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The Journal of Neuroscience, December 1, 1998, 18(23):10136-10149
Immunocytochemical Localization of the Postsynaptic Density
Protein PSD-95 in the Mammalian Retina
Peter
Koulen1,
Erica L.
Fletcher1,
Sarah E.
Craven2,
David S.
Bredt2, and
Heinz
Wässle1
1 Max-Planck-Institut für Hirnforschung, D-60528
Frankfurt, Germany, and 2 Department of Physiology,
University of California at San Francisco, San Francisco, California
94143
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ABSTRACT |
Synapse-associated proteins are the scaffold for the selective
aggregation of ion channels at synapses; they provide the link to
cytoskeletal elements and possibly are involved with the
regulation of synaptic efficacy by electrical activity. The
localization of the postsynaptic density protein PSD-95 was studied in
different mammalian retinae (rat, monkey, and tree shrew) by using
immunocytochemical methods. Immunofluorescence for PSD-95 was most
prominent in the outer plexiform layer (OPL). The axon terminals of
rods and cones, the rod spherules and cone pedicles, were strongly
labeled. Electron microscopy, using preembedding immunocytochemistry,
showed PSD-95 localized presynaptically within the photoreceptor
terminals. Distinct PSD-95 labeling was also present in the inner
plexiform layer (IPL). It had a punctate appearance suggesting the
synaptic clustering of PSD-95 in the IPL. Electron microscopy showed
that PSD-95 was concentrated in processes that were postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two
postsynaptic members of the dyad was labeled for PSD-95.
Double-labeling experiments were performed for PSD-95 and for SAP 102 or PSD-93, respectively, two other members of the family of
synapse-associated proteins. All three were found to be colocalized in
the synaptic hot spots in the IPL. In the OPL, however, PSD-95 and
PSD-93 were found presynaptically, whereas SAP 102 was located
postsynaptically at photoreceptor synapses. Double-labeling experiments
also were performed for PSD-95 and for the NR1 subunit of the
NMDA receptor. They were found to be colocalized in synaptic hot
spots in the IPL.
Key words:
PSD-95; SAP 102; PSD-93; NMDA receptors; NR1 subunit; postsynaptic density; receptor clustering; cone pedicle; rod
spherule
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INTRODUCTION |
The postsynaptic density (PSD) is a
complex network of transmitter receptors, anchoring proteins, and
cytoskeletal elements (Kennedy, 1997 ). The selective concentration of
transmitter receptors in PSDs not only is essential for the reliable
and rapid synaptic transmission but also plays an important role in
synaptic plasticity and possibly in memory formation (Mammen et al.,
1997; Rao and Craig, 1997; Kirsch and Betz, 1998).
The postsynaptic density protein PSD-95 belongs to a family of ion
channel clustering molecules (for review, see Gomperts, 1996; Sheng,
1996; Kennedy, 1997). In its N-terminal part PSD-95 contains three
highly homologous regions of ~90 amino acids. They have been called
PDZ domains because they have been found in postsynaptic density
proteins (PSDs), in the Drosophila tumor suppressor gene "disks large" (Dlg), and in cell-cell adhesion proteins
like zonula occludens-1 (ZO1). In mammals this family of postsynaptic
density proteins includes four closely related proteins: PSD-95/SAP 90 (Cho et al., 1992; Kistner et al., 1993), SAP 97/hdlg (Lue et al.,
1994; Müller et al., 1995), PSD-93/Chapsyn-110 (Brenman et al.,
1996a; Kim et al., 1996), and SAP 102 (Müller et al., 1996).
The second PDZ domain in PSD-95 has been shown by the yeast two-hybrid
method to bind tightly to the COOH tail of the NMDA receptor subunit 2 (NR2) and of the Shaker-type K+ channels,
respectively (Kim et al., 1995; Kornau et al., 1995; Niethammer et al.,
1996). Brenman et al. (1996b) described the PDZ domain of neuronal
nitric oxide synthase (nNOS) as binding to the second PDZ domain of
PSD-95. An interaction of the third PDZ domain of PSD-95 with the
neuronal cell adhesion molecule neuroligin has been found by Irie et
al. (1997). These binding studies suggest that PSD-95 is a modular
scaffold that aggregates ion channels and links them to several
intracellular and transmembrane proteins.
The localization of PSD-95 to various parts of the CNS has been
studied with specific antibodies both by light and electron microscopy.
Cho et al. (1992) described a punctate immunofluorescence in the
hippocampus. Kornau et al. (1995) showed that on the dendrites of
cultured hippocampal neurons the puncta are in register with NR2B
clusters. Hunt et al. (1996) demonstrated by electron microscopy that
PSD-95 in the forebrain was associated with postsynaptic densities. In
contrast, a presynaptic localization of PSD-95 was found in the
cerebellum (Kistner et al., 1993; Laube et al., 1996).
In the past we have studied the localization of various transmitter
receptors in the mammalian retina and found that they are clustered at
synaptic sites (for review, see Brandstätter et al., 1998;
Wässle et al., 1998). The clusters were highly specific with
respect to their subunit composition and the type of synapses at which
the subunits were expressed. Therefore, selective sorting mechanisms
and protein interactions must be involved. The PDZ domain-containing
proteins are primary candidates for targeting transmitter receptors to synapses.
In a preceding study (Koulen et al., 1998) we have investigated the
localization of SAP 102 that, like PSD-95, also has three PDZ domains.
It was clustered at postsynaptic sites in the outer plexiform layer
(OPL) and inner plexiform layer (IPL), and weak extrasynaptic
expression was found in bipolar cells and ganglion cell axons. In the
present study we applied specific antibodies against PSD-95 to the rat
retina and studied the immunolabeling in the OPL and in the IPL both by
light and electron microscopy. To show a possible colocalization of
PSD-95 with the closely related synapse-associated proteins PSD-93 and
SAP 102, we performed double-labeling experiments. The possible
association of PSD-95 and NMDA receptors was studied by double-labeling
experiments for PSD-95 and the NR1 subunit of the NMDA receptor.
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MATERIALS AND METHODS |
All experiments were performed according to the guidelines for
the welfare of experimental animals issued by the Federal Government of Germany.
Antisera
Two mouse monoclonal antibodies (clone 7E3-1B8 and clone
6G6-1C9; Affinity Bioreagents, Golden, CO) were used to detect PSD-95 immunoreactivity in the rat retina. The specificity of both antibodies was described in Kornau et al. (1995). Because of its specific interaction with PSD-95, clone 7E3-1B8 was chosen for all of the experiments that have been described (diluted 1:200). Although clone
6G6-1C9 was reported to recognize a slightly larger band in addition to
a 95 kDa band in Western blots (Kornau et al., 1995), we found
identical results in immunocytochemical experiments and no difference
in staining specificity when compared with clone 7E3-1B8, as reported
previously for other CNS tissues (Kornau et al., 1995). A polyclonal
antiserum raised in rabbit against the first 119 amino acids of SAP 102 was used in this study (diluted 1:1500; kindly provided by Dr. C. C. Garner University of Alabama at Birmingham, AL). The antiserum was
characterized in detail in Müller et al. (1996). A guinea pig
polyclonal antiserum against PSD-93 (diluted 1:2500) was used to
visualize the distribution of the protein in the rat retina. The
specificity of the antiserum was described in Brenman et al.
(1996a,b). Rod bipolar cells were labeled with a polyclonal
rabbit antiserum against protein kinase C (PKC) (Greferath et
al., 1990) (diluted 1:10,000; Sigma, Saint Louis, MO). A rabbit
polyclonal antiserum directed against a synthetic peptide corresponding
to the C terminus of the NR1 subunit of the NMDA receptor was used to
detect the NR1 subunit (diluted 1:25; Chemicon, Temecula, CA).
Tissue preparation
Retinae of six adult albino rats 6-8 weeks of age, of one adult
Macaca mulatta, and of one adult Tupaia belangeri
were investigated. Rats were anesthetized deeply with halothane and
decapitated. Eyes of M. mulatta and of T. belangeri were taken from animals that were killed for
experiments unrelated to this study by a lethal dose of pentobarbital
sodium (Nembutal). After the optic nerve was cut, the superior part of
the eye was marked with a felt pen, and the eye was removed. For light
microscopy the eyes were opened along the ora serrata, and the eyecups
were immersion-fixed for 5, 15, or 30 min in 4% (w/v) paraformaldehyde
in phosphate buffer (PB; 0.1 M, pH 7.4). For experiments
involving detection of the NR1 subunit, rat retinae were
immersion-fixed for 30 and 60 min in 4% 1-ethyl-3-(3
dimethylaminopropyl) carbodiimide in PB. The vitreous body was removed,
and the retinae were dissected free. The marking of the superior part
of the retina was conserved by making radial incisions. The retinae
were cryoprotected in 10% (w/v) and 20% (w/v) sucrose in PB for 1 hr
each and in 30% (w/v) sucrose in PB overnight at 4°C. Pieces of
retinae were frozen in freezing medium (Reichert-Jung, Bensheim,
Germany), sectioned vertically at 12 µm thickness on a cryostat, and
collected on gelatin-coated slides.
For electron microscopy the eyecups were fixed in 4% (w/v)
paraformaldehyde and 0.05% (v/v) glutaraldehyde in PB for 10 min, followed by an additional 40 min in 4% (w/v) paraformaldehyde in PB.
After the retina was dissected out and cryoprotected in 10, 20, and
30% (w/v) sucrose, the tissue was frozen and thawed repeatedly to
enhance the penetration of the antibodies. After the retina was washed
in PBS (0.01 M, pH 7.4), small pieces of retina were
embedded in agar, and vertical sections (60 µm thick) were cut with a
vibratome for preembedding electron microscopic immunocytochemistry.
Depending on the fixation time, differences in the appearance of
PSD-95, PSD-93, SAP 102, and especially of NR1 were found. With short
and mild fixation (5-10 min), immunofluorescence in the IPL had a
bright punctate appearance suggesting that single synapses were
stained. Unfortunately, such short fixation times resulted in very soft
retinal tissue, and in the cryostat sections the tissue often appeared
to be ruptured (see Figs. 6, 8, 9, 10). With longer fixation times the
quality of the sections was better, but the punctate labeling was much
less intense. We exclude that fixation causes a redistribution of
synapse-associated proteins. We assume that cross-linking the densely
packed synapse-associated proteins by the fixative reduces the
accessibility of relevant epitopes. We have found such fixation
sensitivity in the past for glycine receptors,
GABAA, GABAB, and
GABAC receptors, and ionotropic and metabotropic glutamate
receptors, all of which had a punctate distribution in the IPL (for
review, see Brandstätter et al., 1998; Wässle et al.,
1998).
Immunocytochemistry
Light microscopic immunocytochemistry. A specific
mouse monoclonal antibody against PSD-95 (clone 7E3-1B8; diluted 1:200) was used in this study. In double-labeling experiments, antibodies known to stain distinct populations of retinal neurons or specific proteins [primary antibodies (pAbs) from rabbit against PKC, SAP
102, and NR1, respectively, and pAbs from guinea pig against PSD-93],
were coapplied with the antibody against PSD-95 to characterize its
cellular distribution.
Immunocytochemical labeling was performed via the indirect fluorescence
method (for details, see Grünert and Wässle, 1993; Sassoè-Pognetto et al., 1994). After being blocked in PBS
containing 10% normal goat serum (NGS), 1% bovine serum albumin, and
0.05% Triton X-100, the binding sites of the primary antibodies were revealed by fluorescently labeled secondary antibodies: goat anti-mouse IgG, goat anti-guinea pig IgG, or goat anti-rabbit IgG conjugated to
Alexa 564, Alexa 594 (red fluorescence), or Alexa 488 (green fluorescence) (Molecular Probes, Eugene, OR) diluted in PBS containing 3% NGS, 1% bovine serum albumin, and 0.05% Triton X-100.
In double-labeling experiments the sections were incubated in a mixture
of the primary antibodies, followed by a mixture of secondary
antibodies. To check for antibody specificity, we prepared controls by
omitting one of the two primary antibodies during the incubation. In
this case only the immunoreactivity for the remaining primary antibody
and unspecific background staining were detected, as in the case of
single labeling experiments, in which the primary antibody was omitted.
Electron microscopic immunocytochemistry. The vibratome
sections were collected in cold PBS, immersed for blocking for 2 hr in
10% NGS (v/v) in PBS, and then incubated in the primary antibody against PSD-95. The antibody was diluted 1:200 in the same medium as
that used for light microscopy, but without Triton X-100, for 4 d
at 4°C. Thereafter, the sections were rinsed in PBS several times,
and immunolabeling was detected with a biotinylated goat anti-mouse IgG
(diluted at 1:100; Vector Laboratories, Burlingame, CA) and a
peroxidase-based enzymatic detection system (Vectastain Elite ABC kit,
Vector Laboratories). After washes in PBS and in 0.05 M
Tris-HCl, pH 7.6, the sections were preincubated for 10 min in
3,3'-diaminobenzidine [DAB; 0.05% (v/v) in 0.05 M
Tris-HCl, pH 7.6] and then reacted in 0.05% (v/v) DAB with 0.01%
(v/v) H2O2. The staining reaction was stopped
by rinsing the sections in Tris-HCl. Subsequently, the sections were
rinsed in 0.1 M cacodylate buffer, pH 7.4, post-fixed in
2.5% (v/v) glutaraldehyde in cacodylate buffer (2 hr at 4°C), and
washed in cacodylate buffer overnight at 4°C. After several washes in
distilled water the DAB reaction product was silver-intensified by
incubation for 10 min at 60°C in a solution containing 2.6%
hexamethylene tetramine, 0.2% AgNO3, and 0.2%
borax. Then the thoroughly washed sections were treated with a 0.05%
HAuCl4 solution for 3 min at room temperature and, after
further washings in distilled water, were incubated for 3 min at room
temperature in a 2.5% Na2S2O3
solution. The sections then were post-fixed with 0.05% (w/v)
OsO4 in cacodylate buffer for 30 min, dehydrated in a
graded series of ethanol (30-100%) followed by propylene oxide, and
flat-embedded in Glycidether 100-based resin (Serva, Heidelberg,
Germany). Ultrathin sections were cut and then stained with uranyl
acetate and lead citrate. Control vibratome sections were processed as
described above, except that the primary antibody was omitted,
resulting in no specific staining. Ultrathin sections were examined and
photographed with a Zeiss (Oberkochen, Germany) EM10 electron microscope.
Microscopic analysis
For light microscopic analysis the sections were examined and
photographed with a Zeiss photomicroscope (Axiophot, Zeiss), using
40×, 63×, and 100× objectives and the appropriate fluorescence filter (Alexa 564; Alexa 594: BP 546, FT 580, LP 590; Alexa 488: 450-490, FT 510, LP 520). The fluorescence filters were
wedge-corrected, so shifting from one filter to the other did not cause
any displacement of the image. No adjustment of focus was necessary
when switching from one filter set to another because the objectives
(Zeiss, Neofluar) were corrected for at least three different
wavelengths. Black and white photomicrographs were taken on Kodak TMY
400 film and color photomicrographs on Kodak Ektachrome color reversal film. To visualize colocalizations of puncta in double-labeled sections, we took black and white micrographs with red and green fluorescence. One of the negatives was printed the correct way; the
other negative was turned around for printing. The resulting prints
were, therefore, mirror images. They were sectioned along a common
border and mounted side by side. This made it easier to visualize
colocalizations, because corresponding points were found at equal
distances from that border (see Fig. 8).
Quantification of colocalizations of immunofluorescent puncta
Two micrographs of the sections that were double-labeled for
PSD-95/SAP 102, PSD-95/PSD-93, and PSD-95/NR1, respectively, were taken
with the 100× objective, using red and green fluorescence filters, and
printed at a final magnification of 1800×. The immunofluorescent puncta of the micrographs were transferred onto tracing paper. Already
this first step has an intrinsic failure rate, because puncta are
difficult to detect if they are weakly stained or if they are covered
by a cloudy background. To test the positional errors that may have
been made, we transferred puncta of the same micrograph twice onto
separate transparencies. The two images were superimposed at their
correct position, and the number of puncta that coincided was counted.
In theory the coincidence rate should be 100%; however, the
coincidence rate measured in this way was 85 ± 5% (mean and
SD) in the case of PSD-95/PSD-95; 79.8 ± 2.8% in the case
of SAP 102/SAP 102; 82.3 ± 5% in the case of PSD-93/PSD-93;
81 ± 5% in the case of NR1/NR1. Each percentage is the mean of
>1000 puncta taken from four different sections. The two images also
were superimposed randomly, and the number of puncta that coincided was
counted. The coincidence rates for random superpositions varied between
18.7 and 22.1%. After these two test trials the two images of the
micrographs of the double-labeled sections were superimposed, and the
number of coincidences was counted.
We also analyzed the punctate immunofluorescence of double-labeled
sections, using confocal microscopy. However, for this particular task
we found that conventional microscopy with high-power objectives was
superior. Because immunofluorescence in the present experiments is
punctate, small changes in focus in a conventional microscope cause the
puncta to disappear; therefore, optical sectioning is very precise. The
punctate fluorescence acts like the pinhole of the confocal microscope.
Because of the scanning mode of the confocal microscope, individual
puncta will be represented by several pixels, thereby tending to blur
the punctate immunofluorescence. For these reasons conventional
microscopy was the method of choice for the present analysis.
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RESULTS |
Light microscopic localization of PSD-95
In vertical sections of the rat retina that were immunostained
with the antiserum against PSD-95 (Fig.
1), very intense immunofluorescence was
present in the OPL, and comparably moderate immunolabeling was observed
in the IPL. It was impossible to capture this enormous difference in
intensity between OPL and IPL on a single negative. Figure 1 is
therefore a montage printed from two negatives in which the IPL had
been exposed eight times longer than the OPL. In the outer retina the
immunofluorescence was concentrated in the axon terminals of the
photoreceptors; however, weak immunolabeling also was observed in the
microscope all along the descending photoreceptor axons. In the IPL the
immunofluorescence had a punctate appearance, suggesting that PSD-95
was clustered at synaptic sites.
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Figure 1.
Photomicrograph of a vertical cryostat section
through a rat retina that was immunolabeled for PSD-95. The retinal
layers are shown in the Nomarski micrograph (right).
ONL, Outer nuclear layer; OPL, outer
plexiform layer; INL, inner nuclear layer;
IPL, inner plexiform layer; GCL, ganglion
cell layer. PSD-95 immunofluorescence is prominent in the OPL, where
the axon terminals of rods, the so-called rod spherules, are labeled
intensively. In the IPL punctate immunofluorescence is found,
representing a concentration of PSD-95 at synapses. Scale bar, 25 µm.
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Localization of PSD-95 in rod spherules and cone pedicles
The OPL of a rat retina that was immunolabeled for PSD-95 is shown
at higher magnification in Figure
2A. Immunofluorescence is concentrated in the membrane of rod spherules, which have a diameter
of 1-2 µm and which are staggered irregularly at the outer margin of
the OPL. Because cones are only a small fraction of the photoreceptors
of the rat retina (Szél et al., 1996), no typical cone pedicle is
visible in Figure 2A. However, when a macaque monkey
retina (Fig. 2B) or a tree shrew retina (Fig. 2C) was immunolabeled for PSD-95, it became apparent that
cone pedicles also express PSD-95. Four cone pedicles of 5-7 µm
diameter are interspersed between the rod spherules in the monkey
retina (Fig. 2B, arrows). The tree shrew retina is
strongly cone-dominated (Müller and Peichl, 1989); consequently,
exclusively labeled cone pedicles are found in Figure 2C.
The localization of PSD-95 in photoreceptor axon terminals was
confirmed by electron microscopy (Fig.
3). Figure 3, A and
C, shows the labeling of cone pedicles and Figure 3,
B and D, shows the labeling of rod spherules. Two presynaptic ribbons are indicated in the cone pedicle of Figure 3A by arrowheads, and three are indicated in Figure
3B. Several invaginating postsynaptic processes are marked
with stars. Grains of the silver-intensified reaction product are
restricted to the cone pedicles, and no traces of labeling are found in
horizontal cell or bipolar cell processes contacting the cone pedicles.
The same holds true for the two rod spherules illustrated in Figure 3,
B and D. The spherules containing the ribbons are
labeled; the postsynaptic rod bipolar cell dendrites and the horizontal cell processes (stars) are not labeled. In all, 10 cone
pedicles and 44 rod spherules were photographed on the electron
microscope, and many more were inspected; in no case were bipolar or
horizontal cell processes immunoreactive for PSD-95. The light
microscopic appearance of labeled cone pedicles and rod spherules
suggests a concentration of PSD-95 at the cellular membrane. This is
not so evident from the silver-intensified reaction product in the electron micrographs. However, if one concentrates instead on the
fluffy diaminobenzidine reaction product in Figure 3, A and C, the aggregation of the labeling along the membrane
becomes more apparent. In general, because of the short fixation times that are necessary, the use of a very low concentration of
glutaraldehyde in the fixative, and the large amounts of reaction
product, the quality of the electron micrographs is compromised
somewhat.
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Figure 2.
High-power fluorescence micrographs of vertical
cryostat sections through rat (A), macaque monkey
(B), and tree shrew (C)
retinae that were immunostained for PSD-95. A, Rod
spherules at the outer margin of the OPL are labeled in the rat.
B, Four cone pedicles (arrows) and
several rod spherules are labeled in the monkey retina.
C, Exclusively, cone pedicles are labeled in the tree
shrew retina. The labeling in rod spherules and cone pedicles is
concentrated in their outer membranes.
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Figure 3.
Electron micrographs of sections through cone
pedicles (A, C) and rod spherules
(B, D) of a rat retina that was
immunolabeled for PSD-95. A, The labeled cone pedicle
base (top half) contains two synaptic ribbons
(arrowheads) and receives several invaginating processes
(stars) from unlabeled bipolar or horizontal cell
dendrites (bottom half). B,
Section of the synaptic triad within a labeled rod spherule. The
arrowhead indicates the ribbon; the three invaginating
processes (stars) are unlabeled. C, The
labeled cone pedicle base (top half) contains three
synaptic ribbons and receives several unlabeled invaginating processes.
D, Section of the synaptic triad within a labeled rod
spherule. The arrowhead indicates the ribbon; the
stars mark unlabeled invaginating processes. Scale bars,
0.3 µm.
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Localization of PSD-95 in the IPL of the rat retina
PSD-95 in the IPL has a punctate appearance (see Fig. 1). Such
punctate immunofluorescence suggests a clustering of PSD-95 in synaptic
hot spots. This is shown more directly by the electron micrographs in
Figure 4. The nine micrographs show the
axon terminals of different types of bipolar cells: three OFF cone
bipolar cells, which terminate in the outer half of the IPL (see Fig.
3A-C), three ON cone bipolar cells, which terminate in the
lower part of the IPL (see Fig. 3D-F), and three rod
bipolar cells, which terminate close to the ganglion cell layer (see
Fig. 3G-I). Within the bipolar cell axon terminals
the presynaptic ribbons are indicated by arrowheads. Usually two
postsynaptic processes are opposed to the ribbons; therefore, this
synaptic complex has been named a dyad (Dowling and Boycott, 1966). In
all nine dyads illustrated in Figure 4, only one of the postsynaptic
processes expressed PSD-95 immunoreactivity. This labeling pattern is
reminiscent of the localization of glutamate receptors at the dyads: a
given glutamate receptor or subunit is found only in one postsynaptic process at bipolar cell dyads [NR2A subunit, Hartveit et al. (1994); metabotropic glutamate receptors, Brandstätter et al. (1996) and
Koulen et al. (1997); kainate receptor subunits, Brandstätter et
al. (1997); for review, see Brandstätter et al. (1998)]. In all,
76 bipolar cell ribbon synapses were photographed at the ultrastructural level, and in all cases PSD-95 was restricted to one
postsynaptic process of the dyad (15 OFF cone bipolars, 19 ON cone
bipolars, and 42 rod bipolars). We did not observe PSD-95
immunoreactivity in the IPL associated with conventional chemical
synapses. Because such synapses have amacrine processes as presynaptic
partners (Dowling and Boycott, 1966), this makes important predictions
with respect to the transmitter receptors that might be associated with
PSD-95 at postsynaptic sites. Bipolar cells release glutamate, whereas
amacrine cells release GABA, glycine, or acetylcholine (Massey and
Redburn, 1987; Massey, 1990). PSD-95, which was found postsynaptic to
bipolar cells and not to amacrine cells, therefore is associated most
likely with glutamate receptors in the IPL.
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Figure 4.
Electron micrographs of sections through the IPL
of a rat retina that was immunolabeled for PSD-95. A-C,
Shown are the axon terminals of putative OFF cone bipolar cells from
the outer part of the IPL. The presynaptic ribbons are marked by
arrowheads. Only one of the two postsynaptic processes
at this ribbon synapse (dyad) appears to be labeled
(star). D-F, Shown are the axon
terminals of putative ON cone bipolar cells from the inner part of the
IPL. The presynaptic ribbons are marked by arrowheads.
Labeling is found in only one of the two postsynaptic processes.
G-I, Shown are the axon terminals of putative rod
bipolar cells close to the ganglion cell layer. The presynaptic ribbons
are opposed to two postsynaptic processes, of which one
(star) appears to be labeled. Scale bars, 0.2 µm.
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The postsynaptic clustering of PSD-95 at rod bipolar cell axon
terminals (Fig. 4G-I) also was confirmed by light
microscopy. Vertical sections of rat retinae were double-labeled for
protein kinase C (PKC) and for PSD-95. The high-power light micrograph in Figure 5A shows the PKC
immunoreactive axon terminals of rod bipolar cells in the inner IPL
(Greferath et al., 1990). The micrograph in Figure 5B shows
the PSD-95 immunoreactive puncta. As indicated by the small arrows in
Figure 5, groups of PSD-95 immunoreactive puncta are in register with
axonal varicosities of PKC-immunolabeled rod bipolar cells. Individual
varicosities are decorated by three to five puncta. In some cases the
whole axon terminal could be studied, and between 10 and 15 puncta per
rod bipolar cell axon terminal were found. Chun et al. (1993) estimated
that rat rod bipolar cells make ~15 ribbon synapses. It is,
therefore, possible that PSD-95 immunoreactive clusters are found at a
major portion of rod bipolar cell ribbon synapses.
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Figure 5.
Vertical cryostat section through a rat retina
double-labeled for PKC (A) and PSD-95
(B). The fluorescence micrographs show the inner
one-third of the IPL. A, PKC-immunolabeled axon
terminals of rod bipolar cells form small varicosities 2-3 µm in
diameter. Three are indicated by arrows.
B, PSD-95 immunofluorescence is concentrated in "hot
spots" 0.25-1 µm in diameter. The hot spots are aggregated in
groups (arrows) that correspond to the varicosities in
A. Careful inspection of this high-power light
micrograph shows that neighboring hot spots can still be resolved if
their distance is ~0.3 µm.
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Colocalization of PSD-95 and SAP 102 in the
rat retina
The family of synapse-associated proteins that have
three PDZ domains includes four closely related proteins: PSD-95/SAP 90 (Cho et al., 1992; Kistner et al., 1993), SAP 97/hdlg (Lue et al.,
1994; Müller et al., 1995), chapsyn-110/PSD-93 (Brenman et al.,
1996a; Kim et al., 1996), and SAP 102 (Müller et al., 1996).
PSD-95, PSD-93, and SAP 102 have been shown to interact with NMDA
receptors in vivo (Kornau et al., 1995; Brenman et al., 1996a,b; Müller et al., 1996). It is interesting,
therefore, to determine whether they are localized at the same synapses
or whether they are involved with different circuits. In a preceding publication (Koulen et al., 1998) we have studied the retinal distribution of SAP 102 in the rat retina. In this study we performed double-labeling experiments with specific antibodies against PSD-95 and
SAP 102, respectively. A section that was double-labeled for both
proteins is shown in Figure 6. In the OPL
PSD-95 (Fig. 6A) is found in rod spherules, whereas
SAP 102 (Fig. 6B) shows a punctate distribution. In
the IPL PSD-95 and SAP 102 show a punctate fluorescence, suggesting
that both are aggregated in synaptic hot spots. In the optic nerve
fiber layer (NFL) SAP 102 (Fig. 6B) is strongly expressed by nerve fiber bundles. The retinal expression patterns of
the two synapse-associated proteins therefore are different.
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Figure 6.
Vertical cryostat section through a rat retina
double-labeled for PSD-95 (A) and SAP 102 (B). The Nomarski micrograph in C
shows the retinal layers. A, PSD-95 immunofluorescence
is most prominent in rod spherules of the OPL. In the IPL the
fluorescence is concentrated in "hot spots." B, SAP
102 immunofluorescence is sparse in the OPL, and weak extrasynaptic
labeling is present in the INL; in the IPL the labeling is concentrated
in hot spots, and prominent labeling is also present in the optic nerve
fiber layer (NFL). Because of the short fixation the
tissue was very soft, and the cryostat sections were of uneven
thickness and appeared to be ruptured.
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The localizations of PSD-95 and SAP 102 in the OPL are shown in Figure
7 at higher magnification. As described
earlier, PSD-95 is localized in rod spherules. This is shown, albeit a
bit blurred, in Figure 7A. SAP 102 has a punctate appearance
in Figure 7B. In Figure 7C the SAP 102 immunoreactive puncta have been inserted into the micrograph of
PSD-95-labeled rod spherules at their correct position, and it becomes
apparent that the puncta are encircled by the rod spherules. This is
consistent with the localization of PSD-95 and SAP 102 as revealed by
electron microscopy. PSD-95 is found in photoreceptor terminals (see
Fig. 3), whereas SAP 102 has been localized to invaginating processes
at photoreceptor ribbon synapses and in particular to horizontal cell
processes (Koulen et al., 1998). PSD-95 and SAP 102 therefore are found at opposite sides of the photoreceptor synapses and most likely fulfill
different functions there.
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Figure 7.
High-power fluorescence micrograph of a vertical
section through the OPL of a rat retina that was double-labeled for
PSD-95 (A) and SAP 102 (B).
A, Slightly blurred rod spherules express PSD-95
immunofluorescence. B, Small puncta express SAP 102 immunoreactivity. We previously have shown by electron microscopy that
these puncta represent terminals of horizontal cell processes (Koulen
et al., 1998). C, The same micrograph as in
A, with the puncta, taken from B,
inserted as black dots.
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In the IPL both PSD-95 labeling and SAP 102 labeling have a punctate
appearance (see Fig. 6A,B), suggesting that they are localized in synaptic hot spots. This is supported by the electron micrographs in Figure 3, which show that PSD-95 is aggregated in one of
the postsynaptic processes at bipolar cell dyads. Exactly the same
localization has been described for SAP 102 (Koulen et al., 1998),
which raises the interesting question as to whether they are found in
the same process associated with the dyad.
Micrographs of the IPL of a section that was double-labeled for PSD-95
and SAP 102 are shown in Figure
8A. To visualize
possible colocalizations, we have printed mirror images of the PSD-95
and the SAP 102 immunoreactivities side by side in Figure
8A. A comparison of puncta across the midline shows
that many are at symmetrical positions, which suggests they are
colocalized. However, the white blur in Figure 8A,
which is caused partly by out-of-focus puncta and partly by the uneven
thickness of the section, makes it difficult to compare the puncta
across the midline. The puncta of Figure 8A therefore
are shown in isolation in Figure 8B.
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Figure 8.
The high-power photomicrographs (left
column, A, D, G) show the IPL of double-labeled sections
through rat retinae. PSD-95 immunofluorescence is shown in the
left side of each micrograph. Immunofluorescence for SAP
102 (A), PSD-93 (D), and
NR1 (G) are shown on the right
side and are printed as mirror images. The midline is cut along
a common border, and puncta at symmetric positions with respect to the
midline represent colocalizations. Drawings of all puncta are shown in
the middle column (B, E, H).
Drawings of puncta at symmetrical positions, representing
colocalizations, are shown in the right column
(C, F, I). The numbers of the
labeled puncta are inserted. Each frame represents
40 × 40 µm.
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Figure 8B contains 247 puncta immunoreactive for
PSD-95 and 198 puncta immunoreactive for SAP 102. It is possible that
the different numbers reflect the better staining quality of PSD-95. In
all, 134 puncta (68%) were found to be colocalized, and these are in
isolation shown in Figure 8C. Their symmetric positions with
respect to the midline become apparent. This result also was analyzed
more quantitatively, as described in Materials and Methods. High-power
micrographs were printed, and the puncta were transferred onto tracing
paper. When puncta of the same SAP 102-labeled section were transferred
twice onto separate sheets of tracing paper and when the coincidences
were counted, only 79.8 ± 2.8% instead of the theoretical 100%
was found. The reason for this lower count is the jitter introduced by
tracing the puncta and the uneven staining of the sections. When the
drawings of the SAP 102-labeled puncta were superimposed onto the
drawings of a PSD-95-labeled puncta, 68 ± 1% of the puncta
coincided. However, allowing for the intrinsic failure rate, the actual
colocalization of SAP 102 and PSD-95 must be higher than the 68%
measured, and we therefore conclude that in most instances SAP 102 and
PSD-95 are found in the same postsynaptic densities within the IPL. The optical resolution for neighboring puncta in the light micrographs was
found to be 0.3 µm (see, for example, Fig. 5B), which is
good enough to decide whether SAP 102 and PSD-95 label the same
processes at the dyads or two adjacent processes. The close
correspondence of the puncta suggests that SAP 102 and PSD-95 are
present in the same processes.
Colocalization of PSD-95 and PSD-93 in the retina
PSD-93 has been cloned recently (Brenman et al., 1996a), and its
overall amino acid identity with PSD-95 is 70%. More importantly, like
SAP 102 and PSD-95, PSD-93 contains three PDZ domains, suggesting comparable functions. However, in the cerebellum PSD-93 was found postsynaptically in Purkinje cells, whereas PSD-95 was found
presynaptically (Brenman et al., 1996b; Laube et al., 1996), suggesting
different functions of the protein. We therefore looked in
colocalization experiments (Fig. 9) for
the expression of PSD-95 and PSD-93 in the mammalian retina. As can be
seen in the low-power micrographs (Fig. 9A,B), PSD-95 and
PSD-93 have a similar distribution both in the OPL and in the IPL. In
the OPL the rod spherules show a prominent expression. In the IPL
punctate immunofluorescence is found for both PSD-93 and PSD-95. The
only qualitative difference we could detect was an expression of PSD-93
in the optic nerve fiber layer (Fig. 9B), which was absent
in the PSD-95 immunofluorescence (Fig. 9A). A similar
staining of the optic nerve fiber layer also was observed in the case
of SAP 102 (see Fig. 6). High-power micrographs of the puncta labeled
in the IPL are shown in Figure 8D. They are printed
as mirror images; hence puncta at symmetric positions with respect to
the midline represent colocalizations. It is obvious that many such
symmetric puncta are present in Figure 8D. To show this in better detail, we have drawn the puncta in isolation in Figure
8E. In Figure 8F, finally, only
those puncta that are at symmetric positions are inserted. Figure
8E contains 221 PSD-93 and 294 PSD-95 immunoreactive
puncta. Of these, 165 (75%) were found to be colocalized (see Fig.
8F).
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Figure 9.
Vertical cryostat section through a rat retina
double-labeled for PSD-95 (A) and PSD-93
(B). The labeling in the OPL is much more
prominent for PSD-95; however, close inspection in the microscope shows
that both PSD-95 and PSD-93 label rod spherules. Punctate labeling is
present in the IPL both in A and B, and
comparison of the puncta suggests that they are colocalized. This is
demonstrated in more detail in Figure 8D. The
optic nerve fiber layer expresses PSD-93 (labeling at the bottom of
B), but not PSD-95. Because of the short fixation the
IPL appears to be ruptured. Scale bar, 25 µm.
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This result also was analyzed more quantitatively, as described before.
Superposition of PSD-93 immunoreactive puncta drawn from identical
micrographs resulted in a 82.3 ± 5% coincidence rate instead of
the theoretical 100%. When drawings of PSD-93-labeled puncta were
superimposed onto the drawings of PSD-95-labeled puncta, a coincidence
rate of 65 ± 8% was found. This leads to the conclusion that
PSD-93 and PSD-95 are aggregated at the same synapses in the IPL.
Colocalization of PSD-95 and NMDA receptors
The expression of NMDA receptors (NRs) in mammalian retinae has
been studied by in situ hybridization (Brandstätter et
al., 1994; Watanabe et al., 1994). In the ganglion cell layer virtually every ganglion cell or displaced amacrine cell expressed the receptor subunits NR1, NR2A, NR2B, and NR2C. In the inner nuclear layer the
receptor subunit NR1 was distributed homogeneously and therefore possibly is expressed by all cell types in this layer. The NR2A, NR2B,
and NR2C subunits were expressed in subsets of amacrine cells; the NR2C
subunit also was found at the location of bipolar cell perikarya
(Brandstätter et al., 1994). The NR1, the NR2A, and the NR2D
subunits have been localized immunocytochemically (Hartveit et al.,
1994; Wenzel et al., 1997a; Lo et al., 1998; Wong-Riley, 1998). Strong
punctate NR2A immunofluorescence was observed in the IPL, and the
application of electron microscopy showed that the NR2A subunit is
aggregated at postsynaptic sites (Hartveit et al., 1994). In the case
of the NR2D subunit, Wenzel et al. (1997a) reported extrasynaptic
labeling of rod bipolar cells. NR1 immunoreactivity was observed in
ganglion cells, in amacrine cells, and in both plexiform layers
(Wong-Riley et al., 1998). Lo et al. (1998) reported the presence of
four different splice variants of NR1 and described the
immunocytochemical labeling of rod bipolar and Müller cells.
In other parts of the CNS, SAP 102 or PSD-95 has been found to be
associated with NMDA receptors (Kornau et al., 1995; Müller et
al., 1996; Niethammer et al., 1996). The NR1 subunit is the most widely
distributed NMDA receptor subunit in the brain (Hollmann and Heinemann,
1994) and in the retina (Brandstätter et al., 1994). Only when
they are coexpressed with the NR1 subunit do NR2 subunits form
functioning channels in artificial expression systems. This suggests
that NMDA receptors at synapses contain either homomeric combinations
of NR1 or heteromeric combinations of NR1 and other subunits (Hollmann
and Heinemann, 1994). Therefore, double-labeling experiments were
performed for NR1 and PSD-95 to show the possible colocalization of
NMDA receptors and PSD-95 in the mammalian retina. We found a high
degree at colocalization between PSD-95 and NR1. However, it should be
emphasized that this may not represent the direct binding of PSD-95
with NR1; rather, it may be the consequence of the association of NR1
with NR2, which has been shown recently to be the binding partner of PSD-95 (Kim et al., 1995; Kornau et al., 1995).
The results are shown in Figure 10. The
most prominent labeling for the NR1 subunit is found in the IPL. The
labeling there is punctate (Fig. 10B), with some
indication of separate layers. The perikarya of ganglion cells and
possibly of displaced amacrine cells are labeled in the ganglion cell
layer. Some amacrine cells (Fig. 10B, arrow) are
labeled distinctly; others are labeled more faintly. Weak labeling is
also present in the OPL and in some cell bodies at the outer margin of
the inner nuclear layer (Fig. 10B, arrowhead).
Clearly, this weak labeling in the OPL is not related to the prominent
labeling of rod spherules and cone pedicles by PSD-95 (Fig.
10A).
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Figure 10.
Vertical cryostat section through a rat retina
double-labeled for PSD-95 (A) and the NR1 subunit
of the NMDA receptor (B). In addition to the
punctate labeling in the IPL, NR1 immunofluorescence is also present in
cell bodies of the ganglion cell layer and in some amacrine cells
(arrow). Weak and sparse labeling is also present in the
OPL; a putative horizontal cell body is indicated by the
arrowhead. Because of the short fixation the IPL appears
to be ruptured. Scale bar, 25 µm.
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To show the possible colocalization in the IPL, we have shown
high-power micrographs of PSD-95 and the NR1 subunit in Figure 8G. They are printed as mirror images for better
visualization of colocalizations. Many puncta appear at symmetrical
positions with respect to the midline. This becomes more obvious from
Figure 8H, where the puncta are shown in isolation:
332 PSD-95 immunoreactive puncta are opposed to 221 NR1 immunoreactive
puncta. In Figure 8I only the 148 puncta that would
coincide are shown, and this suggests that 67% of the NR1 puncta also
express PSD-95.
As described before, the colocalizations also were measured more
quantitatively by transferring puncta from double-labeled sections onto
tracing paper and creating superpositions of the images. When puncta of
the same NR1-labeled micrograph were transferred twice onto separate
sheets of tracing paper and when the coincidences were counted, this
result should have been a coincidence rate of 100%; however, only
81 ± 5% was found. The staining quality of the NR1 puncta and
the resulting positional errors cause this deviation from the
theoretical prediction. When the NR1 puncta were superimposed onto the
PSD-95 puncta at their correct position, 65 ± 3% of the puncta
coincided. This shows that PSD-95 is a constituent of the postsynaptic
density in the majority of NR1-expressing synapses.
| |
DISCUSSION |
Localization of PSD-95 in the outer retina
There was a strong expression of PSD-95 in both
cone and rod photoreceptors. Labeling was observed all along their
descending axons but was most prominent in cone pedicle and rod
spherule membranes. We have never observed before, with any antigen,
such a strong fluorescent signal. The labeling was confined to the presynaptic site of the photoreceptor synapse; however, it was not
restricted there to the region around the ribbons but was found all
over the pedicle or spherule. One might argue that the invaginating
processes of horizontal cells provide a feedback signal, and thus the
PSD-95 in photoreceptors would be located postsynaptically to
horizontal cells (for review, see Piccolino, 1995; Yazulla, 1995). We
are not in favor of this view, because PSD-95 is not clustered at the
contacts between photoreceptors and horizontal cell terminals but is
found all around rod spherules and cone pedicles.
A variety of K+ channels,
Ca2+-activated chloride channels, and
Ca2+ channels has been described in photoreceptor
inner segments (for review, see Barnes, 1994), and they are candidates
to be associated with PSD-95. However, recent immunocytochemical
localizations of Shaker-like K+ channels did not
describe a labeling of cone pedicle or rod spherule membranes (Klumpp
et al., 1995a,b). Therefore, PSD-95 may not be associated in the
photoreceptor terminals with Shaker-like K+ channels
as has been found in other parts of the CNS (Kim et al., 1995; Laube et
al., 1996). Similarly, an association of PSD-95 with NMDA receptors in
photoreceptor terminals can be excluded. We did not observe any
significant staining of photoreceptors with the antibodies against the
NR1 subunit (see Fig. 10). A comparable negative result was found for
the NR2A subunit (Hartveit et al., 1994), the NR2B subunit (our
unpublished observations), or the NR2D subunit (Wenzel et al., 1997a).
Hence proteins other than NMDA receptors or K+
channels may be associated with PSD-95 in photoreceptor terminals. Neuronal nitric oxide synthase (nNOS) has been found to be colocalized with PSD-95 in many neuronal populations (Brenman et al., 1996b). NADPH
diaphorase histochemistry, an indicator of nNOS-containing neurons,
applied to the retina did not reveal a preferential staining of
photoreceptor terminals (Sagar, 1986). Recently, macaque monkey retinal
sections have been immunostained for nNOS (Wong-Riley et al., 1998),
and strong immunoreactivity was observed in the OPL. Unfortunately, the
resolution in the published micrographs is too low to detect the
labeling of cone pedicles or rod spherules. Our lab recently has
studied the calcium extrusion from photoreceptor terminals and found
that this is mediated preferentially by the plasma membrane calcium
ATPase, which was enriched in rod and cone terminals [see Morgans et
al. (1998), their Fig. 1B]. However, in cone
pedicles only the neck region showed strong staining, whereas the
synaptic region, the pedicle base, was labeled only weakly. The pedicle
base was strongly immunoreactive for Ca2+ channels.
This shows that we may have to consider more exotic partners to be
associated with PSD-95 in photoreceptor terminals or that PSD-95 may
fulfill separate roles in different compartments within the terminals.
At the pedicle neck it could be associated with one sort of molecule
and at the pedicle base with some other type.
Localization of PSD-95 in the inner retina
The distribution of PSD-95 in the IPL had a punctate appearance,
which was shown by electron microscopy to represent a clustering of
PSD-95 in terminals postsynaptic to bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members at the
dyad was labeled. Because of the short fixation time and the low
concentration of glutaraldehyde in the fixative, the tissue preservation was compromised. Therefore, we were not able to decide whether at cone bipolar cell dyads both ganglion and amacrine cell
dendrites were labeled (Dowling and Boycott, 1966). Similarly, we could
not recognize the two postsynaptic processes at rod bipolar cells, the
AI and AII amacrine cells (Famiglietti and Kolb, 1975). However, the
colocalization of PSD-95 immunoreactive puncta with NR1 immunoreactive
puncta could provide the answer. In Figure 10B
amacrine and ganglion cell perikarya were found to be immunoreactive for the NR1 subunits. They almost certainly represent the parent cell
bodies for the NR1 puncta in the IPL. Consequently, we suggest that
both amacrine and ganglion cells may express PSD-95 at the synapses
they receive from bipolar cells.
Double-labeling experiments were performed to determine whether PSD-95
participates in the clustering of NMDA receptors in the retina, as has
been suggested by biochemical studies and immunostaining in other parts
of the brain (Kim et al., 1995; Kornau et al., 1995; Niethammer et al.,
1996). The NR1 subunit is not only the most abundant subunit in the
brain (Wenzel et al., 1995, 1997b) but is also a necessary constituent
of functioning NMDA receptor channels (Hollmann and Heinemann, 1994).
Although the direct interaction between NMDA receptor channels and
PSD-95 is supposed to be through the NR2 subunits (Kennedy, 1997), such
channels also must contain NR1 subunits. After many failures with other
fixation protocols we finally succeeded in immunolabeling retinal
sections for the NR1 subunit by using a carbodiimide fixation. This
resulted in prominent punctate (synaptic) labeling in the IPL and in
weaker extrasynaptic labeling of the parent cell bodies. This is in
contrast to the widely distributed and diffuse labeling that Wong-Riley et al. (1998) reported for the NR1 subunit in the primate retina. It
also differs from the labeling rod bipolar and Müller cells reported by Lo et al. (1998) for the mouse retina. This difference might be caused by the different fixation protocols that were applied
and by different specificities of the antibodies that were used. Our
double-labeling experiments for PSD-95 and the NR1 subunit showed
that the majority of NR1 immunoreactive clusters also expressed
punctate PSD-95 immunoreactivity. Unfortunately, we cannot be more
precise at present for the following reason. The punctate staining for
PSD-95 was always crisp, and it was easy to recognize and draw the
puncta. The definition of puncta was not so clear in the case of NR1
immunolabeling (see Fig. 8G). More puncta were found that
express PSD-95 immunoreactivity, and fewer expressed NR1
immunoreactivity. Whether this reflects merely the staining quality or
whether PSD-95 is associated in the IPL with channels other than NMDA
receptors is presently uncertain. It is also unclear whether there is a
subpopulation of NMDA receptors that is not associated with PSD-95.
However, only one of the two postsynaptic members at the bipolar cell
ribbon synapses expressed PSD-95. Both postsynaptic processes must
express glutamate receptors, which leads to the conclusion that PSD-95
is associated only with a subset of the glutamate receptors.
Localization of PSD-95, PSD-93, and SAP 102 in the
mammalian retina
In the IPL punctate immunolabeling was found for all three
synapse-associated proteins. This suggests that all three are
aggregated at synaptic sites. A postsynaptic localization has been
shown by electron microscopy in our previous study for SAP 102 (Koulen et al., 1998) and in the present study for PSD-95. Because most of the
PSD-93 immunoreactive puncta are colocalized with PSD-95, this also
implies a postsynaptic localization of PSD-93. The colocalization experiments PSD-95/PSD-93, PSD-95/SAP 102, and PSD-95/NR1 suggest that
in many postsynaptic sites these proteins are aggregated together. This
raises the interesting question as to why there are three proteins that
are structurally and functionally so similar in the same postsynaptic
density and suggests that they fulfill different roles. This is
supported further if one compares the localization of the three
proteins outside the IPL.
In the OPL PSD-95 is strongly expressed in the photoreceptor terminals.
PSD-93 also is found in photoreceptor terminals, but the intensity of
the fluorescence is substantially lower than for PSD-95 (compare Fig.
9, A with B). SAP 102 was found in a postsynaptic
position in the OPL (see Fig. 7). A weak expression of SAP 102 also was
found in extrasynaptic membranes of cells in the inner nuclear layer,
where it possibly is associated with K+ channels
(Koulen et al., 1998). A difference also was observed with respect to
the labeling of the optic nerve fibers. PSD-93 and SAP 102 were
expressed in the nerve fiber layer, whereas PSD-95 could not be
detected there. A possible function of PSD-93 and SAP 102 in the nerve
fiber layer is the anchoring of K+ channels. The
nerve fibers in the retina are not myelinated, and a smooth
distribution of K+ channels along the fibers
therefore is expected, which is in agreement with the labeling pattern
observed for SAP 102 (see Fig. 6B) and PSD-93 (see
Fig. 9).
We also have studied the postnatal development of PSD-95 in the rat
retina by light microscopy. The punctate immunofluorescence was
observed immediately after birth (P0) and apparently precedes the
formation of ribbon synapses (Sassoè-Pognetto and Wässle, 1997). This would suggest that PSD-95 not only is involved with the
aggregation of transmitter (NMDA) receptors at synaptic sites or the
modulation of synapses but also plays an important role in the
generation of synapses.
| |
FOOTNOTES |
Received July 8, 1998; revised Sept. 8, 1998; accepted Sept. 15, 1998.
This work was supported by Deutsche Forschungsgemeinschaft Grant SFB
269/B4, National Health and Medical Research Council (Australia),
C. J. Martin Fellowship (to E.L.F.), and National Institutes of
Health Grant RO1 GM36017. We thank F. Boij, A. Hildebrand, W. Hofer,
and G. S. Nam for excellent technical assistance; Dr. C. C. Garner for providing antibodies; and I. Odenthal for typing this manuscript.
Correspondence should be addressed to Dr. Heinz Wässle,
Max-Planck-Institut für Hirnforschung, Neuroanatomie,
Deutschordenstrasse 46, D-60528 Frankfurt, Germany.
| |
REFERENCES |
-
Barnes S
(1994)
After transduction: response shaping and control of transmission by ion channels of the photoreceptor inner segment.
Neuroscience
58:447-459[Web of Science][Medline].
-
Brandstätter JH,
Hartveit E,
Sassoè-Pognetto M,
Wässle H
(1994)
Expression of NMDA and high-affinity kainate receptor subunit mRNAs in the adult rat retina.
Eur J Neurosci
6:1100-1112[Web of Science][Medline].
-
Brandstätter JH,
Koulen P,
Kuhn R,
van der Putten H,
Wässle H
(1996)
Compartmental localization of a metabotropic glutamate receptor (mGluR7): two different active sites at a retinal synapse.
J Neurosci
16:4749-4756[Abstract/Free Full Text].
-
Brandstätter JH,
Koulen P,
Wässle H
(1997)
Selective synaptic distribution of kainate receptor subunits in the two plexiform layers of the rat retina.
J Neurosci
17:9298-9307[Abstract/Free Full Text].
-
Brandstätter JH,
Koulen P,
Wässle H
(1998)
Diversity of glutamate receptors in the mammalian retina.
Vision Res
38:1385-1397[Web of Science][Medline].
-
Brenman JE,
Christopherson KS,
Craven SE,
McGee AW,
Bredt DS
(1996a)
Cloning and characterization of postsynaptic density 93, a nitric oxide synthase interacting protein.
J Neurosci
16:7407-7415[Abstract/Free Full Text].
-
Brenman JE,
Chao DS,
Gree SH,
McGee AW,
Craven SE,
Santillano DR,
Huang F,
Xia H,
Peters MF,
Froehner SC,
Bredt DS
(1996b)
Interaction of nitric oxide synthase with the synaptic density protein PSD-95 and -1 syntrophin mediated by PDZ motifs.
Cell
84:757-767[Web of Science][Medline].
-
Cho K-O,
Hunt CA,
Kennedy MB
(1992)
The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.
Neuron
9:929-942[Web of Science][Medline].
-
Chun M-H,
Han S-H,
Chung J-W,
Wässle H
(1993)
Electron microscopic analysis of the rod pathway of the rat retina.
J Comp Neurol
332:421-432[Web of Science][Medline].
-
Dowling JE,
Boycott BB
(1966)
Organization of the primate retina: electron microscopy.
Proc R Soc Lond [Biol]
166:80-111[Medline].
-
Famiglietti EV,
Kolb H
(1975)
A bistratified amacrine cell and synaptic circuitry in the inner plexiform layer of the retina.
Brain Res
84:293-300[Web of Science][Medline].
-
Gomperts SN
(1996)
Clustering membrane proteins: it's all coming together with the PSD-95/SAP90 protein family.
Cell
84:659-662[Web of Science][Medline].
-
Greferath U,
Grünert U,
Wässle H
(1990)
Rod bipolar cells in the mammalian retina show protein kinase C-like immunoreactivity.
J Comp Neurol
301:433-442[Web of Science][Medline].
-
Grünert U,
Wässle H
(1993)
Immunocytochemical localization of glycine receptors in the mammalian retina.
J Comp Neurol
335:523-537[Web of Science][Medline].
-
Hartveit E,
Brandstätter JH,
Sassoè-Pognetto M,
Laurie DJ,
Seeburg PH,
Wässle H
(1994)
Localization and developmental expression of the NMDA receptor subunit NR2A in the mammalian retina.
J Comp Neurol
348:570-582[Web of Science][Medline].
-
Hollmann M,
Heinemann S
(1994)
Cloned glutamate receptors.
Annu Rev Neurosci
17:31-108[Web of Science][Medline].
-
Hunt CA,
Schenker LJ,
Kennedy MB
(1996)
PSD-95 is associated with the postsynaptic density and not with the presynaptic membrane at forebrain synapses.
J Neurosci
16:1380-1388[Abstract/Free Full Text].
-
Irie M,
Hata Y,
Takeuchi M,
Ichtchenko K,
Toyoda A,
Hirao K,
Takai Y,
Rosahl TW,
Südhof TC
(1997)
Binding of neuroligins to PSD-95.
Science
277:1511-1515[Abstract/Free Full Text].
-
Kennedy MB
(1997)
The postsynaptic density at glutamatergic synapses.
Trends Neurosci
20:264-268[Web of Science][Medline].
-
Kim E,
Niethammer M,
Rothschild A,
Jan YN,
Sheng M
(1995)
Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases.
Nature
378:85-88[Medline].
-
Kim E,
Cho KO,
Rothschild A,
Sheng M
(1996)
Heteromultimerization and NMDA receptor-clustering activity of chapsyn-110, a member of the PSD-95 family of proteins.
Neuron
17:103-113[Web of Science][Medline].
-
Kirsch J,
Betz H
(1998)
Glycine-receptor activation is required for receptor clustering in spinal neurons.
Nature
392:717-720[Medline].
-
Kistner U,
Wenzel BM,
Veh RW,
Cases-Langhoff C,
Garner AM,
Appeltauer U,
Voss B,
Gundelfinger ED,
Garner CC
(1993)
SAP 90, a rat presynaptic protein related to the product of the Drosophila tumor suppressor gene dlg-A.
J Biol Chem
268:4580-4583[Abstract/Free Full Text].
-
Klumpp DJ,
Song EJ,
Pinto LH
(1995a)
Identification and localization of K+ channels in the mouse retina.
Vis Neurosci
12:1177-1190[Web of Science][Medline].
-
Klumpp DJ,
Song EJ,
Ito S,
Sheng MH,
Jan LY,
Pinto LH
(1995b)
The Shaker-like potassium channels of the mouse rod bipolar cell and their contributions to the membrane current.
J Neurosci
15:5004-5013[Abstract].
-
Kornau H-C,
Schenker LT,
Kennedy MB,
Seeburg PH
(1995)
Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95.
Science
269:1737-1740[Abstract/Free Full Text].
-
Koulen P,
Kuhn R,
Wässle H,
Brandstätter JH
(1997)
Group I metabotropic glutamate receptors mGluR1 and mGluR5: localization in both synaptic layers of the rat retina.
J Neurosci
17:2200-2211[Abstract/Free Full Text].
-
Koulen P,
Garner CC,
Wässle H
(1998)
Immunocytochemical localization of the synapse-associated protein SAP 102 in the rat retina.
J Comp Neurol
397:326-336[Web of Science][Medline].
-
Laube G,
Röper J,
Pitt JC,
Sewing S,
Kistner U,
Garner CC,
Pongs O,
Veh RW
(1996)
Ultrastructural localization of Shaker-related potassium channel subunits and synapse-associated protein 90 to septate-like junctions in rat cerebellar Pinceaux.
Mol Brain Res
42:51-61[Medline].
-
Lo W,
Molloy R,
Hughes TE
(1998)
Ionotropic glutamate receptors in the retina: moving from molecules to circuits.
Vision Res
38:1399-1410[Web of Science][Medline].
-
Lue RA,
Marfatia SM,
Branton D,
Chishti AH
(1994)
Cloning and characterization of hdlg: the human homologue of the Drosophila discs-large tumor suppressor binds to protein 4.1.
Proc Natl Acad Sci USA
91:9818-9822[Abstract/Free Full Text].
-
Mammen AL,
Huganir RL,
O'Brien RJ
(1997)
Redistribution and stabilization of cell surface glutamate receptors during synapse formation.
J Neurosci
17:7351-7358[Abstract/Free Full Text].
-
Massey SC
(1990)
Cell types using glutamate as a neurotransmitter in the vertebrate retina.
In: Progress in retinal research (Osborne N,
Chader G,
eds), pp 399-426. Oxford: Pergamon.
-
Massey SC,
Redburn DA
(1987)
Transmitter circuits in the vertebrate retina.
Prog Neurobiol
28:55-96[Medline].
-
Morgans CW,
El Far O,
Berntson A,
Wässle H,
Taylor WR
(1998)
Calcium extrusion from mammalian photoreceptor terminals.
J Neurosci
18:2467-2474[Abstract/Free Full Text].
-
Müller B,
Peichl L
(1989)
Topography of cones and rods in the tree shrew retina.
J Comp Neurol
282:581-594[Web of Science][Medline].
-
Müller BM,
Kistner U,
Veh RW,
Cases-Langhoff C,
Becker B,
Gundelfinger ED,
Garner CC
(1995)
Molecular characterization and spatial distribution of SAP97, a novel presynaptic protein homologous to SAP90 and the Drosophila discs-large tumor suppressor protein.
J Neurosci
15:2354-2366[Abstract].
-
Müller BM,
Kistner U,
Kindler S,
Chung WJ,
Kuhlendahl S,
Lau L-F,
Veh RW,
Huganir RL,
Gundelfinger ED,
Garner CC
(1996)
SAP 102, a novel postsynaptic protein that interacts with the cytoplasmic tail of the NMDA receptor subunit NR2B.
Neuron
17:255-265[Web of Science][Medline].
-
Niethammer M,
Kim E,
Sheng M
(1996)
Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases.
J Neurosci
16:2157-2163[Abstract/Free Full Text].
-
Piccolino M
(1995)
Cross-talk between cones and horizontal cells through the feedback circuit.
In: Neurobiology and clinical aspects of the outer retina (Djamgoz MBA,
Archer SN,
Vallerga S,
eds), pp 221-248. London: Chapman and Hall.
-
Rao A,
Craig AM
(1997)
Activity regulates the synaptic localization of the NMDA receptor in hippocampal neurons.
Neuron
19:801-812[Web of Science][Medline].
-
Sagar SM
(1986)
NADPH diaphorase histochemistry in the rabbit retina.
Brain Res
373:153-158[Web of Science][Medline].
-
Sassoè-Pognetto M,
Wässle H
(1997)
Synaptogenesis in the rat retina: subcellular localization of glycine receptors, GABAA receptors, and the anchoring protein gephyrin.
J Comp Neurol
381:158-174[Web of Science][Medline].
-
Sassoè-Pognetto M,
Wässle H,
Grünert U
(1994)
Glycinergic synapses in the rod pathway of the rat retina: cone bipolar cells express the 1 subunit of the glycine receptor.
J Neurosci
14:5131-5146[Abstract].
-
Sheng M
(1996)
PDZs and receptor/channel clustering: rounding up the latest suspects.
Neuron
17:575-578[Web of Science][Medline].
-
Szél A,
Röhlich P,
Caffé AR,
van Veen T
(1996)
Distribution of cone photoreceptors in the mammalian retina.
Microsc Res Tech
35:445-462[Web of Science][Medline].
-
Wässle H,
Koulen P,
Brandstätter JH,
Fletcher EL,
Becker C-M
(1998)
Glycine and GABA receptors in the mammalian retina.
Vision Res
38:1411-1430[Web of Science][Medline].
-
Watanabe M,
Mishina M,
Inoue Y
(1994)
Differential distributions of the NMDA receptor channel subunit mRNAs in the mouse retina.
Brain Res
634:328-332[Web of Science][Medline].
-
Wenzel A,
Scheurer L,
Künzi R,
Fritschy JM,
Möhler H,
Benke D
(1995)
Distribution of NMDA receptor subunit proteins NR2A, 2B, 2C, and 2D in rat brain.
NeuroReport
7:45-48[Web of Science][Medline].
-
Wenzel A,
Benke D,
Möhler H,
Fritschy J-M
(1997a)
N-methyl-D-aspartate receptors containing the NR2D subunit in the retina are selectively expressed in rod bipolar cells.
Neuroscience
78:1105-1112[Web of Science][Medline].
-
Wenzel A,
Fritschy JM,
Möhler H,
Benke D
(1997b)
NMDA receptor heterogeneity during postnatal development of the rat brain: differential expression of the NR2A, NR2B, and NR2C subunit proteins.
J Neurochem
68:469-478[Web of Science][Medline].
-
Wong-Riley MTT,
Huang Z,
Liebl W,
Nie F,
Xu H,
Zhang C
(1998)
Neurochemical organization of the macaque retina: effect of TTX on levels and gene expression of cytochrome oxidase and nitric oxide synthase and on the immunoreactivity of Na+ K+ ATPase and NMDA receptor subunit I.
Vision Res
38:1455-1477[Web of Science][Medline].
-
Yazulla S
(1995)
Neurotransmitter release from horizontal cells.
In: Neurobiology and clinical aspects of the outer retina (Djamgoz MBA,
Archer SN,
Vallerga S,
eds), pp 249-271. London: Chapman and Hall.
Copyright © 1998 Society for Neuroscience 0270-6474/98/182310136-14$05.00/0
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Top
Abstract
Introduction
