Histochemical Demonstration of Glycogen in Neurons of
the Cat Retina
Elisabeth Rungger-Brdndle,* Helga Kolb,\ and Gunter Niemeyer%
Purpose. To demonstrate histochemically the cellular distribution of particulate glycogen in
the cat retina and to correlate it with glucose sensitivity of neuronal electrical activities.
Methods. Free-floating, ultrathin sections of cat eyes (without glucose challenges) were stained
by the periodic acid-thiocarbohydrazide-silver proteinate procedure and examined by electron microscopy.
Results. Miiller cells were filled uniformly withfine-grainglycogen throughout all retinal layers.
Particle density was higher in Muller cells of the peripheral retina than in those of central
retina. Astrocytes contained little, if any, particulate glycogen. Alpha and beta ganglion cells
had a heavy content of clumped glycogen granules. Rod bipolar and A17 amacrine cells of
the rod pathway were stained intensely with particulate glycogen. No glycogen was seen in
photoreceptor cells, cone bipolar cells, and the majority of amacrine cells, including All cells
of the rod pathway. However, one type of cone bipolar-driven amacrine cell was intensely
glycogen positive. Its ultrastructural morphology, stratification pattern, and synaptology suggest that it is a wide-field, axon-bearing type called A22.
Conclusions. Except for the cone bipolar-driven ON-OFF A22 amacrine cell, it appears that
glycogen staining preferentially labels neurons of the rod pathway. These observations are
compatible with the reported sensitivity of the rod-driven electroretinogram and optic nerve
response to glucose in the cat retina. Invest Ophthalmol Vis Sci. 1996;37:702-715,
plasma can be manipulated, have indicated that the
rod-driven responses, b-wave, scotopic threshold responses,7 and optic nerve action potential (in vitro)
are affected by changes in plasma glucose concentration to a greater extent than the cone-driven responses
(Figs. 1 and 2 and refs. 8 to 10). Figure 1 shows the
effect on both the ERG b-wave and the optic nerve
response (ONR) in the isolated perfused cat eye of a
step increase in glucose content from control values of
5.5 mM to 10 mM. At increased glucose concentration,
scotopic threshold response is larger than normal.
Similarly, higher intensities of light elicit larger b-wave
amplitudes at increased plasma glucose concentrations. The compound action potential of the optic
nerve is correspondingly increased in amplitude unFrom, the ^Electron Microscopy laboratory, Department of Ofthtkalmalogy, University der elevated glucose concentration, and the correHospital, Geneva; the %NeuTophysiolagy ljilxrraUrry, Department of Ophthalmology,
sponding data from in vitro experiments are summaUniversity Hospital, Zurich, Switzerland; and the ^John Moran Eye Center,
University of Utah, School of Medicine, Salt Lake City.
rized in Figure 2. Clearly, the rod-driven ERG b-wave
Supported by the Swiss bonds for Prevention of Blindness, Swiss National Science
and ONR amplitudes are more affected by both inFoundation grant 31-32047.91, National Institutes of Health grant EY03323, and
Research to Prevent Blindness, Inc.
creased (positive values) and decreased (negative valSubmitted for publication August 23, 1995; revised December 4, 1995; accepted
ues) glucose concentrations than cone-driven b-waves
January II, 1996.
Proprietary interest category: N.
and ONRs.
Reprint requests: Elisabeth Rungger-Brdndle, Laboratoire de Microscopie
To determine which glial or neuronal compoelectronique, Clinique d'Ophtalmologie, HCUG, 22 rue AlcideJentzer, CH-I2II
nents of the cat retina store glycogen and thus are
Geneve 14, Switifrland.
A he dark-adapted function of the mammalian retina
is accompanied by an increase in oxygen consumption 1 " 3 and glucose use. 45 Energy in neuronal cells
arises from both glycolysis and oxidation of glucose
through the Krebs cycle.6 Glycogen stores in neuronal
and glial cells indicate high glucose metabolic and
oxidative need of the retina. The importance of glucose to normal electrical activity of the retinal neurons
is now becoming appreciated in the context of retinal
diseases of metabolic origin.
Electroretinogram (ERG) recordings from the arterially perfused isolated cat eye or from the anesthetized cat in which the glucose content of the blood
702
Investigative Ophthalmology 8c Visual Science, April 1996, Vol. 37, No. 5
Copyright © Association for Research in Vision and Ophthalmology
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703
Glycogen in the Cat Retina
ERG
likely to be most affected by glucose challenge, we
performed histochemical staining of glycogen in retinal thin sections from cat eyes that had not been challenged by increased glucose content before sacrifice.
Ultrastructural findings reveal that under these experimental conditions, neurons and glia thought to contribute to the scotopic components of the ERG response are filled with glycogen stores. In addition, we
found unexpectedly that a relatively rare amacrine cell
of the cone system is heavily glycogen positive. This
finding has allowed us a glimpse into its synaptology. A
preliminary report of this study has been presented."
METHODS
Experimental Animals
Cats were obtained from a breeding colony at CIBAGEIGY AG (Basel, Switzerland) kept at the animal
facility of the University Hospital and were treated in
accordance with the ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research and with
the Veterinary Authority of Zurich. Enucleation was
carried out on deeply anesthetized animals as described in detail previously.10'1213
Electrophysiology
For details of changing plasma glucose and recording
of electrophysiological light-evoked signals in isolated,
arterially perfused cat eyes, the reader is referred to
Niemeyer12 and Macaluso et al.9 As a typical example,
original recordings of an intensity series from threshold to 2.4 log units higher in a fully dark-adapted
preparation are shown in Figure 1 to illustrate effects
of increase in plasma glucose on the direct current
electroretinogram (ERG) and on the optic nerve action potential (briefly, the ONR). This intensity range
covers rod function selectively. The ERG was recorded
between a vitreal silver-silver chloride salt bridge and
an identical scleral reference electrode. The ONR was
recorded by means of a suction and a silver-silver
chloride surface electrode. A summary of the results
of a previous electrophysiological study, closely related
to the question addressed herein, is shown in Figure
2, in which data points represent maximal changes in
the amplitude of the ERG b-wave and of the ONR
under conditions of stepwise increases and decreases
in plasma glucose.
Electron Microscopy
Eyes were enucleated from animals that had received
no challenge with glucose or insulin before sacrifice.
The eyes were hemisected, and the posterior eyecups
were immersed in 0.1 M phosphate buffer (pH 7.0)
containing 2.5% glutaraldehyde and 0.2 mg/ml tannic acid for 30 minutes. The retinas attached to pigment epithelium and choroid were removed, cut into
ONR
-7.0
-6.9
-6.6
400 ms
FIGURE l. Effect of increase in plasma glucose on light-evoked
electrical field potentials recorded in a perfused cat eye. Original recordings of electroretinogram (ERG) and ONR7 of a
dark-adapted preparation at increasing light intensities (top to
bottom, indicated at left margin in log units of attenuation by
neutral-density niters). The thin traces are controls (c) at 5.8
mM glucose concentration, and the superimposed heavy traces
(e, for effect) were recorded at 10 minutes at an increased
glucose concentration of 10.3 mM. The ERG reveals at threshold a cornea-negative signal, the scotopic threshold response
(STR), that increases much like the b-wave in amplitude at
elevated glucose concentration (third trace from top, arrow).
The ONR also increases in all its components at elevated glucose concentration. Vertical calibration: 10 //V. The light pulse
of a duration of 400 msec is indicated as an upward deflection
of the lowermost traces.
small pieces, and left in fixative at room temperature
for an additional 5 hours. The tissue pieces were then
transferred to a 0.5% glutaraldehyde solution in phosphate buffer and stored overnight at 4°C. After extensive washing in 0.1 M cacodylate buffer (pH 7.4) at
4°C, the tissue was incubated in 0.8% potassium ferrocyanide in 50 mM cacodylate buffer for 30 minutes
and postfixed in 0.5% OsO4 in the same buffer for
1.5 hours at 4°C. Rinsing in distilled H2O, dehydration
in increasing ethanols, and rinsing in propylene oxide
preceded embedding in Epon using standard procedures. En bloc staining with uranyl acetate was omitted.
Some eyes were prefixed by arterial perfusion12
with a mixture of 1 % glutaraldehyde and 0.8% formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 6 min-
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704
Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5
c
CD
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change in glucose [mM]
change in glucose [mM]
FIGURE 2. (a) Effects of changes in plasma glucose concentration on electroretinogram
(ERG) and (b) optic nerve response (ONR) in the isolated cat eye. The scatter plot represents maximal changes in signal amplitude of rod-driven (filled symbols) and cone-driven
responses (open symbols). Control amplitudes of the responses (100% on the ordinate) at
the control glucose concentration of approximately 5 mM (0 on the abscissa, vertical line)
meet at the crossed lines. Response amplitudes are expressed (^100%) as a function of
decreased (negative values on abscissa) or increased (positive values on abscissa) glucose
concentration. Each data point represents the maximal change in one experimental trial
at the corresponding glucose concentration. Note that rod-driven signals are more glucose
sensitive than cone-driven signals. Modified from reference 9, with permission of LippincottRaven Publishers, Philadelphia, PA.
utes, opened at the ora serrata, cut into pieces, and
further fixed by immersion in 2.5% glutaraldehyde in
the same buffer for 4 hours. After extensive rinsing
in 0.1 M cacodylate buffer (pH 7.4), the tissue was
incubated in 0.8% potassium ferrocyanide in 50 mM
cacodylate buffer before osmication (30 minutes at
room temperature), dehydrated, and embedded as
above.
Glycogen Staining
Ultrathin vertical sections of pieces of Epon-embedded central and peripheral retina were cut on a
Reichert ultramicrotome (Merck, Zurich, Switzerland) and processed for glycogen particle staining.14
Free-floating sections were treated with 1% periodic
acid for 25 minutes, washed in distilled water (3 times
at 5 minutes each) and refloated on 0.2% thiocarbohydrazide in 10% acetic acid for 1 hour. The sections
were rinsed in decreasing series of acetic acid into
several changes of distilled water before being floated
on 1% aqueous silver proteinate in the dark for 1
hour. After final washes in distilled water, the sections
were mounted on copper grids, dried, and stained
with uranyl acetate and lead citrate. Sections were examined in a Philips CM10 (Geneva, Switzerland) or a
Hitachi H500 (Salt Lake City, UT) electron microscope.
RESULTS
Glycogen particles, as revealed by the histochemical
method we used, are visible in cells only at the magnification afforded by the electron microscope. Single
glycogen particles appear electron dense and measure
approximately 10 to 20 nm across. They correspond
in size to /^-particles according to the nomenclature
of Drochmans. 15 Typically in the retina, glycogen either is distributed as fine single particulate matter or
clumped into larger irregular electron densities measuring up to 50 nm in diameter.
Miiller Cells and Astrocytes Contain Glycogen
to Different Degrees
Single glycogen granules are found through all parts of
Muller cells. As shown in Figure 3, particles are present
in the Muller cell bodies and in their fine processes
ensheathing neuronal profiles in the nuclear (Figs. 3a,
3b) and plexiform layers (Fig. 3c). Where Muller cells
appose to form the inner and outer limiting membranes
of the retina, scattered fine-grain glycogen particles are
clear markers of this type of glia (Figs. 3a, 3b, 3d). At
the outer limiting membrane, it is particularly conspicuous that the glycogen is confined to Muller cell processes
and absent from inner segments of the photoreceptor
cells (Figs. 3a, 3b). At the inner limiting membrane, the
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705
Glycogen in the Cat Retina
FIGURE 3. Electron micrographs showing the histochemical localization and typical arrangement of glycogen particles {arrowheads) in Mtiller cells (M) of the peripheral retina. (a,b)
Miiller cell processes but not rod inner segments (RIS) contain glycogen granules at the
outer limiting membrane (OLM). Higher magnification inset shows the area of adherens
junctions (AJ) of the OLM. (c) Miiller cell process stretching through inner plexiform
layer (IPL). Fine glycogen particles are scattered throughout the Miiller cell cytoplasm and
occasionally are associated with membranes, (d) Subcellular compartments of Miiller cell
end feet enriched in either intermediate filaments (IF) or endoplasmic reticulum (ER).
Note that both subcompartments contain glycogen particles. ONL = outer nuclear layer;
ILM = inner limiting membrane. Magnification bars = 1 fj,m.
Miiller cell end feet again contain scattered glycogen
particles in subcellular compartments enriched in either
intermediate filaments or endoplasmic reticulum (Fig.
3d). The abundance of glycogen particles in Muller cell
end feet shows topographic variation with a much higher
density in the peripheral (Figs. 4a, 4b) than in the central (Fig. 4c) retina. Similar observations on regional
differences in glycogen content have been reported"'
earlier using another staining technique on the light
optical level.
In contrast to Muller cells, astrocytes surrounding
part of the blood vessels within the innermost retinal
layers17 virtually are devoid of particles in the peripheral retina (Figs. 4a, 4b) but appear to contain some
glycogen in the central retina (Fig. 4c).
Rod Bipolar Cells and A17 Amacrine Cells
Contain Glycogen
A vertical view of die inner nuclear layer of the cat retina,
where the cell bodies of the rod and cone bipolar cells
are packed together, showstfiatrod bipolar cells contain
an abundance of small glycogen granules whereas the
cone bipolar cells appear to be entirely devoid of glycogen (Fig. 5). Rod bipolar cell bodies are the most com-
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706
Investigative Ophthalmology 8c Visual Science, April 1996, Vol. 37, No. 5
— b
ILM
FIGURE 4. Electron micrographs showing glycogen granules {some of them denoted
by arrowheads) in glial celts in
the inner peripheral (a,b) and
central (c) retina. (a,b) Miiller
end feet (M) are densely
packed with glycogen particles, whereas astrocytic processes (A) appear empty, (c)
Both Muller end feet and
astrocytes contain small
amounts of glycogen particles.
V = vessel; Ax = axones; ILM
= inner limiting membrane;
IF = intermediate filaments.
Magnification bars = 1 fim.
mon bipolar cell bodies in the inner nuclear layer because the cat retina is a rod-dominated and rod pathwayrich retina. They are distinguished easilyfromcone bipolar cells by their darker cytoplasmic content and more
irregular nucleus with clumped chromatin (Fig. 5; ref.
18). In the outer plexiform layer, the dendrites of the
rod bipolar cells pass up to the rod spherules and enter
the spherules to become central elements at synaptic
ribbons. Many of these characteristic rod bipolar cell
dendrites are marked with glycogen granules (Figs. 5a,
5b). By contrast, die cone bipolar cell dendrites invaginating into cone pedicles or making basal junctions
show no sign of glycogen granules (Fig. 6c). In Figure
6c, we illustrate a rare observation in this material of
a heavily glycogen-filled lateral element (large arrow),
normally considered a horizontal cell dendritic ending,19 entering a cone pedicle to end alongside a synaptic
ribbon. We note this curious finding here, but otherwise
we found no sign of glycogen in any horizontal cell body,
dendrite, or axon terminal in the outer plexiform layer
(see Fig. 5).
Rod bipolar cell axons end in club-shaped expansions in the lowermost portion of the inner plexiform
layer (IPL) neuropil close to ganglion cell bodies. This
type of axon terminal also is noted to contain glycogen
particles, although perhaps not as abundantly as den-
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fit
Glycogen in the Cat Retina
rb
INL
M
FIGURE 5. Electron micrograph of the neurons of the
inner nuclear layer. Rod bipolar cells (rb) contain scattered glycogen granules (arrowheads) in cytoplasm, but
to a lesser degree than do
Muller cell processes (M).
Cone bipolar cell (cb) cytoplasm is free of glycogen
granules as are horizontal
processes (A-type horizontal
cell [HC] dendrite is shown)
in the outer plexiform layer.
Magnification bar = 2 //m.
drites (Figs. 6d, 6e). Thus, the whole rod bipolar cell,
from finest dendrites to axonal endings, contains glycogen in this histochemically stained material.
Two amacrines are commonly postsynaptic to ribbons in rod bipolar axon terminals, namely, the small
bistratified rod amacrine known as All and the widefield amacrine A17 which, by its reciprocal synapse
with rod bipolar axon terminals, commonly is characterized as the other partner of the postsynaptic dyad
with the All amacrine.l8'20~a4 Of these two amacrine
cells postsynaptic to rod bipolar axons, the A17 is
stained with the histochemical procedure for glycogen
(Figs. 6d, 6e). The A17 is recognized by its large, bulbous, relatively electron-lucent synaptic endings with
clear clustering of synaptic vesicles at the reciprocal
synapse it makes to the rod bipolar axon terminal
(Figs. 6d, 6e). By contrast, the All amacrine cell's dendrites postsynaptic to the rod bipolar axon do not
contain any glycogen granules, nor do All amacrine
cb
X•
V
cell bodies, lobular appendages, or main dendritic
trunks in distal IPL show any sign of glycogen staining.
On the other hand, the A17 cell body in the amacrine
cell layer of the inner nuclear layer does contain a
light peppering of glycogen granules, as can be seen
in Figure 8. Based on this staining pattern, we have
calculated the fraction of A17 cell bodies in one particular cat retina as between 15% and 16% of the total
amacrine cell population (Table 1).
Ganglion Cells Contain Abundant Glycogen
Granules
Figure 7a shows a montage of electron micrographs
of a ganglion cell body and primary dendrite passing
into the IPL. The cell body is consistent with its being
a beta ganglion cell type of the cat retina1825"27 and
is characterized by a large nucleus and clear cytoplasm
containing abundant Nissl substance and clusters of
small mitochondria. The glycogen granules are dis-
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Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5
708
l. Frequency of Glycogen-stained
Amacrine Cells
TABLE
Frequency (%) of Staining Pattern
n
Light
Dense
Cells Unlabeled Dispersed? Clumpedf
Central retina]:
Peripheral retina
402
307
80.8
78.8
14.9
16.3
4.2
4.9
Frequency was determined on ultrathin sections of six tissue
blocks from different regions of one cat retina. *Al7-type; fA22type; Jwithin 3 mm of area centralis.
tributed fairly evenly throughout the cytoplasm and
often appear to be clumped into larger masses. Vertically running Muller cell processes, filled with glycogen, can be seen surrounding the ganglion cell body
(Fig. 7). The inset (Fig. 7b) shows similar-appearing
ganglion cell dendrites, one of which is postsynaptic
to a cone bipolar ribbon synapse in the distal neuropil
(sublamina a) of the IPL, a distinctive feature for recognizing ganglion cell dendrites.18 Again, the ganglion cell secondary dendrites are filled with glycogen
granules (Fig. 7b). The larger, easily recognizable ganglion cell dendrites belonging to the alpha- and betatype ganglion cells running in all parts of the IPL
neuropil, have similar appearances with similar complements of glycogen granules. We suspect all ganglion cells in cat retina contain glycogen although it
is those belonging to alpha and beta types that are
most easily discernible in the tissue.
A Rare Cone System Amacrine Cell Has a
Particularly Large Glycogen Content
In contrast to the light content of fine glycogen particles seen in amacrine cells that can be recognized as
A17 cells,21'28 another less commonly occurring amacrine cell type (frequency 4% to 5%, Table 1) contains
a heavy content of glycogen (Fig. 8a). Note that this
amacrine cell has a large cell body, dark cytoplasm,
and many wavy endoplasmic reticulum-like membranes arranged in parallel. The glycogen commonly
is clumped into larger irregular densities measuring
as much as 50 nm across compared with the smaller,
discrete, single particles (10 nm size) found in rod
bipolar cells, Muller cells, and A17 amacrine cell profiles (Fig. 8b).
Large-diameter dendrites running in the neutropil of the IPL have a similar cytologic appearance as
the above cell bodies, with a dark cytoplasm, collapsed
parallel membranes, and a huge mass of clumped glycogen. The longest horizontally coursing dendrites
are seen in stratum 4 of the IPL (the IPL can be
conveniently divided into five strata starting with stratum 1 at the amacrine cell layer-IPL border and ending with stratum 5 at the IPL-ganglion cell layer bor-
der; ref. 29). Such profiles are seen in Figures 9b and
10a, 10b, and lOd. Very large cross-sectioned profiles
in stratum 1 (Figs. 9a, 10a), resembling those in stratum 4, are interpreted as being the major primary
dendrites coming from the cell body and ending up
in stratum 4. The large cell body size, large caliber of
the dendrites, and lack of spines on these dendrites,
taken together with their eventual stratification in stratum 4 of the IPL, prompted us to identify this heavily
glycogen-positive amacrine cell as an A22 cell, described originally in a Golgi study.25 This wide-field
amacrine cell type is also axon bearing and has been
recorded as an ON-OFF amacrine cell in the cat retina.3031
Further details of this putative A22 amacrine cell's
synaptology are revealed in Figures 9 and 10. In stratum 4 of the IPL, the putative A22 major dendrites
appear to be presynaptic and postsynaptic to cone
bipolar cells (Figs. 9b, lOd) and to unknown amacrine
cells (Fig. lOd). We also have seen A22 dendrites in
stratum 4 to be presynaptic themselves to other profiles of a similar sort, possibly other A22 dendrites
(Fig. 9b). We can recognize two different types of cone
bipolar cell axons to be presynaptic to the major A22
dendrites in stratum 4. One type runs horizontally in
stratum 4 (Fig. 9b, cone bipolar cell [cb]6) and does
not appear to have invaginations into its axon terminal, whereas the other type, seen in strata 4 and 5, is
characterized by these invaginations (Figs. 9a, lOd,
cb5). The former we interpret as cb6 and the latter
as cb525V2 (see Discussion).
In stratum 1 of the IPL, the large-caliber A22 dendrites are postsynaptic to caliber profiles of similar
appearance. In other words, they appear to be other
A22 dendrites, characterized by a small cluster of synaptic vesicles along an unspecialized, parallel pair of
synaptic membranes (Fig. 10b). By contrast, another
glycogen-containing amacrine cell profile is more
electron lucent and contains a definite cluster of vesicles directed at a long, stiff-looking parallel synaptic
membrane assembly (Figs. 10a, 10c). We have not
been able to identify this larger electron-lucent amacrine profile with certainty. However, because we
know A22 cells have long, axon-like processes that run
up stratum 1 for long distances (measured in millimeters) away from the parent cell dendritic field,31it is
possible that they represent the axon terminals of A22
cells from another part of the retina.
DISCUSSION
If energy stores become exhausted, for example, in
prolonged hypoglycemia, the rod-driven components
of the ERG diminish and ultimately collapse.4'10'33 In
the brain, it has been shown that glycogen content
decreases under extreme hypoglycemia and correlates
with an accompanying deterioration in the EEG.34 It
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Glycogen in the Cat Retina
RS
FIGURE 6. Electron micrographs of rod bipolar cell dendrites and axons. (a,b) Rod bipolar
dendrites, entering rod spherules (RS) to make synapses at ribbons, contain glycogen particles (arrowheads). (c) In comparison, dendrites entering cone pedicles (CP), to end at ribbon
synapses, do not contain glycogen. A heavily labeled horizontal cell process (arrow) ending
as a lateral element at a ribbon, occasionally is filled with glycogen. (d,e) Rod bipolar axon
terminals (rbat) contain a smattering of glycogen particles and make characteristic ribbon
synapses (small arrows) to nonglycogen-containing All amacrine cell dendrites and to glycogen-positive A17 amacrine cell profiles. The latter make reciprocal synapses to the rbats
[arrows). Magnification bar = 0.5 /zm.
seems reasonable, therefore, to expect a similar decrease in glycogen stores in the retina under conditions of blood glucose drop, hypoglycemia, or lower
oxygen availability. Even the change in metabolic activity of the retina from light to dark can be measured
as increased turnover of glycogen in frog retina,35 differences in glucose uptake,5 or increased oxygen consumption. '•"
Although the absolute glycogen content of the
mammalian retina has not yet been measured after
hypoglycemia, oxygen challenge, or even dark adaptation, the electrical responses of the retina have been
studied under experimental changes in plasma glucose. The curious finding emerges that the rod-driven
components of the ERG b-wave, scotopic threshold
responses, and ONR are more affected than the conedriven components under these experimental conditions (Fig. 2, refs. 8-10, 36). During dark adaptation,
there is known to be a dramatic increase in oxygen
consumption1 and glucose use'1'5 at the photoreceptor
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710
Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5
FIGURE 7. Electron micrographs to show glycogen content of ganglion cells, (a) A
beta ganglion cell contains
clumped glycogen granules
{arrotoheads) in the cytoplasm
of the cell body, extending
into the dendrites in the inner
plexiform layer (IPL). M =
Muller cell processes, (b) The
glycogen-containing ganglion
cell dendrites are found
throughout the IPL and are
postsynaptic to cone bipolar
cell axons (cone bipolar cell
[cb], small thick arrow) and to
amacrine cell profiles (A, tiny
thin arrmv), both of which are
nonglycogen containing. Magnification bars = 2 ^m (a), 0.5
j3GC
fim
level of the outer retina. Thus, one might expect the
PHI component of the ERG to be most affected under
glucose challenge rather than the b-wave generated
in the inner retina. The findings of greater sensitivity
of the rod-driven b-wave of the ERG to low plasma
glucose levels suggests that the energy requirements
of the neurons in the middle and inner retina barely
are met under dark-adapted conditions.10'33
To investigate this issue, we performed histochemical labeling of glycogen and found that both Muller cells
and neurons of the middle and inner retina contained
stores of glycogen granules. Muller cells have long been
implicated in the generation of die b-wave,a7~41 although
probably secondary to the rod bipolar cells of the middle
retina42"45 in a rod-dominant mammalian species such
(b).
as the cat. The fact diat Muller cells contain heavy stores
of glycogen in the peripheral retina may be indicative
of their major contribution to energy supply in these
poorly vascularized regions. We only can speculate why
neurons did not display such regional differences. During development, the capacity to store glycogen is imperative for neurons, such as ganglion cells, that differentiate early and long before Muller cells are generated and
the retina becomes vascularized. Although on vascularization Muller cells might adapt to abundant energy supply through the vascular bed by eventually reducing their
glycogen stores in highly irrigated regions, neurons that
are not in intimate contact with blood vessels may maintain their capacity to store glycogen independent of vascular density.
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Glycogen in the Cat Retina
•7U
A17
A22
^
am
FIGURE 8. Electron
•••?*
micro-
graphs to show the A22 amacrine cell, (a) A22 cell body
in the inner nuclear layer is
stained heavily for glycogen
granules. A neighboring A17
cell is stained lightly, and another unidentified amacrine
cell (am) is totally devoid of
glycogen. (b) Higher-magnification micrograph shows
the clumped nature of the
glycogen staining in the A22
cells cytoplasm compared
with the particulate glycogen
in Milller cell (M) processes.
Magnification bars = 2 fim
M
(a), 0.5 ^m (b).
In this material, neither rod nor cone photoreceptors were glycogen positive, whereas rod bipolar cells
were labeled preferentially over cone bipolar cells.
This is in agreement with the concept that the photoreceptors are supplied energy primarily by oxidative
metabolism of glucose from the choriocapillaris.33 4(i
In addition, the concept put forth by Ames et al33 that
energy is supplied to the inner retina by glycolysis is
consistent with our observations in the cat of strong
staining for glycogen in certain bipolar, amacrine, and
ganglion cells. This concept is supported further by
the marked sensitivity of the inner retina (expressed
. Mi it
- - - - - u—
fit
in the light-evoked scotopic threshold response and in
the optic nerve action potential) to changes in glucose
concentration.9
Our surprising finding that the A17 amacrine cell
of the rod system is strongly glycogen positive (Figs.
5 to 8) suggests as large a role in the generation of
the scotopic b-wave as that of the rod bipolar cell.
Both are depolarizing units, as recorded intracellularly,21:1AA1 and both could thus contribute to the
massed potential of the b-wave. The contribution of
the rod bipolar cell response to the b-wave certainly
is expected from the intracellular recording studies
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Investigative Ophthalmology & Visual Science, April 1996, Vol. 37, No. 5
FIGURE 9. Electron micrographs showing A22 primary
dendrites in the inner plexiform layer (IPL). (a) A largediameter primary dendrite
on the strata '/ a border has
die cytological characteristics
of the stained cell body (asterisk; see Fig. 8a, A22) and is
filled widi large clumps of
glycogen. No synapses occur
on this dendrite in this section, (b) Two A22 major dendrites run parallel to each
odier in stratum 4 of die IPL.
They contain glycogen and
are synaptic (large arrows) on
other A22 dendrites and a
cone bipolar cell (cb)6 type
of cone bipolar cell. One of
the A22s receives a ribbon
synapse from die cb6 bipolar
A22
M
M
A22
cb6
1
cell (small thick arrow). Two
A22
nonglycogen-containing
amacrine cells (a) are presynaptic to the cb6 (arrowheads).
Magnifications bars = 2 fim
(a), 0.5 //m (b).
and pharmacologic effects of ON-center antagonists
such as APB (2-amino 4-phosphonobutyric acid) on
the b-wave,42"45 but to our knowledge the contribution
of the A17 cell has never been considered.
The A17 is a GABAergic and serotonin-accumulating neuron of the mammalian retina that appears in
high density in rod-dominated retinas such as cat, rabbit, and human.22'48*49 Its only synaptic interactions are
A serendipitous finding of this electron microscopic study was the prominent histochemical reaction
for glycogen in a putative A22 amacrine cell. The
Golgi-impregnated appearance of A22 is that of a
wide-field cell with a large cell body and sturdy dendrites running in stratum 4 of the IPL.25 This cell type
is axon bearing and electrophysiologically is considered an ON-OFF type.30'31 We found here that the
the reciprocal synapses with its input neuron, the rod
A22 was a cone system-driven amacrine cell, and its
bipolar cell, as seen in Figures 6d and 6e. However, it
is probably under dopaminergic amacrine cell control
because synapses from the tyrosine hydroxylase immunoreactive cell in the cat retina are common on A17's
cell body and on fine dendrites in stratum 1 and 2 of
the IPL.2850 An effect of low dopamine levels on the
size of the ERG b-wave has been recorded in human
patients with Parkinson's disease,51 'aZ experimental
Parkinsonism in monkeys,53'54 and perfused cat eyes.55
The dopamine-compromised effect on the ERG bwave has always been assumed to be caused by an
effect on the neurons responsible for the proximal
negative response and/or oscillatory potential in the
ERG, putatively the All amacrine cells.5'3 Perhaps in
the future we shall have to consider A17 amacrine cell
involvement as well.
major bipolar input was from what appeared to be two
morphologically distinct cone bipolar cell axons: one
putatively a cb6 type, which we know from intracellular
recordings to be an OFF-center type,32 and the other
a cb5 type,18'32'57'58 which we know to be ON-center.32
Thus, the ON-OFF physiology of the glycogen-rich
A22 cell is explained most easily by its bipolar input.
We could find no clear output to ganglion cells from
this cell type, so it does not appear to be able to drive
ganglion cells direcdy.
Judging by their synaptic interactions and putative
intraretinal axonal connections on one another, we
propose that A22 cells are involved in long-range communication from one side of the retina to another.
Their reciprocal synapses on cone bipolar cells might
lead to frequency doubling characteristics of ganglion
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713
Glycogen in the Cat Retina
FIGURE 10. Electron micrographs of synaptic relationships of the A22 dendrites in the inner
plexiform layer (IPL). (a) A large A22 primary dendrite in stratum 1 of the IPL is postsynaptic
to a vesicle-filled amacrine profile (arrows) that could be an axon of another A22 cell (A22?).
(b) Another large diameter A22 dendrite in stratum 2 of the IPL is postsynaptic to both
another A22 major dendrite (arrow) and to another putative A22 axon terminal (A22?).
The characteristic curved, stiff synapse of the putative axon is cut en face (arrowheads), (c)
A putative A22 axon terminal makes a long, curved synapse (arrow) on a similar-appearing
putative A22 axon in stratum 3 of the IPL. (d) A cross-section of an A22 primary dendrite
running in stratum 4 of the IPL. A cone bipolar type with imaginations of postsynaptic
dendrites into its outer surface, which is characteristic of a cone bipolar cell (cb)5 type of
cone bipolar cell, is presynaptic to the A22 dendrite (arrow). Anodier nonglycogen-containing amacrine profile also makes a synapse on the A22 dendrite (a, arrmvhead). Magnification bars = 0.5 fj,m.
cells postsynaptic to these cone bipolar cells as well.
Because they are transient, spiking neurons of the
retina,31 A22 cells might be expected to have high
energy requirements, as do ganglion cells—hence,
their high glycogen stores. More complete evaluation
of intracellularly labeled A22 cells, with full axon-like
processes, eventually will have to confirm the initial
interpretations presented here.
In conclusion, we have described a specific pattern of glycogen stores in Muller cells and subclasses
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Investigative Ophthalmology 8c Visual Science, April 1996, Vol. 37, No. 5
of neurons of the cat retina. High glycogen levels are
confirmed in energy-rich spiking neurons, such as
ganglion cells and ON-OFF amacrine cell types. Our
findings point out the particularly high content of
glycogen in the second-order neurons subserving the
rod system. These observations corroborate the electrophysiological evidence of a marked sensitivity of
the rod system to small changes in glucose concentration in vitro and in vivo. By contrast, the virtual absence of glycogen in cone bipolar cells correlates well
with the relative insensitivity to changes in plasma glucose, under the same experimental conditions, of the
cat's cone system.
Key Words
12. Niemeyer G. Neurobiology of perfused mammalian
eyes. /Neurosci Meth. 1981;3:317-337.
13. Niemeyer G. The isolated perfused mammalian eye.
In: Kettenmann H, Grantyn R, eds. Practical Electrophysiological Methods: A Guide for in vitro studies in Vertebrate
14.
15.
16.
17.
18.
amacrine cell, bipolar cell, electron microscopy, ganglion
cell, glycogen
19.
Acknowledgments
The authors thank Franziska Werren, Urszula Englert, Luisa
Iuliano-Dello Buono, and Alain Conti for skillful technical
assistance, and they thank Laura DeKorver for excellent
help with the photography.
20.
21.
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