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MICROSCOPY RESEARCH AND TECHNIQUE 36:43–56 (1997)
Light Adaptation Affects Synaptic Vesicle Density
But Not the Distribution of GABAA Receptors
in Goldfish Photoreceptor Terminals
STEPHEN YAZULLA* AND KEITH M. STUDHOLME
Department of Neurobiology and Behavior, State University of New York, Stony Brook, New York 11794
KEY WORDS
GABAA receptors; immunocytochemistry; retina; photoreceptors; light adaptation; monoclonal antibodies
ABSTRACT
GABA is a likely feedback transmitter from H1 horizontal cells to cone photoreceptors in fish retinas. Spinules arise from H1 cell dendrites in light-adapted retinas, are correlated
with responses attributed to feedback, and have been proposed to be the GABA release sites. We
used mAb 62-3G1, an antibody against the b2/b3 subunits of the GABAA receptor complex, to
visualize GABAA receptor immunoreactivity (GABAr-IR) in photoreceptors as a function of light and
dark adaptation at the electron microscopical level. Regardless of adaptation, GABAr-IR was
restricted to the synaptic terminals of all cones and most rods; synaptic vesicular membrane and
plasma membrane exhibited GABAr-IR. Contrary to expectations, the density of GABAr-IR was
least on the plasma membrane within the invagination, regardless of the presence or absence of
spinules. Dense GABAr-IR was observed on the lateral surface of cone pedicles, on cone processes
proximal to the invagination, and on presumed telodendria from nearby cones. There was no
difference in GABAr-IR of rod plasma membranes within or outside of the invagination or with
adaptation. The only novel effect of adaptation was in regards to the density of synaptic vesicles.
Cones showed a 29% increase in vesicle density with dark adaptation, whereas rods showed a 17%
decrease. We conclude that all goldfish photoreceptors will be GABA-sensitive and that the
sensitivity is distributed over the surface of the synaptic terminal rather than localized to within the
invagination. The role of spinules in GABA release remains to be determined, but we conclude that
spinules are not related to the GABA sensitivity of goldfish photoreceptors. Microsc Res Tech
36:43–56, 1997. r 1997 Wiley-Liss, Inc.
INTRODUCTION
There is overwhelming evidence supporting the hypothesis that H1 horizontal cells of the fish retina are
GABAergic, providing a feedback inhibition onto cone
photoreceptors (for review see Yazulla, 1986). This
inhibition appears to be mediated by a bicucullinesensitive, GABA-activated chloride conductance
(Djamgoz and Ruddock, 1979; Murakami et al., 1982a,b;
Toyoda and Fujimoto, 1983), indicative of GABAA receptors. The actual sites of GABA feedback from horizontal
cells to cones have not been identified, and the function
of this feedback still generates considerable controversy (for review see Burkhardt, 1993; Wu, 1992).
Horizontal cell dendrites that invaginate cone pedicles
do not contain synaptic vesicles or other specializations
typical of chemical synapses (Stell, 1967; Witkovsky
and Dowling, 1969), although small conventional synapses have been observed in the horizontal cell body
(Marc and Liu, 1985; Witkovsky and Dowling, 1969).
There is strong evidence indicating that H1 horizontal
cells release GABA by a sodium-dependent transport
carrier (Ayoub and Lam, 1984; Schwartz, 1982, 1987;
Yazulla and Kleinschmidt, 1982, 1983), a process that
would eliminate the need for synaptic vesicles. H1
horizontal cell dendrites contain electron-dense spinelike protrusions that invaginate the cone pedicle (Stell,
r 1997 WILEY-LISS, INC.
1967). These spinules display a striking degree of
plasticity with changes in ambient illumination. Numerous spinules protrude from the distal ends of horizontal
cell dendrites in the light-adapted state but retract and
eventually disappear with dark adaptation (Raynauld
et al., 1979; Wagner, 1980). The presence and disappearance of the spinules is directly correlated with light
adaptive electrophysiological events (i.e., chromaticity
responses in C-type horizontal cells), leading to the
suggestion that the spinules may be the sites of feedback interaction (Djamgoz et al., 1988; Weiler and
Wagner, 1984). Cones of all spectral classes are innervated by the GABAergic H1 horizontal cells, (Stell and
Lightfoot, 1975; Stell et al., 1975), indicating that all
cones should be GABA-sensitive.
GABAA receptors have been localized to the fish outer
plexiform layer (OPL) using 3H-muscimol binding (Lin
and Yazulla, 1994) and immunocytochemical detection
of monoclonal antibodies (62-3G1) (Vitorica et al., 1988)
against the b2/b3 subunit of the GABAA receptor/
benzodiazepine receptor/Cl2 (GABAA/BZDr/Cl2) chan-
*Correspondence to: Stephen Yazulla, Dept. Neurobiology and Behavior,
SUNY, Stony Brook, NY 11794-5230.
Received 27 June 1994; Accepted in revised form 12 October 1994.
44
S. YAZULLA AND K.M. STUDHOLME
nel complex (Yazulla et al., 1989). GABAA receptor
immunoreactivity (GABAr-IR) was found on the plasma
membrane and intracellularly on synaptic vesicle membrane of all cones and to a lesser extent in rods.
Curiously, there was no relative increase in GABAr-IR
on cone membranes in the region of horizontal cell
dendrites, the area of presumed GABA feedback. There
are several possible explanations. First, the retinas
used in that study were obtained from dark-adapted
animals and dissected in a mesopic state. As a result of
this procedure, there were very few horizontal cell
spinules in the cone pedicles. Since spinules have been
implicated in GABAergic feedback, there could have
been a reduction in the level of GABAA receptors on the
cone plasma membrane in the absence of the spinules.
Second, the large amount of presumed intracellular
GABAr-IR on synaptic vesicles could have been due to
diffusion of the diaminobenzidine (DAB) reaction product from the plasma membrane. Third, since mAb
62-3G1 had not been characterized in any retinal
tissue, it was possible that 62-3G1 did not recognize
GABAA receptors in goldfish retina. However, this
appears not to be the case, because Lin and Yazulla
(1994) recently showed that mAb 62-3G1 immunoprecipitated 3H-muscimol binding activity from detergentsolubilized goldfish retinal membranes and, on immunoblots, reacted with 55–57.5 kDa Mr polypeptides, similar
to bovine brain (Park and De Blas, 1991). Therefore, we
have reinvestigated the distribution of mAb 62-3G1–
derived immunoreactivity in goldfish photoreceptors
using pre- and postembedding immunocytochemical
techniques but as a function of light and dark adaptation to determine if there is a relationship between the
distribution of GABAr-IR in the outer plexiform layer
and the state of horizontal cell spinule formation.
MATERIALS AND METHODS
Subjects
Goldfish (Carassius auratus), approximately 10–13
cm standard body length, were obtained from Mt.
Parnell Fisheries (Mercersburg, PA), maintained in
aerated tanks at 22°C filled with tap water circulating
through a polyester fiber/charcoal filter system, and fed
crushed Purina trout chow. The major light source was
a 60 W tungsten lamp about 1 m over the fish tanks. A
12/12 h light/dark cycle was controlled by a timer which
turned the light on at 7 AM and off at 7 PM. Fish were
either light-adapted under room light fluorescence or
dark-adapted for 3 h, after which dissections took place
in the early afternoon. Goldfish were decapitated (in
compliance with procedures for the sacrificing of small
animals). The eyes were enucleated and hemisected.
Dissections of dark-adapted fish occurred under indirect red illumination (25 W, BCJ red safety light).
Immunoreagents
The production and characterization of mAb 62-3G1
has been reported in detail elsewhere (Vitorica et al.,
1988). This monoclonal antibody was raised against the
GABAAr/BZDr/Cl2 complex from bovine brain that
purified by affinity chromatography on the immobilized
benzodiazepine R07-1986/1. In goldfish retina, mAb
62-3G1 reacts with 55–57.5 kDa Mr polypeptides on
immunoblots and immunoprecipitates 3H-muscimol
binding activity but not 3H-flunitrazepam binding activity (Lin and Yazulla, 1994). Goat anti-mouse IgG and
mouse peroxidase-antiperoxidase (PAP) were purchased from Dako (Carpinteria, CA); goat anti-mouse
IgG conjugated to fluorescein isothiocyanate (FITC)
was obtained from Boehringer-Mannheim (Indianapolis, IN).
Immunocytochemistry
Preembedding Procedures. Following enucleation,
half-eyecups were placed vitreous-side down on a type
HA Millipore filter (0.45 µm) on a Swinnex filter holder.
Light suction was applied to remove the vitreous humor
and to provide a stable mechanical support for slicing
and subsequent handling of the retina. Procedures for
preembedding immunocytochemistry were as described
in Eldred et al. (1983) and Yazulla et al. (1989). In brief,
retinas were fixed for 1 h in 4% paraformaldehyde and
0.15% glutaraldehyde in 0.1 M sodium phosphate buffer
(pH 7.4) at 20°C. After 15 additional minutes in the
same fixative without glutaraldehyde, retinas were
fixed overnight in 4% paraformaldehyde in 0.1M sodium bicarbonate buffer (pH 10.4) at 4°C. Retinas were
then washed briefly, postfixed in 2% OS O4 at 4° for 1 hr
in 0.1 M sodium phosphate buffer (pH 7.4) with 4%
sucrose and 0.15 M CaCl2 (rinse buffer). The tissue was
washed for 30 min in rinse buffer, incubated for 30 min
in 1% sodium borohydride in rinse buffer, and washed
in several changes of rinse buffer for at least 1 h (until
bubble formation ceased) in order to restore much of the
immunoreactivity which would otherwise have been
masked following glutaraldehyde fixation. The retina
was cryoprotected (30 min in rinse buffer with 5%
glycerin 1 15% sucrose, 1 h in rinse buffer with 10%
glycerin 1 15% sucrose, 12 h in rinse buffer with 10%
glycerin 1 20% sucrose), frozen on dry ice, and thawed
to enhance the penetration of immunological reagents.
Tissue samples were then incubated for 48 h at 4°C
with hybridoma culture media containing the mAb
62-3G1 at a 1:50 dilution. Visualization of the antibody
labeling was accomplished with a standard peroxidaseantiperoxidase technique (PAP). Conditioned medium
of the parental myeloma P3X63Ag8.6.5.3 was used as a
control. Controls showed no immunocytochemical staining. Previous studies showed displacement of mAb
62-3G1 immunoreactivity after prior incubation of the
antibody with 9 µg of affinity-purified GABAr/BZDr
complex (De Blas et al., 1988).
Retinal slices were dehydrated and embedded in
Durcupan A.C.M. resin. Ultrathin sections were collected on formvar-coated slot grids and, unless stated
otherwise, were viewed, photographed, and presented
in this paper without heavy metal counterstaining so
that the immunoreaction product would not be confused with electron density imparted to the membranes
by heavy metal staining.
Postembedding Procedures. Serial 1 µm sections
were collected on Fisher Superfrost slides. The resin
was etched in saturated sodium ethanolate solution for
30–45 min at room temperature (RT). Slides were
rinsed in 100% ethanol (3 3 5 min) followed by distilled
H2O rinse (3 3 5 min) and then incubated in 1% sodium
periodate for 7 min at RT to remove OsO4. Sections
were rinsed in distilled H2O and incubated in 1%
sodium borohydride for 30 sec to reduce autofluores-
GABA RECEPTORS IN PHOTORECEPTORS
cence of the tissue due to aldehyde fixation. Sections
were incubated in mouse monoclonal mAb 62-3G1
antibodies at concentrations of 1:10 to 1:100 for 48 h at
4°C. Tissues were incubated in goat-anti-mouse IgGs
conjugated to FITC for 30 min at 37°C.
RESULTS
General Observations
Consistent with our previous findings (Yazulla et al.,
1989), GABAr-IR was concentrated in the distal portion
of the axon and synaptic terminals of rods and cones;
this was the case regardless of whether the retinas
were light- or dark-adapted (Fig. 1). The state of
adaptation at the time of fixation could be verified at
the light microscopical level in that cone myoids are
fully contracted and rods fully extended in the lightadapted state (Fig. 1A); the reverse situation is observed for dark adaptation (Fig. 1B) (Ali, 1971; Burnside and Nagel, 1983). In well-oriented sections,
GABAr-IR could be followed from the synaptic terminals along the axon into the layer of rod nuclei (Fig. 1,
arrowheads).
It was clear from Figure 1 and from Yazulla et al.
(1989) that GABAr-IR is contained within the photoreceptor terminals rather than confined to the plasma
membrane. It was possible that the intracellular
GABAr-IR was due to diffusion of DAB reaction product
during processing. However, visualization of GABAr-IR
with a postembedding technique on 1 µm thick resin
sections (Fig. 2) clearly shows that the photoreceptor
terminals are labeled uniformly with GABAr-IR rather
than outlined as would be expected if receptors were
confined to the plasma membrane. Notice that
GABAr-IR can be followed along the cone axon for short
distances into the outer nuclear layer (ONL) with both
preembedding and postembedding procedures (compare Figs. 1 and 2). Thus, the intracellular distribution
of GABAr-IR following prembedding procedures is not
an artifact of DAB processing but represents an accurate view of GABAr-IR in photoreceptor terminals.
At the ultrastructural level, GABAr-IR was found
intracellularly and on the plasma membrane of all
cones and most rods in both the light-adapted and
dark-adapted states (Fig. 3). Rod spherules, although
more variable in their staining for GABAr-IR, tended to
stain more intensely than cone pedicles in the lightadapted state (Fig. 3A) but not in the dark-adapted
state (Fig. 3B). Most of the intracellular GABAr-IR was
associated with the membrane of synaptic vesicles,
with occasional clumps of GABAr-IR scattered throughout the terminal. For the most part, synaptic vesicles
were not filled with GABAr-IR but appeared hollow
with GABAr-IR concentrated around the cytoplasmic
surface of the synaptic vesicle membrane (e.g., see Figs.
6B and 8A). GABAr-IR synaptic vesicles were distributed throughout the receptor terminal, extending into
the photoreceptor axon where their numbers and consequently GABAr-IR decreased rather sharply (Fig. 3,
arrowheads). It was our impression that the differential
GABAr-IR staining in rod and cone terminals could be
due to differences in the density of synaptic vesicles.
45
Adaptation Affects Synaptic Vesicle Density
To test this idea, retinas from light-adapted fish and
those dark-adapted for 3 h were processed in parallel
with those reacted for GABAr-IR, but they were not
subjected to immunocytochemical processing (Fig. 4).
This was done to increase the visibility of synaptic
vesicles that would be obscured by the DAB reaction
product. It was obvious from even a casual inspection of
the micrographs that the density of synaptic vesicles
was less in light-adapted cone pedicles than rod spherules (Fig. 4A), whereas the densities appeared more
comparable in the dark-adapted state (Fig. 4B). A grid
was used to count the number of synaptic vesicles in
four separate regions of each synaptic terminal. The
four samples were pooled to produce the vesicle density
for that terminal. At least ten synaptic terminals each
of rods and cones were tabulated for the light- and
dark-adapted conditions. Three pair of retinas were
analyzed in this fashion. Synaptic vesicle densities
were quantified, and the results are presented in
Figure 5. The ratio of synaptic vesicle density for
rods:cones was 1.86:1 (P Ò 0.001) for the light-adapted
state and 1.2:1 (P , 0.05) for the dark-adapted state.
The reduction in this ratio was accounted for by two
factors: a 29% increase (P , 0.001) in cone synaptic
vesicle density and a 17% decrease (P , 0.001) in rod
synaptic vesicle density.
Cone Pedicles
It should be pointed out again that the micrographs
to be described were obtained from tissue sections that
were not counterstained with heavy metals. The electron density of plasma membranes and synaptic vesicle
membranes was due largely to GABAr-IR. However,
the electron density of synaptic ribbons and the intracellular surface of horizontal cell spinules and processes is
observed with osmication but without counterstaining
and thus is not due to GABAr-IR.
Our original rationale was based on the hypothesis
that horizontal cell spinules were the sites of GABA
release (Djamgoz et al., 1988; Weiler and Wagner,
1984), and thus GABAr-IR should be enriched on cone
membrane facing the spinules. However, despite an
extensive survey of numerous cone pedicles, obtained
from several retinas, we observed no obvious increase
in GABAr-IR on cone membranes opposing the spinules
(Fig. 6). GABAr-IR appeared on the plasma membrane
of the entire synaptic terminal, with dense patches of
GABAr-IR on the perimeter of the pedicle (Fig. 7B,
small arrows). Although cone membrane opposite horizontal cell spinules was weakly GABAr-IR (Fig. 6B),
the density of GABAr-IR opposite the spinules was no
greater and often appeared less than GABAr-IR on
other regions of the cone pedicle membrane (Fig. 7).
Similarly with dark-adapted cones, GABAr-IR did
not appear more dense on plasma membrane within the
invagination than in other regions (Fig. 8). Dendrites of
horizontal cells contain very few spinules in the darkadapted state. Instead, the dendrites are smooth and
mostly associated with synaptic ribbons of photoreceptors. It often appeared as if GABAr-IR increased in
density as one proceeded along the cone membrane
away from the synaptic ribbon to the more lateral
46
S. YAZULLA AND K.M. STUDHOLME
Fig. 1. Light micrographs of GABAr-IR in light-adapted (A) and
dark-adapted (B) goldfish retinas; 1 µm sections of retinas embedded
in Durcupan resin after preembed immunocytochemistry. GABAr-IR
was found in both rod and cone synaptic terminals regardless of
adaptive state. Arrowheads indicate GABAr-IR in the connecting axon
of cones that are extending into the rod nuclear layer. Cones (C) are
fully contracted in the light-adapted state, and rods (R) are fully
contracted in the dark-adapted state. Calibration bar 5 20 µm.
GABA RECEPTORS IN PHOTORECEPTORS
47
Rod Spherules
Regardless of level of adaptation, GABAr-IR of rod
spherules was far more variable than that observed
with cone pedicles, ranging from very weak to intense.
Intensely labeled rod spherules are illustrated in Figure 9. Unlike with cone pedicles, the plasma membrane
of rod spherules was stained uniformly, both within and
outside the invagination. There seemed to be no differential distribution of GABAr-IR along the plasma membrane of rod spherules nor were any differences in
GABAr-IR vis-à-vis horizontal cell dendrites noted.
Also, except for a decrease in the density of synaptic
vesicles, there were no apparent differences in
GABAr-IR of rod spherules with light or dark adaptation.
Fig. 2. Light micrograph of 1 µm resin section of light-adapted
goldfish retina processed for GABAr-IR by postembedding immunofluorescence (1:25 dilution of mAb 62-3G1). GABAr-IR was most intense
in the synaptic terminals of rods and cones in the outer plexiform
layer. Arrowheads indicate GABAr-IR in cone axon terminals as they
enter the region of rod nuclei, the same as observed in Fig. 1.
Calibration bar 5 20 µm.
aspect of the horizontal cell dendrites (Fig. 8A, arrowheads).
Regardless of the state of adaptation, membranes
with the most intense GABAr-IR were found on processes that were just proximal to the invagination of the
cone pedicle. In some cases, these were continuous with
the overlying pedicle and appeared to encapsulate a
horizontal cell process (Fig. 8b, arrowheads). However,
in most examples, these processes with GABAr-IR were
observed in isolation, but they had cytological features
that were identical to the cone pedicles (Figs. 6A, 7,
large arrows), indicating that they were part of cone
pedicles. In some cases, these processes with GABAr-IR
were within, though not deeply, the invagination (Figs.
6A, 7B, arrows); whereas in other cases they were
proximal to the pedicle (Fig. 7A, arrows). Other intense
processes with GABAr-IR were smaller in diameter and
occasionally were observed projecting from a pedicle
(Fig. 8C). Curiously, when cut longitudinally, these
processes did not exhibit GABAr-IR uniformly along
their length. Identifiable bipolar cell dendrites within
the cone pedicle did not show GABAr-IR (Fig. 8C, B
arrowheads), and it seems likely that these all of these
intense GABAr-IR processes were cone telodendria.
Note the similarity in appearance between the intense
GABAr-IR processes continuous with or projecting from
a pedicle (Fig. 8C,T, arrowheads) with the disconnected
GABAr-IR process proximal to the pedicle. We did not
determine the origin of the cone telodendria—that is,
whether the telodendria derived from the overlying or
nearby cone pedicles. There was no obvious presynaptic
structure adjacent to these GABAr-IR processes.
DISCUSSION
We have corroborated our earlier finding that
GABAr-IR is found on the plasma membrane of all cone
pedicles and many rod spherules and is associated with
the membrane of synaptic vesicles (Yazulla et al., 1989).
In addition, we found that despite the light-dependent
plasticity in the formation of horizontal cell spinules,
there was no relative increase in GABAr-IR on cone
plasma membrane opposing the spinules, nor was there
any corresponding change in the density of GABAr-IR
on the cone plasma membrane opposing the horizontal
cell dendrites. On the contrary, the density of GABAr-IR
was weakest on cone membrane within the invagination whether or not spinules were present, as opposed
to patches of high GABAr-IR density on the perimeter
of the pedicle and on cone processes proximal to the
invagination. The only novel effect of light adaptation
was on synaptic vesicle density: an increase in rod
spherules and a decrease in cone pedicles. Our findings
raise several issues: 1) how the distribution of GABArIR, determined by mAb 62-3G1, relates to GABAA
receptors in fish retina, 2) what the locations are of
GABA receptive elements in the fish OPL, and 3) how to
explain the effect of light and dark adaptation on
synaptic vesicle density in photoreceptor terminals.
mAb 62-3G1 Localizes GABAA Receptors
in Fish OPL
The validity of mAb 62-3G1 as an indicator of GABAA
receptors in the fish retina has been treated in detail by
Lin and Yazulla (1994). However, this paper specifically
relates to the outer plexiform layer. The presence of
intracellular GABAr-IR in photoreceptor terminals appears valid and not an artifact of the HRP/DAB reaction process because it persists with postembedding
ICC procedures (see Fig. 2) that are not subject to
diffusion of reaction products. Lin and Yazulla (1994)
showed that 3H-muscimol binding sites were immunoprecipitated by mAb 62-3G1 and in addition were
localized to the OPL in goldfish retina by dry autoradiography, indicating that at least some of the GABAr-IR,
as determined by mAb 62-3G1, is representative of
GABAA receptors. It is possible that there are GABAA
receptor sites in the OPL that are not recognized by
mAb 62-3G1. For example, the synaptic terminals of
the large mixed rod/cone bipolar cells receive massive
GABAergic input of the GABAA type (Heidelberger and
Matthews, 1991; Marc et al., 1978; Tachibana and
48
S. YAZULLA AND K.M. STUDHOLME
Fig. 3. Electron micrographs of GABAr-IR in photoreceptor terminals of light-adapted (A) and dark-adapted (B) retinas. Cone pedicles
are indicated (C); other GABAr-IR profiles are rod spherules. Arrowheads indicate the connecting axon of a cone in A and rod in B,
illustrating the decrease in GABAr-IR as the axon enters the outer
nuclear layer. Note that cone terminals show less GABAr-IR than rod
terminals with light adaptation (A) but appear comparably stained
with dark adaptation (B). Calibration bar 5 1 µm. Note that sections
were not counterstained with heavy metals.
GABA RECEPTORS IN PHOTORECEPTORS
Fig. 4. Electron micrographs of goldfish photoreceptor terminals
after light adaptation (A) and dark adaptation (B). The density of
synaptic vesicles appears higher in rods (R) than cones (C) with light
adaptation but not with dark adaptation. Also, note the presence of
49
horizontal cell spinules(S) invaginated into the cone pedicle that was
light-adapted (A); dark-adapted cone pedicles (B) do not contain
spinules. Sections were lightly counterstained with lead citrate and
uranyl acetate. sr, synaptic ribbons. Calibration bar 5 0.5 µm.
50
S. YAZULLA AND K.M. STUDHOLME
Fig. 5. Histobars illustrating the density of synaptic vesicles of rod
and cone terminals as a function of light and dark adaptation
(X 6 s.e.m.). All differences were statistically significant: light adapta-
tion—rod/cone: t 5 19.7, P Ò 0.0001; dark adaptation—rod/cone: t 5
2.5, P , 0.05; light/dark adaptation—cones: t 5 4.2, P , 0.001;
light/dark adaptation—rods: t 5 4.0, P , 0.001.
Kaneko, 1987; Yazulla et al., 1987), yet mAb 62-3G1
stains very few of the numerous GABAergic amacrine
cell synaptic contacts onto the bipolar cell synaptic
terminal (Yazulla et al., 1989). We conclude that mAb
62-3G1 localizes GABAA receptors on goldfish photoreceptor terminals, but the possibility remains that additional epitopes exist that are not recognized by this
antibody. Another monoclonal antibody (bd-17; reportedly against the same b2/b3 subunits of the GABAA
receptor complex (Ewert et al., 1992) does not label the
goldfish OPL (Lin and Yazulla, 1994), a further indication of potential epitope differences.
within this environment. Additionally, there were smalldiameter processes within the pedicle that showed
intense GABAr-IR. We believe these to be cone telodendria rather than bipolar cell dendrites for two reasons.
First, they had similar cytology, and, on occasion, we
could identify the initial portion of a GABAr-IR telodendrite emanating from a pedicle (see Fig. 8C). Secondly,
no GABA sensitivity has been observed on the dendrites of fish bipolar cells (Tachibana and Kaneko,
1987). Telodendria are a common feature of vertebrate
cones (Ramon y Cajal, 1933), and, in teleost fish, they
extend about 15 µm from each cone where they make
gap junctions and shallow invaginating contacts with
nearby cones of similar and different spectral classes
(i.e., Kraft and Burkhardt, 1986; Scholes, 1975; Stell,
1980; Witkovsky et al., 1974). Thus, H1 horizontal cells
could provide GABAergic feedback to cones at encapsulating basal processes of the overlying cone and to
nearby cones via telodendria. Although this feedback
may be involved in spatiotemporal and color coding in
the outer retina, its role is still the subject of much
debate (for critical reviews of this topic see Burkhardt,
1993; Wu, 1992).
Another conclusion is that rods should be sensitive to
GABA. Such a situation would require diffusion of
GABA from horizontal cells to the rods because there is
no evidence for contact between GABAergic H1 horizontal cells and rods in the goldfish retina (Marc et al.,
1978; Stell and Lightfoot, 1975). This so-called action at
a distance is feasible because the high affinity uptake of
GABA is not 100% efficient, considering that it is
GABAA Receptor Localization in the OPL
What are the locations of GABA receptive elements in
the fish OPL? With respect to cone pedicles, we suggest
that GABA sensitivity is distributed all over the cone
pedicle plasma membrane. Intense GABAr-IR was found
on pedicle processes that were proximal to the invagination rather than deeper within the invagination near
the synaptic ribbons. This is not to say that cone
membrane opposing spinules or the lateral face of
horizontal cell dendrites is devoid of GABAr-IR. Figures 6B and 8A clearly showed GABAr-IR on cone
membrane within the invagination, indicative of GABA
sensitivity. The point is that GABA sensitivity may be
broadly distributed and even higher at other areas of
the pedicle. The cone pedicle comprises a highly enclosed space of interdigitating neuropil, analogous to a
glomerulus. It is highly likely that GABA released into
this space would affect membrane receptors anywhere
GABA RECEPTORS IN PHOTORECEPTORS
51
Fig. 6. GABAr-IR in cone pedicles from light-adapted retinas.
GABAr-IR was found on the membrane of synaptic vesicles and on the
cone plasma membrane and opposing horizontal cell dendritic spinules
(S, arrowheads). There was no relative increase of GABAr-IR on the
cone membrane within the invagination opposing the spinules. B is a
higher magnification view (rotated 90° counterclockwise) of a portion
of the upper horizontal cell dendrite and spinules in A. The arrow in A
indicates intense GABAr-IR of a cone pedicle. Sections were counterstained for 10 sec in lead citrate. H, horizontal cell dendrites; S,
spinules. Calibration bar 5 0.5 µm (A) or 0.25 µm (B).
possible to detect a stimulated release of 3H-GABA as
low as 0.03% and as high as 20% of the total 3H-GABA
content of the fish retina (Yazulla, 1983, 1985). Although the small size of goldfish rods has precluded any
test of whether rods are GABA-sensitive, we suggest
that future studies will find GABA sensitivity of rods. If
so, goldfish would differ from turtle, in which isolated
red and green cones showed a high sensitivity to GABA,
whereas blue cones and rods showed a low sensitivity
(Tachibana and Kaneko, 1984).
that GABAr-IR occurs on the cytoplasmic surface of
both the plasma membrane and synaptic vesicles,
which would be expected for vesicles formed by endocytosis. Also, the distribution of GABAr-IR synaptic
vesicles throughout the synaptic terminals is similar to
that reported for HRP-containing synaptic vesicles,
formed by endocytosis during darkness, in frog, skate,
and turtle photoreceptor terminals (Ripps et al., 1976;
Schacher et al., 1974, 1976; Schaeffer and Raviola,
1978). Perhaps the rapid turnover of plasma membrane, due to vesicular fusion, within receptor invaginations precludes the concentration of GABA receptors
directly opposing the horizontal cell dendrites. In this
way GABAA receptors could be located on plasma
membranes that were less active endocytotically in
order to present a more stable environment for GABA
reception by photoreceptors. Unfortunately, this explanation provides no hint as to the site GABA release
from horizontal cells.
GABAr-IR and Synaptic Vesicle Membrane
Consistent with our previous report (Yazulla et al.,
1989), we found that GABAr-IR was associated with the
membrane of synaptic vesicles. Since the synaptic
vesicles appeared clear and not filled with reaction
product, we conclude that GABAr-IR was associated
with the cytoplasmic surface of the synaptic vesicle
membrane. The plasma membrane of photoreceptor
terminals undergoes considerable endocytosis, probably to retrieve membrane added by vesicular exocytosis (Ripps and Chappell, 1991; Ripps et al., 1976;
Schacher et al., 1976; Schaeffer and Raviola, 1975).
This endocytotic activity could explain the presence of
GABAr-IR synaptic vesicles in view of the observation
Adaptation and Synaptic Vesicle Density
We found that light and dark adaptation affected the
density of synaptic vesicles of both rods and cones in
contrast to previous studies on teleost fish retinas that
have not reported such effects (e.g., Kohler et al., 1990;
52
S. YAZULLA AND K.M. STUDHOLME
Fig. 7. GABAr-IR in cone pedicles from light-adapted retinas. The
most intense GABAr-IR was in patches on the perimeter of the cone
plasma membrane (B, small arrows), on cone processes proximal to
the pedicle (large arrows), and on presumed cone telodendria (T,
arrows). There was no relative increase of GABAr-IR on the cone
membrane within the invagination opposing the spinules (arrowheads). Sections were not counterstained. Calibration bars 5 0.5 µm.
Wagner, 1980; Weiler and Wagner, 1984). This discrepancy is puzzling, considering that we have corroborated
their observations on horizontal cell spinule formation
and convolutions in cone terminals. One possible explanation is that these previous studies employed 2.5%
glutaraldehyde in the fixative, whereas we used 0.15%
glutaraldehyde prior to overnight fixation in 4% paraformaldehyde at pH 10.4. We have found that our procedure maximizes tissue preservation that is compatible
with preembedding immunocytochemistry for a wide
variety of antigens, including enzymes as well as
transmitter/glutaraldehyde conjugates (Eldred et al.,
1983; Yazulla and Studholme, 1991). Since our goal was
to investigate the source of the apparent changes in
intracellular GABAr-IR with adaptation, all retinas
were fixed identically in low glutaraldehyde whether or
not they were processed for GABAr-IR. The answer to
the discrepancy may lie in a parametric study on the
effects of fixation on synaptic vesicle density. This,
however, is beyond the scope of the present investigation that was directed towards GABAr-IR and horizontal cell spinules. Given these qualifications, some comments as to possible implications of these data are
warranted.
Goldfish cones showed a dramatic 29% increase in
synaptic vesicle density with 3 h of dark adaptation.
The plasma membrane of goldfish cone terminals would
be expected to show an increase in surface area with
light adaptation due to invagination by horizontal cell
spinules (Raynauld et al., 1979; Wagner, 1980). With
dark adaptation and retraction of the spinules, there
may be an increase in the recovery of cone plasma
membrane (Wagner, 1980) and a consequent increase in
synaptic vesicle density. Also, cone neurotransmission
in teleost fish is suppressed markedly during prolonged
dark adaptation (e.g., Raynauld et al., 1979; Yang et al.,
1988), perhaps resulting in an excess accumulation of
synaptic vesicles.
Reported effects of lighting conditions on synaptic
vesicles have been inconsistent and appear to be speciesdependent. An early report of a dark-induced decrease
in synaptic vesicle size in rat retina (De Robertis and
Franchi, 1958) was not corroborated (Mountford, 1963;
Cragg, 1972), and more recent studies in rat did not
address specifically effects on synaptic vesicle density
(Brandon and Lam, 1983; Case and Plummer, 1993).
Studies in amphibian retinae also have yielded varying
results. Ball and Dickson (1983) reported, in newt
GABA RECEPTORS IN PHOTORECEPTORS
Fig. 8. GABAr-IR in cone pedicles from dark-adapted retinas.
GABAr-IR is found on the membrane of synaptic vesicles and on the
cone plasma membrane. H, horizontal cell dendrites. A: GABAr-IR is
very light on cone membrane in the region of the synaptic ribbon but
appears to get denser with increasing distance from the ribbon
(arrowheads). Calibration bar 5 0.25 µm. B: Dense GABAr-IR was
found on cone membrane away from the synaptic ribbon but adjacent
53
to horizontal cell dendrites (arrowheads). Notice that this portion of
cone pedicle appears to encapsulate the invaginating processes.
Calibration bar 5 0.5 µm. C: Dense GABAr-IR also was observed on
presumed cone telodendria (T, arrowheads). Bipolar cell dendrites (B)
make basal contacts with cone terminals and are not GABAr-IR.
Calibration bar 5 0.5 µm.
54
S. YAZULLA AND K.M. STUDHOLME
Fig. 9. GABAr-IR in rod spherules from light-adapted (A) and dark-adapted (B) retinas. GABAr-IR
appears on the membrane of synaptic vesicles and on the plasma membrane. There does not appear to be
any differential staining with GABAr-IR inside or outside the invagination or with states of adaptation.
H, horizontal cell dendrites. Calibration bars 5 0.25 µm.
55
GABA RECEPTORS IN PHOTORECEPTORS
retina, that synaptic vesicle density increased during
the day cycle, but this increase could be accounted for
by a decrease in synaptic terminal volume because the
total number of synaptic vesicles per terminal did not
change. Schacher et al. (1976) found no effects of light
adaptation on synaptic vesicle density in frog photoreceptors. However, dense-core vesicles appeared and
increased in frequency in Xenopus photoreceptors following prolonged light (Monaghan and Osborne, 1975) and
in newt photoreceptors during the day cycle (Ball and
Dickson, 1983); this finding was interpreted to represent ‘‘supercharging’’ of vesicles with a neurotransmitter during a period of low transmitter release.
Rod spherules showed a modest (17%) reduction in
synaptic vesicle density with dark adaptation. Perhaps
this reduction was due to increased transmitter release
by rods in the dark. Rod spherules are not invaginated
by horizontal cell spinules, unlike cone pedicles. Rather,
the reverse situation occurs in that processes of rod
spherules protrude into horizontal cell processes during dark adaptation (Brandon and Lam, 1983). Thus,
reduced synaptic vesicle density in goldfish cones and
rods is correlated with increased membrane convolutions. This is the opposite of that reported for the newt
(Ball and Dickson, 1983), in which increased synaptic
vesicle density was an epiphenomenon of decreased
volume.
Light-dependent plasticity of fish photoreceptor terminals includes not only invagination by horizontal cell
spinules, changes in synaptic vesicle density, and
changes in surface contour but also effects on synaptic
ribbons. The length and number of synaptic ribbons per
cone terminal are reduced by dark adaptation and
during the night phase (Wagner, 1973, 1975; Wagner
and Ali, 1977). We did not quantify effects on synaptic
ribbons. However, it is evident from the representative
micrographs in Figure 3 that synaptic ribbons in lightadapted cones (Fig. 3A) were longer than in darkadapted cones (Fig. 3B). This corroborative finding
means that during periods of inactivity (prolonged
darkness) (Raynauld et al., 1979; Yang et al., 1988),
there is a concomitant decrease in synaptic ribbon
length and an increase in synaptic vesicle density in
goldfish cones.
Summary and Conclusions
We have shown that GABAr-IR on the plasma membrane of photoreceptor terminals appears independent
of the state of light adaptation and is not concentrated
within the invagination, the site of hypothesized GABA
release. We conclude that GABA sensitivity of photoreceptors is broadly distributed on the synaptic terminal
and is not affected by adaptation. We suggest that
GABA released into the invagination of a pedicle can
affect not only the overlying cone but also nearby cones
via action on telodendria. The proposed widespread
distribution of GABA sensitivity raises questions as to
the role of horizontal cell spinules in GABA release.
Horizontal cell spinules have been studied intensively
over the last few years, yet their function remains
controversial (i.e., Burkhardt, 1993; Downing and
Djamgoz, 1989; Wagner and Djamgoz, 1993). Perhaps
the spinules are involved in the efficacy of signal
transmission from horizontal cells to cones, without
being the actual site of GABA release. An analogous
situation may be found in the ampulla of Lorenzini of
elasmobranch fishes, in which transmission from firstto second-order neurons is correlated with the degree of
invagination between the processes (Fields and Ellisman, 1985). The invaginating spinules may serve a
similar role for horizontal cell to photoreceptor transmission, although the mechanism for this action remains to
be determined.
ACKNOWLEDGMENTS
We thank Dr. A.L. De Blas, who generously supplied
us with mAb 62-3G1 against the GABA receptor. This
study was supported by NIH grant EYO1682 to S.Y.
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