k n o w n . Nevertheless, D o w l i n g a n d B o y c o t t 8 a n d D o w l i n g 7 r e p o r t u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s similar to those o f s o m e

Correspondence: Prof. Dr. Francisco A. Prada Elena, Instituto de Biologia del Desarrollo, Facultad de Medicina, Avda. Sanchez Pizjuan no. 4, 41009-Sevilla, Spain.

0168-0102/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.

330 types of amacrine and ganglion cells in amphibian and mammalian retinas. Hogan et al. 16 also describe perikaryon ultrastructural characteristics similar to those of the ganglion cells in the human eye. However, an ultrastructural study of avian DGCs is not available. In this study, the shape, distribution and ultrastructure of the DGCs in the chick retina are analyzed by means of argentic impregnation and electron microscopy techniques. MATERIALS AND METHODS

We used 70 White Leghorn chick adult retinas of 1 month after hatching. Twenty eye globes were immersed in Colonnier's solution s. The fixation time varied between 7 and 8 days. Then they were washed before staining with 0.5% silver nitrate. Another 20 eye globes were processed by the pyridine-silver method described by Goldberg and Frank 12 Some of these retinas were flat mounted. Others were serially sectioned (100/xm thick), collected in a clearing medium and mounted with dammar resin, following the procedure described elsewhere 21, in order to prevent fading of the preparations. The stained retinas were scanned, under the light microscope, checking by over- and underfocusing that all relevant cells were wholly included within the section. Microphotographs were made from the most representative fields using a Leitz Orthoplan microscope and Copex Pan film (35 mm). Ten retinas were fixed by immersion in 1% glutaraldehyde, 1% paraformaldehyde in a 0.1 M phosphate buffer, and then postfixed in a 1% osmium tetroxide solution. Pieces of retina, 1-3 mm in size, were embedded in Epon 812. Ultrathin serially sections were cut in a LKB-III ultramicrotome and stained with uranyl acetate and lead citrate. They were observed in a Jeol 100-C transmission electron microscope. The counting of DGCs was conducted in 10 retinas, fixed in 1% glutaraldehyde, 1% paraformaldehyde in a 0.1 M phosphate buffer, serially sectioned at 100 /~m thickness and observed by the interferential contrast method of Nomarsky. The stereometric analysis was made with a graphic table connected to an Apple lie computer. RESULTS

The DGCs are sparsely located in the inner border of the INL (Fig. 6, DGC). The argentic methods used in our study stained DGCs whose cellular bodies ranged in size

Fig. 1. Vertical section through the central retina showing a displaced ganglion cell stained by the Golgl method. The arrowheads indicate different levels of dendritic arborization in the inner plexiform layer. The tong arrow shows the origin of the axon from the body of the cell. x 425. Fig. 2. Displaced ganglion cell located in the central retina. The long arrow shows that the axon of this cell originates from a dendrite. The outline arrow shows the axon at the optic nerve fiber layer level x425. Fig. 3. The zone of origin of the displaced ganglion cell axon of Fig. 4. × 765. Fig. 4. Photographic m o u n t i n g of a Golgi-stained displaced ganglion cell observed in a flat m o u n t e d retina. The dendritic arborization occupies an area of 5000/~. x 765. Fig. 5. Displaced ganglion cell stained by the Goigi method. The long arrow shows the origin of the axon. x 425. Fig. 6. Vertical section through the peripheral retina showing a displaced ganglion cell observed by Nomarsky's interferential contrast technique, x 425.

331 from 18 to 42 #m in their largest diameter. Two to 4 main dendritic processes, 1.8-3.3. /~m in diameter, arise from the inner pole of the cell body and subsequently give rise to secondary and tertiary branches, forming a radiated dendritic pattern which expands in a mean area of 5000 /~m2 (Figs. 4, 8, 10, 11). Though the dendritic tree of the DGCs generally extends through the first strata of the IPL (Figs. 2, 5), we have found some cells

332

Fig. 7. Vertical section of peripheral retina showing a displaced ganglion cell stained by the pyridine-silver method (Goldberg and Frank). This cell corresponds with that labeled with an asterisk in Fig. 8. The arrowhead points to the structure with the appearance of neurofibrillar framework observed in all displaced ganglion cells: the long arrow points to the axon origin; the outline arrow indicates the arrival of the displaced ganglion cell axon at the optic nerve fiber layer, x 170. Fig. 8. The photographic mounting gives a general view of displaced ganglion cells observed in a flat mount of peripheral retina stained with the pyridine-silver method (Goldberg and Frank). The number of displaced ganglion cells stained in this area (0,20 mm 2) is 10. The cell labeled with an asterisk is shown vertically sectioned in Fig. 7. x 170.

333 which have dendrites expanding through the first 3 strata of the IPL (Fig. 1, arrow heads). The axon of the DGCs mainly arises from the inner pole of the cellular body (Figs. 1, 7, 15, long arrows), but axons emerging from the proximal portion of a dendritic trunk are sometimes observed (Figs. 2, 5, 9, 14, long arrows). They traverse the IPL and GCL and join the pathway of the ganglion cell axons in the ONFL (Figs. 2, 7, open arrows). An interesting fact is that the soma of all DGCs shows a characteristic ring structure with the appearance of a neurofibrilar framework (Figs. 7, 9, 12, 13, arrow heads) when stained with the pyridine-silver method of Goldberg and Frank 12. This neurofibrilar-like structure is located at the outer pole of the cell body (Figs. 9, 13, solid arrow), and seems to correspond to the ring of rough endoplasmic reticulum observed inside the plasma membrane at the electron microscopy level. The counting of the DGCs and their topographic distribution is shown in Figs. 20 and 21. The chick retina has a total population of 10 397 + 100 DGCs (Table I), which represents 0.43% of the total population of ganglion cells. Their distribution is not homogeneous throughout the retina (Fig. 20): 66% of the DGCs are located in the peripheral retina, 44% in the central retina. The DGC density is shown in Table I. In the peripheral retina it is 55 cells/mm 2 against 20 cells/mm 2 in the central retina. With regard to the quadrantic distribution (Fig. 21), DGCs appear to have a much higher concentration in the temporal than in the nasal retina. The plasma membrane of the DGCs appears to be surrounded by neuroglial cytoplasm (Fig. 16, M) belonging to Miiller cells, whose dark appearance is due to the content of abundant glycogen particles. The body of the DGC is characterized by a large nucleus, generally half-moon shaped, which is located in the apical pole of the perikaryon. The RER (Nissl bodies) appears in large amounts surrounding the nucleus and also concentrated inside the plasma membrane, forming a ring bordering the cell body (Fig. 16, NB, ER). Other organdies are located in the center of the ring of RER (Fig. 16, Go, Ly). The chromatin in the DGCs is homogeneous, and it is thinly dispersed throughout the karyoplasm. The nucleolus is a dense conglomeration of granules (not shown), which collectively form an irregular and large body, usually lying near the center of the nucleus, but occasionally near the nuclear envelope. Dispersed chromatin and a large granulated nucleolus are well known characteristics of very active cells, synthesizing large m o u n t s of RNA and proteins 1. This is in correlation with the large amounts of RER, Golgi apparatus and other organelles observed, as well as with the cell size. The Golgi apparatus appears as a complicated framework which is mostly located near the nucleus, in the central zone of the perikaryon. It is formed by a considerable accumulation of flattened cisterns whose 'cis' faces are towards the plasma membrane (Fig. 16, Go). The DGCs are particularly rich in lysosomes (20-30 per section). These inclusions are abundant in the

TABLE I NUMBER OF DISPLACED GANGLION CELLS (DGCs) IN THE WHOLE CHICK RETINA A N D THEIR DISTRIBUTION IN THE CENTRAL A N D PERIPHERAL RETINA Area

DGCs

m m2

%

Total

C e U s / m m2

Retina

298

100

10 397

35

100

Central retina

175

58.7

3 550

20

34

Peripheral retina

123

41.3

6 846

55

66

334 perikaryon where the Golgi apparatus is located and also at the beginning of the axon (Fig. 16, Ly). Their diameter is 0.2 ~m and they have a round shape. All the lysosomes have a dense and thinly granular material enveloped by a limiting membrane. Larger and

335 more complex lysosomes are scarcely found. There are also many mitochondria in the DGCs. Their shape is round at the perikaryon level and elongated at the axon. Their diameter varies between 0.3 and 0.7/~m (Figs. 16, 19, * ). In the peripheral cytoplasm, the endoplasmic reticulum is continued by many subsurface cisterns, which are highly developed in these cells (Fig. 16, arrow). We cannot describe the ultrastructural characteristics of the dendrites of the DGCs since they are not differentiated from the dendrites of amacrine or other ganglion cells. They can all present microtubules, microfilaments, scattered mitochondria and some cisterns. Neither can we know the type of synapses received by the dendrites of the DGCs. A combined Golgi and electron microscopy study is necessary to get this information. With reference to the synapsis of the DGCs, we note the existence of Gray type I synapses (Figs. 17, 19, small rectangle) and of synaptic complexes (Figs. 18, 19, large rectangle) which are located in the portion of the axon that goes through the IPL. These synaptic complexes are formed by a Gray type I chemical synapse (Fig. 18, *) and by a small electric synapse also called a 'gap junction' (Fig. 18, arrow). DISCUSSION

Though we cannot deny the existence of small DGCs (10-15/~m) in the chick retina such as those described by Heaton et al. 14, the argentic techniques used in our study have stained only large DGCs (18-42 /~m). These cells seem to correspond to the DGCs described by LaVail and LaVail 18 in the chick retina, which specifically project to the BORn 23. The number of DGCs counted by us (10397/retina) is slightly higher than that proposed by Reiner et al. 23, a maximum of 7700/retina, by injecting horseradish peroxidase in the BORn. The difference could be explained by the use of different techniques. However, the cellular size and topographic distribution, according to the retinal quadrants, found by Reiner et al. 23 is very similar to ours. The fact that the pyridine-silver technique of Goldberg and Frank used in our study stained a similar Fig. 9. Vertical section of the retina. Displaced ganglion cells stained by the pyridine-silver method (Goldberg and Frank); the arrowheads point to the neurofibfillar frameworks; the solid arrow points to the ring-like neurofibrillar framework; the long arrow points to the origin of displaced ganglion cell axon. x 765. Fig. 10. Displaced ganglion cells with 4 dendritic trunks stained by the pyridine-silver method (Goldberg and Frank). Flat mounted retina, x 765. Fig. 11. Displaced ganglion cell with 2 dendritic trunks, stained by the pyridine-silver method (Goldberg and Frank). Flat mounted retina. × 765. Fig. 12. Shows the body of a displaced ganglion cell stained by the pyridine-silver method (Goldberg and Frank). Flat mounted retina. The arrowhead indicates the neurofibrillar framework, x 765. Fig. 13. The bodies of 2 displaced ganglion cells stained by the pyridine-silver method (Goldberg and Frank). Flat mounted retina. The arrowhead indicates the neurofibrillar framework. × 765. Fig. 14. Displaced ganglion cell observed in a flat mounted retina, stained by the pyridine-silver method (Goldberg and Frank). The arrow shows the axon origin from the dendritic trunk. × 765. Fig. 15. Displaced ganglion cell observed in a flat mounted retina, stained by the pyridine-silver method (Goldberg and Frank). The arrow shows the axon origin from the body of the cell. x 765.

t.~

337

lOG

[ ] Peripheral Retina ~Central Retina

5(

Fig. 20. Histogram representing the density of displaced ganglion cells (DGCs) in the central and peripheral retina of the chick.

S

N

_5/ Fig. 21. Quadrantic distribution of displaced ganglion cells in the chick retina. S, superior; I, inferior; T, temporal; N, nasal.

Fig. 16. An electron micrograph of a displaced ganglion cell. NB, Nissl body; Go, Golgi apparatus; Ly, lysosomes; ER, endoplasmic reticulum; *, mitochondria; arrow, subsurface cistern; a, amacrine cells; M, Miiller cell; IPL, inner plexiform layer. × 7100. Fig. 17. Synapse aml~lified from Fig. 19 (small rectangle), x 18460. Fig. 18. Synapse amplified from Fig. 19 (large rectangle). The arrow indicates a small gap junction (electrical synapse). The asterisk indicates a chemical synapse, x 18460. Fig. 19. Electron micrograph of the axon of one displaced ganglion cell traversing the inner plexiform layer. The asterisk indicates an elongated mitochondrion. The rectangles identify 2 synapses amplified in Figs. 17 and 18. x 3550.

338 number of DGCs to those counted using Nomarsky's interferential contrast (see Figs. 7. 8) might indicate that: (a) our counting technique is trustworthy and (b) the method of Goldberg and Frank selectively stains DGCs. It has been established that in the chick retina, the dendrites of the DGCs are distributed exclusively through the first stratum of the IPL 6.20,22. However, the photographic mounting of Fig. 1 shows that some DGC dendrites can reach several strata of this synaptic layer, in the same manner as occurs in the teleost retina 11.15 Several authors 4,9.19 have described the DGCs as a type of ganglion cell whose ectopic localization is probably due to the fact that they leave the cycle late in development and fail to migrate towards the GCL. Nevertheless, autoradiographic experiments carried out by our group (work in preparation) demonstrate that in the chick retina, the DGCs leave the cycle between days 3 and 7 of incubation. We suggest that the abnormal position is not due to a failure in the migratory process, but that these cells could have specific receptors for the molecules of positional information similar to those of amacrine cells. In this way, the final position of this neuronal population would be specifically marked. The ultrastructure of the DGCs shown in this work is different from that presently accepted. The main difference between DGCs and amacrine cells is that these cells have a smaller perikaryon, darker chromatin, and scarce cytoplasmic organelles, and that they do not show the same distribution of organelles as DGCs. The DGCs are more like the ganglion cells, but the nucleus of these cells is proportionally larger, their organelles are less abundant and less developed, and they never show the ring distribution of RER. On the other hand, the axon of the DGCs shows synaptic contacts at the IPL level which are similar to those described in the BORn by Rio 24 as type II. This synaptic complex is formed by a chemical synapse (Gray type I) and a electric one (gap junction). Taken together the data reported by LaVail and LaVail 18, Brecha and Karten 2 and Reiner et al. 23, and the results presented in this study lead us to propose that DGCs of the chick retina may constitute a specific morphofunctional system. ACKNOWLEDGEMENTS

This study was supported by grants from the Comision Interministerial de Ciencia y Tecnologia GG85-0153 and by the Fundaci6n Ram6n Areces, Madrid. REFERENCES 1 Alberts, B., Bray, D., Lewis, I., Roff, M., Roberts, R. and Watson, I., Molecular Biology of the Cell, Garland Publishing, Inc., 1983. 2 Breclia, N. and Karten, M.J., Accessory optic projections upon oculomotor nuclei and vestibulocerebellum, Science, 203 (1979) 913-916. 3 Bunt, A.H., Lund, R.D. and Lund, J.S., Retrograde axonal transport of horseradish peroxidase by ganglion cells of the albino rat retina, J. Camp. Neurol., 73 (1974) 215-228. 4 Bunt, A.H. and Minckler, D.S., Displaced ganglion cells in the retina of the monkey, Invest. Ophthalmol. Vis. Sci., 16 (1977) 95-98. 5 Colonnier, M., The tangential organization of the visual cortex, J. Anat. (Lond.), 98 (1964) 327-334. 6 Dogiel, A.S., f0ber d ~ Verhah~a der nerv~sen Elemente in der Retina der Ganoiden, Reptitien, V~gel und Sgmgetiere, Anat. Anz., 3 (1888) 133-143. 7 Dowling, J.E., Synaptic organiTJ~tion of the frog retina: an electron microscopic analysis comparing the retina of frogs and primates, Proc. R. Soc. Set. B., 170 (1968) 205. 8 Dowling, J.E. and Boycott, B.B., Neural connections of the inner plexiform layer, CoM Spring Harb. Syrup. Biol., 30 (1965) 393-402.

339 9 Drager, V.L. and Olsen, J.F., Origins of crossed and uncrossed retinal projections in pigmented and albino mice, J. Comp. Neurol., 191 (1980) 383-412. 10 Dreher, B., Sefton, A.J., Ni, S.Y.K. and Nisbeto, G., The morphology, number, distribution and central projections of class I retinal ganglion cells in albino and hooded rats, Brain Behav. Evol., 26 (1985) 10-48. 11 Dunn-Meynell, A.A. and Sharma, S.C., The visual system of the channel catfish (Ictalurus punctatus). I. Retinal ganglion cell morphology, J. Comp. Neurol., 247 (1986) 32-55. 12 Goldberg, S. and Frank, B., Pyridin-silver methods for the study of optic axons in retina whole mounts, Stain Technol., 54 (1979) 21-23. 13 Grandall, J.E., Heaton, M.B. and BrowneU, W.B., Tectal projection of displaced ganglion cells in the avian retina, Invest. Ophthalmol. Vis. Sci., 16 (1977) 774-776. 14 Heaton, M.E., Alvarez, I.M. and Grandall, J.E., The displaced ganglion cells in the avian retina: developmental and comparative consideration, Anat. Embryol., 155 (1979) 161-178. 15 Hitchcock, P.F. and Easter, S.S., Retinal ganglion cells in goldfish: a qualitative classification into four morphological types, and a quantitative study of the development of one of them, J. Neurosci., 6 (1986) 1037-1050. 16 Hogan, M.M., Alvarado, J.A. and Weddel, J.E., Histology of the Human Eye, W.B. Saunders Co., Philadephia, PA, 1971. 17 Karten, H.J., Fite, K.V. and Brecha, N., Specific projection of displaced ganglion cells upon accessory optic system in the pigeon (Columbia livia ), Proc. Nail Acad. Sci. USA, 74 (1977) 1753-1756. 18 LaVail, J.H. and LaVail, M.J., The retrograde intra-axonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopy study, J. Comp. Neurol., 157 (1974) 303-358. 19 Linden, R., Displaced ganglion ceils in the retina of the rat, J. Comp. Neurol., 258 (1987) 138-143. 20 Polyak, S.L., The Retina, University of Chicago Press, Chicago, IL, 1941. 21 Prada, C. and Lopez Mascaraque, L., Dammar resin prevents the fading of celloidin sections of Golgi impregnated embryonic central nervous system, Mikroskopie, 42 (1985) 146-147. 22 Ram6n y Cajal, S., Histologie du Systkme Nerveux de l'Homme et des Vertebras, 11. Maloine, Paris, 1911, pp. 80-106. 23 Reiner, A., Brecha, N. and Karten, J.M., A specific projection of retinal displaced ganglion cells to the nucleus of the basal optic root in the chicken, Neuroscience, 4 (1979) 1679-1688. 24 Rio, J.P., The nucleus of the basal optic root in the pigeon: an electron microscope study, Arch. Anat. Microsc. Morphol. Exp., 68 (1979) 17-27. 25 Robson, J.A. and Hollander, H., Displaced ganglion cells in the rabbit retina, Invest. Ophthalmol. Vis. Sci., 25 (1984) 1376-1381.