Some observations on the fine structure of the retinal
receptors of the American gray squirrel
Adolph I. Cohen
Electron microscopy suggests that the squirrel retina has two types of receptors whose outer
segments possess rodlike and conelike structural details, respectively. They are further distinguished by the length and diameter of their outer segments and by the shapes and location
of their nuclei. Increased membrane density is noted at contact points between the pedicle
plasma membranes of the receptors and suggests interreceptor synapses. These have been noted
between cells possessing rodlike and conelike outer segments and between cells with similar
outer segments. On one occasion such a contact patch appeared to involve a glial and a receptor
cell. Questions are raised relative to the identification and definition of receptors as rods or
cones, the interpretation of "all cone" electroretinograms, as well as the visual pigment content
of this retina. The problem of the retinal attachment mechanism is considered. Other receptor
regions are also described.
R
presence of a pigment generally associated
with "rod" morphology and scotopic vision
not only appears to be superficially inconsistent with the notion that the gray squirrel
posseses an all-cone retina but also does
not agree with either the spectral sensitivity of this retina as determined by
electror etino graphic thresholds2' 32 or with
Weale's34 results employing reflection spectrophotometry. These in vivo techniques are
in fair agreement and show a maximum
sensitivity at 535 m^,. The electroretinographic analysis showed a second peak at
490 mfx.32 Finally, a recent in vivo study
consisting of a spectral sensitivity analysis
by Arden and Silver3 based upon squirrel
behavior yielded a curve resembling that
of the extracted rhodopsin-like pigment,
thus tending to confirm Dartnall's suggestion that at the high illumination levels employed in electroretinography and spectrophotometry another pigment confused
the rhodopsin spectrum.
'ecent studies on the retina and vision
of the American gray squirrel (Sciurus
carolinensis) have led to a curious dilemma.
On one hand, the histology of the retina
and cytology of the receptors suggested to
Walls33 and Arden and Tansley2 that this
species posesses an all-cone retina and this
appeared to be supported by certain physiologic, data of Arden and Tansley2 and Tansley, Copenhaver, and Gunkel.32 On the
other hand the visual pigment extracted
by Dartnall10 consists mainly of a photolabile broad band rhodopsin (abs. max. 502
m/x) together with a small amount of a stabile photo product (abs. max. 480 m/x). The
From the Departments of Ophthalmology and
Anatomy, Washington University School of
Medicine, St. Louis, Mo.
This research supported in part by Grant NB
04816-01 from the National Institute of Neurological Diseases and Blindness of the National
Institutes of Health, United States Public Health
Service.
198
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Fine structure of gray squirrel retinal receptors 199
Accordingly, it was decided to reinvestigate the cytology of the gray squirrel receptors with the use of modern methods
of light and electron microscopy. These
studies led to the finding that from the
morphologic point of view the gray squirrel,
rather than possessing an all-cone retina,
possesses a retina whose receptors are somewhat ambiguous in their structure, but
which on detailed examination appear to
consist of two varieties which exhibit certain rodlike and conelike features. These
occur in almost equal numbers (4:5) but
are separated by an arrangement described
below. In addition, a curious artifact may
bear on the disparity of the results obtained on extracted versus in situ pigments.
Materials and methods
Gray squirrels were obtained by trapping
locally. They were dark adapted overnight, anesthetized with ether to facilitate removal from the
traps, and then immobilized. The animals were
allowed to recover from the anesthesia, or when
recovery was imminent were placed under cold
anesthesia (22° C.) by packing in ice. They were
then decapitated. These procedures were instituted because of the impression obtained that
the dark adapted and nonanesthetized receptors
were more resistant to the brief period of anaerobiosis occurring during the dissection interval
prior to fixation.
As Dartnall10 has noted, the vitreous humor of
this species is viscous and rather adherent to the
retina. After removal of the anterior segment of
the eye an attempt, only occasionally successful,
was made to strip the vitreous from the retina.
In any event, the posterior half of the eye was
submerged in cold Earle's physiologic saline*
containing 2 per cent osmium tetroxide with a
final pH of 7.0 or in Dalton's9 chrome-osmic
fixative (pH 7.6). The eye in the fixative was
placed in a specially constructed fume hood
which contains a dissecting microscope whose
oculars pass through the hood wall, permitting
dissection of the eye in the fixative while protecting the operator from the fumes. Since the
vitreous minimizes fixative penetration from the
inner retinal surface, it was important to provide
access of the fixative from the scleral direction. This
was done by dissecting the sclera from the uvea
"Baltimore Biological Laboratories.
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while the eye was in the fixative. Simply removing
the retina from the eye for fixation was entirely
unsatisfactory in our hands because of the adhesion of most of the outer segments to the pigment epithelium, major portions of the latter
consistently remaining behind when the retina
was withdrawn.
After fixation for 1 hour in the cold and then
30 minutes at room temperature, the retina with
its uveal backing was progressively dehydrated
with ethanol and cut into small fragments while
in the absolute ethanol. During dehydration the
uvea tends to contract at a rate which differs from
that of the retina and often splits the pigment
epithelium from the retina. This can be minimized
by encasing the fragments in 2 per cent agar
prior to dehydration as previously described.5 The
fragments were then stored in warm tertiaiy
butanol until they were embedded in epoxy resin.*
Thick sections for light microscopy and thin sections for electron microscopy were cut with an
LKB ultramicrotome. Sections for light microscopy
were stained by the method of Richardson, Jarett,
and Finke.23 Sections on grids for electron microscopy were stained with uranium acetate or with
lead salts and photographed with an RCA 3F
electron microscope.
Results
Fig. 1 is a light micrograph of the retina
of the gray squirrel. The receptors end in
a single horizontal line of pedicles and the
outer segments of all receptors reach the
pigment epithelium. Beyond these uniformities, two classes of receptors were discerned. The "R" class of receptor, receptors
with rodlike features, possessed nuclei
which occur just sclerad to the pedicles
and which are ellipsoidal in shape but with
the major axis only slightly longer than the
minor. Their ellipsoid regions, containing
mitochondria, lie a short distance sclerad
to the line of the external limiting membrane and their outer segments are long
and cylindrical in appearance. The "C"
class of receptors, which possess conelike
characteristics, have nuclei which are long
ellipsoids and which lie just vitread to the
line of the external limiting membrane. The
ellipsoid region is of somewhat larger
diameter than that of the "R" receptors
°Araldite, Ciba Pharmaceutical Company.
Investigative Ophthalmology
April 1964
200 Cohen
P
e
Fig. 1. A light micrograph of the squirrel retina showing the two receptor classes, one with
an outer segment shorter and of greater diameter than the other. The bounding levels designated
are those of the pigment epithelium (Pe), the external limiting membrane [elm), and the
receptor pedicles (p). (Chrome-osmium fixed, methylene blue-azure B stained. x630.)
Fig. 2. A light micrograph of receptor outer segments adherent to the pigment epithelium
following removal of the retina, illustrating conelike outer segments (c) as well as one with a
rodlike character (r). (Chrome-osmium fixed, methylene blue-azure B stained. x5,700.)
Fig. 3. A light micrograph of receptor portions remaining in a detached retina, illustrating
tlie total absence of the conelike outer segments of one receptor variety whose inner segments
(cis) are shown, as well as remaining portions of the rodlike outer segments of the second
receptor class (ros). (Chrome-osmium fixed, methylene blue-azure B stained. x3,400.)
Fig. 4. A light micrograph of a section tangential to the retina and at a level containing the
upper tier of ellipsoids and the lower tier of outer segments, thus permitting counting the two
receptor classes. (Chrome-osmium fixed, methylene blue—azure B stained. x2,500.)
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Fine structure of gray squirrel retinal receptors 201
ros
Figs. 2, 3 } and 4. For legends see opposite page.
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202 Cohen
V
Fig. 5. An electron micrograph of a conelike outer segment attached to a cell of the pigment
epithelium. The latter cell shows a pigment granule (p), ribosomes (r), its nucleus (n),
processes intimately draping the outer segment tip as well as a small protuberance (arrow)
which indents the outer segment. The villous processes (v) are believed to be artifactual.
(Chrome-osmium fixed, lead stained. x66,500.)
Fig. 6. An electron micrograph of the base of a conelike outer segment. The section has just
missed the ciliary backbone of the outer segment at c. Note the continuity of the cell membrane
and saccule membranes below the arrow. (Chrome-osmium fixed, lead stained. x70,750.)
Fig. 7. Ai\ electron micrograph of the junction of the inner and outer segments of a conelike
outer segment, showing saccules both continuous with or separate from the cell membrane.
{Chrome-osmium fixed, lead stained. x94,000.)
Fig. 8. An electron micrograph of the base of a rodlike outer segment illustrating the typical
absence of evident continuity of the cell membrane with that of the saccules. Rare instances
of continuity involving one or two saccules were encountered. (Chrome-osmium fixed, lead
stained. x79,750.)
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Fine structure of gray squirrel retinal receptors 203
Figs. 6, 7, and 8. For legends see opposite page.
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Investigative Ophthalmology
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Fig. 9. An electron micrograph of the tip of a rodlike outer segment. This tip is not as intimately opposed to the cells of the pigment epithelium although surrounded by pigment cell
processes. (Chrome-osmium fixed: lead stained. x75,300.)
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Fine structure of gray squirrel retinal receptors 205
Fig. 10. An electron micrograph of a cross-section of a rodlike outer segment. Note the somewhat scalloped saccule perimeter and separation of the saccule from the cell membrane.
(Chrome-osmium fixed, lead stained. x50,000.)
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Investigative Ophthalmology
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Fig. 11. An electron micrograph of a cross-section of a rodlike outer segment. Again note the
separateness of the cell membrane from the saccule. In this slightly oblique section the
scalloping is less evident. Cross-sections of pigment containing processes of the pigment
epithelium are also seen. (Chrome-osmium fixed, lead stained. x5(X,000.)
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mv
Fig. 12, An electron micrograph semitangential to the retina at the level of the external limiting membrane, illustrating the Golgi region (g), the glial cells of Miiller (m), and sections of
their microvillous processes (mv). Note the absence of a gearlike cross-section as reported
elsewhere. (Chrome-osmium fixed, lead stained. xl2,000.)
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208 Cohen
m
Fig. 13. An electron micrograph illustrating the three types of presumptive synaptie arrangements involving receptors. One is an external synapse (arrow, upper left), the second an
interreceptor synapse (arrow, central), and the last the complicated enveloped synapse below
the synaptie lamella (si). The nucleus of a receptor ( n ) 3 a curious cytoplasmic cistern (c),
and glial cells of Midler ( M ) are also shown. (Chrome-osmium fixed, lead stained. x27 J 000.)
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Fine structure of gray squirrel retinal receptors 209
Fig. 14. An electron micrograph of receptor (r) cytoplasm adjacent to a Miiller cell (m).
Note the cisterns formed by paired membranes and synaptic vesicles, a few of which (arrows)
are surrounded by a halo of particles. (Chrome-osmium fixed, lead stained. x8l,000.)
Fig. 15. An electron micrograph illustrating cistern formation by folding of the membranes
of collapsed vacuoles. The continuity of the cistern with the general pedicle cytoplasm is indicated by an arrow. (Chrome-osmium fixed, lead stained. x72,250.)
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Investigative Ophthalmology
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r
m
Fig. 16. An electron micrograph showing details of a desmosomal-contact- between two receptors. Miiller cell cytoplasm {in) does not intrude. (Chrome-osmium fixed, lead stained.
x70,000.)
Fig. 17. An electron micrograph of a single instance where membrane of a glial cell of Miiller
(m) does appear to be involved in a receptor contact characterized by a desmosome. (Osmiumfixed, lead stained. x41,500.)
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Fine structure of gray squirrel retinal receptors 211
and appears to contain a greater mitochondrial mass. The outer segment is shorter,
varies in shape from bulbous to tapering,
and has a slightly larger diameter than that
of the "R" receptors. These relative dimensions vary somewhat with retinal regions.
It is noteworthy that the villous processes
of the cells of the pigment epithelium drape
the major portion of the outer segments of
the "C" receptors and only about the outer
third of the outer segments of the "R"
receptors. Following the slightest manipulation of the retina, the outer segments
tore, those of the "C" receptors breaking at
the junction of the outer and inner segments, those of the "R" receptors breaking
somewhere in the length of the outer
segments (Figs. 2 and 3). For this reason
retinas isolated without the pigment epithelium contained only "R" receptor outer
segment portions.
This finding also suggests that the attachment of the receptors to the pigment
epithelium involves forces acting between
their intimately applied membranes. Fig.
5 shows an electron micrograph of an outer
segment of a presumptive cone at its junction with the pigment epithelium. Note that
the gap between the dark lines designating
some component of the outer membranes
of the receptor and that of the pigment cell
or its processes is of the order of 200 A, i.e.,
as close as such lines ever approach in
electron micrographs of contiguous cells
of any tissue fixed with osmium tetroxide.
Note also how the receptor membrane is
molded at one point around an irregularity
in the pigment cell membrane. "C" type
outer segments tended to have their apices
firmly applied to the cell body of the cells
of the pigment epithelium, whereas "R"
receptors often had their contacts confined
to the processes from these cells. Similar
intimacy of such membranes has been
shown in the rhesus monkey0 and man.14
It is obvious that a section in the plane
of the ellipsoids of the "C" receptors would
reveal cross-sections of the outer segments
of "R" receptors set among them and permit counting their relative numbers. Such
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a section is shown in a light micrograph
(Fig. 4) and gives a ratio of 4 "R" to 5 "C.'
This ratio was remarkably similar in the
few retinal areas thus far sampled.
The membranes of the receptor outer
segments seemed to be exquisitely sensitive
to conditions of fixation. With delayed
fixation, the flattened saccules, whose stacking results in the lamellar appearance of
the outer segments, exhibited a deterioration into fingerlike microvillous processes
seen as circular cross-sections (Fig. 5).
Another presumptive artifact seen was a
highly ordered rippling of the saccules
(Fig. 7). The mean thickness of the
saccules in the "R" receptors was 130 A
and in the "C" receptors 140 A. The respective frequencies were 45 and 40 sacs per
micron. These differences are not significant.
At the base of the "C" receptors in a
typical section about 7 to 15 of the flattened
saccules were seen to result from infoldings
of the cell membrane (Figs. 6 and 7) and
above this level for a short interval both infolded and noninfolded saccules were seen
(Fig. 7). The arcs of infolding are probably not in precise register and at least
some apparently noninfolding saccules
probably represent cases where the arc of
infolding was not in the plane of section.
Finally, in the remaining and major portion
of the length of the outer segment of the
"C" receptor, no infoldings were observed.
As the ability to observe an infolding relates to the probability of a plane of section
passing through the arc over which the
membrane infolds to form a saccule, it is a
function of the length of the arc. Accordingly, the rapid transition observed to
saccules exhibiting no connection with the
cell membrane suggests that the arc is
greatest basally and narrows rapidly. This
raises the question as to whether the process
proceeds to the point where the membranes
of the flattened sacs are no longer continuous with the cell membrane. Unfortunately, our cross-sections of "C" receptors
are not interpretable because of membrane
rippling and other distortions which have
212 Cohen
thus far prevented our obtaining good sections in the planes of the saccules.
The "R" receptors, on the other hand,
typically show no connection of the cell
membrane and the saccule membranes at
any point (Figs. 8 and 9). In two instances
one or two of the most basal saccules were
seen in longitudinal sections to be continuous with the cell membrane. Crosssections of "R" receptor outer segments
(Figs. 10 and 11) show no obvious connection of the saccules with the cell membrane and a shallow scalloping of the outer
perimeters of the saccules. In revealing two
classes of receptors differing in degree of
membrane infolding, these observations are
qualitatively similar to those previously
reported for the outer segments of extrafoveal rods and cones of the rhesus monkey," the only other mammal for which
such studies have been made. Here infoldings were observed in the basal third
of the outer segments of cones and only
at the very base of rod outer segments.
Monkey receptors show more infoldings
than do those of the squirrel, with monkey
rods approaching the number of infolds
seen in squirrel "C" receptors.
The outer segments of both receptor
varieties are connected with the inner segments by ciliary stalks. In both receptors,
calycal processes, fingerlike projections
from the apices of the inner segments, surround the bases of the outer segments. Both
ellipsoid regions are rich in mitochondria,
and the inner segments present no notable
distinctions from those of other mammals.4' G> 27 Contrary to the report of Pedlar
and Tansley,22 no fins or ridges of the inner
segment base (Fig. 12) were observed.
There was little noteworthy about the
receptor nuclei. Below this level, however,
the cytoplasm of the remainder of the cell
was rich in the small vesicles of 250 A
dimension (Fig. 13), now generally termed
synaptic vesicles.11'21> 2S Some of these
showed a halo of a fine granule (Figs. 14
and 15). In addition, curious membranous
cisternae, with walls consisting of either
two, four, or six membranes were observed
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Investigative Ophthalmology
April 1964
(Figs. 13, 14, and 15). This appearance
apparently results from sectioning domeshaped, loosely folded aggregations of
membranous cytoplasmic saccules (Fig.
15). Similar aggregations, similarly interpreted, were reported in the pedicles of the
lizard Phelsuma inunguis by Pedlar and
Tansley.22
The synaptic bases of the pedicles resemble those of other vertebrates described7
in the presence of invaginated or enveloped
synapses as well as superficial synapses
and synaptic ribbons or lamellae and their
associated structures (Fig. 13). What is
most striking in the squirrel is the presence
of what may be interreceptor synapses
(Figs. 13 and 16). Sjostrand2S described
these in the guinea pig and suggestions of
them were seen in the pigeon.s Lateral contacts between pedicle bases characterized
by an increased density of both contacting
membranes are common (Fig. 16). They
satisfy the conditions for Grays17 type 2
synapse. Sometimes these are evident at
the end of a process from one pedicle ending upon another, more often the contacts
occur between the flattened lateral aspects
of the bases, even as high as the nuclear
region. Contacts occur between cells having
their nuclei at different levels (receptors
with rodlike and conelike outer segments),
or between cells whose nuclei are at the
same level. On one occasion, however, but
perhaps significantly, a desmosome appeared to involve a receptor and a glial
cell of Miiller (Fig. 17), the latter identified by the presence of characteristic
fibrillar and particular material in its cytoplasm.5* G> 2S It is possible, however, that
this is a section through a tongue of Miiller
cell which slightly separates the edge of a
more deeply lying contact region involving
two receptors. In any event it must be
firmly noted that a "synapse" is a physiologic concept and can only be suggested
by morphology, and that where increased
membrane densities or desmosomes are involved in junctions of two cells, such findings are not exclusive to probable synaptic
sites.
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Fine structure of gray squirrel retinal receptors 213
Discussion
The use of the terms "rod" and "cone
in the literature is plagued by a confusing
evolution of what these are meant to designate. Although in many instances referring
to receptors with columnar or conical outer
segments, extensions of meaning involving
both physiologic and additional morphologic criteria are now common. These are
doubtlessly useful in particular cases, but
are not universally applicable. Thus, for
"rod" and "cone," the idea of scotopic and
photopic receptor was suggested by Walls33
on the grounds that clear columnarity and
conicity are not evident in many forms and
that the shapes may not universally indicate
these physiologic functions. However,
Walls' position is not satisfying. Ignoring
distinctions of form may retard the achievement of insight into other functional meanings of the widespread form differences in
receptors. Moreover, as Weale35 has emphasized, it is by no means clear that the
basis of scotopic versus photopic retinal
response lies in the receptors. In addition,
from terms referring to the outer segment
form, "rod" and "cone" have been extended
to refer to the shape of the entire receptor.
Thus receptors whose inner segments
possess diameters greater than those of the
outer segments may reveal an over-all
conical appearance even when the outer
segment is columnar rather than conical.
One is occasionally placed in the awkward
position of making statements such as, "the
foveal cones (i.e., photopic receptors) of
the monkey are rodlike (i.e., inner segment
diameters are similar to outer segment
diameters)." In addition, because the
synaptic bases of receptors which make
numerous synapses are pedicle-like whereas those making few synapses tend to be
knoblike or spherular, some such as
Granit16 have proposed that this feature is
the most reliable way to distinguish cones
from rods. That this aspect is not universally applicable is evident from the fact
that foveal photopic receptors of primates,
making few synaptic contacts, have endings
which are similar to spherules. Finally, the
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association of rhodopsins and porphyropsins
with rodlike scotopic receptors has occasionally led to the definition of a rod as a
receptor containing one of these pigments.
Because of the growing interest in the
fiber-optical properties of receptors,13 a
nomenclature related to form would appear
most desirable, where clear-cut distinctions
exist.
Turning to the gray squirrel, the overall shape of the receptors is conical because
the inner segments exceed the outer segments in diameter. As is usual in such cases,
all the receptors end in pedicles. These
aspects usually, but not always, have been
associated with photopic receptors. The
outer segments of all the receptors are
essentially columnar. This usually but not
always has been associated with scotopic
receptors. Rhodopsin has been extracted
from the retina. This has generally been
associated with scotopic receptors. The examination of the fine structure of the outer
segments of those receptors whose nuclei
occupy the outer (more sclerad) tier of
receptor nuclei shows that in their basal
portions there are many more infoldings of
the cell membrane to form the flattened
saccules than are evident in the outer segments of the receptors whose nuclei lie in
the inner (i.e., vitread) tier. Here infoldings are rarely seen. These features have
been associated with cones and rods, respectively, in frogs,20 toads,12-19 perch,29'31
monkeys,6 and pigeons.s Cross-sections of
outer segments of the lower tier receptors
reveal no contiguity or continuity with
the cell membrane. This has tended to be
associated with scotopic receptors.4' s
The evidence of the squirrel electroretinogram2' 32 was said to indicate the presence
of an "all cone" retina in that no Purkinje
shift was evident. In fact, such findings are
not inconsistent with the presence of substantial numbers of rhodopsin-containing
receptors, morphologically rodlike, or conelike. It is not yet clear whether a rod like
a frog rod is a scotopic receptor because
of its receptor properties per se or because
of the organization of this receptor in the
214 Cohen
frog retina. The presence of a Purkinje
shift relates in part to the presence of (1)
two or more classes of receptors characterized by different light absorption characteristics, and (2) differential sensitivity of the
retinal response initiated by one of these
classes. The sensitivity of response, in addition to relating to receptor properties, also
depends in part on (a) the relative numbers
of receptors in the classes, (b) the relative
degrees of convergence of neuronal chains
in the classes, and (c) the resultants of
inhibitory interactions of signals originating
from the receptor classes. Thus if 45 per
cent of the receptors in the gray squirrel
contained rhodopsin and these receptors
led to no greater degree of neuronal convergence than other squirrel receptors, the
absence of a Purkinje shift would not be
surprising. The squirrel retina, physiologically speaking, would be better described as a "photopic" rather than "all
cone" retina. This is not to argue however
that receptors participating in photopic or
scotopic vision may not have evolved in
physiologic, biochemical, or morphologic
directions which enhance their functional
efficiencies in a photopic or scotopic role.
However, the behavioral study of Arden
and Silver3 raises the strong possibility that
a more sensitive electroretinographic system would detect a Purkinje shift, and require that this retina not be regarded as
purely photopic.
A more difficult matter is the disparity
between the characteristics of the pigment
extracted from the squirrel retina by Dartnail10 (a rhodopsin-like pigment with an
absorption maximum of 502 m^), and supported by Arden and Silver's3 study of the
spectral sensitivity of squirrel behavior
versus those pigment properties inferred
from the reflection spectrophotometry difference spectrum of Weale34 (maximum
535 m/i.) and the electroretinographic response (maxima 490 and 533), as discerned
by Tansley, Copenhaver, and Gunkel.32
The possibility has already been indicated
that in removing the retina from the eye
one may selectively discriminate against
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Investigatioc Ophthalmology
April 1964
one type of outer segment which tends to
adhere to the pigment epithelium. Dartnall
has noted that, based on Weale's pigment
densities, his extraction yield was poor,
and the above observation may explain a
part of this loss although it is conceivable
that Dartnall's procedures avoid the artifact
consistently noted in this investigation. A
distinctive class of outer segment remaining behind may also contain a distinctive
pigment. This possibility requires investigation before evaluation of Dartnall's interesting and important hypothesis10 based on
the retinal sensitivity being a modulus of
the difference curve between two photosensitive pigments of the squirrel.
The presence of what appear to be interreceptor synapses is a finding of great
interest and tends to confirm the previous
report of such structures in the guinea
pig.2S Suggestions of such arrangements in
the pigeon were also seen in the form of
branching processes extending from the
synaptic bases and passing horizontally in
the retina.s Since the receptors are epithelial
cells, the possibility must be considered
that these desmosomes are epithelial desmosomes not concerned in the establishment
of synapses. By virtue of occurrence in a
region of the cell filled with synaptic
vesicles related to other synaptic functions,
such a structure would falsely meet the
morphologic criteria of a synapse. Indeed,
although a structure included in the broad
term desmosome appears to be associated
with all synaptic terminals,11' 1Ti 21 there is
no evidence as to whether it plays a role
in synaptic transmission or simply anchors
the synapse. The desmosomes seen in the
squirrel correspond to those of type 2
synapses of Gray17 and are the only kind
thus far associated with known inhibitory
synapses.1 On the other hand they also resemble desmosomes seen between glial cells
of Miiller in the monkey retina5 and one
example of a desmosome between a receptor and a Miiller cell has been presented in this report. It would be highly
desirable to see electrophysiologic evidences
bearing on whether functional interrecep-
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Fine structure of gray squirrel retinal receptors 215
tor synapses exist. It might be expected, by
analogy with interreceptor activity in
Limulus18 that they would be inhibitory, as
Sjostrand28 has already suggested. I have
recently observed similar rod-cone and
cone-cone contacts in peripheral human
retina.
The intimacy of the attachment of the
squirrel receptors to the pigment epithelium does not appear to leave much room
for a cementing substance unless it be a
component of the cell membrane. As
Sjostrand and Rhodin,20 Sjostrand,29 and
Robertson2'1 have observed, in osmium-fixed
materials the dark lines probably do not
represent the cell surfaces. Current
thoughts on membrane structure, based
largely on comparative studies of membranes fixed in either osmium tetroxide or
potassium permanganate suggest that the
dark line of the osmium-fixed material is
but the innermost component of the cell
membrane, probably protein. External to it
is a lipid layer and then probably a layer
of protein or mucoprotein. Thus the 200 A
gap between the dark lines of the receptor
and pigment cell of Fig. 5 may be partly
occupied by unstained cell membrane components. Unless the acid mucoprotein which
Sidman25 and Fine and Zimmerman15 noted
in the fluid of the ventricular space between the receptors and the pigment epithelium enters into the cell surfaces, it would
seem unlikely that it would play a role in
attachment of the retina. It is possible that
its role relates to the regulation of the refractive index of the medium bathing the
receptors.
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Discussion
Dr. George K. Smelser, New York, N. Y. Since
Max Schultze reported that retinas contained two
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Investigative Ophthalmology
April 1964
classes of photoreceptors, the rods and cones,
many modifications of his concept have been
necessary. Some were based on the shape of
these structures, so that "rodlike cones" were
noted in the fovea. Other classifications were based
on the type of synapses or photopigment which
the retina contained. The evidence for the
existence of two types of photoreceptors in the
squirrel retina has been contradictory. Now Dr.
Cohen's study has demonstrated that there are
two types of photoreceptors, one rodlike and the
other conelike. Apparently the "duplicity theory"
is safe once again. Dr. Cohen's paper is particularly good because he has carefully related
functional and chemical studies, which have been
done on the retinas of this species, with the
morphology of the visual elements, which he has
very expertly demonstrated.
It would be of interest to hear a microscopist
explain how he distinguishes artifact from reality,
with respect to the regularity of the arrangements of these ultramicroscopic membranes. How
does one decide that a technical procedure which
produces "rippling" is not as good as a technique
which does not do so? What is the significance
of the formation of saccules, as infoldings of the
outer membranes of the rod? Does the numerical
proportion of this type of saccule relate to the age
of the individual, or is it a character which is
more or less primitive, as determined by comparison of squirrel visual elements with those of
more primitive animals? What significance may
be ascribed to the fact that the cones are more
firmly attached to the pigment epithelium than
are the rods in this and other species?
I appreciate very much the opportunity of reading Dr. Cohen's manuscript. It is an excellent
study.
Dr. Cohen (closing). I thank Dr. Smelser for
his kind remarks. With reference to his first
questions, microscopists employ a parsimonious
approach, in that order is more likely to be real
than disorder, and that a procedure yielding
simple order is more likely to be revealing the
real than one yielding complex order. Certainly,
probability judgments based on accumulated experience are involved, and these are not easy to
codify. With reference to his second group of
questions, nothing is known regarding infoldings
except that they appear to be relatively more
numerous in photopic system receptors in all of
the few studies made. Finally, regarding the
relative degrees of attachment of the outer segments to the pigment epithelium, I have nothing
to suggest.