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-
fluorescence signal due to presence of PSD-95 appears as a 3.1. PSD-95 is present in the OPL of normal and
thick band in the OPL Fig. 2a. Furthermore, double- transgenic swine
labeling of PSD-95 and cone transducin reveals two separate but adjacent ‘layers’ in this thick band of the
Consistent with its expected localization in rod and cone OPL: a layer of rod terminals in the outer margin red-
Fig. 1. PSD-95 is present in the OPL of normal and transgenic swine retinas. Cross sections of 2-week-old normal A and P347L B retinas stained with anti-PSD-95 antibodies using immunoperoxidase cytochemistry. RPE, retinal pigment epithelium; PRL, photoreceptor layer; ONL, outer nuclear layer;
OPL, outer plexiform layer; INL, inner nuclear layer. Scale550 mm.
56 S
Fig. 2. PSD-95 is selectively lost from the rod terminals of young transgenic swine. Cross sections of 2-week-old normal A,B,C and P347L D,E,F retinas stained with anti-PSD-95 red and anti-cone transducin green antibodies using confocal microscopy. Arrowhead marks a cone pedicle. Arrowhead
with asterisk marks a cluster of rod terminals. Refer to Fig. 1 for other symbols. Scale550 mm.
staining only because rods do not express cone transducin The rod terminal layer consists of irregularly staggered
and a layer of cone terminals in the inner margin yel- individual rod spherules, which have diameters on the
lowish-green staining due to colocalization of the red and order of 1 mm [17]. At this magnification Fig. 2a, they
green signals derived from the presence of both PSD-95 appear as a row of red-stained clusters. One such cluster is
and cone transducin. identified by the upper arrow marked by an asterisk in Fig.
S .M. Blackmon et al. Brain Research 885 2000 53 –61
57
Fig. 3. Cones continue to express PSD-95 in late stages of retinal degeneration. Cross sections of 10-month-old normal A and P347L B retinas stained with anti-PSD-95 and anti-cone transducin antibodies using confocal microscopy. IPL, inner plexiform layer; refer to Fig. 1 for other symbols. Scale550
mm.
Fig. 4. The loss of PSD-95 from the rod terminals of transgenic swine is observable early in the neonatal period. Cross sections of newborn normal A, newborn P347L B, and 5-day-old P347L C retinas stained with anti-PSD-95 and anti-cone transducin antibodies using confocal microscopy. Refer to
Fig. 1 for symbols. Scale550 mm.
Fig. 5. Rod cell bodies are still present in 2-week-old transgenic retinas. Cross section of 2-week-old P347L transgenic swine retina stained with anti-rhodopsin antibody. Arrows point to rod outer segments, many of which remain. Arrowhead indicates the presence of rhodopsin in the rod synaptic
terminal. Asterisk denotes the outer limiting membrane. Refer to Fig. 1 for other symbols. Scale520 mm.
58 S
2a. For comparison, a cone terminal is marked by the comparing 2-week-old Fig. 2 and 10-month-old Fig. 3
lower arrow in Fig. 2a. Individual red-stained rod retinas, then, PSD-95 is selectively and completely lost
spherules can be resolved at a higher magnification, as from rod terminals early in postnatal life but continues to
explained in Section 2.4 of Materials and methods and be expressed in cone terminals, even in late stages of
illustrated in Fig. 2c upper arrow marked by an asterisk. retinal degeneration.
Identification of rod and cone terminals is further demonstrated by comparing Fig. 2b and Fig. 2c, which
3.3. Loss of PSD-95 immunoreactivity occurs before correspond to showing the two immunofluorescence labels
substantial rod degeneration separately and at a higher magnification of the same
2-week-old normal retina as shown in Fig. 2a. The upper Studies performed in neonatal swine show that the loss
arrows in Fig. 2b and Fig. 2c point to a cluster of rod of PSD-95 is observable even in newborn animals. In the
spherules immunoreactive for PSD-95 Fig. 2c but not for normal retina, both rod and cone terminals express PSD-95
cone transducin Fig. 2b. The lower arrows in Fig. 2b and Fig. 4a. Double-labeling immunofluorescence signals
Fig. 2c point to a cone terminal that is immunoreactive for show a pattern typical of normal retinas Figs. 2a and 3a.
both cone transducin and PSD-95. The signal due to The rod terminals are stained red only whereas the cone
presence of PSD-95 is stronger in the outer portions of the terminals appear yellowish-green. In contrast, in the trans-
cone terminal. genic retina, while there are still a small number of rod
In the 2-week-old transgenic porcine retina, similar terminals expressing PSD-95, most of them are no longer
double-labeling immunofluorescence studies using PSD-95 PSD-95 immunoreactive Fig. 4b. By postnatal day 5,
and cone transducin as markers identified very few red- even fewer PSD-95 immunoreactive rod terminals are
staining rod terminals; instead, only a single row of observed Fig. 4c. In these neonatal transgenic retinas,
yellowish-green photoreceptor terminals remains Fig. 2d. almost all the terminals appear yellowish-green, suggesting
Since these terminals appear yellowish-green due to the that they belong to cones.
presence of both the red and green signals derived from Results shown in Figs. 2 and 4 indicate an early,
PSD-95 and cone transducin, respectively, they must selective loss of PSD-95 from rod terminals. At the ages
belong to cones. The presence of only cone terminals in when loss of PSD-95 occurs, it is expected that most of the
the transgenic retina is further demonstrated by comparing rods still remain and, at least morphologically, appear
Fig. 2e and f, which correspond to showing the two healthy [20,26,29]. Immunofluorescence labeling with
immunofluorescence labels separately and at a higher antibodies specific for rhodopsin illustrates the condition of
magnification of the same 2-week-old transgenic retina as the 2-week-old transgenic retina Fig. 5. In normal
shown in Fig. 2d. As shown in Fig. 2e and f, for every porcine retinas, intense rhodopsin immunoreactivity is seen
PSD-95-staining terminal, there is corresponding immuno- in the rod outer segments with relatively weak staining
reactivity for cone transducin. Therefore, PSD-95 immuno- elsewhere in the rod cell bodies. These data are very
reactivity is lost from virtually all the rod terminals but similar to those shown in Ref. [20]. In transgenic retinas,
continues to be expressed in cone terminals. in contrast, rhodopsin is delocalized, appearing in outer
As illustrated by the immunofluorescence signal in Fig. and inner segments, nuclei, and synaptic terminals. This
2a and c normal retina, PSD-95 appears to concentrate in expected pattern of rhodopsin immunoreactivity [20] is
the outer portions of the cone pedicles, leaving a non- shown in Fig. 5. A large number of rod cell bodies are
stained interior. In comparison, the non-stained interior is present, and these rods still retain their outer segments,
much smaller in the cone pedicles of a transgenic retina of which are generally shortened. Numerous rod synaptic
the same age Fig. 2d and f; thus, the cone pedicles of the terminals can also be observed, although they are no longer
transgenic retina appear more compacted than those in PSD-95 immunoreactive.
normal retinas. This finding is consistent with results Results such as those shown in Figs. 2, 4 and 5, were
obtained from previous electron microscopic studies, in used to establish the quantitative relationship between the
which shrinking of the cone terminals in the transgenic loss of PSD-95 immunoreactivity and the number of
swine was observed [20,29]. remaining rod photoreceptors by the procedures described
In rhodopsin P347L transgenic swine, the cones undergo in Section 2.4 of Materials and methods. For standardiza-
protracted degeneration. By the age of 10 months, trans- tion in comparison, the values given in Table 1 are
genic retinas are virtually devoid of rods and the cones are expressed as percent remaining relative to the value in the
grossly abnormal, having lost their outer segments due to newborn normal retina. The data presented in Fig. 6 and
severe degeneration [20,26,29]. In spite of these con- Table 1 support our interpretation that loss of PSD-95 from
ditions, immunofluorescence signal due to the presence of rod terminals occurs at a much faster pace than the loss of
PSD-95 is still observed in the terminals of the surviving rod cell bodies in the transgenic retina. For example, by
cones Fig. 3b. These terminals belong to cones because postnatal day 5, greater than 80 of rod cell bodies still
they are stained yellowish-green, which provides evidence remain but there is an approximately 90 reduction in
for the presence of both PSD-95 and cone transducin. In PSD-95 immunoreactive rod terminals.
S .M. Blackmon et al. Brain Research 885 2000 53 –61
59 Table 1
The loss of PSD-95 immunoreactivity in transgenic retinas is much greater than the concurrent loss of rod cell bodies in the same retinas PSD-95
Rod cell bodies Rod terminals stained as
Rod cell bodies remaining as of newborn normal n57
of newborn normal n56 Region of retina
Mid peripheral Central
Mid peripheral Central
Newborn normal 100
100 100
100 Newborn transgenic
30.5 22.5
92.9 92.4
5-day-old transgenic 11.2
10.8 82.4
81.2
4. Discussion loss of PSD-95 from the rod terminals appears to be
