54 S
created a new conceptual framework to address the conse- serves as a rod-specific marker, and anti-G protein-gc
quences of the rhodopsin P347L mutation, such as discern- subunit polyclonal antibody CytoSignal, referred to
ing the mechanisms that prevent the formation of the hereafter as anti-cone transducin. G protein-gc is the g
rod–rod bipolar cell synaptic connection. subunit of cone transducin and thus serves as a cone-
Synapses are highly specialized structures, the formation specific marker [23].
of which requires the interactions of dozens of unique synaptic proteins. As a preliminary screen, we performed
2.3. Immunocytochemistry PCR to identify, in normal and transgenic porcine retinas,
some of the expressed genes that encode for proteins For immunoperoxidase cytochemistry, the retinal sec-
known to be associated with synapses [31]. Among the tions were incubated with mouse monoclonal anti-PSD-95
genes identified unpublished results of Blackmon and antibody 1:200 overnight at 48C, followed by three washes
Wong, PSD-95 is present in both normal and transgenic in PBS for 15 min each. Triton X-100 0.3 was added to
porcine retinas. This finding is consistent with recently all incubation and wash buffers. The sections were then
published results showing the presence of PSD-95 in the incubated with a biotin-conjugated anti-mouse secondary
mammalian retina, including the rat, monkey, and tree antibody 1:200, Vector Laboratories in PBS for 2 h at
shrew [17]. room temperature and then washed three times in PBS for
In the studies reported here, we have used specific 15 min each, followed by incubation with an avidin–
antibodies and immunocytochemical methods to determine biotin–peroxidase complex 1:100, Vector Laboratories in
the localization of PSD-95 in rod and cone terminals in the PBS for 1 h. After three more washes of 15 min each in
porcine retina. We discovered that the rod terminals of the PBS, the staining was developed with a substrate solution
transgenic swine lose PSD-95 during early postnatal life, of 20 ml of PBS, 0.1 ml of 3 hydrogen peroxide and 10
before substantial rod degeneration has taken place. Since mg of diaminobenzidine Sigma. After washing with PBS
in the central nervous system CNS, PSD-95 is known to to terminate the staining reactions, the sections were then
play an important role in synapse formation and or coverslipped with 50 glycerol in PBS.
maintenance, our results suggest that early loss of PSD-95 For fluorescence microscopy, the retinal sections were
from the rod terminals may be causally related to the incubated with mixed primary antibodies rabbit polyclonal
absence of rod–rod bipolar cell synapses in the rhodopsin anti-cone transducin antibody 1:1000 and mouse mono-
P347L transgenic retina. clonal anti-PSD-95 antibody 1:100 overnight at 48C. After
two washes in PBS for 15 min, sections were incubated with mixed secondary antibodies rhodamine-conjugated
2. Materials and methods goat anti-mouse immunoglobulin secondary antibody 1:50
and fluorescein isothiocyanate-conjugated goat anti-rabbit 2.1. Tissue preparation
immunoglobulin secondary antibody 1:50 for 1 h at room temperature.
Transgenic swine from the line Pig-TgN1Pet [26] and For single-labeling immunocytochemistry using anti-
non-transgenic littermates were used in this study. Porcine rhodopsin antibodies, the procedure was the same except
eyes of different ages were removed from the animals that one primary antibody mouse monoclonal anti-rhodop-
killed under deep anesthesia. After removal of the anterior sin antibody 1:500 and one secondary antibody fluores-
segments, the posterior eyecups were fixed in 4 parafor- cein isothiocyanate-conjugated goat anti-mouse immuno-
maldehyde in 100 mM sodium phosphate buffer PBS, pH globulin secondary antibody 1:50 were used.
7.3 at 48C overnight. The eyecups were then frozen in For microscopic analysis, the sections were examined
isopentane cooled in liquid nitrogen, and embedded in and photographed using a 633 objective with a Zeiss
O.C.T. embedding medium Triangle Biomedical Sciences, LSM-410 confocal microscope with rhodamine isothio-
Durham, NC. Retinal sections, 10-mm thick, were pre- cyanate- and fluorescein isothiocyanate-fluorescence exci-
pared from a cryostat and mounted on gelatin-coated tations.
slides. One normal and one transgenic pig of each of the following ages were used in this study: newborn ,24-h-
2.4. Quantifications of immunoreactive rod terminals old, 5-day, 2-week, and 10-month.
and nuclei 2.2. Antisera
Fourteen micrographs of each of the sections that were double-labeled for PSD95 cone transducin were taken
A mouse monoclonal antibody clone 7E3-1B8; Affinity using the 633 objective seven from the mid-peripheral
Bioreagents, Golden, CO was used to detect PSD-95 retina and seven from the central retina. Rod synaptic
immunoreactivity in the swine retina. The specificity of terminals were counted per 200 mm of retinal section at
this antibody has previously been described [16,17]. Other high magnification using a combination of single- and
antibodies used were: anti-rhodopsin monoclonal antibody double-labeled views to differentiate rods from cones. The
a gift from P. Hargrave, University of Florida, which plane of focus in confocal imaging was finely adjusted to
S .M. Blackmon et al. Brain Research 885 2000 53 –61
55
determine the numbers of rod terminals present per high terminals also known as spherules and pedicles, respec-
magnification field. An example of this technique is shown tively, PSD-95 immunoreactivity appears as a very strong
in Fig. 2c, which identifies seven rod synaptic terminals band in the outer plexiform layer OPL of a normal retina
present within the cluster marked by the upper arrow Fig. 1a. The cellular localization of PSD-95 was further
denoted by an asterisk. determined in experiments using antibodies specific for the
Twelve micrographs of the sections that were single synaptic vesicle protein synaptophysin, which is known to
labeled for rhodopsin were taken with the 633 objective localize in the rod and cone terminals in the OPL [3].
six from the mid-peripheral retina and six from the central Using double-labelling immunocytochemistry with anti-
retina. The numbers of rod cell bodies labeled with bodies specific to PSD-95 and synaptophysin, respectively,
rhodopsin were then counted per 200 mm of retinal we showed virtually complete colocalization of these two
section. Quantitative studies of the numbers of rod cells in proteins in the OPL of normal porcine retinas at all the
transgenic porcine retinas were achieved by counting the ages tested data not shown.
rhodopsin immunoreactive nuclei Fig. 5. Staining by PSD-95 immunoreactivity is also present in the OPL of
anti-rhodopsin antibodies allowed excellent visualization the 2-week-old transgenic porcine retina Fig. 1b. Con-
of the rod cell bodies in the transgenic swine due to sistent with the expected reduction in the number of
delocalization of rhodopsin [20]. The rod cell bodies in the photoreceptors due to degeneration [20,26,29], the band of
normal retina were only weakly labeled by anti-rhodopsin PSD-95 immunoreactivity is thinner than that of a normal
staining. Therefore, the intensity of the confocal image was retina of the same age.
artificially increased so that the rod nuclei could be quantified in a similar fashion to allow comparisons
between the normal and transgenic retinas. 3.2. PSD-95 is selectively lost from the rod terminals in
Differences between the numbers of PSD-95-expressing transgenic swine
rod terminals or rhodopsin-positive rod cell bodies in normal vs. transgenic swine are reported in this study as
Rod and cone photoreceptor terminals were identified by mean6standard error. Statistical analyses were performed
double-labeling immunofluorescence confocal microscopy using the independent t-test SigmaPlot.
using antibodies specific for PSD-95 and cone transducin a cone-specific marker, respectively. In the 2-week-old
normal porcine retina, as expected from the results ob-
3. Results tained from immunoperoxidase cytochemistry, immuno-
