Antibodies against neurofilament subunits label retinal ganglion cells but not displaced amacrine cells of hamsters

Life Sciences, Vol. 64, No. 19, pp. lT3-1?78,1999 GJpyright @ 1999 EIsevier scicncc Inc. Printed in the USA. All righk rercMd cm324-3205/99/s--see front matter

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ANTIBODIES AGAINST NEUROFILAMENT SUBUNITS LABEL RETINAL GANGLION CELLS BUT NOT DISPLACED AMACRINE CELLS OF HAMSTERS W.C. Kong and E.Y.P. Cho Department

of Anatomy, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. (Received in final form February 3, 1999)

Summary Although neurofilament (NF) antibodies have been used to visualize ganglion cells and their axons in the retina, it is not known, however, how many ganglion cells contain NF, and how the various NF subunits are distributed in the ganglion cells. Moreover, it is not known whether displaced amacrine cells in the ganglion cell layer are also labelled. In order to see whether NF antibodies can be used as a specific marker for ganglion cells, antibodies raised against the low @IF-L), middle (NF-M) and high @IF-H) molecular weight subunits of NF were employed to stain retinal whole-mounts of adult hamsters after pre-labelling the ganglion cells with Granular Blue. It was found that NF-L and NF-H antibodies labelled 38,777 and 17,750 cells in the ganglion cell layer respectively. By co-localization with GB-labelled cells, 88% of NF-L positive cells and 91% of NF-H positive cells were found to be ganglion cells. In contrast, the NF-M antibody labelled only very few ganglion cells (418 per retina) although robust staining of axonal bundles was observed. Thus, NF antibodies may prove useful in studying this population of ganglion cells. Key Words: neurofilament subunits, retinal ganglion cells, displaced amacrine cells, neurofilament antibodies, hamster

Neurotilaments (NFs) are intermediate filaments which are specifically associated with the cytoskeleton of neurons (1). They are comprised of 3 polypeptide subunits having molecular weights of 200 kD (NF-H), 160 kD @IF-M) and 68 kD (NF-L) which are synthesized in the neuronal cell bodies and become assembled into the polymerized filamentous form (2). The recent availability of well-characterized antibodies raised specifically against the 3 subunits has allowed a detailed study of their distribution in different parts of the nervous system to be made (3-5). Results from these studies reveal a unique and consistent pattern of localization of NF proteins in different neuronal populations, and thus highlight their usefulness as specific cell markers. In the retina, for example, NF antibodies label distinct populations of horizontal cells, bipolar cells and retinal ganglion cells (RGCs), depending on the species examined (5-9). In these past studies, although NF antibodies have been shown to label some RGCs, there is little quantitative distribution

information on the number of RGCs that contain NF, as well as the pattern of of the 3 NF subunits in RGCs. Moreover, it is not known whether displaced

Correspondence:

E.Y.P. Cho. Department of Anatomy, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China. Phone: (852)26096842; Fax: (852)2603503 1; e-maileric-cho@c&k.edu.hk

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amacrine cells which make up as much as 50% of the neurons in the ganglion cell layer (10, 11) also contain NF. By pre-labelling RGCs of adult hamsters before subjecting the retina to immunostaining with antibodies against different NF subunits, we found that NF antibodies label mainly RGCs but not displaced amacrine cells. Moreover, a differential distribution of the 3 NF subunits in RGC somata was observed. Methods All experiments were performed with adult Syrian golden hamsters (Mesocricetus aura&s) 6-8 weeks old, using chloral hydrate (40mg/lOOg body weight) as the anaesthetic. In the first set of experiments, the number and distribution of cells in the ganglion cell layer which contained the various NF subunits were determined by staining retinal whole-mounts with antibodies against the different subunits: clone N52 (1:80) which recognizes NF-H independent of phosphorylation state, clone NN18 for NF-M (1:40), and clone NR4 (1:40) for NF-L. All NF antibodies were mouse-monoclonals purchased from Boehringer-Mannheim and diluted in PBS containing 1% Triton, 0.5% BSA and 0.9% sodium chloride (PBS-T-BSA). A total of 4 retinas were examined for each NF subunit. The animal was perfused transcardially with PBS, and the right retina dissected and fixed in 4% pamformaldehyde (in O.lM phosphate buffer, pH 7.4) for lh at room temperature. After extensive washing in PBS, the retina was placed in 10% fetal bovine serum for 1 h to block non-specific staining, and then incubated in the NF antibody overnight at 4°C. The following day, the retina was washed in PBS-T-BSA and then incubated with a secondary anti-mouse IgG antibody conjugated to peroxidase (Jackson Lab., 1:500 in PBS-T-BSA) overnight at 4°C. Staining was developed by diaminobenzidine (DAB), using the nickelenhanced glucose oxidase method (12). After DAB staining, the retina was dehydrated and coverslipped with Permount. The number and distribution of cells in the ganglion cell layer that contained NF were determined by a systematic sampling method. In each of the 4 quadrants of the retina, an ocular grid square delineating an area of 0.245XO.245mn-1~ was used to sample points located at 1, 2 and 3mm from the optic disc. At each point, the number of NF-positive cells in the ganglion cell layer which were enclosed within the grid was counted. Altogether 6 points (2 for each distance) in each quadrant were sampled, giving a total of 24 points for the whole retina. The area of the retina was traced and measured by an image analysis software developed by our department. The total number of NF-positive cells in the whole retina was calculated by pooling the data from all 24 points to obtain the mean density of labelled cells and multiplying it by the retinal area. All values were expressed as the mean number f standard error of the mean. In the second set of experiments, the proportion of NF-positive cells in the ganglion cell layer which belonged to RGCs was addressed. For each NF subunit, 3 retinas were examined. RGCs in the retina were labelled by the fluorescent dye Granular Blue (GB) applied at the superior colliculus. The left superior colliculus was exposed by craniotomy and suction removal of the overlying cerebral cortex, and a piece of gelatin sponge soaked in 3% GB was placed in contact with the colliculus. After a survival period of 3 days to allow for retrograde axonal transport of GB to label the RGCs, the animal was sacrificed by an overdose of chloral hydrate and transcardially perfused with PBS. The right retina was dissected as a whole-mount and fixed in 4% paraformaldehyde for 1h. The retina was incubated with the NF subunit antibody overnight at 4°C as in the first series of experiments, followed by anti-mouse secondary antibody conjugated to rhodamine (Jackson Lab, 1:200 diluted in PBS-T-BSA) overnight at 4°C. The retina was subsequently mounted in glycerol and examined under a Zeiss epi-fluorescence microscope using different filter sets for GB and rhodamine. Using the same sampling method

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as in the first series of experiments (except that 12 areas were sampled in each retina), the number of GB labelled RGCs, NF-positive cells, and NF-positive cells which also contained GB within the area delimited by the grid were separately recorded. Results

Fig. 1 Photomicrographs of mid-superior temporal region of retinal whole-mounts after staining with anti-NF-L (A), anti-NF-M (B), and anti-NF-H (C) antibodies. Axonal bundles (*) as well as cells of various soma sizes located in the ganglion cell layer were revealed. On some of the labelled cells, the presence of an axon (indicated by an arrow) emerging from the cell body indicated that they are RGCs. The “mottled” background appearance in B and C was due to the labelled horizontal cell plexus which appeared out of focus because of its different plane of location in the retina. Scale bar same for A-C. The axon bundles of RGCs in the nerve fibre layer were heavily labelled by all 3 NF subunit antibodies (Fig. l), but only the NF-H and NF-M antibodies stained the horizontal cell plexus in the outer plexiform layer (data not shown). No cell bodies in the outer or inner nuclear layer were stained, but some neuronal cell bodies together with their proximal dendritic branches in the ganglion cell layer as well as occasional cells in the inner plexiform layer were labelled. The

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labelled cells in the ganglion cell layer exhibited a range of soma sizes, but many of them corresponded to those of the large and intermediate-sized RGCs. The presence of an axon originating either from the cell body or proximal dendrite of some of these cells indicated that they were RGCs (Fig. 1A and C). Similarly, the appearance of the labelled cells (as well as their co-localization with the retrograde marker GB in the second set of experiment) in the inner plexiform layer suggested that they were displaced RGCs. The number of cells labelled tend to vary with the NF subunit antibody used: the number of NF-L positive cells was highest (mean number per retina = 38,777*4081, n=4) followed by NF-H cells (mean number per retina = 17,75&3724, n=4). In contrast, very few cells were labelled by the NF-M antibody (Fig. lB), so that an accurate determination of the total cell number using the sampling method would not be feasible due to the extremely low cell count per sampling area. Instead, the total number of NFM positive cells distributed throughout the whole retina was counted and the mean number calculated for the 4 retinas. This gave a mean number per retina = 4 18* 195.

Fig. 2 Paired photomicrographs illustrating the incidence of co-localization of GB prelabelled RGCs (A, C, E) with NF subunit-positive cells (B, D, F): panels A and B for NF-L, panels C and D for NF-M, and panels E and F for NF-H. Most NF-L and NF-H positive cells were co-localized with GB (some examples were indicated by arrows), while GB-labelled RGCs that were NF-L or NF-H negative were denoted by arrowheads. NF-M positive cells were very rare so that the field in D showed only axon bundles. Scale bar in E applied to all other panels.

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In order to determine whether the majority of NF-positive cells were RGCs, the incidence of colocalization of NF-positive cells with RGCs pre-labelled by GB was examined (Fig. 2). Application of GB to the superior colliculus resulted in a mean number per retina of 110,101~2706 (n=S) RGCs being labelled, a value which agreed favourably with RGC axon counts in the optic nerve (13); thus validating the use of this fluorescent dye as a reliable marker for the whole RGC population. In 3 retinas stained with anti-NF-L, 87.5% (1951 out of 2291) of the sampled NF-L positive cells contained GB, and thus were RGCs. In the case of anti-NF-H staining, 90.8% (726 out of 800) of the NF-H positive cells contained GB. The 12% of NF-L and 9% of NF-H positive cells which lack the GB label may indicate that they are displaced amacrine cells, or they could be RGCs which either failed to become labelled by GB or lost the GB during the immunostaining procedure. Further evidence which suggests that they are in fact RGCs comes from the observation that 28 days after cutting the optic nerve, only about 1% of the NF-L or NF-H positive cells remained (unpublished results). For anti-NF-M staining, due to the fact that the antibody labelled very few cell bodies, the systematic sampling procedure was not performed. However, random observations of a number of NF-M positive cells distributed throughout the retina indicated that they all contained GB, and thus were RGCs. The proportions of RGCs which contained either NF-L or NF-H were also estimated by sampling the cells at retinal eccentricities of 2 and 3mm (cells at the lmm eccentricity were not examined because the bright fluorescence of axonal bundles tended to make examinations of the labelled cells difficult). The proportions of RGCs which contained the NF-L subunit were 45.2% (708 out of 1566 cells) and 50.4% (692 out of 1374 cells) at 2 and 3mm of retinal eccentricities, respectively. In the case of NF-H-positive RGCs, they comprised 17.3% (258 out of 1494 cells) and 25.1% (290 out of 1156 cells) of the RGC population, at 2 and 3mm of retinal eccentricities respectively. Discussion

Although previous studies have shown that NF antibodies stain some RGC somata and their axons (5, 6, 8, 9), it is not known how the 3 subunits are distributed in the whole RGC population and whether displaced amacrine cells also contain NF. Coupled with double labelling of the whole population of RGCs by GB, the present results highlight that the 3 NF subunit antibodies produce distinct patterns of labelling of RGC somata in hamster. All 3 subunit antibodies stain intra-retinal axons of RGCs, but whereas the NF-L and NF-H antibodies label a certain population of RGCs, very few RGC somata are revealed by the NF-M antibody. Moreover, the number of NF-L containing RGCs is twice that labelled by the NF-H antibody. This differential pattern of localization of the different NF subunits in RGC somata cannot be attributed to the antibodies specifically recognizing epitopes related to phosphorylation, as have been reported for some of the antibodies raised against NF-H and NF-M subunits (14, 15), since the antigenic binding sites of the 3 antibodies used in this study have been characterized as phosphorylation independent (16). Although it is generally believed that all 3 NF subunits coexist in the same cellular compartment in mature neurons (4), exceptions have been reported (17). If we are to assume that RGC somata contain all 3 NF subunits, other post-translational modifications distinct from phosphorylation of the NF subunits may occur as they are in transit from the RGC somata into the axons in order to explain the present results. Although further studies are required to address this issue, our findings that NFs are localized to particular subpopulations of RGCs of adult hamster provide an opportunity to study this well defined group of neurons with respect to the role played by NFs in relation to neuronal injury and degeneration

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(6, IS), an especially relevant example being that the mechanisms RGCs in Alzheimer’s disease (19).

responsible

for the loss of

Acknowledgments This study was supported by RGC Earmarked Grant CUHK 254/96M. References 1. K.H. FLIEGNER and R.K.H. LIEM, Int. Rev. Cytol. 131 109-167 (1991). 2. R.A. NIXON, S.E. LEWIS, D. DAHL, C.A. MAROTTA and U.C. DRAGER, Mol. Brain Res. 5 93-108 (1989). 3. G. SHAW, M. OSBORN and K. WEBER, Eur. J. Cell. Biol. 26 68-82 (1981). 4. J.Q. TROJANOWSKI, N. WALKENSTEIN and VM-Y. LEE, J. Neurosci. 6 650-660 (1986). 5. J.C. VICKERS and M. COSTA, Neurosci. 49 73-100 (1992). 6. U.C. DRAGER and A. HOFBAUER, Nature 309 624-626 (1984). 7. L. PEICHL and J. GONZALEZ-SORIANO, J. Neurosci. 13 4091-4100 (1993). 8. G. SHAW and K. WEBER, Eur. J. Cell. Biol. 33 95-104 (1984). 9. C. STRAZNICKY, J.C. VICKERS, R. GABRIEL and M. COSTA, Brain Res. 582 123-128 (1992). 10. R. LINDEN and C.E.L. ESBERARD, Vis. Res. 27 1071-1076 (1987). 11. V.H. PERRY, Neurosci. 6 931-944 (1981). 12. S. SHU, G. JU and L. FAN, Neurosci. Lett. 85 169-171 (1988). 13. D. TAY, K.-F. SO, L.S. JEN and K.C. LAU, Brain Res. 395 268-273 (1986). 14. G. BENNETT and C. DILULLO, J. Cell. Biol. 100 1799-1804 (1985). 15. L.A. STERNBERGER and N.H. STERNBERGER, Proc. Natl. Acad. Sci. U.S.A. 80 61266130 (1983). 16. G. SHAW, M. OSBORN and K. WEBER, Eur. J. Cell. Biol. 42 l-9 (1986). 17. J. HARRIS, S. MORENO, G. SHAW and E. MUGNAINI, J. Neurocytol. 22 1039-1059 (1993). 18. J.H. MORRISON, D.A. LEWIS, M. J. CAMPBELL, G. W. HUNTLEY, D.L. BENSON and C. BOURAS, Brain Res. 416 331-336 (1987). 19. J.C. BLANKS, Y. TORIGOE, D.R. HINTON and R.H. BLANKS, Neurobiol. Aging 17 377-384 (1996).