Download Estimation of the Ratio of Cones to Neurons in the Fovea of

Reports 1045
Volume 13
Number 12
vestigation applying this method to the experimentally damaged retina (monkey) is in progress
in our laboratory.
In the present study, the Miiller cells, seen
in the horizontal section of the retina, were often
found in a small mass in close contact with the
neighboring cells. This tendency is more noticeable
in the posterior than in the peripheral part of the
retina. Larger masses of the Miiller cell processes
have been observed in the extremely thickened
nerve fiber layer in the juxta-optic nerve region
of the human retina.s These facts may represent
functional aspects of the Miiller cells such as
mechanical support of the retina. The present observations also showed quite clearly that there is
a close relationship between the retinal capillary
and the Miiller and accessory glial cells and are
in accordance with those of earlier investigators.
The astrocytes, terminating on the surface of the
capillary, always showed an end-foot structure
comprising bundled processes. The light microscopic studies"- 1() of the retinal glia have revealed
the presence of an additional two types of glial
cells, perivascular glia and lemmocyte (cell of
Remak), but these glial elements have not been
pursued in this study.
We would like to extend our sincere thanks to
Victoria Ozanics for her valuable advice and
reviewing this manuscript.
From the Department of Ophthalmology,
Faculty of Medicine, Kyushu University, Fukuoka,
Japan. Submitted for publication April 26, 1974.
Reprint requests: Dr. S. Uga, Department of
Ophthalmology, Faculty of Medicine, Kyushu
University, Fukuoka, japan.
REFERENCES
1. Stell, W. K.: Correlation of retinal cytoarchitecture and infrastructure in Golgi preparations, Anat. Rec. 153: 389, 1965.
2. Dowling, J. E.: Organization of vertebrate
retinas, INVEST. OPHTHALMOL. 9: 655, 1970.
3. Matsusaka, T.: The fine structure of the inner
limiting membrane of the rat retina as revealed by ruthenium red staining, J. Ultrastruct. Res. 36: 312, 1971.
4. Peyman, G. A., Spitznas, M., and Straatsma,
B. R.: Peroxidase diffusion in the normal and
photocoagulated retina, INVEST. OPHTHALMOL.
10: 181, 1971.
5. Ashton, N., and Tripathi, R.: The argyrophilic
mosaic of the internal limiting membrane of
the retina, Exp. Eye Res. 14: 49, 1972.
6. Magalhaes, M. M., and Coimbra, A.: The
rabbit retina Miiller cell. A fine structural
and cytochemical study, J. Ultrastruct. Res.
39: 310, 1972.
7. Luft, J. H.: Fine structure of capillary and
endocapillary layer as revealed by ruthenium
red, Fed. Proc. 25: 1773, 1966.
8. Uga, S.: Some structural features of the
retinal Miillerian cells in the juxta-optic nerve
region, Exp. Eye Res. 19: 105, 1974.
9. Wolter, J. R.: Perivascular glia of the blood
vessels of the human retina, Am. J. Ophthalmol. 44: 766, 1957.
10. Lessel, S., and Kuwabara, T.: Retinal
neuroglia, Arch. Ophthalmol. 70: 133, 1963.
Estimation of the ratio of cones to neurons
in the fovea of the human retina. L. MisSOTTEN
The ratio of pedicles over neurons has been
determined in thin sagittal and -flat sections of the
rod-free central area of the human retina. The
real amount of neurons has been calculated from
the number of transections of nuclei. Results show
that for each pedicle two to three bipolar cells
are found, ± 0.6 horizontal cells, t 0.7 amacrine
cells, and 0.9 ganglion cells.
The central rod-free area of the fovea is especially well-suited for a study of the synaptic
contacts of the cones. Only one class of receptor
cells is present in this area and the neurons of
the second and third echelon are small and, for
this reason, easier to investigate by electron
microscopy. In order to translate the findings of
the analysis of single cells in a general schema, it
would be helpful to know the ratio between the
different neurons and the receptor cells.
A comparison of the data available from the
literature on the number of cones1 and the number
of ganglion cells- does not give the answer, because the inner layers of the retina are shifted
centrifugally in respect to the visual cells (Fig. 1).
In front of the central bouquet of cones, in the
foveola, the inner layers of the retina are absent.
No synaptic connections are found in this area.
The synaptic pedicles of these cones are located
in an annular zone surrounding the central area;
they are connected to their parent cells with long
fibers "Henle's fibers." The centermost pedicle
is usually found at 100 microns distant from the
center of the fovea. The area between 100 and
300 microns contains pedicles at irregular intervals; from about 300 microns outward the pedicles
form a continuous and uniform layer to about
800 to 1,000 microns from the center, where rod
spherules are found in increasing numbers.
The centrifugal shift of the neurons connected
to the cones of the fovea hinders the correlation
of the numbers of receptor cells and neurons, because the layers are displaced in respect to each
other, and because the area available to the
neurons is much larger than the surface occupied
by the cones, as is shown in Fig. 1.
The neurons of the inner layers are oriented
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 06/18/2017
1046
Investigative Ophthalmology
Deceviber 1974
Reports
Qfi
500M.
Fig, 1. The centrifugal shift of the pedicles of the cones of the fovea as seen in flat section
(upper drawing) or in sagittal section (below). A, the 2,500 cones of the central bouquet
(dark area) have their pedicles situated in an annular area (grey) extending from 125 microns
to 260 microns from the center. B, the 25,000 cones of the fovea externa (dark) and their
pedicles (grey area). C, the 100,000 cones of the slope of the fovea (dark area) and their
pedicles (grey area). This schematic representation shows how the area covered by the pedicles
is several times larger than the area of the receptors. The lower schematic drawing shows
how the neurons of the second and third echelon are placed in front of the pedicles. The
scale applies to retina fixed in glutaraldehyde dichromate and embedded in epon.
perpendicular to the surface of the retina, as can
be seen very clearly in a Golgi-stained preparation.
The proportion of neurons to cones may thus be
determined by counting pedicles and neurons in
the area between 300 and 800 microns from the
center of the fovea. This approach has been used
by Vilter,a but this author does not mention the
horizontal cells nor the amacrine cells in his paper
and does not take into account the stereologic
problems.
Methods. Human retinae have been obtained
from eyes with normal visual function enucleated
for melanomas of the iris root or the ciliary body.
They have been prepared for Golgi staining by
the glutaraldehyde-dichromate method,4 dehydrated, and embedded in Epon, and cut in 60micron thick sections on a sliding microtome after
softening the superficial layers of the block with
heat.5
The fovea was located, and Golgi-stained cells
recorded in optical micrographs. The central area
was then remounted and cut on an ultra microtome
in thin sections for electron microscopy and in
one micron-thick sections for optical microscopy.
Sagittal sections through the center of the fovea
have been made, together with "flat" sections
parallel to the surface of the retina from an area
situated at 600 to 800 microns from the center.
Measurements and counting was done on micrographs made with a Zeiss Photomicroscope with
a 40x oil immersion planapochromate and studied
at a final magnification of l,000x (Fig. 2).
Pedicles and neurons have been counted in tangential and flat sections. The number of neurons
has been estimated by counting their nuclei. All
dimensions apply to embedded retina. No correction was made for the shrinkage during fixation
and embedding. The real number of nuclei in a
unit volume (Ni) has been calculated from the
apparent number of nuclei (Na) in a unit area
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 06/18/2017
Volume 13
Number 12
Reports
-wt«
Fig. 2. A sagittal section of human retina, situated at 550 microns from the center of the
fovea. From top to bottom: the outer plexiform layer with Henle's fibers and pedicles, the inner
nuclear layer, the inner plexiform layer, the layer of ganglion cells, the layer of optic nerve
fibers, and the inner limiting membrane. The mark measures 50 microns.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 06/18/2017
1047
Investigative Ophthalmology
December 1974
1048 Reports
Table I. Number of transections of pedicles and
nuclei in sagittal sections
Pedicles
Nuclei of the inner
Horizontal cells
Bipolar cells
Amacrine cells
Ganglion cells
nuclear layer
172 (15.5%)
731 (66%)
207 (18.5%)
298
1.110
495
Table II. Number of neurons in a volume of foveal
retina containing 100 pedicles
Neurons in the inner nuclear layer
Horizontal cells
45.5- 71
Bipolar cells
194 -303
Amacrine cells
54.5- 85
Ganglion cells
294-458
90
of the thin sections by means of the relation0:
Ni =
Na 3/2
K
(Vi/Vt) 1/2
Vi/Vt is the ratio of the volume occupied by the
nuclei of the neurons to the total volume of the
inner nuclear layer or, for the ganglion cells;
the ratio of the volume of their nuclei to the volume of the ganglion cell layer. These ratios have
been determinated by measuring their surface
in the sections by means of the "point" method
proposed by Glagolev.7
P equals 1.38 for spherical bodies0 and is not
appreciably different for ellipsoids up to a ratio
of width over a length of 0.6.s
K = 1 for a population of bodies of identical
size.
Results. Apparent numbers of pedicles and
neurons. One micron-thick sagittal sections through
the center of the fovea have been studied and
the number of pedicles and neurons has been
determined in 19 areas, each 100 microns long,
situated between 300 and 800 microns from the
center. No significant differences have been found
between these areas. The results have been pooled
as shown in Table I. Pedicles and neurons have
also been numbered on 47 flat sections, chosen at
different levels in a region situated at 600 to 800
microns from the center of the fovea.
Differentiation. The nuclei of different types of
neurons show different forms and staining patterns
(Fig. 2). Horizontal cells have dark nuclei with
a few black spots, amacrine cell nuclei are
grayish and have indentations, Miiller cells have
very dark angular nuclei. The characteristics are
not much influenced by the fixative used, be it
osmium tetroxide, glutaraldehyde-osmium tetroxide,
or glutaraldehyde dichromate.
The bipolar cells show different aspects accord-
ing to the fixative used. In osmium-fixed tissues
all the bipolar cells have grayish nuclei; with
glutaraldehyde-dichromate fixation two kinds of
nuclei are seen in about equal numbers in bipolar
cells: some nuclei are uniform grayish and others
are pale with dark spots (Fig. 2). This differentiation is not equally well visible in all retinae;
its significance is still unclear.
The identification of the neurons by their nuclei
is based on: (1) the preferential location of each
type of nucleus, (2) the study of nuclei of Golgistained neurons, and (3) the correlation with
electron micrographs. In some instances, the aspect
of a nucleus was ambiguous: especially the
amacrine cell nucleus whose indentation is not
visible in the section is hard to differentiate from
a gray bipolar cell nucleus.
Pedicles. In sagittal sections 15.5 pedicles are
counted per 0.1 mm., corresponding to 15.52 or
240 pedicles per unit volume. This number could
be verified on flat sections, where 250 pedicles are
found per 0.01 mm.2. The latter determination is
more direct; it will be used as a base for further
calculations.
Inner nuclear layer. Weibel and Gomez'0 equation has been used to calculate the real number
of neurons in the inner nuclear layer. In the
foveal area this layer is approximately 50 microns
thick. In sagittal sections the relative surface of
nuclei equals 39.5 per cent and 29 transections
are seen per (50 microns),2 as may be deduced
from Table I. In flat sections, 41 transections are
counted per unit area; their relative surface
measures 43 per cent. As an approximation, we
assume that the nuclei are spherical and all of the
same size. The introduction of these values in the
equation shows that a unit volume of (50
microns):t contains between 184 nuclei (calculated
from measurements on sagittal sections) to 286
nuclei (based on flat sections). The 50 times 50
micron outer surface of this unit volume is adjacent to 62.5 pedicles.
The number of neurons per 100 pedicles, and
the number of different kinds of neurons is shown
in Table II. The lowest number results from
measurements on sagittal sections, the highest
number from flat sections.
Ganglion cell layer. The ganglion cell layer in
sagittal sections has a mean thickness of 70
microns and 18.2 transections of nuclei are found
in (70 microns).- They occupy a relative surface of
25.8 per cent. The analysis of flat sections gave
identical results. Calculation of the Equation 1
shows that a unit volume of (70 microns ):t contains 110 ganglion cell nuclei or ganglion cells.
The outer surface of this unit volume corresponds
to an area containing 122 pedicles. Transformation
of this result for a volume of retina containing
100 pedicles is shown in Table II.
Discussion. Stereologic problems. Pedicles are
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 06/18/2017
Volume 13
Number 12
best visualized and numbered in one micron-thick
sections. They form a single layer and may be
counted in sagittal and flat sections. The estimation of the number of neurons from thin sections,
however, requires the transformation from apparent to real numbers by means of a principle
developed by Weibel and Gomez.(i These authors
enumerate four conditions that must be satisfied.
The investigated bodies must be randomly distributed, they must be well defined, the bodies
must be small compared to the total volume, and
the section must be thin. The first condition
means that any section in whatever direction
should pass through the same number of bodies.
Although the inspection of sections such as Fig. 2
suggests that the distribution of the nuclei in the
nuclear layers is more or less random, comparison
to flat sections shows that in the inner nuclear
layer this condition is not satisfied, whereas in the
ganglion cell layer the distribution is random. For
this reason, the calculated number of ganglion
cells is fairly accurate. In the inner nuclear layer,
calculations based on flat sections, showing the
highest concentration of transections, and on
sagittal sections that have the lowest density, set
an upper and lower limit to the number of
neurons.
The coefficient fi in the equation reflects the
configuration of the bodies to be counted. Coefficient /3 equals 1.382 for spheres, and grows with
increasing lack of sphericity. It has been shown
that, for ellipsoids with a ratio of diameter over
length of 0.6 or more, the increase of fi is small,8
less than was originally calculated. As the nuclei
of the retinal neurons are only slightly elongated,
the value 1.382 for /S has been used. This results
in a slight overestimation of the number of
neurons.
The coefficient K equals one for bodies of equal
size, and increases with increasing heterogeneity
of the sample. It has been shown that in biological
material the coefficient K is usually between 1.02
and 1.1.!) The nuclei in the inner nuclear layer
and those in the ganglion cell layer each form a
fairly homogenous population, as can be seen in
thick sections. For this reason, the coefficient K
has been assumed to be one. This approximation
results in a slight underestimation of the number
of neurons. The incertitude introduced by these
two approximations, however, is much less than
that due to the lack of randomness.
The synoptic connections of the cones. The discovery of the flat midget bipolar10 in addition to
the invaginating midget bipolar has given rise to
the hypothesis that each cone might be connected
to one neuron of each type. In addition, contacts
are made with the diffuse cone bipolars also
present in the fovea.
Reports 1049
The ratio of bipolar cells over pedicles being
approximatively 2.5 confirms that this is a possibility. Direct confirmation may be obtained from
electron microscopic study of Golgi-stained retina,
combined with reconstruction of the unstained
neurons from serial sections. However, the present
amount of information accumulated by this method is still insufficient to permit generalizations.
The author thanks Mrs. A. Geysen for her expert
technical assistance and Miss Chr. Van Rijmenant
for typing and proofreading the manuscript.
From the Department of Ophthalmology, University of Louvain, K.U.L., Belgium. This investigation was supported by Grant No. 1201 from
the Belgian Fund for Medical Scientific Research.
Submitted for publication May 7, 1974. Reprint
requests: Prof. L. Missotten, Ophthalmology,
Academisch Ziekenhuis St. Rafael, Capucienenvoer,
B 3000 Leuven, Belgium.
Key words: retina, fovea, cones, horizontal cells,
bipolar cells, amacrine cells, ganglion cells.
REFERENCES
1. Oesterberg, G. A.: Topography of the layer
of rods and cones in the human retina, Acta
Ophthalmol. 6: 1, 1935.
2. Oppel, O.: Untersuchungen iiber die Verteilung und Zahl der retinalen Ganglienzellen
beim Menschen, Albrecht v. Graefes Arch.
Klin. Exp. Ophthalmol. 172: 1, 1967.
3. Vilter, V.: Recherches biom6triques sur l'organisation synaptique de la retine humaine,
C.R. Soc. Biol. 143: 830, 1949.
4. Colonnier, M.: The tangential organization
of the visual cortex, J. Anat. 98: 327, 1964.
5. West, R. W.: Superficial warming of epoxy
blocks for cutting of 25 to 150 micron sections
to be resectioned in the 40 to 90 nm. range,
Stain Technol. 47: 201, 1972.
6. Weibel, E. R., and Gomez, D. M.: A principle
for counting tissue structures on random sections, J. Appl. Physiol. 17: 343, 1962.
7. Glagolev, A. A.: Quantitative analysis, with
the microscope by the "point" method, Eng.
Mining J. 15: 399, 1934.
8. Baudhuin, P.: L'analyse morphologique quantitative de fractions subcellulaires, These Universite de Louvain, 1968.
9. Weibel, E. R., Kistler, G. S., and Scherle,
W. F.: Practical stereological methods for
morphometric cytology, J. Cell Biol. 30: 23,
1966.
10. Kolb, H., Boycott, B. B., and Dowling, J. E.:
Organization of the primate retina, light
microscopy. Appendix: A second type of
midget bipolar cell in the primate retina,
Phil. Trans. B. 255: 109, 1969.
Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/932884/ on 06/18/2017